Laboratory Studies in Glaucoma

Aqueous humor is a colorless fluid which nourishes the front part of the eye and drains out of the eye into the blood vessels through the trabecular meshwork. The aqueous humor meets resistance while crossing through the layers of the trabecular meshwork which causes a normal eye pressure to exist. In primary open angle glaucoma (POAG), the resistance to fluid flow through this meshwork is abnormally high, causing an elevated intraocular pressure. The increased pressure in the eye can lead to irreversible damage of the optic nerve and blindness. POAG has been difficult to study, since it is not known what factors cause the trabecular meshwork to become more resistant to fluid flow. No clear cause for this blockage to fluid flow, has been found either microscopically or biochemically in glaucomatous eyes. Furthermore, the determinants of this resistance in normal eyes are not completely understood. It is therefore, necessary to learn more about the cells which reside in this tissue: the trabecular meshwork cells. All cells contain an extensive cytoskeleton, an internal “skeleton” made up of different type of proteins. This protein skeleton is essential to many cellular functions, such as determining the shape of the cell, cell movement, and movement of material in and out of the cell. Changes in cytoskeletal proteins can alter the behavior of the cell significantly. Lately it has been found that many drugs which are used clinically to treat glaucoma, have an effect on cytoskeletal proteins of cells grown on a plastic plate for research purposes (tissue culture). However, exactly how these drugs produce changes in the cytoskeleton is largely unknown. We propose to study the cytoskeleton in normal and glaucomatous eyes. We will investigate the cytoskeleton of cells grown in tissue culture and in normal and glaucomatous donor eyes. In this manner, we can perform many types of experiments and directly observe the results. Our group has taken a multidisciplinary approach to studying these problems, as we study the anatomy, physiology, pharmacology, biochemistry, and fluid dynamics (engineering aspects) of this complex tissue. During the past year we have made progress in understanding several aspects of fluid flow through the trabecular meshwork. We have learned more about the composition of the aqueous humor itself. If protein composition of the aqueous is altered, it may damage the trabecular meshwork cells as the fluid flows through this tissue. We have successfully employed sophisticated nuclear magnetic resonance (NMR) techniques to measure the accumulation of proteins in the aqueous. There has been exciting progress made in the investigation of a new class of naturally occurring hormones which regulate the flow of fluid through the wall of blood vessels. One of these hormones is called endothelin. This agent lowers eye pressure by decreasing the resistance to fluid flow out of the eye in living monkeys and in donor human eyes in the laboratory. Not only will this discovery be extremely useful in our ongoing investigations on the normal mechanism of fluid flow in the eye, it may be a first step to developing a whole new class of drugs to treat glaucoma. During the past year we have found evidence that cells within this outflow pathway contain several types of specialized contractile proteins. These proteins are only found in muscle-like cells. Until now, there was little evidence that this type of cell existed in the trabecular meshwork. If these cells function as muscle cells, they may be involved in the regulation of fluid flow through this tissue. This new information is altering long held views of how the trabecular meshwork functions. After the aqueous humor traverses the trabecular meshwork, it must finally cross a cellular lining before it reaches the circulatory system. The aqueous does so through “holes” (pores) in this cellular sheet. In microscopic studies we have shown that there are fewer “holes” in glaucomatous eyes than in normal eyes. Ye are currently investigating whether less fluid crosses this cellular sheet, resulting in fewer holes, or whether an abnormality exists in the transfer of fluid in this lining. The multidisciplinary approach of our group has enabled us to make significant progress in understanding the cause of POAG. Much work remains and the continuous generous funding from National Glaucoma Research will provide essential support for our search for a cure for primary open angle glaucoma.

2nd Year

During the past year, we have accomplished many of the objectives outlined in our last grant application. This has been accomplished through research conducted at our laboratory and through collaborative research performed with colleagues at other laboratories. These efforts have produced much new insight into the structure and function of the aqueous outflow system in man. We have previously shown that there are cells within the trabecular meshwork, the tissue which functions as a drain within the eye, which contains a muscle protein utilized in contraction of muscle tissue. It was postulated that these cells may open or close the drainage channels within the trabecular meshwork. More recently, another major contractile protein, called actin, was localized to the same areas. In related research, cells containing a specific protein for nerve tissue, called neuron specific enolase, was localized to areas adjacent to cells containing muscle proteins. These findings support the theory that there may be an organized system which regulates the flow of aqueous humor out of the eye. This could be an important approach to understanding how glaucoma develops. Some people develop high eye pressure if given steroids (glucocorticoids) either by mouth or as an eye drop. These same people are at higher risk of developing the most common form of glaucoma. For this reason it is felt that steroids may play a role in the cause of this disease. However, there has been no model of this form of glaucoma to study. This year, through our collaboration with Dr. Abott Clark at Alcon Laboratories, we have studied human donor eyes perfused with steroids in an organ culture system. These organ cultured eyes develop an elevated eye pressure much like that seen in patients. This exciting research is providing a model to study many aspects of glaucoma, which has not been possible in the past. In previous work we have shown that changes in the cells skeleton (cytoskeleton) produced by a variety of pharmacological agents can significantly decrease the resistance to aqueous flowing from the eye, thus altering eye pressure. One drug known to lower eye pressure is called ethacrynic acid. Through computerized reconstructions of individual cells, we have been able to examine changes in the skeletal proteins in three dimensions. In particular, a protein called tubulin appears to be affected in a manner not previously described. Currently we are investigating how tubulin may be involved in the control of aqueous flow. We have also compared the openings in the lining of the drainage canal (Schlemm’s canal) of donor eyes from normal individuals and those with glaucoma. The number of these openings, which are called pores, was significantly decreased in glaucomatous eyes. It also appeared that the cells lining the drainage canal were functioning less efficiently in other ways. In another study we examined the changes in the dimensions of this canal in normal and glaucomatous eye. We found that the canal was diminished in size in glaucomatous eyes, which may play a significant role in the increased resistance to outflow found in glaucomatous eyes. In a collaborative study with Dr. Ross Ethier, a biomechanical engineer at the University of Toronto, we are analyzing the microscopic flow channels through the trabecular meshwork to compare differences between normal and glaucomatous eyes. Finally, we are beginning to analyze normal and glaucomatous eye tissue using laser microscopy technology. Using powerful computers, we can build three-dimensional pictures of the trabecular meshwork. With this technology we can examine specific parts of tissue in three dimensions. We feel that our accomplishments of the past year have added to our understanding of the disease glaucoma. Much work remains to be done, and we hope that in future years we will be able to provide valuable new insight into the development of this disease.