2017 Shaffer Research Grants

Loss of vision in glaucoma results from the irreversible death of retinal ganglion cells (RGCs). A crucial step towards circuit repair in glaucoma is to promote damaged RGCs to regenerate not only axons, but also dendrites to successfully reconnect with their synaptic partners. In this study, we tested the hypothesis that insulin will stimulate dendrite regeneration and the reestablishment of synaptic connections thus improving survival and function in injured RGCs. Using a range of genetic, pharmacological, imaging, and electrophysiological in vivo approaches, we show that insulin promotes striking RGC dendrite and synapse regeneration in injured RGCs. Importantly, insulin promoted robust neuronal survival and rescued light-triggered retinal responses.

Our study reveals that adult RGCs are endowed with the ability to effectively regenerate dendrites and synapses once they have been lost, and identifies insulin as a powerful strategy to restore dendritic morphology and enhance the function and survival of these neurons. Collectively, our data support the rationale for using insulin and its analogues as proregenerative therapeutic targets to counter progressive RGC neurodegeneration and vision loss in glaucoma. In summary, our findings are innovative and a major scientific advancement in the field.

This work was accepted for publication in the prestigious journal Brain (July 2018 issue).

Dr. Adriana Di Polo was awarded the 2019 Shaffer Prize for Innovative Glaucoma Research. The Shaffer Prize, presented annually by Glaucoma Research Foundation, recognizes a researcher whose project best exemplifies the pursuit of innovative ideas in the quest to better understand glaucoma.

Glaucoma is a leading cause of blindness and is associated with degeneration of nerves in the retina of the eye. We have discovered that in the normal eye small molecules called lipoxins, are released by cells that support and maintain the nerves. Under stress, as happens in glaucoma, these cells appear to stop producing enough of the neuroprotective lipoxins and the neural cells and their axons start to die. We propose to study the role of lipoxins in protecting the nerves of the eye and how they might be involved in the development of glaucoma. To do this we will use a newly developed rodent model that enables the pressure in the eye to be moderately raised over several months. We will also use specially bred mice that are unable to normally use the lipoxin molecules. This will allow us to understand the pathways and mechanisms by which lipoxins can protect the eye, and potentially develop new approaches to the treatment of glaucoma.

Despite astounding recent advances in technological capabilities that enable earlier and more accurate diagnosis of glaucoma, the fundamental events that lead to progressive axon degeneration in glaucoma remain incompletely understood. Thus, even when glaucoma is accurately diagnosed at an early stage, there is still a significant risk for progressive loss of vision, which can be severe in some cases despite successful treatment to lower intraocular pressure. A fuller understanding of the sequence of physiological events that lead to axon damage should provide new targets for both diagnostic technology and therapeutic intervention to be applied during a stage when axons are susceptible, prior to the stages of irreversible degeneration upon which current diagnostic paradigms are based. In this project, we plan to develop an assay of axon transport of mitochondria that can be applied in the living eye to study early pathological events in experimental models of glaucoma. Mitochondria are the motile power plants that supply the fundamental source of energy all along each axon for maintenance of its basic functions, most importantly, conduction of electrical signals to the brain. Evidence suggests that abnormalities of mitochondrial function and transport are among the earliest events after axon injury. There is even evidence from clinical studies suggesting that human beings with better mitochondrial function are less susceptible to glaucoma. Thus, having a reliable assay of mitochondrial transport will be beneficial for future studies to investigate the role of this critical function in the early sequence of glaucomatous axon damage.

The retina and the optic nerve are populated by microglia, a cell type supporting neurons. In glaucoma activation of these cells is known to result in the production of toxic molecules that lead to neuronal destruction. However, our preliminary data suggest that suppressing the activity of these cells may not be a beneficial therapeutic strategy. We propose that the response of microglia to glaucoma damage may have two stages. There is clear evidence that activity of microglia can induce damage in glaucoma, but we propose that this is only true in late-stage disease and that during the early stages of the disease microglia exert a protective effect. We will also determine the level of pro-inflammatory cytokines during this process.

Glaucoma patients rarely report receiving instruction on eye drop technique from their doctors, and doctors have little time to instruct patients on eye drop technique. A short educational video that can be watched online at home or on mobile devices may help patients learn correct eye drop technique more easily. This study will be the first randomized trial of an educational video for improving eye drop technique. The Meducation® eye drop technique video from Polyglot Systems instructs patients on all the critical steps of proper eye drop technique in easily understandable language with animations to demonstrate each step. This project will be significant because patients who successfully learn better eye drop technique can have a greatly improved chance of performing the crucial skill of eye drop instillation correctly, without added burden to overworked clinicians. By learning better eye drop technique, patients can avoid vision loss and blindness, as well as painful medication side effects and eye infections from contaminated eye drop bottles. The video is easy to disseminate to patients nationwide and does not take any clinician time to deliver.

This work was accepted for publication in the journal Patient Education and Counseling (December 2018 issue).

Glaucoma is a leading cause of blindness affecting millions of Americans. However, current treatment strategies are not sufficient to prevent progressive injury to specific nerve cells and continuous loss of visual function. To better understand and treat this blinding disease, our proposed project specifically aims to determine the disease-causing importance of a specific molecular process (named autophagy) in experimental glaucoma models. For this purpose, we will model glaucoma in specific mouse strains lacking the activity of specific molecules in nerve cells or glia (another important cell type that are adjacent to nerve cells and play diverse roles to support nerve cells or contribute to inflammatory nerve injury) and analyze specific responses of neurons and glia using up-to-date analysis techniques. We expect that this project will enable us to value whether therapeutic manipulation of autophagy provides protection against nerve inflammation and injury in glaucoma. The new information should help develop new treatment possibilities for patients suffering from this disease.

Our proposed research sought to understand how movement of the muscle within the fluid drainage pathway of the eye (ciliary muscle) affects the eye pressure. This muscle has two functions; it allows us to change our focus to clearly see near or far objects, and it is a pathway for fluid drainage from the eye (uveoscleral outflow). We can change our focus at will, which means we have conscious control over this muscle. Our study supported the idea that the more we move the muscle (change our focus), the more fluid is squeezed out of the eye and the lower the eye pressure. This idea was tested in three groups of adult volunteers aged 20-25 years, 40-49 years and 60-69 years. We tested how much the eye pressure changed when staring at a distance, when focusing up close, or when alternating between near and far vision. This was done for 10 minutes per test with a 20-minute rest in between tests. The results showed that alternating accommodation lowered eye pressure significantly and surprisingly the age of the person did not make a difference. This project helped to better understand how the ciliary muscle drains eye fluid and controls eye pressure. In a future study we will investigate glaucoma patients with high pressure with the ultimate goal of finding better treatments for this blinding disease.

In glaucoma, there is extracellular matrix (ECM) remodeling of the optic nerve head (ONH). The ONH astrocytes and lamina cribrosa cells synthesize ECM proteins to support the ONH. However, in glaucoma, these cells cause the detrimental changes to the ONH. We want to understand the regulation involved in glaucomatous ECM remodeling of the ONH. In this study, we examined microRNAs (miRNAs) which are small molecules that silence gene expression. In this project, we treated human astrocytes and lamina cribrosa cells with or without the profibrotic cytokine TGFβ2 to compare miRNA profiles. Our results identified profibrotic and anti-fibrotic miRNAs that are dysregulated in both astrocytes and lamina cribrosa cells with TGFβ2 treatment. We also observed by using small molecules that act as miRNA mimics, we can block the effects of TGFβ2 induced ECM expression or ECM related proteins that are associated with glaucoma. We believe miRNAs are of interest to help us understand the cellular mechanisms that occur in the glaucomatous ONH and may provide a novel therapy to treat glaucoma patients.