Radiation Retinopathy Research Report

Radiation Retinopathy (RR) is a common side effect following radiation therapy (such as brachytherapy or proton beam therapy) in ophthalmic tumor treatments. RR presents as delayed and progressive microvascular alterations, ischemia, and macular edema, which may ultimately lead to vision loss, neovascular glaucoma, and even enucleation of the eyeball in extreme cases. Although anti-vascular endothelial growth factor (VEGF) drugs, steroids, and laser photocoagulation therapy show some efficacy for RR, their effects are limited, and the role of retinal inflammation in RR and its contribution to microvascular damage are not fully understood. This paper aims to explore the timeline of cellular and vascular events following radiation therapy through experimental research.

Research Origin

This paper is co-authored by Cécile Lebon, Denis Malaise, Nicolas Rimbert, Manon Billet, Gabriel Ramasamy, Jérémie Villaret, Frédéric Pouzoulet, Alexandre Matet, and Francine Behar-Cohen. These authors are from various research institutes in Paris, France, including Centre de Recherche des Cordeliers, Institut Curie, Université Paris Saclay, and Sorbonne Université. The paper was published in the 2024 issue of the Journal of Neuroinflammation.

Research Background

RR mostly occurs after ocular tumor treatments, such as brachytherapy or proton beam therapy for choroidal melanoma. Typical RR usually appears 6 months to 3 years, or even later after radiotherapy, manifesting as gradually developing vascular alterations. Clinical symptoms include retinal hemorrhage, neovascular glaucoma, etc., which may lead to severe vision impairment. The incidence and severity of RR are closely related to cumulative radiation dose, dosage distribution, and exposure range. Currently, the treatment options for RR are limited, mainly relying on VEGF inhibitors and laser photocoagulation therapy. Although their effects are positive, they are not curative. Therefore, RR remains an unmet medical need.

Research Method

To address the cause of RR, the research team established a rat model and analyzed the timeline of cellular and vascular events after 45 Gy X-ray irradiation at 1 week, 1 month, and 6 months.

1. Animal Model Construction: Six-week-old male Long-Evans rats were used as experimental subjects, with all experimental steps following the statement of the Association for Research in Vision and Ophthalmology (ARVO). Experimental animals were sampled for analysis at one week, one month, and six months post-injection with 45 Gy of X-rays.

2. Retinal Hypoxia and Vascular Detection: The Hypoxyprobe-1 kit was used to detect retinal perfusion and hypoxic state. Rats were injected with the probe three hours before sacrifice, followed by enucleation of the eyes, and immunofluorescence staining was used to detect hypoxic areas and vascular status.

3. Flatmount Retina and RPE Samples: The retina and Retinal Pigment Epithelium (RPE) of the sacrificed animals were flatmounted and stained, using different antibodies for cellular and vascular analysis.

4. Cryosections and Western Blot: 10 μm thick cryosections were prepared to detect cell death and GFAP (Glial Fibrillary Acidic Protein) expression. Results were analyzed statistically using Prism 8 software.

Research Results

1. Model Construction and Morphological Changes: A RR rat model was established through three cycles of 15 Gy X-ray irradiation, totaling 45 Gy, which caused slight hair loss and skin pigmentation changes around the eyes, with no significant early damage observed in the retina.

2. Retinal Hypoxia and Vascular Status: Pimonidazole staining showed significant retinal hypoxia in the rats at 6 months, with a significant reduction in small blood vessel numbers. This is similar to patients with RR in the rat model, presenting chronic retinal hypoxia and vascular alterations.

3. Cell Death: Radiation-induced retinal cell death was detected using TUNEL technology, starting from one week after irradiation and persisting over the following months. It was primarily manifested in the Outer Nuclear Layer (ONL) cell death, suggesting persistent stress caused by radiation.

4. Glial Cell Activation and Vascular Damage: GFAP staining detected rapid activation of retinal glial cells after radiation, which gradually infiltrated into the inner retinal layer. Further verified by Western Blot, the expression of GFAP was significantly increased from one week to six months. At the same time, Iba1 (Ionized Calcium-Binding Adaptor Molecule 1) staining showed that microglial cells invaded the entire retina at 6 months and were in close contact with blood vessels.

5. Outer Retinal Blood Vessel Barrier (Blood-Retinal Barrier, BRB) Disruption: The structure of the RPE started showing significant changes one month after irradiation, including changes in cell morphology and size, the reorganization of the cytoskeletal F-actin, reduced expression of ZO-1 (Zonula Occludens-1), “zigzag” patterning of the RPE layer, and intercellular openings indicating radiation-induced BRB leakage.

Research Conclusion

This study highlighted the damage caused by radiation to the RPE and retina, especially the impact on the integrity of the inner and outer BRB. The research proved the importance of a persistent inflammatory mechanism in the development of RR. This study in a rat model found high relevance to clinical observations in RR patients, which helps further research into the pathological mechanisms of RR. Future research should delve deeper into choroidal/retinal inflammatory mediators to better target harmful inflammation for treatment and prevent irreversible retinal damage after radiation.