TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS

SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

INTRODUCTION:
Diabetic retinopathy (DR) is a major complication of diabetes. Early diagnosis, monitoring, and
suitable management of this ocular disease are primordial in order to reduce the rate of blindness in
diabetic patients. The classic pathophysiology of DR postulated that it’s primarily a vascular disease,
in which changes in the retinal vessel endothelium cells leads to the blood–retina barrier breakdown
and so an increase in the vascular permeability1. Recent research have postulated that there is a
neurodegenerative process that preceded vascular changes in diabetic patients with neuronal cell
loss concerning especially retinal ganglion cells 2. Is the micro vascular changes causes the retinal
neuropathy, or neurodegeneration is the primary feature of chronic hyperglycemia or both
mechanisms are simultaneous and independents?
Optical Coherence Tomography (OCT) is an indispensable tool in ophthalmology nowadays, actually
the most precise method to measure retinal thickness with high accuracy. It is also routinely used in
all patients with diabetes to rule out diabetic maculopathy. OCT becomes an essential tool to assess
macular and peripapillary retinal layers. The purpose of this study is to measure the peripapillary
retinal nerve fiber layer (RNFL) thickness and the ganglion cell-inner plexiform layer (GCIPL)
thickness with OCT, in diabetic patients (with and without DR) and compare them to normal age and
sex-matched controls. The correlation with systemic metabolic control will be evaluated.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

Above image shows Intra-retinal layer segmentation of OCT B-scans (A) Exemplary B-scan of spectralis SD-OCT with
manually reviewed and corrected segmentation of retinal layers as marked in image (B) Exemplary Cirrus HD- OCT B-scan
with segmentation in the RNFL and combined ganglion cell and inner plexiform (GCIP) layer. 3

Neuroretinal degeneration also seems to occur in different optic nerve diseases, such as glaucoma4.
Interestingly, GCIPL and RNFL thinning has been found in retinal illnesses, such as non-proliferative
idiopathic macular telangiectasia5.
In recent studies, it was reported that GCIPL and RNFL thickness values were markedly reduced in
diabetic eyes without DR and the authors concluded that neuroretinal alterations may precede
micro-vascular abnormalities in diabetic eyes6789.
Molecular mechanisms involved in retinal neurodegeneration in diabetes are complex. The exact
relationship between vascular DR and diabetic retinal neuropathy is not yet well known8.
It’s admitted that diabetes induces neural apoptosis of ganglion, amacrine, and Müller cells, as well
as increased expression of glial fibrillary acidic protein in Müller cells, activation of microglia possibly
caused by chronic glutamate toxicity10, inflammatory glial activation, and increased expression of
other neurotrophic factors, including basic fibroblast growth factor and ciliary neurotrophic factor.
Altered glutamate metabolisms have been noted recently in diabetic retinas11.
Chronic hyperglycemia, even without clinically detectable microvascular complications, can lead to
the impairment of retinal ganglion cell layer and so to a reduction of GCIPL and RNFL thicknesses.
Objective assessment of GCC is important in DR as it will help in detection of inner retinal loss
associated with the disease and help in developing neuroprotective therapeutic regimens12.
Current study is in accordance with other studies.13 14 15
Superior RNFL loss in diabetic retina has been related to lower perfusion in superior retina and the
ONH which may lead to greater ischemia and so to structural damage in the ganglion cells
superiorly15.

Significance of this study:

Detecting early impairment of neurologic tissue in DR can allow a preventive rather than
interventional approach in treatment and then a better visual prognosis for these patients. This study
will enable us to find whether RNFL and GCIPL thinning occurs before the onset of clinical DR (diabetic
retinopathy) or not. Moreover, the study will also help to identify and establish the relation of
diabetic duration, grade of DR and HbA1c levels with RNFL and GCIPL thickness. At risk patients group

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

for example those with long duration of diabetes or advanced grade of DR or high HbA1c levels can
be identified who require earlier neuroprotective therapeutic treatment.
The SD-OCT (Spectral domain OCT) may represent an indispensable tool for identifying early signs of
neurodegeneration in diabetic eyes.

BRIEF REVIEW OF LITERATURE:

Jonsson KB, Frydkjaer-Olsen U, Grauslund J. Vascular Changes and Neurodegeneration in the Early Stages of
Diabetic Retinopathy: Which Comes First? Ophthalmic Res. 2016 ;56(1):1. A systematic literature search was
performed to detect neuro-degeneration in patients with no or Mild Diabetic retinopathy as compared to
healthy controls. Eleven studies which used mfERG (multi-focal electroretinogram) and/or OCT to detect
neurodegeneration were included and it was concluded that retinal neurodegeneration is an early
component of DR, which can precede visible vasculopathy.

Van Dijk HW, Verbraak FD, Kok PH, Garvin MK, Sonka M. Decreased retinal ganglion cell layer thickness in
patients with type 1 diabetes. Invest Ophthalmol Vis Sci. 2010. In this observational cross-sectional study,
normal control subjects were age-matched with paients There was a significant linear correlation (R = 0.53, P
< 0.01) between GCL thickness and diabetes duration in the pooled group of patients. Multiple linear
regression analysis (R = 0.62, P < 0.01) showed that DR status was the most important explanatory variable.
Thus this study demonstrate GCL (Ganglion cell layer) thinning in the pericentral area and corresponding loss
of RNFL thickness in the peripheral macula in patients with type 1 diabetes and no or minimal DR compared
with control subjects. These results support the concept that diabetes has an early neurodegenerative effect
on the retina, which occurs even though the vascular component of DR is minimal.

Afef M, Asma K, Chaker B, Faida A, Riadh R (2019) Retinal Fiber Layer and Macular Ganglion Cell Layer
Thickness in Diabetic Patients. J Clin Exp Ophthalmol 10: 785. In this comparative case-control study two
groups of 58 eyes were examined by SD-OCT with peripapillary RNFL, and macular GCIPL assessment. The
first group includes 58 eyes of type 2 diabetic patients (33 eyes without DR, 25 eyes with moderate DR,
without diabetic macular edema), the second group included 58 eyes of non-diabetic, non-glaucomatous
patients. A significant differences between the two groups was found for the average, superior and inferior
RNFL thickness (p<0.001, p=0.005 and p=0.01 respectively). Concerning the GCIPL thickness, it was
significantly less in the diabetic eyes (p<0.001), but no significant difference was noted between the diabetic
patient's eyes without DR and the healthy controls in all quadrants for the RNFL and the GCIPL. Multivariate
linear regression analysis concluded that the diabetic duration, the HbA1c were correlated to RNFL and
GCIPL loss in diabetic patient's eyes and the presence of arterial hypertension as well as lipid disorders were
found as risk factors in the non-diabetic group. It concludes that RNFL and GCIPL loss seems to be the
earliest retinal changes in diabetic patients, and associated with diabetic duration and poor control.

Frydkjaer-Olsen U, Hansen RS, Peto T, Grauslund J. Structural neurodegeneration correlates with early
diabetic retinopathy. Int Ophthalmol. 2018: In this cross-sectional study, 103 patients were examined with
type 2 diabetes mellitus. It found that the duration of diabetes increased with higher levels of DR (p = 0.04),

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

but patients with different level of DR did not differ according to age, sex, blood pressure, HbA1c, and mf-
ERG or OCT parameters. In a multiple logistic regression model, macular ganglion cell layer thickness was
associated with the presence of DR (OR 1.73 per 5 μm increase, 95% CI 1.06-2.85, p = 0.03). Conversely,
retinal nerve fibre layer thickness at optic disc was inversely related to DR (OR 0.69 per 5 μm increase, 95%
CI 0.51-0.95, p = 0.02. Conclusion: In patients with type 2 diabetes, structural neurogenic characteristics
were associated with DR. If confirmed by larger prospective studies, these results may indicate that a
complex neurovascular interaction is an early event in the pathogenesis of DR.

Wan ZQ, Gao Y, Cui M, Zhang YJ. Association between risk factors and retinal nerve fiber layer loss in early
stages of diabetic retinopathy. Int J Ophthalmol. 2021 concluded that RNFL loss, especially in the superior
and inferior quadrants, may be the earliest structural change of the retina in diabetic patients, and is also
associated with diabetic duration, HbA1c, and systolic blood pressure.

Shi R, Guo Z, Wang F, Li R, Zhao L, Lin R. Alterations in retinal nerve fiber layer thickness in early stages of
diabetic retinopathy and potential risk factors. Curr Eye Res. 2018 concluded that RNFL loss might be the
earliest structural change of retina in diabetic patients, and associated with diabetic duration, BMI (Body
Mass index), TG (Triglyceride levels) and HbA1c/

Ng DS, Chiang PP, Tan G, Cheung CG, Cheng CY, Cheung CY, Wong TY, Lamoureux EL, Ikram MK. Retinal
ganglion cell neuronal damage in diabetes and diabetic retinopathy. Clin Exp Ophthalmol. 2016
RGC loss is present in subjects with diabetes and no DR, and is progressive in moderate or severe DR. RGC
neuronal damage in diabetes and DR can be clinically detected using OCT.

AIMS AND OBJECTIVE:

Primary objective: To compare the peripapillary Retinal Nerve Fibre Layer (RNFL) and Ganglion cell
Inner Plexiform Layer (GCIPL) thickness in eyes of patients of Diabetes Mellitus (with and without
Diabetic- retinopathy) & Normal eyes.
Secondary objective: To study relationship of RNFL and GCIPL thickness with:
A) Duration of diabetes
B) Diabetic-retinopathy grade
C) Glycosylated hemoglobin (HbA1c) level of diabetic patients.
Research hypothesis: In patients suffering from diabetes mellitus, retinal neurodegeneration as
measured by decreased RNFL and GCIPL thickness occurs before the appearance for retinal vascular
changes.
Aim of the study: To investigate the relationship of RNFL and GCIPL thickness in between normal
healthy eyes and those affected by diabetes mellitus and also associate it with the extent of the disease.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

Inclusions:
i) Healthy patients who have no systemic diseases.
ii) Patients diagnosed with Diabetes Mellitus (both type-1 and type-2 DM)
iii) Patients with normal fundoscopic examination or with mild- moderate diabetic retinopathy.
iv) All the patients included belong to age group 20-60 years (both genders included)

Exclusions:

i) Glaucomatous patients and patients with raised IOP.

ii) Patients having severe NPDR (Non-proliferative DR), proliferative DR, diabetic macular
edema.
iii) High myopia ( > -6.0 Dioptre)
iv) Epiretinal membrane
v) Intra-ocular inflammation
vi) Optic nerve pathology or neurological illness
vii) History of ocular surgeries like cataract / laser/ injections.
viii) Media opacities like cataract impeding SD-OCT functions.
ix) Patients who are unwilling to participate in study or those who are uncooperative for
indirect ophthalmoscopy or OCT.

MATERIALS AND METHOD:

Type of study: Cross-sectional study (Observational study)
Sample size:
Following is the formula used to calculate the estimated sample size in my cross-sectional model:
𝑍 2 (𝑆.𝐷)2
𝑛= Where,
𝑑2

n = estimated sample size,

S.D = Standard deviation. It is taken on the basis of following reference study:
Afef M, Asma K, Chaker B, Faida A, Riadh R (2019) Retinal Fiber Layer and Macular Ganglion Cell Layer
Thickness in Diabetic Patients. J Clin Exp Ophthalmol 10: 785.{Details of this study has been mentioned
above in the section of Review of literature (page 3)}.
The mean RNFL thickness reported in the normal healthy subjects in this study is: 99.7 ± 20.6 μm
(Mean ± SD)
Z = Value for selected alpha level here taken 1.96. It corresponds to 95% Confidence Interval
which will be suitable for this study.
d = Margin of error, value of 5% will be appropriate in this study

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

Substituting the above the values in the equation, we get an estimated normal patients sample
size as 62.

All patients with diabetes will be put in A-group. All normal subjects (non- diabetics) will be
put in B-group.
Group A: Patients with Diabetes Mellitus: With and without DR (Diabetic retinopathy) on
fundus examination. 62 eyes of 31 diabetic patients will be enrolled in this group; out of which
31 eyes without DR (Group A-NDR) and 31 eyes with mild-moderate DR (Group A-DR) will be
enrolled.
Group B: Normal patient’s eyes; 62 non-diabetic age-matched control eyes will be enrolled.

Diabetic subjects Normal subjects (Non-Diabetics)

GROUP-A GROUP-B

Evidence of mild-mod DR No evidence of DR

Group A- DR Group A-NDR

*Allotment of patients in Group A-DR , Group A-NDR and Group B will be according to inclusion and
exclusion criteria as mentioned above

Patients attending the tertiary care ophthalmic OPD will be recruited under Group A and Group B as per
the inclusion and exclusion criteria and the following will be done:
- Infomed written consent will be taken from all patients before enrollment
- Detailed history will be elicited. All the recent blood investigations report including the FBS and
HbA1c of all the subjects will be noted from the patients chart and kept record. RBS of the
patient will be done only if no prior history of Diabetes and not possessing recent (within 3
months) RBS report. It is done routinely and free of cost.
FBS and HbA1c is routinely done for all patients with Diabetic retinopathy.
- Best corrected Visual Acuity (BCVA) will be assessed for all patients on snellens chart
- SLE will be done for anterior segment evaluation
- IOP measurement will be done by Applanation Tonometery (AT).
- Dilated fundus examination by Indirect Ophthalmoscopy and S/L bio-microscopy will be
performed by 78D lens. Diabetic retinopathy changes if present will be noted.
- SD- OCT will be performed and peripapillary RNFL and GCIPL thickness will be measured.
i) RNFL thickness will be measured using the ONH and RNFL Analysis using the Optic Disc
Cube 200x200 protocol. The main parameters to be analyzed will be average RNFL
thickness, temporal, nasal, inferior and superior RNFL thickness.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

ii) GCIPL thickness will be measured using the Macular Thickness OU Analysis using the
Macular Cube 512x128 protocol. The main parameters to be analyzed will be average,
superior, inferior, superonasal, inferonasal, superotemporal and inferotemporal GCIPL
thickness.
CIRRUSTM HD-OCT (500-22069) Version 10.0.0.14618 manufactured by Carl Zeiss Meditec is
used for OCT examination of all patients enrolled in this study. OCT examination is a routinely
conducted and free of cost test done in our department.

Statistical analysis:
-Data will be analyzed using : mean and SD ( for quantitative variables) & frequencies and relative
frequencies, percent ( for categorical variables).
-Unpaired t-tests will be used for comparing groups. For comparing categorical data, Chi-square test
will be used and P-value will be calculated.
-Multivariate linear regression analysis will determine the risk factors of RNFL and GCIPL loss in
dabetics and healthy patients.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

Diabetic retinopathy classification used in this study is according to International Clinical

Disease Severity Scale for DR16

Patients with Severe NPDR and PDR are excluded from this study. Patients with no DR, mild
NPDR and Moderate NPDR are included in study.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

By definition severe NPDR is present: i) If hemorrhages are of at least the magnitude shown in
standard photograph A in all 4 quadrants, ii) If two quadrants or more have venous beading of same
magnitude or greater than standard photograph B. iii) If one or more quadrants has intra-retinal
microvascular abnormalities of the same or greater magnitude than standard photograph. The image
1A shows severe NPDR with diabetic macular edema, the corresponding OCT image is shown in
image 1B
The standard photographs are taken from the following article: “Classification of diabetic retinopathy and
diabetic macular edema”
Lihteh Wu, Priscilla Fernandez-Loaiza, Johanna Sauma, Erick Hernandez-Bogantes, and Marissé Masis. World
J Diabetes. 2013 Dec 15; 4(6): 290–294. Published online 2013 Dec 15. doi: 10.4239/wjd.v4.i6.290 PMCID:
PMC3874488. PMID: 24379919.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

Exemplar images of two main SD-OCT functions used in this study. Image in left is for calculation of GCIPL thickness
using Ganglion cell OU Analysis: Macular Cube 512x128. Image in right for calculation of RNFL thickness using ONH and
RNFL Analysis: Optic Disc Cube 200x200 1718

REFERENCES

1 Antcliff RJ, Marshall J (1999) The pathogenesis of edema in diabetic maculopathy. Semin
Ophthalmol 14: 223-232.

2 Lynch SK, Abràmoff MD (2017) Diabetic retinopathy is a neurodegenerative disorder. Vision Res
139: 101-107.

3 Oberwahrenbrock T, Weinhold M, Mikolajczak J, Zimmermann H, Paul F, Beckers I, Brandt AU.

Reliability of intra-retinal layer thickness estimates. PloS one. 2015 Sep 8;10(9):e0137316.

4 Rao HL, Yadav RK, Addepalli UK, Begum VU, Senthil S, et al. (2015) Comparing spectral-domain
optical coherence tomography and standard automated perimetry to diagnose glaucomatous optic
neuropathy. J Glaucoma 24: e69-e74.

5 Chhablani J, Rao HL, Begum VU, Jonnadulla GB, Goud A, et al. (2015) Retinal ganglion cells
thinning in eyes with nonproliferative idiopathic macular telangiectasia type 2A. Invest Ophthalmol
Vis Sci 56: 1416-1422.

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TITLE: RETINAL NERVE FIBRE LAYER AND MACULAR GANGLION CELL LAYER THICKNESS IN SUBJECTS
SUFFERING FROM DIABETES MELLITUS: AN OBSERVATIONAL STUDY.

6 Harrison WW, Bearse MA, Ng JS, Jewell NP, Barez S, et al. (2011) Multifocal electroretinograms
predict onset of diabetic retinopathy in adult patients with diabetes. Invest Ophthalmol Vis Sci 52:
772-777.

7 Reis A, Mateus C, Melo P, Figueira J, Cunha-Vaz J, et al. ( 2014) Neuroretinal dysfunction with
intact blood-retinal barrier and absent vasculopathy in diabetes type 1. Diabetes 63: 3926-3937.

8 Evre P, Gökhan T, Hüseyin K, Alper K, Gökhan D, et al. (2017) Assessment of optic disc and
ganglion cell layer in diabetes mellitus type 2. Medicine 96: 29(e7556).

9 Carpineto P, Toto L, Aloia R (2016) Neuroretinal alterations in the early stages of diabetic
retinopathy in patients with type 2 diabetes mellitus. Eye (Lond) 30: 673-679.

10 Barber AJ (2003) A new view of diabetic retinopathy: a neurodegenerative disease of the

eye. Prog neuropsychopharmacol Biol Psychiatry 27: 283-290.

11 Feng Y, Wang Y, Stock O, Pfister F, Tanimoto N, et al. (2010) Vasoregression linked to neuronal
damage in the rat with defect of polycystin-2. PLoS One 4: e7328

12 Van Djik HW, Verbraak FD, Abramoff MD (2010) Decreased retinal ganglion cell layer thickness in
patients with type 1 diabetes. Invest Ophthalmol Vis Sci 51: 3660-3665.

13 Maalek Afef, Khallouli asma, Bouguerra Chaker, Ajili Faida, Rannen Riadh [2019] Retinal Fiber
Layer and Macular Ganglion cell layer thickness in Diabetic patients. J Clin Exp Ophthalmol 2019,
10:1 DOI: 10.4172/2155-9570.1000785

14 Park HY, Kim IT, Park CK (2011) Early diabetic change in the nerve fibre layer at the macula
detected by spectral domain optical coherence tomography. Br J Ophthalmol 95: 1223-1228.

15 Rodrigues EB, Urias MG, Penha FM, Badaró E, Novais E, et al. (2015) Diabetes induces changes in
neuroretina before retinal vessels: a spectral-domain optical coherence tomography study. Int J
Retina Vitreous 1: 4.

16 Lihteh Wu, Priscilla Fernandez-Loaiza, Johanna Sauma, Erick Hernandez-Bogantes, and Marissé
Masis
Classification of diabetic retinopathy and diabetic macular edema World J Diabetes. 2013 Dec 15;
4(6): 290–294.

17 Jang, Jun & Lee, Myung & Cho, Kyong. (2018). Comparative analysis of mean retinal thickness
measured using SD-OCT in normal young or old age and glaucomatous eyes. International
Ophthalmology. 38. 10.1007/s10792-017-0744-7.

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