Complement peptide C3a inhibits neurodegeneration induced by neonatal hypoxic-

ischemic brain injury

Andrea Pozo-Rodrigálvarez1, Yixian Li1, Jingyun Wu1,2, Verena Dehm1, Hana

Sourkova2, Harry Steinbusch3,4, Anders Ståhlberg2, Carina Mallard5, Henrik Hagberg6,7,
Milos Pekny2,8, Marcela Pekna1,8*

1
Laboratory of Regenerative Neuroimmunology, Center for Brain Repair, Dept. of
Clinical Neuroscience, Sahlgrenska Academy at the University of Gothenburg,
Gothenburg, Sweden
2
Laboratory of Astrocyte Biology and CNS Regeneration, Center for Brain Repair, Dept.
of Clinical Neuroscience, Sahlgrenska Academy at the University of Gothenburg,
Gothenburg, Sweden
3
Department of Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht
University, the Netherlands
4
Department of Brain & Cognitive Sciences, DGIST, Daegu, Republic of Korea
5
Centre of Perinatal Medicine & Health, Institute of Neuroscience and Physiology,
Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
6
Centre for the Developing Brain, King’s College, London, UK
7
Centre of Perinatal Medicine & Health, Inst of Clinical Sciences, Sahlgrenska Academy,
University of Gothenburg, Gothenburg, Sweden
8
Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.

*Corresponding author:
Prof. Marcela Pekna, MD, PhD
Institute of Neuroscience and Physiology
Dept. of Clinical Neuroscience
The Sahlgrenska Academy at University of Gothenburg
Box 440, SE-405 30 Gothenburg
Sweden
Tel: +46-31-786 3581
Fax: +46-31-416 108
E-mail: Marcela.Pekna@neuro.gu.se

Article type: Original research

Running title: C3aR and post-HI neurodegeneration

Keywords: neonatal encephalopathy / hypoxia-ischemia / complement system /

neurodegeneration / reactive gliosis

Summary

Hypoxic-ischemic neonatal encephalopathy (HIE) due to perinatal asphyxia is the leading

cause of brain injury in term infants. The long-term neurological consequences of

perinatal asphyxia are caused by acute neuronal cell death as well as progressive cerebral

atrophy and range from mild behavioral deficits to severe intellectual disability, epilepsy

and cerebral palsy. While experimental HIE research has mainly focused on the acute

phase after injury, clinical data suggest that brain inflammation induced by perinatal

insults may be detectable even years later. Here we show that mild neonatal hypoxic-

ischemic (HI) brain injury on postnatal day (P) 9 triggers in the hippocampus a secondary

neurodegenerative process and pronounced and persisting reactive gliosis. Transgenic

over-expression of a complement activation-derived peptide C3a led to over 60%

reduction in hippocampal neurodegeneration and reduced reactive gliosis on P31. In

contrast, neurodegeneration and microglial cell density were increased by over 150% and

57%, respectively, in mice lacking the C3a receptor (C3aR-/-) 7 weeks post HI. Intranasal

administration with C3a starting 1 hour after HI induction reduced neurodegeneration and

reactive gliosis in the hippocampus of wild-type mice analyzed on P52. Thus, neonatal

HI brain injury leads to long lasting secondary neurodegeneration that is substantially

reduced by intranasal treatment with C3aR agonists, conceivably through the modulation

of reactive gliosis.
Introduction

Neonatal hypoxic–ischemic encephalopathy (HIE) affects 1-3 of 1000 live term infants

(Kurinczuk et al., 2010) and as many as 50% of the survivors develop neurological

complications such as intellectual disability, epilepsy and cerebral palsy, despite the

substantial advances in critical care (Hagberg et al., 2016). Therapeutic hypothermia

reduces mortality without increasing major disability in survivors and is the only

intervention that improves outcomes in newborns with moderate to severe HIE (Jacobs

et al., 2013). However, even in hypothermia treated infants, the incidence of death and

neurological impairment is around 40%, and additional therapies to further improve

outcomes of infants suffering from acute encephalopathy are therefore urgently needed

(Papile et al., 2014).

The long-term neurological impairment after neonatal hypoxia-ischemia (HI) correlates

with the extent of brain damage (Ten et al., 2004). HI and other perinatal brain insults

lead to brain cell death in the acute and secondary phase that last for hours to days as well

as delayed neuronal cell death in the so called tertiary brain damage phase that can persist

for weeks to years, prevents repair and regeneration, disturbs the development and

function of the affected brain networks, or sensitizes them to dysfunction and cell death

due to a subsequent inflammatory challenge (Fleiss and Gressens, 2012). Even a mild to

moderate ischemic insult to the perinatal brain was shown to result in progressive

cerebral atrophy with delayed infarction and long-term cognitive impairment in rodent

HIE models (Geddes et al., 2001; Järlestedt et al., 2011; Järlestedt et al., 2013; Moran et

al., 2017; Stone et al., 2008; Ten et al., 2004).

Inflammation is considered an important contributor to the acute tissue injury as well as

to secondary neurodegeneration (Hagberg et al., 2015) and tertiary brain damage (Fleiss

and Gressens, 2012). Microglia, the principal CNS resident immune cells, and astrocytes

are essential for normal CNS development, the maintenance of CNS tissue homeostasis

as well as regulation of neuronal functioning by e.g. controlling the number and function

of neuronal synapses (Pekny and Pekna, 2014, 2016). Reactive gliosis, the highly

orchestrated response of microglia and astrocytes to any type of CNS injury, is necessary

for neuroprotection, repair and recovery, but maladaptive reactive gliosis can hamper

neural plasticity and accentuate tissue damage (Pekny et al., 2019). Reactive gliosis is

also one of the underlying mechanisms and hallmarks of tertiary brain damage triggered

by neonatal HI (Fleiss and Gressens, 2012).

The complement system has multiple functions in the developing as well as adult CNS.

C3a, a 77 amino acid peptide generated through proteolytic activation of the third

complement component (C3), exerts its functions through C3a receptor (C3aR). C3aR is

a seven transmembrane G-protein-coupled receptor (Ember et al., 1998) that is widely

expressed in many tissues including rodent and human neurons (Benard et al., 2004;

Davoust et al., 1999; Pedersen et al., 2007; van Beek et al., 2000), adult and embryonic

neural progenitor cells (Coulthard et al., 2018; Rahpeymai et al., 2006), microglia

(Möller et al., 1997), and astrocytes (Davoust et al., 1999; Shinjyo et al., 2016; van Beek

et al., 2000). C3aR signaling regulates neural progenitor cell proliferation (Coulthard et

al., 2018), and neuronal migration during brain development (Gorelik et al., 2017),

stimulates neurogenesis in naïve as well as post-stroke adult brain (Rahpeymai et al.,

2006; Stokowska and Pekna, 2018) and facilitates functional recovery after brain

ischemia by promoting peri-infarct neural plasticity (Stokowska et al., 2017). We

previously showed that C3aR signaling is protective against neonatal HI-induced

cognitive impairment (Järlestedt et al., 2013; Moran et al., 2017). However, C3aR

signaling was also implicated in driving synapse loss in virus-induced cognitive

impairment (Vasek et al., 2016) and Alzheimer type neurodegeneration (Lian et al., 2016;

Lian et al., 2015). Thus, the functions of C3aR in the CNS appear to be rather diverse and

the consequences of C3aR activation in the brain may depend on the developmental stage

and disease context.

The objectives of the present study were to determine the long-term effects of mild

neonatal HI brain injury on neurodegeneration and glial responses in the hippocampus

and to investigate the role of C3aR signaling in these processes.

Results

Even mild HI leads to long lasting neurodegeneration

To investigate whether mild HI triggers neurodegeneration and to determine the temporal

profile thereof, we used FJC to label degenerating neurons (Schmued et al., 2005). We

observed that 1 week after HI, FJC+ cells were abundant in the CA of the ipsilesional

hippocampus but absent in the contralesional hippocampus and in the sham operated

animals. At later time points, FJC+ cells were detected only in the CA3 and this region
was therefore selected for the quantification of HI-induced neurodegeneration and glial

responses throughout the study. Three and 7 weeks after HI, FJC+ cells were found in the

CA3 of 58% and 64% of the HI mice, respectively, and their numbers peaked at 7 weeks

post HI. At 12 and 16 weeks after HI, the FJC+ cells were very rare and were found only

in 2 out of 34 (6%) mice (Fig. 1A-B). To estimate the half-life of the FJC+ cells in the

hippocampus, we quantified these cells at 24 and 72 hours after HI. At both time points

we detected FJC+ cells in all the mice analyzed (4 out of 4), however, at 72 hours post HI,

there were about 50% less FJC+ cells in the CA3 compared to the earlier time point

(107±18 versus 198±21, P<0.05). These findings indicate that the FJC labeled neurons

have a half-life of about 48 hours.

At all the time points assessed, the number of NeuN+ cells in ipsilesional CA3 was

dramatically reduced (to 50% or less) compared to the sham-operated animals and

compared to the contralesional CA3. There was no difference between the number of

NeuN+ cells in the contralesional CA3 in the HI mice and the left CA3 of the sham

operated mice (Fig. 1A, C). We did not find any difference in the left/right hippocampus

ratio or the number of NeuN+ cells in the left CA3 of sham operated mice at 3 or 7 weeks

after surgery (Suppl. Fig. 1A-B). There was no difference in the number of NeuN+ cells

in the CA3 between the mice in which we found FJC+ cells in hippocampus and HI mice

that were FJC negative at 3 and 7 weeks after HI (Suppl. Fig. 2A). There was a negative

correlation between the number of FJC+ cells and the number of NeuN+ cells in the CA3

of HI mice at 3 weeks but not at 7 weeks after HI induction (r= -0.74, p<0.01 and r=0.23,

p=0.39, respectively); Suppl. Fig. 3A, D.

Compared to the sham-operated animals, the ipsilesional hippocampus was smaller in the

HI mice at every time point examined (Fig. 1D-E). We did not find any difference in the

ipsilesional/contralesional hippocampus ratio between the mice in which FJC+ cells were

detected in the hippocampus and those that were FJC negative (Suppl. Fig. 2B).

These results demonstrate for the first time that even a mild neonatal HI triggers a

secondary neurodegenerative process in the hippocampus. CA3 is particularly vulnerable

to this type of post-HI injury neurodegeneration. The HI-induced neurodegeneration is

most pronounced in the first days after injury, can persist for at least 4 months and can be

one of the mechanisms contributing to the atrophy / lack of growth of the affected tissue.

Mild hypoxia-ischemia leads to long lasting reactive gliosis

Next, we assessed reactive astrogliosis and the density of microglia/macrophages by

using immunostaining with antibodies against GFAP and Iba-1, respectively. We found

that compared to the contralesional hippocampus, the relative GFAP+ area as well as the

density of Iba-1+ cells were increased in the ipsilesional compared to contralesional CA3

for at least 16 weeks after HI induction (Fig. 2). There was no difference in the relative

GFAP+ area or the density of Iba-1+cells between the HI mice that had FJC+ cells in

hippocampus and the HI mice that were FJC negative (Suppl. Fig. 2C-D). However, we

found a positive correlation between the number of FJC+ cells and the relative GFAP+

area in the CA3 of HI mice at 3 weeks as well as 7 weeks after HI induction (r= 0.56,

p<0.05 and r=0.66, p<0.01, respectively); (Suppl. Fig. 3 B, E). The number of FJC+ cells
showed a positive correlation with the density of Iba-1+ cells in the CA3 of HI mice at 3

but not 7 weeks after HI induction (r= 0.63, p<0.05 and r=-0.14, respectively); (Suppl.

Fig. 3C, F-G). Combining data from all the time points, we also observed a negative

correlation between the number of NeuN+ cells and the relative GFAP positive area in the

CA3 (r= -0.33, p<0.05); (Suppl. Fig. 3G).

These findings show that even mild neonatal HI triggers long lasting reactive gliosis in

the affected hippocampus.

Over-expression of C3a in reactive astrocytes reduced neurodegeneration and

reactive gliosis after HI

We previously showed that mice over-expressing C3a under the control of the GFAP

promoter (C3a/GFAP) are protected against HI-induced loss of hippocampal tissue,

however the underlying mechanisms were elusive (Järlestedt et al., 2013). To test the

hypothesis that C3a limits HI-induced neurodegeneration, we quantified FJC+ cells,

NeuN+ cells and reactive gliosis in the CA3 of C3a/GFAP mice and their wild-type (WT)

littermates 3 weeks after HI induction. We found that the number of FJC+ cells in the

hippocampus of C3a/GFAP mice was reduced by 62% compared to WT mice (p<0.01),

(Fig. 3A-B). In addition, both the relative GFAP+ area and the density of Iba-1+cells in

the ipsilesional CA3 were lower in the C3a/GFAP mice compared to WT mice

(p<0.0001; Fig. 3A,D-E). The proportion of mice containing FJC+ cells in CA3 (54%

versus 44% in C3a/GFAP and WT mice, respectively), and the number of NeuN+ cells in

the CA3 did not differ between the groups (Fig. 3C). Jointly, these results show that
transgenic over-expression of C3a in reactive astrocytes ameliorates HI-induced

hippocampal neurodegeneration and reduces reactive gliosis.

C3aR deficiency aggravates HI-induced neurodegeneration

As the above findings in the C3a/GFAP mice point to the neuroprotective role of C3aR

signaling, we next quantified neurodegeneration and reactive gliosis in the CA3 of C3aR

deficient (C3aR-/-) and WT mice. We found that seven weeks after HI induction the

number of FJC+ cells in the hippocampus of C3aR-/-mice was increased by more than

150% compared to WT mice (p<0.005), but there was no difference between the groups

with regard to the proportion of mice with detectable FJC+ cells (26% versus 23% in

C3aR-/- and WT mice, respectively), (Fig. 4A-B), or the number of NeuN+ cells in the

CA3 (Fig. 4C). The relative GFAP+ area was also comparable between the groups but the

density of Iba-1+ cells in the ipsilesional CA3 was higher in the C3aR-/-mice (p<0.0005;

Fig. 4D-E). Jointly, these results support the contention that C3aR signaling inhibits HI-

induced hippocampal neurodegeneration and reactive microgliosis.

Intranasal C3a treatment reduced neurodegeneration and reactive astrogliosis in

the ipsilesional CA3

To determine the effect of HI on the expression of C3aR, we next quantified C3aR

mRNA in the hippocampus at several time points after HI and found that HI leads to a

transient increase in C3aR expression in the ipsilesional hippocampus (Fig. 5A). To

determine whether intranasal delivery of C3a affects HI-induced neurodegeneration, we

treated WT mice with intranasally administered C3a or PBS starting 1 hour after HI
induction and then once daily for additional 2 days. Seven weeks after HI induction, the

number of FJC+ cells in the ipsilesional CA3 was reduced by 30% in the C3a treated

compared to PBS treated mice (p<0.05), but there was no difference between the groups

with regard to the proportion of mice containing FJC+ cells (47% versus 64% in C3a and

PBS treated mice, respectively), (Fig. 5B-C) or the number of NeuN+ cells in the CA3

(Fig. 5D). The relative GFAP+ area in the ipsilesional CA3 was reduced in the C3a

treated mice (p<0.01) but the density of Iba-1+ cells did not differ between the groups

(Fig. 5B, E-F). These results provide further evidence for the protective role of C3aR

signaling after HI by inhibiting secondary neurodegeneration and modulating reactive

gliosis. They also show that both processes can be targeted pharmacologically by

intranasal treatment with C3aR agonists.

Discussion

Here we show that even mild HI injury to the neonatal brain triggers region specific

neurodegenerative process that continues for months after the initial insult, and

conceivably contributes to both the atrophy/lack of growth of the affected CNS tissue and

injury-induced functional impairment that is still prominent in the adulthood. In the

hippocampus, the CA3 region, known to be particularly sensitive to HI induced damage-

(Sheldon et al., 2001), is especially vulnerable to such secondary neurodegenerative

changes that are accompanied by pronounced reactive gliosis. Our findings further show

that transgenic overexpression of C3a in reactive astrocytes reduces the magnitude of the

HI-induced neurodegeneration, whereas genetic deficiency of C3aR has the opposite

effect. Jointly, these results provide a strong evidence for the inhibitory role of signaling

through the C3aR in secondary neurodegeneration after HI. Finally, we demonstrate that

the HI-induced neurodegeneration can be pharmacologically reduced by intranasal

administration of C3a.

These findings raise several questions. First, do the HI-induced astroglial responses and

neurodegeneration eventually subside or are they life-long? No long term-studies of

reactive gliosis in animal models or human HIE have been reported, however, results

from a clinical study in children with cerebral palsy that in this patient population suggest

that perinatal insults may lead to inflammatory changes in the brain that are detectable at

least 7 years later (Lin et al., 2010). Northington et al. described degenerating neurons in

post-mortem brain tissue from full‐term human infants with HIE leading to death within

3 days to months after birth (Northington et al., 2011). In animal models of HI,
neurodegenerative changes were observed up to 9 weeks after the insult (Geddes et al.,

2001; Stone et al., 2008; Ten et al., 2004). Using a slightly more severe model of HI,

Stone et al. observed that hippocampal degeneration peaks 8 days after injury with

limited recovery noted 6 weeks after HI induction (Stone et al., 2008). Our results

demonstrate that the tissue loss is indeed most pronounced at around 1 week after HI,

there appears to be some degree of recovery thereafter, but the consequences of the injury

are clearly detectable even 16 weeks after the insult. Importantly, ours is the first study

providing evidence for HI-induced neurodegeneration and reactive gliosis as late as 4

months after HI. The very prominent reactive astrogliosis at 4 months after HI implies

that even at this late time point after injury, the HI-induced changes are far from

subsiding and may continue for the rest of the life of the animal. More long-term studies

addressing this important issue are therefore warranted.

Secondly, why do some of the mice at the later time points do not exhibit any active

neurodegeneration as visualized by FJC? As there was no difference between FJC+ and

FJC- groups with regard to the relative size of the affected hippocampus, number of

neurons and reactive gliosis, a possible explanation for the dichotomy with regard to the

presence of FJC+ cells could be that the HI-induced neurodegeneration is not a

continuous process but rather occurs in bouts / waves. This concept is compatible with

the notion that FJC labels neurons that are irreversibly damaged (Poirier et al., 2000), and

is supported by our findings of the FJC+ cell half-life of 48 hours or less as well as by our

observation that the variation in the numbers of degenerating neurons at any given time

point is rather small. Notably, 3 weeks after HI there was a negative correlation between
NeuN+ and FJC+ cells but this correlation was lost at the later time point. Altogether,

these findings may suggest that during the first weeks after the insult, the

neurodegenerative process is at least partially dependent on the number of neurons in the

CA3. Over time, there is some degree of recovery and growth, while the

neurodegenerative process gradually becomes less pronounced and more sporadic. Based

on our results from the genetic and pharmacological modulation of C3aR signaling, we

would argue that C3aR inhibits the magnitude of HI-induced neurodegeneration but not

its dynamics, i.e. the occurrence or the propagation of the HI-induced neurodegenerative

waves.

Thirdly, what is the mechanism underlying post-HI neurodegeneration? As reported

previously, the over-expression of C3a in the C3a/GFAP mice is rather transient with a

peak in the first 24 hours after HI induction (Järlestedt et al., 2013). Although the half-life

of C3a in the brain parenchyma is not known, it is conceivable that in both the

C3a/GFAP mice and the mice that received C3a through the intranasal route, the

activation of C3aR signaling was limited to the first days after HI. It is therefore

noteworthy that despite the short exposure to C3a, both interventions reduced the

numbers of FJC positive cells and reactive gliosis assessed several weeks later. Given the

modulatory effects of C3aR on astrocyte activation (Shinjyo et al., 2016), the positive

correlation between the number of FJC positive cells and GFAP expression, and the

negative correlation between GFAP expression and the number of NeuN+ cells, the long-

term effects of C3a-based interventions point to the reactive astrocytes as an important

driver of the HI-induced neurodegenerative changes.

Lastly, why did none of the genetic or pharmacological interventions alter the number of

NeuN cells in the affected CA3? After HI, C3aR is predominantly expressed by microglia

(Shah et al., 2017), and therapeutic hypothermia, the only clinically used intervention that

improves outcomes in newborns with HIE (Jacobs et al., 2013), increased the expression

of C3aR in the rat brain 24 and 48 hours post-HI (Shah et al., 2017). Notably, in the same

study, therapeutic hypothermia did not affect the density of neurons, microglia or

astrocytes in the first days after HI (Shah et al., 2017). Microglia respond to C3a

stimulation with an upregulation of nerve growth factor (NGF) (Heese et al., 1998),

which is globally neuroprotective in a neonatal model of HI (Holtzman et al., 1996) and

promotes axonal growth and branching in vitro and in the adult brain (Lukoyanov et al.,

2003; Madduri et al., 2009). C3a itself stimulates neurite outgrowth and neuronal

maturation (Shinjyo et al., 2009). Most importantly, the reduced neurodegeneration was

observed after intranasal treatment with C3a which ameliorates HI-induced cognitive

impairment (Moran et al., 2017). The positive net effect of C3aR signaling in the

neonatal brain is conceivably due to the combination of direct and indirect glia-mediated

effects that involve both neuronal survival and neuronal functioning in CA3 as well as

other affected brain regions. While the number of neurons in CA3 may not be the single

most important determinant of functional outcome, and neurodegeneration may represent

only one of many factors that affect neuronal numbers in this region after neonatal HI,

understanding of the mechanism controlling brain cell responses to HI and the temporal

profile thereof may inform the development of much needed effective therapies and their

therapeutic window.
In summary, our findings show that neonatal HI brain injury leads to long lasting reactive

gliosis and secondary neurodegeneration, provide the evidence for the inhibitory role of

C3aR in this process and put forward intranasal administration of C3a as an attractive and

clinically relevant strategy to inhibit secondary neurodegeneration and improve

neurological outcome after neonatal HI brain injury.

Acknowledgments

This work was supported by Swedish Research Council (2017-00991), the Swedish state

under the agreement between the Swedish government and the county councils, the ALF

agreement (716591), The Gothenburg Medical Society, The Swedish Brain Foundation,

T. Söderberg’s Foundation (M169/14, MT5/16), E. Jacobson’s Foundation, and R. and U.

Amlöv’s Foundation.

Author Contributions

Conceptualization: M.Pa., M.Py; Data acquisition and analysis: A.P.R.; Y.L. J.W.,

V.D., H.Sa., A.S. Data interpretation: A.P.R., M.Pa., H.H., C.M., M.Py. Manuscript

writing: M.Pa., M.Py.; Manuscript editing and approval: all the authors; Funding

acquisition: M.Pa., H.H., M.Py.

Declaration of Interests

The authors declare no competing interests.

Material and methods

Animals

Subjects were male and female mice expressing C3a under the control of glial fibrillary

acidic protein promoter (C3a/GFAP) (Boos et al., 2004) on a C57BL6/J / C57BL6/CNr

background and their wild type (WT) littermates, C3aR deficient (C3aR-/-) mice

(Kildsgaard et al., 2000)on a C57BL6/J / C57BL6/CNr background, and C57BL6/CNr

mice (Charles River Laboratories, Sultzfield, Germany). All animal experiments were

approved by the Animal Ethics Committee in Gothenburg (29-2006, 48-2009, 308-2012;

41-2015, 2735-2020). Mice were housed at Experimental Biomedicine (EBM),

Sahlgrenska Academy, University of Gothenburg under specific pathogen free

conditions, standard temperature (20°C), and relative humidity (45%), on an artificial

light-dark cycle of 12 h (lights on at 07:00) with free access to food and water.

HI injury induction

Neonatal HI injury was induced on postnatal day 9 (P9), as previously described

(Hedtjärn et al., 2002; Järlestedt et al., 2013; Rice et al., 1981; Sheldon et al., 1998).

Briefly, mice were anesthetized with 3.5% isoflurane (Baxter Medical, Kista, Sweden)

for induction and 1.5% thereafter, in 1:1 oxygen and nitrous oxide or nitrogen. The left

common carotid artery was dissected and permanently ligated with a prolene suture. The

incision was closed and infiltrated with lidocaine (Xylocain®, Astra Zeneca, Gothenburg,

Sweden). Mice were returned to the dam for 1 hour, thereafter placed in a chamber with

humidified air at 36° C for 10 min, then exposed to humidified 10% oxygen in nitrogen

for 30 min at 36° C, and then kept in humidified air at 36° C for 10 min before being
returned to the dam. This mild HI injury affects the left hippocampus and leads to

persistent cognitive impairment without any apparent motor function deficit (Järlestedt et

al., 2013). Sham animals were subjected to anesthesia and an incision in the neck on P9,

removed from the dam for 50 min, but remained in a warming tray at 36°C under normal

oxygen conditions. At postnatal day 21 (P21) mice were weaned and group housed with

same sex littermates. The sham animals were killed 3 (n=3) or 7 (n=6) weeks after the

sham surgery. As we did not observe any differences between the two groups of sham

mice in any of the parameters assessed (Suppl. Fig. 1), the data from the sham animals

were pooled.

Intranasal C3a administration

Purified human C3a (Complement Technology Inc., Tyler, TX, USA) was diluted in

sterile phosphate buffered saline (PBS) to a concentration of 200 nM, and a total of 8 µl,

i.e. 1.6 pmol (4 µl /nostril; corresponding to ca. 2.56 µg/kg body weight) of peptide

solution or PBS was given intranasally to awake and hand-restrained mice held in a

supine position. Solutions were administered through a pipette tip, drop-wise in 2 µl-

portions divided by 1 min intervals to allow for absorption. This method of

administration to one nostril at a time does not affect breathing. C3a or PBS was given

every 24 hours for three days starting 1 h after HI induction, i.e. between P9 and P11.

Mice in each litter were randomly assigned to C3a or PBS treatment. The investigators

analyzing data were blinded to treatment group.

Brain collection and processing

On P16-130, mice were deeply anesthetized with thiopental [Penthothal Sodium (0.01

ml/g body weight), Hospira, Illinois, USA)] and transcardially perfused with 0.9% saline,

followed by 4% paraformaldehyde (PFA) in 0.1 M PBS. Brains were removed and post-

fixed in 4% PFA at 4°C for 24 h and thereafter in 70% ethanol for 24 h. After processing

in an automatic tissue processor (SAKURA Tissue TeK VIP 3000, Tournai, Belgium),

the brains were embedded in paraffin, cut into 8-μm serial coronal sections on a sliding

microtome (Microm HM 450, Thermo Scientific, Massachusetts, USA), attached to

silane coated slides and dried at room temperature (RT).

Morphometric analysis

After 1 h incubation at 65°C, slides were stained with hematoxylin and eosin. A wide-

field microscope (Nikon Eclipse 80i; Nikon Instruments Inc., Tokyo, Japan) equipped

with a color camera (Axiocam 506c, Carl Zeiss Jena GmbH, Jena, Germany) was used to

obtain images of brain sections 208 μm apart between -1.60mm and -2.10 mm relative to

Bregma; 3 sections/mouse. ImageJ 1.46r software was used to trace around the

ipsilesional and contralesional hippocampus and hemisphere to determine the area. As the

hemisphere area was not altered by HI at any time point assessed and the contralesional

hippocampus/contralesional hemisphere area did not change with age, the

ipsilesional/contralesional hippocampus area ratio was used to quantify HI-induced

atrophy / lack of growth of the ipsilesional hippocampus throughout the study.

FluoroJade C (FJC) staining

Degenerating neurons were visualized by FluoroJade® C (AG325, Sigma-Aldrich,

Stockholm, Sweden). Following deparaffinization, sections were incubated for 1h at

room temperature (RT) in PBS containing 0.3% Triton X-100, rinsed in dH2O and then

incubated in0.06% potassium permanganate in water for 10 min. After rinsing indH2O,

Sections were incubated in 0.0002% FJC in 0.1% acetic acid for 30 minutes, washed, air-

dried at 50ºC for 5 min, cleaned in xylene and coverslipped with DPX non-fluorescent

mounting medium (Sigma-Aldrich, Stockholm, Sweden).

Immunohistochemistry

Neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP) and ionized calcium-

binding adapter molecule 1 (Iba-1) were visualized in the cornu ammonis (CA) of the

dorsal hippocampus by immunohistochemistry. Briefly, following deparaffinization and

heat-induced antigen retrieval with 0.01 M citrate buffer (pH 6, 0.05% Tween 20) for 3x5

min, sections were washed 3x5 min with PBS-T (0.05% Tween 20), non-specific protein

binding was reduced by incubation with blocking buffer [4% normal donkey serum

(NeuN and Iba-1), or 1% bovine serum albumin (GFAP) in PBS-T] for 1h at RT. Tissue

was then incubated with the primary antibody [anti-NeuN biotinylated (1:100, MAB

377B, Millipore, MA, USA), anti-GFAP (1:200, Z0334, Dako, Stockholm, Sweden),

anti-Iba-1 (1:500, 019-19741, Wako, Osaka, Japan)] in blocking buffer overnight at 4°C.

One section per slide was incubated only with blocking buffer without primary antibody

and used as a negative control. Next, sections were washed 3x5 min with PBS-T and

incubated with the secondary antibody [Alexa Fluor 488 goat-anti rabbit (1:2000, GFAP,

A11034, Molecular Probes, Oregon, USA), donkey-anti rabbit biotinylated (1:500, Iba-1,

711-065-152, Jackson ImmunoResearch Inc., PA, USA), in blocking buffer for 1h at RT.

After washing 3x5 min with PBS-T, sections were incubated with Streptavidin-Cy3 1:300
(NeuN), S6402, Sigma-Aldrich, Missouri, USA) in blocking buffer for 1h at RT. Then,

sections were washed 3x5 min with PBS-T, mounted with ProLong Gold (P36931, Life

Technologies, CA, USA) and cover slipped for 24h before being sealed with nail polish.

For Iba-1 staining, following secondary antibody, sections were incubated with an

avidin/biotin complex (VECTASTAIN® Elite ABC kit, PK-6100, Vector Laboratories

Inc., CA, USA) followed by diaminobenzidine (DAB) Substrate Kit (SK-4100, Vector

Laboratories Inc., CA, USA) according to manufacturer's instructions. Next, sections

were washed 3x5min with PBS-T, dehydrated (70% EtOH 2 min, 95% EtOH 2 min,

100% EtOH 2 min) and cleared with xylene for 5 min. Slides were mounted with

VectaMount medium (H-5000, Vector Laboratories Inc., CA, USA) and cover slipped.

Image acquisition and analyses

FJC positive cells, NeuN positive cells and GFAP positive relative area were quantified

using ImageJ 1.46r software on images of CA3 obtained with a 20x (NeuN, FluoroJade C

and GFAP) objective (Nikon Eclipse 80i; Nikon Instruments Inc., Tokyo, Japan)

equipped with a color camera (Axiocam 506c, Carl Zeiss Jena GmbH, Jena, Germany).

Iba-1 positive cell somata were counted on bright field images obtained with a 20x

objective. Three sections per animal (208 μm apart) were used for the analysis and the

data were presented as either number of positive cells for NeuN and FJC, density

(positive cells / μm2) for Iba-1, and positive area relative to the total area (%) for GFAP.

C3aR mRNA measurement

Hippocampus was dissected and quickly frozen on dry ice at 0 hours, 6 hours, 24 hours, 3

days, 7 days, and 21 days after HI and stored at -80º C. Total RNA was extracted using

RNeasyLipid Tissue Mini Kit, including DNase treatment (Qiagen). A NanoDrop ND-

1000 spectrophotometer (NanoDrop Technologies) was used to measure RNA

concentrations. RNA integrity was checked on randomly selected tissue samples using

the Agilent 2100 bioanalyzer (Agilent Technologies). Reverse transcription was

performed with SuperScript III (Invitrogen) using a mixture of 2.5 μMoligo (dT) and 2.5

μM random hexamers (Invitrogen) as primers. Temperature profile for reverse

transcription was 25º C (5 minutes), 50º C (60 minutes), 55º C (15 minutes), and 70º C

(15 minutes). LightCycler 480 (Roche Diagnostics) was used to perform the real-time

PCR measurements. Temperature profile for real-time PCR was 95º C (3 minutes), and

then 50 cycles at 95º C for 20 seconds, 60º C for 20 seconds, and 72º C for 20 seconds.

Ten-microliter reactions contained iQ SYBR Green Supermix (Bio-Rad) and 400 nM of

each primer (Eurofins MWG Operon). The following validated primers for mouse C3aR1

were used: 5’-TGTTGGTGGCTCGCAGAT-3’ (forward), 5’-

GCAATGTCTTGGGGTTGAAA-3’ (reverse). The Mouse Endogenous Control Gene

Panel (TATAA Biocenter, Gothenburg, Sweden) and NormFinder (Andersen et al., 2004)

were used to evaluate reference genes. All data were geometrically averaged against

Pgk1 and B2m. Correctly sized PCR product formation was confirmed by agarose gel

electrophoresis (2%) and melting curve analysis for all samples. Data analysis was

performed as described previously (Nolan et al., 2006; Ståhlberg et al., 2005).

Blinding
All experiments were performed with investigators blinded to the conditions at each

stage.

Statistical analysis

Data were analyzed with GraphPad Prism 6.0f (GraphPad Software Inc., CA, USA).

Two-tailed unpaired Student’s t-test was used for comparison between two samples, One-

way ANOVA with Dunnett’s post-hoc test was used for comparison of the number of

FJC positive cells and hippocampal IL/CL ratio at the time points with sham group. For

comparison of the NeuN positive cell numbers, relative GFAP positive area and Iba-1

positive cell density at different time points after HI induction with the sham mice, Two-

way ANOVA with Sidak’s posthoc test was used. Two-way ANOVA with Tukey’s

multiple comparisons post-hoc test was used for comparison of the NeuN positive cell

numbers, relative GFAP positive area and Iba-1 positive cell density in WT vs

C3a/GFAP mice, WT vs C3aR-/- mice, and PBS vs C3a treated mice. Pearson’s linear

correlation was used to determine the associations between the number of FJC, NeuN and

Iba-1 positive cells, and between the number of positive cells and the relative GFAP

positive area. Data are presented as mean ± SEM. P values < 0.05 were considered

statistically significant. N values for all experiments are provided in the figures and figure

legends.
Figure legends

Figure 1. Mild hypoxia-ischemia leads to long lasting neurodegeneration in the

ipsilesional hippocampal CA3.

(A) Representative images of ipsilesional CA3 in which neurons are visualized with

antibodies against NeuN and degenerating neurons are visualized with FluoroJade C 1-16

weeks after hypoxia-ischemia induction or 3 weeks after sham surgery. Scale bar: 50µm.

(B) Number of FluoroJade C positive cells, and (C) NeuN in the CA3 of mice 1-16

weeks after neonatal hypoxia-ischemia induction or 3 weeks after sham surgery. (D)

Representative hematoxilin-eosin stained brain sections of mice 1-16 weeks after

hypoxia-ischemia induction or 3 weeks after sham surgery. (E) Relative size of the

ipsilesional hippocampus in mice 1-16 weeks after neonatal hypoxia-ischemia induction

or 3-7 weeks after sham surgery.

IL, ipsilesional; CL, contralesional. **p<0.005, ***p<0.001, ****p<0.0005 for

comparison with sham; #p<0.05, ##

p<0.005, ###p<0.001 for comparison between

ipsilesional and contralesional. One-way ANOVA followed by Dunnett’s posthoc

analysis was used to analyze the data in (B, E). Two-way ANOVA followed by Sidak’s

posthoc analysis was used to analyze the data in (C). Numbers in the bars indicate

number of positive mice / total number of mice in each group (B). (C) n=9 (sham);

n=3,9,14,10, and 10 for 1,3,7,12, and 16 weeks after HI induction, respectively. (E) n=9

(sham); n=3,12,14,16, and 18 for 1,3,7,12, and 16 weeks after HI induction, respectively

Data are presented as mean ± SEM.

Figure 2. Mild hypoxia-ischemia leads to long lasting reactive gliosis in the CA3.
(A) Representative images of ipsilesional CA3 in which astrocytes are visualized with

antibody against GFAP and microglia are visualized with antibody against Iba-1 1-16

weeks after hypoxia-ischemia induction or 3-7 weeks after sham surgery. Scale bar:

50µm.

(B) Relative GFAP positive area, and (C) the density of Iba-1 positive cells in CA3 of

mice 1-16 weeks after neonatal hypoxia-ischemia induction or 3 weeks after sham

surgery.

IL, ipsilesional; CL, contralesional. **p<0.005, ***p<0.001, ****p<0.0005 for

comparison with sham; #p<0.05, ###p<0.001, ####p<0.0005 for comparison between

ipsilesional and contralesional. Two-way ANOVA followed by Sidak’s posthoc analysis.

n=8-9 (sham); n=3,9-10,13-14,9, and 9 for 1,3,7,12, and 16 weeks after HI induction,

respectively. Data are presented as mean ± SEM.

Figure 3. Over-expression of C3a in reactive astrocytes reduced neurodegeneration,

astrocyte activation and microglia proliferation in the ipsilesional CA3 three weeks

after hypoxia-ischemia.

(A) Representative images of ipsilesional CA3 in which degenerating neurons are

visualized with FluoroJade C, astrocytes are visualized with antibody against GFAP and

microglia with antibody against Iba-1 3 weeks after hypoxia-ischemia induction. Scale

bar: 50µm. (B) Number of FluoroJade C positive cells, (C) NeuN positive cells, (D)

relative GFAP positive area, and (E) the density of Iba-1 positive cells in CA3 of wild-

type (WT) and C3a over-expressing mice (C3a/GFAP) 3 weeks after neonatal hypoxia-

ischemia induction.
IL, ipsilesional; CL, contralesional. **p<0.005, ***p<0.001 for comparison between

WT and C3a/GFAP; ###p<0.001, ####

p<0.0005 for comparison between ipsilesional and

contralesional. Two-tailed unpaired Student’s t-test was used to analyze the data in (B).

Two-way ANOVA followed by Tukey’s posthoc analysis was used to analyze the data in

(C-E). Numbers in the bars in (B) indicate number of positive mice / total number of

mice. (C-E) n=7-8 (WT) and 11-13 (C3a/GFAP). Data are presented as mean ± SEM.

Figure 4. Hypoxia-ischemia induced neurodegeneration in the ipsilesional CA3 is

increased in mice lacking C3aR.

(A) Representative images of ipsilesional CA3 in which degenerating neurons are

visualized with FluoroJade C, 7 weeks after hypoxia-ischemia induction. Scale bar:

50µm. (B) Number of FluoroJade C positive cells, (C) NeuN positive cells, (D) relative

GFAP positive area, and (E) the density of Iba-1 positive cells in CA3 of wild-type (WT)

and C3aR deficient mice (C3aR-/-) 7 weeks after neonatal hypoxia-ischemia induction.

IL, ipsilesional; CL, contralesional; *p<0.05; ***p<0.001 for comparison between WT

and C3aR-/-; ###p<0.001, ####p<0.0005 for comparison between ipsilesional and

contralesional. Two-tailed unpaired Student’s t-test was used to analyze the data in (B).

Two-way ANOVA followed by Tukey’s posthoc analysis was used to analyze the data in

(C-E). Numbers in the bars in (B) indicate number of positive mice / total number of

mice. (C-E) n=7 (WT) and 5 (C3aR-/-). Data are presented as mean ± SEM.

Figure 5. Intranasal C3a treatment reduced neurodegeneration and reactive

astrogliosis in the ipsilesional CA3.

(A) Relative expression of C3aR mRNA after hypoxia ischemia. n=3. (B) Representative

images of FluoroJade C stained, and GFAP and Iba-1 immunostained ipsilesional CA3 7

weeks after hypoxia-ischemia induction in mice treated with PBS or C3a. Scale bar:

50µm. (C) Number of FluoroJade C positive cells, (D) NeuN positive cells, (E) relative

GFAP positive area, and (F) the density of Iba-1 positive cells in CA3s 7 weeks after

hypoxia-ischemia induction in mice treated with PBS or C3a.

IL, ipsilesional; CL, contralesional.*p<0.05, **p<0.005 for comparison between PBS

and C3a; #p<0.05, ###

p<0.001, ####p<0.0005 for comparison between ipsilesional and

contralesional. Two-tailed unpaired Student’s t-test was used to analyze the data in (C).

Two-way ANOVA followed by Tukey’s posthoc analysis was used to analyze the data in

(D-F). Numbers in the bars in (C) indicate number of positive mice / total number of

mice. (D-F) n=13-14 (PBS) and 8-15 (C3a). Data are presented as mean ± SEM.

Supplementary figures

Supplementary figure 1. Mice at 3 and 7 weeks after sham surgery do not differ in

brain histomorphology.

(A) Relative size of the ipsilesional hemisphere, (B) relative size of the ipsilesional

hippocampus, (C) number of NeuN positive cells, (D) relative GFAP positive area, and

(E) the density of Iba-1 positive cells in CA3 of mice 3 and 7 weeks after sham surgery.

Two-tailed unpaired Student’s t-test. Numbers in the bars indicate number of mice. Data

are presented as mean ± SEM.

Supplementary figure 2. Mice with and without FJC positive cells in CA3 do not

differ in brain histomorphology after hypoxia-ischemia.

(A) Relative size of the ipsilesional hippocampus, (B) number of NeuN positive cells,

(C) relative GFAP positive area, and (D) the density of Iba-1 positive cells in CA3 of

mice 3 weeks after hypoxia-ischemia induction. (E) Relative size of the ipsilesional

hippocampus, (F) number of NeuN positive cells, (G) relative GFAP positive area, and

(H) the density of Iba-1 positive cells in CA3 of mice 7 weeks after hypoxia-ischemia

induction. Two-tailed unpaired Student’s t-test was used to analyze the data in (A) and

(E). Two-way ANOVA followed by Tukey’s posthoc analysis was used to analyze the

data in (B-D) and (F-H). Numbers in the bars indicate number of mice. Data are

presented as mean ± SEM.

Supplementary figure 3. Correlation between the number of FluoroJade C positive

cells and reactive gliosis in CA3 after hypoxia-ischemia. Correlation between the

number of FluoroJade C positive cells and the number of NeuN positive cells (A),

relative GFAP positive area (B), and the density of Iba-1 positive cells in the CA3 3

weeks after hypoxia-ischemia (C), based on data from WT and C3a/GFAP mice.

Correlation between the number of FluoroJade C positive cells and the number of NeuN

positive cells (D), relative GFAP positive area (E) and the density of Iba-1 positive cells

in the CA3 3 weeks after hypoxia-ischemia (F), based on data from WT mice treated with

PBS and C3a. (G) Correlation between the number of NeuN positive cells and relative

GFAP positive area, based on combined data from all time points after hypoxia-ischemia

induction as shown in Fig. 1C and Fig. 2B.

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