Effects of Oxidative Stress on Trabecular Meshwork

It has been proposed by a number of investigators that the disease of glaucoma is related to oxidative damage which occurs with age in the trabecular meshwork (TM). The TM resides in a potentially damaging oxidative environment as a result of continuous exposure to oxidants present in the aqueous humor. For example the aqueous contains significant concentrations of hydrogen peroxide and ascorbic acid, both of which can lead to generation of highly reactive oxygen species. The aqueous humor also contains oxygen and photosensitizer which in combination with light are capable of producing a highly reactive, potentially damaging compound called superoxide anion. It is well established that active species of oxygen such as those which may challenge the TM can be damaging to a number of cell constituents including protein-lipids and DNA. However little is known about the possible harmful effect of these reactive compounds on the TM and the possible relationship to glaucoma. One reason that oxidation is believed to play a possible role in the pathogenesis of glaucoma is the fact that healthy TM possess a number of mechanisms, to defend against oxidative damage. For example TM has been shown to have an active glutathione redox cycle which is capable of detoxifying potentially harmful compounds such as hydrogen peroxide and superoxide anion. Similarly, TM has high activities of antioxidant enzymes such as catalase and superoxide dismutase. Although the TM is known to possess significant antioxidant activity the functions and relative roles of the anion antioxidant systems in the tissue are not clearly understood. A significant portion of the aqueous outflow pathway is lined by cells of the TM It has been suggested that the metabolic activities of these cells may be important in regulating aqueous outflow dynamics and that the cells may be associated with underlying causes of glaucoma. Trabecular cell cultures are a useful means of evaluating antioxidant systems present in TM and of investigating oxidative effects which may be related to the development of glaucoma. By culturing cells from calf and cow TM it may be possible to investigate age-related oxidative effects on TM. An additional reason for using cultured bovine trabecular cells in the planned studies is that a significant amount of information has been collected during the past several years on the biochemical composite of isolated bovine trabecular tissue. Very little information is available on oxidant detoxification mechanisms present in cultured trabecular cells. Studies are needed concerning the levels of activity of the glutathione redox cycle and other antioxidant systems in cultured TM cells. It will be important to compare values in young and old TM cells with corresponding results for cultured lens epithelium which is known to possess exceptional antioxidant activity. In addition it will be important to evaluate the ability of the TM cells to tolerate oxidative stress. Oxidative effects on DNA in TM cells and the possible relationship to the development of glaucoma has also not been studied. DNA of cultured lens epithelial cells has been reported to be damaged by H2O2 concentration only slightly higher than normal physiological levels found in the aqueous humor. The first goal to this research project is to culture TM cells from calf and cow eyes and establish parameters which will be employed in oxidation experiments such as passage number, cell density and cell number. Baseline values will be determined for various antioxidant mechanisms present in the cells including levels and activities of components of the glutathione redox cycle. The ability of TM cells to tolerate oxidative stress will be evaluated by exposing the cells to maintained and pulsed levels of hydrogen peroxide, various pressures of oxygen and various concentrations of ascorbic acid. The ability of the cells to maintain normal biochemistry under oxidative stress will be determined. Cell viability will be monitored by various techniques including scanning electron microscopy. Factors evaluated will include cell growth, activity of the enzyme Na+-K+ ATPase, cation transport, synthesis of proteins and DNA, DNA damage and effects on soluble, membrane and cytoskeletal proteins as evaluated by electrophoretic techniques. Our laboratory has considerable research experience concerning oxidative processes taking place in the ocular lens. We believe that similar mechanisms may be operative in other types of cells and it is opportune to extend our knowledge in this area to TM which has a crucial role in the outflow mechanism of the aqueous humor and hence to the pathogenesis of glaucoma. Our experience is this general area offers a unique opportunity to extend these studies to TM cells. The broad, long-term objective of this project is to investigate a possible link between oxidative damage to the TM and the progression of glaucoma. A discovery of this type would stimulate development of antioxidant drugs which might lead to a cure for the disease.