An Abnormality in the Nitric Oxide System in Glaucoma

A major factor which predisposes a person to develop glaucoma is the presence of increased intraocular pressure (IOP). Increased IOP in glaucoma is caused by a partial or total blockage of aqueous humor as it leaves the eye through the trabecular meshwork (TM). Until now it had not been known why a blockage or increase in outflow resistance, as it is sometimes referred to, occurred in certain eyes. As a result of research carried out in a previous one-year grant which our Laboratory received from the AHAF, we have identified what appears to be a major biochemical defect in the eyes of glaucoma patients and which may help explain why there is an increase in outflow resistance. If this defect can be confirmed, it will represent the first major biochemical abnormality ever found in glaucoma and may point the way toward new treatments for the disease. The defect we have found is in a recently-discovered cellular messenger called nitric oxide, or NO. Once considered only as a toxic gas, NO has been found, in recent years, to act as a key regulator between cells acting both in the nervous system and in other tissues such as smooth muscle. For example, NO causes muscles in peripheral blood vessels to relax, lowering blood pressure. In previous experiments, we had found that drugs which mimic the action of NO, decrease IOP in animal eyes. We hypothesized that NO might therefore occur naturally in eyes. At the time that we carried out our prior AHAP grant, the only marker suitable for identifying NO within small amounts of tissue was an indirect method which used a special stain called NADPH-diaphorase. Previous investigators had used this stain to look at the eyes of rats but were disappointed to find little evidence for the presence of NO in the areas where aqueous humor leaves the eye. However, rats do not have a TM nor do they possess a special muscle, called the ciliary muscle (CM) a part of which seems to be involved in regulating resistance of aqueous humor passing out of the eye through the TM. Accordingly, in our studies, we used donor human eyes which become available for research when they are judged unsuitable for corneal transplantation. Utilizing these eyes and the diaphorase staining method, we discovered that, unlike the rat, human TM and CM contain an large amount of NO-producing tissue. While the diaphorase method provided indirect evidence only, it nonetheless suggested a possibly important role for NO in regulating pressure in the normal human eye. Of even greater interest was our preliminary finding that donor eyes from patients with primary open angle glaucoma (POAG), the major form of glaucoma in adults, have a defect in diaphorase-containing tissues in two places. The first was in the TM and the second was in the special part of the CM (the longitudinal potion or Long.CM) that normally regulates resistance in the TM and whose normal movement may help to keep the TM free of debris. In the Long.CM of POAG patients we found what appeared to be a degeneration of diaphorase-containing Long.CM fibers near the point where they attach to the TM. Unfortunately, we do not know whether the change in diaphorase staining which we have found reflects a true change in synthesis of NO (which is made by an enzyme called NOS) and, if so, which particular forms of NOS are affected (since there are known to be at least three different forms of the enzyme). This is important as it would tell us more about how the normal eye regulates IOP and also which particular drugs might be effective in altering pressure in POAG. Even though diaphorase is altered in POAG, we also do not know if NO synthesis itself is altered and if the changes we see are specific for NO or are part of a more generalized abnormality in glaucoma eyes. For example, there might be a more general decrease in the structure or number of cells or nerves (including nerves known to contain NO) that are normally present in the TM and CM. In our proposed two-year AHAF application, we intend to answer these questions and thereby determine definitively whether there is a defect in NOS in the TM and Long.CM of POAG eyes. We plan to do this by using some new techniques that we have recently developed and, in preliminary testing, have found be be quite effective. For example, using special antibodies which can identify specific forms of NOS, we have discovered that, in normal eyes, the form of NOS enzyme present is one similar to that normally found in the endothelium or lining of blood vessels. We plan to use that technique to discover the types and abnormal changes in NOS present in glaucoma eyes. We will also use special stains to determine if the defect in NOS is specific for NO or is part of a more general defect. We have also discovered how to use in the eye a newly developed microscopic probe which will allow us to measure directly the formation of NO in specific parts of the CM and TM and compare this production between normal eyes and those with glaucoma. These experiments will allow us to definitively define and characterize the defect in NOS and NO in POAG. This defect may be a cause of glaucoma or could be a result of the disease. In either case, the planned experiments will have great relevance to potential new drug and surgical treatments for POAG.