Progress in Retinal and Eye Research xxx (xxxx) xxx

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/preteyeres

Studies of the retinal microcirculation using human donor eyes and

high-resolution clinical imaging: Insights gained to guide future research in
diabetic retinopathy
Chandrakumar Balaratnasingam a, b, c, 1, 2, *, Dong An a, b, 1, 2, Martin Hein a, b, 1, Paula Yu a, b, 1,
Dao-Yi Yu a, b, 1
a
Lions Eye Institute, Nedlands, Western Australia, Australia
b
Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Australia
c
Department of Ophthalmology, Sir Charles Gairdner Hospital, Western Australia, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: The microcirculation plays a key role in delivering oxygen to and removing metabolic wastes from energy-
Retinal capillary plexus intensive retinal neurons. Microvascular changes are a hallmark feature of diabetic retinopathy (DR), a major
Retinal microcirculation cause of irreversible vision loss globally. Early investigators have performed landmark studies characterising the
Diabetic retinopathy
pathologic manifestations of DR. Previous works have collectively informed us of the clinical stages of DR and the
Retinal histology
retinal manifestations associated with devastating vision loss. Since these reports, major advancements in his­
Multimodal imaging
Optical coherence tomography angiography tologic techniques coupled with three-dimensional image processing has facilitated a deeper understanding of
the structural characteristics in the healthy and diseased retinal circulation. Furthermore, breakthroughs in high-
resolution retinal imaging have facilitated clinical translation of histologic knowledge to detect and monitor
progression of microcirculatory disturbances with greater precision. Isolated perfusion techniques have been
applied to human donor eyes to further our understanding of the cytoarchitectural characteristics of the normal
human retinal circulation as well as provide novel insights into the pathophysiology of DR. Histology has been
used to validate emerging in vivo retinal imaging techniques such as optical coherence tomography angiography.
This report provides an overview of our research on the human retinal microcirculation in the context of the
current ophthalmic literature. We commence by proposing a standardised histologic lexicon for characterising
the human retinal microcirculation and subsequently discuss the pathophysiologic mechanisms underlying key
manifestations of DR, with a focus on microaneurysms and retinal ischaemia. The advantages and limitations of
current retinal imaging modalities as determined using histologic validation are also presented. We conclude
with an overview of the implications of our research and provide a perspective on future directions in DR
research.

to the retinal circulation can result in devastating vision loss by limiting

1. Introduction
The retinal microcirculation is the chief source of nutrient supply to
inner retinal neurons. The choroid plays a vital role in oxygenating outer
retinal structures such as photoreceptors (Yu and Cringle, 2001; Yu
et al., 2007). Per gram weight, oxygen consumption measured within
the retina places it as one of the most metabolically active tissues in the
human body (Ames, 1992). As such, even relatively minor perturbations
energy supply to retinal neurons. Unlike most other vascular systems in
the human body, the retinal microcirculation is highly specialised and
demonstrates morphologic alterations that are correlated to the unique
energy demands of each retinal layer (Country, 2017; Yu et al., 2019a).
The organisation of the retinal microcirculation has remained conten­
tious with ongoing debate regarding the inflow and outflow pathways
that connect the superficial and deep capillary beds (Campbell et al.,
2017; Freund et al., 2018; Nesper and Fawzi, 2018). One of the reasons

* Corresponding author. Lions Eye Institute, University of Western Australia, 2 Verdun Street, Nedlands, WA, 6009, Australia.
E-mail address: chandrabalaratnasingam@lei.org.au (C. Balaratnasingam).
1
Percentage of work contributed by each author in the production of the manuscript is as follows: Chandrakumar Balaratnasingam (35%); Dong An (35%); Martin
Hein (20%); Paula Yu (5%); Dao-Yi Yu (5%).
2
CB and DA contributed equally to the work presented here and should be regarded as equivalent first authors.

https://doi.org/10.1016/j.preteyeres.2022.101134
Received 23 May 2022; Received in revised form 18 September 2022; Accepted 3 October 2022
1350-9462/© 2022 Elsevier Ltd. All rights reserved.

Please cite this article as: Chandrakumar Balaratnasingam, Progress in Retinal and Eye Research, https://doi.org/10.1016/j.preteyeres.2022.101134
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Abbreviations ICGA – Indocyanine green angiography

ICP Intermediate capillary plexus
AMD – Age-related macular degeneration ILM – Inner limiting membrane
AO – Adaptive optics INL Inner nuclear layer
AOSLO – Adaptive optics scanning laser ophthalmoscope IPL – Inner plexiform layer
BRB – Brain retinal barrier IRMA – Intra-retinal microvascular abnormality
CNS – Central nervous system MRI – Magnetic resonance imaging
DCCT – Diabetes control and complications trial NFL Nerve fibre layer
DM – Diabetes mellitus NPDR – Non-proliferative diabetic retinopathy
DMO – Diabetic macular oedema OCT – Optical coherence tomography
DPN – Diabetic peripheral neuropathy OCTA – Optical coherence tomography angiography
DR – Diabetic retinopathy OLM – Outer limiting membrane
DRS – Diabetic retinopathy study OPL – Outer plexiform layer
DCP – Deep capillary plexus PAMM – Paracentral acute middle maculopathy
DVC – Deep vascular complex PDR – Proliferative diabetic retinopathy
ETDRS – Early treatment diabetic retinopathy study RPCP – Radial peripapillary capillary plexus
FA – Fluorescein angiography RPE – Retinal pigment epithelium
FAF – Fundus autofluorescence SCP – Superficial capillary plexus
FAZ – Foveal avascular zone STZ Streptozotocin
FMD – Flow mediated dilation SVC – Superficial vascular complex
GBM – Glomerular basement membrane SVP – Superficial vascular plexus
GCL – Ganglion cell layer UWF – Ultra wide field
GFAP – Glial fibrillary acidic protein VEGF – Vascular endothelial growth factor
HbA1c – Glycated haemoglobin VSMC – Vascular smooth muscle cells
ICAM-1 – Intracellular adhesion molecule 1 αSMA – Alpha smooth muscle actin

for the lack of agreement regarding the connectivity patterns of retinal
capillary beds is the wide variations in histologic and in vivo imaging
techniques that have been used to model the structure of the retinal
capillary circulation.
Diabetes mellitus (DM) is an epidemic disease that is projected to
increase in prevalence over the next three decades. It is estimated that
up to 1 in 3 adults in the United States alone may have DM by 2050
(Boyle et al., 2001). Many patients with DM will eventually suffer some
form of vision loss due to diabetic retinopathy (DR). Microcirculatory
disturbances are a key feature of DR and can progress in a step-wise
manner over time. Our understanding of the histopathology of DR
should be credited to the detailed bodies of histopathologic work of
previous investigators that have provided a comprehensive characteri­
sation of the varying manifestations of DR such as cotton wool spots,
retinal haemorrhages and retinal neovascularisation using post-mortem
human tissue (Ashton, 1949; Cogan et al., 1961a). Their collective work
has also been instrumental in defining the key causes of vision loss in
DR.
Over the past two decades there have been rapid advances in histo­
logic and clinical imaging techniques for visualising the retina. As an
example, immunohistochemistry coupled with high-resolution confocal
scanning laser microscopy has enabled precise studies of the retinal
microcirculation in health and disease (Yu et al., 2010b). We have
leveraged these developments with isolated perfusion techniques of
human donor eyes to provide new quantitative information regarding
the spatial relationships between capillary beds in the retina. These
techniques have been used to expand our understanding of the patho­
physiologic mechanisms that underpin DR. Histologic validation of
widely used imaging tools such as fluorescein angiography (FA) and
optical coherence tomography angiography (OCTA) using human donor
eyes has also been performed by our research group.
This article provides an overview of our research in the field of
retinal microvascular disease spanning over 20 years. We commence by
summarising the cytoarchitectural characteristics of the human retinal
circulation as defined by our research and propose a standardised his­
tologic lexicon for classifying the vascular elements of the retinal
microcirculation. We subsequently discuss the pathophysiologic
mechanisms that underlie the development of key manifestations of DR
including microaneurysms and retinal ischaemia. The strengths and
limitations of current imaging tools as determined using histologic
validation will then be presented. The final part of this article will
discuss current gaps in our understanding of DR and key areas of future
research that will help improve our understanding and reduce the
morbidity due to DR. Although cotton wool spots, intra-retinal micro­
vascular abnormalities (IRMA), venous beading and retinal neo­
vascularisation are other important manifestations of DR, those features
will not be covered in great detail in this report. Furthermore, the re­
lationships between choroidal vascular changes and retinal function in
DR will not be covered. This report will focus on the role of the inner
retinal microcirculation in the pathophysiology of DR.
2. Retinal vascular apparatus
The organisation of the retinal vascular apparatus ensures that the
momentary and rapid fluctuations in the energy demands of retinal
neurons are met. The retinal vascular apparatus is comprised of a range
of cells that collectively serve to fine tune retinal perfusion. The density
and spatial organisation of these elements are topologically varied. In
this section the key elements of the retinal vascular apparatus, with an
emphasis on their structure, function and histologic properties, will be
provided.
2.1. Retinal endothelia
Retinal endothelial cells form the inner most lining of retinal
vasculature. Compared to choroidal endothelial cells, retinal endothelial
cells are non-fenestrated and inter-connected by tight junctions, or
zonula occludens to form part of the blood retinal barrier (BRB) (Cun­
ha-Vaz et al., 2011). The central retinal artery has adrenergic autonomic
innervation to the level of the lamina cribrosa in the optic nerve head.
Beyond this point, retinal arterioles and venules lack innervation by the
autonomic nervous system (Laties, 1967). Autoregulation of retinal
perfusion is crucially dependent on endothelial release of vasoactive
substances including endothelin-1 and nitric oxide to modulate vascular

2
C. Balaratnasingam et al.
tone in response to systemic blood pressure fluctuation, hypoxia and
lactate levels (Riva et al., 1986; Yu et al., 2004, 2016). Retinal endo­
thelial cells are non-uniform in nature with respect to morphology (An
et al., 2020b; Yu et al., 2010a). Arterial endothelia are long, spindle
shaped cells with the long axes oriented parallel to the direction of blood
flow. The cell nuclei of endothelia, which are centrally located within
the cell body, are also elongated, and have the same orientation
(Fig. 1A). Higher order retinal artery endothelia have longer and rela­
tively narrower cell bodies compared to lower order arteries and arte­
rioles (Fig. 1B). Capillary endothelia typically form a 360◦ tubular
structure on their own, with elongated oval shaped nuclei that form
focal areas of inward bulge within the lumen (Fig. 1C). Endothelia of
small venules resemble that of capillaries, but with two or more endo­
thelial cells forming the vessel tube (Fig. 1D). Larger venules and veins
have polygonal shaped endothelial cells and rounded nuclei (Fig. 1E).
Within the cells, actin cytoskeleton or stress fibres can be seen under
high magnification (Fig. 2). Higher densities of stress fibres are found in
arteries and arterioles compared to other vessels, likely related to the
degree of shear stress that these endothelial cells need to withstand (Yu
et al., 2005). Actin cytoskeleton plays a key role in maintenance of the
structure and morphology of the cells, stabilise intracellular organelles
localisation and intracellular transport. They also anchor transcellular
proteins which form barrier functions of endothelial cells. These include
cadherin, occludin and claudin (Bogatcheva and Verin, 2008).
Retinal vessel endothelium can be compromised in multiple path­
ways in DR. Vascular endothelial growth factor (VEGF) driven endo­
thelial dysfunction leads to leakage of vessels, which can be appreciated
Fig. 1. Endothelial cell morphology of retinal vasculature. Retinal arteries have
long spindle shaped endothelial cells with elongated nuclei located centrally
within the cell (A; yellow ar). Their long axes are oriented in the same direction
as blood flow. A few vascular smooth muscle cells are visible within this thin
section (green ars). Endothelial cells of arterioles have similar spindle
morphology as arteries but are typically shorter in overall length (B; yellow
dotted outline). Capillary endothelial cells have a 360◦ tubular morphology and
typically only border two adjacent cells. Their nuclei have elongated oval shape
and bulge into the lumen (C; yellow ars). Endothelial cells of venules also have
oval shaped nuclei but do not form a tube on their own (D; yellow ars). In
retinal veins, endothelial cells have variable polygonal morphology with
rounded nuclei (E; yellow ar). Sparse vascular smooth muscle cells are also
visible in this section (E; Green ars). Red = F-actin. Blue = Nuclei. All scale bars
= 30 μm.
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Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 2. Intracellular stress fibres. Stress fibres labelled using F-actin are shown
within endothelial cells of an artery with a luminal diameter of 80 μm from a
donor with systemic hypertension. The outline of an endothelial cell is manu­
ally traced to highlight its spindle shaped morphology. Examples of endothelial
stress fibres are marked with white ars. Sections of vascular smooth muscles are
also visible in this section (green ar) Red = F-actin. Blue = Nuclei. Scale bar =
30 μm.
clinically on FA (Section 6). ICAM-1 related inflammatory pathway via
activated microglia can lead to leukostasis, chronic inflammation, BRB
disruption and endothelial cell death (Gardner et al., 2002; Kinuthia
et al., 2020). Damaged endothelium along with activated leukocytes
lead to thromboembolism, which may lead to capillary nonperfusion
and retinal ischaemia, a key pathologic feature of DR (Altmann and
Schmidt, 2018; Joussen et al., 2004; Kern, 2007). In chronic disease,
surviving endothelial cells may show alterations such as gaining ability
to contract in response to a diseased environment. In a study performed
using streptozotocin (STZ) rats, It has been shown that rats with early
diabetes had dysregulation and over-expression of F-actin in endothelia
of retinal arteries and veins compared to non-diabetic rats (Yu et al.,
2005). Similarly, in a human donor eye study, we found increased alpha
smooth muscle actin (αSMA) expression in capillaries and venules in
donors with DR and donors with pre-clinical DR (An et al., 2022a).
Furthermore, significant endothelial proliferation is seen in micro­
aneurysms and within larger vessels (Aguilar et al., 2003). Endothelial
proliferation also plays a key role in large vessel occlusions in DR. These
topics are further discussed in Section 5.
2.2. Pericytes
Retinal pericytes, previously termed mural cells, are found on the
abluminal aspect of lower order retinal vessels, namely capillaries, small
venules, and terminal arterioles (Buzney et al., 1983; Caporarello et al.,
2019; de Oliveira, 1966). In higher order vessels, vascular smooth
muscle cells (VSMC) are present instead, though the exact point of
transition between these two related cell types are often difficult to
distinguish (Fig. 3A). In the healthy retina, the ratio between pericytes
and endothelial cells is 1:1 (Huang, 2020; Yanoff, 1966). Additionally,
Frank and colleagues found that pericyte processes cover more than 85%
of the capillary circumference (Frank et al., 1990). Pericyte functions
include reinforcement of the BRB via angiopoietin-1/Tie-2, platelet
derived growth factor pathways and modulation of immune responses
via cytokine release (Caporarello et al., 2019; Huang, 2020). Previous
studies have also attributed contractile properties to retinal pericytes,
which are thought to play a critical role in retinal perfusion
C. Balaratnasingam et al.
autoregulation (Alarcon-Martinez et al., 2019; Das et al., 1988; Hamil­
ton et al., 2010). The function of pericytes in the retina share some
similarities to that in the brain where they are one of the key regulators
of blood flow (Gonzales et al., 2020; Sengillo et al., 2013). It is important
to note that unlike the choroid, the retinal vasculature is devoid of
autonomic innervation and regional blood flow is predominantly
controlled by autoregulation (Alarcon-Martinez et al., 2019; An et al.,
2020b; Kureli et al., 2020). It is difficult to isolate pericytes from VSMC
using immunohistologic markers (Huang, 2020). In our recent studies,
we found that a combination of Hoechst (nucleus), αSMA and F-actin
labelling allowed for accurate visualisation of pericyte structures (An
et al., 2020b, 2022a).
Ultrastructural morphology can also be used to differentiate peri­
cytes from other cell types. In 2D illustrations, retinal pericytes typically
appear as dome shaped extrusions on the surface of small retinal vessels.
The majority of the cell body is occupied by the nucleus (Fig. 3B). Three-
dimensional rotation of the vessel allows for accurate visualisation of
pericytes and their nuclei (Fig. 3C). Pericyte morphology has been
investigated more extensively in the brain compared to the retina. Three
subtypes of pericytes were discovered in the brain. In proximal capillary
branches or terminal arterioles, ensheathing pericytes are found to
contain ovoid soma with circumferential processes enveloping the ves­
sels and express αSMA (Smyth et al., 2018). This morphology closely
resembles pericytes found in the retina’s superficial and intermediate
plexuses (An et al., 2020b, 2022a). In mid-capillaries and post-capillary
segments, mesh-like pericytes and helical pericytes are present in the
brain (Grant et al., 2019; Hartmann et al., 2015). Both of these subtypes
express minimal αSMA, which is also characteristic for pericytes found
in the deep capillary plexus (DCP) of the retina (An et al., 2022a).
The role of pericyte loss in DR and microaneurysm formation has
been widely accepted as a hallmark feature of the disease based on
several previous papers (Garner, 1993; Stitt et al., 1995; Yanoff, 1969).
De Oliveira postulated that pericyte loss in DR could be attributed to
migration, rather than cell death (de Oliveira, 1966). However, he found
that pericyte migration occurred not only in DR, but also in central
retinal vein thrombosis, polycythaemia and macroglobulinaemia.
Further evidence of pericyte migration was presented by Pfister, who
found that the migration process is angiopoietin-2 dependent and
occurred along straight capillary segments (Pfister et al., 2008). Quan­
titative studies of the degree of pericyte loss are limited. In our recent
study, 21% of microaneurysms in DR were observed to still contain
pericytes, which implies that several distinct mechanisms may underlie
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Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 3. Retinal pericyte morphology. Retinal peri­
cytes are located on the abluminal aspect of small
retinal vessels (A; red ars). In this image, a small
retinal arteriole measuring 15 μm further branches
into two smaller arterioles each measuring 11 μm.
Pericyte morphology viewed in two dimensions may
resemble a saddle shape (B) or dome shape (C). This
is in contrast to smooth muscle cells which have a
much larger cell volume and elongated morphology
as seen in arteries and larger arterioles (A; green ar).
Yellow = αSMA. Blue = Nuclei. All scale bars = 30
μm.
the formation of microaneurysms in DR (An et al., 2022b). This topic is
further discussed in Section 5.
2.3. Retinal vascular smooth muscle cells
Retinal VSMCs belong to the same embryologic lineage as pericytes
and are found within larger retinal arterioles, venules, arteries and
veins. VSMC contraction allows for regulation of blood flow throughout
the retinal microvasculature. Arterial VSMC also control retinal artery
diameter and elasticity in order to withstand fluctuating systemic blood
pressure and maintain perfusion. This is reflective of the autoregulation
properties of retinal vasculature (Delaey et al., 2000; Kornfield and
Newman, 2014; Yu et al., 2019a; Yu et al., 2010a).
Arterial VSMC form dense interconnecting muscle fibres surrounding
the vessels arranged in a ring like pattern, with the orientation of the
muscle fibres perpendicular to the direction of flow and the orientation
of the endothelia (Fig. 4). We observed that large arteries over 60 μm,
such as branch retinal arteries, may contain two layers of VSMC whilst
smaller arteries contain a single layer of VSMC. VSMC nuclei are usually
elongated with the long axis oriented in the same direction as the cells.
(Fig. 2). However, some irregularly shaped nuclei are also observed in
healthy retina (Fig. 4B). Muscle fibres may also have irregular branching
patterns and are interconnected to function as a syncytium. Venous
VSMC are sparse and less organised compared to arterial VSMC (Fig. 1).
Previous animal histologic studies showed VSMC loss in early stages
of DR, prior to the onset of classic vascular pathologies such as micro­
aneurysm formation (Gardiner et al., 1994; vom Hagen et al., 2005). In
human eyes, selective loss of VSMC and pericytes were found in in­
dividuals with only short periods of DM disease duration, and with
normal endothelium and well preserved macroscopic vascular struc­
tures. Gardiner et al. found that excessive autophagy of mural cells
occurred in these cases (Gardiner and Stitt, 2022).
2.4. Astrocytes
Retinal astrocytes, or astroglia, are glial cells that are located su­
perficially within the retina, primarily in the ganglion cell layer (GCL)
and nerve fiber layer (NFL) (Reichenbach and Bringmann, 2020). They
are thought to have migrated into the retina via the optic nerve during
embryogenesis and retinal development (Provis, 2001; Watanabe and
Raff, 1988). In the GCL, astrocytes typically have a stellate morphology
with long processes that accompany and encircle blood vessels of the
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 4. Retinal vascular smooth muscle cells (VSMC) morphology. Retinal arteries are densely enveloped by one or two layers of VSMCs (green ars; A). The
orientation of these cells is perpendicular to the direction of blood flow. Adjacent VSMCs are often interconnected to function as a syncytium. An example of a cell is
manually traced in (B). Note that VSMCs have variable cellular and nuclear morphology when viewed under high magnification. VSMC nuclei can also have an
irregular morphology. An example of a crescent shaped nucleus is traced in (C) and this can be seen in individuals without retinal vascular disease. Red = F-actin.
Blue = Nuclei. All scale bars = 30 μm.

superficial vascular plexus (SVP) whilst supporting nearby axons of
retinal ganglion cells (RGC), forming the neurovascular unit (Fig. 5A)
(Yu et al., 2010b). Another population of astrocytes are located within
the radial peripapillary capillary plexus (RPCP) of the NFL. They man­
ifest a small oval shaped cell body and nucleus. Their cellular processes
are arranged parallel to the ganglion cell axons and retinal peripapillary
capillaries (Fig. 5B). The structural and functional properties of this
subtype of astrocytes is less well understood than the stellate astrocytes
of the GCL.
Glial fibrillary acidic protein (GFAP) is a glial intermediate filament
commonly used as an astrocyte marker in experimental studies (Hol and
Pekny, 2015; Schnitzer, 1987). Astrocytes express GFAP under physio­
logic state and upregulate GFAP expression when astrocytes proliferate
and undergo gliosis (Nagayach et al., 2014). Astrocytes have also been
recognised to partake in suppression of neuronal proliferation in the
central nervous system (CNS) and regulation of blood flow following
ischaemia (Brenner, 2014).
A key function of astrocytes is to maintain ganglion cell homeostasis
and form part of the BRB together with pericytes and endothelial cells
(Newman, 1999). In diseased states, astrocytes can undergo gliosis as
part of an inflammatory proliferative response (Luna et al., 2010). In DR,
astrocytes were found to upregulate VEGF and downregulate
anti-permeability factors, which contribute to the development of
retinal oedema (Rollin et al., 2005).
In DR, astrocyte proliferation may be an important part of a micro­
aneurysms life cycle. Imaging studies show that microaneurysms may
enlarge or regress in a temporal sequence during their natural life cycle
(Chui et al., 2016; Dubow et al., 2014). Recent works performed by our

Fig. 5. Retinal astrocyte morphology. Retinal astrocytes within the ganglion cell layer (GCL) have processes which form interconnecting networks and assume a
classic stellate morphology (A). Some of these processes are in close contact with vessels of the superficial vascular plexus located within the GCL. Astrocytes within
the nerve fibre layer (B, elderly human donor; C, young pig) have a more linear/radial orientation with fibres communicating with nerve fibres of ganglion cells and
the similarly oriented retinal peripapillary capillary plexus (not shown). Astrocyte nuclei are labelled with white ars. Red = F-actin. Yellow/Green = GFAP. Blue =
Nuclei. All scale bars = 30 μm.

5
C. Balaratnasingam et al.
laboratory show that extensive gliosis are sometimes seen around large
microaneurysms and such a change may be an attempt to physically
tamponade microaneurysm leakage. At a later phase of the micro­
aneurysms lifecycle, complete regression occurs leaving only traces of
glial scar. The details of these observations are provided in the latter
section of this manuscript (Section 7).
2.5. Muller cells
Muller cells, or Muller glia, are the main type of retinal glial cells in
vertebrates. Structurally, Muller cells differ from astrocytes as they are
large cells that span the entire thickness of the neuroretina, from the
vitreoretinal interface to the photoreceptor layer, with their cell bodies
and nuclei located in the inner nuclear layer (INL) (Bringmann et al.,
2006; Kolb, 1995). Muller cell end-feet form the internal limiting
membrane at the vitreoretinal junction. Muller cell adherent junctions
with photoreceptor inner segment contribute to a membrane-like
appearance and is known as the outer limiting membrane (OLM). Both
features contribute to the retina’s structural integrity. Given the trans­
retinal nature of Muller cells, they are well placed to detect and respond
to retinal mechanical tension by regulating calcium uptake and release
(Lindqvist et al., 2010).
Muller cells also perform a large number of non-structurally related
functions, one of which is maintaining the homeostasis of the retinal
extracellular environment by mediating ion, water and bicarbonate
transport (Reichenbach and Bringmann, 2013). In addition, Muller cells
participate in the metabolic cycle of photoreceptors by performing
glycolysis, and its product lactate is used as the primary energy source
within photoreceptors using the Kreb cycle. Muller cells are also
involved in regulation of neuronal synaptic activities by regulation the
reuptake of glutamate neurotransmitter. A newly recognised physiologic
function of Muller cells is their role in light transmission through the
retina. Due to their transretinal cell structure, they act as fibreoptic
cables which enable light to reach the photoreceptors with minimal
scatter (Reichenbach and Bringmann, 2013).
The structural and outer retinal physiologic functions of Muller cells
are distinctly different to the roles performed by astrocytes. However,
there is less distinction between Muller cell and astrocyte’s roles in inner
retinal BRB and inner retina extracellular space homeostasis (Reich­
enbach and Bringmann, 2013; Tout et al., 1993). Studies have found that
Muller cells release VEGF and tumour necrosis factor in response to
hypoxia and inflammation and are involved with the neovascularisation
process and retinal oedema seen in DR (Eichler et al., 2004). In contrast
to astrocytes, it is understood that Muller cells under physiologic con­
ditions either do not or only minimally express GFAP. Following Muller
cell activation under pathologic conditions such as DR, significant
upregulation in GFAP expression is seen (Aartsen et al., 2010; Fernan­
dez-Sanchez et al., 2015).
Evidence of Muller cell activation in DR can be detected by direct
GFAP staining using an immersion technique. Increased inner limiting
membrane (ILM) gliosis can be seen in eyes with advanced DR with a
highly irregular appearance (Snead et al., 2004). The ILM may also be
focally disrupted by superficially located microaneurysms. This is
further discussed in Section 7.
2.6. Microglia and circulating inflammatory cells
Microglia are resident macrophages of the CNS and retina. Quiescent
microglia in their ramified form reside in the inner and outer plexiform
layers (IPL; OPL) and serves a key role in maintaining retinal neuronal
homeostasis and phagocytosis of cellular debris whilst balancing be­
tween its pro and anti-inflammatory states (Karlstetter et al., 2015;
Takahashi et al., 2005). In a chronic disease state such as DR, hyper­
glycemia and increased oxidative stress leads to microglia activation and
reset of the balance that favours a pro-inflammatory state (Gonza­
lez-Scarano and Baltuch, 1999; Kinuthia et al., 2020). When this occurs,
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Progress in Retinal and Eye Research xxx (xxxx) xxx
microglia transition from a ramified form into amoeboid form and can
migrate away from the plexiform layers into other parts of the retina
(Vujosevic et al., 2013). Pro-inflammatory cytokines are released,
including Interleukin-1, Interleukin-6 and Tumour necrosis factor α
which lead to retinal tissue inflammation and vascular cell dysfunction,
and may ultimately contribute to vascular leakage or occlusion.
In addition to microglia, circulating leukocytes are also involved in
retinal inflammation, vascular dysfunction and occlusion. Noda et al.
reported leukostasis within retinal vessels in diabetic rat models where
adhesion of leukocytes to arterial and venous walls were observed (Noda
et al., 2014). Human donor eye studies performed in our laboratory
using perfusion labelling method found that intracapillary leukostasis
may play a key role in the mechanism of capillary nonperfusion in DR,
and that leukostasis within microaneurysms suggest an inflammatory
component of microaneurysm pathophysiology (An et al., 2022b).
Although inflammation is an established pathogenic mechanism in
DR the role of microglia in controlling DR progression is not well un­
derstood (Joussen et al., 2004). Intraretinal hyper-reflective foci is a
recognised feature of DR seen on optical coherence tomography (OCT)
and thought to represent microglia aggregates (Bolz et al., 2009). This
signature can have predictive value in determining treatment response
to intravitreal corticosteroids in diabetic macular oedema (DMO). By
extension, this indicates that microglia are involved in breakdown of the
BRB in a complex manner.
3. Isolated retinal perfusion
Precise labelling and clear visualisation of elements of the deep
retina has remained a great challenge for histologists in the field of
retina diseases particularly when it comes to the study of retinal vascular
disorders. Commonly employed methodologies for studying retinal
vasculature include trypsin digest, retinal thin section staining, whole
retina immersion staining, endovascular injection of dye and electron
microscopy (Ashton, 1953; Cogan et al., 1968). Each method has its
distinct advantages and shortfalls. Trypsin digest is a technically chal­
lenging method that involves digesting non-vascular tissue with trypsin
solution in Tris buffer and carefully isolating the more resistant vascular
tissue (Ashton, 1963; Chou et al., 2013). It enables clear unobstructed
microscopy of the vasculature tree and its endothelial cells. However, it
has several distinct limitations. Firstly, the vasculature is flattened and
loses its three-dimensional angioarchitecture, making spatial correlation
difficult to perform. Secondly, digestion and physical handling of the
tissue may inadvertently damage external vascular structures such as
VSMC and pericytes, leading to a misconception of cell loss due to a
pathologic process.
Whole retina immersion staining with a permeating agent such as
Triton X-100 allows labelling of both vascular and extravascular tissues.
Specific antibodies for vascular labelling therefore need to be carefully
selected to minimise extravascular tissue labelling to enable visual­
isation of vessels. Background staining is often inevitable despite great
efforts to rinse the tissue. Additionally, we found that the depth of tissue
penetration is generally less than 100 μm. The thickness of the central
macula is greater than 200 μm, which makes visualisation of the deeper
retinal plexuses more difficult.
Retinal cryosections or paraffin sections are perhaps the most
commonly employed methodologies in retinal research. This method
offers a number of advantages: 1) It enables labelling using a variety of
antibodies; 2) Positive and negative control stain section can be per­
formed for every donor retina; 3) Every layer of the retina can be
studied. This bypasses retinal tissue thickness related antibody pene­
tration issues, and high noise signals related to confocal microscopy
imaging of deeper structures. Additionally, retinal sections can be
imaged using transmission electron microscopy, which provides
unparalleled resolution of small cellular structures that is not achievable
using light microscopes. The tradeoff of retinal sectioning is that struc­
tures are difficult to track across serial sections for both the investigators
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

and the readers and does not provide a good 3D overview of the vascular neuro-glia elements to the retinal vasculature. A list of antibodies/stains
tree. that have been used for retinal labelling is provided in Table 1.
Dye injection method was widely employed in the mid 20th century. A schematic of the perfusion system is provided in Fig. 6. Syringe
The substances injected include Indian ink and periodic acid-Schiff stain pumps are used to deliver an adjustable flow of perfusate, and the
(Ashton, 1953). The advantage of this is that it allows precise visual­ perfusion pressure is continuously monitored through conventional
isation of nonperfusion patterns in retinal vascular diseases. Disadvan­ transducers that are connected to a bridge amplifier and recorded on a
tages include the technical challenge of injection at an appropriate chart recorder. We use a baseline flow rate of 50 μL/min as it results in
pressure and rate to ensure complete labelling of the vasculature, whilst an average transmural pressure in the central retinal artery close to 50
protecting the vessels from rupture as a result of high pressure. Air mmHg. This flow rate ensures that the intravascular pressures do not
bubble within the perfusate may also strip the endothelium along the exceed the physiologic range causing vessel rupture and leakage.
path and may artefactually produce a “ghost vessel”. Continual measurement of perfusion pressure is a critical aspect of this
Over the past 20 years our research group has refined the technique technique as it allows us to monitor for the occurrence of vascular oc­
of isolated ocular perfusion using human donor eyes. Data collected clusion due to post-mortem thrombus or introduced air bubbles that in
from over 500 eyes using this technique comprises much of the histo­ turn may influence completeness of retinal vascular labelling. Such
logic information discussed in this article. Isolated perfusion of human perfusion pressures also ensure the integrity of the BRB and minimises
donor eyes is a technique that is akin to intracardiac perfusion utilised in extravasation of perfusate beyond the vascular tree. It is important to
small animal studies. As the human retina is supplied by a single central note that although we are able to control perfusion pressure the exper­
retinal artery it is possible to achieve complete labelling of the retinal iments are not conducted under physiologic intraocular pressure and
vasculature through cannulation of the central retinal artery or vessels ocular perfusion pressure.
that supply the central retinal artery. As the choroidal circulation is Following perfusion, the eyes are de-cannulated and dissected along
supplied by over 20 ciliary arteries it is not possible to achieve complete the equator. Vitreous is carefully removed from the vitreous cavity using
choroidal labelling through cannulation of a single ciliary artery (Weiter a dissecting microscope. In eyes without existing posterior vitreous
and Ernest, 1974; Yu et al., 2014a, 2014b). As perfusion labelling is a detachment, vitreous is peeled or dissected from the retina. The poste­
method of intravascular labelling its efficacy is not dependent on the rior segment is then immersed in 4% paraformaldehyde for 12 h. Post
thickness of retinal tissue and is capable of labelling even the deepest fixation, the neuro-retina is detached from the retinal pigment epithe­
vascular structures of the retina whilst extravascular tissue labelling is lium (RPE). The optic nerve head is transected to be continuous with the
minimized. retina. The retina is subsequently flat mounted on a glass slide by
making several radial incisions along the edge. Glycerol is added to
3.1. Methodology enhance the optical quality of the tissue before placement of the
coverslip.
An exhaustive description of our technique of isolated ocular
perfusion of the human eye has been published in our previous reports
Table 1
and only an overview of the methodology details in this section will be
List of antibodies and stains.
provided (An et al., 2018; Tan et al., 2012; Yu et al., 2003, 2012, 2014a,
2019a, 2019b). Human tissue for our research has been obtained Antibody/stain Target Concentration in Manufacturer
0.1M phosphate
through DonateLife Western Australia, the organ and tissue retrieval
buffer
authority in Western Australia, Australia, and the Lions Eye Bank of
Western Australia. In the majority of cases the human eyes are used for Hoechst DNA 1 μg: 400 μl Sigma-Aldrich
Product No. H6024,
research purposes only following the removal of corneal buttons for Missouri, USA
corneal transplantation. In some instances, when there are medical Lectin FITC Endothelial 1 μg: 10 μl Sigma-Aldrich
contraindications for transplantation, the entire human donor eye is glycoprotein Product No. H6024,
perfused. In our experience, the removal of corneal buttons does not Missouri, USA
Phalloidin Filamentous 1 μg: 1000 μl Sigma-Aldrich,
adversely affect the quality of the retinal tissue or the completeness of
TRITC -actin Product No. P1951,
vascular labelling despite the intraocular pressure being effectively 0 Missouri, USA
mmHg. We have found that if the vitreous body is preserved and not Phalloidin FITC Filamentous 1 μg: 1000 μl Sigma-Aldrich,
disrupted during removal of corneal buttons then the integrity of retina -actin Product No. P5282,
is also maintained. Excellent vascular labelling can usually be achieved Missouri, USA
Rabbit anti Collagen IV in 1 μg: 100 μl Sigma-Aldrich,
if eyes are perfused within 24 h post-mortem. After 24 h, a high rate of collagen IV vascular Product No.
extravasation of antibody labels are commonly found and may indicate basement SAB4300738,
post-mortem related tissue breakdown. This significantly increases membrane Missouri, USA
artefactual labelling of the surrounding tissues. Mouse anti- αSMA 1 μg: 50 μl Sigma-Aldrich,
αSMA Product No. A2547,
Following enucleation, eyes are transported in oxygenated Ringer’s
Missouri, USA
lactate solution. Eyes are placed in a custom-built holder and the central Mouse anti- GFAP 1 μg: 100 μl Sigma, Product No.
retinal artery is cannulated for perfusion labelling. The eyes are can­ GFAP G6171, Missouri,
nulated using a custom-built glass micropipette (100 μm tip diameter) USA
and secured with a suture (Supplementary Fig. 1). Key aspects of the Goat anti-rabbit Rabbit IgG 1 μg: 50 μl Abcam, Product No.
antibody ab150077,
technique of central retinal artery perfusion include precise cannulation Alexa Fluor Cambridge, UK
of the central retinal artery lumen without the formation of a false 488
passage and introducing air bubbles. It is essential to secure the cannula Goat anti-rabbit Rabbit IgG 1 μg: 50 μl Abcam, Product No.
using a double suture to prevent cannula migration. Washout of residual antibody ab150078,
Alexa Fluor Cambridge, UK
blood cells and clots is initially performed using heparinised Ringer’s
555
lactate solution. The retinal circulation is subsequently perfused with Donkey anti- Mouse IgG 1 μg: 50 μl Abcam, Product No.
antibodies that target various elements of the retina. Post perfusion mouse ab150111,
labelling, extravascular components of the retina such as Muller cells antibody Cambridge, UK
and astrocyte can also be labelled using a traditional immersion method Alexa Fluor
647
of immunohistochemistry. Such a technique allows co-localisation of

7
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 6. Setup for perfusion of a human donor eye.

(A1) The human eye is placed on an eye holder and
cannulated for perfusion staining of retinal micro­
vasculature. (A2) A microphotograph taken after
cannulation shows the posterior globe of the eye ball
(EB), optic nerve (ON), and surrounding tissues. The
central retinal artery (CRA) is cannulated and secured
by double sutures, as is shown in (A1). Perfusate flow
is delivered by the syringe pump and the perfusion
pressure monitored by chart recorder through pres­
sure transducers (P) attached at the outflow end of
the syringes. (B) Typical chart recordings (a, b, c, d)
reflecting the pressure change during perfusion of
various solutions: Ringer’s BSA, Ringer’s with 0.5%
bovine serum albumin; 4% PFA, 4% para­
formaldehyde in 0.1 M phosphate buffer; Triton X-
100, 0.1% Triton X-100 in 0.1 M phosphate buffer;
and PB wash, 0.1 M phosphate buffer wash). Perfu­
sion pressure rose to a constant level on Ringer’s BSA
perfusion, indicating unobstructed outflow free from
blood clots. There was an increase in perfusion pres­
sure in response to fixative (4% PFA), indicating
vessel contraction. The pressure dropped on exposure
to detergent (Triton X-100). The PB wash at the end
of staining reflected a constant unobstructed outflow
at the end of the perfusion protocol.

Perfused tissue was visualised using a confocal scanning laser mi­

croscope (Nikon Eclipse 90i and C1, Nikon Corporation, Tokyo, Japan)
equipped with four solid state lasers at wavelengths of 405 nm, 488 nm,
561 nm and 635 nm and a range of objective lenses from 4× to 100×
magnification. Confocal image files are usually processed with Nikon
NIS-Elements (Nikon, Tokyo, Japan), IMARIS (v7.42, Bitplane, Zurich,
Switzerland) and/or Fiji, ImageJ (Rasband, W.S., ImageJ, U. S. National
Institutes of Health, Bethesda, Maryland, USA).

3.2. Limitations and benefits

As stated above, the major advantage of perfusion vascular labelling

is that it allows precise visualisation of deep retinal structures, partic­
ularly the DCP. This has been particularly useful for quantifying the
topographic properties of the macular circulation in health and also
diseases that have a predilection for the deep circulation such as DR (An
et al., 2020a, 2021; Carnevali et al., 2017; Furino et al., 2019). The
application of the three-dimensional image reconstruction software has
particularly aided the investigation of spatial relationships between
retinal vascular elements that would otherwise have been missed had
the images been visualised in two dimensions (Fig. 7).
The other advantage of the perfusion labelling technique is that it
allows some correlation between clinical imaging and histology and
assists in the validation of emerging retinal visualisation technologies.
This is particularly important in the era where retinal imaging plays a
key role in clinical management and in an era where it is difficult to
validate such technologies due to the dearth of human tissue that have
been adequately imaged pre-mortem. We have performed a number of
studies validating the technique of OCTA using ex vivo perfusion label­ Fig. 7. Two and three dimensional histologic characteristics of the human
ling of human and porcine eyes (An et al., 2018; Balaratnasingam et al., perifoveal circulation. A 1.27 × 1.27 mm confocal Z-stack microscopy section
2018; Yu et al., 2021). More details of the findings of this work are shows dense perifoveal microvasculature with the retinal artery of the super­
otemporal arcade located at the centre of the image (A). The stack has been
provided in subsequent sections but this work has helped identify the
stratified and colour-coded into the superficial vascular plexus (SVP; Green),
vascular beds that can be clearly visualised using OCTA as well as
intermediate capillary plexus (ICP; Yellow) and deep capillary plexus (DCP;
highlighting vascular elements that may not be seen completely using Red). Magnified three-dimensional (B) and cross-sectional (C) views of the
OCTA. This has informed clinicians regarding the limitations and ad­ perifoveal artery demonstrate a capillary-free zone immediately adjacent to the
vantages of OCTA. lumen at the level of the superficial plexus. Note that in the two dimensional
The limitations of the current perfusion labelling technique include: image (A) this spatial relationship of the arterial capillary free-zone is less
1) High technical demand of retinal artery cannulation technique and apparent. Capillaries of the intermediate and deep plexus are seen to clearly
precise perfusion pressure; 2) Requires relatively short post-mortem bridge under the retinal arteries when the tissue is studied in cross-section and
time between death and CRA cannulation to ensure precise endothe­ three-dimensions. Vasculature labelled using Lectin. Modified with permission
lial labelling; 3) Deeper retinal structures are more difficult to image due from Balaratnasingam et al. (Balaratnasingam et al., 2018).

8
C. Balaratnasingam et al.
to the limitations of confocal microscopy laser penetrance, scatter, noise
and objective working distance; 4) A limited selection of stains can be
utilised for each eye compared to performing retinal sections. However,
the latter issue can be addressed by combining perfusion labelling with
conventional immersion labelling techniques.
4. Organisation of the retinal circulation
The topographic characteristics of the retinal circulation are not
homogenous and demonstrate significant specialisation per eccentricity.
As an example, the cytoarchitecture and morphology of capillary plexus
beds that supply the cone-dominant macula is significantly different to
the capillary beds that supply the rod-dominant peripheral retina.
Reconciling the morphologic characteristics of retina capillary beds is
important for several reasons: 1) It will add to the body of knowledge
concerning the role of the microcirculation in retinal physiology and
disease; 2) It will aid the clinical interpretation of emerging technologies
that facilitate high-resolution visualisation of retinal capillaries in vivo as
a means of detecting retinal disease and monitoring disease progression.
Using isolated perfusion labelling the morphologic characteristics of the
retinal circulation has been defined through a series of reports. We will
summarise the contents of those studies in the sections below.
4.1. The macular circulation
The human macula is approximately 5.5 mm in diameter. Hogan
subdivided the macula into four concentric annuli from centre: foveola
(0.35 mm diameter), fovea (1.85 mm diameter), parafovea (2.85 mm
diameter) and perifovea (5.85 mm diameter) (Hogan et al., 1971). The
macula is responsible for high-resolution vision. It is the site within the
retina where the density of cone photoreceptors and RGCs is the great­
est. There are competing interests between the structural organisation of
the macular circulation that serve to optimise nutrient-waste exchange
and the functional demands of the macula that requires a transparent
light pathway to optimise visual acuity. An increase in the distribution of
capillary density as a means of improving energy delivery can
compromise visual acuity and introduce aberrations by interfering with
the pathway of incident light. However, a scant capillary density within
the macula as a means of increasing image resolution is unlikely to
satisfy the immense energy demands of densely packed RGCs and other
neurons.
The foveal avascular zone (FAZ) is the central portion of the fovea
that is devoid of any vascular elements. It is a physiologic landmark that
can be identified in almost every normal human eye. The size of the FAZ
is highly variable between individuals (Balaratnasingam et al., 2016;
Falavarjani et al., 2018). Interestingly, in the normal eye, a significant
relationship between the size of the FAZ and best corrected visual acuity
has not been established (Balaratnasingam et al., 2016). However, in
diseases such as albinism, DR and retinal vein occlusion (Balar­
atnasingam et al., 2016; Lu et al., 2018; Mansour et al., 2021) there is a
significant relationship between the size of the FAZ and best corrected
visual acuity. The relationship between macular vessels density and
visual acuity is therefore different in health and disease.
4.1.1. Two-dimensional organisation
Snodderly, Weinhaus and Choi published a study in 1992 that
defined the spatial organisation of the vascular network of the primate
retina (Snodderly et al., 1992). Their work was instrumental in under­
standing the organisation of the macular circulation. Using eyes of
cynomolgus monkeys (Macaca fascicularis) the vasculature in whole­
mounts were drawn by using drawing tubes mounted on microscopes.
The precision and detail in their approach was able to provide a greater
understanding of the laminar distribution of the macular vasculature. In
their study they concluded that the macular circulation could be sepa­
rated into 4 planes – a sclerad network that brackets the INL and a vit­
read network that brackets the GCL or lies within the GCL and NFL.
9
Progress in Retinal and Eye Research xxx (xxxx) xxx
We expanded upon the findings of Snodderly et al. and investigated
the morphometric characteristics of the perifoveal circulation using
human eyes. In our previous study we showed that the macula is on
average, supplied by 9 paired arteries/veins that are radially arranged
around the fovea (Yu et al., 2010a). We further characterised the
capillary circulation of the human macula (An et al., 2020b; Chan et al.,
2012) and showed that, similar to monkeys, it can also be separated into
4 different plexuses that can be localised to the retina layers as follows
(Fig. 8):
1. NFL network/retinal peripapillary capillary plexus (RPCP): charac­
terised by long capillary segments that are predominantly oriented
parallel to the direction of retinal ganglion cell axons. A small
number of shorter capillary segments that interconnect long radial
capillaries are also present in this network. Interconnecting capil­
laries are oriented either diagonal or orthogonal to long segments.
The retinal peripapillary capillary network can only be found at the
outer reaches of the perifovea, bordering the optic disc nasally, as
well as the superotemporal and inferotemporal vascular arcades.
2. GCL and the superficial portion of the IPL (Superficial vascular
plexus; SVP): this is characterised by a dense meshwork of three-
dimensional vessels of various diameters that are arranged in a lat­
tice pattern. Capillaries in this network demonstrate long hairpin
turns that project vertically. Small retinal arteries and veins run
within the SVP that divide into arterioles and venules that supply
other plexuses.
3. Deep portion of the IPL and superficial portion of the INL (Inter­
mediate capillary plexus; ICP) – this is characterised by a three-
dimensional capillary network composed of vertical and oblique
segments that result in an irregularly shaped loop configuration.
Capillaries in this network demonstrate great tortuosity.
4. Deep portion of the INL and OPL (DCP) – this is characterised by
capillaries arranged in a one-dimensional laminar configuration.
Capillaries run a linear trajectory with little tortuosity. The DCP is
characterised by a large number of capillary loops and prominent
draining venules.
Our work provided evidence that the macular capillary networks are
specialised and arranged in accordance with the unique metabolic de­
mands of each neuronal layer. We also provided spatio-morphometric
structural information that could be used to infer knowledge
regarding oxygen exchange within the retina. As an example, when we
measured intercapillary areas and used those measurements as indices
of oxygen diffusion we found significant heterogeneity between retinal
layers (Chan et al., 2012). More specifically, we found that capillary
loop area was lowest in the RPCP and SVP networks suggesting that
oxygen diffusion may play a key role in ATP synthesis in these layers
relative to other regions of the retina.
Interestingly we did not find a significant variation in capillary
diameter between networks (An et al., 2021). This is contrary to the
brain microcirculation where capillary diameter in addition to capillary
loop area plays a key role in influencing the rate of capillary oxygen
exchange (Pawlik et al., 1981).
Direct oxygen measurements have shown that the IPL is one of the
greatest consumers of oxygen within the retina due to the immense
energy required to maintain synaptic activity (Ahmed et al., 1993; Yu
and Cringle, 2006; Yu et al., 2011). However our studies found that the
networks that supply the IPL, namely the SVP the ICP, are located
adjacent to the IPL rather than the mid-strata of this layer. Although
there are numerous small connecting vessels bridging the SVP and ICP
by traversing through the IPL it is unlikely that the retinal vasculature is
the sole source of the immense nutrient requirements of the IPL. It is
plausible that the retinal circulation is supported by other elements of
the retina. A putative candidate for this additional mechanism is retinal
glia as discussed above.
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 8. Morphology of vascular layers of the macula

region. The retinal peripapillary capillary plexus,
located within the nerve fibre layer, is characterised
by multiple long capillary loops oriented radially
along the superotemporal and inferotemporal
vascular arcades (A). This plexus is not present within
the foveal region. The superficial vascular plexus
(SVP) contains retinal arteries (red ar) and veins (blue
ar; B) which originate from the vascular arcades.
Capillaries of the intermediate capillary plexus (ICP)
are highly tortuous (C). Terminal arterioles which
supply the deep capillary plexus (DCP) originate from
the ICP. Venules which originate from the DCP can
also be seen traversing through the ICP, prior to
joining a retinal vein in the SVP (blue ar). The DCP is
characterised by a single laminar layer of vessels
which contains numerous capillary loops (D; yellow
asterisks). Capillaries converge to form venules,
which drain towards the retinal veins in the SVP (blue
ars). Inflow of DCP is dependent on a limited number
of terminal arterioles (magenta ars). Vasculature
labelled using Lectin.

4.1.1.1. Deep capillary plexus. The DCP is a thin, single laminar plexus
that manifests a very different morphology to the other capillary plex­
uses of the retinal circulation. The stimulus for the formation of the DCP
is physiologic ischaemia during the development of the middle and
outer retina (Michaelson, 1936). Once formed, the DCP provides
nutrient and oxygenation to the outer part of the INL and the highly
energy demanding OPL. Although within close proximity to the ONL, it
has been shown that ONL derive its oxygen supply mostly via the
choriocapillaris instead of the DCP, likely due to the OPLs oxygen con­
sumption rate and a relatively limited vasculature of the DCP compared
to other plexuses (Yu and Cringle, 2001, 2006).
The layout of the DCP vasculature is dependent on the particular
region’s proximity to 4th order (v4) retinal veins, which drains the DCP
into the SVP (An et al., 2020b). DCP regions adjacent to retinal veins
form short segment 3rd order venules (v3) which run an almost vertical
course from DCP to SVP before joining V4. Surrounding capillaries

Fig. 9. Illustration of deep capillary plexus (DCP) morphology. Two types of v3 venule (white arrows) morphologies are found in the DCP (A). Short segment v3 are
located directly underneath v4 (marked as v) which are located within the superficial vascular plexus (SVP; B). They ascend in a near vertical fashion. Long segment
v3 travel a longer course within the DCP and ascend obliquely and join a v4 in a more distant location. The v3/v4 junctions are indicated with magenta circles. Note
that the drainage zone indicated by inset AI (short segment v3) resembles a vortex and inset AII (long segment v3) resembles a fern. No other v4 are found within the
drainage zone of the long segment v3 as indicated by inset BII. Panel C shows a full thickness projection of the retina which also includes the intermediate capillary
plexus (ICP). Red = DCP; Green = SVP; Yellow = ICP. a = artery, v = vein. Scale bar = 300 μm.

10
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

merge to 2nd order venules(v2), and the pattern of v2 merging into v3

from all directions gives an appearance of a vortex. DCP regions distant
from v4 retinal veins form long segment v3 within the DCP and even­
tually run an oblique course through the ICP, before joining v4 in the
SVP. v2 join either side of v3 along its flat course within the DCP. This
pattern of outflow resembles a river or fern tree, and not a vortex
(Fig. 9).

4.1.1.2. Macular capillary density changes with eccentricity and age. The
thickness of the macula varies remarkably from the boundaries of the
perifovea to the central fovea due to the variation in cell types between
these eccentricities. As an example, the density of cone photoreceptors
increases in a centripetal direction towards the fovea whilst the density
of RGCs demonstrates the opposite relationship (Jonas et al., 1992). In
our previous report we showed that vascular density increased from the
fovea to the parafovea in accordance with changes in retinal thickness
(Yu et al., 2018). We also showed that the increase in vascular density
occurs in the superficial and deep vascular networks. The vascular
density between various quadrants of the retina can be ranked in
increasing order as follows: inferior > superior > temporal > nasal (Yu
et al., 2018). That latter findings are particularly important as it in­
dicates that the macula is not a homogenous structure and distinct
quadrants may be protected (or more vulnerable) to neuronal injury due
to the inherent topological properties of the vasculature within that site.
Such a natural arrangement of the macular circulation may help un­
derstand conditions such as macular telangiectasia type 2 where the
insult to the macula is often highly focal.
We investigated the changes in macular vascular density with age
and unveiled surprising results. We compared the findings between
young donors (mean age 27.3 years) and older donors (mean age 66.9
years) and found that there was a significant increase in density of the
superficial layer with age that was attributed to an increase in the
diameter of macular microvasculature (Yu et al., 2018). The density in
the deep layer did not change with age (Fig. 10). There is evidence to
indicate that there is a reduction in RGC density, rod photoreceptor
density and rod bipolar cell density with age so it would be intuitive to
assume that vascular density will also diminish accordingly due to the
presumed reduction in metabolic demands within retinal tissue.
(Aggarwal et al., 2007; Harwerth et al., 2008; Panda-Jonas et al., 1995).
Our findings therefore indicate that the relationship between macular
vascular density and retinal metabolic function is not linear and may not
change in the expected manner with age as particular retinal cells
decrease in density. It also highlights the importance of accounting for
age-related variations in the assessment of any clinical measurement of
macular vascular density such as that derived using OCTA.
An increase in vascular density therefore does not necessarily
represent a greater availability of energy to regional neuronal pop­
ulations and vice versa. Such a relationship will also explain why pro­
found changes in macular function can occur in the setting of relatively
normal structural vascular measurements. In this setting, profound
Muller cell or glia dysfunction may account for energy depletion and an
inability to maintain retinal homeostasis. This point has great applica­
tion in the assessment of macular conditions such as Macular Telangi­
ectasia Type 2 and DR where significant perturbations in visual function
can be observed in the context of relatively normal vascular density
measurements as determined using OCTA.
4.1.2. Three-dimensional organisation
A point of great conjecture regarding the organisation of the macular
circulation is the arrangement and connectivity of the different capillary
beds. Although there has been great focus on the morphology of the
individual capillary networks there has been less study on the three-
dimensional organisation of the entire circulation in toto. It is not
straight forward to reconcile this research question using non-primate
animal models as most animals do not manifest a macula and have a
Fig. 10. Age related capillary density comparison in two individuals. Projected
images of the superficial and deep vascular layers are presented. Images for the
young patient is provided in the left panel and those for the elderly patient are
provided in the right panel. Significant increase in capillary density is seen in
the macula of elderly individual in only the superficial circulation (A vs B)
whilst there is no discernible change in vessel density of the deep layer (C vs.
D). The intermediate capillary plexus is segmented within the superficial layer
in this illustration. Vasculature has been labelled using F-actin. Scale bars =
300 μm.
remarkably different vascular arrangement to the human eye. This is a
critical area of research however that requires precise clarification as it
ultimately will inform us of the mechanisms concerning human macular
perfusion. The findings will have direct relevance for conditions such as
DR, macular telangactasia type 2, retinal vein occlusion, acute macular
neuroretinopathy and the spectrum of diseases that can manifest with
paracentral acute middle maculopathy (PAMM) lesions on OCT. In all
these disease entities there is a preponderance for local vascular network
injuries within the macula that may be the cause or effect of abnor­
malities to the macular circulation either upstream or downstream from
the site of alteration.
Rapid advances in high resolution microscopy and image analysis
software coupled with supercomputers has allowed the study of histo­
logic data in three-dimensions. This has not been possible previously and
cumulatively has been an important “game changer” in this field of
research.
4.1.2.1. Defining elements of the retinal vascular tree using histology. A
standardised lexicon that distinguishes the different compartments and
segments of the macular circulation is urgently required. Currently,
there is no robust consensus regarding the definition of each element of
the vascular tree and this has led to some inconsistency in the proposed
models of inflow and outflow pathways within the macula. Size and
diameter of vessels can be used to distinguish arteries from arterioles
and capillaries, but such definitions omit vital information regarding the
mechanisms that control the dynamic properties of the vascular
segment. For instance, it might be implied that flow within a large
diameter arteriole is high however if such a structure lacks an apparatus
to regulate flow, such as an encircling smooth muscle cell, then its
function within the vascular circuit is most likely different to a similar
caliber vessel that is enveloped by smooth muscle cells expressing αSMA.

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C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Integration of data regarding vascular diameter and the characteristics

of perivascular elements such as smooth muscle cells and pericytes could
allow a more standardised categorisation of the retinal vascular system
(An et al., 2020b). Such a construct provides information about flow and
resistance but also takes into consideration the structures that can
control retinal perfusion on a momentary basis.
Using established definitions of endothelial morphology (Yu et al.,
2005, 2010a) (Fig. 1), smooth muscle cell morphology and orientation
(An et al., 2020b; Yu et al., 2010a) (Fig. 4) as well as lumen diameter for
artery, arteriole, capillary, venule and vein we have previously proposed
a histologic lexicon for the macular circulation as follows.

• Artery – As per Hogan’s definition (Hogan et al., 1971; Hogan and

Feeney, 1963a), retinal arteries are vascular structures with a
diameter >15 μm. Retinal arteries can also be distinguished by
endothelial cells where the nuclei and long axis of the cell are ori­
ented parallel to the direction of blood flow (Fig. 1). Intracellular
microfilament (stress fibers) are seen as short and thick bundles
within endothelial cells of arteries (Fig. 2). Arteries are also char­
acterised by a coat of smooth muscle cells (usually one or more
layers), that are perpendicularly oriented and circumferentially
Fig. 11. Schematic representation of the human parafoveal circulation. The
envelop the endothelial cells of retinal arteries (Fig. 4). Retinal ar­ superficial vascular plexus (SVP) is located at the level of the ganglion cell layer
teries are surrounded by a capillary-free zone that is localised to the (GCL). The intermediate capillary plexus (ICP) is located between the inner
level of the SVP (Fig. 3). plexiform layer and the inner aspect of the inner nuclear layer (INL). The deep
• Arteriole – As per Hogan’s definition (Hogan and Feeney, 1963b) capillary plexus (DCP) is located within the outer aspect of the inner nuclear
retinal arterioles are vascular structures with a diameter between 8 layer (INL) and outer plexiform layer. Both SVP and ICP receive direct arteriolar
and 15 μm that serve as a conduit between arteries and capillaries. supply from a retinal artery. The SVP and ICP are interconnected by numerous
Arterioles are characterised by a surrounding capillary-free zone that vessels on both the arteriolar and venular aspects. In contrast, the DCP is
are not seen in venules of comparable lumen diameter. An arteriole supplied only by small arterioles that originate from the ICP. All three plexuses
can also be differentiated from a venule by its thicker wall with a form venules that drain into the retinal vein located at the level of the SVP. A
venule may drain directly into a vein or converge with venules from another
circular pattern of smooth muscle cells (usually one layer) encircling
plexus prior to draining into a vein. Reproduced with permission from An et al.
it (Fig. 1).
(An et al., 2020).
• Capillary – These are vascular structures with a diameter less than 8
μm (Hogan and Feeney, 1963b). Capillaries are characterised by a
was always the ICP and this occurred in the form of an a1 terminal
single layer of endothelium and basement membrane. Capillary
arteriole segment that connected the ICP to the DCP. Our work revealed
endothelia are characterised by long peripheral border intracellular
that blood flow to the DCP is critically dependent on the ICP and any
microfilament distributions that are oriented parallel to the long axis
insult to the ICP (or upstream to the ICP) can render the deeper layers of
of the cell as well as large oval shaped nuclei which were oriented
the retina ischaemic or anoxic.
parallel to the direction of blood flow (Fig. 1). A capillary loop is a
There is significant asymmetric arrangement of arterial inflow and
fully enclosed two-dimensional structure formed by two or more
venous outflow of the DCP. As discussed previously, inflow of the DCP is
capillaries located within the same plexus (Fig. 8). These capillaries
derived via a limited number of a1 terminal arterioles arising from the
share a common arterial supply and venous drainage.
ICP with a mean diameter of 8.3 μm (An et al., 2020b). In contrast,
• Venule – These are vascular structures between 8 and 20 μm in
outflow of the DCP occurs via significantly larger v3 order venules with
diameter that adjoin capillaries to retinal veins (Hogan and Feeney,
an average diameter of 18.8 μm. Such an arrangement of inflow and
1963b). The endothelium of venules are similar to capillaries but
outflow pathways renders the DCP vulnerable to ischaemic insults.
each cell does not form the entirety of the vessel tube on its own due
Previous investigators have modeled capillary-tissue exchange kinetics
to a larger vessel diameter (Fig. 1).
in the retina using the Krogh cylinder (McLeod, 2010; Schumacker and
• Vein – Vessels larger than 20 μm in diameter that are connected to
Samsel, 1989). Such modelling has been based on experimental data
venules can be defined as veins (Hogan and Feeney, 1963a). The
derived from muscle tissue. It assumes that the microvasculature is
morphology of venous endothelia is very different to arteries, arte­
comprised of independent circulatory units, each consisting of a feeding
rioles and capillaries and demonstrate a polygonal shape with round
arteriole, a capillary bed and a draining venule. In the retina, the venule
nuclei (Fig. 1). Veins are encircled by only a sparse number of smooth
of the DCP has been proposed to act as the major venous outflow system
muscle cells that are irregular in shape and orientation.
equivalent to the draining venule in a Krogh’s cylinder. However, such
as assumption fails to recognise the parallel nature of the retinal
4.1.2.2. Histology supports a series and parallel connectivity pattern of the
microvasculature, that is, every plexus of the retina contains its own
macula. Applying the definitions above and using a histology-based
draining venules (An et al., 2020b). Using our experimental data derived
categorisation system we found evidence of both series and parallel
from three-dimensional modelling of human donor eyes, we propose
arrangement of vascular networks in the normal human macula (An
that the DCP is not the primary venous outflow channel of the entire
et al., 2020b; Cabral et al., 2022). A schematic representation of the
retinal circulation as previously described (Henkind and Wise, 1974;
arrangement is provided in Fig. 11. Importantly, we did not find any
Michaelson, 1936).
evidence of direct connections from the retinal arteries located within
The asymmetric and parallel arrangement of human macular capil­
the SVP to the DCP. The predominant inflow arrangement was that of
lary beds has relevance for understanding the pathophysiology of a
arterioles feeding the SVP first, and subsequently the ICP, and this
number of important clinical entities. Firstly, conditions which pri­
occurred at a rate of 79%. A direct connection between the arterioles and
marily cause an absolute reduction or impaired retinal perfusion, such as
ICP was seen in 21% of cases. The origin of vascular inflow for the DCP
retinal artery occlusion, ocular ischaemic syndrome and DR, can lead to

12
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

severe hypoperfusion of the DCP prior to affecting the ICP and the SVP.
Therefore, PAMM may be considered a manifestation of mild to mod­
erate retinal ischaemia where partial retinal perfusion may still be
present at the level of the superficial capillary beds. Secondly, conditions
which primarily cause an elevation of distal pressure, such as retinal
vein occlusion, leads to elevation of venular pressure across all 3 retinal
plexuses due to the parallel arrangement of the capillary plexuses. An
asymmetric inflow and outflow arrangement can lead to nonperfusion of
the DCP, due to its inability to maintain a positive perfusion pressure
gradient, and lead to PAMM. In summary, it is important to remember
that PAMM is a manifestation of many aetiologies. PAMM may represent
a primary insult to inflow, outflow or both pathways between the
macular circulation.
It is well established that smooth muscle cells, pericytes and glia
regulate vascular tone and control perfusion (Hill et al., 2015; Kornfield
and Newman, 2014; vom Hagen et al., 2005; Yu et al., 2010b). They are
important elements of the apparatus that control local flow by
responding to vasoactive agents and autoregulation. There is an abun­
dance of these elements along large diameter vessels such as arteries and
arterioles (Fig. 12). The manner in which retinal perfusion to the smaller
capillary beds is controlled on a momentary basis has remained more
contentious.
αSMA is a major contractile protein in the retinal circulation that
mediates vasoregulation (Kim et al., 2020; Yu et al., 2019b). It is
expressed by smooth muscle cells, pericytes and endothelia (An et al.,
Fig. 13. Topographic patterns of αSMA staining within the parafoveal circu­
2020b). An examination of the distribution of αSMA between the SVP,
lation. An en face view of a projected confocal stack comprised of all capillary
ICP and DCP provides important clues to the mechanisms that control
plexuses reveals an observable variation in αSMA staining between the arterial
local perfusion in the macula at the level of the microcirculation. A and venous portions of the parafoveal circulation. The intensity and distribu­
striking observation regarding the pattern of αSMA stain in the macula is tion of αSMA staining is observably greater at the arterial side of the circulation.
the physiologic and sharp delineation of αSMA stain between the arterial Red = F-actin. Yellow = αSMA. Blue = Nuclei. Reproduced with permission
and venous portions of the circulation. More simply, the pattern of αSMA from An et al. (An et al., 2020).
stain alone, allows reliable differentiation between macular arterial and
venous systems. The pattern of prominent αSMA staining involves all inconsistent between the SVP, ICP and DCP. The DCP is the outlier with
elements of the arterial system including arteries, arterioles and capil­ respect to αSMA expression as it is virtually devoid in this layer in
laries (Fig. 13). Importantly however, this pattern of staining is normal human eyes. In addition to the incongruous pattern on αSMA
stain in the x-y axis there is also marked heterogeneity along the z-axis
and as a function of retinal depth. Most notably, there is an abrupt
transition in stain in the a1 arteriole segment that connects the ICP to
DCP (Fig. 14). Such a pattern of αSMA stain is remarkably similar to
circulatory beds within the brain (Fig. 15). αSMA termini have been
reported in brain studies and denote areas of sharp SMA termination
(Nehls and Drenckhahn, 1991). They represent sites where there is a
shift in pericyte coverage and most commonly occur at larger order
branches. There is a slight preponderance for these termini to be
observed at approximately 400 μm in cortical depth which corresponds
to the 4th layer in the cortex.
The DCP is devoid of smooth muscle cells that typically surround
large order vessels. Pericytes can be detected in the DCP however they
do not typically express αSMA, along with endothelial cells(An et al.,
2020b, 2022a). Collectively, the histologic evidence suggests that the
DCP has reduced capacity to autoregulate blood flow relative to other
vascular beds in the macula. The mechanisms that control perfusion to
the DCP predominantly originate at the level of the ICP as supported by
our three-dimensional angioarchitectural anatomic model(An et al.,
2020b). As discussed previously, the prevailing hypothesis regarding the
role of the DCP is that it is a predominant venous circulation and this
observation has been largely formed on the basis that shunt vessels in
retinal vein occlusion occur in the deeper retinal bed (Henkind and
Wise, 1974) (Fig. 16) However, evidence from our studies suggest that
the dilation of deep capillaries may be due to the inherent lack of αSMA
and vascular contractility in this layer and therein a reduction in
Fig. 12. Spatial relationship between retinal astrocytes and artery in a normal vascular capacitance.
human donor eye. Astrocyte processes (green ars) are found in close proximity
to an artery. Horizontally arranged ovoid nuclei belong to vascular smooth 4.1.2.3. OCTA supports a series connectivity pattern of the macula. The
muscle cells (VSMC). Note that several VSMC nuclei have irregular morphology introduction of OCTA has revolutionised the study of retinal vascular
(white ars). Red = F-actin. Yellow = GFAP. Blue = Nuclei. Scale bar = 30 μm.

13
C. Balaratnasingam et al.
Fig. 14. Staining patterns of arteries, arterioles, and capillaries with F-actin
and αSMA. All orders of vasculature stained positive for F-actin. Arterioles and
arteries stained intensely for αSMA. Capillaries on the arteriolar aspect showed
moderate staining in the superficial vascular plexus (SVP) and Intermediate
capillary plexus (ICP; A). Inset (B) illustrate the variation in staining pattern of
αSMA between the deep capillary plexus (DCP) and intermediate capillary
plexus (ICP). Separate images for F-actin, αSMA, and merged channels are
presented. Note that a1 segments, which are small arterioles that connect the
ICP to the DCP, consistently demonstrated an abrupt reduction in intensity
along their trajectory between the ICP and DCP (green arrows). Pericytes were
identified as αSMA positive, external to vessel walls of small arterioles and
capillaries, as highlighted in inset (C). Red = F-actin. Yellow = αSMA. Blue =
Nuclei. Reproduced with permission from An et al. (An et al., 2020).
Fig. 15. Example of αSMA termination in adult mouse brain cortex arterioles.
αSMA labelling intensity (green) is found to decrease sharply when the diam­
eter of the arteriole reaches less than 10 μm (white arrows). The arterial tree is
labelled according to vessel order system defined by the authors with order
0 representing the large artery. Reproduced with permission from Grant et al.
(Grant et al., 2019).
14
Progress in Retinal and Eye Research xxx (xxxx) xxx
diseases by facilitating clear visualisation of retinal capillary elements.
This has not been possible with FA that has been the longstanding gold
standard technique for imaging retinal vessels. The other advantage of
OCTA is the ability to stratify the capillary networks of the macula
relative to retinal depth, another feature that has not been consistently
possible with FA.
Several investigators have attempted to resolve the three-
dimensional connectivity pattern of the macular circulation using
OCTA. Nesper and Fawzi imaged 20 eyes from 10 healthy subjects and
reported that the arterioles and venules within the SVP had vascular
connections to the superficial capillary plexus (SCP), ICP and DCP
(Nesper and Fawzi, 2018). They also reported that arteriolar connec­
tions to the SCP and ICP capillaries were more easily identified than the
connections to the DCP. Campbell and colleagues imaged 9 eyes from 9
normal subjects using projection-resolved OCTA (Campbell et al., 2017).
They described that a diving arteriole feeds the SCP, ICP and DCP and a
diving venule drains the SCP, ICP and DCP. Freund et al. imaged 23 eyes
of 23 patients with retinal vein occlusion and applied volume rendering
to study the anatomic relationships between collateral vessels and
retinal capillary plexuses (Freund et al., 2018). They reported that
collateral vessels were predominantly seen at the level of the DCP and
because they could not find an example of collaterals that were exclu­
sively localised to the level of the SCP, they concluded that the circu­
lation conforms to an in-series vascular arrangement. The schematic
diagrams of the arrangement of the capillary circulations as proposed by
the three groups are summarised in Fig. 17.
4.1.2.4. Reconciling histology and OCTA differences. There are several
reasons that explain the discrepancy in the three-dimensional organi­
sation of blood vessels that are derived using high resolution histology
and OCTA-based information. Firstly, as previously stated, there is no
agreed upon lexicon for defining the different segments of the vascular
tree using histology or OCTA. For OCTA studies of the retinal circula­
tion, the size or caliber of vessels are commonly used to distinguish
arterioles from smaller-order vessels. Using OCTA, it is currently not
possible to discern the presence of smooth muscle cells, pericytes or glia
that control macular perfusion. Inherent differences in the classification
of capillaries, arterioles and venules between histology and OCTA can
lead to differences in three-dimensional constructs. Secondly, there are
limitations in the capabilities of OCTA to visualise deep capillary net­
works. This topic will be discussed further in Section 6. One important
issue that contributes to imprecision in DCP visualisation include faults
in automated layer segmentation. It is also difficult to visualise vertically
oriented vessels that connect the different plexuses using OCTA using
conventional approaches (Yu et al., 2021). A major advantage of his­
tologic approaches is that it is possible to apply immunohistochemical
techniques to co-localise vessels to different retinal layers and thereby
improve the accuracy of vascular modelling along the z-axis. The spatial
resolution (axial and lateral) of confocal scanning laser microscopy is
also significantly greater than the commercially-available OCTA devices
that were used to describe the previous models of macular microvascular
arrangement. Other reasons why it is not possible to extract a full
three-dimensional network of the retinal vasculature using OCTA
include artefact due to bulk movement of the head or eye or due to poor
fixation and microsaccades. Many OCTA devices also utilise fixed
interscan times between successive B-scans and this can limit the
detection of blood vessels with low flow rates. Changing the interscan
time may facilitate differentiation of vessels with low flow rates and fast
flow rates (Ploner et al., 2016). The oversampling ratio can also influ­
ence visualisation of retinal blood vessels. Oversampling can be used to
improve the quality of images but can also increase the presence of bulk
motion artefacts. Furthermore, the oversampling ratio can influence
lateral resolution such that smaller retinal vessels may not be detected
for larger scan areas compared to lower scan areas. Quantification of
blood flow velocity using phase resolved Doppler OCT also requires
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 16. Shunt vessels in the deep capillary circula­

tion. A case of chronic superior branch retinal vein
occlusion (BRVO) with shunt vessels is presented.
Colour fundus photography (A) and fluorescein
angiography (B) captured at 0:36 s showing extent of
disease with correlating loss of normal vascular ar­
chitecture. The site of the BRVO is characterised by
sheathed retinal vessels and some retinal haemor­
rhages. The macula of this case is also imaged with
optical coherence tomography angiography (OCTA);
C, D, E). Segmentation slabs of the superficial, deep
and all layers of the retina are presented. Segmenta­
tion of layers were performed automatically using
manufacturer settings (RTVue XR AngioVue). Some
degree of imprecision in segmentation as well as the
presence of projection artefact is seen in the deep
layer segmentation. However, the presence of dilated
shunt vessels are evident and predominantly seen to
be localised to the deep retinal layers (yellow ars).
Note the preserved vasculature in the unaffected
inferior quadrants of the OCTA images.

Fig. 17. Schematic representations of the macula

vasculature as modeled using OCTA from three
different studies. Panel A: Schematic representation
of hypothesised retinal vascular structure and blood
flow illustrating a predominantly series arrangement,
particularly on the venous aspect. There is a radiating
pattern in the superficial vascular plexus (SVP) and a
vortex-oriented pattern in the deep vascular complex
(DVC) specialised for outflow. Reproduced with
permission from Freund et al. (Freund et al., 2018).
Panel B: The anatomical relationships between arte­
rial and venous systems in the three vascular plexuses
and the interconnecting layers. Reproduced with
permission from Campbell et al. (Campbell et al.,
2017). Panel C: Proposed illustration of connectivity
and configuration of retinal vascular plexuses. The
superficial capillary plexus (SCP), middle capillary
plexus (MCP) and deep capillary plexus (DCP) receive
arteriolar supply from SCP arterioles and have
distinct drainage in SCP venules. Anastomotic con­
nections between each layer are seen in both arteri­
olar and venular aspects of the capillary beds.
Reproduced with permission from Nesper et al.
(Nesper and Fawzi, 2018). DCP, deep capillary
plexus; GCL, ganglion cell layer; ICP, intermediate
capillary plexus; IPL, inner plexiform layer; INL inner
nuclear layer; NFL, nerve fibre layer; OPL, outer
plexiform layer; HFL, Henle fibre layer; ONL, outer
nuclear layer; OLM, outer limiting membrane.

measurement of the angle between the OCT detection beam and retinal OCT is able to exploit the difference in contrast between vasculature and
blood vessels. This method cannot be applied when the Doppler angle is surrounding retina tissue to visualise the ICP and DCP (Gattoussi and
90◦ (Li et al., 2019; Spaide et al., 2018). Freund, 2017). Such a technique reduces the error due to projection
Previous work by Gattoussi and Freund showed that high-resolution artefact that is encountered when the ICP and DCP is visualised using

15
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

OCTA alone. In our most recent study we combined high resolution OCT
(axial optical resolution of 3 μm) with OCTA data to visualise the
three-dimensional properties of the macular circulation (Cabral et al.,
2022). Utilising such a technique yielded results that were similar to our
histologic findings namely that it supported a series and parallel con­
nectivity patten within the macular circulation.

4.1.2.5. Physiologic implications of series and parallel circuits. The rea­

sons for reconciling the organisation of the macular circulation are
manifold from a physiologic standpoint. For instance, the arrangement
has great consequence for modelling resistance within the macular
vascular tree. Given that the resistance of blood vessels is a major
component of what dictates tissue perfusion the magnitude of vascular
resistance is expected to have a direct impact on nutrient/waste ex­
change in retinal tissue. A series arrangement of networks will have
greater vascular resistance than one where the networks are largely
arranged in parallel.
Another important reason for distinguishing series from parallel
networks is that it provides valuable information regarding upstream
and downstream control mechanisms of blood flow. In the model con­
structed using histology data, it is proposed that the ICP is intrinsically
linked to the control of blood flow in the DCP as there is no direct
communication between arteries and the DCP. In contrast the OCTA
models of the macula circulation suggest that the DCP may have an Fig. 18. Organisation of capillary networks at different eccentricities viewed in
independent circuit for blood flow control through a direct communi­ 3D via the X-Z axis (left panel) and in 2D via full z-stack projection (right
cation with arteries. These anatomic considerations have direct rele­ panel). The parafovea (A) consists of three separate plexuses, including the
vance for understanding the pathophysiologic mechanisms underlying superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep
any entity that preferentially involves selected capillary beds such as capillary plexus (DCP). The 3-mm location (B) along each of the vascular ar­
PAMM, acute macular neuroretinopathy and collateral vessels in retinal cades also contains an additional layer—the retinal peripapillary capillary
vein occlusion. plexus (RPCP). Retina thickness reduces peripherally, where the 6-mm region
(C) typically contains two distinct retinal layers, with arteries and veins running
within the superficial layer. Communicating vessels are evident between the
superficial and deep layers. The 9-mm region (D) typically contains only a
4.2. The peripheral retinal circulation
single capillary plexus, with arteries and veins located in a slightly more su­
perficial plane than the capillaries. Vasculature labelled using Lectin.
The peripheral retinal circulation supports rod-dominant vision. The
tissue is thinner in the periphery and the neurons increase in size and are
Another important observation regarding the far peripheral retinal
lower in density in the peripheral retina compared to the posterior pole
circulation in DR is that it is most often devoid of microaneurysms.
(Toussaint and Danis, 1970). The structural organisation of the pe­
Microaneurysms are typically localised to the posterior pole and mid­
ripheral retinal vasculature have arguably lower optical constraints as it
periphery retina in DR. The reason for this is unclear. In Supplementary
is not responsible for high-acuity vision. The complexity and number of
Fig. 2, we present the ultrawide field (UWF) angiographic appearance
capillary layers diminishes in a centrifugal direction from the macula to
between a patient with DR due to Type 2 DM and Coats disease. His­
the far peripheral retina. In our previous studies we have shown that the
topathology studies have shown that pericyte loss accounts for the for­
eccentricity 3 mm from the macula is comprised of four plexuses, the
mation of peripheral retinal aneurysmal dilations in Coats disease
eccentricity 6 mm from the macula is comprised of two plexuses and the
(Tripathi and Ashton, 1971). The preferential involvement of the pos­
eccentricity 9 mm from the macula is comprised of a single plexus (An
terior pole in the formation of microaneurysms in DR provides further
et al., 2021) (Fig. 18). This is associated with a reduction in total
evidence that the pathogenic mechanisms acting on the vascular ele­
capillary density between the 3 mm plexus (91.5 ± 24.3), 6 mm plexus
ments of the peripheral circulation are possibly different compared to
(64.9 ± 15.7) and 9 mm plexus (44.0 ± 11.3).
DR.
The peripheral and mid-peripheral circulation is important as it can
be preferentially involved in ischaemic retinal vascular disorders such as
5. Manifestations of diabetic retinopathy
DR with sparing of the posterior pole (An et al., 2021). When vascular
occlusion is observed in the peripheral retina it can very often involve
5.1. Vascular endothelia dysfunction
the entire vascular bed extending from the level of the arteriole to the
venule. Furthermore, when there is evidence of peripheral vascular
Vascular endothelial cells play a vital role in maintaining cardio­
occlusion it typically involves all capillary beds between the arteriole
vascular homeostasis (Rubanyi, 1993). From a cellular standpoint,
and venule. However, we have histologic evidence to show preferential
vascular endothelial dysfunction is proposed to be one of the earliest
involvement of the deeper capillary beds in some instances of peripheral
pathogenic alterations to occur in DM (Avogaro et al., 2011; De Meyer
retinal ischaemia using human donor eyes (Fig. 19). This observation
and Herman, 1997; Tousoulis et al., 2012). Endothelial dysfunction re­
suggests that the pathophysiologic mechanisms underlying vascular
fers to the loss of endothelia capability to perform physiologic function
occlusion in the macula is, in important ways, different to the peripheral
such as fibrinolysis, vasodilation and anti-aggregation of platelets
retina (Bek, 2013). By extension, if the pathophysiologic mechanisms
(Avogaro et al., 2011). Changes to the vascular endothelia in DM are
underlying vascular occlusion is different between the macula and pe­
diffuse and involve multiple organs within the cardiovascular system
ripheral retina the risk of developing sight-threatening complications
including heart, brain and the carotid vascular system. Endothelial
such as macular oedema and retinal neovascularisation will also be
dysfunction characterises the genesis of atherosclerosis that can progress
different. Patterns of peripheral retinal vascular occlusion will be dis­
to myocardial infarction, stroke, peripheral vascular disease and carotid
cussed in more detail in subsequent sections of this article.

16
C. Balaratnasingam et al.
stenosis (Daiber et al., 2017; Libby et al., 2006). The pathogenic
mechanisms that lead to endothelial dysfunction in DM are complex and
not due to a single process. More specifically, although hyperglycemia
can alter endothelia function there is evidence to indicate that endo­
thelial dysfunction can progress despite restoration of the biochemical
environment to a normo-glycemic state (El-Osta et al., 2008). Mecha­
nisms other than hyperglycemia that mediate vascular endothelial
dysfunction in DM include oxidative stress, localised inflammatory
cascades and depletion of nitric oxide levels (Funk et al., 2012).
Given that endothelial dysfunction is one of the earliest vascular
alterations to occur in DM, a number of clinical tests have been devel­
oped to measure endothelia activity as a means of detecting and pre­
dicting the risk of cardiovascular morbidity in DM. Some of the
commonly utilised tests include:
1. Brachial artery imaging with high resolution ultrasound during
reactive hyperemia - This test is often referred to as flow mediated
dilation. In this test, forearm or hand ischaemia is induced by
interrupting arterial blood supply with a cuff inflated to supra-
systolic pressure. The magnitude of the change in vessel diameter
from the baseline period to the peak observed during reactive hy­
peremia is used as a measure of endothelial function. This test is not
well tolerated by all subjects and is also dependent on the skill of the
ultrasound operator (Deanfield et al., 2007; Verma et al., 2003).
2. Peripheral artery tonometry - Pulse wave amplitude is assessed
before and during reactive hyperemia, which is induced by occluding
blood flow of the brachial artery using an inflatable cuff. The
calculated index between the flow in the arm with reactive hyper­
emia and the control arm represents a measure of the endothelial
function. This test suffers the same limitations as flow-mediated
dilation as described above (Deanfield et al., 2007; Verma et al.,
2003).
3. Venous occlusion plethysmography with or without administration
of vasoactive agents such as nitric oxide. In this test, measures of
early volume change following occlusion of an anatomic compart­
ment in the forearm or leg is used to estimate the rate of arterial
inflow and endothelial dysfunction. This test can be invasive and
sometimes requires brachial artery cannulation c(Deanfield et al.,
2007; Verma et al., 2003).
4. Measuring serologic levels of thrombomodulin, von Willebrand fac­
tor, E-selectin, asymmetric dimethylarginine, intracellular adhesion
17
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 19. Capillary density comparison of the mid-
peripheral retina between control (non-DR; panel A)
and two patterns of capillary nonperfusion in DR
(panels B and C). The superficial vascular plexus (SVP;
top row) and deep capillary plexus (DCP; bottom row)
of mid-periphery retina 6 mm nasal to the optic nerve
are presented. The intermediate capillary plexus is not
present at this location. Control retina shows normal
density of perfused vessels (A). Panel B illustrates the
most common pattern (pattern 1) of peripheral
capillary loss where both the SVP and DCP show areas
of significant nonperfusion (dotted line encircled re­
gions). Panel C illustrates a less common pattern
(pattern 2) where the degree of nonperfusion in the
DCP is profoundly more severe compared to the
overlying SVP. Sites of arteriolar occlusion can be
identified via vessel tracing in 3D and are indicated
using red ars. Note that the peri-arterial capillary-free
zone is a normal physiological finding and are indi­
cated using yellow asterisks. Vasculature labelled
using Lectin. Scale bar = 300 μm.
molecule 1 (ICAM-1), C-reactive protein and copper/zinc superoxide
dismutase. The disadvantages of these tests for endothelia dysfunc­
tion is the occurrence of false-positive and false-negative results as
well as the limited specificity and sensitivity of positive findings.
Such tests are invasive, costly and characterised by a delay between
when the test is performed and when the results are returned (Con­
stans and Conri, 2006; Leite et al., 2020).
The eye is one of the few systems in the human body where the
capillary circulation can be directly visualised using in vivo state-of-the-
art clinical imaging. Despite the perceived advantages of using the
ocular vasculature (specifically the retina) as a biomarker for assessing
diffuse systemic microvascular dysregulation there has been only a
limited number of studies that have leveraged and validated this concept
such that it can be applied in daily clinical practice in a meaningful way.
It is firmly established that perturbations to the retinal endothelia are
one of the earliest features of DR (Gui et al., 2020; Yu et al., 1998b,
2005). Given that it is possible to resolve retinal vessels up to a diameter
of 7–8 μm using OCTA it should be possible to leverage this technology
to investigate endothelial dysfunction in DM(Yu et al., 2018, 2021). The
perceived advantages of evaluating microvascular dysfunction in the
retina instead of systemic macrovascular diseases are many. Major ad­
vantages of OCTA techniques are that it is a safe, rapid and reproducible
test and has already been shown to overcome many of the limitations of
the current clinical tests for assessing endothelial dysfunction described
above. In a subsequent section of this article (Section 7.5) we propose a
novel way to detect endothelial dysfunction in DM using OCTA.
5.2. The retinal microaneurysm
Although endothelial dysfunction is one of the earliest functional
disturbances to occur within the systemic circulation in DM it is the
retinal microaneurysm that is the hallmark structural change that
characterises the earliest retinal abnormality in DR (Ballantyne and
Loewenstein, 1944; de et al., 1976; Hausler and Sibay, 1961; Pope,
1960). In other words, the retinal microaneurysm is the manifestation
that is globally recognised in the clinical setting to be the first structural
change to characterise the onset of DR. Since the first description by
Nettleship in 1879, several controversies have surrounded the patho­
physiology of diabetic microaneurysms (Mackenzie and Nettleship,
1879). One of the prevailing controversies regarding microaneurysms is
C. Balaratnasingam et al.
whether they are a fundamental structural abnormality due to out­
pouchings of the vascular wall or whether they represent abortive at­
tempts of retinal neovascularisation (Ashton, 1963).
Putative pathogenic mechanisms that underlie microaneurysm for­
mation include pericyte loss, basement membrane injury, endothelial
proliferation, increases in capillary haemodynamic forces and upregu­
lation of regional vasoproliferative growth factors (Aguilar et al., 2003;
Ashton, 1963; Cogan et al., 1961a; Pope, 1960; Tolentino et al., 2002). It
is important to understand the pathophysiology of microaneurysms for
several reasons:
1) As they are one of the earliest clinically recognisable manifestation of
DR, delaying or preventing the formation of microaneurysms will
possibly halt the development of other retinal vascular complications
such as ischaemia and retinal neovascularisation.
2) Understanding why some microaneurysms regress (Fig. 20) may
provide novel insights into how the structural alterations of DR can
be reversed without a legacy of permanent structural damage or
vision loss.
3) The lifecycle and quantitative properties of microaneurysms such as
rate of turnover and microaneurysm count are closely linked to the
risk of retinopathy progression (Nunes et al., 2009; Pappuru et al.,
2019; Santos et al., 2021). The pathophysiologic mechanisms un­
derlying the microaneurysm is therefore likely to be an exemplar for
the other structural manifestations of DR such as IRMA, venous
beading and possibly retinal neovascularisation.
Fig. 20. Growth and regression of microaneurysms (MA). Fluorescein angiog­
raphy (FA; A and B) and image-registered optical coherence tomography (OCT;
C and D) imaging from a 52-year-old male subject with Type 2 Diabetes mellitus
at baseline visit. Two different microaneurysms (MA1 and MA2) are evaluated
using each of these imaging modalities over a two year period. MA1 and MA2
are seen in close vicinity within the microcirculation of the superior macula.
MA1 is seen on FA and OCT at baseline visit in 2020. On OCT, the occurrence of
a shadow projection confirms the presence of MA1 in 2020. By 2022, MA1 is
not seen on FA or OCT and does not leave any observable legacy of retinal
structural change. MA2 has increased in size over the same time span and this
observation is most evident on the OCT images. By 2022, other new micro­
aneurysms are seen on the FA (green arrows). The time stamps for each FA
image are provided in the lower right hand corner of each panel.
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Progress in Retinal and Eye Research xxx (xxxx) xxx
4) Leakage from microaneurysms can result in retinal thickening,
distortion of the layered organisation of retinal layers, and accu­
mulation of fluid in the subretinal compartment (An et al., 2022b;
Blair et al., 2008; Reznicek et al., 2011); all of which can culminate in
irreversible vision loss.
In the subsequent sections we will discuss the most recent clinical-
and laboratory-based research on retinal microaneurysms and how they
have aided our understanding of DR. An emphasis will be given to our
research studies.
5.2.1. Histology of retinal microaneurysm
Given the great variability in the size, natural course and functional
properties of retinal microaneurysms it is likely that a number of
different, and possibly distinct, biologic pathways modulate their
morpho-functional characteristics. Histology is a powerful means of
studying the cellular abnormalities associated with microaneurysms and
by extrapolating the findings from histologic reports it has been possible
to generate biological models that underlie the microaneurysm life
cycle. The advantage of histology and electron microscopy is that it
provides unparalleled, high-resolution analysis of cellular detail. The
major drawback however is that only a single time-point in the natural
course of the disease process can be studied using histology. Correlations
between in vivo multimodal clinical imaging and histopathology may
help overcome some of these limitations.
Previous histologic investigations regarding diabetic micro­
aneurysms have focussed on the role of pericyte loss, endothelial
dysfunction, ischaemia and inflammation (Aguilar et al., 2003; Cogan
et al., 1961; Hausler and Sibay, 1961; Stitt et al., 1995). Many of these
previous studies considered each of these elements in isolation with less
focus on the effects of pathogenic interactions and how the loss of one
element could influence the other. As an example, the elegant studies by
Cogan and colleagues were one of the earliest reports to make the
observation that pericyte loss was associated with the formation of
microaneurysms in DR (Cogan et al., 1961a). Myron Yanoff expanded
upon this observation using a quantitative study of endothelia:pericyte
count in DR (Yanoff, 1966). He showed that the mean ratio of pericytes:
endothelia in a control group was 1:1 while that in DR was significantly
reduced being 1:3.5. However, both Cogan and Yanoff did not investi­
gate the role of ischaemia or inflammation in microaneurysm formation
and their works were not designed to explain how microaneurysms
could form without pericyte depletion. It should be noted that many of
the pivotal studies concerning the histopathology of DR were performed
nearly five decades ago. Trypsin digest was commonly used in the his­
tologic preparation in many of these studies and such techniques may
have influenced some of the conclusions through artefactual cellular
changes/loss that can occur during tissue preparation with trypsin. Such
methods of tissue preparation may have also artefactually influenced
spatial relationships between vascular structures.
Subsequent to the work by Cogan and Yanoff, Yamashita and Rosen
were one of earliest investigators to apply transmission electron mi­
croscopy techniques to study microaneurysms (Yamashita and Rosen,
1962). They described two types of microaneurysms, the saccular type
with a thin walled balloon shape associated with surrounding haemor­
rhage, and the hyalinised type with a thick periodic acid-schiff positive
wall. They postulated that all microaneurysm develop as the saccular
type and subsequent hyalinisation occurs over time. Importantly, pro­
liferation of endothelial cells was noted in a number of microaneurysms.
Subsequent to their work, Stitt and colleagues evaluated the ultra­
structure of diabetic microaneurysms and proposed four stages of
microaneurysm formation (Stitt et al., 1995). The early stage (type 1
microaneurysm) was characterised by luminal occlusion due to the
accumulation of monocytes and leucocytes. Type 2 and 3 micro­
aneurysms were characterised by endothelial and pericyte loss and
variable quantities of red blood cells infiltrating the microaneurysm
lumen. Type 4 microaneurysms were typified by dense walls of
C. Balaratnasingam et al.
thickened basement membrane with proliferation of extracellular ma­
trix within the lumen.
5.2.2. Microaneurysm histology informs the pathophysiology of DR
In an earlier section of this article, we provided reasons why ocular
perfusion techniques coupled with three-dimensional microscopy con­
fers a distinct advantage for investigating spatial relationships between
cells within the retina. We have applied this technique extensively to
study the structural characteristics of microaneurysms. In our most
recent study we utilised human donor tissue from patients with DR to
investigate the pathogenic mechanisms associated with the occurrence
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Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 21. Microaneurysm located within non-
ischaemic and ischaemic regions. Panel A shows a
microaneurysm (yellow ar) located within an
observably normal circulation with respect to
vascular density and minimal evidence of capillary
non-perfusion. This is referred to as a non-ischaemic
region. However, some capillary structures with this
region demonstrated vessel staining and leakage
(blue ar). Panel B shows a large cluster of micro­
aneurysms located in an ischaemic region. Ischaemia
is represented by focal areas of capillary non-
perfusion (yellow asterisks) which results in a
reduction in capillary density. Vasculature perfused
with Lectin.
Fig. 22. Pericytes and microaneurysm (MA). Panel A
is a z-projected image of a 60 μm diameter MA
without pericytes. Panel B is an internal cross-section
of the MA shown in Panel A, demonstrating the
preservation of endothelial cells (yellow ars) within
the MA. Panel C is a z-projected image of a 20 μm
diameter MA and Panel D is an internal cross-section
of the same MA demonstrating the presence of
endothelial cell (yellow ar) within the basement
membrane (green ar) and pericyte (red ar) outside the
basement membrane of the MA. Grey = Lectin. Blue
= Nuclei.
of microaneurysms (An et al., 2022b). Arguably, our report constituted
the largest histologic study of microaneurysms in DR. We performed
three-dimensional quantitative histologic analysis of 636 micro­
aneurysms from 20 donor eyes and focused on three putative factors that
have previously been associated with microaneurysm formation:
ischaemia, inflammation and pericyte loss. The major findings of that
report are as follows:
1) 33.6% of microaneurysms were found in areas of observably normal
capillary perfusion, that is, nearly one third of microaneurysms
formed at sites of normal capillary density (Fig. 21).
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 23. Presence of inflammatory cells in microaneurysm (MA). Panel A shows a MA in cross-section without inflammatory cells within its lumen. Panel B shows a
MA with two nucleated inflammatory cells adhering to the MA endothelium (green ar). In Panel B, the morphology and nuclear pattern of inflammatory cells are
consistent with neutrophils. Grey = Lectin. Blue = Nuclei.

2) Pericyte loss was found in 60.8% of microaneurysms, that is, nearly
40% of microaneurysms demonstrate preservation of pericytes
(Fig. 22).
3) 31.3% of microaneurysms demonstrate histologic evidence of
intraluminal inflammatory cells (Fig. 23).
Collectively, our findings indicate that the pathophysiologic mech­
anisms underlying microaneurysms are complex and cannot be attrib­
uted to a single factor. When we evaluated the interaction between
different pathogenic mechanisms and the frequency at which one factor
occurred in the presence of the other we reached two important con­
clusions: 1) Pericyte loss is nearly twice as likely to occur within
microaneurysms in areas of capillary nonperfusion compared to those
regions where capillary density is observably normal; 2) Inflammatory
cells are nearly twice as likely to be found in microaneurysms within
areas of nonperfusion than those regions where capillary density is
preserved.
We then studied the predictive factors that were significantly asso­
ciated with microaneurysm size using the assumption that an increase in
microaneurysm size is indicative of a more-progressive stage of the
microaneurysm lifecycle (Moore et al., 1999; Stitt et al., 1995). The
results of that analysis are summarised in Table 2 (An et al., 2022b).
In summary, our analysis suggested that the pathways governing
microaneurysm formation and progression in areas of capillary non­
perfusion are likely to be different to those areas where perfusion is
relatively preserved. We found that within areas of capillary non­
perfusion the chronology of microaneurysm progression was likely
characterised by an initial loss of pericytes after which luminal
enlargement and inflammatory cell infiltration occurred. Within areas of
capillary nonperfusion, only 1.4% of microaneurysms were found to
manifest inflammatory cells as well as preservation of pericytes. The
largest microaneurysms were those that manifested luminal inflamma­
tory cells and an absence of pericytes.
In areas where perfusion was preserved, the mean diameter of
microaneurysms was much smaller than regions with nonperfusion.
Additionally, in regions where perfusion was preserved, absence of
pericytes alone and presence of inflammatory cells alone were not sig­
nificant determinants of microaneurysm size. This finding was different
to the properties of microaneurysms within areas of capillary non­
perfusion. Based on these results we propose several different patho­
genic mechanisms, that possibly overlap and underlie the lifecycle of
microaneurysms. It is possible that these mechanisms are also relevant
to the pathophysiology of other vascular changes that characterise DR
such as IRMA. Irrespective of the pathogenic mechanism that propagates
the microaneurysm lifecycle, from a cellular standpoint, endothelial cell
proliferation is frequently seen within microaneurysms implicating the
key role of endothelial dysfunction in these structural abnormalities.
Our histologic surveys and the work by Aguilar et al. have clearly shown
the occurrence of endothelial proliferation within microaneurysms
(Aguilar et al., 2003; An et al., 2022b). The relevance of these findings in
the context of retinal vascular occlusion are discussed in a subsequent
section of this manuscript (Section 5.3.1.2).
5.2.3. Relevance of microaneurysm morphology
Microaneurysms represent outpouchings of the retinal capillary wall
but their morphologies can vary (Dubow et al., 2014). It is unclear if
microaneurysm morphology correlates to a distinct pathogenic mecha­
nism in DR or if it plays a role in modulating the natural course of DR. In
broad terms, the morphologies of microaneurysms can be divided into
the following categories (Fig. 24):
• Focal bulge – Small microaneurysm where the combined width of the
microaneurysm and the feeding capillary measures less than twice
the width of an unaffected segment of the feeding capillary.

Table 2
Univariate and multivariate analyses for determinants of microaneurysm diameter.
Microaneurysm Diameter Analysis Univariate Analysis Multivariate Analysis

Coeffici-ent (μm) Standard Error (μm) P-Value Coeffici-ent (μm) Standard Error (μm) P-Value

Capillary nonperfusion 7.89 1.95 <0.001 4.91 1.59 0.002

Pericyte (present) − 12.64 2,57 <0.001 − 4.99 − 1.98 0.005
Inflammatory cells (present) 17.62 1.89 <0.001 16.22 21.56 <0.001
Leakage (present) − 12.01 1.83 <0.001 − 6.48 0.92 <0.001
Age 0.08 0.06 0.175 0.57 0.55 0.300
Sex − 1.31 1.84 0.229 − 10.85 12.43 0.383

20
C. Balaratnasingam et al.
(caption on next column)
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Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 24. Microaneurysm (MA) morphology. Histologic illustration of the five
different MA morphologies in diabetic retinopathy. The left panel represents
full thickness z-projection image to highlight each of the MA morphologies. The
right panel represents a single X–Y cross-section slice with nuclear stain to
illustrate the internal structure of each of these MA. A: Focal bulge MA is
typically less than 20 μm in size. B: A pericyte nucleus is found beyond the
basement membrane (red asterisk) C: The lesion on the right represents a large
fusiform MA with dilation on both sides of the capillary. D: An inflammatory
cell is attached to the endothelium within the lumen (yellow asterisk). E: a large
saccular MA with thrombus which contains non-nucleated red blood cells (red
asterisk). F: Inflammatory cells (yellow asterisk) are also found within the
lumen. G: A pedunculated MA showing only one feeder vessel (red ar). The
outflow vessel is occluded. H: thrombus is found within MA chamber (yellow
asterisk). Endothelial cells are found on the inner aspect of the basement
membrane (green asterisks), one of which appears to be hypertrophic. Pericytes
are found on the outer aspect of the basement membrane (red asterisks). I:
Irregular MA are usually large (>80 μm diameter) with complex surface con­
tour as well as internal structure. J: multiple chambers are seen and separated
by fibrous material (green ar). Grey = Lectin. Blue = Nuclei. All scale bars =
30 μm.
• Fusiform – Microaneurysm with dilation on both sides of the capil­
lary, with less than 75% of the total area of the microaneurysm on
either side of the capillary.
• Saccular – Microaneurysm with dilation towards one side of the
capillary with more than 75% of the total area. Saccular micro­
aneurysms have a relatively rounded morphology in the en face view.
• Pedunculated – Microaneurysm with a single associated capillary
segment and an occluded distal segment.
• Irregular – Microaneurysm with dilation towards one side of the
capillary with more than 75% of the total area, but with a complex
surface morphology that is irregular.
It is unclear if there is a difference in the frequency of each of the
microaneurysm morphologies in DR. For the purposes of this manuscript
we analysed the 636 microaneurysms from our previous histologic study
and categorised microaneurysm morphology with respect to widest
diameter and frequency of occurrence (An et al., 2022b). We provide the
results of the unpublished histologic data henceforth. That analysis
showed that the most common microaneurysm morphologies included
focal bulge (20.1%; 24.9 ± 8.9 μm), fusiform (36.0%; 47.7 ± 24.4 μm)
and saccular (35.7%; 43.3 ± 22.0 μm). Fusiform and saccular micro­
aneurysms were similar in diameter (P = 0.394), and both were larger
than focal bulge (both P < 0.001). Pedunculated (4.9%; 54.6 ± 24.0 μm)
and irregular (3.3%; 74.4 ± 27.4 μm) microaneurysms are observed
infrequently. There is no difference in morphology distribution between
central and peripheral locations of the retina (P = 0.468), and between
capillary plexuses within the circulation where more than two plexuses
are present (central location, P = 0.802; mid peripheral location, P =
0.939). There is also no difference in microaneurysm morphology be­
tween regions with and without capillary nonperfusion (P = 0.969).
Data for microaneurysm morphology across different retinal layers is
provided in Table 3.
Given the absence of clear relationships between microaneurysm
morphology, the topologic properties of the retinal circulation, and the
presence/absence of regional ischaemia it is possible that other patho­
genic elements in the retinal circulation dictate microaneurysm
morphology. The role of haemodynamic alterations within the retinal
microcirculation in DR is poorly understood but may be inherently
linked to microaneurysm morphology. The neurosurgical literature de­
scribes clear links between cerebral vascular geometry, blood flow dy­
namics and the development of aneurysms. More precisely, there is
strong evidence that aneurysm-to-parent- vessel size ratio is an impor­
tant determinant of cerebral aneurysm formation and the risk of rupture
(Zhang et al., 2014; Zheng et al., 2016). As an example, the International
Study of Unruptured intracranial Aneurysms (ISUIA) found that
perpendicular height was the strongest predictor of risk of rupture of
C. Balaratnasingam et al.
Table 3
Microaneurysm morphology and prevalence across different retinal layers.
Central Retina
Superficial (n = 49) Intermediate (n = 50)
Focal Bulge 11 (22.4%) 8 (16.0%)
Saccular 19 (38.8%) 22 (44.0%)
Fusiform 15 (30.6%) 18 (36.0%)
Pedunculated 3 (6.1%) 1 (2.0%)
Irregular 1 (2.0%) 1 (2.0%)
cerebral aneurysms (Mocco et al., 2018). A schematic illustrating how
perpendicular height was calculated in the study is presented in Fig. 25.
It is plausible that parallel pathophysiologic mechanisms underscore the
development of cerebral aneurysms and retinal microaneurysms. Clearly
there are important distinctions between the two vascular abnormal­
ities. Most importantly, cerebral aneurysms commonly originate from
large order arteries such as the anterior communicating artery, posterior
communicating artery and the carotid artery. Such large arteries consist
of a tunica intima, tunica media and tunica externa and are quite
different to the vascular anatomy of retinal capillaries. Large arteries are
also prone to atherosclerosis (Greig, 1956), which is an important pre­
cursor for the formation of aneurysms whereas retinal capillaries do not
form atheromatous plaques but can be subject to inflammatory and
immune-complex mediated damage to the vascular wall (Altmann and
Schmidt, 2018; An et al., 2022b; Kern, 2007).
Although there are inherent differences between the structural
characteristics of cerebral arteries and retinal capillaries, the concept
that focal alterations in flow patterns due to disease-induced modifica­
tions in vascular geometry may be shared. We have performed pre­
liminary work to investigate the relationships between retinal
microaneurysms and parent vessel geometry in DR. There are significant
methodologic challenges with such work as it requires computational
three-dimensional modelling and mathematical assumptions.
Our preliminary analysis supports the concept that there are signif­
icant differences in geometric relationships between parent vessels and
microaneurysm morphology subtypes in DR. As an example, we
22
Progress in Retinal and Eye Research xxx (xxxx) xxx
Peripheral Retina
Deep (n = 50) Superficial (n = 143) Deep (n = 206)
6 (12.0%) 33 (23.1%) 50 (24.3%)
16 (32.0%) 50 (35.0%) 65 (31.6%)
26 (52.0%) 51 (35.7%) 72 (35.0%)
1 (2.0%) 5 (3.5%) 14 (6.8%)
1 (2.0%) 4 (2.8%) 5 (2.4%)
illustrate the markedly different microaneurysm and parent vessel ge­
ometry for a saccular and focal bulge microaneurysm in Fig. 26.
Other lines of evidence suggest that the haemodynamic properties of
the retinal microcirculation may be associated with microaneurysm
morphology. Cai and colleagues used in vitro microfluidic experiments to
study the mechanics of blood flow in diabetic microaneurysms (Cai
et al., 2021). Microchannels were designed to mimic various shapes of
saccular microaneurysms. Velocity fields and stress fields were inferred
from two dimensional images of red blood cell flow using artificial in­
telligence techniques. Using such a technique they were able to show
that microaneurysms with different body-to-neck ratio were associated
with changes in flow velocity and wall shear stress. One of the limita­
tions of this technique was the in vitro setup that did not account for the
influence of capillary branching patterns and surrounding supportive
cells, such as pericytes and glia, that may influence the intraluminal
haemodynamic parameters. Ocular perfusion technique is the most
precise means for labelling the luminal wall of the microaneurysm
whilst limiting the amount of noise. By increasing the quality and
magnitude of experimental data using an ocular perfusion setup in a
human eye, it may be possible to apply artificial intelligence velocimetry
to perform a more nuanced study of the haemodynamic parameters that
influence the pathophysiology on microaneurysms.
Rotational three-dimensional visualisation of microaneurysms using
OCTA has also provided novel insights into microaneurysm geometry
and their relationship with parent vessels. Using OCTA, Borrelli et al.
showed that microaneurysms were associated with 1–4 parent vessels
Fig. 25. 3D model of cerebral aneurysm depicting
morphological variables previously studied in the
literature. The aspect ratio (AR) is obtained by
dividing the perpendicular height by the neck diam­
eter. Size ratio (SR) is calculated by dividing the
maximum height (Hmax) by the average composite
diameter of the all vessels (BAv, RPCAv, LPCAv,
RSCAv, LSCAv) involved with the aneurysm. Com­
posite diameters are obtained by averaging the initial
diameter of the vessel (BA1, RPCA1, LPCA1, RSCA1,
LSCA1) at the vessel branching point by the aneurysm
neck with the diameter of the vessel 1.5 away from
the initial diameter (BA2, RPCA2, LPCA2, RSCA2,
LSCA2). Aneurysm angle is defined as the angle be­
tween the vectors formed by the maximum height of
the aneurysm with the aneurysm neck. The vessel
angle is defined as the angle between the vector of
flow and the neck of the aneurysm. The flow angle is
defined as the angle between the vector of flow and
the vector formed by the maximum height of the
aneurysm. (Ho, AL et al. 2014).
C. Balaratnasingam et al.
Fig. 26. Morphologic parameters that may predict risk of microaneurysm (MA)
growth and leakage in diabetic retinopathy (DR). Three-dimensional rendered
images of MA from human donor eyes are presented. Confocal scanning laser
microscope images of MA histology have been rendered using Imaris micro­
scopy image analysis software. The morphology of a focal bulge MA (A) and
saccular MA (B and C) are presented. The approximate boundaries of neck
diameter (red dashed line) and maximum height (yellow dashed line) are
indicated for each MA. Note that the ratio of maximum height to neck diameter
is vastly different between the three lesions. The composite diameters of feeding
and draining vessels and the vessel angles are also different between the three
MA images. Letters embedded into each figure panel denote the anatomic
viewpoints used to analyse the image. A = anterior; L = left; P = posterior; R =
right; S = superior.
and that nearly 60% of microaneurysms were associated with two ves­
sels (Borrelli et al., 2019). Collectively, the above bodies of work give us
confidence to propose that regional microcirculatory haemodynamic
disturbances and flow shear stress are linked to the morphologic sub­
types of microaneurysms. Validating the inherent role of haemodynamic
alterations in the pathophysiology of DR is important and may provide a
tangible pathogenic factor that can be targeted to reverse the
23
Progress in Retinal and Eye Research xxx (xxxx) xxx
complications of DR. Restoration of focal retinal haemodynamic alter­
ations back to the physiologic range may be one reason to explain
regression of microaneurysms. It is currently not possible to directly
quantify the haemodynamic characteristics of the retinal microcircula­
tion however it may be possible to make assumptions regarding regional
wall shear stress and flow rates in DR based on microaneurysm
morphology. If we were to acquire a series of three-dimensional struc­
tural data regarding the retinal microcirculation over time it may be
possible to speculate upon blood flow changes within microaneurysms.
In the field of neuroradiology, four-dimensional imaging has been used
to describe changes in three-dimensional structural data over time with
time being the fourth dimension. Specifically, neuroradiologists have
used four-dimensional flow magnetic resonance imaging (MRI) to assess
the risk of aneurysm rupture. Four-dimensional MRI is a set of
three-dimensional volumes that are captured over time. Such data is
used to quantify the haemodynamic environment of cerebral aneurysms
and identify vortex cores that predispose to rupture (Futami et al.,
2019). Rather than the use of MRI, high-resolution retinal imaging could
possibly be leveraged to increase our understanding regarding micro­
aneurysm behaviour in DR in four-dimensions. Dubow et al. described
the use of adaptive optics (AO) scanning light ophthalmoscope FA to
classify microaneurysm morphology in vivo (Dubow et al., 2014).
Although a time consuming and invasive technique, it is possible to
apply such modalities to study longitudinal changes in microaneurysm
morphology and their relationship to the long-term complications of DR.
5.2.4. Tissue oedema due to microaneurysms
The anatomic disturbance that characterises a retinal capillary
microaneurysm is unlikely to induce significant visual alterations
however it is the functional consequence of leakage and tissue oedema
that can lead to drastic vision loss. Loss of the integrity of the BRB is a
fundamental precursor for microaneurysm leakage but, similar to
microaneurysm formation, there are many cellular and haemodynamic
alterations that can account for this. We recently investigated the sig­
nificant determinants of microaneurysm leakage using histologic
perfusion labelling with immunofluorescent lectin (An et al., 2022b).
We assessed the influence of retinal topology, focal cellular changes as
well as microaneurysm morphology on the occurrence of leakage and
reached the following conclusions:
1. Microaneurysm size/diameter is the strongest predictor of leakage.
Smaller microaneurysms have a greater risk of leakage than larger
microaneurysms (Fig. 27).
Fig. 27. Relationship between microaneurysm (MA) diameter and leakage.
Comparisons between leaking and non-leaking MA, per diameter grouping,
from a cohort of 636 MA from human donor eyes is presented. Smaller dimeter
MA have a greater risk of leakage. Reproduced with permission from An et al.
(An et al., 2022b).
C. Balaratnasingam et al.
2. There is no difference in the rate of leakage between microaneurysms
in the deep capillary bed and those in the superficial capillary bed.
3. Microaneurysms with preserved pericytes have an increased risk of
leakage.
4. Microaneurysms with inflammatory cells have a lower risk of
leakage.
5. Of the 636 microaneurysms that were studied we found evidence of
leakage in 58% of cases. We did not find a significant difference in
rates of microaneurysm leakage between areas of capillary non­
perfusion (55.8%) and normal perfusion (63.6%).
These results indicate that microaneurysm leakage is governed by
the cumulative effect of several pathogenic factors. It can be assumed
that VEGF is upregulated in areas of non-perfusion (Marsh et al., 2000;
Schultz et al., 1999). VEGF also increases the permeability of retinal
capillaries by altering the BRB (Antonetti et al., 1998; Joussen et al.,
2002; Murata et al., 1996). It is interesting that capillary nonperfusion
alone does not increase the risk of microaneurysm leakage. One inter­
pretation of this finding is that interactions between VEGF and other
elements of the vascular tree are required for microaneurysm leakage.
We found that a lower microaneurysm diameter and preservation of
pericytes increases the risk of microaneurysm leakage (An et al., 2022b).
It suggests that leakage is more likely to occur in the earliest stages of the
microaneurysm lifecycle. The reduced risk of leakage in the presence of
inflammatory cells may be explained by the role of these cells in regu­
lating thrombosis and controlling platelet activation and essentially the
formation of a “plug” that blocks leakage.
For this paper we undertook an original analysis of the histologic
data of 636 microaneurysms to evaluate relationships between micro­
aneurysm morphology and leakage. Results are summarised in Table 4.
We found that microaneurysm morphology was significantly associated
with rates of leakage (P = 0.010). Both focal bulge and fusiform
microaneurysms had significantly greater frequency of leakage than
saccular microaneurysms (P = 0.046 and P = 0.044, respectively).
There is expected to be an overlap in the pathophysiologic mecha­
nisms that govern microvascular leakage and those that underlie DMO,
the latter being a major cause of vision loss in DR (Das et al., 2015; Miller
and Fortun, 2018). From a clinical standpoint, DMO can be consequent
to microaneurysm leakage or diffuse leakage within the macular capil­
lary circulation due to breakdown of the BRB (Blair et al., 2008).
Intravitreal anti-VEGF is widely considered the first line treatment of
DMO but several clinical trials have shown that anti-VEGF has a sub­
optimal response in nearly 40% of patients with DMO (Gonzalez et al.,
2016). The reason for this is unclear. Our findings concerning the
pathophysiology of microaneurysm leakage may have wider implica­
tions for understanding treatment failure in DMO. It is plausible that
anti-VEGF treatment failure is possibly more common in patients with
DMO that have a relatively well-preserved retinal circulation with
limited ischaemia. Anti-VEGF treatment does not modulate the density
of the pericyte population and may therefore be less efficacious when
the integrity of the BRB is compromised despite the presence of peri­
cytes. In vivo methods for imaging pericytes and white cells may allow
nuanced selection of patients that could benefit from anti-VEGF therapy
for DMO. Our results however do not precisely explain why intraocular
corticosteroids may have efficacy in some eyes with DMO that do not
respond well to anti-VEGF therapy (Chawan-Saad et al., 2019; Iglicki
Table 4
Microaneurysm morphology and leakage rates.
Morphology Total number (n = 636) No leak (n) Leak (n) Leak (%)
Focal bulge 128 44 84 65.6
Fusiform 229 78 151 65.9
Saccular 227 108 119 52.4
Pedunculated 31 20 11 35.5
Irregular 21 11 10 47.6
24
Progress in Retinal and Eye Research xxx (xxxx) xxx
et al., 2019). Given that the occurrence of white cells and, by extension
the increased activation of thrombosis, reduces the risk of micro­
aneurysm leakage, it would appear counter-intuitive that the
anti-inflammatory effects of intraocular corticosteroids would reduce
tissue oedema. It is possible that the efficacy of corticosteroids in the
management of DMO may be independent of its anti-inflammatory ac­
tivities. Alternative ways by which intraocular steroids may reduce tis­
sue oedema is by promoting fluid clearance through aquaporin-4 and
potassium channels of Muller cells (Bonnin et al., 2015; Dugel et al.,
2015).
5.2.5. Clinical imaging of microaneurysms in diabetic retinopathy
Retinal imaging forms the cornerstone of modern DR management.
Unlike histology, a major advantage of retinal imaging is that it facili­
tates time-dependent (four-dimensional) study of a disease. Colour
photography has historically been the mainstay technique for imaging
microaneurysms however in the setting of DR it can be difficult to
differentiate a small retinal haemorrhage from a capillary micro­
aneurysm using colour photography (Friberg et al., 1987; Hellstedt
et al., 2006). This has clinical implications if microaneurysm counting
using colour photography is utilised as a biomarker to prognosticate the
risk of DR development and progression, as has been done in the past
(Nunes et al., 2009; Pappuru et al., 2019; Santos et al., 2021). FA has
long been considered the gold standard for visualising the retinal cir­
culation, but there is a 10% risk of adverse reaction/complication
associated with this technique (Yannuzzi et al., 1986). Additionally,
some retinal microaneurysms are not detectable by FA if they are
comprised of a large thrombus that completely obstructs blood flow. The
introduction of OCT techniques has facilitated visualisation of retinal
structures at greater spatial resolution than colour photography. How­
ever, assessment of B scans that comprise an OCT volume is
time-consuming, and OCT is more expensive and less widely available
than colour photography. OCTA is an extension of the OCT technique
and provides unprecedented visualisation of retinal capillaries. The
drawback of this technique however is that the dynamic properties of
retinal blood flow can result in microaneurysms not being visualised if
OCTA scans from only a single time point are assessed (Fig. 28).
Abnormally low blood flow rates within microaneurysms due to
thrombus and turbulence may also preclude visualisation of micro­
aneurysms on OCTA. These points are readily appreciated when FA and
OCTA images of microaneurysms are compared (Fig. 29). In most in­
stances, the FA will highlight more microaneurysms within the same
field of view than the OCTA.
Several investigators have used high-resolution AO scanning laser
ophthalmoscopy (AOSLO) to study diabetic microaneurysms and
vascular disease as a whole (Paques et al., 2018). Dubow and colleagues
used AOSLO to categorise microaneurysms into 6 different types based
on morphology (Dubow et al., 2014). Hafner et al. applied AO-OCT to
investigate the changes in microaneurysm morphology over time (Haf­
ner et al., 2018). Using such techniques they were able to demonstrate
growth and regression of microaneurysms over time. Lammer and col­
leagues evaluated the association between the imaging characteristics of
microaneurysms as seen on AOSLO and structural OCT features such as
disorganisation of retinal inner layers, intraretinal cysts and retinal
volume (Lammer et al., 2018). They found that hyperreflectivity of
microaneurysm walls as seen on AOSLO was associated with the pres­
ence of disorganisation of retinal inner layers. The theoretic resolution
of AOSLO systems is close to 2.5 μm and provides superior spatial res­
olution than most OCTA systems. However, the narrower field of view,
the greater reliance on patient fixation to attain high quality images and
the greater length of time required to capture an image has limited its
widespread use in clinical practice.
Multimodal retinal imaging involves the integration of imaging data
from multiple sources and addresses some of the limitations of assessing
structural data acquired from a single modality. Microaneurysms are
well-demarcated, round or oval lesions that appear as red or white dots
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Table 5
Demographic details and specifications of optical coherence tomography angiography (OCTA) devices.
OCTA Device Wavelength A-scan rate Algorithm Motion Artefact Total Males Age Range Total Regions
(nm) (Khz) Minimisation Subjects (years) Imaged

RTVue XR 840 70 SSADA Orthogonal Registration 5 3 24–51 10

Heidelberg 870 85 FS-ADA Eye tracking (Truetrack) 5 3 24–51 10
OCT2
PLEX Elite 9000 1040–1060 100 OMAG Eye tracking (FastTrac) 3 1 27–34 6
DRI OCT Triton 1050 100 OCTARA Eye tracking (SMARTTrack) 3 1 27–34 6

FS-ADA = Full-spectrum amplitude decorrelation algorithm; OCTARA = OCTA ratio analysis; OMAG = Optical Microangiography; SSADA = Split spectrum amplitude-
decorrelation angiography.

Fig. 29. Multimodal imaging of diabetic microaneurysms (MA). Images ac­

quired during the same visit illustrate the differences in MA morphology and
frequency between various modalities. Colour image illustrates a prominent
Fig. 28. Time-dependent variations in microaneurysm (MA) characteristics as
saccular MA that appears as a red dot nasal to the fovea (A; yellow ar). Red free
seen on optical coherence tomography angiography (OCTA). Images from the
photography illustrates numerous more discrete round lesions in the macula (B;
macular circulation from a human subject with Type 2 Diabetes Mellitus are
yellow ars). Early (C) and late (D) phase fluorescein angiogram (FA) images
presented. Images that were acquired 1 min apart demonstrate the alterations
allow distinction of MA (hyperfluorescent) from haemorrhages (hypofluor­
in the appearance of MA on OCTA. Three MA originating in the deep vascular
escent). Note that some MA seen in the early frame of the FA are not discernible
plexus have been highlighted with insets. There is a decrease in signal intensity
in the late frame. Inset provides a magnified view of the macula acquired during
in MA 1, a relative increase in signal intensity in MA 2 and minimal change in
the early frame of the FA (E). Only some of the MA seen on FA are evident on
MA 3 over the 1 min. Signal intensity in non-aneurysmal vascular structures
optical coherence tomography angiography (OCTA; red ars; F). The dimensions
remains relatively unchanged between images. Note that flow signal is localised
of MA depicted on the two modalities are also different. Some MA that are
to a focal point in MA 1 with a large portion of the MA void of signal (red
clearly visualised on FA are not seen on OCTA (blue ars).
arrows). Note that projection artefact is evident in the OCTA slabs of the deep
vascular layer.
on colour photography and red-free imaging (Fig. 29). Microaneurysms
are denoted by circular or ovoid lesions with a hyperreflective capsular
structure and/or hyperreflective spots on en face slab B-scan and cross-

25
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

microaneurysms that span across 2 retinal plexuses are a common

occurrence. However, it is very uncommon to find large micro­
aneurysms that extends across 3 plexuses, or into the outer retina. In our
previous histologic studies we also did not find a significant difference in
the density of microaneurysms between the SVP/ICP, SVP/DCP and the
ICP/DCP in donor eyes with diabetes (An et al., 2021, 2022b). If we were
to assume that histology represents the ground truth then it would
appear intuitive that comparing new imaging techniques against his­
tology would be the most precise way to highlight the accuracies and
flaws of any emerging imaging techniques. The discrepancies between
findings from our histology reports and previous OCTA studies implicate
some important limitations associated with OCTA (Balaratnasingam
et al., 2019).
One advantage with multimodal imaging of the retinal circulation is
that it is possible to identify some of the structural correlates of a
microaneurysm that have been validated using histology. This is much
more difficult if a single modality was assessed in isolation. We provide
some examples to highlight this point. In Fig. 31 we illustrate a large
microaneurysm that appears compartmentalised on colour

Fig. 30. Correlations between microaneurysm (MA) structure and optical

coherence tomography angiography (OCTA). Patient A demonstrates a MA with
a complete ring structure as seen on the cross-sectional OCT without adjacent
cystoid spaces (red ar). The MA is clearly delineated on the en face B scan
however there is no observable flow signal on en face OCTA projections. Patient
B presents with OCT features that are associated with MA leakage including the
presence of hyperfluorescent spots (green ar) and adjacent cystoid spaces. The
leaking MA is clearly visible on the en face B scan image however an observable
flow signal is not evident on OCTA images. Note the relatively high flow signal
that correlates to another aneurysm in the deeper vascular network (blue ar)
without adjacent cystoid spaces. Insets on en face images denote the position of
the MA.

sectional B-scan OCT images (Fig. 30). One of the major advantages of
integrating OCT and OCTA data in the study of microaneurysms is that it
allows co-localisation of microaneurysms to individual retinal layers.
Clinical studies that have co-localised microaneurysms to retinal capil­
lary plexuses have suggested that the DCP has a greater propensity for
the development of microaneurysms (Khatri et al., 2021; Stattin et al.,
2020). However, there are several caveats with performing such OCTA
studies: 1) Microaneurysm count and localisation may be
under-estimated due to time-dependent variations in microaneurysm
flow properties (as discussed above) that precludes their visualisation.
2) Microaneurysm count can be over-estimated using OCTA as micro­
aneurysms can be confused with normal arterioles of vertical trajectory
Fig. 31. Compartmentalisation and non-uniform flow within microaneurysms
that connect the SVP to the ICP and ICP to the DCP. In a later portion of (MA). A saccular MA that is evident on red free imaging is magnified in the
this article, we outline some of the difficulties in visualising the deep inset (A). A pale fibrous rim is seen around the margins of the MA. Two distinct
retinal circulation using OCTA. Another important reason that micro­ lesions are seen within the lumen; a purple coloured structure that is consistent
aneurysms can be falsely attributed to the DCP is due to errors in seg­ with the appearance of a thrombus (red arrow) and a bright red structure that is
mentation of the OCTA slab. most likely red blood cell filled space (green arrow). Each of these compart­
OCTA studies that have investigated the spatial relationships of ments have a different flow signature on en face optical coherence tomography
microaneurysms relative to retinal layers have described the occurrence angiography (OCTA). Cross sectional structural OCT and OCTA images (C, D E
of microaneurysms that span a number of retinal layers, including the and F) through each of these aneurysmal components demonstrates the dif­
ference in flow. A strong OCTA flow signal correlates to the site occupied by red
avascular outer retinal layers (Schreur et al., 2018b; Wang et al., 2012).
blood cells (OCTA slice 1; D) while a flow void correlates to the site of thrombus
Our observations based on histologic studies is that large
(OCTA slice 2; F).

26
C. Balaratnasingam et al.
photography. Cross sectional OCT and OCTA scan through the two
compartments reveal flow within one compartment that is likely to be
the perfused portion of the microaneurysm. In the second compartment
there is a flow void that most likely represents the presence of a
thrombus. In contrast, similar multimodal imaging assessment of a much
smaller microaneurysm within an ischaemic macula due to DR is noted
to have complete perfusion within the microaneurysm dome (Fig. 32).
If multimodal imaging could be leveraged to predict the risk of
microaneurysm leakage and vision loss due to foveal oedema it would
have powerful translational application. However, to date, such infor­
mation is lacking. Given that some microaneurysms will fall within the
resolving limits of OCT, previous authors have investigated the struc­
tural characteristics of microaneurysms that may predict the risk of
leakage (Horii et al., 2010). Positive findings that predict the risk of
leakage include the presence of luminal hyperreflective spots, adjacent
cystoid spaces and the absence of a capsular structure. One of the issues
with using such metrics is that they are subjective with inter-observer
variation. They are also not quantitative measurements. Furthermore,
these OCT measurements lack sensitivity and specificity and may not be
readily applied to artificial intelligence techniques.
It would seem plausible that the presence of a flow signal as seen on
OCTA within a microaneurysm may predict the risk of leakage. The logic
being that microaneurysms without a flow signal may have suffered
Fig. 32. Microaneurysms (MA) with OCTA flow signatures throughout the
entire lumen. Red-free imaging (A) demonstrates a MA temporal to the foveal
avascular zone in a 38-year-old patient with type 1 diabetes mellitus. On colour
imaging (Inset) the lumen of the MA has a homogeneous appearance. En face
OCTA (B) demonstrates a flow signal at the site of the MA and comparisons
between structural OCT and cross-sectional OCTA reveal flow signal through
the superior (C and D) and inferior (E and F) portions of the MA. The green
arrow identifies the MA in each image panel. This patient has previously
received laser photocoagulation therapy and continues to have significant
macular ischaemia.
27
Progress in Retinal and Eye Research xxx (xxxx) xxx
total occlusion due to infiltration by white cells and formation of
thrombus. We performed a pilot study to explore this hypothesis but the
preliminary data (unpublished work) did not find an observable asso­
ciation between the presence of a flow signal and the risk of leakage as
shown in Fig. 30. The results of this preliminary work were consistent
with our histologic data where only 58.3% of perfused microaneurysms
demonstrated leakage (An et al., 2022b).
In summary, by using the microaneurysm as an exemplar to under­
stand the pathophysiology of DR it is clear that there remains a signif­
icant mismatch between the knowledge that has been attained using
histology and the data that has been acquired using high-resolution
clinical imaging.
5.3. Retinal ischaemia
One of the most devastating sequelae of DR is the development of
retinal ischaemia. It is important to remember that vascular injury in DR
predominantly occurs at the level of the microcirculation and the most
frequent site within the retina to suffer occlusive injury is the capillary
system (Ashton, 1963; Cogan et al., 1961). In this regard, the patho­
physiology of DR is very different to diseases of the large retinal vessels
such as retinal artery or vein occlusion. Retinal ischaemia is a risk factor
for the development of DMO and retinal neovascularisation, both of
which are major sight-threatening complications of DR (Ramirez et al.,
2011; Wessel et al., 2012). Plausibly, this is because the occurrence of
retinal ischaemia initiates a cascade of biochemical and pathologic
pathways that eventuates in upregulation of VEGF and other
pro-angiogenic factors. As discussed in the previous section, upregula­
tion of VEGF plays a putative role in disrupting the BRB (Murata et al.,
1996; Ozaki et al., 1997). It also plays a key role in pathologic angio­
genesis and the development of new blood vessels (Aiello et al., 1995;
Miller et al., 1994; Ozaki et al., 1997). Although retinal neo­
vascularisation is one of the most serious sequelae of retinal ischaemia,
it should be highlighted that other pathogenic mechanisms also under­
pin the formation and progression of retinal neovascularisation in DR.
As an example, retinal neovascularisation can progress in eyes with
proliferative DR (PDR) despite the administration of comprehensive
panretinal photocoagulation therapy (Figs. 33 and 34). Therefore,
similar to DMO, multiple biochemical pathways drive retinal neo­
vascularisation in DR. Retinal ischaemia however is a key factor in this
process.
There are few studies that have investigated the relationships be­
tween ischaemia, retinal topography and the complications of DR
(Famiglietti et al., 2003). More specifically, is it the magnitude of retinal
ischaemia or the retinal eccentricity where retinal ischaemia occurs that
is the primary driver for ischaemia-related retinal complications? It is
unlikely that the complications due to a macular ischaemia and pe­
ripheral retinal ischaemia will be of similar magnitude. There are several
reasons that underpin this hypothesis: 1) The major retinal cell types
that are responsible for VEGF production include the RPE, RGC, retinal
endothelia and retinal glia (Amin et al., 1997; Ijichi et al., 1995; Le,
2017; Pollreisz et al., 2013). These cell types are unevenly distributed
within the retina with great variations in subtypes and densities between
the central macula and retinal peripheries. Therefore, the concentration
and isoforms of VEGF production between peripheral and central retinal
ischaemia is expected to be different. 2) Growth factors other than VEGF
are involved in the pathophysiology of DR (Grant et al., 2004). There is
likely to be differential upregulation of the spectrum of growth factors
following macular ischaemia and peripheral retinal ischaemia.
The onset of retinal capillary non-perfusion and ischaemia in DR is
irreversible. Preventing or prolonging the onset of retinal ischaemia is
therefore a key management goal of DR. Although anti-VEGF therapy
can reverse some of the microangiopathic complications of DR such as
retinal haemorrhages and regression of microaneurysms it does not
restore perfusion to areas of vascular occlusion (Manousaridis and Talks,
2012; Pongsachareonnont et al., 2020). This point is highlighted by
C. Balaratnasingam et al.
Fig. 35. Ischaemia is therefore a hallmark feature of irreversible injury
due to DR and a detailed understanding of the pathology and histologic
characteristics of this important manifestation will aid the development
of new therapies that can prevent vision loss due to DR. Characterisation
of the functional and structural retinal alterations that precede the
development of retinal ischaemia will play a key role in preventing
28
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 33. Development of retinal neovascularisation
in diabetic retinopathy despite laser therapy to treat
retinal ischaemia. Ultra-wide field fluorescein angi­
ography (FA; A and B) and posterior pole FA (C and
D) images from a 54-year-old male with type 2 dia­
betes mellitus. Images captured in 2018 appear in left
panel and images from 2022 appear in right panel.
Note that despite the application of more extensive
panretinal photocoagulation therapy over the period
of 4 years there has been the development of retinal
neovascularisation (red arrows). The area of far pe­
ripheral non-perfusion has not observably changed on
the FA however there has been a point of new arte­
riolar occlusion (blue arrow) at the latest visit.
Fig. 34. Progression of retinal neovascularisation in
diabetic retinopathy despite comprehensive pan­
retinal photocoagulation therapy to address retinal
ischaemia. Images from a 32 year old female with
Type 1 diabetes mellitus is presented. Colour
photography and ultrawide field fluorescein angiog­
raphy images from baseline visit (A) and 2 years later
(B) are provided. At baseline visit there is profound
capillary nonperfusion with associated optic disc and
retinal neovascularisation. Patient was immediately
treated with comprehensive panretinal photocoagu­
lation therapy. Despite treatment of the entire retina
with laser there has been progression of both optic
disc and retinal neovascularisation over time. The
boundaries of the areas of retinal neovascularisation
have been delineated by red arrows. Despite the onset
of some fibrosis after laser the retinal neo­
vascularisation is still characterised by active prolif­
eration. The patient eventually required vitrectomy
surgery to treat recurrent vitreous haemorrhage and
tractional macular elevation due to the
neovascularisation.
vision loss due to DR. Our work in the field of retinal ischaemia will now
be discussed and placed in the context of previous investigations.
5.3.1. Histopathology of retinal vascular occlusion
There is great variation in the phenotype of retinal ischaemia in DR
and this can be readily appreciated by comparing the retinal angiograms
C. Balaratnasingam et al.
Fig. 35. Effects of intravitreal anti-vascular endothelial growth factor (VEGF)
on the retinal circulation in diabetic retinopathy. Colour photography (A and
B), fluorescein angiography (FA; C and D) and optical coherence tomography
angiography (OCTA; E and F) images are presented of a 42-year-old female with
Type 1 DM and diabetic macular oedema. The left panel comprises images at
baseline visit and the right panel comprises images following 6 anti-VEGF
treatments over 6 months. Note that there has been significant regression of
microangiopathy and retinal haemorrhages as seen on colour photography after
treatment. FA demonstrates a reduction in vascular staining and leakage after
treatment providing evidence of an improvement in blood-retina-barrier func­
tion. The macula OCTA however does not demonstrate a significant difference
in capillary density after anti-VEGF therapy. Areas of perifoveal capillary loss
(cyan asterisk) still persist. In this case there is evidence of ongoing capillary
loss (red arrows) despite treatment. The OCTA images at each visit represents
an averaged projection of 10 consecutively acquired OCTA images as a means
of improving signal:noise ratio. The average projection also accounts for tem­
poral variations in retinal perfusion and improves capture of capillaries that
may be momentarily non-perfused.
from a cohort of patients with DM (Fig. 36). Some patients manifest
predominant ischaemia of the mid-peripheral circulation while others
demonstrate preferential involvement of the peripheral retinal circula­
tion and in rare cases diffuse ischaemia of the entire retinal circulation is
evident. The site of ischaemia frequently involves the capillary circu­
lation but in some instances, it can involve the large order vessels
resulting in a clinical picture that resembles occlusive vasculitis due to
autoimmune diseases (Fig. 36E and F). The pattern of vascular occlusion
may reflect the predominant pathophysiologic mechanism underlying
DR in that patient. In other words, it is possible that the pattern of
vascular occlusion in an eye with a primarily VEGF-mediated mecha­
nism may be different to that of an eye where an inflammatory mech­
anism is the predominant pathophysiologic mediator. Histologic studies
29
Progress in Retinal and Eye Research xxx (xxxx) xxx
may help test and reconcile such hypotheses.
5.3.1.1. Retinal capillary occlusion. To date, we have performed flat-
mount perfusion based histologic studies utilising over 100 donor eyes
from patients with DM. With respect to the relationship between areas of
capillary bed closure and the large order vessels we have found that
capillary ischaemia most frequently occurs on the arterial-side of the
retinal circulation (Fig. 37). Ashton was one of the earliest investigators
to make this observation and it is a concept that has been validated by
subsequent clinical studies including the use of OCTA (Ashton, 1953,
1963; Ishibazawa et al., 2019). It is unclear why preferential involve­
ment of capillaries in the arterial side of the circulation would occur.
Hypotheses that have been put forward to explain this observation is
that loss of arteriolar pericytes and arterial smooth muscle cells pref­
erentially increases shear stress in the arterial side of the capillary bed
and initiates a cascade of cellular changes that predisposes it to occlu­
sion. Such changes are less profound on the venous aspect of the cir­
culation where shear stress is presumably lower.
There is overwhelming histopathologic evidence to indicate that
endothelial dysfunction is the primary cellular change that predisposes
to retinal capillary occlusion (Hofman et al., 2001). In our specimens it
has been consistently found that endothelial hypertrophy characterises
sites of capillary occlusion in DR (Fig. 38). Hofman performed an
experimental study using two monkeys where intravitreal VEGF injec­
tion was used to generate a model of retinal capillary nonperfusion
(Hofman et al., 2001). Light and electron microscopic analysis of retina
specimens from these experiments showed significant reduction in the
diameter of retinal capillary lumina due to endothelial hypertrophy.
Basement membrane thickening has been well documented in DM (Roy
and Kim, 2021). Bek and Ledet performed a histopathologic study of
donor eyes from 7 DM patients and found evidence of basement mem­
brane thickening at sites of capillary occlusion (Bek and Ledet, 1996).
However they identified other cellular elements at areas of occlusion
and concluded that “basement membrane thickening cannot fully ac­
count for vascular occlusion in diabetic retinopathy”. Based on our
histologic studies we propose that basement membrane thickening in DR
is more an epiphenomenon while endothelial hypertrophy is the key
change accounting for luminal occlusion.
One of the consequences of endothelia hypertrophy and a reduction
in lumina diameter is the mechanical constriction of red and white blood
cells during their passage through the microcirculatory bed. The mean
diameter of a retinal capillary lumen is 7–10 μm (An et al., 2020b, 2021;
Tan et al., 2012) while the mean diameter of an erythrocyte (Ponder and
Millar, 1924) and leucocyte (Bain et al., 2016) is 8 μm and 12 μm,
respectively. Velocity of capillary flow is proportional to the fourth
power of the radius of the capillary as defined by Poiseuille’s law (Feke
et al., 1989). Although leucocytes and erythrocytes are deformable cells,
a reduction in luminal diameter will favour a state of cellular stagnation
and capillary occlusion (Chibber et al., 2007; Fu et al., 2016). In addition
to the mechanical reduction in capillary diameter, parallel disease
mechanisms that increase the adhesive characteristics of leucocytes such
as the increased regional expression of chemotactic factors and also
increased expression of adhesion molecules by endothelia and leuco­
cytes will favour capillary occlusion (Danton and Dietrich, 2003; Jous­
sen et al., 2002). Our histologic surveys have provided ample evidence
of leucocytes and their close apposition to endothelial at sites of capil­
lary occlusion (Fig. 38A and D). The interaction between endothelia and
leucocytes play a key role in atherosclerosis but the retinal capillary
lacks a tunica intima and therefore cannot suffer atherosclerotic plaques
like large order arteries in the human body. However, immune complex
and inflammatory damage can result in capillary occlusion without
plaque formation (Chibber et al., 2007; Fu et al., 2016).
5.3.1.2. Large vessel occlusion. The morphology and pattern of endo­
thelia damage at sites of vascular occlusion in large order vessels within
C. Balaratnasingam et al.
the retina is remarkably different to what is seen in capillaries. There is
strong evidence that endothelia dysfunction plays a putative role in
large vessel occlusions but rather than demonstrate hypertrophy signs of
endothelial proliferation in these larger diameter vessels have been
observed (Fig. 39). These findings are consistent with the concept that
the haemodynamic properties of the retinal circulation are altered in DR
as proposed by modelling studies of the retinal circulation (Patel et al.,
1992). Clinical studies that have also shown an increased prevalence of
systemic hypertension in patients with DM, suggesting that retinal
vascular resistance and flow may be significantly altered in such patients
(Chase et al., 1990; Davis, 1986). Alterations in retinal blood flow and
pressure will alter the shear stress experienced by retinal endothelia.
Disturbed shear stress, that is more profound at sites of arterial division,
is a major risk factor for endothelial hyperplasia (Li et al., 2005; Russo
et al., 2020). Signaling pathways that underlie endothelia proliferation
include the transcriptional signaling pathways mediated by ERK1/2 and
the translational signaling pathways mediated by mTOR (Jin et al.,
2013; Yao and van Wijngaarden, 2020). The United Kingdom Prospec­
tive Diabetes Study showed that tight blood pressure control resulted in
a 34% reduction in the risk of two-step progression in DR (King et al.,
1999). One explanation for this clinical finding may have been the
favourable effects of tight blood pressure control on retinal endothelia
function.
Endothelial proliferation has also been identified within retinal
microaneurysms. Aguilar and colleagues labelled donor eyes from
30
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 36. Varying phenotypes of retinal ischaemia in
diabetic retinopathy. Pseudo-colour images and ul­
trawide field fluorescein angiogram (UWF-FA) images
from 3 different patients. Patient 1 (A and B) is a 34-
year-old female with Type 1 diabetes mellitus. She
manifests predominantly peripheral retinal ischaemia
with angiographic preservation of the circulation in
the posterior pole. Patient 2 (C and D) is a 54-year old
male with Type 2 diabetes mellitus. He manifests a
concentric pattern of ischaemia that is predominantly
confined to the mid-peripheral retina. His UWF-FA
demonstrates relative preservation of the far periph­
eral and macular circulation. Patient 3 (E and F) is a
41-year-old male with Type 1 DM that has diffuse
ischaemia involving the entire retina. The capillary
beds throughout the retina are not discernible in this
case.
patients with DR with cell-cycle-specific nuclear antigen Ki-67 and
identified positive labelling within most microaneurysms but rarely
outside retinal vessels (Aguilar et al., 2003). The Ki-67-positive cells
were most frequently seen in the middle and outer portions of the
microaneurysm wall. The authors concluded that basement membrane
and pericyte changes within microaneurysms resulted in weakening of
the vessel wall with subsequent dilatation. They proposed that aneurysm
dilation induced a biomechanical stimulus that activated angiogenic
pathways. They hypothesised that, similar to retinal neovascularisation,
endothelial proliferation was a response within the spectrum of angio­
genesis. In our cohort, there is evidence of endothelial cell proliferation
within microaneurysms (Fig. 40).
In addition to endothelial alteration, occlusion of large retinal vessels
in diabetes is also associated with marked changes in the morphology
and expression of intermediate filaments by regional astrocytes and
muller cells. Bek performed immunohistochemical labelling of glia using
10 eyes of 6 DM patients (Bek, 1997). An important finding in that study
was the increased expression of GFAP and Vimentin in the perivascular
cells at sites of occlusion as well as the material that was causing the
occlusion. Our studies reaffirm this observation (Fig. 41). It is likely that
changes to retinal glia at sites of vascular occlusion is a secondary
response to ischaemia rather than a primary pathophysiologic mecha­
nism underlying the occlusion. It has been previously shown that in the
acute phase of retinal ischaemia there is no change in GFAP expression
within the NFL (Balaratnasingam et al., 2010). Reactive astrocytosis is
C. Balaratnasingam et al.
Fig. 37. Capillary non-perfusion preferentially affecting the arterial side of the
retinal circulation in diabetic retinopathy (DR). A section of flat mount retina
with DR showing multiple microaneurysms (MA) represented by hyper­
fluorescent dots as well as regions of capillary non-perfusion (yellow asterisks).
Note that capillary non-perfusion typically occurs adjacent to arteries or arte­
rioles (A). Notch in retina seen inferiorly is due to radial incision for flat
mounting purpose (blue asterisk). A magnified inset of a capillary non-
perfusion site is provided in B. Sites of arteriolar and capillary occlusion are
indicated (yellow ars). Arteries and veins (V) can be differentiated via the
presence of periarterial capillary free zone around arteries (Magenta dashed
line; C). Vasculature in this specimen was labelled using Lectin.
characterised by increased expression of GFAP (Chang et al., 2007). It
has been documented in a plethora of CNS diseases including stroke and
multiple sclerosis (Abdelhak et al., 2018; Choudhury and Ding, 2016). In
the CNS literature, reactive astrocytosis has been proposed to serve a
protective or compensatory function in several ways: 1) Forming a
physical barrier that limits the influx of peripheral immune cells; 2)
repairing the blood retina barrier; and 3) minimizing neuronal damage
(Pekny and Nilsson, 2005).
An example of GFAP changes at sites of vascular occlusion in a
human donor eye with DR is demonstrated in Fig. 41. Note that there is
intraluminal infiltration of GFAP positive process through a compro­
mised BRB that extends to the site of intraluminal occlusion. These
processes originate from GFAP positive perivascular cells. The processes
extend to the site of intraluminal occlusion. It is difficult to determine if
these GFAP changes are akin to reactive astrocytosis reported in CNS
diseases. The morphology and pattern of GFAP changes suggest that
they consolidate the clot and possibly provide biomechanical rein­
forcement to this site thereby inhibiting clot dislodgement and vascular
reperfusion.
In summary, the evidence suggests that the pathophysiology of
vascular occlusion and ischaemia in DR is complex and varied. The
histopathologic studies outline at least two therapeutic targets that
could reverse the cellular consequences of vascular occlusion. Targeting
endothelial dysfunction, hyperplasia and proliferation may inhibit the
earliest stages of vascular occlusion by inhibiting thrombosis and leu­
costasis. Targeting the activity of retinal glia may prevent maturation of
the occlusion in large order vessels and increase the success of vascular
reperfusion through medical intervention.
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Progress in Retinal and Eye Research xxx (xxxx) xxx
5.3.2. Increased vulnerability of deep capillary beds
In an earlier section of this manuscript the variation in the angio-
architectural characteristics of the retinal capillary beds were dis­
cussed. We have shown that the conduits that feed and drain each of the
retinal capillary beds are vastly different. In addition, the biochemical
milieu and the energy demands of the neuroglia populations supplied by
each capillary bed is also different. It is therefore likely, that a complex
metabolic condition such as DM will have an unequal effect on the
structure and function of each of the retinal capillary beds.
In particular, there has been considerable attention paid to the DCP
of the macula and how it is altered in disease. The role of the DCP in
physiology and disease has remained contentious. The DCP has long
been regarded as a predominantly venous network as it was observed to
be the site of venous-venous collateral vessel formation following retinal
vein occlusion (Henkind and Wise, 1974). We have performed a number
of histologic studies to evaluate the differential changes to capillary beds
in DR and have reached the following consistent conclusions (An et al.,
2020a, 2021):
1) Capillary density in the DCP is frequently reduced in non-
proliferative DR (NPDR) before a quantifiable change in capillary
density occurs within the SVP or the ICP. Changes to the DCP are
therefore early and preferential in DR.
2) There is no difference in capillary diameter measurements between
the capillary plexuses in NPDR. Capillary diameter increase is found
in preclinical DR however following the onset of DR there is no
change in capillary diameter measurements between eyes with DR
and age-matched control eyes for any of the plexuses (An et al.,
2021).
The above findings were observed in studies of the peripapillary
circulation as well as the macula circulation (An et al., 2020a, 2021)
(Fig. 42). Qualitative studies in our laboratory examining the capillary
beds of the mid-peripheral retina have shown that the deep circulation
has greater vulnerability to ischaemic injury and capillary loss in DR
(Fig. 19). The greater vulnerability of the DCP to ischaemia may be
explained by its dependency on the SVP and ICP for blood flow. As we
have shown in our three-dimensional histologic reconstructions of the
normal retinal circulation, the DCP does not derive a direct flow of blood
from retinal arteries, instead, arteriolar supply originate from the ICP
(An et al., 2020b). Vascular changes that occur upstream may culminate
in profound changes to the microcirculation of the DCP. Another reason
that could explain the differential vulnerability of the DCP is the change
in retinal oxygen consumption that occurs in DR. Using oxygen-sensitive
microelectrodes, Cringle and Yu measured oxygen tension in the vitre­
ous and retina in STZ rats (Cringle et al., 1992). They found that diffu­
sion of oxygen from large retinal arteries to supply neurons in the
peri-arterial capillary free zone is lost after five weeks of experimental
diabetes. This may be due to changes in oxygen consumption within the
capillary-free zone or the superficial layers of the retina. Finally, the
blood from patients with diabetes is more viscous when deoxygenated in
contrast to that of normal eyes (Smith, 1990). This measure is known as
the hypoxic viscosity ratio. In the absence of a rise in the pressure
gradient within the retinal microcirculation, the consequence of an
increased hypoxic viscosity ratio could lead to a state of chronic mild
hypoperfusion of which the DCP is most vulnerable.
The vulnerability of the deep capillary beds to retinal ischaemia
exemplifies the importance of assessing this aspect of the retinal circu­
lation in the clinical assessment of DR. The DCP is involved earlier and
more extensively in DR and serves as a valuable biomarker for detecting
onset and severity of disease. The advent of OCTA technology has
improved our ability to detect the DCP in the clinical setting but there
remain some limitations in applying this technology for that purpose, as
discussed later.
C. Balaratnasingam et al.
5.4. Alterations to macular blood flow patterns in diabetic retinopathy
Given that retinal ischaemia is permanent and a hallmark feature of
irreversible injury in DR an understanding of the vascular changes that
precede the development of ischaemia will be key for designing any
intervention that mitigate the development of ischaemia. It has been
shown that macular perfusion abnormalities precede the development of
retinal ischaemia (Cringle et al., 1993). Plausibly, if microcirculatory
disturbance is detected during a state of reversible cellular injury, then
the application of appropriate therapeutic intervention may prevent or
delay the development of ischaemia. Currently, it is very difficult to
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Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 38. Capillary narrowing and occlusion mecha­
nisms in diabetic retinopathy. Images from a perfused
human donor eye with diabetic retinopathy is pre­
sented. Thin section through a capillary demonstrates
a narrowed lumen secondary to adhesion of an in­
flammatory cell to the endothelium but without
complete occlusion (Yellow ar; A). In panel B, adja­
cent hypertrophied endothelial cells contain nuclei in
close proximity (yellow ar) which lead to significant
narrowing of the lumen. Panel C demonstrates a
significantly hypertrophic endothelial cell (red ar)
which has led to significant narrowing of the lumen.
On the right, an occluded capillary segment is seen
with thickening of the basement membrane and loss
of endothelial cell (green ar). A microaneurysm is also
seen (yellow ar). Panel D demonstrates occlusion of a
narrowed capillary segment due to an inflammatory
cell (yellow ar). Adjacent endothelial hypertrophy
have resulted in critical stenosis such that total oc­
clusion is now possible by sequestration of inflam­
matory cells. Grey = Lectin. Blue = Nuclei.
Fig. 39. Retinal artery occlusion due to endothelial
cell proliferation in diabetic retinopathy. Images from
a perfused human donor eye in a patient with diabetic
retinopathy are presented. Early stage endothelial
proliferation is demonstrated in panel A. In a local­
ised part of the artery endothelium, multiple layers of
endothelial cells are seen (Inset C; white asterisks). In
panel B, images from a different eye are presented.
Significant intraluminal proliferation of endothelial
cells is found to completely occlude the artery at the
site of bifurcation. Arterial endothelial cells can be
recognised via their spindle cellular morphology and
elongated, ovoid shaped nuclei. C and D show
magnified insets of A and B, respectively. Red = F-
actin. Green = αSMA. Blue = Nuclei. Both scale bars
= 30 μm.
visualise and quantify retinal perfusion in vivo. However, it is possible to
speculate upon how retinal perfusion is disturbed in DR using histo­
logical information. The following sections will discuss macular perfu­
sion abnormalities in DR in the context of our histologic studies in this
field.
5.4.1. The “steal” phenomenon in preclinical diabetic retinopathy
The early works of Ashton, Kuwabara and other investigators have
been instrumental in defining the histologic findings of DR once the
onset of observable ophthalmoscopic changes have occurred (Ashton,
1963; Kuwabara and Cogan, 1963). Much less is known about the
C. Balaratnasingam et al.
Fig. 40. Endothelial proliferation within microaneurysms (MA). Full stack z-projection image of an irregular MA measuring 100 μm in diameter is shown in panel A.
A single XY slice shows complex internal structures (B). Several chambers are found within the MA, divided by septa. Endothelia proliferation characterised by
atypical nuclei morphology are found to line the interior of the MA (yellow ars). Grey = Lectin. Blue = Nuclei. All scale bars = 30 μm.
vascular changes that characterise preclinical DR. We define preclinical
DR as the stage of retinal alteration due to DM prior to the onset of
clinically visible retinal changes such as the microaneurysm, which can
be readily detected on the wholemount retinal micrograph. From a
clinical standpoint, preclinical DR is defined as the state of retinal
alteration prior to the earliest stage as graded by the early treatment
diabetic retinopathy study (ETDRS) classification of DR.
In our recent study we performed a histologic assessment of alter­
ations in vascular inflow and outflow pathways within the macula in
donor eyes from patients with DM with preclinical DR (An et al., 2022a).
These findings were compared to age-matched control eyes from healthy
subjects and also age-matched eyes from patients with clinical DR. We
used αSMA stain to characterise changes to the contractile elements of
the macular circulation in preclinical DR. The main findings of this work
were as follows:
1. In preclinical DR, there is no change in capillary density within any
of the capillary plexuses compared to control eyes.
2. In preclinical DR, there are significantly more connections between
retinal arteries and the ICP than control eyes.
3. In preclinical DR, there is increased αSMA stain in the DCP within
close vicinity to the a1 inflow site (Fig. 43). The increase in expres­
sion of αSMA is seen in both pericytes and endothelia in the DCP
(Fig. 43).
The significance of the above findings are manifold. Firstly, it in­
dicates that a “steal phenomenon” characterises preclinical DR with
preferential diversion of blood flow to the deeper capillary beds. The
principles of this phenomenon are analogous to the “steal” phenomenon
that can lead to stroke following carotid stenting (Kim et al., 2012).
Kornfield and Newman demonstrated a physiologic “steal” effect in rat
eyes using flicker stimulation that induced significantly greater dilation
of intermediate layer capillaries than in capillaries of the superficial and
deep vascular layers (Kornfield and Newman, 2014). Given that inflow
to the DCP in the human eye is almost entirely derived from the ICP it
can be inferred that in preclinical DR there is a greater demand for blood
flow to the DCP. Earlier, we provided evidence that the earliest struc­
tural changes to occur within the macular circulation in DR is at the level
of the DCP (An et al., 2020a, 2021). The steal phenomenon may be a
compensatory mechanism that occurs in the earliest stages of preclinical
DR in an attempt to prevent damage to this part of the retina. The
preferential diversion of blood flow to the ICP may also reflect a
disproportionate increase in energy demands in these retinal compart­
ments in diabetes.
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Progress in Retinal and Eye Research xxx (xxxx) xxx
The other important observation from our recent work is that the
expression of αSMA in pericytes play a key role in increasing blood flow
to the DCP in preclinical DR. Many studies have focused on pericyte loss
in DR (Beltramo and Porta, 2013; Spencer et al., 2020). Animal studies
and histologic reports have shown that the consequences of pericyte
death include vascular occlusion and ischaemia, microaneurysm for­
mation, loss of the integrity of the BRB with subsequent tissue oedema
and loss of control of capillary endothelial proliferation resulting in
retinal neovascularisation (Hammes et al., 2002; Kuwabara and Cogan,
1963; Robinson et al., 1991). One mechanism by which the regional
pericyte population is depleted by disease is through the migration of
pericytes away from sites of capillary injury (Pfister et al., 2008). There
is strong evidence from studies of the brain and retina that signaling
through trophic factors such as angiotensin-2 also control pericyte
migration (Nadal et al., 1999; Toth et al., 2013). In healthy human eyes,
pericytes of the DCP are largely devoid of αSMA but in preclinical DR
αSMA expressing pericytes are found. This provides evidence of a
compensatory response via functional alterations in protein expression
by the pericytes within the DCP to increase or preserve blood flow in
order to prevent the development of ischaemia. The function of pericytes
within capillaries is akin to the role of smooth muscle cells in large order
vessels. Through the expression of αSMA they are major regulators of
blood flow by controlling vascular diameter. Although retinal arterioles
have been shown to be the major control point for blood flow within the
retinal circulation, our studies indicate that the control point for blood
flow shifts to the level of the capillary in preclinical DR (Hill et al., 2015;
Kornfield and Newman, 2014). The increased expression of αSMA may
confer greater auto-regulatory capacity within the DCP. Exploration of
this concept and strategies to prevent alteration or loss of capillary
pericytes may serve a key role in preventing the development of macular
ischaemia in DR.
5.4.2. The “no-reflow” phenomenon may propagate vascular injury
following ischaemia
As discussed above, one of the changes that characterise clinical DR
is the occurrence of ischaemia in the DCP. This can occur in the setting of
otherwise normal capillary density measurements in the SVP and ICP
and therefore may not be recognised. In addition to this, our recent
studies using human donor eyes from patients with DR has revealed the
following:
1. In clinical DR, there is an increase in the number of direct connec­
tions between retinal arteries and the ICP when compared to controls
but no difference was found when compared to preclinical DR.
C. Balaratnasingam et al.
Fig. 41. Glial process infiltration at sites of vessel occlusion in a donor retina
with diabetic retinopathy. Panel A shows a site of small vessel bifurcation. The
pathway indicated by the large green arrow is patent (by examining the z-stack)
and represents the direction of blood flow. Note the presence of endothelial cell
linings along this vessel. The capillary branch on the bottom right side of the
image is occluded. Thickened basement membrane material is seen at the site of
occlusion (magenta asterisk). This occluded capillary segment is acellular in
contrast to the patent capillary. Glial process infiltration within the occluded
capillary lumen can be seen (white ar) to run in parallel with regular extra­
vascular glial process, forming a tram-track appearance. Panel B shows an
alternate slice through the same occluded capillary segment and highlights the
site of glial penetration (magenta ar) into the capillary lumen that then extends
towards the occlusion site. In comparison, sites of arterial occlusion due to
endothelial proliferation (green ars; Panel C) show no significant evidence of
glial infiltration into the lumen of arteries. Grey = F-actin. Yellow = GFAP.
Blue = Nuclei. All scale bars = 30 μm.
2. In clinical DR there is an increase in αSMA staining within vascular
structures of the deep macular circulation. Specifically, an increase
in αSMA stain occurs in the DCP both downstream of a1, and at
venular junctions adjacent to v3 (Fig. 44). Additionally, distal
capillary segments of the DCP away from inflow and outflow sites
may also have increased staining of αSMA (Fig. 44).
The first observation implies that an increase in blood flow to the
DCP by forming greater vascular connections between arteries and the
ICP is sustained in the face of retinal ischaemia. Given that there was no
difference in the number of connections between preclinical DR and DR,
34
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 42. Capillary density comparison in the parafovea between diabetic reti­
nopathy (DR; left panel of images) and age-matched controls (right panel of
images). Full stack parafovea confocal histology image is captured and strati­
fied into the superficial vascular plexus (SVP; A and B), intermediate capillary
plexus (ICP; C and D) and deep capillary plexus (DCP; E and F). In this donor
eye, note that there is no significant reduction in capillary density in DR (left
panel) for both the SVP and ICP, compared to controls (right panel). In the DCP,
capillary density of DR parafovea is significantly reduced compared to control,
evidenced by sites of capillary nonperfusion (yellow asterisks) and subsequent
increase in intercapillary distance. Vasculature labelled using Lectin. Scale bar
= 100 μm.
our results suggest that there may be an upper limit to this compensatory
mechanism for increasing blood flow and that there is likely to be a finite
amount by which the number of connections can increase. Decompen­
sation, in the most severe cases of macular ischaemia, is characterised by
almost complete absence of capillary segments in the DCP (Fig. 45).
Schematic diagrams illustrating the relationship between capillary
density and αSMA stain in preclinical DR and DR is provided in Fig. 46.
We were surprised to find that αSMA stain was increased in the DCP
in DR despite the presence of capillary loss and ischaemia. Given that
pericyte loss is considered an integral step in retinal ischaemia in DR one
would expect a decrease in αSMA in clinical DR. It is possible that an
increase in αSMA within surviving endothelia and pericytes serves a
compensatory response such as that seen in preclinical DR. However,
such a finding also implicates a “no-reflow” phenomenon that propa­
gates retinal vascular injury in DR.
The “no-reflow” phenomenon has been extensively described in the
cardiovascular literature (Ames et al., 1968; Kloner et al., 2018; Rezkalla
and Kloner, 2002). It is a phenomenon whereby reperfusion within large
order vessels beyond a defined period of ischaemia will not increase
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 43. αSMA stain patterns of the deep capillary plexus (DCP) in a human donor eye with preclinical diabetic retinopathy. Strong staining of αSMA is found
surrounding the a1 arteriole inflow site (A and B). There was no significant αSMA stain around v3 venule (venous outflow site of DCP; orange ar; B). αSMA was
expressed by both endothelial cell (magenta ar) and pericytes (green ars) at the arteriolar inflow site (inset; C). ICP, intermediate capillary plexus. Yellow = αSMA.
Red = F-actin. Blue = Nuclei. All scale bars = 100 μm.

Fig. 44. αSMA stain patterns of the deep capillary plexus (DCP) in a human donor with diabetic retinopathy. Strong staining of αSMA is found throughout the DCP
vasculature (A). Both a1 arterioles and DCP capillaries contain αSMA positive pericytes (inset B; green ars) and endothelial cells (yellow ar). Similar findings are
present at the venular outflow site (inset C). The superficial vascular plexus (SVP; D) and intermediate capillary plexus (ICP; E) are also shown. αSMA stain is
increased within the ICP capillaries, as well as within segments of the venule (V). A retinal cyst which is present throughout all 3 plexuses is indicated with a yellow
asterisk. Red = F-actin. Yellow = αSMA. Blue = Nuclei. All scale bars = 100 μm.

Fig. 45. Advanced stage of diabetic retinopathy in a human donor eye illustrating marked reduction in parafoveal capillary density across all plexuses (A, B and C).
In particular, capillaries of the deep capillary plexus (DCP) are almost completely absent (C). SVP, superficial vascular plexus; ICP, intermediate capillary plexus.
Vasculature labelled using Lectin.

35
C. Balaratnasingam et al.
blood flow to ischaemic tissue due to the occurrence of deleterious
microcirculatory changes. In the setting of myocardial ischaemia,
capillary pericytes have been clearly shown to be one important
component that mediates the “No-reflow” phenomenon. The study by
O’Farrell and colleagues showed that cardiac pericytes reduce micro­
vascular flow after ischaemia despite reperfusion of the coronary artery
(O’Farrell et al., 2017). Similarly, as shown in our work, the increase in
expression of αSMA in the DCP may result in enhanced capillary
constriction and the reduction in capillary diameter below a critical
limit, such that there is obstruction of the passage of leucocytes and
erythrocytes. In the cardiac circulation, a reduction in capillary luminal
diameter beyond 37% is sufficient to completely obstruct the flow of
these cells (O’Farrell et al., 2017). Complete occlusion of the lumen is
not required. A similar change in luminal diameter in the DCP may result
in capillary occlusion that propagates the ischaemic injury that is
already present within the DCP. Taken together, it is likely that the
strategies to preserve the function of pericytes and the activity of cells
expressing αSMA will need to account for the stage of DR. In preclinical
DR, the key will be to prevent pericyte alterations and preserve pericyte
function. However, following the onset of ischaemia, the same strategy
may have a deleterious effect on the retinal circulation by limiting blood
flow to an already ischaemic region.
5.5. Pathologic differences between central and peripheral ischaemia
Histologic studies indicate that pathology of the peripheral
ischaemia differs to macular ischaemia. The 1953 report by Norman
Ashton showed that peripheral ischaemia in DR was frequently due to
occlusion of large order arterioles resulting in diffuse non-perfusion of
36
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 46. Schematic representation of alpha smooth
muscle actin (αSMA) distribution and changes in the
development of diabetic retinopathy (DR). Venous
circulation is depicted in blue and arterial circulation
in red. Insets provide magnified views of regions of
interest, at the inflow sites of deep capillary plexus
(DCP). The locations of each vascular plexus are
indicated in the retinal layers panel. (A) The pre­
clinical DR group showed moderate αSMA (yellow
dots) expression along veins, venules, and capillaries
on the venous aspect of the circulation. In addition,
αSMA expression along the a1 arteriole spanned its
entire course and extended into the DCP (inset I;
black ar). The DR group (B) was characterised by
microaneurysms and capillary dropout within the
DCP (asterisks; inset II). Compared to the preclinical
DR group, there was significantly more αSMA
expression along veins, venules, and capillaries on the
venous aspect. Within the DCP, αSMA expression was
more extensive. They were found distal to the a1 ar­
terioles and at venular junctions (inset II; blue ar).
Superficial vascular plexus (SVP), Intermediate
capillary plexus (ICP). Reproduced with permission
from An et al. (An et al., 2022).
capillary beds (Ashton, 1953). He also found that on the arterial side of
the capillary network that was undergoing obliteration, some eyes
demonstrated dilated vascular connections between the artery and the
venous capillary system. These histologic observations were verified in
the clinical angiographic report by Bresnick et al. that showed
obstruction at the level of retinal arterioles in DR with profound oc­
clusion of capillary beds within the peripheral retina, downstream to the
site of occlusion (Bresnick et al., 1975).
In contrast, our histologic assessments suggest that vascular occlu­
sion within the macula predominantly involve selective capillary seg­
ments and/or capillary beds. In our survey of 100 human donor eyes
with DR we have not yet seen a pattern of diffuse non-perfusion of
macular capillary beds due to occlusion at the level of the arteriole, i.e.
there is almost always some capillary segments in the SCP, ICP or DCP
that are observably perfused in the face of macular ischaemia. Clinical
studies of diabetic macular ischaemia using OCTA and FA have also
supported a non-uniform pattern of capillary occlusion that is similar to
histologic studies (Falavarjani et al., 2021; Fan et al., 2017; Fang et al.,
2019; Park et al., 2018; Sim et al., 2014). The report by Bresnick and
colleagues described a select number of cases of retinal arteriolar oc­
clusion within the posterior pole that resulted in diffuse macular
ischaemia however such a pattern of macular non-perfusion appears to
be uncommon. In their survey, Sim et al. studied the patterns of macular
and peripheral retinal ischaemia in DR using UWF-FA (Sim et al., 2014).
They found that a high peripheral ischaemic index was associated with a
larger foveal avascular zone, a surrogate marker of macular ischaemia.
However, peripheral retinal ischaemia did not influence central macular
thickness measurements in that study. Taken together, the clinical and
histologic data suggest that the pathogenic mechanisms underlying
C. Balaratnasingam et al.
peripheral retinal ischaemia may be different to central ischaemia. The
histologic changes associated with peripheral and macular vascular
occlusion have been provided above. Treatment of vascular occlusion in
DR may therefore need to be tailored to the phenotypic pattern of
vascular occlusion.
6. Clinical imaging of the retinal circulation
Precise retinal imaging forms the cornerstone of DR management.
Over the past two decades there has been significant expansion in the
clinical imaging tools that can be used to evaluate retinal disease. It
should be emphasised that the improvements have predominantly
increased our ability to assess retinal structure with greater precision.
The clinician is still very limited in their ability to assess retinal function
using non-invasive technology. This limitation needs to be urgently
addressed in order to expand our understanding of the temporal nature
of DR and to account for the significant inter-individual variations of the
natural disease course and development of irreversible complications.
As described in the preceding sections, there are parallel pathogenic
mechanisms that underlie DR. Much of the multimodal imaging studies
have focused on assessing the vasculogenic mechanisms involved in DR
with less focus on the inflammatory and neuro-degenerative pathways.
We will now discuss the key imaging tools that are widely employed to
assess the vasculogenic mechanisms in DR. The advantages and limita­
tions of these technologies will be described.
6.1. Colour photography
With the exception of the ophthalmoscope, colour photography is the
oldest modality for detecting and documenting the complications of DR.
The major advantage of colour photography is that it is safe, rapid,
relatively inexpensive and widely available. It is also an excellent tool
for documenting the temporal sequence of retinal changes over time and
serves as an educational tool for patients.
One of the earliest committees to promote the application of colour
photography in an effort to characterise DR in a standardised manner
was the Airlie House symposium in 1968 (ETDRS, 1969). The classifi­
cation schema was subsequently recognised as the Airlie-House classi­
fication (Goldberg and Jampol, 1987). The seven standard photographic
fields were modified and subsequently used in the Diabetic Retinopathy
Study (DRS) and the ETDRS (ETDRS, 1981). The results of the ETDRS
study remains the gold standard for classifying the severity of DR in the
clinical setting. In this system, colour photographic standards were
established for the presence of haemorrhages, hard exudates, micro­
aneurysms, IRMA and neovascularisation of the optic disc and/or retina.
Photographic fields were selected on their importance to visual function,
such as the optic disc and macula, and also for their involvement in early
DR such as the area temporal to the macula.
Although the 7-field colour photography allows a standardised
method of grading DR it is associated with a number of limitations. Most
importantly, the spatial resolution of colour photography does not allow
clear visualisation of retinal capillaries. In this paper we provide strong
evidence to highlight the importance of visualising the capillary circu­
lation to allow early detection of DR. The limitations associated with
colour photography for evaluating capillaries is exemplified by the
manner in which colour photography has been utilised in clinical trials.
In the DRS, colour photography was used to define high-risk PDR by the
occurrence of neovascularisation or vitreous haemorrhage (ETDRS,
1987). The ETDRS series of reports evaluated various treatment strate­
gies, predominantly laser, for the management of macular oedema.
There has been limited application of colour photography in the man­
agement of the very earliest stages of DR where intervention is expected
to provide a greater chance of reducing the occurrence of
sight-threatening complications.
As will be reasoned in a later section, the gold-standard management
of DR should be to prevent the onset of major sight-threatening retinal
37
Progress in Retinal and Eye Research xxx (xxxx) xxx
complications such as macular ischaemia, macular oedema and retinal
neovascularisation. Currently, much of the treatment paradigms in DR
involve limiting the extent of structural damage once it has already
occurred rather than preventing it. In the case of panretinal photoco­
agulation therapy that is widely used to treat the neovascular compli­
cations of DR, it can be argued that some destruction of healthy tissue is
required to ameliorate vision loss.
Unlike FA, it can be very difficult to assess retinal nonperfusion using
colour photography. This is a key feature that needs to be precisely
evaluated in every eye with DR as it has importance in prognosticating
the development of vision-threatening complications. The “featureless
retina” is a clinical term that denotes the appearance of an ischaemic
retina as seen on colour photography (Shukla et al., 2021). However,
this term in itself is poorly defined in the ophthalmic literature with little
information provided as to what clinical features would constitute this
appearance. Some authors have described a featureless retina as one that
manifests sheathed appearance to retinal vessels or one that manifests a
thin retina. Both of these clinical features can be missed on clinical
assessment. Shukla and colleagues showed that the extent of capillary
nonperfusion and the rate of neovascularisation in eyes with featureless
retina was significantly greater when visualised using FA compared to
colour photography (Shukla et al., 2021). In Fig. 47 we provide a case of
a patient with a featureless retina that illustrates the mismatch between
the degree of ischaemia as judged using colour photography and FA.
6.2. Fluorescein angiography
Dye angiography of the retina has long been considered the gold-
standard technique for evaluating the retinal vasculature. Novotny
and Alvis were two medical students that developed this technique and
reported the application of this method in clinical practice in their
publication in Circulation in 1961 (Novotny and Alvis, 1961). Since that
report, fluorescein has been the most widely used dye in retinal angi­
ography, with indocyanine green angiography (ICGA) having much less
clinical application in the study of retinal vascular diseases.
Other than illuminating the structural characteristics of the retinal
circulation there are many other aspects of the FA that collectively
convey vital information regarding the state of the retinal and systemic
circulation (Fig. 48). As an example, a significant delay in arm-to-retina
transit time can indicate concomitant carotid artery disease and the need
to investigate the carotid vascular system using carotid ultrasonography
or contrast-based angiography as a measure to prevent stroke (Sarkies
et al., 1986). Additionally, FA is a precise way of studying the integrity
of the BRB particularly in the macula. Leakage of fluorescein in the
macula is frequently seen in macular oedema and can be easily missed if
OCTA or colour photography images of the macula were assessed.
Diffuse staining of retinal vessels and leakage from the deep capillary
circulation may be indicative of raised VEGF concentrations in the
posterior segment and may predict greater response to intravitreal
anti-VEGF therapy in these patients. FA has also provided a reliable way
of detecting capillary ischaemia in the setting of DR as well as quanti­
fying the extent of ischaemia. For the above reasons there are significant
advantages associated with FA that have not been surpassed by the
newer angiography techniques including OCTA. FA therefore continues
to serve an important role in the management of DR. Despite the
introduction of OCTA techniques, FA is not an obsolete investigation and
remains widely used by ophthalmologists.
An important development that has facilitated significant progres­
sion in our understanding of the pathophysiology of DR is UWF-FA. This
technique allows visualisation of nearly 200 degrees of the retina in a
single image. Detailed assessment of the peripheral retina using UWF-FA
has outlined new and important relationships between peripheral retina
changes and the natural course of DR. It has exemplified the importance
of assessing retina tissue outside the ETDRS 7-field when making de­
terminations about the severity of DR and the risk of disease progression.
Silva and colleagues showed that the presence of DR lesions outside the
C. Balaratnasingam et al.
ETDRS 7-field increased the risk of PDR development and DR progres­
sion over 4 years by 4.7 and 3.2 times, respectively (Silva et al., 2013).
The work by Oliver et al. showed that the occurrence of peripheral
retinal ischaemia as seen on UWF-FA is associated with an increased risk
of retinal neovascularisation and macular ischaemia (Oliver and
Schwartz, 2010). Tan and colleagues leveraged the capabilities of
UWF-FA for visualising the peripheral retina and described a novel
technique for quantifying the area of retinal non perfusion using ste­
reographic projection software (Tan et al., 2016). Accurate quantifica­
tion of UWF-FA images requires stereoscopic projection to correct for
peripheral distortion due to the curvature of the eye. Following this
correction, it is possible to calculate the area of retinal ischaemia and
express it as a percentage of the total retinal area that is perfused. This
technique has been useful for investigating quantitative relationships
between peripheral ischaemia and macular structure and function in DR
and retina vein occlusion. Fan and colleagues made the interesting
observation that retinal vascular bed area, not retinal ischaemia, as seen
on UWF-FA was correlated with DMO (Fan et al., 2022). The in­
vestigators of the DAVE trial demonstrated that targeted retinal photo­
coagulation to areas of nonperfusion as seen on UWF-FA did not reduce
the treatment burden with respect to anti-VEGF therapy over three years
in patients with concomitant DMO (Brown et al., 2018). Their findings
exemplified the complex relationships between ischaemia, VEGF and
DMO and provided further evidence that non-VEGF mediated pathways
underlie the development of DMO and may not be addressed by laser
photocoagulation. Taken together, there is overwhelming evidence that
UWF-FA plays an important, if not an essential, role in the management
of DR.
The major drawback of FA is that it is an invasive technique that is
associated with a series of potential adverse effects. Yannuzzi et al.
surveyed the outcomes of 221,781 FAs and reported the frequency of
death to be 1:222,000 (Yannuzzi et al., 1986). Kornblau and El-Annan
performed a meta-analysis of 78 articles reporting adverse reactions to
FA and reported an overall adverse reaction rate of 0.083–21.69%
(Kornblau and El-Annan, 2019). The frequency of mild reaction was
38
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 47. Featureless retina in diabetic retinopathy.
Clinical images from a 41-year-old male patient with
Type 1 diabetes mellitus is presented. Colour
photography and fluorescein angiography (FA) im­
ages from the posterior pole (A and B) and peripheral
retina (C and D) are presented. Assessment of colour
imaging, particularly the ultrawide field modality,
reveals a relative paucity of retinal haemorrhages and
microangiopathy. Areas of neovascularisation and
fibrosis are seen in the posterior pole. The FA images
clearly highlight areas of neovascularisation (red ar­
rows) that appear to be greater in number than what
is seen on colour photography. Furthermore, FA
demonstrates profound ischaemia of the peripheral
retinal circulation that is not readily appreciated
using colour photography, or would be expected
given the relative paucity of pathology on colour
photography.
1.24–17.65% and included the occurrence of nausea, vomiting, extrav­
asation of dye, sneezing, pruritus, paresthesia of the tongue as well as
abdominal cramping. The risk of a severe reaction was encountered in
nearly 1:200 patients and included myocardial infarction, anaphylaxis,
laryngeal oedema, bronchospasm, respiratory arrest and seizure. Due to
these potential risks, FA is only carried out in a supervised medical
setting where medical staff are trained in cardiopulmonary resuscitation
and have available the necessary equipment to manage medical
emergencies.
Despite FA providing excellent visualisation of large order retina
vessels it is limited in its capacity to image the fine retinal capillaries.
There are several reasons for this including the presence of choroidal
hyperfluorescence that diminishes contrast of the fine order vessels. In
the presence of media opacity the quality of the FA can be degraded
further obscuring assessment of retina detail. Another important limi­
tation of FA is that it does not clearly stratify the superficial and deep
capillary networks of the retina. In our previous report we compared the
appearance of the retinal capillary circulation as seen on histology and
high-resolution fundus FA. The histology and clinical cohorts were
different but both were comprised of normal healthy subjects (mean age
27 years) with clear ocular media. We found that meaningful interpre­
tation of capillary detail was only possible in 30% of cases (Mendis et al.,
2010). Furthermore, our study showed that the deep capillary circula­
tion of the retina was poorly visualised using FA. Weinhaus et al. have
performed an in-depth comparison of microvascular information
attained from FA and retinal histology using macaque retinas (Weinhaus
et al., 1995). Their experiments demonstrated that FA capillary detail
declined proportionally with distance away from the FAZ. In addition,
they clearly showed that capillary visibility on FA was a joint function of
retinal capillary diameter and retinal depth, with large diameter capil­
laries more clearly visualised on FA than small diameter capillaries.
Weinhaus et al. also reported that capillary segments in the NFL were
visualised more than four times as effectively as segments of comparable
diameter in the deepest vascular plane. In their work, >50% of super­
ficial capillaries were visualised out to an eccentricity of 1200 μm,
C. Balaratnasingam et al.
Fig. 48. Salient features of fluorescein angiography (FA) in diabetic retinop­
athy. Images from a 52 year-old male with Type 1 diabetes mellitus is pre­
sented. Arm-to-retina transit time denotes the time it takes for fluorescein to
appear in the retinal circulation. In this patient it is delayed and may be due to
stenosis within the carotid circulation. Macular ischaemia (B) and peripheral
retinal ischaemia (C) can also be appreciated (blue asterisk) in the posterior
pole and also in ultrawide field images of the peripheral retinal circulation.
Areas of retinal neovascularisation (red arrows) are clearly highlighted using
FA as are disruptions to the blood-retina-barrier. In this case there is a diffuse
increase in permeability of the retinal circulation that is best appreciated on
panel C. The inset illustrates a magnified view of the macula during the late
stages of the angiogram and denotes the presence of macular oedema. Features
such as arm-to retina transit time and increased vascular permeability cannot
be readily appreciated using other vascular imaging modalities such as optical
coherence tomography angiography. Time stamps (minutes:seconds) of the FA
sequence for each image are provided.
39
Progress in Retinal and Eye Research xxx (xxxx) xxx
whereas <20% of deep capillaries were visualised in equivalent retinal
regions. In Fig. 49 we provide original data from a histology-FA com­
parison from the same human donor eye. It clearly outlines the limita­
tions associated with FA and the inability to provide histology-like
detail.
Therefore, despite, FA being an excellent clinical tool, there remains
significant limitations related to adverse events and an inability to
reliably visualise all retinal capillary beds. These are important limita­
tions that restrict the usefulness of FA for evaluating the earliest stages of
capillary and endothelial dysfunction in DR that we have shown in
histologic studies to have a predilection for the deep retinal capillary
beds.
6.3. Optical coherence tomography angiography
OCTA is the latest imaging technology that is being used in clinical
practice to image the retinal circulation. Developed on the platform of
OCT, a major advantage of OCTA is that it provides depth-resolved
images of the retinal circulation. There are a number of excellent arti­
cles outlining the principles of OCTA and for that reason we will not be
repeating the same information in this report (Kashani et al., 2017;
Makita et al., 2006; Spaide et al., 2018). In short, blood flow using OCTA
is visualised by comparing successively acquired OCT B-scans and
computing differences between the scans on a pixel-by-pixel basis,
highlighting the areas of change which in theory should only be the
vasculature. The OCTA is based on the premise that the only structural
changes that occur within a region of tissue within a very small interval
of time is the movement of cells within the retinal circulation. A major
advantage of OCTA is that it allows label-free visualisation of the retinal
circulation and that is the visualisation of retinal vasculature without
the administration of fluorescein or other dyes. It is therefore a risk-free
procedure that is rapid and well-tolerated by patients. Vascular infor­
mation derived using OCTA can be visualised in the en face and trans­
verse planes.
An important development in the field of OCTA is the introduction of
UWF systems. Some commercial OCTA systems have facilitated visual­
isation of up to 80 degrees of field of view in a single fundus image
(Khalid et al., 2021). The major advantage of UWF-OCTA is that it
captures regions of ischaemia and neovascularisation that would
otherwise have been missed on clinical assessment alone. This is a sig­
nificant advantage, for example, in the evaluation of pregnant patients
as pregnancy is a significant risk factor for DR progression and because
of the preference to avoid procedures in pregnancy (Chew et al., 1995;
Temple et al., 2001). Sawada et al. compared retinal non-perfusion and
neovascularisation as seen on UWF-FA and UWF-OCTA in DR (Sawada
et al., 2018). Using UWF-FA as the gold standard they reported that the
sensitivity of UWF-OCTA for detecting non-perfusion was 98% and the
Fig. 49. Direct comparison between fluorescein
angiography (FA; A) and histology (B) from the same
human donor eye. FA was performed pre-mortem and
the image of the temporal peripapillary retina was
captured at 23 s. The eye was retrieved following
patient death and used for histologic analysis. Cor­
responding retinal arteries are marked with red ars,
and retinal veins are marked with blue ars. For any
given region, the histology section demonstrates
greater density of capillary structures as well as
greater clarity of capillary boundaries than the FA.
Histology vasculature labelled using lectin.
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

sensitivity of UWF-OCTA for detecting neovascularisation was 100%.
Over the past decade we have performed a range of reports that have
validated the technique of OCTA using human donor eyes and animal
eyes (An et al., 2018; Balaratnasingam et al., 2018, 2019; Tan et al.,
2015; Yu et al., 2021). Most of these studies have compared the
morphology of vascular structures as seen on OCTA with that of his­
tology. The collective data from the cohort of human donor eyes studied
using histology and age-matched and sex-matched control eyes imaged
with OCTA has revealed the following: 1) OCTA provides unprecedented
visualisation of retinal capillaries, arterioles and venules; 2) OCTA fa­
cilitates depth-resolved study of the retinal circulation and stratified
visualisation of different capillary beds.
Despite the obvious strengths of OCTA that have been demonstrated
by a number of investigators, there remain some questions regarding
this technique and its application in daily clinical practice. Importantly,
it remains uncertain if all the vascular structures within a defined vol­
ume of tissue are visualised using OCTA. As OCTA uses motion and flow
characteristics to detect vascular structures, it is plausible that vessels
with low flow will not be visualised using OCTA. Earlier in this report we
highlighted how variations in blood flow within microaneurysms can
modify their appearance on OCTA on a momentary basis. Additionally,
several reports have shown that artefacts and errors in image segmen­
tation could lead to incorrect interpretations of OCTA images (Fala­
varjani et al., 2017; Spaide et al., 2015). Accurate automated
segmentation is particularly challenging in cases where the layered
organisation of the retina is disrupted by disease processes such as
macular oedema. Comparing the appearance of the macular circulation
as seen on OCTA against the histologic gold standard would help
reconcile some of these issues. We were able to achieve this comparison
using a human donor eye that was retrieved in 2018. Unpublished and
published data from this donor eye will be used henceforth to validate
the technique of OCTA as well as highlight some potential limitations of
this technique.
decorrelation angiography algorithm. This instrument has an A-scan
rate of 70,000 scans per second using a light source centered on 840 nm
and a bandwidth of 45 nm. Multiple OCTA studies of the left eye were
acquired, and only scans that were deemed to be of sufficient image
quality were included in the analysis (n = 9; mean scan quality = 6.6 and
median scan quality = 7). The multimodal imaging features of the eye
are shown in (Supplementary Fig. 3).
6.3.1.1.1. Tissue preparation and image analysis. The patient was
managed palliatively due to the advanced nature of the malignancy. The
left eye was enucleated 54 min after death and immediately prepared for
histologic analysis. Our established and previously described perfusion
techniques were employed to achieve retinal vascular endothelial
labelling (as outlined in Section 3). The eye was labelled using lectin. We
observed complete labelling of the entire peripheral retinal circulation
(Fig. 50) and also the macular circulation (Fig. 51) when the specimen
was examined with microscopy. This finding reaffirmed the absence of
artefactual occlusion of the retinal circulation.
The segmentation boundaries for the various manual and software-
defined OCTA projections acquired pre-mortem are illustrated in (Sup­
plementary Fig. 4). We used a similar technique to that previously
described by Uji et al. to generate en face averaged images of OCTA data
(Uji et al., 2017). We used 9 single OCTA images (frames) to generate an
averaged en face image. Fiji, Image J (Rasband, W.S., ImageJ, U. S.
National Institutes of Health, Bethesda, Maryland, USA, https://imagej.
nih.gov/ij/) was used to align and average the OCTA frames. Histology
and OCTA images of the macula were cropped so that the region of

6.3.1. Direct Histology-OCTA comparison of a human donor eye

6.3.1.1. Details of human donor eye. An 84-year-old female was referred

to the principal author of this article (CB) for assessment of subacute
right vision loss. Best-corrected visual acuities on presentation were
hand motion in the right eye and 6/6 in the left eye. Dilated retinal
evaluation of the right eye revealed a total retinal detachment due to a
large choroidal mass in the posterior pole and therefore was not exam­
ined histologically. Left retinal examination revealed mild RPE changes
within the superior and inferonasal macula (Supplementary Fig. 3).
Drusen were not evident. Optic disc and foveal examination of the left
eye were normal. Peripheral retinal examination demonstrated reticular
pigmentary degeneration that was most prominent in the nasal retina.
There was no history of hypertension or cigarette smoking, and DM had
been excluded by her physician approximately four months before
presentation. She was not taking any systemic medications. Blood
pressure on presentation was normal at 128/74 mmHg.
Retinal imaging of the left eye included colour photography (Canon
CF-60DSi, Canon Inc., Japan) and red-free photography (Canon CF-
60DSi, Canon Inc., Japan). Ultra-widefield pseudocolour, fundus auto­
fluorescence (FAF), FA and ICGA images were captured with the Optos
California (Optos, Dunfermline, Scotland, United Kingdom). Near-
infrared reflectance, FAF, FA, and ICGA images were acquired on the
Heidelberg HRA + OCT2 (Heidelberg Engineering, Heidelberg, Ger­
many) at 30◦ . Using the HRA + OCT2, the macula was imaged with a
30◦ × 25◦ raster pattern of 61 horizontal B-scans, each averaged 10
Fig. 50. Peripheral retinal perfusion of the donor eye. A low-magnification
times, with an interscan spacing of 115 μm. The Heidelberg HRA +
image of the flat-mount histologic specimen as well as magnified views (In­
OCT2 is a confocal scanning laser ophthalmoscope system. sets I, II and III) of the peripheral retina demonstrate complete labelling of the
OCTA images of the left eye were acquired with the Optovue RTVue retinal circulation. Note that the peripheral retinal circulation is clearly
XR Avanti (Optovue, Inc., Freemont, CA, with AngioVue version perfused and demonstrates excellent preservation of vascular detail making it
2017.1.0.155 software) which uses the split spectrum amplitude unlikely that the retinal circulation was obstructed by thrombus during perfu­
sion labelling. Scale bars = 2.5 mm.

40
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 51. Retinal layer co-localisation and histologic characteristics of the macular capillary plexuses in the donor eye. Flat mount (A) and cross-sectional (B) his­
tologic images of the donor eye, depicting all vascular plexuses, demonstrate the density and complexity of the macular capillary circulation. On the tissue cross-
section, nuclei appear in red stain and that of the vascular endothelium appear in green stain. The cross-section demonstrates that the superficial plexus is pre­
dominantly localised to the level of the ganglion cell layer, the intermediate plexus to the inner aspect of the inner nuclear layer and the deep plexus to the outer
aspect of the inner nuclear layer. Capillary plexuses on histologic flat-mounts have been false-coloured cyan (superficial plexus), yellow (intermediate plexus) and red
(deep plexus). The region where the cross-sectional image has been attained is represented by a white-fenestrated line on Panel A. Note that the retinal arterioles and
venules are localised to the superficial and intermediate plexus and are not seen at the level of the deep plexus. The deep vascular complex is comprised of the
intermediate and deep capillary plexuses. Note that there is complete perfusion of the macular circulation without any artefactual occlusion. Vasculature labelled
using lectin.

interest was comparable. The topologic characteristics of large-order
retinal vessels were used to define the boundaries of the region of
interest.
Different OCTA projections of the region of interest, including
manufacturer recommended automated segmentation, manual
anatomical-based OCTA slab segmentation, single OCTA images and
averaged OCTA images were compared to histology. Comparisons of
microcirculatory morphology were performed for the SVP, ICP, DCP and
the FAZ.
OCTA and histologic images were also studied in three-dimensions
using volume rendering techniques (Videos 1 and 2). Quantitative
measurements of OCTA data were attained using en face averaged im­
ages. The following calculations were determined:
1. Capillary density - Capillary counting was used to measure capillary
density as shown in (Supplementary Fig. 5) (An et al., 2021; Yu et al.,
2015). Projected images of histologic confocal volumes depicting the
superficial vascular complex (SVC), Deep vascular complex (DVC)
and DCP were used for quantitative analysis. For OCTA data, mea­
surements were attained of the SVC and DVC using manufacturer
recommended settings. Measurements of the DCP were attained after
manual anatomical-based OCTA slab segmentation. For the SVC,
circles centered at the foveola, with radii of 350, 700 and 1050 μm
were drawn and the number of intersections between the boundary
of the circle and capillary segments were manually counted. For the
DVC and DCP, circles of 700 and 1050 μm radii were used (Supple­
mentary Fig. 5)
2. Area of the FAZ - Manual tracing of the terminal foveal capillary ring
was used to determine the area of the FAZ. Separate calculations of
FAZ area were made at the level of the SVC and DCP. The area of the
FAZ was also determined using the OCTA projection depicting all
vascular plexuses. For OCTA data, manufacturer recommended set­
tings were used to stratify the superficial FAZ and manual
anatomical-based OCTA slab segmentation (as described above) was
used to stratify the deep FAZ and the area of the FAZ in the projection
depicting all vascular plexuses (Fig. 52E and F).
6.3.1.2. Comparisons between OCTA, histology and other imaging modal­
ities. The appearance of the macular circulation as visualised by colour
photography, red-free imaging, FA, ICGA and OCTA are provided in
Fig. 53. The histologic correlate from a similar region of interest is also
provided in Fig. 53 panel F. Large-order arterioles and venules were seen
on colour, red-free and ICGA images, but it was not possible to clearly
discern capillary structures or the FAZ on these modalities. The FA
provided some detail about the capillary circulation but due to fluo­
rescence from the choroidal circulation, it was not possible to clearly
discern individual capillary lumens in some parts of the macula. For the
same reason, it was not possible to clearly define the terminal foveal
capillary ring on FA. The size of the FAZ as seen on FA appeared larger
than the histologic flat-mount.
OCTA provided the closest histologic representation of the macular

41
C. Balaratnasingam et al.
Fig. 52. Comparisons of the foveal avascular zone (FAZ) as seen on optical
coherence tomography angiography (OCTA) and histology. Images of the su­
perficial FAZ (A and B) and deep FAZ (C and D) as seen on OCTA following
image averaging and histology are presented. OCTA segmentation was per­
formed manually to match the anatomical layers of vascular plexuses found in
histology. There is a close resemblance in the morphology of the superficial FAZ
between histology and OCTA. In the OCTA image of the superficial FAZ, the
intensity of the flow signature in many capillary segments comprising the ter­
minal foveal ring is less than that of other vascular structures in the fovea.
Despite image averaging, one capillary segment (red arrow) is not clearly seen
on OCTA whilst being clearly visualised in histology. The size of the deep FAZ
as seen on OCTA (C) is significantly less than histology (D). Note that some
capillary segments of the superficial foveal capillary ring are seen in the deep
FAZ (green arrow) despite manual segmentation. Panels E and F demonstrate an
overlay of the two plexuses following manual tracing of the superficial FAZ
(false-coloured yellow) and deep FAZ (false-coloured orange). The ratio be­
tween the deep and superficial FAZ is greater in histologic images than OCTA.
Histology specimen was labelled using lectin.
circulation as it provided clear visualisation of all orders of the vascular
tree including arterioles, capillaries, and venules. Vessels represented on
single OCTA scans, however, frequently appeared fragmented with
discontinuous lumens (Fig. 54). En face image averaging resulted in
vascular segments having sharper borders and a reduction in the number
of discontinuous segments (Fig. 54B). Image averaging also improved
visualisation of vascular segments that were sometimes not readily
appreciated on single OCTA scans (Fig. 54).
6.3.1.3. Comparisons between histology and OCTA of the superficial and
deep vascular complexes. Comparisons between histology and an aver­
aged OCTA image of the SVC are provided in Fig. 55. Retinal arterioles
and venules were clearly evident on OCTA images depicting the SVC.
Similar to histology, retinal arterioles seen on OCTA were also
42
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 53. Multimodal imaging of the donor macular circulation. Colour (A), red-
free (B), fluorescein angiography (FA; C), indocyanine green angiography
(ICGA; D), optical coherence tomography angiography (OCTA; E) and histology
(F) images of a similar region of interest. The OCTA image represents an
average of 9 vascular slabs, each slab representing all vascular plexuses. The
larger arterioles (red arrow) and venules (blue arrow) are labelled on the his­
tology panel. Aside from OCTA, structural information regarding the capillary
circulation is poorly represented on the other imaging modalities. It is also
difficult to precisely discern the boundaries of the FAZ using imaging modalities
other than OCTA. Note that it is not possible to differentiate arterioles from
venules using the intensity of OCTA flow signatures. Modified from Balar­
atnasingam et al with permission (Balaratnasingam et al., 2019).
characterised by a peri-arterial capillary-free zone. Most of the retinal
capillaries seen on histology were also evident on OCTA; however, we
observed that some capillaries were not readily appreciated on OCTA
despite image averaging (Fig. 55 Panel II). These capillaries typically
had a smaller diameter than the vessels that were consistently visible on
OCTA. The number of capillary intersections measured on histology and
OCTA, respectively, were 18 vs. 16 (for circles of radii 350 μm), 56 vs. 47
(for circles of radii 700 μm) and 98 vs. 84 (for circles of radii of 1050
μm).
Comparisons between histology and an averaged OCTA image of the
DVC using manufacturer recommended automated segmentation is
provided in Figs. 56 and 57. Using this segmentation protocol, we
identified capillaries that were histologically verified to be in the SVC to
be displayed as well in the projected image of the DVC. Capillaries of the
SVC were more frequently projected closer to the FAZ than capillaries of
the intermediate plexus. The number of capillary intersections measured
on histology and OCTA, respectively, of the DVC was 66 vs. 76 (for
C. Balaratnasingam et al.
Fig. 54. Impact of optical coherence tomography angiography (OCTA) image
averaging. A single OCTA image (A) and average of 9 OCTA images (B)
comprising all vascular plexuses is provided. A histologic flat mount (C) of the
same region of interest is also shown for comparison. Insets I, II and III provide
a magnified view of the same region of interest. Many capillaries appear frag­
mented and discontinuous on the single OCTA image. However, following
image averaging they are predominantly continuous with smooth borders.
OCTA image averaging also results in visualisation of some capillary segments
(red arrow) that are not readily seen in a single OCTA image. Note that in areas
distal to the FAZ (green arrow) it can be difficult to clearly discern the
boundaries of capillary segments despite image averaging.
circles of radii 700 μm) and 105 vs. 115 (for circles of radii of 1050 μm).
Comparisons between histology and an averaged OCTA image of the
DCP following manual segmentation are provided in Figs. 57 and 58.
Following manual segmentation, there were many similarities in the
topologic characteristics of the deep plexus between OCTA and histol­
ogy. Many of the closed loops and capillary segments attributed to the
deep plexus evident on histologic flat-mounts were visible on OCTA.
Despite manual anatomical-based OCTA slab segmentation, capillary
segments that were verified histologically to arise from the superficial
and intermediate capillary plexuses were seen in the OCTA image of the
DCP. This was more apparent closer to the FAZ. The number of capillary
intersections measured on histology and OCTA, respectively, of the deep
plexus following manual segmentation was 49 vs. 69 (for circles of radii
700 μm) and 74 vs. 93 (for circles of radii of 1050 μm).
6.3.1.4. Comparisons between histology and OCTA of the foveal avascular
zone. Images of the FAZ as seen on histology and OCTA are provided in
Fig. 52. There was close resemblance in the size and morphologic
43
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 55. Comparisons of the superficial vascular complex (SVC). The SVC as
seen on an averaged optical coherence tomography angiography (OCTA; A)
image closely resembles the histology (B) image. In both images, there is clear
representation of the larger order arterioles and venules as well as the peri-
arterial capillary-free zone. Insets I and II provide a magnified view of the
same region of interest. Although most of the capillaries of the SVC are seen on
OCTA, some capillary segments that are visible on histology (green arrow), are
not clearly seen on OCTA. Note that the vessel not seen on OCTA has a lower
diameter than the capillaries that are readily visualised on OCTA. Selective
arterioles (red arrow) and venules (blue arrow) are labelled on the histol­
ogy panel.
characteristics of the FAZ at the level of the SVC between histology and
OCTA. The intensity of capillaries comprising the terminal foveal
capillary ring was observably less than the remaining vasculature in the
SVC on OCTA. Despite image averaging, a capillary segment of the
terminal capillary ring that was visible histologically was not clearly
seen on OCTA (Fig. 52). The area of the superficial FAZ was measured as
0.257 mm2 on histology and 0.272 mm2 on OCTA.
A comparison of the topologic characteristics of the FAZ at the level
of the DCP as seen on histology and following manual anatomical-based
OCTA slab segmentation are provided in Fig. 52C and D. Some capillary
segments arising from the superficial FAZ were seen in the deep FAZ on
OCTA. The area of the deep FAZ on OCTA were observably smaller than
on histology. The area of the deep FAZ was measured as 0.539 mm2 on
histology and 0.363 mm2 on OCTA. The ratio between the area of the
deep FAZ and superficial FAZ for histology and OCTA was 2.1 and 1.3,
respectively.
Volume rendering and evaluation of the deep FAZ in three-
dimensions also demonstrated significant mismatch between histology
and OCTA in the morphology of the deep FAZ. The size of the deep FAZ
appeared larger on histology than OCTA at all angles of projection
(Supplementary Videos 1 and 2).
Comparisons of the appearance of the FAZ at the level of the SVC,
DVC, DCP (following manual anatomical-based OCTA slab segmenta­
tion) and after projection of all capillary plexuses are provided in
Fig. 59. The area of the FAZ measured from the projection depicting all
capillary plexuses was 0.257 mm2 on histology and 0.265 mm2 on
OCTA.
6.3.1.5. Summary of knowledge gained from direct human OCTA-histology
correlation. To our knowledge, there are no other studies of direct
C. Balaratnasingam et al.
Fig. 56. Comparisons of the deep vascular complex as seen on optical coherence tomography angiography (A) and histology (B). The deep vascular complex
projection in both images is comprised of the intermediate capillary plexus and deep capillary plexus. Note that the foveal avascular zone is significantly larger in the
histology image.
histology-OCTA correlation using human donor tissue. We have pro­
vided an exhaustive summary of the findings above. The time from pre-
mortem clinical imaging to post-mortem histologic assessment in our
donor was short and this was a key reason why we were able to perform
precise validation of the structural information visualised using OCTA.
The main findings from the histology-OCTA correlation are as follows:
1) OCTA provides clearer and more precise visualisation of retinal
capillary structures than FA or ICGA.
2) The morphologic characteristics of the SVC as seen on OCTA are
comparable to histology.
3) Capillary density of the DVC measured using OCTA is greater than
with histology due to the inadvertent inclusion of capillary structures
from the SVC.
4) There is significant mismatch between OCTA and histology in the
topologic and quantitative properties of the FAZ at the level of the
DCP.
6.3.2. Evidence from other OCTA-Histology correlation studies
Other ways by which we have sought to validate the technique of
OCTA is by performing OCTA-histology correlations using isolated
ocular perfusion. We have performed these studies using intact human
donor eyes that have not had their corneas removed for transplantation
as well as porcine eyes obtained from the local abattoir (An et al., 2018;
Yu et al., 2021). The experimental set up is illustrated in Fig. 60. In brief,
we performed ex vivo imaging using a graphics processing unit accel­
erated OCT clinical prototype. The system uses a 1060 nm swept source
(Axsun Inc, Massachusetts, USA) with 200 kHz A-scan rate, and a
coherence length of ~6.5 μm in tissue. The cannulated donor eye was
perfused with 25 mL of human packed red blood cells (obtained from the
human blood bank at Redcross Blood Service) with 58% haematocrit.
The final haematocrit after dilution with heparin solution was 48%
which is similar to the average haematocrit in human males (Thirup,
2003). Ex vivo OCTA images were acquired during perfusion.
Although ex vivo perfusion studies can overcome some of the diffi­
culties associated with attaining donor eyes with high-quality pre-
mortem retinal imaging there are some important limitations associated
with this technique. Due to post-mortem tissue oedema, the quality of
the OCTA images can be compromised in some cases due to corneal
swelling, pupil miosis, formation of early cataract and retinal oedema.
Post-mortem neurosensory retinal detachment can also preclude accu­
rate segmentation of retinal layers. Other limitations with an ex vivo
approach is the formation of retinal vascular thrombi that can result in
incomplete perfusion of some capillary beds and artefactual flow voids.
This issue is often detected when adequate outflow of perfusate through
44
Progress in Retinal and Eye Research xxx (xxxx) xxx
the central retinal vein is not observed or when perfusion pressure in­
creases on the chart recorder. Histologic assessment of tissue with
confocal microscopy after perfusion-based imaging will also readily
identify sites of artefactual vascular occlusion.
Despite these limitations, an ex vivo approach still allows a valuable
correlation between histology and OCTA. A number of consistent results
were identified in ex vivo studies of both human and pig eyes that
implicate some potential limitations in using OCTA for quantitative
studies. In human eyes, we found that vascular density as measured
using OCTA was consistently lower than histology (Fig. 61). The diam­
eter of blood vessels as seen on OCTA was also significantly greater than
histology. A larger ex vivo study comparing histology to OCTA using
porcine eyes revealed the following findings (Figs. 62 and 63) (Yu et al.,
2021):
• OCTA provides accurate histology-like representation of the large
caliber retinal vessels.
• Retinal vessels smaller than 10 μm in diameter and branch points in
all four retinal vascular plexuses are under-estimated on OCTA when
compared to histology. The under-estimation is most profound in the
ICP.
• Vessels associated with an increased angle of descent are poorly
visualised on OCTA.
• With increased retinal depth there is a reduction in the visibility of
vessels on OCTA.
Experimental reasons that may influence the differences in capillary
diameter between OCTA and histology include the specific settings of
the OCTA set up. In our in vitro experiments, the tissue pressures in an
isolated eye are expected to be different to that of a live human eye with
physiologic intraocular pressure. This may be another reason influ­
encing the in vitro OCTA measurements of retinal capillary diameter.
In a separate study we used the peri-arterial capillary free zone as an
exemplar to investigate the capacity of several commercially available
OCTA devices to image the DCP in the human eye (Balaratnasingam
et al., 2018). The capillary-free zone along retinal arteries is an area of
physiologic avascularisation that forms during embryogenesis. Histo­
logic studies have clearly shown that the capillary-free zone is confined
to the plane of the SVP and does not involve the deeper capillary beds in
the human perifovea (Fig. 7) (Balaratnasingam et al., 2018; Michaelson
and Campbell, 1940). Commercial OCTA devices used for patient im­
aging including the RTVue XR Avanti (ver. 2016.1.0.26; Optovue, Inc,
Fremont, California, USA), PLEX Elite 9000 (ver. 1.5.0.15909; Zeiss
Meditec, Inc, Dublin, California, USA), Heidelberg Spectralis OCT2
(Family acquisition module 6.7.21.0; Heidelberg Engineering,
C. Balaratnasingam et al.
Fig. 57. Comparisons between the superficial (A), intermediate (B) and deep
(C) capillary plexuses as seen on histology and the deep vascular complex
(DVC) as seen on optical coherence tomography angiography (OCTA; D). An
averaged OCTA image of the DVC (D) was generated using manufacturer-
recommended settings. The DVC is comprised of the intermediate and deep
capillary plexuses. Insets of the same region of interest demonstrate that cap­
illaries of the superficial plexus (red arrows), the origin of which have been
verified histologically, are projected in the OCTA image of the DVC. Capillary
segments of the superficial capillary plexus are typically projected at the
boundary of the foveal avascular zone. Capillary segments of the intermediate
capillary plexus (green arrows) are seen in the DVC. Note that due to the high
density of capillary structures in the DVC it can be difficult to discern individual
capillary segments of the deep capillary plexus.
Heidelberg, Germany), and DRI-OCT Triton (Ver. 1.1.1; Topcon Corp,
Tokyo, Japan). Software and hardware specifications for these devices
are summarised in Table 5. All OCTA devices acquired a 3 mm × 3 mm
volume scan.
We examined the appearance of the peri-arterial capillary free zone
in vascular slabs depicting all capillary beds within the retina. We
observed that in the commercial devices that were used for this study a
prominent area of avascularisation was seen at the level of the DCP,
despite histologic comparisons revealing the presence of a formed
vascular network at this level. This study reaffirmed the results of our
previous in vivo and ex vivo donor human studies and demonstrated that
vascular structures at the level of the deep capillary circulation may be
45
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 58. Comparisons between the superficial (A), intermediate (B) and deep
(C) capillary plexuses as seen on histology and the deep capillary plexus as seen
on optical coherence tomography angiography (OCTA; D). Manual anatomical-
based slab segmentation was used to generate an averaged OCTA image of the
deep capillary plexus. Insets of the same region of interest demonstrate that
some of the capillaries of the superficial plexus (red arrows) and intermediate
plexus (green arrows), the origin of which have been verified histologically, are
visualised in the OCTA image of the deep plexus despite manual segmentation.
The morphologic characteristics of capillary loops in the deep capillary plexus
(yellow arrows) are relatively clearly appreciated on OCTA using this seg­
mentation protocol.
incompletely visualised using OCTA. This limitation is important to bear
in mind when OCTA is used to perform quantitative measurements of
vascular structures or form hypotheses regarding the role of the DCP in
retinal vascular diseases.
It is important to highlight that our histology-OCTA studies were
performed over the past four years. Since that time there have been some
improvements in OCTA software and hardware that may address some
of the limitations described above. Furthermore, we also highlight that
there are several commercially available OCTA devices that were not
used in our histologic comparisons which may offer greater visualisation
of the DCP than what we have found.
C. Balaratnasingam et al.
Fig. 59. Foveal avascular zone (FAZ) morphology following different optical
coherence tomography angiography (OCTA) segmentation protocols. Images of
the FAZ at the level of the superficial vascular complex (A) and deep vascular
complex (B) using manufacturer (RTVue XR Avanti, Optovue Inc.) recom­
mended settings are shown. Manual anatomical OCTA-based slab segmentation
was also used to generate images of the FAZ at the level of the deep capillary
plexus (C) and following projection of all capillary plexuses (D). Note that area
of the deep FAZ increases following manual segmentation but still bears close
resemblance to the superficial FAZ.
6.3.3. Artefact in OCTA
In the clinical setting, acquisition of a high-quality OCTA image re­
quires a degree of patient co-operation below which an unacceptable
rate of artefact may be present within the OCTA image. Any form of
motion that is introduced during image acquisition will result in arte­
fact. These can occur in the form of doubling of retinal vessels, blink
artefact, criss-cross artefact and stretch artefact. Some of these artefacts
Fig. 60. Experimental setup for post-mortem perfusion imaging using optical coherence tomography angiography (OCTA). Donor eye cannulated using a glass
micropipette (blue ar) via the central retinal artery is placed in a custom built eye holder (red ar). The eye holder is secured in front of the OCTA sample arm. Red
blood cells with 48% haematocrit (not shown) are then perfused through the eye using a syringe pump during OCTA imaging.
46
Progress in Retinal and Eye Research xxx (xxxx) xxx
such as stretch artefact are due to software correction that attempt to
correct for motion.
Media opacity artefacts which can occur in the context of dry eye
syndrome, severe astigmatism, cataract, vitreous syneresis and vitreous
haemorrhage and can further degrade the quality of the OCTA image. In
some instances, the quality of a severely degraded image quality pro­
hibits any form of qualitative or quantitative assessment of retinal
vascular detail.
Although OCTA facilitates depth-resolved study of the retinal cir­
culation, the ability to precisely stratify the superficial, intermediate and
DCP remains an important limitation of this technique that needs to be
recognised. In the above section we have provided original clinical-
histologic correlation data to support this claim. This is particularly
problematic when OCTA data is used to perform quantitative measure­
ments of retinal vascular detail as a means of detecting disease or
monitoring progression. Increased or reduced retinal vascular density
measurements due to segmentation error can erroneously be interpreted
as a disease state such as macular ischaemia. Segmentation artefact is
particularly profound in conditions that disrupt the normal anatomic
organisation of retinal layers such as macular oedema. Despite the use of
manufacturer recommended settings to stratify vascular layers or
manual techniques to outline retinal layers there still persists a degree of
inaccuracy in the segmentation boundaries in eyes with macular
oedema. In such instances, the most accurate measure of vascular den­
sity would be to consider the vascular slab that incorporates all the
vascular layers within the retina.
There are many more artefacts that can be associated with OCTA and
these have been reported in depth in previous review articles (Anvari
et al., 2021; Falavarjani et al., 2017; Spaide et al., 2015). Appreciation of
these artefacts is important when interpreting vascular data as seen
using OCTA.
6.3.4. Improving quantitative measurements using OCTA
Over the past 5 years there have been rapid advancements in OCTA
software and hardware that have in resulted in higher-resolution retinal
imaging of capillary structures. We emphasise that the data presented in
this paper is largely derived from a single OCTA device that is
commercially available. Although we have provided some generalisa­
tions regarding the limitations of OCTA technology in this report some
of the issues relevant to image segmentation and visualisation of the
DCP is encountered in multiple devices. As an example, in our 2018
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 61. Direct comparison of optical coherence to­

mography angiography (OCTA) performed using an
isolated, red blood cell perfused human donor eye (A)
versus histology of the same region of interest pre­
pared using perfusion of lectin (B). A large retinal
vein of the superotemporal arcade is seen (blue ar) in
both images. A smaller retinal artery (red ar) is seen
crossing the vein. The artery can be differentiated
from the vein via the presence of periarterial capillary
free zone on the histology image (red ar). Smaller
arterioles (a) and venules (v) are evident on both
OCTA and histology. A significant number of capil­
laries evident on histology are not seen on OCTA
(yellow ars). Scale bar = 500 μm.

Fig. 62. Direct comparison of perfused porcine

retinal histology and optical coherence tomography
angiography of the same region. (A) and (B) show the
respective histology and OCTA images from the same
study region in porcine eye 1. Both images were
colour coded according to depth (the colour indicates
the z-position at which the maximum intensity value
occurs) as indicated by the colour scale bar showing
yellow-green vessels in the superficial half of the
stack and brown-orange vessels in the deep half of the
stack. Red ars point to corresponding locations in the
two stacks, showing vessels that were visible in the
confocal image stack either branching from the su­
perficial half of the stack or located in the deep half of
the stack but not visible on the OCTA images stack.
(C) and (D) show the respective histology and OCTA
images from a study region of porcine eye 2. Both
images were depth colour coded using a green-blue-
magenta-red scale as shown. Yellow ars point to
vessels that were visible in the confocal image stack
but not visible on the corresponding OCTA stack. The
indicated vessels were either branching from the su­
perficial layers of the stack or in the deeper layers of
the stack. Reproduced with permission from Yu et al.
(Yu et al., 2021).

Fig. 63. Vessel tracking through porcine retinal

vasculature. Perfusion labelled histology (A) and op­
tical coherence tomography angiography (OCTA; B)
images are presented. Each image shown is a snap­
shot of a scrollable z-stack for which points can be
placed at any x, y and z position. An arteriole –
capillary – venule pathway is traced via green dots,
which begins at 1 (arteriolar aspect), and ends at 12
(venular aspect) on the histology image. This vessel
has a diameter of 7.73 μm at the point of branching
from the main arteriole (dot point 2 on the histology
image) and can be tracked through a total depth of
13.75 μm for a total track distance of 572 μm within
the superficial vascular plexus. Total length on the
OCTA stack can only be tracked 338 μm through depth of 16.5 μm and was not visible from mid-track onwards (dot point 4 on OCTA image). Reproduced with
permission from Yu et al. (Yu et al., 2021).

study, we used the peri-arterial capillary free zone as an exemplar to truth.

investigate the accuracy of OCTA for visualising the DCP in the human Swept source technology and improvements in A-scan rates recently
eye (Balaratnasingam et al., 2018). We found that all 4 of the com­ have allowed quicker imaging and better resolution of deeper vascular
mercial devices used in that study were unable to provide complete structures. Additionally, image averaging using multiple OCTA scans
visualisation of the DCP that was comparable to the histologic ground that are acquired within a relatively short period of time can also

47
C. Balaratnasingam et al.
improve signal:noise ratio as shown in Fig. 54 and reduce artefact. This
is another measure by which the accuracy of quantitative measurements
can be improved. Other measures that may improve the accuracy of
quantitative measurements include the use of Artificial Intelligence
techniques such as machine learning that can be trained to identify and
remove artefact (Hormel et al., 2021). Also using volumetric OCTA data
to segment and classify the retinal circulation in three-dimensions may
also allow a more accurate and complete vascular analysis than the
current use of two-dimensional projections (Borrelli et al., 2021). Ana­
lysing OCTA data in three-dimensions may also remove errors due to
segmentation that are encountered when distinct capillary plexuses are
analysed in two dimensions. Switching between fast and slow scanner
axis on the OCTA device may also improve capillary visibility and in­
crease the accuracy of quantitative OCTA measurements.
7. Future directions
So far, we have presented several lines of evidence to demonstrate
that changes to the retinal microcirculation are inherently linked to the
pathophysiology of DR. Therefore, preserving capillary and endothelial
function is a key strategy in preventing vision loss in DR. In this section
48
Progress in Retinal and Eye Research xxx (xxxx) xxx
we will highlight areas in the field of DR that require greater research
focus to improve our understanding of this complex disease and also
enable earlier detection of disease-induced structural and functional
changes that are responsible for vision loss.
7.1. Importance of clinical-histology correlation studies
Multimodal retinal imaging is a vital component of DR management.
The key imaging tools routinely employed in clinical practice include
structural OCT, colour photography, FA and OCTA. An example of an
eye with DMO that has undergone multimodal imaging is illustrated in
Fig. 64. The key features of DMO that is currently evaluated by each of
the technologies are highlighted. There have been major improvements
in imaging technology such that it is now possible to study aspects of the
retina in almost cellular-like detail. For instance, the spatial resolving
power of many commercially available structural OCT devices have
improved beyond 6 μm and permit precise visualisation of distinct
macular bands. This property of OCT technology has been leveraged to
improve our understanding of age-related macular degeneration (AMD).
Much of this recent work in the field of AMD has been completed by
Christine Curcio’s laboratory in the University of Birmingham in
Fig. 64. Current multimodal clinical imaging of dia­
betic macular oedema. Contemporaneous imaging
with colour photography (A), fluorescein angiogram
(B), structural optical coherence tomography (OCT;
C), macular thickness map generated using OCT (D)
and OCT angiography (E) from a single eye is pre­
sented. The key features that are evaluated from each
modality and used to assess visual morbidity and
predict response to treatment are provided. The his­
tologic correlates for many of these imaging features
remain unresolved.
C. Balaratnasingam et al.
Alabama, USA. Their collective work has improved our understanding of
the RPE, drusen and neovascularisation in AMD progression and the
development of atrophy (Gambril et al., 2019; Li et al., 2018; Tan et al.,
2018; Tong et al., 2016). Most importantly, that research has identified
ways by which clinicians are able to recognise the precursors to the
development of macular atrophy. Their work has also validated OCT
correlates for different types of macular neovascularisation and has
greatly enhanced the clinician’s ability to interpret imaging technology
with great precision (Dolz-Marco et al., 2018; Litts et al., 2016; Tong
et al., 2016). Much of the clinical-histology correlation work by Curcio’s
team has employed ex vivo OCT imaging of human donor eyes followed
by post-mortem tissue processing. Their work has facilitated precise
point-to-point correlation between OCT features and high-resolution
histology. Similar clinical-histology correlation work utilising OCT
would significantly benefit the field of DR but is currently lacking.
One of the difficulties in performing such research is that it requires
intricate methodologic technique which is achievable by only a select
number of research groups across the globe. These groups need to be
equipped with the knowledge and niche instrumentation to perform
these experiments. The other important limitation in completing such
work is access to human donor tissue. The Curcio team for instance, have
a strong collaborative link to the Alabama Eye Bank that has been a
ready source of human donor tissue over the years. To complete this
work in a precise manner, in addition to accessing tissue, the donor
tissue needs to be retrieved shortly after death, thereby minimizing the
effects of post-mortem tissue digestion. With regard to the donor eye
that we have presented in Section 6, it was retrieved less than 1 h after
death and this was the key feature that facilitated very precise
Fig. 65. Spectrum of macular ischaemia in diabetic retinopathy (DR). Averaged projection images of 9 eyes with DR using optical coherence tomography angi­
ography (OCTA) are presented. The spectrum of capillary density change and alterations in the size and morphology of the foveal avascular zone are shown. Macular
ischaemia is one of the most devastating complications of DR that is currently irreversible.
49
Progress in Retinal and Eye Research xxx (xxxx) xxx
correlation between histology and OCTA. Finally, high quality pre-
mortem retinal imaging that is acquired relatively close to the time of
death is another feature that is of paramount importance when refining
the histologic correlates for retinal imaging. It is therefore clear that
there are a multitude of challenges associated with performing such
work.
Despite the challenges associated with such an experimental design,
the information attained would be invaluable to the field of DR, as it has
been to the field of AMD. Some of the key questions that it may help
answer include:
1. What is the histologic correlate for intra-retinal hyperreflective foci
seen on OCT? In AMD, the histologic correlate for intraretinal
hyperreflective foci has been attributed the RPE cell but in DR there
is conjecture as to whether it is the RPE cell or microglia (Christen­
bury et al., 2013; Folgar et al., 2012; Framme et al., 2012; Vujosevic
et al., 2013, 2016). There is evidence that intraretinal hyper­
reflective foci can help prognosticate visual outcomes in DMO and
also predict response to different intravitreal therapies (Uji et al.,
2012; Vujosevic et al., 2016; Yoshitake et al., 2020). Further
knowledge regarding this OCT feature therefore has great clinical
relevance.
2. In which tissue compartment is there accumulation of fluid in DMO?
Also, what is the role of vascular and glia changes in the development
of DMO? DMO is one of the most important sight-threatening com­
plications of DR yet it still remains unclear if the accumulation of
fluid is intracellular (such as that in retinal glia in cystoid macula
oedema) or if the accumulation is in the extracellular space (Yanoff
C. Balaratnasingam et al.
et al., 1984). Given that up to 40% of eyes with DMO will exhibit a
suboptimal response to anti-VEGF therapy, an improved under­
standing of the histology of DMO will aid the development of better
therapeutic strategies to address this complication (Gonzalez et al.,
2016). It will also help stratify patients that should receive intra­
vitreal anti-VEGF or intravitreal corticosteroids as first line therapy
for DMO.
3. There is immense variation in the phenotype of macular ischaemia in
DR as shown in Fig. 65. It is unknown why some eyes develop
macular ischaemia following a relatively short duration of DM while
others do not develop any observable macular microcirculatory
changes despite over 20 years of DM. Some eyes will also demon­
strate changes in retinal capillary calibre such as greater attenuation
and tortuosity (Sasongko et al., 2011). The reason for these changes
are unclear and whether or not it is linked to regional pericyte,
smooth muscle, glia and haemodynamic alterations needs to be
reconciled. Such information will improve our understanding of
macular ischaemia and help develop treatment strategies to prevent
its occurrence. Macular ischaemia remains the most important cause
of central vision loss in DR. We still do not have a treatment for
macular ischaemia (Cheung et al., 2022).
De Venecia et al. and Bresnick et al. provided some of the few
clinical-histology studies in DR (Bresnick et al., 1977; de Venecia et al.,
1976). Both reports used the same donor eye and compared the histo­
logic characteristics of microaneurysms to that of the clinical features as
seen on colour photography and FA. The findings of this work had major
relevance to clinical practice as it clearly showed that microaneurysms
are often not clearly visualised using colour photography. The impli­
cation of that finding is that colour photography may not be the most
appropriate screening tool for DR. The data was also used to propose the
life cycle of microaneurysms. They proposed that the initial stages of
microaneurysm formation is due to endothelial proliferation which
initially results in a thin-walled microaneurysm. They proposed that the
late stages of the microaneurysm lifecycle is characterised by hyalini­
sation of the vessel wall, an increase in wall thickness and total oblit­
eration in the late stages.
7.2. Reconciling renal-retinal pathogenic links in diabetes mellitus
Diabetic nephropathy is a major cause of morbidity worldwide.
Nearly 45% of patients with Type 1 DM will develop clinically evident
diabetic nephropathy during their lifetime (Gross et al., 2005). There is
significant overlap in the risk factors that predict the development of
diabetic nephropathy and DR. The risk factors that are common to both
disease manifestations include systemic hypertension, poor glycaemic
control, duration of disease and pregnancy (Davis, 1992; Gross et al.,
2005). There are also two key histologic features seen in diabetic ne­
phropathy that overlap with DR:
1. Glomerular basement membrane (GBM) thickening – This is one of
the earliest changes to characterise diabetic nephropathy and usually
occurs within 1–2 years of disease onset (Tsilibary, 2003). GBM
thickening precedes the onset of microalbuminuria. Akin to the
retinal pericyte, the renal podocyte surrounds renal capillaries and is
inherently linked to GBM thickening (White et al., 2002). Injured
podocytes are proposed to have a key role in disturbing the balance
between the synthetic and degradative pathways of the GBM.
2. Hyalinisation of renal afferent and efferent arterioles and endothelial
injury – Hyalinosis is due to insudation and accumulation of plasma
proteins between the glomerular endothelium, the GBM, the
glomerular tuft and Bowmans capsule (Smith, 1955).
Given the overlap in clinical risk factors and histologic features be­
tween DR and diabetic nephropathy it would appear intuitive that the
natural course of one may modulate the other. Klein and colleagues
50
Progress in Retinal and Eye Research xxx (xxxx) xxx
studied the relationship between the histologic measures of diabetic
nephropathy and the severity of DR that was graded using colour
photography (Klein, 2006). After controlling for the duration of diabetes
and glycated haemoglobin (HbA1c) levels they found that severity of DR
was significantly correlated with GBM width and glomerulopathy index
graded histologically. Decreased glomerular filtration rate is associated
with severe visual impairment in the advanced stages of diabetic ne­
phropathy (Fig. 66). Park and colleagues evaluated the relationship
between clinical DR as seen on colour photography with serial mea­
surements of renal function and albuminuria (Park et al., 2019). Their
study revealed two important findings: 1) A greater proportion of pa­
tients with chronic kidney disease demonstrated progression of DR; 2)
After adjusting for DM duration, HbA1c, baseline glomerular filtration
rate and use of angiotensin converting enzyme (ACE) inhibitors the risk
of chronic kidney disease progression was independently associated
with DR severity. Specifically, eyes with NPDR had 2.9 times and PDR
had 16.6 times greater risk of chronic kidney progression compared to
eyes with no DR. An illustrative case is provided in Fig. 66. Additionally,
in such cases, there is quite often progression of retinopathy despite laser
or surgical intervention that would ordinarily serve to reduce the risk of
retinopathy progression (Fig. 67).
Given that the pathogenesis of renal disease is linked to retinal dis­
ease in a complex manner in DM it could be possible to predict the risk of
developing the complications of renal disease using retinal biomarkers
or vice versa. Although we have presented several lines of evidence to
demonstrate a link when irreversible end-organ damage has already
occurred such as in PDR or chronic kidney disease there are few renal or
retinal biomarkers that predict early organ dysfunction. The optical
properties of the eyes enable visualisation of retinal capillaries and by
extension, detection of endothelial dysfunction. Linking indices of
endothelial dysfunction and retinal perfusion derived from state-of-the-
art high-resolution retinal imaging to novel biomarkers of diabetic ne­
phropathy may further expand our understanding of retinal-renal
pathogenic links in DM. More specifically, if retinal endothelial
dysfunction as detected using clinical imaging can be used to predict the
risk and degree of renal dysfunction it may provide a non-invasive
means for detecting diabetic nephropathy. This is an important
concept that is worthy of further exploration as it could obviate the need
for invasive, expensive and time-consuming urine and blood tests to
detect diabetic nephropathy. Ultimately, such techniques can help
reduce morbidity, reduce healthcare costs and improve renal and visual
outcomes.
7.3. Reconciling relationships between diabetic retinopathy and diabetic
peripheral neuropathy
Diabetic peripheral neuropathy (DPN) is one of the most prevalent
complications of DM (Dyck et al., 1993). Significant risk factors for the
development of DPN include duration of DM, increasing age, HbA1c
level and DR (Liu et al., 2019). Over 23 years of mean follow-up, 33% of
patients in the Diabetes Control and Complications Trial (DCCT)
developed DPN (Braffett et al., 2020). The pathophysiology of DPN is
complex and multifactorial (Feldman et al., 2017). The importance of
microvascular disturbances in the development of DPN is exemplified by
histologic studies that have revealed evidence of endothelial cell hy­
perplasia and hypertrophy, basement membrane thickening, luminal
narrowing and pericyte loss within the endoneurial capillaries in human
specimens of DPN (Malik et al., 1989, 1992; Thrainsdottir et al., 2003;
Yasuda and Dyck, 1987).
Pathogenic links between DPN and DR have been reported. Deghani
et al. studied the relationship between DPN and NFL thinning in patients
with Type 1 DM. They found that significantly greater thinning of the
NFL occurred in the superior part of the optic nerve in patients with DPN
when compared to patients without DPN (Dehghani et al., 2017). Sri­
nivasan and colleagues showed that retina thickness as measured using
OCT was significantly lower in the parafovea and perifovea in eyes with
C. Balaratnasingam et al.
DPN (Srinivasan et al., 2017). In the cross-sectional study, Rasheed et al.
analysed 500 patients with Type 2 DM and found that 86% of patients
with DR also manifested DPN (Rasheed et al., 2021). Barr et al. evalu­
ated over 1000 subjects with impaired glucose tolerance and impaired
fasting glucose and found that neuropathy score was independently
associated with albuminuria and retinopathy after adjusting for the ef­
fects of age, sex, hypertension, lipid lowering medication and total
cholesterol (Barr et al., 2006).
51
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 66. Association between progression of diabetic
nephropathy and diabetic retinopathy (DR). Colour
photography and optical coherence tomography
(OCT) images of the central macula for each visit is
presented. A 42-year-old female with Type 1 diabetes
mellitus presented in January 2020 for baseline
assessment at which point her retinopathy was
graded as severe non-proliferative disease with
concomitant diabetic macular oedema. During the
course of 19 months she developed rapid progression
of chronic renal failure and reduction in glomerular
filtration rate necessitating renal dialysis by July
2021. Note that during that same period of time she
manifests rapid progression of DR with development
of a tractional macular detachment at the July 2021
visit. Sites of retinal neovascularisation and fibrosis
are highlighted by red arrows. Areas of vascular oc­
clusion and sheathing are indicated by blue arrows
and the site of vitreous haemorrhage by green arrows.
The OCT scans provided are image-registered to the
baseline visit.
Fig. 67. Progression of diabetic retinopathy (DR)
despite treatment in a patient with advanced ne­
phropathy. A 32-year-old female developed rapid
progression of proliferative DR between January and
December 2021. This was associated with a rapid
decline in glomerular filtration rate during the same
period of time. The boundaries of areas of neo­
vascularisation (red arrows) and vitreous haemor­
rhage (green arrows) are highlighted. She underwent
vitrectomy and delamination surgery to remove the
areas of retinal neovascularisation with a good
anatomic result. Note that there is progression of
vascular occlusion (blue arrows) despite successful
surgery.
The link between DR and DPN may not be intuitive, as NFL and RGC
thinning in the retina represent changes in the central nervous system
whilst DPN is a disease of peripheral nerves. From an anatomic stand­
point, the NFL is also an unmyelinated structure while DPN typically
involves nerves that are myelinated by Schwann cells, such as the sural
nerve. The association between microvascular disturbances in DR and
DPN is difficult to study as the severity of DPN is also confounded by
other disease pathways such as the polyol pathway activation and
C. Balaratnasingam et al.
accumulation of advanced glycation end products in the nerve (Pang
et al., 2020). The microcirculation of the skin can be directly visualised
by videocapillaroscopy and similarly the microcirculation of the retina
can be visualised using OCTA and other vascular imaging modalities.
This provides a unique opportunity to study associations in microcir­
culatory changes between the two systems. However, such detailed
studies are yet to be performed. Uyar et al. investigated the association
between nailfold videocapillaroscopy and DR as determined using
colour photography and OCT (Uyar et al., 2016). Abnormalities seen on
nailfold capillaroscopy were found to be significantly higher in patients
with PDR than those with NPDR or no DR. However, this study did not
specifically study structural or dynamic changes to retinal capillaries. Li
and colleagues showed that dynamic measures of endothelial dysfunc­
tion such as flow mediated dilation (FMD) may be correlated with the
severity of DPN (Li et al., 2022). They found that only 2% of patients
with an FMD measurement of >7% demonstrated severe nerve injury
while 70% of patients with a FMD measurement of <4% demonstrated
severe nerve injury. As outlined in a previous section of this report, FMD
studies can be subjective and invasive. By studying associations between
retinal capillary imaging biomarkers of endothelial dysfunction and
severity of DPN it may be possible to identify novel retinal biomarkers
that predict the onset and natural course of some subtypes of DPN.
7.4. Defining the role of gliopathy in diabetic retinopathy
Glia are key cells that support neurons in the CNS. The density of glia
outnumber neurons by several orders of magnitude, in the human cortex
there are approximately 60 billion glia to 16 billion neurons (Azevedo
et al., 2009). The overabundance of glia in comparison to neuronal tis­
sue is also a property of the retina and optic nerve (Balaratnasingam
et al., 2014).
The human retina contains three types of glial cells: astrocytes,
Muller cells and microglia (Reichenbach and Bringmann, 2020; Vecino
et al., 2016). Astrocytes and Muller cells are classified as macroglia.
Astrocytes are predominantly localised to the NFL and GCL. Muller cells
are radial glial cells that span the entire thickness of the retina between
the ILM and OLM. There are nearly 5 million Muller cells in the human
retina and the cell bodies of Muller cells are located in the INL. As the
name suggests, microglia are much smaller cells than macroglia that are
analogous to blood-borne phagocytes (Vecino et al., 2016). They are
found in greatest concentration in the GCL, IPL, OPL and around blood
vessels. Collectively, retinal glia maintain neuronal viability and support
visual function. In broad terms, retinal macroglia maintain the health
and metabolic activity of retinal neurons while the microglia are pri­
marily innate immune cells that maintain retinal homeostasis. The
ophthalmic literature comprises a number of detailed reports that pro­
vide very specific information regarding the structure and function of
retinal glia (Coorey et al., 2012; Reichenbach and Bringmann, 2020;
Sorrentino et al., 2016; Vecino et al., 2016). That information will not be
repeated in this section and is beyond the scope of this article.
Given the key function of retinal glia in maintaining retinal ho­
meostasis it is expected that they also play an integral role in the
pathophysiology of DR. Yet, there is a relative dearth of scientific in­
formation on this matter. A systematic evaluation of functional and
structural changes to retinal glia at different stages of DR will be a
fundamental next step in progressing our understanding of this complex
disease. We will now provide a summary of what has been reported in
this field.
In advanced retinal disease such as PDR, strong staining of GFAP and
Vimentin within resected epiretinal membranes provide evidence that
glia-mediate contraction is one factor that underlies the development of
tractional retinal detachment (Guidry et al., 2009; Lee et al., 2020; Nork
et al., 1987). Ly et al. utilised STZ rodent models of DM and found that
Muller cell gliosis was also a late feature of DR and was coincident with
functional deficits of photoreceptor cells and amacrine cells as measured
using electroretinography (Ly et al., 2011). Zeng et al. performed a
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Progress in Retinal and Eye Research xxx (xxxx) xxx
histologic analysis of 21 eyes from 13 patients and observed that areas of
retinal neovascularisation due to DM were heavily infiltrated by
microglia (Zeng et al., 2008). This finding led them to propose that
microglia play a significant role in the angiogenesis of proliferative
vitreoretinopathy.
There is experimental evidence to indicate that glia changes also
occur in the earliest stages of DR. Using rodent models of DR, Ly et al.
showed that Astrocyte connexin 26 and 43 gene and protein expression
preceded loss of astrocytes (Ly et al., 2011). Rungger-Brandle et al. also
used a rodent model of DR and observed a gradual increase in Muller cell
number in the first 4 weeks of hyperglycaemia compared to controls
(Rungger–Brändle et al., 2000). Developments in retinal imaging has
also allowed exploration of the concept that retinal glia are inherently
linked to the pathogenesis of DR. One of the earliest clinical papers to
demonstrate this association is the work by Lopes de Faria et al. that
showed a significant reduction in the thickness of the superior NFL in
patients with Type 1 DM without DR (Lopes de Faria et al., 2002). In
their paper, the NFL measurements were acquired using a confocal
scanning laser ophthalmoscope. The widespread availability of OCT
since then has facilitated the completion of studies that have reinforced
this original finding. Most recently, Orduna-Hospital et al. examined 90
eyes from patients with Type 1 DM without DR and an age-matched
control group using spectral-domain OCT over 8 years (Orduna-Hospi­
tal et al., 2021). They found that total retinal thickness was significantly
greater at baseline visit in the Diabetes group but gradually reduced over
time such that there was no difference between the two groups by the
8-year visit. The reduction in total retinal thickness in the DM group
occurred within the inner retinal layers (defined in the study as the
tissue located between the ILM and OLM). These findings were used to
speculate that neurodegeneration plays a prominent role in early DR.
In the CNS, glia response to neuronal injury is varied and is often
very specific to the stage of the disease course. As an example, using the
optic nerve as a model to study brain injury we have previously shown
that the initial response of astrocytes following pressure elevation is
decreased expression of GFAP (Balaratnasingam et al., 2008). In that
study we also demonstrated bulbous morphologic changes to astrocytes
that was consistent with intracellular swelling (Fig. 68). In contrast,
following prolonged intraocular pressure elevation such as in glaucoma
there is gliosis of astrocytes and increased expression of GFAP with loss
of astrocyte cell bodies (Hernandez et al., 2008). Targeting glia
dysfunction prior to the onset of gliosis is likely to prevent the occur­
rence of permanent structural and functional retina changes. Clinical
measures that identify the precursors of retinal gliosis will be key for
achieving this objective.
Several parallel pathogenic pathways define the pathogenesis of DR.
However, there is some degree of overlap between these pathways.
These pathways include: 1) Vasculogenic pathway; 2) Neurodegenera­
tive pathway (Barber, 2003; Lieth et al., 2000; Sohn et al., 2016); 3)
Immune-mediated and inflammatory pathways (Altmann and Schmidt,
2018; Joussen et al., 2004; Kern, 2007; Pan et al., 2021). A significant
proportion of studies have examined each of these pathways in isolation
and it has been difficult to discern if these pathways are indeed separate
or if changes characterising one pathway is the cause/effect of changes
due to another. Feit-Leichman evaluated the chronology of glia and
vascular changes in C57BI/6J mice with experimental diabetes induced
by STZ injection (Feit-Leichman et al., 2005). They found that retinal
neurons undergo a small but transient apoptosis initially after the
development of DM but this is not maintained during the chronic stage
of diabetes. They also found increased GFAP labelling within astrocytes
of the ganglion cell layer in the first two months after induction of
diabetes but after 6 months the intensity of GFAP stain had returned to
the normal level. When vascular changes were examined in the same
eyes they noted a progressive increase in the number of acellular cap­
illaries, pericyte ghosts and vascular cell apoptosis over time despite
normalisation of GFAP expression and absence of neuronal apoptosis.
Their study provided evidence that vasculogenic pathways in DR may
C. Balaratnasingam et al. Progress in Retinal and Eye Research xxx (xxxx) xxx

Fig. 68. Astrocyte changes following acute optic

nerve injury. Porcine optic nerve astrocytes were
labelled using glial firbrillary acidic protein (GFAP).
Montaged confocal microscopy images of eyes with
normal (10 mmHg; A) intraocular pressure (IOP) and
high-IOP (B) after 12 h of IOP elevation are pre­
sented. Longitudinal cryosection-prepared images of
the optic nerve including the pre-laminar, lamina
cribrosa and post-laminar tissue are presented. The
appearance of stain in normal-IOP eyes was homo­
geneous, demonstrating a lattice like appearance in
astrocyte morphology. This is particularly evident in
magnified images of proximal post laminar (I) and
lamina cribrosa (II) tissue. In high-IOP eyes the neural
tissue demonstrated a spectrum of morphological
changes. Nodular enlargements were seen in the pe­
riphery of many neural bundles (III) while some
neural bundles demonstrated these enlargements in
the peripheral and central parts of the nerve (IV).
Some neural bundles demonstrated a total loss of
GFAP architecture with an amorphous appearance in
astrocyte morphology. This is evident in the nerve
bundles located on the left of the high-IOP eye
montage. Nodular enlargement of GFAP-positive cells
is most likely representative of astrocyte swelling.
Scale bar = 50 μm.

occur independent of neurodegenerative pathways.
Earlier in this manuscript we discussed the pathophysiologic com­
plexities of the microaneurysm lifecycle (Fig. 20). An important aspect
of this lifecycle that is frequently observed but poorly understood is that
of microaneurysm regression i.e. an observable disappearance of the
capillary microaneurysm without a legacy of permanent structural and
functional retinal alteration. Although such a pathway in the micro­
aneurysm lifecycle is the exception rather than the rule if we were to
understand the mechanisms that drive the pathway of regression it could
help reveal new ways to prevent irreversible retinal injury in DR. There
is clear evidence that astrocytes and Muller cells modulate the
mechanical properties of vessels (Metea and Newman, 2006; Vecino
et al., 2016). It is therefore plausible that aneurysmal dilations to retinal
capillaries are consequent to localised glial changes. Similarly, given
that there is continual cross talk between glia, retinal pericytes and
SMCs, it is also possible that depletion of pericytes within micro­
aneurysms is an epiphenomenon that characterises localised glia injury
(Nakahara et al., 2013; Yu et al., 2010b). By direct and indirect effects of
the capillary wall, local glia may play a vital role in modulating the
natural course of microaneurysms and determining whether they
progress or regress.
Most recently we have documented the histologic changes to the

Fig. 69. Glial infiltration into the microaneurysms (MA) lumen in diabetic retinopathy. Glial processes can be seen within a MA (red ar) with complex internal
structures evidenced by multiple septae separating the lumen into chambers. There is no evidence of pericytes and there is also a paucity of endothelial cells (green
ar). Panel B shows one MA (top left) at an advanced stage where the external wall is infiltrated and replaced by glial material. The centre of the MA contains indistinct
cell nuclei and infiltrated GFAP positive material. The MA on the bottom right of Panel B has completely regressed and stains negatively for lectin, indicating that the
MA is no longerperfused. A residual glial scar is seen to surround the MA. Panel C is a three-dimensional view of the remnants of a MA located within the inner
nuclear layer (INL). Note the space which was occupied by the MA is replaced by glial scar and the neurons of the INL are disrupted. The hyperfluorescent material
(magenta ar) adjacent to the space may represent haemorrhage or exudate. Yellow = GFAP. Grey = Lectin. Blue = Nuclei. All scale bar = 30 μm.

53
C. Balaratnasingam et al.
inner retina in human donor eyes with DR with an emphasis on studying
how the relationships between the vasculature and glia are modified.
The findings support the hypothesis that glia changes are intricately
related to microaneurysms. Some original observations from pre­
liminary experiments will now be provided:
1. Astrocyte processes can infiltrate the wall of microaneurysms to
enter the lumen and the astrocyte processes can embed within other
aneurysmal contents such as red blood cells and white cells (Fig. 69).
2. The response of astrocyte processes can remain predominantly
external to the capillary wall to form a gliotic ‘ball’ that encapsulates
the microaneurysm (Fig. 70).
3. An observable localised GFAP alteration is not associated with some
microaneurysms (Fig. 71).
The first step in formulating a relevant clinical classification of glial
change in DR would be to develop a robust pathologic classification of
diabetic gliopathy. In this regard, lessons can be learnt from the field of
neurology (Matias et al., 2019). Alzheimer’s disease is the most common
cause of dementia and although the hallmark histopathologic features of
this condition are extracellular amyloid plaques and intracellular
neurofibrillary tangles localised glia changes have also been shown to be
intimately associated with the stage of Alzheimer’s disease. More spe­
cifically, astrocyte atrophy occurs in the early stage of Alzheimer’s
disease before plaque formation (Matias et al., 2019). In this stage of the
disease there are reduced number of astrocytes, thinner astrocyte pro­
cesses and reduced volume in cell soma. In the late stages of Alzheimer’s
disease, astrocyte hypertrophy is seen around amyloid plaques. Such a
time-dependent relationship between amyloid plaques and astrocytes in
Alzheimer’s disease could be analogous to the relationship between
retinal vascular changes and astrocytes in DM. In Alzheimer’s disease,
there is emerging evidence that addressing glia reactivity through
therapeutic strategies such as α2-NKA (sodium, potassium, ATPase) in­
hibition can prevent tau pathogenesis and brain atrophy (Mann et al.,
2022). Similarly, understanding the role of gliopathy in DR may unravel
glia targets the can be exploited to reduce the onset of DR and the
development of vision-threatening complications such as DMO.
7.4.1. Role of microglia in diabetic retinopathy
Microglia are resident macrophages of the CNS and retina. As dis­
cussed previously in Section 2, microglia are predominantly located
within the plexiform layers under quiescent state and are responsible for
localised retinal neuron homeostasis and clearance of metabolic debris.
Microglia activation and polarisation in DR is represented by a transition
from its anti-inflammatory form to a pro-inflammatory form (Arroba
and Valverde Á, 2017; Li et al., 2021). Elevated levels of inflammatory
cytokines including TNF-α, IL1β, IL-6 and IL-12 were found in the
54
Progress in Retinal and Eye Research xxx (xxxx) xxx
aqueous humour of eyes with NPDR and PDR and microglia were
considered to be one of the main sources of pro-inflammatory cytokine
release together with Muller cells (Adamiec-Mroczek et al., 2009; Boss
et al., 2017; Bromberg-White et al., 2013; Mao and Yan, 2014; Wu et al.,
2017). The analysis of vitreous humour has similarly been proposed
(Nawaz et al., 2019). These inflammatory cytokines recruit circulating
inflammatory cells such as neutrophils and macrophages to infiltrate
injured areas and incite further damage.
Microglia related neuronal injury is considered to be a contributary
mechanism to several clinical manifestations of DR. Retinal neuro­
degeneration characterised by thinning of the GCL and retinal NFL
during early stages of DR was shown to precede microvascular impair­
ment and visual symptoms (Barber, 2003; Kim et al., 2019; Simó and
Hernández, 2014; van Dijk et al., 2012). In addition to inner retinal
changes, microglia associated IL-6 upregulation was found to facilitate
microglia recruitment into the RPE layer. In turn, this is proposed to
cause disruption of the outer BRB and the development of subretinal
fluid in DR (Jo et al., 2019).
Intraretinal HRF are found on OCT in a number of retinal diseases,
including macular oedema associated with DR and AMD (Bolz et al.,
2009; Coscas et al., 2013; Framme et al., 2012; Uji et al., 2012). They are
small lesions <30 μm with moderate reflectivity which differentiates
them from hard exudates. In DR, HRF may be detected prior to the onset
of diabetic vasculopathy and are thought to represent proliferating ag­
gregates of microglia (Vujosevic et al., 2013). HRF are observed to
originate from the inner retina but distribute throughout the retina
overtime (Vujosevic et al., 2017). In the context of DMO, HRF is
considered a useful clinical biomarker in assessment of intravitreal
anti-VEGF and steroid treatment response. The reduction of HRF
numbers were positively correlated with visual acuity gain (Bonfiglio
et al., 2019; Framme et al., 2012; Schreur et al., 2018a). Compared to
anti-VEGF therapy, intravitreal dexamethasone was found to be more
effective in reducing the number of HRF thus supporting the concept
that the pro-inflammatory effects of microglia underlie the development
of some retinal changes in DMO (Bonfiglio et al., 2019; Vujosevic et al.,
2017).
Direct visualisation of microglia offers the advantage of potentially
detecting disease progression at an even earlier stage compared to using
HRF as a clinical biomarker. Current AOSLO technology allows in vivo
label-free, high-definition visualisation of retinal surface microglia
population in rats (Joseph et al., 2021). Another study using AO-OCT
achieved visualisation of ILM macrophages in human subjects
(Hammer et al., 2020). Potential biomarkers for DR severity using AO
technology include baseline microglia density, short term cellular pro­
cess motility and long term cellular migration. Leveraging emerging
imaging techniques such as AO to better visualise microglia could
improve the management of DR.
Fig. 70. Gliotic ball structure surrounding large
microaneurysm (MA). 3-Dimensional view of a large
MA measuring 100 μm is shown in the XZ plane (A).
The MA is located between the ganglion cell layer and
the inner nuclear layer (not shown) and the feeding
vessels originate from the superficial vascular plexus
(red ars). The entire MA is enveloped in glial pro­
cesses proliferated in a ball-like configuration. Only
glial processes are shown in panel B to highlight the
pattern of gliosis. Yellow = GFAP. Grey = Lectin.
Scale bar = 30 μm.
C. Balaratnasingam et al.
7.5. Perfusion and oxygen handling in the retina
Information regarding perfusion and blood flow rates in the retinal
microcirculation is essential for understating oxygen delivery to retinal
neurons. A major challenge in studying retinal perfusion in the human
eye is that there is significant variation in the spatial and temporal
properties of retinal perfusion (Yu et al., 2019a). One reason for the
immense momentary variations in retinal perfusion is the rapidly fluc­
tuating and disparate energy requirements to maintain visual function
under photopic and scotopic conditions. Unlike much of the vasculature
in the CNS, the retina lacks autonomic innervation and relies on local
mechanisms to regulate blood flow. Neurovascular coupling, a term that
is frequently used inter-changeably with functional hyperemia, refers to
a mechanism by which immediate changes in local blood flow can occur
within neural tissue in response to a change in functional activity
(Hamilton et al., 2010; Iadecola, 2017; Lecrux and Hamel, 2011). A
combination of feedforward signals as well as metabolic negative feed­
back signals contribute to neurovascular coupling. Retinal glia are major
contributors to neurovascular coupling (Metea and Newman, 2006;
Paemeleire, 2002; Reichenbach and Bringmann, 2013).
The precise location of functional hyperemia in the human retinal
vascular tree remains contentious. Kornfield and Newman performed
studies of functional hyperemia using rodent retina and observed that it
is primarily mediated by arterioles (Kornfield and Newman, 2014).
Curtis and colleagues have also performed an extensive body of ex vivo
work using isolated retinal vasculature to demonstrate that retinal ar­
terioles play a key role in regulating blood flow by modulating luminal
diameter (Curtis et al., 2018). The work by Mishra and Newman showed
that functional hyperaemia is attenuated in Type 1 DM in a
time-dependent manner (Mishra and Newman, 2010). Using an
STZ-model of rodent DR they showed that light-evoked arteriole dilation
was reduced by 58% after 7 months of disease. There was no change
when compared to controls after 4 months of disease. By studying the
vasomotor response of muller cells and astrocytes following a rise in
intracellular calcium they were able to conclude that abnormal glial
regulation of the vasculature was a key mechanism for the loss of
functional hyperaemia. They were able to show that glia-evoked vaso­
dilation decreased and glia-evoked vasoconstrictions increased in dia­
betic retinas. In a subsequent report, Mishra and Newman showed that it
was possible to reverse some of the loss of functional hyperaemia in
diabetes through the intravenous injection of aminoguanidine (Mishra
and Newman, 2011). As aminoguanidine is an inducible nitric oxide
synthase inhibitor that lowers nitric oxide concentrations they
concluded that this treatment restored neurovascular coupling mecha­
nisms in the retina.
However, there are important anatomic differences between the
rodent and human retina, namely that the rodent retina lacks a
55
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 71. Microaneurysm (MA) without an observable
change in localised GFAP staining or morphology. En
Face view of a MA measuring 35 μm in largest
diameter shows no significant proliferation of glial
processes surrounding the structure. Normal glial
processes are seen surrounding and running parallel
to the feeding capillary (red ars). There is significant
leakage from the MA resulting in hyperfluorescence
of the surrounding tissue (green asterisk). 3-dimen­
sional view of the MA shows glial processes in close
contact with the capillary (red ars) as well as verti­
cally oriented glial structures which represents Muller
cell fibres (green ars) extending from the inner nu­
clear layer to the nerve fibre layer (B). Yellow =
GFAP. Grey = Lectin. Scales bars = 30 μm.
specialised macula. In an earlier section of this report, histologic evi­
dence has been provided to suggest that in the human eye there may be
other control points for retinal perfusion as evidenced by the distribu­
tion of αSMA proteins (An et al., 2020b). The greater concentration of
αSMA in the SVP and ICP suggest that these microcirculatory beds have
greater capacity for autoregulation than the DCP. The increased con­
centration of αSMA at junctions between venules and veins also suggest
that this site may serve a sphincter-like function and regulate blood flow
out of the vascular beds.
Currently, it is not possible to reliably visualise in vivo the individual
cellular components of the vascular apparatus that controls retinal
perfusion such as endothelia, pericytes, VSMCs and glia. However, in
this paper, we have provided compelling histologic evidence that these
cells may undergo significant morphologic changes in the earliest stages
of DR. Quantifiable measures of retinal perfusion could therefore serve
as a surrogate clinical marker of structural injury to the elements of the
vascular apparatus i.e. rather than image the pericyte or smooth muscle
cell directly we may be able to indirectly derive the function of these
cells by measuring retinal perfusion.
If it is possible to measure retinal perfusion, it may also be possible to
use it as a quantifiable metric to gauge disease onset and progression.
Additionally, it may be possible to test and develop systemic pharma­
cologic therapies that prevent the progression of DR by improving
retinal perfusion. The field of cardiovascular medicine has clearly shown
that systemic therapies that improve myocardial perfusion such as ACE
inhibitors and Angiotensin II receptor blockade can reduce cardiac
morbidity by delaying or preventing the risk of a major cardiovascular
events such as myocardial infarction (Buus et al., 2004; Mancia et al.,
1990). A similar beneficial effect will be expected if retinal perfusion is
preserved in DR. Our group has performed an extensive number of
studies utilising isolated properties of ocular vasculature to investigate
the vasoactive response of retinal vessels to a range of pharmacologic
agents (Yu et al., 2003). As an example, nimodipine, betaxolol and
timolol can induce significant dilation of human retinal arterioles in the
ex vivo setting (Yu et al., 1998a). In short, if we can preserve retinal
perfusion with such therapies, we may be able to prevent the onset of
retinal ischaemia and damage to retinal vascular structure which are
quite often irreversible complications of DR.
Few investigators have been able to successfully measure retina
blood flow in vivo. Kornfield and Newman’s work in this field has greatly
enhanced our understanding of retinal perfusion. Using fluorescently
labelled red blood cells together with confocal microscopy and ultrafast
line scans in live rodent animals they were able to measure absolute
blood flow and velocity in the retina. In the rat retina they estimated that
blood flow rate was 3.14 μl/min (Kornfield and Newman, 2015).
However such measurements require complex methodology, specialised
equipment and for these reasons cannot be performed in human
C. Balaratnasingam et al.
subjects.
A variety of clinical methods have been used to evaluate capillary
perfusion and retinal oxygenation in vivo. Burns and others have used
the AOSLO to study retinal structure in patients with DM (Burns et al.,
2019). A major advantage of the AO technique is the high spatial reso­
lution of approximately 2 μm that allows study of cellular and
sub-cellular structures in the live human eye. A combination of retinal
vascular imaging using AO-based techniques together with perfusion
mapping can facilitate differentiation of perfused and non-perfused
capillaries. Such techniques would be particularly useful for detecting
the non-proliferative stages of DR as shown by Burns and colleagues.
Using 7 subjects with NPDR, Burns et al. clearly showed that it was
possible to detect vascular changes such as capillary remodeling,
branching abnormalities of capillary beds and flow abnormalities within
microaneurysms using AOSLO (Burns et al., 2014). They also showed
that it was possible to detect non-perfused capillaries using AOSLO
techniques within areas of retina that appeared normal on structural
OCT B-scans. However, due to the relatively lengthy time required to
capture a retinal image (usually more than 10 min) within a narrow field
of view, AOSLO methods have not gained widespread application in the
clinical setting for studying the retinal circulation.
For over four decades, various investigators have applied Doppler
techniques to investigate the dynamic properties of the retinal circula­
tion (Wei et al., 2018). Grunwald et al. published one of the earliest
reports where laser Doppler velocimetry was employed to study blood
flow rates at various stages of DR (Grunwald et al., 1986). Measurements
attained from the temporal retinal artery and vein using a bidirectional
laser revealed that the maximum or centre-line velocity of red blood
cells is reduced in DR. In a separate report, Grunwald et al. applied a
similar method of Doppler velocimetry to study blood flow rates in 19
patients with Type 1 DM of less than 4 years duration and no retinop­
athy. In that report they showed that total volumetric blood flow rate
increased by nearly 12% in the very earliest stages of DM (Grunwald
et al., 1996). Their results however differed from the subsequent report
by Nagaoka et al. that also used laser Doppler velocimetry to investigate
retinal blood flow in 139 eyes without DR and 55 eyes with mild NPDR
(Nagaoka et al., 2010). They found that retinal blood flow was signifi­
cantly lower in the retinal arterioles in patients with Type 2 DM
compared with the non-diabetic control subjects. One of the limitations
of laser Doppler velocimetry studies is that they have largely been used
to measure blood flow rates in retinal arteries and veins as they operate
on the assumption that vessels less than 50 μm in diameter are not major
contributors to total retinal blood flow. As such, laser Doppler veloc­
imetry has been predominantly confined to the research settings and has
not focused on blood flow changes in the retinal microcirculation in live
subjects.
Retinal oximetry imaging provides information regarding oxygen
saturation of haemoglobin within retinal blood vessels (Stefánsson et al.,
2019). The difference in absorption of light between oxyhaemoglobin
and deoxyhaemoglobin is exploited in the technique of non-invasive
retinal oximetry. Hardarson, Steffanson and Bek used the commer­
cially available Oxymap T1 oximeter to investigate oxygen saturation
changes in large retinal vessels over a range of 0.76–6.8 years in subjects
with DR (Hardarson et al., 2021). They found a significant increase in
oxygen saturation in both arterioles and venules over time. In that same
study they found that 83% of patients did not demonstrate a change in
retinopathy grade as per the ETDRS and Danish guidelines for screening
DR. Although a large number of studies have applied retinal oximetry to
study oxygen saturation changes in various disease states the measure­
ments were largely confined to that of larger order arteries and veins.
Most recently, Pi and colleagues used visible light OCT to measure ox­
ygen saturation in various capillary beds of the rat retina under hypoxic
and hyperoxic conditions (Pi et al., 2020). They found that the averaged
oxygen saturation values in the SVP, ICP and DCP were similar and all
were lower than in arteries. They also found that oxygen saturation
decreased with increasing capillary order. Additionally, oxygen
56
Progress in Retinal and Eye Research xxx (xxxx) xxx
extraction (calculated as the difference between arterial and venous
oxygen saturation) was approximately 20% in normoxia and hypoxia
and reduced to almost 14% in hyperoxia. The study by Pi and colleagues
was a major leap forward in our understanding of oxygen saturation
changes during physiology and disease and with further application may
provide novel insights into microcirculatory oxygen saturation changes
in the very earliest stages of DR.
In the sections above we have outlined the limitations of FA as an
imaging tool to measure retinal perfusion. OCTA is a major break­
through in retina imaging that facilitates stratification of retinal capil­
lary beds and provides high-resolution imaging of retinal capillaries.
Most recently, OCTA has been used to quantify and characterise the
functional properties of the retinal microcirculation (Yu et al., 2019a).
This is a novel concept that utilises structural OCTA data to derive a
proxy heat map of retinal perfusion. Specifically, point-to-point com­
parisons of pixel intensity between sequential OCTA images are used to
quantify and detect sites within the retinal circulation where significant
variations in blood flow occur (Fig. 72). Using this technique, spatial and
temporal variations in retinal perfusion can be detected using OCTA. By
projecting sequentially acquired OCTA images we generate a single
two-dimensional image where pixel intensity can be false coloured to
provide an en face heat map depicting spatial and temporal variations in
retinal capillary perfusion (Fig. 73). The mean co-efficient of variation of
pixel intensity in such a heat map can be used to quantify and compare
variations in capillary perfusion between large order retinal vessels and
capillaries.
Implementation of such techniques to quantify and monitor changes
in retinal perfusion will allow a nuanced functional characterisation of
DR. It may also serve as a valuable biomarker to predict the risk of DR
progression. Other authors have also investigated novel ways to quantify
retinal blood flow non-invasively in the experimental setting. The
elegant work by Joseph and colleagues measured single blood cell ve­
locity in mouse retina using the confocal mode of AOSLO (Joseph et al.,
2019). They provided a complete functional map of the arteriolar tree
and venular system and performed absolute flow measurements across
all vessel generations. Limitations acknowledged in their work included
the constrained field of view that could be studied as well as the influ­
ence of eye movements on the line-scan technique. Developing new
clinical methods to measure retinal perfusion is an important and
promising area of DR research.
7.6. Reconsidering the clinical classification of diabetic retinopathy
In an earlier section of this manuscript we provided a brief historical
overview of the clinical classification of DR. We outlined an important
limitation of the current classification schema and that is the use of
colour photography as a screening tool for DR, a modality that is widely
available but severely constrained by spatial resolution. In this article
we have also provided evidence regarding the value of newer technol­
ogies such as FA, OCTA, structural OCT and UWF imaging for charac­
terising the structural manifestations of DR. Laboratory and clinical
studies have clearly shown that microvascular abnormalities and
endothelial dysfunction characterise the earliest states of DR yet colour
photography cannot precisely detect these alterations. A newer schema
with an expanded classification of the earliest stages of DR prior to what
can be detected using colour photography is therefore needed.
There is evidence that retinal perfusion abnormalities occur in the
earliest stages of DR prior to the occurrence of microaneurysms (Cringle
et al., 1993). It is noted in the literature that there is a need for an
imaging-based marker of early disease (Stitt et al., 2016). If it was
possible to use coefficient of variation measurements derived from
OCTA to detect and quantify spatial and temporal retinal perfusion ab­
normalities it may be possible to detect the retinal changes induced by
DM at an earlier stage than currently possible using colour photography.
This concept can be directly applied to DR screening programmes where
co-efficient of variation measurements from OCTA could be used to
C. Balaratnasingam et al.
Fig. 72. Dynamic macular perfusion. Three sequentially acquired OCTA images
from a normal subject are compared (A,B,C). Two isolated capillaries are
selected to illustrate differences in intensity changes between time frame due to
variations in perfusion parameters. Note that there is marked variation in the
intensity of the capillary of the terminal foveal ring (blue ar) while the other
capillary (red ar) remains unchanged between the three frames. Marked spatial
and temporal variations in retinal perfusion are present in the normal
human macula.
stratify risk of developing DR. However, before such a significant change
to the current methodologies for screening DR is undertaken, it is
important to first validate that coefficient of variation measurements
from OCTA are reliable surrogate markers of retinal perfusion. The next
steps will be a population-based study of a normal cohort to quantify
retinal perfusion per retinal eccentricity after taking into consideration
the influence of age, sex, ethnicity and the presence of common systemic
co-morbidities such as hypertension.
The chief purpose of developing a classification scheme is to provide
a standardised means of assessing the severity of retinal disease for
application in clinical practice and research trials. Although this is
possible to some degree with colour photography as shown in the DRS
57
Progress in Retinal and Eye Research xxx (xxxx) xxx
Fig. 73. Generating a heat map of retinal perfusion using OCTA. (A) En face
projection of sequentially acquired OCTA images of the same subject illustrated
in Fig. 72. Note that projection of the image results in the generation of a crisper
OCTA image where the borders of vessel segments can be clearly delineated. (B)
Coefficient of variation heat map generated using OCTA data shown in panel A.
Vessels coded by dark blue indicate they have no variation between sequential
OCTA images while vessels coded red have significant variation. Note the vessel
marked by the blue ar has significant variation and is coded red on the heat
map. The vessel marked with the red ar has less variation and is coded green on
the heat map. All larger arterioles and venules in this macular region are coded
blue and indicate that their blood flow is consistent within all time-points while
most of the variation occurs at the level of the capillaries and tiny arterioles.
Inserted colour bar indicates the variation from blue (no variation) through to
red (most variation).
(Rand et al., 1985) the concept can be nuanced further through the
integration of multimodal imaging data. Given the great phenotypic
variability of DR at baseline visit, the great inter-individual variability in
natural course and risk of developing sight-threatening complications,
the generation of a more precise predictive model will help identify
patients that require frequent ophthalmic assessment and define those
that require less frequent follow up than currently recommended.
Collectively, this could serve to decrease the burden of eye care on the
health system while improving visual outcomes.
The other aspect of the DR classification scheme that is lacking is the
integration of dynamic data regarding the function of retinal cells and
quantifiable data regarding retinal oxygenation. Although retinal
perfusion maps may provide some data regarding the delivery of blood
and nutrients to retinal layers it does not convey how oxygen exchange
is handled by retinal cells. The rheologic properties of the retinal cir­
culation are altered in DM such as the hypoxic viscosity ratio (Kelly
et al., 1993; Rimmer et al., 1990). It is therefore likely that abnormalities
in oxygen exchange is also a manifestation of DR and quantifying such
may provide a novel means of detecting retinal dysfunction and moni­
toring disease progression.
There is clinical and experimental evidence to demonstrate that glia
and astrocyte dysfunction characterise inner retinal changes in the
earliest stages of DR (Barber et al., 2000; Lieth et al., 1998). There is
compelling evidence that retinal glia are inherently linked to the
development of retinal microvascular changes such as microaneurysms
and ischaemia. The glia is therefore an important target for under­
standing the pathophysiology of DR. Clinical data regarding the struc­
ture and function of retinal glia would provide invaluable information
regarding the extent of retinal dysfunction in DR. Powerful imaging
methods have been used to visualise retinal glia in vivo. Srienc et al. used
a calcium indicator to label retinal glia and studied spontaneous calcium
C. Balaratnasingam et al.
waves, photolysis-evoked calcium waves and ATP-evoked calcium
waves to assess astrocyte and Muller cell function (Srienc et al., 2012). A
confocal microscope was used for retinal imaging. This technique is
however invasive and cannot be applied in human eyes in a routine
clinical setting. Given that the glia plays a key role in controlling mac­
ular hydration, clinical imaging of macular glia is likely to have great
relevance for understanding the risk of developing DMO and for pre­
dicting response to different intravitreal agents used for the manage­
ment of DMO.
Finally, through research we need to determine if the clinical clas­
sification of DR should incorporate the types of DM, that is, Type 1 DM
versus Type 2 DM. Although both types of DM induce a state of hyper­
glycaemia, there is significant experimental evidence to demonstrate
that hyperglycaemia alone does not underlie the changes that charac­
terise DR (Cunha-Vaz et al., 2014; El-Asrar et al., 2004; Sasaki et al.,
2010; Sohn et al., 2016; van Dijk et al., 2009). Given that there are
several important distinctions in the pathogenesis of Type 1 and Type 2
DM it could be expected that the clinical manifestations of DR, the rate
of progression and response to treatment will also differ. Some impor­
tant distinctions regarding pathogenesis include:
1) Type 1 DM is an autoimmune disorder that is characterised by
destruction of insulin-producing pancreatic beta cells. In contrast,
patients with Type 2 DM demonstrate an initial stage of insulin
resistance that is compensated for by hypersecretion of insulin by
beta cells.
2) Familial clustering of Type 1 DM is greater than Type 2 DM. The
overall genetic risk ratio for Type 1 DM is 15 while that of Type 2 DM
is 2 (Rich, 2006).
These differences translate in various ways with respect to the
complication profile seen in Type 1 and Type 2 DM. As an example,
cardiovascular disease in Type 1 DM is more likely to present at a
younger age, involve males and females equally, and is associated with a
diffuse and concentric alteration to the coronary circulation. Romero-
Aroca et al. examined the incidence of DR between Type 1 and Type 2
DM over a period of 9 years (Romero-Aroca et al., 2017). They found
that the annual incidence of any DR in Type 1 DM was 15.2% and that of
Type 2 DM was 13.3%. Although the duration of disease was greater in
the Type 1 DM group and may explain the greater incidence of any type
of DR in that study, it is possible that pathogenic mechanisms and the
natural course distinct to Type 1 DM may have accounted for some of
these differences. A deeper analysis of the differences in clinic course
and rate of complication in Type 1 DM and Type 2 DM is therefore
required.
8. Conclusion
The social and economic burden due to DR is expected to signifi­
cantly increase over the next 30 years (Boyle et al., 2001). Mitigating
vision loss due to DR is therefore a key research priority for vision sci­
entists globally and specialised groups towards this mission have been
proposed (Levine et al., 2022). State-of-the-art management in DR is to
recognise the earliest microcirculatory disturbances prior to the onset of
irreversible structural manifestations such as microaneurysms. There is
clinical and experimental evidence to indicate that retinal perfusion
abnormalities signify the very earliest stages of diabetes-related retinal
injury, focused research to better understand and quantify retinal
perfusion is therefore urgently needed.
Retinal ischaemia is a devastating complication of DR that frequently
precedes the development of macular oedema and retinal neo­
vascularisation (Ramirez et al., 2011; Wessel et al., 2012). There is great
phenotypic heterogeneity in the pattern of retinal ischaemia in DR
implicating a wide range of pathogenic mechanisms underlying this
manifestation (Bresnick et al., 1975) (Fig. 36). Because retinal ischaemia
is an irreversible complication, a key management strategy in DR should
58
Progress in Retinal and Eye Research xxx (xxxx) xxx
be to prevent the development of retinal ischaemia. Endothelial
dysfunction is a hallmark feature of retinal ischaemia, but there is his­
tologic evidence that reactive gliosis also contributes to permanent
vascular occlusion in DR (Bek, 1997) (Fig. 41). As such, treatment
strategies that mitigate the development of retinal ischaemia need to
address the endothelial and glial mechanisms that underlie vessel
occlusion.
Advancements in retinal imaging have greatly aided the detection
and monitoring of retinal vascular diseases. OCTA has been a major leap
forward for studying the role of capillary disturbances in the patho­
physiology of DR. However, the limitations of OCTA should be recog­
nised when using this modality to perform quantitative measurements of
the retinal circulation and generate hypotheses concerning the patho­
physiology of DR.
CRediT
Chandrakumar Balaratnasingam: Conceptualization, Formal anal­
ysis, Investigation, Writing – Original draft, Writing – Review & editing,
Visualisation, Project administration, Funding acquisition, Dong An:
Conceptualization, Methodology, Formal analysis, Data Curation, Re­
sources, Writing – Original Draft, Writing – Review & Editing, Visual­
isation, Software, Martin Hein: Visualisation, Software, Writing –
Review & Editing, Paula Yu: Data Curation, Writing – Review & Editing,
Dao-Yi Yu: Conceptualization, Project administration, Funding
acquisition.
Declaration of competing interest
None.
Data availability
No data was used for the research described in the article.
Acknowledgement
The authors thank the Lions Eye Bank of Western Australia, and
DonateLife Western Australia for facilitating the donor eye retrieval
process. We also thank Dr Andrew Mehnert for his expert advice on
image processing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.preteyeres.2022.101134.
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