Analysis of JCT Permeability Using Quick-Freeze Deep-Etch

Primary open-angle glaucoma is one of the leading causes of blindness in the United States and is particularly common among Black-Americans. One of the major risk factors for the development of this disease is elevation of the pressure within the eye. In order to be transparent so that vision is possible, some of the tissues of the eye have no blood supply. These tissues depend upon a clear nutritive fluid called aqueous humor in order to survive. Aqueous humor must be constantly replenished to provide the required nutrients. Used aqueous humor leaves the eye through a specially designed filtration tissue known as the trabecular meshwork and the part of this filter with the finest mesh is called the juxtacanalicular region or simply the JCT. In order for the soft-walled eye to maintain its shape and keep its optical elements aligned for vision, a pressure is maintained with in the eye by interposing a resistance in the pathway for aqueous humor outflow. Most clinicians and most researchers believe that the principal site of this resistance resides within the trabecular meshwork and most likely, within the JCT region. But the precise location and the mechanisms that control this resistance remain uncertain. Ample evidence suggests that an abnormal increase in this resistance causes the elevation of intraocular pressure that is so often associated with glaucoma. Virtually all forms of therapy for glaucoma are aimed at reducing this elevated pressure. Finding the site of outflow resistance and identifying the tissue constituents that regulate resistance has proven difficult. In part this is because standard methods for preparing tissue for examination by electron microscopy alter and even remove several classes of compounds from the meshwork. The result is that the picture of the meshwork we end up with may be missing the most critical elements in solving the puzzle of glaucoma. The goal of the proposed studies is to exploit a recently developed tissue preparation method called “Quick-freeze/Deep-etch”. As the name would imply, the tissue is quick-frozen. While frozen, the trabecular meshwork is cracked open under a high vacuum and water is removed by sublimation without the specimen being allowed to melt. The result is that the underlying matrix structure, usually altered during tissue preparation, remains largely intact. By shadowing the surface of the specimen with a fine layer of platinum and carbon, a detailed replica of these structures is made that can be examined at very high magnification in an electron microscope. Our intention is to use this new method for the first time to examine the JCT region of the trabecular meshwork in normal and glaucomatous eyes. These specimens should permit us to make very accurate measurements of the size of the filter mesh in this region and use those measurements to complete computational models of flow resistance to determine whether the JCT region truly is the site of resistance.