Under Pressure: The Role of Retinal Astrocytes and the Response to Ocular Hypertension

The vertebrate retina – a thin layer of photosensitive tissue that forms from a protrusion of the early brain during development – is a highly conserved structure essential for functioning sight. Although there are modest variations from species to species, across taxa the overall structure is largely the same: photoreceptor cells at the back of the retina sense light, multiple types of interneurons in the intermediate layers perform computational comparisons that sharpen the image, and the retinal ganglion cells at the front of the retina aggregate these signals, transmitting them to the brain through their axons, which form the optic nerve. The retina is a highly energy intensive structure, however, and it is in part through their adaptations to provide additional blood flow to this tissue that the retinas of distinct groups of vertebrates differ./nIn humans and other mammals, a unique type of astrocyte – a class of non-neuronal support cell that maintains homeostasis in the central nervous system and responds to injury or infection via a process known as reactivity – emerges from the optic nerve head during development to facilitate the direct vascularization of the retina, which is a sharp departure from most other vertebrate species. These astrocytes tile the surface of the retina, and like their counterparts in the optic nerve, are closely associated with both blood vessels and the delicate axons of the retinal ganglion cells, although their full range of functions is yet to be elucidated. During glaucoma, a disease of the retina and optic nerve head that causes progressive and irreversible vision loss, the vulnerable axons of retinal ganglion cells degenerate, leading to the death of these irreplaceable cells. Although some risk factors such as age and elevated intraocular pressure have been identified, and treatments have been developed that can slow this degeneration to an extent, much remains unknown about this condition, which affects tens of millions around the world and is a leading cause of blindness./nNotably, the known risk factors are only modestly predictive of development or progression of glaucoma, suggesting that other elements of the visual pathway may contribute, whether positively or negatively, to retinal ganglion cell survival. A number of studies have indicated a role for retinal and optic nerve head astrocytes in the loss of retinal ganglion cells during the most common form of glaucoma, primary open angle glaucoma. Recent research has suggested that these astrocytes undergo changes that result in a loss of neuroprotective behavior, a gain of neurotoxic behavior, or some combination of the two. Understanding the contributions of retinal astrocytes to this debilitating disease, and determining whether they are a suitable target for treatment, requires close study of these incompletely understood cells./nAs the primary obstacle to investigating these cells has been their sparseness – retinal astrocytes are estimated to represent less than 0.1% of cells in the retina – I have developed a novel method in order to rapidly isolate these cells. This method, which relies on the characteristic layering of the retina, involves directly removing the astrocytes by first treating the retina with an enzyme to breakdown the extracellular matrix, then mechanically removing the astrocyte layer as a single, thin sheet. It possesses a number of advantages, including speed and the versatility of the samples generated, which can be used both for molecular biology techniques as well as immunostaining./nBy combining this retinal astrocyte isolation technique – which I termed the ‘astrocyte pulloff’ in reference to earlier techniques that were attempted on retinal ganglion cells – with an in vivo model of ocular hypertension, I investigated the changes undergone by these cells in response to changes in intraocular pressure that mimic some elements of glaucoma, such as the death of retinal ganglion cells. This model, performed by treating the episcleral veins with brief laser pulses to induce photocoagulation, drives an in increase in pressure inside the eye that lasts for approximately one week, during which a significant fraction of retinal ganglion cells are lost. In order to study the changes that retinal astrocytes undergo as a result of this process, I employed a technology called RNA sequencing, which is used to generate a profile of the genes being expressed by cells at the time of isolation. This allowed me to identify key genes that undergo changes in response to this treatment relative to untreated controls./nThe initial phase of these results was characterized by a massive amount of information, with expression changes being logged at the sites of nearly 25,000 genes and other genetic loci. After an initial filtering to remove marginal or questionable results, 1129 genes were found to be upregulated, while another 44 were downregulated. Further analysis revealed that many of the upregulated genes had been previously linked to astrocyte reactivity in brain injury and neurodegeneration, and additional clusters of genes had been found in a separate type of cell, known as microglia, in models of Alzheimer’s disease. These results were of considerable interest, as they suggested a linkage between other neurodegenerative disorders and glaucoma, which has been speculated on at length but for which the evidence is modest./nThe second phase of the experiment was to reprise the laser treatment with immunostaining techniques as the downstream assay. These techniques are fickle and low throughput relative to RNA sequencing, but when done correctly have the major advantage of being able to identify the cell types driving changes in RNA sequencing data. This follow up investigation revealed that many of the observed changes were instead driven by other cell types, including Müller cells, that are typically excluded by this isolation technique; their inclusion, and those of other additional cell types, was driven by focal damage to the retina during in vivo treatment, creating scarring that resisted enzymatic treatment. As one of the chief aims of my approach was to allow for the analysis of the retinal astrocyte response without contributing signals from Müller cells, which are a similar but distinct cell type, this was something of a disappointment. However, the aforementioned versatility of the technique demonstrated a major advantage of this approach, as it was the ability to use identically prepared samples for both phases of the experiment that allowed us to detect this artifact. This was of particular import, as many commonly employed assays would have failed to detect the heterogeneity within the samples, complicating the task of interpretation. In the future, I aim to employ this technique to investigate alternative in vivo treatments that may produce clearer retinal astrocyte responses.

Advisor: Flanagan, John G