Combined Morphologic and Physiologic Studies of Proteoglycans

The cause of the most common form of glaucoma, called primary open angle glaucoma, is unknown. What is known is that an elevation of pressure occurs in the glaucomatous eye which can lead to a permanent loss of vision if left untreated. Currently many experts agree that the abnormal rise in pressure results from some form of obstruction within the tissue through which fluid within the eye must pass to exit the eye and enter the bloodstream. The tissue of the eye suspected to harbor this obstruction is the trabecular meshwork. More specifically, many believe that it is a small portion of this tissue, the juxtacanalicular connective region, which is principally involved. From biochemical studies we know that various substances are normally found in this region, including certain proteins and other classes of compounds called proteoglycans and glycosaminoglycans, or simply GAGs. Unfortunately, until recently, it was impossible to visualize these latter two classes of compounds with the electron microscope. As a result the spaces in which these compounds resided in the trabecular meshwork appeared entirely empty. With so much of the puzzle missing, it becomes less surprising that very few differences have been found between normal and glaucomatous trabecular meshwork using microscopic methods. Recently, a new method has become available which, for the first time, permits relatively specific visualization of some proteoglycans. In other tissues, this new method has been used simply to identify proteoglycan constituents by pre-treating sections with enzymes having varying degrees of specificity for certain proteoglycans and using the staining method to disclose those that remain. In these instances no corroborative information on tissue physiology has been obtainable. In the trabecular meshwork however, we are presented with a unique opportunity to obtain corroborative physiological data by simply modifying the basic protocol. If, instead of using the enzymes to treat tissue sections of meshwork, we perfuse them through the meshwork, their effects on the ability of fluid to flow out of the eye can be objectively assessed. Following the perfusion, these same eyes can be prepared using the new method described above, allowing us to localize the remaining proteoglycans and provide correlates for the flow data as they relate to these compounds.

Hypothesis

Our hypothesis regarding the role played by proteoglycans in aqueous humor dynamics is as follows. One function served by proteoglycans is to provide a hydrophilic covering over the normally hydrophobic surfaces of tissue constituents such as elastin and collagen fibers. Although several investigators have presumed that increased amounts of proteoglycans or GAGs in the trabecular meshwork could block fluid flow accounting for the increased aqueous outflow resistance in glaucoma, more recent evidence suggests that, at least certain classes of the proteoglycans are actually decreased in amount in glaucomatous eyes. Indeed, a reduction in the proteoglycan/collagen ratios has been demonstrated to occur with aging in various tissues outside of the eye. Placed in the context of our hypothesis, a loss of proteoglycans would be expected to result in exposure of the underlying hydrophobic moieties, increasing the potential for binding serum proteins which are known to be present in aqueous humor. Our postulate is that an abnormal accumulation of serum proteins ultimately accounts for the concomitant decrease in permeability of the juxtacanalicular region and the rise in intraocular pressure. Even if we accept this premise, it would be reasonable to question whether the low levels of protein normally found in aqueous humor could ever be sufficient to produce the postulated obstruction. Recent work by our group however, has shown that much of the plasma-derived protein found in the aqueous humor diffuses in from the ciliary body stroma via the ciliary body band and the root of the iris. Given that the bulk of this protein reaches the anterior chamber just beneath the trabecular meshwork, there is reason to suspect that the concentration of protein entering the trabecular meshwork may be much higher than would be predicted based upon the protein levels measured in aqueous humor samples obtained from the anterior chamber. This unexpected additional source of potentially obstructive material would, in our hypothesis, be sufficient to produce an amount of obstruction which could lead to glaucoma.

Specific Aims

1. Determine the distribution of, and preliminarily identify, the various proteoglycans present in the normal human trabecular meshwork at different ages and in glaucoma using the cationic phthalocyanine-like dye Cuprolinic Blue in the critical electrolyte concentration method.
2. Assess the proteoglycan/collagen ratios in these specimens as an indirect indicator of the relative hydrophobicity present on the surfaces exposed to aqueous flow in the trabecular meshwork and specifically it’s juxtacanalicular region.
3. Perfuse bovine and human eyes with selected enzymes which digest GAGs to deplete the proteoglycans of the trabecular meshwork (and JCT), following this with a perfusion of aqueous humor with normal or elevated serum protein concentrations to determine the potential for accumulation of proteins following proteoglycan depletion. Determine the effect of these alterations on the ability of aqueous humor to flow out of the eye.

Long-Term Goal

Determine if tissue changes which would be expected to result in increased hydrophobicity of the trabecular meshwork represent a potential contributing factor in the development of glaucoma.