2016 Shaffer Research Grants

In glaucoma, the optic nerve which sends visual information from eye to brain gets damaged. Once damaged, the optic nerve does not regrow back to the brain, resulting in permanent blindness. Therefore, to restore visual function in glaucoma patients, it might be necessary to promote injured optic nerves to regenerate and reconnect to their original targets.

In the last several years, researchers have identified gene therapies that can promote optic nerve regeneration. However, there is a still major problem. Optic nerves are mostly incapable of growing straight back to the brain, and often fail to reach the brain. In our research, we seek to understand the cellular and genetic factors that prevent these nerves to correctly find their targets. Towards this goal, we first discovered that optic nerves regenerate physically on the surface of astrocytes which are the support cells in the optic nerve. Therefore, we reveal that optic nerve regeneration and navigation are in fact shaped by astrocytes.

Second, we discovered that certain genes, namely Ncad expressed in the astrocytes, are important for optic nerve interaction with astrocytes, and for optic nerve regeneration. Our study identified the key cellular and genetic players that shape optic nerve regeneration and navigation. Ultimately, our research will help elucidate factors that prevent proper optic nerve regeneration and guidance, and to find strategies that promote reconnection of damaged optic nerves and restore visual functions following optic nerve damage.

To date, the only way to stop vision loss from glaucoma is intraocular pressure (IOP) reduction by daily medication use, laser procedure, or incisional surgery. In some patients IOP reduction is difficult to accomplish or glaucomatous vision loss occurs despite substantial IOP reduction.

These clinical situations highlight the need for development of IOP-independent, glaucoma treatment strategies otherwise known as neuroprotection. One promising neuroprotective therapeutic for glaucoma treatment is the blood pressure medication losartan. Losartan’s protective activity is due to prevention of remodeling processes that occur in the wall of the eye (the sclera) during glaucoma.

In these studies we have shown that losartan treatment targets specific cells within the sclera called fibroblasts. Fibroblasts exposed to losartan are prevented from becoming “activated” and remodeling the scleral tissue. In addition, we have developed long-lasting, drug releasing microparticles to prevent scleral remodeling in glaucoma.

Elevation of pressure in the eye is the only risk factor for glaucoma that can be modified. Improved understanding of how the eye’s natural drain is damaged can provide insights to new treatments and prevention of this blinding condition.

We have measured oxygen levels inside the eyes of patients undergoing eye surgery with a small probe and found increased oxygen levels in patients who have had removal of the gel in the back of the eye, a procedure performed for various retinal diseases. Patients who have had this procedure nearly always require cataract surgery and this combination of procedures lead to an increased risk of glaucoma. This excess oxygen may be the source of molecules that cause damage to the cells of the natural drain of the eye. In addition, the level of antioxidants, compounds that protect the cells from this damage, are decreased following this combination of surgeries.

By performing these two procedures (removal of gel and then lens removal), we predicted increased oxygen levels in the front of the eye in the area of the natural drain in a glaucoma model. We were unable to duplicate these findings in this model, but did enhance our techniques utilizing a laser to dissect these specific cells in the drain of the eye to study changes associated with damage and then study the how these cells may have altered programming of their genetic code. In this manner, we can now learn more precisely how these cells are damaged and potentially identify patients who are at risk for damage and new ways to treat glaucoma.

For her research project exploring the role of oxygen and antioxidant levels in the eye, Dr. Siegfried was awarded the 2018 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.

In this project we have optimized techniques to isolate and characterize exosomes from lamina cribrosa cells. Exosomes are small vesicles that are released by cells to perform a variety of functions. In the lamina cribrosa, like the trabecular meshwork we hypothesize that exosomes participate in the turnover of extracellular matrix and homeostatic signaling with cell neighbors, particularly in response to elevations in intraocular pressure/pulsations. In the present study we mimicked pressure pulsations by cyclically stretching lamina cribrosa cells and collecting/purifying released exosomes from the cell culture media. We observed that exosomes from glaucomatous lamina cribrosa cells were different than exosomes from normal cells. These differences hold the potential to provide information about abnormal remodeling of the optic nerve head in early stages of glaucoma.

Many optic nerve diseases result in permanent loss of vision. This occurs in part because the intrinsic growth capacity of retinal ganglion cells rapidly declines after birth, and injured central nervous system axons fail to regenerate. The scientific community has learned a great deal about molecular signals that can support regeneration of damaged neural connections, but there is an urgent need to identify as many pro-regenerative signals as possible because it is not clear which of these might actually translate to use in patients.

Generous support from the Glaucoma Research Foundation allowed us to develop a strategy to comprehensively assess an entire class of biomolecules called lipids for differences that occur during optic nerve regeneration. We hope to use this approach to identify candidate molecules that might represent previously unknown pro-growth signals.

Findings from this research study were published in the January 2018 issue of IOVS (Investigative Ophthalmology & Visual Science), “Optic Nerve Regeneration After Crush Remodels the Injury Site: Molecular Insights From Imaging Mass Spectrometry.”

Glaucoma is a silent disease that, over time, kills the nerve cells of the retina leading to irreversible blindness. Current treatment options are restricted to non-specific interventions aimed at lowering intraocular pressure (IOP). For many glaucomatous patients, however, pharmacological and surgical management of IOP does not always help. The development of targeted therapeutic strategies directed at the cause of elevated IOP is critical for the advanced management of glaucoma. The cause of elevated IOP most likely involves a molecule called transforming growth factor-β2 (TGF-β2). Funding support from the Glaucoma Research Foundation Shaffer Grant has allowed our lab to advance our understanding of exactly how TGF-β2, a multifunctional cytokine, promotes increases in IOP of patients with POAG. We found that specific cells in the eye, called TM cells, constitutively express and secrete TGF-β2, highlighting the TM as a viable targetable source of TGF-β2. This molecule was further found to elicit harmful and pronounced oxidative stress to the TM. Our findings are consistent with other studies also reporting elevated levels of oxidative stress markers in the eyes of POAG patients, along with altered expression of antioxidant defenses in the TM. Results from this Shaffer Grant study also show that targeting antioxidants such as XJB-5-131 to the TM significantly attenuates expression and release of TGF-β2 from cultured human TM cells. Of equal importance, XJB-5-131 protected human primary TM cells against TGF-β2 mediated changes in expression of specific extracellular matrix proteins. These exciting findings are being put to the challenge to see if targeting antioxidants to the TM in porcine and human eyes will lower IOP. To do this, we have encapsulated small beads, called nanoparticles, with various test agents. We are seeing, for the first time, that these nanoparticles can markedly reduce IOP by reducing endogenous expression of TGF-β2. Collectively, our findings support targeted disruption of constitutive TGF-β2 expression within the eye using antioxidant-encapsulating nanoparticles and raises enthusiasm that this strategy will be a clinically useful and effective new therapy by which to better manage IOP in patients with POAG.

Glaucoma is characterized by a gradual loss of retinal ganglion cells (RGCs), which leads to a loss of vision. The most common form of glaucoma, occurring in 70 to 90% of patients, is primary open angle glaucoma (POAG).

One of the most compelling epidemiological features of POAG is that its incidence shows a striking sex-related difference. Women have a significantly lower incidence of POAG, as compared to men, until the age of 80 years. This sex-related difference has been linked to the extent of lifetime estrogen exposure. Indeed, there is a strong assocation between increased estrogen exposure and a reduced POAG risk. Conversely, studies have shown that a decreased exposure (i.e. early loss of estrogens), confers an increased risk of POAG. We hypothesize that an early estrogen deficiency accelerates the aging of the optic nerve and predisposes this nerve to glaucomatous damage.

To test our hypothesis we determined whether early estrogen deficiency is associated with heightened intraocular pressure, RGC loss and glaucoma in an animal model. Our results demonstrate that estrogen deprivation does promote the development of glaucoma in female mice. To continue these studies, we seek to determine whether estrogen administration will serve as a novel preventive treatment for glaucoma, and in particular, POAG. If so, our research will have significantly advanced our understanding of the role of estrogen in the pathophysiology of glaucoma.

We have developed an intraocular pressure sensor based on the principle of interferometry. The sensor is comprised of a diaphragm and a glass substrate. By directing monochromatic light towards the active sensing region and applying pressure, one can observe interference fringes as the diaphragm deflects. Using the principle of interferometry, we were able to back calculate pressure using the images captured by a camera. From our studies, we have found the best results using silicon nitride as the diaphragm material. The sensor read-out can be further optimized by coating a thin layer of silicon nitride onto the glass substrate. In order to further demonstrate proof of concept, our sensor was also tested ex-vivo using a rabbit eye model. Thus far, we have achieved 0.8 mmHg resolution. Our results show that the sensor response is repeatable and agree with mathematical models.