Analysis of the Characteristics of the Most Resilient and Most Vulnerable Retinal Ganglion Cell Subtypes in a Rat Chronic Glaucoma Model

Characterization of the Most Resilient and Most Vulnerable Retinal Ganglion Cell Subtypes in a Rat Chronic Glaucoma Model

Background

Retinal ganglion cells (RGCs) are a class of neurons responsible for transmitting visual signals generated in the retina to the brain; their axons converge to form the optic nerve and establish connections with postsynaptic target regions in the brain. Glaucoma is a neurodegenerative disease characterized by the progressive loss of retinal ganglion cells, causing gradual and irreversible visual field damage that can ultimately lead to blindness. Major risk factors for glaucoma include elevated intraocular pressure (IOP) and increasing age; genetic factors, abnormal vascular regulation, and autoimmune factors are also involved in its pathogenesis. Although the exact mechanisms by which glaucoma causes retinal ganglion cell death have not been fully elucidated, apoptosis is currently considered the main pathway. Retinal ganglion cells (RGCs) are highly heterogeneous. In recent years, transcriptomics-based studies in mice have classified approximately 45 RGC types based on molecular characteristics and confirmed that these molecular classifications correspond to dendritic morphology and visual response properties. Although each RGC subtype has unique and stable characteristics, the exact relationships among these hallmark features have only been partially elucidated. Not all RGC subtypes have the same vulnerability; therefore, this study aimed to comprehensively analyze the differential loss of retinal ganglion cell subtypes using a rat chronic glaucoma model.

Methods

This study was conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals. All experimental protocols complied with European (2010/63/EU) and Spanish (RD53/2013) regulations on the protection of experimental animals and were approved by the Animal Welfare Ethics Committee of the University of the Basque Country (Ethics No.: CEEA-UPV/EHU M20/2024/269). All animal experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

A total of 27 adult female Sprague–Dawley (SD) rats aged 5 months were used. During the experiment, animals were housed under a 12-hour light/dark cycle with constant appropriate temperature and humidity, and had free access to water and standard pellet diet. Before surgery, rats were subjected to general anesthesia with isoflurane (oxygen flow rate 0.8 L/min; 4% isoflurane for induction and 2% isoflurane for maintenance), placed on a heating pad to maintain body temperature, and simultaneously administered a topical anesthetic.

A glaucoma model was established in the left eye of each animal, with the right (contralateral) eye serving as the control. To exclude effects of the surgical procedure on the contralateral eye, retinas from age-matched control rats were compared with retinas from the contralateral eyes of model rats, confirming no difference between them. According to previously reported methods, elevated IOP was induced in rats by cauterizing three episcleral veins of the left eye. Briefly, two dorsal episcleral veins near the superior rectus muscle and one temporal episcleral vein near the lateral rectus muscle were isolated and freed from surrounding tissue. A heat cautery device was precisely applied to the selected veins, taking care to avoid thermal damage to surrounding tissues. After each procedure, an antibiotic-steroid combination ophthalmic ointment containing chloramphenicol and dexamethasone was applied to the ocular surface.

IOP in the left and right eyes of rats was measured using a Tonolab rebound tonometer designed for rats and mice (Icare Finland Oy, Helsinki, Finland), three times per week. The tonometer was positioned perpendicular to the corneal apex, and at least three independent measurement sets were obtained (each value was the average of six readings). All measurements were performed at the same time each day to avoid the influence of circadian IOP fluctuations. IOP results are expressed as mean ± standard error: from the day of surgery to postoperative day 40, data from all rats were pooled and daily averages were calculated. Rats were maintained in a state of ocular hypertension for 40 days; rats unable to sustain elevated IOP were excluded from the study.

Immunofluorescence staining was performed on retinal flat mounts: anti-RBPMS antibody (which labels all retinal ganglion cells) was used to mark RGCs and was co-labeled with the following subtype-specific antibodies: CART, melanopsin (OPN4), Foxp2, Islet1/2, SPP1, and Tbr2.

The percentage of RBPMS-positive cells was used as an indicator to quantitatively analyze RGC loss and cell subtype distribution in different retinal regions.

Figure 1.

Results

In glaucomatous eyes, RGC survival in the peripheral retina decreased, with rates of 65.44% in the dorsonasal region and 76.03% in the ventrotemporal region.

Proportion of CART-positive RGCs:

• Dorsonasal: decreased from 32.9% ± 5.15% to 20.26% ± 2.64%

• Ventrotemporal: decreased from 33.07% ± 4.09% to 22.65% ± 2.65%

This suggests that this subtype is more vulnerable.

In contrast, the proportion of OPN4-positive RGCs in the dorsonasal region increased from 3.27% ± 1.34% to 6.99% ± 2.31%, suggesting that intrinsically photosensitive retinal ganglion cells (ipRGCs) are more resilient.

The proportions of SPP1-, Foxp2-, Islet1/2-, and Tbr2-positive RGCs showed no significant changes, suggesting that their loss occurred proportionally with total RGC loss.

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Conclusion

Different RGC subtypes differ in susceptibility to IOP: OPN4-positive RGCs (ipRGCs) are more tolerant, whereas CART-positive RGCs (ON–OFF direction-selective ganglion cells, ooDSGCs) are highly susceptible.

This result suggests that retinal ganglion cells (RGCs) in the peripheral retina are particularly vulnerable to ocular hypertension injury, consistent with previous reports in various glaucoma animal models, clinical studies of glaucoma patients, and other neurodegenerative diseases such as Alzheimer’s disease. Specifically, RGCs in the dorsonasal and ventrotemporal peripheral regions have higher susceptibility to injury; however, involvement of other retinal regions at later experimental stages cannot be excluded. This phenomenon is consistent with clinical evidence that glaucomatous damage occurs more frequently and progresses more rapidly in the superior visual field, most likely because elevated IOP is more strongly associated with superior retinal nerve fiber layer defects in open-angle glaucoma. In animal models, heat cauterization-induced ocular hypertension first causes impaired retrograde axonal transport in dorsal retinal RGCs, followed by structural loss. Although the susceptibility of the ventral retina remains unclear, elucidating regional differences in retinal vulnerability is crucial for revealing the mechanisms of glaucoma progression.

In summary, there is an urgent need for in-depth research on ooDSGC degeneration and for exploration of targeted neuroprotective strategies. Future studies should be devoted to developing therapies that can protect, regenerate, or replace ooDSGCs.

Publication

Authors: Noelia Ruzafa; Xandra Pereiro; Laura Prieto-López; Aritz Urcola; Arantxa Acera; Elena Vecino

Publisher: Investigative Ophthalmology & Visual Science

October 2025, Vol. 66, 5.

doi: https://doi.org/10.1167/iovs.66.13.5

Original link: https://iovs.arvojournals.org/article.aspx?articleid=2810897&resultClick=1