2023 Shaffer Research Grants

Glaucoma is a disease of the optic nerve and retinal ganglion cells (RGCs) that worsens over time and can result in permanent visual disability and blindness. Increased eye pressure is an important risk factor for glaucoma, and all current treatments are based on reducing eye pressure to slow the speed at which glaucoma worsens. However, there is evidence that there are other ways to prevent glaucoma from worsening that do not involve the reduction of eye pressure. This process, called neuroprotection, may already be occurring when a certain glaucoma medication, brimonidine, is used. Brimonidine achieves it effects by attaching to a specific protein called a receptor, which is present on the cells that are injured in glaucoma – RGCs. This receptor, Adra2a, is likely very important for neuroprotection to happen in RGCs. Despite this, we know little about what Adra2a normally does in the eye, what it does in glaucoma, or how it interacts with brimonidine in the eye. This proposal will study vision, electrical activity, and anatomy of retinas from mice that lack the Adra2a gene. With this approach, we expect to understand, for the first time, the normal function of Adra2a in the eye. Once this is understood, we will further test how reduced or increased amounts of Adra2a impact glaucoma in mice and explore how the addition of brimonidine modifies these effects. If successful, we can unlock one key to neuroprotection, and use this information to develop new ways to slow the speed at which glaucoma worsens.

Lipoxins and their receptors are a recently identified resident neuroprotective circuit in the retina. Our current understanding places astrocytes as the source of lipoxins and retinal ganglion cell as their only known target. Retinal stress and ocular hypertension downregulate the homeostatic formation of lipoxin and treatment with Lipoxin B4 (LXB4) is neuroprotective in a model of glaucoma. The mechanism of action for LXB4 and additional cellular targets in the retina remain to be discovered. Single cell RNA and kinase screening assays identified potent and cell selective actions for LXB4 in healthy retinas. Unexpectedly, the cell type targeted by LXB4 treatment in healthy retinas were microglia, the resident immune cell in the retina and optic nerve. LXB4 down-regulated genes that are upregulated by ocular hypertension. These genes control microglial reactivity, which switches their function from homeostasis to driving inflammation and injury responses. LXB4 treatment reduced microglia reactivity in a model of chronic ocular hypertension. Reactive microglia are a key feature of retinopathy and neurodegenerative diseases such as Alzheimer’s. Hence, LXB4 regulation of microglia may be an important early checkpoint to maintain their homeostatic function. This project aims to establish when retinal and optic nerve microglia switch to a reactive phenotype in glaucoma pathogenesis and how they are regulated by astrocyte-generated lipoxins and LXB4 treatment. The goal is to establish proof of concept that regulation microglia homeostatic function is a novel mechanism for the neuroprotective actions of LXB4 and a potential therapeutic target.

Elevated eye pressure is the most significant and only modifiable risk factor for glaucoma. However, is it largely unknown what and where the pressure sensors are in the eye and how they contribute to glaucoma. Our goal is to discover new strategies for treating glaucoma by understanding the mechanisms of how the eye senses pressure. Using human genetic data, we have identified genetic variants in a mechanosensitive protein that are associated with glaucoma risk. We will use molecular and electrophysiological approaches, as well as mouse models of glaucoma to investigate the role of this protein in the eye, and how genetic variation affects protein function and glaucoma risk. These results will advance our understanding of why high eye pressure leads to vision loss in glaucoma and may identify new targets for glaucoma treatment.

The goal of this project is to develop a new imaging marker for retinal damage in glaucoma. Retinal ganglion cell (RGC) loss is the hallmark of glaucoma, and timely management of RGC survival is vital to preserve vision. Noninvasive imaging techniques have been increasingly applied to assess RGC health and glaucoma progression. Optical coherence tomography (OCT) has been used to measure the bulk thickness of the retinal nerve fiber layer (RNFL) which contains RGC axon bundles and other cells and structures. However, the RNFL thickness measurement is not a direct indicator for RGC damage, and the conventional OCT imaging is not sensitive enough to directly

Glaucoma is characterized by progressive neurodegeneration of the optic nerve that if left untreated leads to blindness. The most important risk factors include elevated intraocular pressure (IOP) and age. Currently, lowering the IOP is the only treatment paradigm. However, many treated patients with glaucoma progress in the disease despite therapy and optimal intraocular pressures. We have recently shown that repeated stress can lead to accelerated tissue aging as measured at the transcriptomic and epigenetic level. Upon repeated insult the young retina responds to stress similarly to the old retina. In the application, we aim to investigate whether this phenomenon is reversible and whether the removal of senescent cells which accumulate with age and in the disease can attenuate the cascade of changes that leads to glaucoma. Ultimately, we plan to research approaches to prevent the impact of high eye pressure on healthy retinal neurons. With our approach, we aim to address gaps in the current state of knowledge and to uncover molecular mechanisms behind the pathogenesis of the disease and the relationship of its biggest risk factors – elevated IOP and age.

Current treatments for glaucoma slow disease progression but are not effective for some patients. The retina has an inbuilt immune system which is supposed to fight off potential infections, clean up waste, and support the retina and optic nerve. In glaucoma this system can become pro-inflammatory, worsening the disease. There is research interest in preventing this inflammation as a way of protecting the retina and optic nerve. Our understanding of these processes has improved in recent years. We know that preventing this inflammation from beginning is protective in models of glaucoma but there is great difficulty in translating this to humans. Since the brain is effective at filling in gaps in vision, people can lose a substantial number of nerve cells before noticing problems with their sight. Often glaucoma diagnoses and treatments begin once the disease is already underway. This means that strategies to stop inflammation occurring are unlikely to be practical. We have previously identified that some anti-cancer drugs can turn off or dampen inflammation in the immune cells of the retina even once these processes are well under way. These drugs may not be very suitable for widespread use in glaucoma patients since they have unpleasant side effects, but we can use them as tools to explore how switching off inflammation may work. We will make use of drug libraries developed by the cancer research community to test the best way to switch off inflammation and determine which genes are important in this process. From this information we can develop more targeted strategies by using computer aided drug discovery to find compounds which can produce similar results but with fewer side-effects. We will then test these drugs to determine if they work as predicted. From this approach we can learn more about the process of inflammation in glaucoma, how it may be suppressed, and potentially find new drugs which may be of benefit to glaucoma patients.

The immune response is protective in models of glaucoma, but how the immune system interacts with the optic nerve to promote neuroprotection is still unknown. In the eye, there are resident T cells that are adjacent to the optic nerve, but the importance of these cells has not been explored. In this project, I will examine the kinetics of the immune cell response in the peripapillary choroid, examine the phenotype of the cells that reside adjacent to the optic nerve in health and in glaucomatous conditions, and explore the functional roles of T cells in this area. There has been recent interest in drug delivery to the choroid which has resulted in the approval of choroidal drug delivery in clinical practice. By better understanding how the choroidal immune system plays into the pathology of glaucoma, we will be better able to target the pathology in a way that is complementary to IOP lowering medications and that does not require additional eye drops.

Although glaucoma has many risk factors, the most important is high pressure inside the eye. High eye pressure compresses the optic nerve, causing it irreversible damage. This damage often remains undetected until significant vision loss has occurred and the ability to perform routine activities becomes severely impaired. Angle-closure glaucoma (ACG) is a common type of glaucoma that affects more than 20 million people worldwide. The angle acts as the drain of the eye; it carries away fluid that the eye produces to maintain its shape and function. In angle closure, the drain is blocked by the iris, which leads to buildup of fluid and pressure inside the eye. In normal eyes, the iris moves and changes shape freely. In ACG eyes, the iris moves slowly and exhibits difficulty changing shape. Therefore, the stiffness of the iris is believed to play a key role in developing angle closure and ACG. If doctors had a tool to measure iris stiffness, it could help them identify patients at high risk of ACG who should receive vision-saving treatments with laser and surgery. Iris stiffness can be measured using a form of technology called ultrasound elastography (USE). USE is safe, non-invasive, and can be performed on humans without causing harm or discomfort. However, little is known about how USE measurements of iris stiffness relate to risk of ACG. Therefore, we propose to use our USE system to: 1) establish that irises in PACG eyes are stiffer than in healthy eyes; 2) understand how iris stiffness interacts with other risk factors to cause PACG. We believe our results will establish iris stiffness measured by USE as a strong risk factor for ACG. This could provide doctors with a diagnostic tool to identify patients with higher risk of ACG who would benefit from prompt treatment to prevent permanent vision loss.