| Author: | Qiu, Chunting |
| Title: | Preserving vision under stress : RGC vulnerability to alkali burn and elevated intraocular pressure |
| Advisors: | Pan, Feng (SO) |
| Degree: | Ph.D. |
| Year: | 2026 |
| Department: | School of Optometry |
| Pages: | 215 pages : color illustrations |
| Language: | English |
| Abstract: | Purpose Retinal ganglion cells (RGCs) serve as the final output neurons of the retina and as essential conduits for transmitting visual information to the brain. Given RGCs' inability to regenerate, any loss of RGCs results in permanent visual deficits, underscoring the need to understand how pathological insults compromise their survival to develop effective therapeutic strategies. This thesis investigates dysfunction and degeneration of alpha retinal ganglion cells (αRGCs) under two clinically essential conditions: chemical injury from corneal alkali burns and elevated intraocular pressure (IOP), which is relevant to glaucoma. Corneal alkali burn can cause irreversible damage to both the ocular anterior segment and posterior segment. Our first study aims to investigate the role of the gap junction subunit connexin 36 (Cx36) in mediating secondary cell death and its impact on the apoptosis of RGCs following ocular alkali burns. Since glaucoma is the most common cause of irreversible blindness, our second aim was to explore the effect on RGCs under different levels of high ocular hypertension and mechanical stress. Methods Wild-type C57BL/6J (WT) mice, KCNG-YFP, and homozygous connexin36-knockout (Cx36 KO HOMO) mice were used in the study. TUNEL staining and confocal microscopy were used to detect apoptotic cells. Intracellular dye injection was performed to confirm the neuronal morphology in the mouse retina. The light-evoked spikes, excitatory postsynaptic currents (EPSCs), and inhibitory postsynaptic currents (IPSCs) of RGCs were recorded using 525nm full-field light as the stimulus to assess the RGC function. Study 1: Corneal alkali burn models were established in WT and Cx36 KO HOMO mice by applying 2µl of 1 M sodium hydroxide solution to the cornea. The gap junction blocker meclofenamic acid (MFA; 200 μM) was administered via intravitreal injection immediately after the corneal alkali burn. Study 2: Pressure-related injury was modeled using acute and sustained ocular hypertension to approximate glaucomatous stress. A sustained ocular hypertension (SOH) model was established in WT mice through 2 µl silicone oil injection into the anterior chamber. An acute ocular hypertension (AOH) model was induced in WT mice by inserting a homemade micropipette connected to a saline-filled container into the anterior chamber for 50 minutes. The container height was calibrated to induce an intraocular pressure of 75 mmHg. Two direct mechanical pressure methods were applied to isolate immediate effects on RGC physiology: one using a pressure controller and another using direct homemade micropipette compression. Behavioral visual performance was evaluated using the optomotor response (OMR) assay, and retinal function was assessed with electroretinography (ERG). Results Study 1: Corneal alkali burn produced rapid damage in RGC number and function in WT mice. The RGC loss was characterized by time-dependent progression, peaking at 24 hours, spreading from peripheral to central retinal regions. Six hours post-injury was determined to be the appropriate time point to assess RGC functional deficits in the corneal alkali burn mouse model. Within 6 hours, transient αRGCs showed significantly reduced light sensitivity, EPSCs, and IPSCs (all p < 0.05). We identified Cx36 gap junctions as key mediators of secondary RGC death in this setting. Both genetic ablation of Cx36 and pharmacological blockade with MFA significantly improved RGC survival after injury. Study 2: In the SOH mice model, ERG recording showed significant decreases in positive scotopic threshold response (pSTR) and negative scotopic threshold response (nSTR), while there were no statistical changes in a-wave and b-wave after 4-week injury. The number of αRGCs was significantly reduced, whereas AII amacrine cell populations remained stable following 4-week injury. All types of αRGCs in 4-week SOH mice exhibited significantly reduced light sensitivities. In the AOH mice model, visual contrast sensitivity started to decline in 2 weeks but was significantly reduced by 4 weeks. There were significant decreases in αRGCs after 2-week and 4-week AOH injury, but no significant difference in morphology. All types of αRGCs in 4-week AOH mice exhibited significantly reduced light sensitivities. To test whether pressure directly perturbs RGC signaling, we applied controlled mechanical pressure to RGCs. Following sequential pressure application to αRGCs via a pressure controller, αRGC light sensitivity decreased. Moreover, micropipette-induced mechanical pressure decreased current amplitudes at various potentials without affecting reversal potentials. Conclusions This work elucidates how two major insults—corneal alkali burns and elevated IOP—compromise RGCs through distinct yet converging mechanisms. Following corneal alkali injury, RGCs underwent rapid early-stage decline involving Cx36 gap junction-mediated secondary death. The gap junction antagonist MFA demonstrated neuroprotective potential by blocking this secondary cell death process. Following IOP elevation, early behavioral deficits could be masked by compensatory mechanisms. However, damage to synaptic function and cell survival could be detected, providing new insights for early diagnosis. Direct pressure manipulation experiments provide a mechanistic link between elevated IOP and visual dysfunction by revealing immediate reductions in light sensitivity and synaptic transmission, potentially due to spurious spiking and baseline fluctuations that mask genuine visual signals. |
| Rights: | All rights reserved |
| Access: | open access |
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