# Vascular dysfunctions during radiation retinopathy

Colin Niaudet, Thibaud Mathis, Thao-Nguyen Pham, Jean-Claude Quintyn, François Paris, Juliette Thariat

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Colin Niaudet, Thibaud Mathis, Thao-Nguyen Pham, Jean-Claude Quintyn, François Paris, et al. Vascular dysfunctions during radiation retinopathy. Radiotherapy & Oncology, 2026, 214, pp.111294. 10.1016/j.radonc.2025.111294. hal-05497664

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## Review Article

# Vascular dysfunctions during radiation retinopathy

<figure><img src="image_3.png" /></figure>

Colin Niaudet<sup>a, ID</sup>, Thibaud Mathis<sup>b,c</sup>, Thao-Nguyen Pham<sup>d ID</sup>, Jean-Claude Quintin<sup>e</sup>,
François Paris<sup>a,f,* ID</sup>, Juliette Thariat<sup>d,** ID</sup>

## ARTICLE INFO

### Keywords:

Eye

Retina

Radiotherapy

Retinopathy

Vasculopathy

## ABSTRACT

Optic tissue toxicity remains a major limitation of radiation therapy for ocular, brain, skull base, and head and neck tumors. Among the most frequent complications, radiation retinopathy (RR) occurs in 6% of patients treated for extraocular tumors and in more than 40% of those with ocular tumors. After an initial asymptomatic stage characterized by vascular remodeling and chronic retinal ischemia, RR may progress to irreversible vision loss and significantly impaired quality of life. Advances in non-invasive, high-resolution imaging have improved our understanding of the molecular and cellular mechanisms underlying RR in both preclinical and clinical models. In particular, multimodal imaging—and especially optical coherence tomography angiography (OCTA)—has enabled earlier detection of RR in humans. Animal studies have highlighted vascular obstruction and rarefaction, together with early neurovascular dysfunction, as central features of the clinical phase. Beyond preventive measures such as dose constraints, biological and imaging biomarkers may help guide targeted interventions. In this review, we summarize current evidence on the sequential endothelial and neuronal alterations that drive RR and discuss molecular and cellular pathways that could inform the development of potential therapeutic strategies.

## Introduction

Radiation therapy (RT) delivered to the head is in general at risk of exposing ocular structures to some incidental dose of irradiation. This is generally the case for ocular tumors such as uveal melanomas, choroidal metastases, ocular lymphomas, and other rare tumors, which are at high risk of optic toxicities [1]. The Collaborative Ocular Melanoma Study (COMS) group was the first to analyze the efficacy and safety of plaque brachytherapy for uveal melanoma in comparison to first-line enucleation. They reported similar survival between the two groups, offering an alternative to enucleation in patients with intraocular malignant tumors [2]. Since then, other RT modalities have been studied in eyes, relegating ablative surgery to second-line treatment. However, extra-ocular tumors (including orbital, central nervous system/brain, skull

base, head and neck tumors) are also at risk depending on their anatomical proximity to optic structures [3]. All these tumors have in common a hierarchical therapeutic challenge that includes tumor control, eyeball preservation, avoidance of vision loss and eye comfort. RT is usually planned to achieve tumor control while limiting toxicity, often requiring complex clinical trade-offs regarding the tolerance of healthy tissues surrounding the tumor. Medically, the risk factors are primarily driven by the tumor location, tumor histology (and its sensitivity to radiotherapy) and tumor bulk as well as individual patient characteristics, such as age, general and ocular comorbidities, and functional/visual status prior to radiotherapy [3–16].

The retina is organized into a series of neurons, starting with photoreceptors, rods (more effective in low light) and cones (active in daylight conditions, enabling photopic vision, color vision, and sharp

**Abbreviations:** RD, retinal detachment; GCL, ganglion cell layer; Gy, Gray (Joules/kg); NV, neurovascular; OCT, optical coherence tomography; OCT-A, optical coherence tomography angiography; RR, radiation retinopathy; RPE, retinal pigmented epithelium; RT, radiotherapy/radiation therapy.

* Corresponding authors at: CRCI2NA, INSERM U1307, CNRS U6075, Université de Nantes, 8 quai Moncousu, 44007 Nantes, France.

** Corresponding authors at: Department of Radiation Therapy, Comprehensive Cancer Centre François Baclesse, 3 Avenue du General Harris, 14000 Caen, France
E-mail addresses: francois.paris@inserm.fr (F. Paris), j.thariat@baclesse.unicancer.fr (J. Thariat).

<footnote><sup>a</sup> Nantes Université, CRCI²NA, INSERM, CNRS, Nantes, France</footnote>

<footnote><sup>b</sup> Ophthalmology Department, Hopital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France</footnote>

<footnote><sup>c</sup> Laboratoire MATEIS, UMR-CNRS 5510, INSA, Université Claude Bernard Lyon 1, Villeurbanne, France</footnote>

<footnote><sup>d</sup> Radiation Therapy Department, Comprehensive Cancer Centre François Baclesse, Caen, France</footnote>

<footnote><sup>e</sup> Ophthalmology Department, Unicaen, University Hospital of Caen, Caen, France</footnote>

<footnote><sup>f</sup> ICO, Saint-Herblain, France</footnote>
vision), that connect to neurons relaying the visual signal all the way to the retinal surface (ganglion cell layer, GCL) before transmitting it to the brain via the optic nerve. The retina relies on a dual vascular supply: a three-dimensional inner vascular network of plexuses at its surface, and the choriocapillaris, from choroidal vascularization, which runs below the photoreceptors and the retinal pigment epithelium (RPE) representing the outer limit of the retina.

Radiation retinopathy (RR) is caused by a slow, progressive vasculopathy after radiation exposure, potentially leading to microaneurysms, dot-blot hemorrhages, capillary closure, exudates, neovascular proliferation, macular edema, and neuroretinal degeneration. Diabetes, vascular comorbidities and exposure to chemotherapy are triggering and aggravating factors of RR in intra- and extra-ocular tumors [17,18]. RR shares several clinical and pathological features with diabetic retinopathy including vascular leakage, capillary dropout, ischemia, as well as pericyte/endothelial imbalance and choriocapillaris compromise [19,20]. In both conditions, the underlying mechanism seems to rely on damage to the retinal microvasculature, just with different triggers: chronic hyperglycemia leads to metabolic and inflammatory changes in diabetic retinopathy and ionizing radiation directly damages endothelial cells and leads to progressive obliterative vasculopathy in RR [21]. However, due to the lower prevalence and the lack of large-scale studies, research in RR is lagging behind. Unlike diabetic retinopathy, international guidelines for detecting, grading and treating RR are still warranted. RR can indeed, similar to diabetic retinopathy, benefit from recent advances in non-invasive, high-resolution imaging. In particular, studies integrating clinical examination and imaging are emerging [22-24] and might provide mechanistic insights, clues for early detection, and early interventions, with potential development of endothelial and neuronal damage mitigators in RR. We here propose an update on the physiopathology of RR, based on evidence from high-resolution imaging data obtained in humans and animal models.

## Radiation parameters

Retinal radiation dose is determined by the proximity of the target to the macula and optic disc, as well as by the fraction of retina encompassed within high dose regions. Consequently, posterior pole tumors and larger irradiated retinal areas are consistently associated with higher rates of radiation retinopathy (RR) [25,26]. The radiation modality further influences retinal dose distributions. Plaque brachytherapy delivers steep inverse-square dose attenuation from the source, with macular dose primarily dependent on tumor-fovea distance [2,13]. Proton beam provides distal Bragg-peak sparing, while the lateral penumbra largely determines retinal exposure [5]. In contrast, stereotactic photon radiosurgery and radiotherapy achieve dose concentration through multiple non-coplanar beams [14]. Across modalities, dose-response relationships and fractionation schedules remain critical determinants of RR risk. Higher mean or maximum retinal doses and larger doses per fraction are associated with shorter latency and higher incidence of RR, whereas hyperfractionation reduces this risk even at high total doses [27-29]. Finally, modality-specific imaging (see Clinical observables) after plaque or proton therapy has confirmed these dose-volume correlations, revealing early macular capillary dropout and choriocapillaris flow voids in irradiated eyes compared with fellow-eye controls, with changes proportional to local dose distribution [30,31].

In a systematic review and *meta*-analysis of 29 studies (n = 2,458), the pooled prevalence of radiation retinopathy was 6 % (95 % CI, 3–11 %) across mixed head-and-neck indications, predominantly sinonasal and nasopharyngeal tumors. Among 20 studies reporting latency, the median time to diagnosis was 39 months (range, 8–111 months) after irradiation. The dose delivered to the retina is a recognized risk factor associated with the occurrence of RR, with maximal incidence for mean doses above 45 Gy, but described from doses of 15 Gy onwards, depending on the fractions used to deliver the total dose [29]. In the *meta*-analysis, RR was observed at tumor prescription doses >50 Gy,

higher by 5 Gy than current guidelines of maximum mean dose to the retina of 45 Gy. However, no significant association was found between retinopathy prevalence and the tumor dose (p = 0.714) or the retinal (p = 0.798) or optic-nerve dose (p = 0.366). In contrast, chiasm dose was significantly associated with retinopathy prevalence (p = 0.009) in their *meta*-analysis, which is indeed intriguing and would warrant further investigations. For extra-ocular tumors, dose fractionation is a predictive factor of RR; higher doses per fraction lead to a higher risk of developing RR [25], while conversely, hyperfractionation (with lower doses per fraction using more fractions) may reduce the incidence of retinal toxicities even at high total doses (>50 Gy) [28]. For these extra-ocular tumors, the overall prevalence of RR reaches 6 %, based on a *meta*-analysis [29]. For intra-ocular tumors, the main risk factors are the location of the tumor at the posterior pole (near the optic nerve and macula), high tumor thickness and diameter at baseline, and large areas of retina irradiated [25,26]. The prevalence of RR in intraocular tumors usually exceeds 40 % and can exceed 80 % in some series of uveal melanomas treated with brachytherapy. In general, reports on the characteristics of the patients diagnosed with RR are rare and it is difficult to address the severity of RR [29].

## Early-stage radiation retinopathy

The initial clinical manifestations of radiation retinopathy often include an exudative–edematous phase that precedes overt ischemic and proliferative changes: OCT can detect increases in central retinal thickness and cystoid edema within months to a year after irradiation, frequently appearing earlier than fundus examination or fluorescein angiography (FA), and correlating with local dose distribution [26,30]. Breakdown of the inner blood–retinal barrier, driven by endothelial dysfunction and Müller-cell stress, provides the mechanistic basis for early edema and leakage, in line with classic descriptions of RR and maculopathy [22,32]. Non-invasive flow studies further suggest a preclinical stage of perfusion compromise that precedes or accompanies edema. Laser speckle flowgraphy has demonstrated reduced retinal and choroidal blood flow after plaque therapy, even before extensive clinical signs are apparent [33]. Similarly, OCT-A reveals early microvascular dropout and choriocapillaris flow-voids in irradiated eyes [34]. Collectively, these findings support the recognition of an early edematous phase in the natural history of RR.

## Progression of radiation retinopathy toward late stage

### Clinical observables

In patients, clinically apparent RR manifests as a wide spectrum of morphological and functional changes on paraclinical exams of the posterior pole, associated with vision loss in case of macular ischemia and edema. After an initial asymptomatic phase ranging from 6 to 18 months, RR is characterized by a progressive accumulation of vascular abnormalities on fundus examination and FA. Vascular abnormalities encompass microaneurysms, dilated or telangiectatic vessels, functional disruptions involving areas of non-perfusion, hemorrhages, and the formation of hard exudates. At this stage, FA can show retinal leakage underlying vascular hyperpermeability [32,34]. Besides, neural tissue exhibits signs of ischemia such as cotton-wool spots. Studies using structural electron microscopy of enucleated eyes can also reveal small clusters of microaneurysms, as dark red spots of 10 to 100 µm in diameter, surrounding compact groups of damaged capillaries. These endothelial lesions coincide with biopsies findings showing areas of capillary dropout, vascular obstruction [35], and endothelial cell loss while pericytes are maintained. In the adjacent neural tissue, cotton-wool spots can form, and peak in this initial phase of RR but may later regress [2].

The severity and short latency of RR correlate with higher radiation total dose and dose per fraction [27]. High dose irradiation can trigger a
widespread vascular closure affecting directly both capillary and arterioles, resulting in the concomitant and rapid development of large ischemic areas [36]. Such observations highlight the central role played by vascular occlusion in precipitating RR.

Historically, fluorescein angiography (FA) has been used to characterize the blood flow in the retina and choroid. Although it remains the gold standard in clinical studies for the detection of RR, more recent approaches are currently used in clinical practice to diagnose and follow patients with RR. Optical coherence tomography (OCT), a high-resolution imaging method has been increasingly used to evaluate RR. OCT uses near-infrared light to capture detailed 2- or 3-dimensional images of ocular tissues, allowing analysis of the retinal architecture. This exam is widely used for the detection of macular edema, being more sensitive than fundus examination or FA [26]. However, OCT alone lacks visualization of blood flow and vascular architecture. Recently, OCT-angiography (OCTA) has emerged as an advanced, non-invasive imaging modality that enables high-resolution visualization of the retinal and choroidal microvasculature. Since its first clinical applications last decade, OCTA has revolutionized retinal imaging by providing detailed vascular mapping without the need for intravenous dye injection. This technology relies on motion contrast, detecting changes in the reflectance signal of moving erythrocytes across sequential OCT B-scans to reconstruct three-dimensional perfusion maps [37]. Multiple individual A-scans are compiled to generate cross-sectional B-scans, which are acquired repeatedly at the same retinal location. By comparing signal differences between successive scans, OCTA distinguishes regions of high blood flow, characterized by significant decorrelation, from areas of slow or absent flow, where minimal or no variation is observed. This ability to segment and quantify perfusion across superficial and deep vascular plexuses has positioned OCTA as a critical tool for both research and clinical practice in retinal vascular diseases.

OCTA enables detailed, non-invasive visualization of radiation-

induced microvascular alterations in the retina [30] (Fig. 1). In early stages of RR, OCTA commonly reveals enlargement of the foveal avascular zone, pronounced capillary dropout in both the superficial and deep capillary plexuses, and decreases vascular density – even before classical clinical or FA signs emerge [24,31,34]. Quantitative OCTA metrics afford sensitive detection of perfusion deficits and retinal ischemia, supporting the re-definition of diagnostic thresholds for radiation retinopathy and opening prospects for prophylactic intervention [24,34]. Artificial intelligence (AI) represents a promising adjunct to enhance OCTA-based diagnostics in retinal disease and radiation retinopathy [38,39]. Deep learning algorithms can automatically segment and quantify vascular biomarkers—including vessel density, FAZ area, and zones of non-perfusion—with accuracy comparable to or exceeding expert graders [38]. Furthermore, predictive AI models may identify patients at highest risk for severe complications and optimize individualized treatment strategies, such as the initiation and monitoring of anti-VEGF therapy. By integrating multimodal imaging data and longitudinal disease progression patterns, AI has the potential to transform management paradigms, enabling earlier diagnosis, improved risk stratification, and precision-based therapeutic planning in patients undergoing ocular radiotherapy. However, current technical limitations of OCTA do not allow assessment of the peripheral retina. Therefore, FA remains the gold standard for whole retina evaluation, until widefield OCTA becomes an option [40].

Laser speckle flowgraphy (LSFG) is a non-invasive imaging technique that provides a quantitative assessment of ocular blood flow by analyzing the speckle pattern generated when coherent laser light is scattered by moving erythrocytes within the eye. This technology enables the evaluation of dynamic blood flow in multiple ocular structures, including the optic nerve head, retina, and choroid, with high reproducibility [41]. In the context of radiation retinopathy, LSFG has demonstrated a significant reduction in retinal and choroidal perfusion

<figure><img src="image_4.png" /></figure>

Niaudet et al.

**Fig. 1.** Radiation maculopathy occurring 20 months after proton beam therapy for choroidal melanoma. A. widefield retinography showing posterior choroidal melanoma. Inset: close-up view of the macular area, showing hemorrhage (arrows) and cotton-wool spots (asterix). B. Corresponding fluorescein angiography demonstrating macular ischemia (foveal avascular zone enlargement), along with vascular telangiectasies. C. Magnification of the macular area depicted on the white square in (B). D. Corresponding OCT angiography, clearly demonstrating non-perfusion areas, telangiectasises and the decrease in vascularity in areas close to ischemia.
in irradiated eyes, suggesting that impaired microcirculation can be detected before overt clinical manifestations [33,41]. These findings indicate that LSFG may serve as an early biomarker of radiation-induced microvasculopathy, potentially aiding in the timely identification of patients at risk of progressive ischemic damage. However, its clinical applicability remains limited due to restricted availability and the need for further validation in larger prospective studies.

Structural changes in radiation retinopathy

Over time, areas of capillary closure extend, defining RR as an occlusive micro-angiopathy, with non-perfused areas, reminiscent of other vascular retinopathies such as diabetic retinopathy and those associated with retinal vein occlusion [32,35]. Accordingly, en-face reconstructions of the superficial and deeper retinal plexi on OCT-A scans reveal an enlargement of the foveal avascular zone, a decrease of the capillary density, and areas of non-perfusion [30]. The vessels surrounding those damaged areas adopt fusiform and saccular dilations, especially after high doses of RT. Microaneurysms can become thrombosed or regress entirely. Electron microscopy has revealed recanalisation processes in which newly formed microvessels grow within the collagenous matrix left behind after endothelial cell loss. These neovessels represent an aberrant adaptation of the retina to ischemia, and are characterized by an increased collagen deposition and the presence of vessel fenestrations [42]. At the sites of microaneurysms, focal leakage becomes progressively apparent by FA at the vessel periphery, and further progressing to an edema on FA and electron microscopy. This edema results in increased retinal thickness detectable by OCT, about one year after radiation and several months before symptoms of maculopathy [26].

Ischemia and edema define end stage RR, where neovessels can grow into the vitreous cavity and result in intravitreal hemorrhage and retinal detachment in case of traction exerted by the retinal fibrovascular complex [32]. This neovascularization is driven by the key pro-angiogenic factor vascular endothelial growth factor (VEGF) secreted by hypoxic ischemic cells, i.e. Muller cells, inflammatory cells recruited in the retinal pigment epithelium, and which promotes angiogenesis. This late stage can be referred to as “proliferative RR”[43]. The ischemic areas appear on imaging as non-perfused vessels on FA, as whitish areas deprived of capillaries on fundus, and as altered hyperreflective appearance in the inner retina on OCT [44]. Retinal neovessels are detectable on fundus views but are better highlighted by FA, as abnormal hyperpermeable neovessels in the vicinity of retinal ischemic areas. In patients’ biopsies, abnormal neovessels appear as fibrovascular structures extending inside the vitreous space, a shared feature with other vascular retinopathies [45]. Besides, observation at higher resolution by electron microscopy deeper have evidenced that neurons suffering from ischemic insult exhibit dilated morphology and are filled with organelles in electron microscopy. Neuronal compartments are often affected in late stages of RR (thinning of inner and outer nuclear layers, detectable on OCT) [46–48]. Ischemia represents a major and rapid cause of neuronal dysfunction in the retina [49]. Macular edema is the accumulation of excess fluid in the extracellular space within the macula. It results from the disruption of the blood-retinal barrier secondary to the secretion of pro-angiogenic and pro-inflammatory factors by the damaged retinal cells. Only significant edema is visible through fundus examination, showing cystoid cavities within the retina, while mild and moderate edema are better seen on OCT imaging [22]. Ischemia, vascular abnormalities and edema continue worsening afterwards, leading to vision loss.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Dynamic pathophysiology of radiation retinopathy. Early cellular events (left) precede clinical observables, which coincide to the phase of damage extension (center). The advanced stage (right) is characterized by deleterious mechanisms precipitating vision loss.</figcaption></figure>
Radiation retinopathy beyond endothelial cell injury

In addition to endothelial damage, other retinal cell types and the perivascular microenvironment likely play key roles in the pathogenesis of RR (Fig. 2). Both clinical and experimental data suggest context-dependent pericyte vulnerability and pericyte-endothelial imbalance, supported by classic angiographic-histopathologic correlations in RR and by dose-response microvascular injury observed in rat retina after clinically relevant proton schedules [35,50]. Long-term primate studies further document progressive vascular remodeling following irradiation [51]. Leukocyte behavior also contributes to early microvascular dysfunction. Intravital imaging in rats has demonstrated radiation-induced leukocyte entrapment and microcirculatory slowing [52]. Moreover, activated microglia modulate neuronal outcomes and have emerged as potential therapeutic targets, consistent with recent findings in RR models [53]. Outer-retinal and choroidal involvement is increasingly recognized. Swept-source OCT-A has revealed early choriocapillaris flow voids after ocular radiotherapy, sometimes preceding overt macular changes, with histologic confirmation of radiation-induced choroidopathy [34,54].

Therapeutic interventions in RR

Despite significant progress in understanding RR, effective therapeutic options remain limited. Current strategies are primarily initiating after the onset of retinal damage to slow disease progression, However, preventive interventions are gaining interest and have shown promising early results.

• Anti-VEGF Therapies

When the ischemic retina surface is large, high VEGF concentration can also result in iris and trabecular meshwork neovascularization, leading to neovascular glaucoma. The treatment of RR has improved with the advent of anti-VEGF therapies, the standard of care for treatment of macular edema associated to RR. VEGF-inhibitors have demonstrated early visual gain and visual acuity preservation without increasing the risk of tumor dissemination [49]. They help reduce macular edema and slow down RR progression. They can be used prophylactically directly after ocular irradiation [50,51]. However, their therapeutic efficacy remains limited by toxicities and resistance mechanisms [55]. Other therapies, such as intravitreal steroid injections also improve macular edema but their effect on RR is more debated [56–58]. While anti-VEGF treatment is effective, concerns regarding atrophy and ceiling effects underscore the need for complementary approaches [59,60]. Anti-VEGF therapy remains the cornerstone of radiation retinopathy (RR) management, but multimodal strategies targeting endothelial specialization, inflammation, and neuronal survival are warranted to effectively address the multifactorial pathophysiology of RR.

• Laser Photocoagulation

Over time, severe vascular dysfunction develops, leading to non-perfusion of extended retinal areas seen on FA. Large areas of non-perfused retinal tissue releases high levels of anti-VEGF in response to hypoxia, which does not improve retinal perfusion but, if prolonged, induces the widespread development of retinal neovessels. Treatment is based on laser photocoagulation of the ischemic retina, thus preventing the development of retinal neovascular proliferation and neovascular glaucoma [61]. Destruction of the ischemic retina will reduce VEGF secretion, but also increase oxygenation of the non-photocoagulated retina [62]. This treatment is indicated when the ischemic surface is large (>25 % of the retina) and therefore at risk of proliferative complications. Precision radioprotection specific to the underlying molecular mechanisms is still warranted.

<table><caption>Table 1
Preclinical models of RR with related references.</caption><thead><tr><th>Model</th><th>Advantage</th><th>Technical range</th><th>Main features of RR progression</th><th>Vascular closure</th><th>Vascular alterations</th><th>Cotton wool spots</th><th>Late RR</th></tr></thead><tbody><tr><td>Monkey</td><td>Holangiectic models (triple vascular plexi radiating from the optical nerve)<br>Macula</td><td>Fundus examination, FA combined with histological observations (trypsin digest and electron microscopy [48])</td><td>Endothelial loss observed simultaneously in multiple foci on trypsin digests and coinciding with small areas of capillary non-perfusion observed by FA</td><td>As in humans, vascular loss and obstruction extending over a period of 4 to 8 months, even though microaneurysms less frequently detected</td><td></td><td>Cotton wool spots appearing from 10 to 20 months post-radiation in the subjacent neural tissue in monkeys [49,50]</td><td>– Spots of intra-retinal NV, coinciding closely with areas of massive leakage observed by FA<br>– Some of the vessels enlarged and surrounded by a thick collagen cast, as in humans [49–51]<br>No evidence of late stage RR except ischemia</td></tr><tr><td>Pig</td><td></td><td>Fundus examination, FA and OCT<br>– Follow-up until 9 months<br>– Unique doses ranging from 0 to 90 Gy [52]</td><td>Capillary dropout, dose-dependent (severity and time)</td><td></td><td>Microaneurysms present by 3 months post-RT</td><td>Cotton wool spots present</td><td></td></tr><tr><td>Rodents (Mouse &amp; Rat)</td><td></td><td>Fundus examination, FA combined with histological observations (trypsin digest and electron microscopy)<br>– Large range of gamma radiation (unique doses ranging from 2 to 20 Gy) and observations times (from 1 h to one year) [34,48,53]</td><td>– Fundus examination, FA and OCT<br>– Follow-up until 9 months<br>– Unique doses ranging from 0 to 90 Gy [52]<br>– Fundus examination, FA combined with histological observations (trypsin digest and electron microscopy)<br>– Large range of gamma radiation (unique doses ranging from 2 to 20 Gy) and observations times (from 1 h to one year) [34,48,53]</td><td>– Vascular changes appearing in long-term (6 months) irradiated animals (15 and 20 Gy).<br>Vascular closure evidenced as closed basement membranes deprived of intact EC, sometimes recognized by glial cells. Further confirmed by acellular capillaries evidenced by trypsin digests. Confirmed using proton therapy [54].<br>– Closure areas surrounded by endothelial cells with altered (fusiform) morphology although little microaneurysms. Rodents in general seem to require much higher levels of capillary damages before developing microaneurysms, and recanalized and telangiectatic vessels could also be observed.</td><td></td><td></td><td>– Increased extracellular space in between neurons while<br>– In the ganglion cell layer, axons swollen with dense bodies, vesicular and mitochondrial accumulation that coincide with cotton wool spots.</td></tr></tbody></table>
* Other therapies

Other therapies had been tried in small, published cases series and included hyperbaric oxygen to increase oxygenation and initiation of cellular and endothelial repair mechanisms [63,64]. Furthermore, oral pentoxifylline, a methylxanthine derivative with vasodilatory and rheological properties, has been shown to increase ocular blood flow in healthy eyes [65]. A single case report described complete reversal of radiation-induced retinopathy and improved visual acuity after 8 months of treatment [66]. However, due to the lack of larger studies, safety and efficacy of these therapies in these contexts of RR with variable evolution, remain unestablished. Other therapeutic strategies extrapolated from radiation-related research, including antioxidants and endothelium-protective agents such as ACE inhibitors and statins, have been proposed as potential options; however, evidence supporting their efficacy in radiation retinopathy remains limited and is primarily based on preclinical data. Mechanistically, the central role of endothelial injury in RR aligns with what happens in other radiosensitive tissues. In the intestine, endothelial apoptosis can initiate radiation-induced damage, while in the heart, microvascular injury and rarefaction are key drivers of radiation-induced pathology [67,68]. Taken together, these findings support a *trans*-organ perspective in which a multi-step cascade, comprising DNA damage, apoptosis or mitotic death, inflammation, and subsequent tissue remodeling also applies to the retinal microvasculature.

## Translational models deciphering neuro-vascular dysfunctions in radiation retinopathy

In complex tumor topologies, e.g. with tumor abutting the retina, it may not be possible to spare the eye from radiation exposure based on beam or particle properties alone. One intense area of research, however, is ultrahigh dose rate RT (FLASH-RT, mean dose rate ≥40 Gy/s), which has been shown to have paradoxical radiobiological effects on brain, lung, skin and gut tissues. In either case of conventional dose rate RT modalities or FLASH-RT, specific data on RR will be warranted.

To investigate these mechanisms, a variety of animal models have been employed. Animal models have been pivotal in identifying pathological features of RR, such as confirming capillary closure as a distinctive feature of RR, and identifying concomitant capillary dropout by electron microscopy. Additionally, cotton-wool spots, indicating neuronal ischemia, can also be observed. In contrast, microaneurysms seem less frequent in most animal models than in human, and RR in animal models seldom progresses to a late stage, with neo-vascularization mostly evidenced in monkeys (Table 1). Among animal models, primates offer the closest approximation to the human retina due to the presence of a well-developed macula, which plays a central role in high-acuity vision. Other smaller species, such as dogs, pigs, and rodents, either lack a macula entirely or rely minimally on cone-dense retinal regions. Furthermore, anatomical differences such as choroidal vascular architecture and the presence of the tapetum lucidum (in pigs and dogs) introduce additional variability. Nevertheless, all these species share a holangiotic retinal vasculature, making them useful for studying general vascular responses to radiation, even if their translational relevance varies [56,57,69]. A detailed characterization of RR progression at the cellular level is still lacking, compared to what has been achieved in other retinal vasculopathies [70,76,77]. Organoid models have proved invaluable in providing human iPSC-derived, large-scale models that are very well suited to reconstitute temporal trajectories of pathologies at the single-cell level [78,79]. Retinal organoids have emerged as powerful tools to recapitulate development and pathological stages, and to support therapy screening [80]. As with other organoid models, retinal organoids are difficult to integrate with the microenvironment (immune and endothelial cells), which poses a major limitation for a vascular retinopathy such as RR. Yet, the rapid improvements in vascularization of organoids are expected to enable swift

progress in modeling RR [81,82].

## Understudied mechanisms in radiation retinopathy

To overcome the limitations of current therapies, particularly anti-VEGF strategies [59,60], attention has turned to underexplored mechanisms that could expand the therapeutic repertoire for RR.

Specifically, three relatively unexplored mechanisms may represent novel targets for therapeutic intervention:

* Early protection against endothelial damage

The vascular compartment has long been proposed as the main target in RR, as multi-focal capillary closure is a central event [71]. The temporal coupling of delayed endothelial proliferation with persistent DNA damage and mitotic death likely drives capillary dropout and lesion progression, consistent with ultrastructural evidence of late endothelial loss following low-dose X-irradiation and established frameworks of programmed cell-death [42,83,84].

Yet, the precise determinants of vascular demise remain elusive. Vessel obstruction seems to be preceded by endothelial elongation. It also coincides with the appearance of empty collagen sleeves in electron microscopy or trypsin digest, a cellular feature marking endothelial loss and identified in diabetic retinopathy [19]. Endothelial loss might result from post-mitotic death, affecting a small sub-population of angiogenic endothelial cells engaged in division. Indeed, the mitotic index of endothelial cells, despite being low, is still higher than the neuronal one [35]. Endothelial-protective interventions derived from ischemic retinopathy models—such as inhibition of endothelial apoptosis—offer direct therapeutic avenues [85]. On the other hand, interphasic death of endothelial cells might also intervene, since the mature retinal network is largely non-proliferative. In fact, high dose of ionizing radiation has been shown to provoke singular death of the endothelium outside of the retinal system, for instance, through the acute generation of the pro-apoptotic sphingolipid ceramide acid sphingomyelinase [67]. Other mechanisms such as cell death following sudden diminution of perfusion have also been evidenced in other types of retinal vasculopathies [85]. Such endothelial loss might coincide with the early vascular effacement of the deep and the superficial plexi on OCT-A in patients. In that case, decreased flow, due to endothelial cell swelling and narrowing the luminal space, detected using laser speckle flowgraphy in rodents, could be promising for early RR detection in patients [39,52]. Finally, patches of living but stressed endothelial cells could contribute to ischemic damages [68]. Besides endothelial cells, the role played by pericytes during RR has not been fully clarified. Pericytes are radioresistant, and maintain themselves even in case of endothelial loss as seen in human and rats [35,50]. However, this may only be true to some point as other data in monkeys suggest that pericyte loss could follow endothelial loss [72]. A systematic analysis of pericytes throughout the entirety of the retinal network might also reveal regional differences [71]. Whether pericyte status impacts the progression of the vascular lesions during RR, as in diabetic retinopathy, remains to be addressed experimentally and translated to detectable features of RR in patients.

* Limiting the propagation of vascular damage

Whether focal endothelial damage can be mitigated at the intermediate stage of RR remains largely unexplored. It could be addressed at three distinct levels: blockade of mitotic death, hemodynamic modulation and Wnt activation. The main hypothesis elegantly formulated by Archer relies on faulty vessel repair of the damaged endothelium: neighboring endothelial cells would re-enter cell cycles, but unresolved DNA damage would precipitate their mitotic death. In rat models, a late proliferation peak occurs in microvascular cells, 6 months after radiation [42,83]. This peak of DNA synthesis coincides with the extension of endothelial damages, as in other irradiated tissues [84]. Impact of
limiting endothelial proliferation outside VEGF inhibition, for instance through mTOR or tyrosine kinase inhibitors, remains unknown. Additional mechanisms might also intervene, such as recruitment of inflammatory cells including leukocytes as evidenced in rats [52]. If there is a consensus that in RR most vascular changes are concentrated at the level of the capillaries, the exact role of higher caliber vessels remains underexplored. Traditionally considered as affected big long-term phenotypes changes [86,87], arterial vessels in the retina display alterations by radiation in some experimental settings possibly fueling RR: in rats, arteries diameters are diminished after radiation and their smooth muscle cell coverage is affected if the animals undergo a double challenge with diabetes and radiation [52,75], and in monkeys, large vessels exhibit shape changes, which may be indicative of a propagation of damages from capillaries to intermediary vessels. Mural cells can get damaged as well [71,72]. It has been suggested that arteriole occlusion is triggered by high doses of radiation [27]. If blood supply gets disturbed at the level of arterioles, damages could spread faster to broad capillary areas. Again, characterization of fine hemodynamic changes in the retina after radiation is just starting [39]. This stage is also marked by microaneurysms and telangiectatic vessels, both visible throughout RR [72]. While the former are bulged microvessels that seem to form preferentially toward the arterial side, where blood flow is higher [35,88], telangiectasiae seem to form preferentially in the deep retinal plexus [88], and to microaneurysms, also affect intermediary-caliber vessels [42]. Laser photocoagulation has been used to prevent aberrant vascular structure, yet therapies used to treat vascular abnormalities are underexplored. Structural analysis by electron microscopy both in humans, monkeys and rats revealed that telangiectatic vessels exhibit a dense collagen lining, but also fenestrations on the endothelial membrane, an interesting observation as it might point out to dedifferentiation of the endothelium [72], described in particular upon over activation of TGFb or loss of function in the Wnt pathway [89]. The Wnt pathway, through the Norrin ligand and its endothelial receptors Frizzled4 and low-density lipoprotein receptor-related protein5, is critically required during development for acquisition of endothelial specialization and in particular barrier properties through its retinal ligand Norrin, and can be diminished during vascular retinopathies [90,91]. Pharmacological restoration of this pathway and of endothelial specialization might therefore represent an unexplored therapeutic avenue [92–94].

* Rescue from neuronal damage

Although RR has been addressed primarily through its vascular component, incorporating neuroprotective strategies alongside endothelial protection may offer additional value. Early neuronal changes remain poorly understood, and investigating them could reveal new avenues to delay or prevent disease progression. As for advanced stages, on which management of RR predominantly focuses, ischemia plays in RR a quite distinct role from common vascular retinopathies. In those, the driving pathological processes are neovascularization and its resulting complications, such as edema. In RR, the direct ischemic damage can be more prevalent than the subsequent neovascularization, highlighting the distinct nature of this condition. This is especially true upon severe radiation damage in patients where the intermediary vessels might get affected [35]. Genome-wide screen in organoid or animal models have the potential to define the regulatory programs mediating insult in neurons, then their protective counterparts leading to drugable targets, as recently evidenced in glaucoma [95,96]. Neuroprotective strategies would benefit from enhanced biomarkers [97]. Evidence of early neuronal dysfunction starts to emerge from advanced clinical imaging of the inner retina [46]. Damage to neurons might also be dependent on the interplay with other non-neuronal cell types: RPE, choroid and microglia. Results from patients exposed to brachytherapy suggest partial or total loss of choroid vessels close to the tumor base [30,54,98], but these lesions seem less frequent in patients treated for

non-ocular tumors [27]. OCT-A analyses even demonstrated recently that choriocapillaris alterations, seen as flow voids within the choriocapillaris architecture occur early after radiation therapy for choroidal melanoma, and even before clinical signs of RR [34]. Damage to the photoreceptors and to a less extent, the choroid vessels were also evidenced in animal models [42,51,72–74]. Damage to the outer segment of the retina could significantly expedite the neuronal damage. In particular, ischemia linked to choriocapillaris damage has also been suggested in diabetic retinopathy [20,34]. Finally, the impact of microglia, a notoriously versatile actor of neuronal damage, remains to be clarified [53,99]. Microglial inflammation emerges as a modifiable driver of neurodegeneration in both degenerative eye diseases and RR models [53,99]. In addition, RPE hypoxia-response plasticity can limit anti-VEGF efficacy by upregulating HIF-regulated angiogenic genes [55].

Insights from other retinal diseases further inform RR biology. Diabetic retinopathy demonstrates pericyte loss, endothelial dedifferentiation, and choriocapillaris ischemia, mirroring microvascular pathology in RR [19,20]. In neovascular AMD, prolonged VEGF antagonism can exhibit ceiling effects and exacerbate atrophy, highlighting the need for complementary pathways beyond VEGF alone [59,60]. Finally, transcriptional survival programs identified in glaucoma provide neuron-intrinsic targets that may be adaptable for post-irradiation retinal neuroprotection [95–97].

## Conclusion and perspectives

Clinical and experimental models have established landmarks in the progression of RR: vascular occlusion, spreading of vascular damage, retinal ischemia and edema mark the early, intermediary and late stage of clinical RR, respectively. These can now be deciphered non-invasively with high resolution imaging with major impact on mechanistic approaches and theragnostic developments. Yet, much remains to be as for the detailed mechanisms leading to these critical steps, a precise understanding of their spatial organization, and the interaction with other cellular actors > organization and their interaction with other cellular actors (inflammatory cells, neuroglial compartment). Equally important is the need to evaluate if specific mechanisms, such as choroidal lesions, can be linked to specific radiation types and doses. This would provide earlier action points and treatment options. Increased understanding of the mechanisms leading to sight impairment is equally needed to tailor management of the late disease.

## Authors contribution

All authors participated in writing the manuscript.

## CRediT authorship contribution statement

Colin Niaudet: Writing – original draft, Conceptualization. Thibaud Mathis: Writing – original draft. Thao-Nguyen Pham: Editing. Jean-Claude Quintyn: Editing. François Paris: Writing – original draft, Conceptualization. Juliette Thariat: Writing – original draft, Conceptualization.

## Funding

The authors acknowledge support for their research work including R2 grant (NExT, CN), PIOTox-SEQ-RTH22 (INCA, JT), PMRT ERDF-FSE 2014–2020 7/16/24 9:09:00 AM18P03532/18 E01765 (UE, JT).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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