Journal of Neuroinflammation

Lebon et al. Journal of Neuroinflammation(2024) 21:162
https://doi.org/10.1186/s12974-024-03151-2

Open Access

RESEARCH

Role of inflammation in a rat modelof radiation retinopathy
Cécile Lebon1, Denis Malaise1,2, Nicolas Rimbert1, Manon Billet3, Gabriel Ramasamy3, Jérémie Villaret4,5,
Frédéric Pouzoulet3, Alexandre Matet1,2,6*† and Francine Behar-Cohen1,7*†
Abstract
Radiation retinopathy (RR) is a major side effect of ocular tumor treatment by plaque brachytherapy or proton
beam therapy. RR manifests as delayed and progressive microvasculopathy, ischemia and macular edema,
ultimately leading to vision loss, neovascular glaucoma, and, in extreme cases, secondary enucleation. Intravitreal
anti-VEGF agents, steroids and laser photocoagulation have limited effects on RR. The role of retinal inflammationand its contribution to the microvascular damage occurring in RR remain incompletely understood. To explore
cellular and vascular events after irradiation, we analyzed their time course at 1 week, 1 month and 6 months
after rat eyes received 45 Gy X-beam photons. Müller glial cells, astrocytes and microglia were rapidly activated,
and these markers of retinal inflammation persisted for 6 months after irradiation. This was accompanied by earlycell death in the outer retina, which persisted at later time points, leading to retinal thinning. A delayed loss of
small retinal capillaries and retinal hypoxia were observed after 6 months, indicating inner blood‒retinal barrier(BRB) alteration but without cell death in the inner retina. Moreover, activated microglial cells invaded the entire
retina and surrounded retinal vessels, suggesting the role of inflammation in vascular alteration and in retinal cell
death. Radiation also triggered early and persistent invasion of the retinal pigment epithelium by microglia andmacrophages, contributing to outer BRB disruption. This study highlights the role of progressive and long-lasting
inflammatory mechanisms in RR development and demonstrates the relevance of this rat model to investigate
human pathology.
Keywords Radiation retinopathy, Microglia, Inflammation, Macrophages, Blood retinal barrier, Microangiopathy,
Hypoxia, Retinal pigment epithelium†

Alexandre Matet and Francine Behar-Cohen contributed equally tothis work.
*Correspondence:
Alexandre Matetalexandre.matet@curie.fr
Francine Behar-Cohenfrancine.behar@gmail.com
1
Centre de Recherche des Cordeliers, INSERM UMRS 1138, Sorbonne
Université, Physiopathology of Ocular Diseases: Therapeutic Innovations,
15 rue de l’école de Médecine, Paris 75006, France

2

Ocular Oncology Department, Institut Curie, 26 rue d’Ulm, Paris
75005, France
3
Translational Research Department, Experimental Radiotherapy Platform,
Institut Curie, Université Paris Saclay, 15 Rue Georges Clemenceau,
Orsay 91400, France
4
Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts,
Paris 75012, France
5
Centre Rétine Gallien, Bordeaux 33000, France
6
Université Paris Cité, 15 rue de l’école de Médecine, Paris 75006, France
7
Assistance Publique, Hôpitaux de Paris, Hôpital Cochin, 27 rue duFaubourg Saint-Jacques, Ophtalmopole, Paris 75014, France© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Introduction
Radiation retinopathy (RR) defines the retinal changes
that occur secondary to ocular exposure to any source ofradiation. Its most frequent cause is irradiation to treat
ocular tumors, such as plaque brachytherapy or proton
beam therapy for uveal melanomas, or collateral irradiation for orbital, facial or cerebral tumors. Clinical signs of
RR are delayed and are usually observed from 6 months
to 3 years after irradiation and most commonly after 2
years. Patients present progressive vasculopathy withmicrovascular occlusion and telangiectasia, areas of nonperfusion and subsequent proliferative complications
that can cause retinal hemorrhages and neovascular glaucoma, which might require the removal of the eye as alast resort. As time progresses, retinal layer thinning canbe associated with visual impairment. Radiation macular edema, a frequent sign of RR, usually precedes any
ischemic retinal changes resulting from areas of capillaryleakage [1,2].
RR onset and severity are closely associated with the
cumulative radiation dose, dose fractioning, and extent
of exposure. The threshold for complications is approximately 30–35 Gy [3,4]. Dose fractionation and localized delivery can limit RR, which still develops in a large
proportion of irradiated patients in the absence of recognized prophylaxis and limited therapeutic options,mostly relying on intravitreal anti-VEGF (vascular endothelial growth factor) and laser photocoagulation, which
delay RR but do not prevent it [5]. RR thus remains anunmet medical need.
The exact mechanisms leading to RR are not fully
understood. It has been postulated that radiation firstalters endothelial cells in retinal capillaries, leading totheir death during the subclinical acute phase [6,7].
Other endothelial cells subsequently die while attempting to divide due to radiation-induced DNA damage.
Focal endothelial cell loss can lead to microvascularaneurysms, occlusions, vessel abnormalities and vasculardamage, which manifests as areas of non-perfusion andvascular leakage [6]. The slow turnover of endothelial
cells could explain the late onset and progressive development of RR. The role of retinal inflammation has beencharacterized in several retinal disorders, including diabetic retinopathy [8–10] and age-related macular degeneration (AMD) [11–13], but retinal inflammation afterradiation has scarcely been explored, although clinicalobservations suggest that anti-inflammatory drugs suchas corticosteroids may reduce RR manifestations [14]. In
addition, the time course of glial and inflammatory cell
activation has not been fully described, despite leukostasis being identified as an early pathogenic event [15], andwhether retinal inflammation contributes to the microvascular damage occurring in RR remains uncertain. To
address this issue, we developed a preclinical model ofPage 2 of 17

RR in rats and analyzed the time course of cellular andvascular events. Our results indicate that after irradiation, retinal inflammation progresses and intensifies andcould play a pathogenic role in irreversible retinal andvascular damage.

Materials and methods
Animals

Six-week-old Long-Evans male rats (Janvier Labs, France)
were used in this study. All experimental procedures
were performed in accordance with the Association for
Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and visionresearch. All animal experiments were conducted following European animal welfare and ethical guidelines andwere approved by the French government (authorization
no. 34397-2021121621111587). The animals were housedin our animal facility, which is accredited for performing
experiments on rodents, under a 12 h light/dark cyclewith free access to standardized pelleted food and water.
After one week of acclimatization, the animals were
irradiated in the Small Animal Radiation Research Platform (SARRP, Xstrahl, USA) under gaseous anesthesia (5% isoflurane for induction and 3% isoflurane formaintenance).
The photon irradiations were realized with a positioning scanner and a set of specific collimators to accurately
target the rat’s bulging eyeball while avoiding the irradiation of organs at risk (lacrimal gland, digestive tractus,
brain). The device’s irradiation parameters were 220 kV
and 13 mA, with an average X-ray tube energy of 75 keV
and a half-value layer (HVL) of 0.89. The irradiation fieldsize was 5 × 5 mm. The dose distribution to the eyeballwas calculated using the “MuriPlan” treatment plan software (Xstrahl, Walsall Wood, UK). One eye per animal
was irradiated for 3 consecutive days with a 15 Gy dose
per day, leading to a total dose of 45 Gy. The contralateraleye was used as a control. The animals were then kept in
the animal facility for 1 week, 1 month or 6 months after
photon irradiation for analysis.
Evaluation of retinal hypoxia and retinal vessels

Retinal perfusion and hypoxia were evaluated using a
Hypoxyprobe-1 kit (Hypoxyprobe, Inc., MA, USA). This
kit contains pimonidazole, a probe classically used todetect hypoxic area. Three hours prior to sacrifice, therats received an intraperitoneal injection of pimonidazole(60 mg/kg body weight). The rats were sacrificed, andthe enucleated eyes were fixed for 30 min in 4% paraformaldehyde (PFA). The eyes were then dissected, and theretinas were flat-mounted, fixed for 10 min in acetone
at -20 °C, blocked with 10% fetal bovine serum in phosphate-buffered saline (PBS, Life Technologies, MA, USA)supplemented with 0.1% Triton for 30 min and incubatedLebon et al. Journal of Neuroinflammation(2024) 21:162

with an anti-pimonidazole antibody at a 1:100 dilution inblocking solution overnight. The retinas were then rinsed
and incubated with secondary antibody (Alexa Fluorconjugated 546 anti-mouse, Invitrogen, USA) together
with FITC-conjugated lectin (Sigma Aldrich) at 1:200
for 2 h at room temperature. Flat mounts were mountedwith fluorescent aqueous mounting medium (Dako Ltd.,UK). Images were acquired with a confocal microscope(LSM 710 Carl Zeiss, France), mostly mosaics with Z
stacks, and measurements were performed using ImageJsoftware with a macro tool developed on Fiji by the CHIC(Centre d’histologie, d’imagerie et de cytométrie, Centre
de recherche des Cordeliers UMR S 1138, Paris, France).
Statistics were performed on Prism 8 software (GraphPad, MA, USA), using one-way ANOVA statistical tests
followed by Tukey’s multiple comparison test, n ≥ 5 eyes.
Retina and RPE flat mounts

The rats were sacrificed, and the enucleated eyes were
fixed for 30 min in 4% PFA. Eyes were then dissected, andboth the retinas and retinal pigment epithelium/choroid
complexes were flat-mounted, fixed for 10 min in acetone
at -20 °C, and blocked with 10% fetal bovine serum in
PBS supplemented with Triton 0, 1% for 30 min. The flat
mounts were incubated with primary antibody at a dilution of 1:100 (anti-podocalyxin #MAB1556, R&D Systems, MN, USA; anti-Iba1 #019-19741 FUJIFILM Wako
Chemicals, VA, USA; anti-CD68 #sc-59103, Santa Cruz,
TX, USA; anti-ZO-1 #40-2200, Invitrogen, MA, USA) inblocking solution for 4 days at 4 °C. After rinsing, the flat
mounts were incubated with the appropriate secondary
antibody at 1:200 (Molecular Probes Alexa Fluor, Invitrogen, MA, USA). RPE flat mounts were then incubatedwith phalloidin (Life Technologies) at a dilution of 1:300
for 1 h. Flat mounts were incubated with 4’,6-diamidino2-phenylindole (DAPI) at a dilution of 1:5000 for 5 minunder constant agitation. They were finally mounted withfluorescent aqueous mounting medium (Dako Ltd., UK).
Images were acquired with a confocal microscope (LSM
710 Carl Zeiss, France), and image analysis of RPE flat
mounts was performed using a macro tool developed on
Fiji software. Statistics were performed on Prism 8 software (GraphPad, MA, USA), using one-way ANOVA statistical tests followed by a Dunnett’s multiple comparison
tests for RPE cell surface and circularity measurements;
and Chi-square test to compare RPE cell area distribution. 7255 cells were analyzed for control RPE, 7283 cells
1 month after irradiation, and 6898 cells 6 months after
irradiation.
We performed 3D reconstructions of z-stack flat
mounts using IMARIS (Oxford Instrument, UK).

Page 3 of 17

Cryosections

After euthanasia, eyes were enucleated, mounted in Tissue-Tek OCT and frozen in liquid nitrogen. 10 μm-thickcryosections were cut on a microtome (Leica CM3050S,
France). A TUNEL assay was performed to evaluate cell
death within the retina. The sections were first fixed for
15 min in 4% PFA and permeabilized for 15 min in 0.3%
Triton X-100/PBS. The TUNEL assay was performed
according to the manufacturer’s instructions (TMR red
protocol, Roche Diagnostics, Germany). For some sections, the TUNEL assay was preceded by a dephosphorylation step according to a protocol described previously[16]. Briefly, sections were dephosphorylated with 10 Uof calf intestinal alkaline phosphatase (CIAP, Invitrogen)in the associated buffer for 30 min at 37 °C and washed.
Nuclei were stained by a 5 min incubation with DAPI at
1:5000 in PBS, and sections were mounted with fluorescent aqueous mounting medium (Dako Ltd., UK).
For GFAP staining, sections were washed in Ca2+/
Mg2+-supplemented PBS, incubated in 4% PFA in PBS
during 15 min for fixation, and washed with PBS. Sections were then incubated in 0.3% Triton X-100/PBS during 30 min for permeabilization and washed with PBS.
Saturation was performed with 1% nonfat milk in PBS for
45 min. Sections were then incubated for 1 h with primary antibody (GFAP, Sigma-Merck, Germany) in 0.1%
nonfat milk in PBS. After several washes, the samples
were incubated with the secondary antibody in PBS for
1 h in a dark chamber. Nuclei were stained by a 5 min
incubation with DAPI at 1:5000 in PBS, and sections were
mounted with fluorescent aqueous mounting medium(Dako Ltd, UK). Images were acquired with a fluorescent
U-25ND25 Olympus microscope, and measurements
were performed with ImageJ software. TUNEL-positive
cells were manually counted twice on cryosections close
to the optic nerve. Statistics were performed on Prism
8 software (GraphPad, MA, USA), using Kruskal-Wallis
statistical tests followed by Dunn’s multiple comparison
test, n = 5 eyes per time point.
Western blotting

After enucleation, neuroretinas and RPE/choroid complexes were dissected, and proteins were extracted inlysis buffer (M-Per supplemented with anti-proteases
and anti-phosphatases; Thermo Fisher Scientific, MA,
USA) with a pellet pestle motor on ice and centrifuged
for 10 min at 15,000 rpm. Protein concentrations were
calculated using a BCA protein assay (Thermo Fisher
Scientific, MA, USA). Western blotting was performed
with precast gels (4–12% Bis-Tris, Bolt, Thermo Fisher
Scientific, MA, USA), a migration tank system (Thermo
Fisher Scientific, MA, USA), a tank transfer system(Bio-Rad, CA, USA) and a nitrocellulose membrane.
Red Ponceau staining was performed immediately afterLebon et al. Journal of Neuroinflammation(2024) 21:162

protein transfer. Nonspecific binding was blocked with
5% nonfat dry milk in PBS. The membranes were thenincubated overnight at 4 °C with the primary antibody
at a dilution of 1:1000 in PBST (0.1% Tween 20). Afterrinsing, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody for 1 h(Vector Laboratories, Eurobio, France). Protein bands
were visualized with Supersignal Pico or Femto Chemoluminescent Substrate (Thermo Fisher Scientific, MA,
USA) using the iBright Imaging System (Thermo Fisher
Scientific, MA, USA). FIJI software was used to quantify
the integrated density of the specific band, and protein
expression was reported on Ponceau staining. Statistics
were performed on Prism 8 software (GraphPad, MA,
USA), using Mann-Whitney statistical tests, with n = 4(M1) or n = 5 (W1, M6).

Results
Development of the radiation retinopathy rat model

Using a SARRP device (Small Animal Radiation Research
Platform) (Fig. 1a and b), a total dose of 45 Gy X-beamphotons delivered over 3 days in 3 daily doses of 15 Gy
was chosen among the other irradiation regimens. The
bulging right eye was held and visible on the positioning
scanner made just before the radiation session (Fig. 1c).
As the photon X-beam trajectory was calculated andadjusted for every animal to minimize irradiation of thelachrymal gland, the brain, and other adjacent structures, after one week, the irradiated eye did not show any
significant damage except for mild periocular hair loss

Page 4 of 17(Fig. 1d). One month after irradiation, hair and lashes
loss, skin and hair depigmentation were visible in theirradiated area, and moderate cataracts were observed in
the irradiated eye (Fig. 1e). Six months after irradiation,skin and hair modifications were still present, but thecataracts were more advanced (Fig. 1f ). In and aroundthe non-irradiated left eye, the lens remained clear, andno skin or hair modifications were visible (Fig. 1g). It is
important to note that no change in animal behavior wasobserved after photon X-beam irradiation. In vivo analyses were planned to follow the kinetics of retinal anatomic and functional changes, but the premature cataract
development did not allow to perform fundus photography, OCT (Optical Coherence Tomography), angiography or ERG (electroretinography) later than 3 weeks after
irradiation. However, we did not observe any significant
anatomic or functional changes between prior to photon
irradiation and 2 weeks after (data not shown).
Delayed hypoxia takes place in the irradiated retina

Local retinal hypoxia is a common feature observed in
RR patients due to microvascular abnormalities and closure. Pimonidazole, a commonly used probe for detecting
hypoxic areas in tissues17,] was used to quantify the areaof the hypoxic retina. As shown in Fig. 2, we observed
a significant increase in retinal hypoxia between 1 and6 months after irradiation, while no significant differences were detected at 1 week or 1 month after irradiation compared to non-irradiated eyes (Fig. 2a and b). It
is to note that pimonidazole was evenly observed in theFig. 1 Photon irradiation animal model. Animals were irradiated into a SARRP under gaseous anesthesia (a,b). A positioning scanner prior to the photon
X-beam irradiation was performed for every animal to adjust the trajectory strictly on the eyeball (c) whilst avoiding peripheral tissues. Irradiated eyes
analyzed after one week (d) did not show any visible modification while hair loss, skin depigmentation, and moderate cataract were observed one monthafter irradiation (e). After six months the depigmentation was still visible, and the cataract was denser (f). Left eyes did not present any visible alteration
six months after irradiation (g)Lebon et al. Journal of Neuroinflammation

Fig. 2 (See legend on next page.)(2024) 21:162

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Lebon et al. Journal of Neuroinflammation(2024) 21:162

Page 6 of 17(See figure on previous page.)
Fig. 2 Retinal hypoxia and vascularization modifications after irradiation. (a) Hypoxic retinal areas are detected by the pimonidazole probe (in red) andthe retinal vascularization is stained with lectin-FITC (in green) on rat flat-mounted retinas at one week, one month and six months after irradiation. Nonirradiated eyes were used as controls. Scale bars represent 1 mm. (b) Quantification of the retinal hypoxia intensity using the pimonidazole staining. Oneway ANOVA statistical test followed by Tukey’s multiple comparison test, n ≥ 5 eyes. (c, d) Quantification of the surface covered by small and large vessels
based on the lectin staining with a dedicated macro. After irradiation, we observed a significant and progressive decrease of small vessels and capillaries
after irradiation along with a significant increase of larger vessels. One-way ANOVA statistical test followed by Tukey’s multiple comparison test, n ≥ 5, 13
images per flat-mounted retina were analyzed. * means p < 0.05; *** p < 0.0005; ****p < 0.0001. (e) Flat-mounted rat retinas stained with pimodinazole
probe (in red) to lectin-FITC (in green) 6 months after irradiation. White squares show area enlarged at the bottom line. White arrows point the retinal
microaneurysms. Scale bars represent 1 mm on merged and pimonidazole pictures, 100 μm on enlargements. (f) Transverse view of vessel blebbing in
retina 6 months after irradiation. Vessel walls are stained with lectin in green and nuclei with Dapi in blue. All images represent the same vessel at different
depth. L means lumen and white arrows show membrane blebs. Scale bar represents 10 μm

entire thickness of the retina after irradiation while it was
mainly observed on the inner surface of the retina in controls (supplementary data).
The density and size of lectin-stained vessels also
appeared to be modified (Fig. 2a and e). Indeed, quantification of the surface occupied by small or large vessels
showed a first increase of small capillaries at 1 week due
to vasodilation, followed by a progressive loss of small
capillaries at 1 month and 6 months, mirrored by anincrease of the surface occupied by larger vessels (Fig. 2cand d). Some microaneurysms were also visible at 6
months (Fig. 2e), as well as blebbing of endothelial cells at
the luminal vessel membrane (Fig. 2f ). A delayed alteration of retinal microvessels and subsequent hypoxia arethus observed after irradiation, similar to human radiation retinopathy.
Retinal cell death is maintained over time in the outer
retinaWe used the widely used TUNEL technique to detect
caspase-dependent apoptotic cells. As expected, we
observed dying nuclei after irradiation, with a peak after
1 week and a decrease over time, but dying cells were stillvisible even after 6 months, suggesting a slow but constant loss of cells after photon irradiation (Fig. 3a andc). Interestingly, when we added a dephosphorylation
step prior to TUNEL to unmask the 3’-P DNA ends, we
detected a greater number of dying nuclei [16] (Fig. 3b
and d) at every time point, suggesting that the mechanisms involved in irradiation-mediated cell death imply
not only caspase-dependent apoptosis but also caspaseindependent apoptosis and possibly necroptosis. It is also
important to note that dying nuclei were localized almost
exclusively in the outer nuclear layer (ONL), which isconsistent with the significant and progressive decreasein the nuclear density of this layer at 1 and 6 months(Fig. 3e). Occasionally, some ganglion cells were stainedwith TUNEL-deP at 6 months. The fact that cell death
was observed at early and later time points after irradiation suggests that it could result from continuous andunregulated stress occurring within the retina.

Glial cells activated after irradiation in the retina
contribute to vascular damage

Glial fibrillary acidic protein (GFAP) is a marker of gliosis
due to the inflammatory response in the retina. In control
retinas, GFAP is expressed by microglia such as astrocytes, as well as by the feet of Müller cells, both of which
are located in the ganglion cell layer (GCL) (Fig. 4a).
After irradiation, we observed a progressive increase in
GFAP expression in the Müller cell body through thedistinctive feature of vertical filaments from the GCL to
the external limiting membrane, which was clearly visible 6 months after irradiation. As soon as a week after
irradiation and throughout the follow-up period, GFAPpositive cells, which could correspond to astrocytes or
microglia, migrated from the most inner retinal layers
toward the internal plexiform and internal nuclear layers (Fig. 4a, white arrows). The GFAP protein expression
rise was validated by Western blot analysis of retinas andwas significant from 1 week to 6 months post irradiation compared with that in non-irradiated eyes (Fig. 4bto d). These results suggested that the glial inflammatory
response appeared quickly after irradiation and seemed
to be amplified with time up to 6 months after photon
beam treatment.
Microglial migration in the retina seemed to startsimultaneously with Müller cell activation. To decipher
the inflammatory cellular response to irradiation, we
stained retinal flat mounts with other types of microglial
markers, such as Iba1 (Ionized calcium-binding adaptor
molecule 1), a pan microglia and macrophage marker
that does not label neurons or astrocytes [18]. As illustrated on Fig. 5a, 6 months after irradiation, the entire
retina was invaded by microglial cells stained with Iba1,
while a 6-month control retina showed only few microglial cells. It is interesting to note that numerous cells werelocated close to the vessels, whose luminal membrane
are stained with podocalyxin [19]. Microglial ramifications seemed to be directly in contact with endothelial
cells (Fig. 5a, magnified area), and in some cases, vessels
were wrapped by those inflammatory cells. Noticeably,
the vessels appeared altered with a loss of podocalyxin
staining in comparison to those in non-irradiated retinas,
with focal lumen constriction showing progressive vesselLebon et al. Journal of Neuroinflammation(2024) 21:162

Page 7 of 17

Fig. 3 Photon irradiation induced different types of cell death in retina. (a) The TUNEL technique (in red) allowed the detection of caspase-dependent
apoptotic nuclei (white arrows) on cryosections of irradiated eyes 1 week, 1 month and 6 months after photon irradiation or non-irradiated eyes used ascontrol (Ctrl). Dapi was used to stain nuclei. The scale bar represents 50 μm. GCL means ganglion cells layer; INL, inner nuclear layer; ONL, outer nuclear
layer; RPE/Ch, Retinal Pigment Epithelium/Choroid. Irradiation induced apoptotic death in the ONL that was quantified on (c), Kruskal-Wallis statistical
test followed by Dunn’s multiple comparison test, n = 5 eyes. (b) The TUNEL staining in red has been preceded by a dephosphorylation step, allowing thedetection of apoptotic nuclei through caspase-dependent and -independent apoptosis. More dying nuclei were visible (white arrows) after irradiation
than with TUNEL alone. Nuclei were stained with DAPI. The scale bar represents 50 μm, the same scale was used for all pictures. Quantification of thenumber of TUNEL-positive cells was made on (d) with Kruskal-Wallis statistical test followed by Dunn’s multiple comparison test, n = 5. (e) We observed
a progressive decrease in the nuclei density in the ONL due to irradiation, quantified by counting the number of nuclei on a constant surface (320*120
pixels), Kruskal-Wallis statistical test followed by Dunn’s multiple comparison test, n = 5, * means p < 0.05; ***p < 0.0005

alterations, possibly mediated by the microglial cells. Tridimensional reconstructions support these observations(Fig. 5b). At one week after irradiation, Iba1-positive cells
were mostly rounded and distant from the vessels. Few
ones harbored extensions that scratched the vessel andcrossed it. One month after irradiation, the Iba1-positive
cell density increased, and the morphology of the cellschanged. They appeared more branched and surroundedthe vessels. At 6 months, almost all cells were spread out
on the vessel with many ramifications, suggesting a rolein post-radiation microvascular changes.
Outer blood–retinal barrier disruption contributes toretinal inflammation

The blood–retinal barrier (BRB) is formed by tight junctions between endothelial cells of the retinal vasculature(inner BRB) and by those between retinal pigment epithelium (RPE) cells (outer BRB) [20]. After exploring the cellular changes associated with inner BRB alterations after
irradiation, we investigated possible changes in the outer
BRB. One week after irradiation, we did not observe
major alterations in the structure of the RPE, which wasstained with phalloidin to visualize the F-actin cytoskeleton (Fig. 6a), despite the small number of missing cells.

However, one month after photon beam irradiation, morphological alterations became visible with modifications
in cellular shape and size: some epithelial cells were nolonger hexagonal but had a more rounded or irregularshape. We also observed stress fibers in most of the cells,
as well as some holes inside the cells, with no surrounding actin belt. Some openings of the actin cytoskeleton
barrier were visible, and the RPE cells were no longer incontact with each other.
After 6 months, all the previous alterations were stillvisible, with a large number of intracellular holes andan important proportion of RPE cells exhibiting alteredmorphology (Fig. 6a). Analysis of the morphologicalparameters of the RPE cells revealed significant changes
in the global cellular area and in the cellular area distribution at 1 and 6 months post irradiation compared tocontrol RPE from non-irradiated eyes. (Fig. 6b and c).
Likewise, the RPE cell morphology changed after irradiation, becoming less angular than those in the controls(Fig. 6d).
The outer BRB is formed by tight junctions between
RPE cells, involving several proteins, including Zonula
occludens-l (ZO-1). ZO-1 localization did not seem to bealtered one week or one month after irradiation (Fig. 6e).

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Page 8 of 17

Fig. 4 Photon irradiation induced GFAP overexpression in the retina. (a) GFAP expression (in red) in Müller cells and microglial cells (white arrows) on
cryosections of irradiated eyes after 1 week, 1 month, 6 months and on non-irradiated eyes used as control (Ctrl). Nuclei are stained with Dapi in blue.
The scale bar represents 50 μm. GCL means ganglion cells layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/Ch, Retinal Pigment Epithelium/
Choroid. (b to d) Western blots of irradiated and control retinas after 1 week (b), 1 month (c), 6 months (d) and their quantification. The integrated densityof the several GFAP specific bands was measured and normalized over the ponceau red staining. n = 4 (M1) or n = 5 (W1, M6), Mann-Whitney statisticaltest. *p < 0.05; **p < 0.005

However, the ‘zigzag’ patterns might suggest a contraction of the cellular membrane (Fig. 6f ). Several abnormalities were observed at one month, with loops, holes
and sometimes disruptions in the ZO-1 pattern. At latestages, ZO-1 expression was reduced and even absent
from some RPE cells in damaged areas (Fig. 6e). Alterations in tight junctions, probably related to actin cytoskeleton remodeling, in addition to missing cells andintracellular holes, could be responsible for outer BRB
leakage.
To determine the role of the holes observed in the
RPE after irradiation, we performed staining for several
microglia, such as Iba1 for microglia or CD68, used as
pan-macrophages marker. One week after irradiation,although the global structure was maintained, we locallyfound missing cells and intracellular holes (Fig. 7a). Surprisingly, we found microglial cells at the exact location where RPE cells were missing, and the shape of the
Iba1-positive cells perfectly fitted with the previous hexagonal cells (Fig. 7a enlarged). In addition, one or several
dendritic microglial cells were found close to or aroundevery intracellular hole (Fig. 7a enlarged). Finally, as soon
as a week after irradiation, the apical surface of the RPE
was covered with numerous dendritic cells, and somewere also found under the RPE in the choroid layer. Six
months after irradiation, we observed the same dendritic cells on the RPE surface, which were often groupedas patches. The same observation of Iba1-positive cells
was made, as shown in Fig. 7b, where several microglial
cells were found in a large hole in the RPE, and some ofthem also presented an RPE-like hexagonal shape (Fig. 7b
enlarged). As observed after one week, every intracellular hole was surrounded by one or several Iba1-positive
cells after 6 months. These dendritic cells were crossing
the RPE layer through the observed intracellular holes.
Hence, as visible on Fig. 7c, a dendrite marked by Iba1
was clearly located under the basal membrane of the
RPE cells (Fig. 7c, dotted arrows on the X and Y projections), while the cellular body was at the level of the RPE.
In various cases, we observed that cellular prolongation of Iba1-positive cells ran along the RPE membrane.
Finally, microglia were also observed under the RPE inLebon et al. Journal of Neuroinflammation(2024) 21:162

Page 9 of 17

Fig. 5 Microglia in contact with retinal vessels. (a) Retinal vessels and microglia invasion 6 months after irradiation. Retina flat mounts stained with antipodocalyxin (in red) to visualize luminal membrane of endothelial cells, anti-Iba1 (in green) to localize microglial cells or macrophages and Dapi (in blue)
to stain nuclei. Scale bars represent 50 μm. (b) 3D-reconstruction of microglia invasion. Images from retina flat mounts stained with lectin (in green) toshow vessel walls and anti-Iba1 (purple) to visualize microglial cells and macrophages. Scale bars represent 20 μm on the upper row, and 5 μm on thebottom row. Confocal microscopy images processed with IMARIS, Oxford Instrument, UK.

the choroid layer. Due to the poor condition of the RPE
at 6 months after irradiation, quantification of Iba1-positive cells on flat mounts was not possible. However, we
counted Iba1-stained cells in the retina on cryosections
and found that the Iba1-positive cell population significantly increased after irradiation, starting as soon as one
week and remaining present after 6 months.
We also examined macrophages on RPE flat mounts
and found that CD68-stained macrophages were present on the RPE as soon as one week after irradiation(Fig. 8a), mostly in the central area of the epithelium.

They looked star-shaped, and their presence decreased
after one month. Few cells were visible at 6 months, similarly to control RPE. However, these macrophages were
mostly on the apical surface of the RPE after irradiation, while they were maintained in the choroid layer incontrol eyes (Fig. 8a, Y projection). On the other hand,
few CD68-positive cells were observed on the peripheral RPE one week after the photon beam (Fig. 8b). Their
number increased slightly after one month, but they
were abundant 6 months after irradiation. Unlike what
was observed in the central RPE, those cells were more

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Page 10 of 17

Fig. 6 (See legend on next page.)round-shaped than star-shaped. However, they were similarly distributed on the apical surface of the epithelium(Fig. 8b, Y projection).

Discussion
Despite the development of several animal modelsof RR, the role of inflammation in the pathogenesis
of RR remains unclear. Among those studies, several

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Page 11 of 17(See figure on previous page.)
Fig. 6 RPE progressive disorganization after irradiation. (a) RPE/Choroid complex flat mounts stained with Phalloidin (in red) to visualize actin-F cytoskeleton. After a week, almost no changes were visible on the RPE integrity. After a month, large rounded cells (white arrows) and holes (blue arrows) withincells were observed. (b) Quantification of RPE cell surface. One-way ANOVA statistical test, followed by a Dunnett’s multiple comparison test. (c) Distribution of the cell area of RPE cells. The statistical analysis was performed using a Chi-square test comparing the distribution of the control (expected) withthe distribution of the 1 month or 6 months (observed). (d) Evaluation of the RPE morphology according to their circularity. Hexagonal cells like RPE cells
have a circularity of 0.8. The more cells become round shaped, the more their circularity tends towards 1. One-way ANOVA statistical test, followed by aDunnett’s multiple comparison test. For all statistics, n = 4 eyes and between 1365 and 2360 cells per eye have been measured on 18 images representative of the whole RPE. **p < 0.005; ***p < 0.0005; ****p < 0.0001. For the control, 1-month and 6-months group, the mean circularity was 0,8012; 0,7985 and
0,8079 respectively with a standard deviation of 0,03860; 0,04185 and 0,04328. (e) RPE flat mounts stained with phalloidin (in red) and ZO-1 (in green) to
localize tight-junctions, one week, one month and 6 months after irradiation. Scale bars represent 50 μm. (f) Enlargement of ZO-1 staining on RPE flat
mounts, associated with the merge image with phalloidin (in red), ZO-1 (in green) and Dapi (in blue). Abnormalities are enlightened with white arrows.
Scale bars represent 20 μm

techniques were used to decipher the molecular mechanisms of RR, with a special focus on the damage occurring in the retinal vascular and structural alterations,but the heterogeneity of irradiation protocols (irradiatedstructure, doses, fractions, and timing of in vivo experiments) makes the understanding of the kinetics of events
challenging [6,21–25]. Here, we characterized the kinetics of retinal and RPE/choroidal damage induced in aphoton beam radiation model. We highlighted vasculardamage and inner and outer BRB alterations leading toa long-lasting inflammatory response and loss of photoreceptors. We chose to irradiate rats at a dose of 45 Gy
delivered in 3 fractions, which was considered comparable to the average dose delivered clinically to patientsaffected by ocular tumors (30 Gy for choroidal metastases, 45 Gy for retinoblastoma and 60 Gy for uveal or conjunctival melanomas). Photon beams have been used dueto their availability in a research setting with SARRP, but
RR occurs after irradiation by all types of sources (proton therapy, photon X-rays, iodine or ruthenium plaquebrachytherapy), suggesting common cellular underlying
mechanisms [3]. We chose 3 time points to explore cellular and molecular mechanisms: 1 week and 1 month, corresponding to early stages, and 6 months, corresponding
to later stage. Due to its small size, the whole rat eyeballwas irradiated, including the lens, which protrudes into
the posterior segment. As this leads to the developmentof a premature cataract, in vivo follow-up of the retinal
vasculature and structure with angiography and OCT
after photon beam became impossible as soon as 3 to 4
weeks following treatment. However, the vascular network was already modified a week after irradiation, withan increase in the surface area occupied by small vessels, suggesting the dilation of small capillaries as a first
response to the photon beam irradiation. This phenomenon seemed to be transient since 1 and 6 months after
irradiation, when the surface occupied by small vessels
and capillaries was significantly decreased compared tocontrol. These results are consistent with previous studies showing a reduction in retinal capillary diameter or
vascular loss after irradiation at lower doses [23,24]. The
loss of capillaries is due to the loss of endothelial cells

through several cell death waves [26]. This is accompanied by global retinal hypoxia at 6 months, which is inaccordance with the reduction in retinal capillary diameter. Surprisingly, it is also accompanied by an increase in
the surface area occupied by larger vessels, as previously
described in several studies [22,26]. This finding might be
explained by the fact that as affected capillaries become
occluded, the blood flow is diverted into nearby vessels,leading to their dilation under the hemodynamic load,
as hypothesized by Archer et al. Moreover, the increased
blood flow in damaged vessels could explain the formation of microaneurysms and capillary abnormalities, suchas blebs, observed here 6 months after irradiation. Membrane blebs have also been observed in a diabetic retinopathy model [27], in which the Rho-associated kinase(ROCK) pathway has been shown to play a role in vascular constriction and microvascular occlusion [17]. Thishypothesis should be further studied in our RR model.
Several studies on both rat and mice models of RR havedescribed a first peak of cell death with apoptotic cells
detected in all retinal layers as early as 12 h after a lower
dose of irradiation [22]. This early cell loss is possibly due
to direct radiation damage to DNA, membrane lipids andproteins, which causes apoptosis through the p53 pathway [28,29]. A second wave of delayed cell death resulting from oxidative damage mediated by reactive oxygen
species (ROS) produced subsequently in irradiated tissue [30] can induce different types of cell death [31] until
one year after irradiation [32]. Chronic hypoxia resulting
from vascular damage can cause inner retinal cell loss
[33] but might not induce direct photoreceptor cell deathsince hypoxia-induced factor 1 (HIF-1) is rather protective for photoreceptors [34], and the oxygen supply forphotoreceptors mostly relies on the choroidal circulation. On the other hand, oxidative stress and persistent
inflammation [35,36], which are mediated by micro- andmacroglia but also by macrophages originating fromthe choroid, can cause photoreceptor cell death. In ourmodel, after the expected peak of cell death observed
at one week, photoreceptor cell death was detected at
one and six months, and was two times greater with amodified protocol of TUNEL staining [16] than with theLebon et al. Journal of Neuroinflammation(2024) 21:162

Page 12 of 17

Fig. 7 Microglia invasion of RPE after irradiation. RPE/Choroid complex flat mounts stained with Phalloidin (in red) allowing the visualization of F-actin
and Iba1 (in green) to identify microglial cells, observed 1 week (a) and 6 months (b) after photon irradiation. Scale bars represent 50 μm. White arrows
show missing RPE cells replaced by Iba1-positive cells. Grey arrows indicate holes inside or in between RPE cells. (c) After 6 months, many holes are visible in the RPE layer, surrounded by one or several Iba1-stained cells going through, see dotted arrows on X and Y projection. Scale bars represent 50 μm

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Page 13 of 17

Fig. 8 Macrophage distribution on RPE after irradiation. RPE/Choroid complex flat mounts stained with Phalloidin (in red) allowing the visualization of
F-actin and CD68 (in green) to identify mature macrophages, observed 1 week, 1 month and 6 months after photon irradiation. Non-irradiated eyes wereused as control. (a) Central RPE. (b) Peripheral RPE. Scale bars represent 50 μm. Images are Z-projections of stack images. Y-images for each panel represent
the Y-projection of the corresponding stackregular protocol, indicating that both caspase-dependent
cell death and caspase-independent apoptosis, as wellas possibly necrosis or necroptosis [37,38], occur alongwith signs of outer retinal inflammation. However, theinvolvement of these cell death pathways remains to beelucidated with a dedicated study.
The progressive but constant loss of photoreceptors,
leading to reduction of nuclear density, but with only aslightly reduction of the ONL thickness suggests thepresence of retinal edema, witnessing retinal inflammation. Retinal edema is a common feature in patients with
RR but also in several retinal disorders, such as diabetic
retinopathy, where the role of microglial and Müller cells
is well established [39]. Müller cells maintain the homeostasis of the retina and can be activated by numerous
stimuli. Reactive Müller cells are first neuroprotective butin the case of homeostatic imbalance, they can contributeto neuronal degeneration, retinal edema [11], and photoreceptor cell death [39]. In our model, Müller cells wereactivated as soon as 1 week after irradiation, as indicatedby an increase in GFAP expression, which continued toincrease progressively until 6 months where the entire
Müller cells body were GFAP positive. This progressive
and persistent inflammatory state might contribute toretinal degeneration and vasculature alterations inducedby irradiation. It has been shown in a diabetic retinopathy model that GFAP overexpression is also an earlyfeature of glial reactivity leading to morphological andstructural changes, such as overexpression of the watertransport channel aquaporin 4 inducing retinal edema[40], secretion of cytokines and inflammatory factors
damaging the BRB [41], and production of VEGF thatin turn increases capillary permeability, vascular leakage
and promotes neovascularization [42]. Targeting specifically Müller cells is a promising therapeutic strategy forretinal neuroprotection [43] as they could directly influence microglial activation and migratory mobilization[44]. GFAP is overexpressed not only by Müller cells incases of retinal inflammation but also by resident microglia and astrocytes [45]. Here, we observed that GFAPpositive dendritic-shaped cells migrated toward the outer
retina, reaching the ONL, suggesting that retinal inflammation maintained by Müller cell gliosis is accompanied
by retinal invasion of astrocytes and/or microglial cells.
This progressive inflammation that persists at late stages
might contribute not only to retinal degeneration but
also to microvascular alterations after irradiation.
The presence of a retinal inflammatory state is also
confirmed by the increase of Iba1-expressing cell density in the retina beginning in the early stages, from one

Lebon et al. Journal of Neuroinflammation(2024) 21:162

week after irradiation until 6 months, when microglia
are found in every layer of the retina and on the apicalsurface of the RPE. Resident microglia are physiologically found mostly in the GCL but also in the inner andouter plexiform layers (IPL and OPL) [46,47]. However,
here, the density of microglial cells was increased compared to control retinas. Interestingly, at 6 months, mostmicroglial cells were close to or in contact with vessels,
and some of them were even wrapped around the vessels. Microglia are increased in areas of retinal hypoxia
and neovascularization where they aggregate in andaround neovascular tufts on the inner surface of the retina [48,49]. Specific markers were used to establish thatthey were activated into macrophages. Indeed, these
inflammatory cells can undergo phenotypic and functional modifications to become macrophages, which areable to release inflammatory cytokines to recruit inflammatory effector cells and thus exacerbate inflammation[50]. Moreover, there are several types of macrophages,
depending on their anti-inflammatory (M2), pro-inflammatory (M1), or perivascular characteristics [47,50,51].
The latter, also called vessel-associated microglia, areinvolved in vessel maintenance and repair as well as in
the clearance of infiltrating cells of the neuro-glial-vascular unit [52], which could be relevant in our RR where
vessels are altered. Interestingly, in diabetic retinopathy,
reactive microglial cells are associated with the perivascular unit. They are also hypothesized to exacerbate
vascular permeability and propagate the inflammatory
response [53]. In RR, endothelial cells are also affected byirradiation, as described in several previous rodent models23,26,] and these alterations have also been establishedin our model as described above.
Our results showed that irradiation also exerts deleterious effects on the outer blood‒retinal barrier formedby tight junctions between RPE cells [54,55], which canmodify the photoreceptor microenvironment and contribute to outer retinal edema and photoreceptor cell
death. After irradiation, the epithelium is rapidly affected,
with some RPE cells missing and with intracellular holes
leading to potential outer BRB breakdown. The numberof these features increased over time, which led to a deteriorated epithelium 6 months after irradiation. This was
accompanied by a global change in the surface and shapeof RPE cells, junctions between cells were locally opened,
cells displayed an increased range of cellular area with anincreasing proportion of small cells, even though larger
multinucleated syncytia-like cells were also detected.
Their hexagonality was also lost in favor of roundshaped cells. These alterations were similar to those
detected in the RPE of several models: Goto Kakizaki
rats, used as a model for diabetic retinopathy [17,56];
OXYS rats, used as an AMD-like retinopathy model [57];and light-induced retinal degeneration murine models

Page 14 of 17[58]. However, the pores or transcellular holes in the
RPE remain poorly described in the literature. In streptozotocin-induced diabetic rats, small holes have been
described only between cells, resulting in junction breakdown [59], which is clearly visible through disrupted
staining of ZO-1 and is a commonly used marker of tightjunctions. In our RR model, these junctions endured
membrane tension, as suggested by the ‘zigzag’ pattern,which was visible as soon as one week after irradiation.
Loops and small holes were also visible after one month,similar to those observed in the above-mentioned diseases. The tortuous ZO-1 pattern has been described asa reorganization of the peri-junctional cytoskeleton, with
ZO-1 being one of the regulators of the cytoskeleton andcell shape at cell–cell contacts [60,61]. Modifications inboth the expression and localization of ZO-1 induced byphoton beam irradiation might play a role in the changes
in cellular shape observed in our model.
Unlike small intercellular holes, larger transcellularpores have only been described in the Goto Kakizaki
model, but they display an actin-F ring, supporting anactive transcellular trafficking hypothesis involving PKCz
(protein kinase C zeta) [62]. These pores could participate to physiological processes facilitating the passage of microglia and macrophages between the retina
and choroid. This also seemed to be the case here since
every hole was surrounded by, or contained, one or several Iba1-positive cells, with some passing through. As
most of the microglia are localized on the apical surfaceof the epithelium, we could hypothesize that microglial
cells were leaving the subretinal space toward the choroid. This finding is also consistent with the increasein microglial density and migration to the outer retina
observed after irradiation. These microglia were likely
activated since CD68-labeled macrophages were similarly observed on the apical surface of the RPE. However,they are not evenly distributed, as they appear ramified
and close to the optic nerve at early stages, while they
are more round-shaped and peripheral at later stages.
Microglial morphology is important since it indicatesthe inflammatory status of microglia. The moderate but
thick ramifications indicate that they are reactive, while
the round or amoeboid shape indicates that they arephagocytic, suggesting a macrophage phenotype [50,63].
This finding is consistent with our observations: after
irradiation, the microglial cells migrate toward the RPE,
differentiate into macrophages upon arrival at the RPE,
where they can phagocytose cellular debris from deadphotoreceptors, RPE or other cells. Nonetheless, it is not
excluded that monocytes-derived macrophages, comingfrom the general circulation and crossing the BRB could
also be involved in radiation-induced retinopathy andphotoreceptor cell death, as described in AMD and RPE
injury models [64]. It would be interesting to investigate

Lebon et al. Journal of Neuroinflammation(2024) 21:162

whether these inflammatory cells are anti-inflammatory M1 and/or pro-inflammatory M2 macrophages orwhether there is a switch in the two populations between
the early and later stages. Specific markers, such as CD86,
iNOS, CD163 or CD206 65, could also be used.
The observation of microglial cells covering the entire
body of a RPE cell which appeared to be missing, is surprising. Indeed, there were no nuclei, no phalloidin
background or stress fibers, and no melanin pigments
detected inside those cells. However, their shape wasconserved, and it appeared that the body of an Iba1-positive cell filled the shape of the missing RPE cell. Microglial cells lying on the RPE have already been describedin various ocular diseases, such as AMD with choroidal
neovascularization [8,66], diabetic retinopathy [67] andRPE injury, but this type of cell shape has never been
described so far. Iba1 cells can be recruited to the injured
area, but the replacement of RPE cells remains obscure.
We could hypothesize that exposure to radiation leads tothe death of some RPE cells and that microglial recruitment to the site is a mechanism to preserve the outer
BRB integrity. Microglial cells could also be attracted to
impaired RPE cells to improve the phagocytosis of cellular debris and the rearrangement of the cells to rapidly recreate the epithelial barrier. It is important to notethat we did not observe CD68-positive cells with thisshape; only Iba1-positive microglia were observed. Previous studies have shown that recruited microglia express
selective inflammatory mediators prior to any pathological sign of disease [68], which is consistent with our
RR model in which microglia are detected at very earlystages. This raises the question of whether the microglia
proliferate once in the subretinal space or the cells are
non-proliferative, and the recruitment is constant andpossibly increasing, which would explain the accumulation of these cells in the outer retina. The role of microglia also seems to depend on the presence or absence ofother types of inflammatory cells, such as infiltrating
monocytes, as well as on the cytokines and inflammatory
or anti-inflammatory factors they can produce [8]. As
they can also produce VEGF in response to necrotic RPE
to contribute to angiogenic development [68,69], it would
be interesting to study angiogenic markers such as VEGF
and angiopoietin 1 and 2 or their receptor Tie2 since vascular proliferative complications are common features
observed in RR.
Whether irradiation has generated choroidopathy [70]and, more specifically, a progressive loss of choriocapillaris blood flow, as suggested by clinical imaging studies[71,72], and whether choroidal inflammation could contribute to progressive damage to the RPE and photoreceptor loss will require further exploration.

Page 15 of 17

Conclusion
This study highlights the deleterious effects of irradiationon the RPE and retina, particularly on inner and outerblood retinal barrier integrity. This finding demonstrates
the paramount implication of progressive and long-lasting inflammatory mechanisms involving different typesof cells. Homologies between this rodent model andclinical observations from irradiated patients seem to be
relevant for investigating RR mechanisms. Further investigations are required to better characterize choroidal/
retinal inflammatory mediators to try and target pathogenic inflammation and prevent irreversible retinal damage after ocular irradiation.
Abbreviations
AMD	Age-related Macular Degeneration
BRB	Blood‒Retinal Barrier
ERG	Electroretinography
GCL	Ganglion Cell Layer
GFAP	Glial Fibrillary Acidic Protein
Iba1	Ionized calcium-binding adaptor molecule 1
INL	Inner Nuclear Layer
IPL	Inner Plexiform Layer
OCT	Optical Coherence Tomography
ONL	Outer Nuclear Layer
RPE	Retinal Pigment Epithelium
RR	Radiation Retinopathy
TUNEL	Terminal deoxynucleotidyl transferase dUTP nick end labeling
VEGF	Vascular Endothelial Growth Factor
ZO-1	Zonula Occludens-l

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03151-2.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Supplementary Material 7
Acknowledgements
The authors gratefully acknowledge Christophe Klein for his help with
confocal microscopy at the Histology, Cell Imaging and Flow Cytometry
Center (CHIC) and with macro developed on ImageJ software, as well as
Aurélien Maillot and the Cell and Tissue Imaging Platform (PICT-IBiSA) at
Institut Curie, a member of the French National Research Infrastructure
France-BioImaging (ANR-10-INBS-04). The authors also express their gratitude
to Laurent Jonet for cryosections; to Anaïs Françon, Marianne Berdugo,
Kimberley Delaunay, Jenny Youale, Lolita Radet and Nathan Abbura for their
help; and to Emilie Picard and Alicia Torriglia for their valuable advice.
Author contributions
Conception of the study: FBC, AM, GR, JV, FP. Design of the work: CL, DM,
GR. Data acquisition: CL, DM, NR, MB. Analysis and interpretation of study
results: CL, NR, DM. Drafting of the manuscript: CL, FBC. Review and editing of
the manuscript: CL, AM, FBC.

Lebon et al. Journal of Neuroinflammation(2024) 21:162

Funding
This work was supported by the Cancéropôle Ile-de-France (RadRet Grant)
and the Institut National du Cancer (INCa) with a Translational Cancer
Research Program (PRT-K 20–127).
Data availability
The datasets supporting the conclusions of this article are available from thecorresponding author upon reasonable request.

Declarations
Ethics approvals and consent to participate
All experimental procedures were performed in accordance with the
Association for Research in Vision and Ophthalmology (ARVO) statement
for the use of animals in ophthalmic and vision research. All animal
experiments were conducted following European animal welfare and ethicalguidelines and were approved by the French government (authorization no.
34397-2021121621111587).
Competing interests
All authors declare they have no competing interests.
Received: 28 April 2024 / Accepted: 11 June 2024

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