A new method for in situ organ culture of human trabecular meshwork will be used to study the cellular biology of the trabecular meshwork. The new technique allows the anterior segment of eyes to maintain a relatively constant intraocular pressure while being perfused with culture media. The media flow through the trabecular meshwork in a normal manner, enter Schlemm's canal, and finally leave the eye through the collector channels. Trabecular meshwork structure and cellularity are maintained for at least four weeks in culture as determined by light and electron microscopy. Cells remain metabolically active, incorporating the amino acid 3H leucine and the proteoglycan precursor 3H glucosamine after four weeks of culture. This technique enables controlled experiments to be performed on intact human trabecular meshwork perfused in a normal fashion. The proposed grant will allow refinement of the present culture technique, development of parameters to assess experimental changes, and study of several basic questions about human trabecular meshwork. The effect of corticosteroid, epinephrine, and pilocarpine on human trabecular meshwork will be studied with this culture technique. Normal human eyes obtained at autopsy will be used, as well as human glaucomatous eyes. Changes induced by these agents will be studied using one eye as an untreated control, the fellow eye as the treated experimental eye. In addition, trabecular biopsies obtained via surgical trabeculectomy will be used to monitor changes during the course of the experiment. Structural appearance will be studied with both light and electron microscopy (transmission and scanning). Changes in protein and extracellular matrix synthesis will be monitored autoradiographically using radioisotope tracers and biochemically using analytical techniques. The effect of intraocular pressure, ascorbate, and growth factor, will be studied in separate experiments. Pairs of eyes will be cultured with one eye receiving the experimental agent, and the fellow cultured eye serving as the untreated control eye. The trabecular response to commonly found intraocular debris (blood, pigment, lens cortex) will be studied in a similar fashion, making special note of cell loss and replication.
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