The long range objective of the ongoing research of this laboratory is to further define the biological role of the keratocyte in establishing and maintaining corneal clarity. Our previous efforts, using a cryolathe freeze injury model of rabbit lamellar keratoplasty (LKP) have documented loss of corneal clarity following keratocyte death resulting from freeze injury. We further demonstrated that the return of clarity correlated temporally with keratocyte repopulation in the LKP button. We developed a working hypothesis that keratocyte repopulation plays an active role in the return of corneal clarity; this hypothesis has been tested further in our laboratory in a series f experiments designed to maximize keratocyte survival. The employment of multiple approaches over the last several years has led to the unifying principle that keratocyte preservation through the freeze injury prevents the loss of corneal clarity seen previously in this animal model. Although these studies have led to improvements in the cryolathe refractive surgical procedures of keratophakia, keratomileusis, and epikeratophakia, more importantly to this proposal they provide a framework for studying various cell biological parameters of the keratocyte. This proposal outlines a research plan which focuses on the cell biology of the keratocyte and its role in establishing and maintaining the ordered extracellular matrix milieu necessary for efficient light transmission and corneal clarity. Using a variety of cell-and matrix-specific probes in conjunction with in vivo tandem confocal microscopy, we will investigate the temporal sequences of the migration of keratocytes into freeze-injured corneas. These studies will document the dynamics of keratocyte activation, migration and mitotic activity as these cells repopulate the LKP buttons. Concurrent studies will also focus on the changes in the extracellular matrix that occur with keratocyte repopulation. These studies will use mono and polyclonal antibody and immunocytochemistry techniques to correlate the appearance of various collagen types, collagenase activity, fibronectin, laminin and glycosaminoglycans in the acellular corneal stroma with repopulation. Further studies will examine these same parameters as they relate to inflammation, surface epithelialization, and exposure to various growth factors. Additionally, studies using lectin cytochemical techniques will focus on further characterization of a novel fibrillar network which we have identified recently in the posterior corneal stroma in a variety of species. Preliminary evidence suggests that this posterior corneal fibrillar network (PCMS) is altered by our LKP model suggesting that it represents a unique macromolecular structure related perhaps to the structure of the cornea. We will study the dynamics of PCMS using the LKP model and attempt to isolate and characterize this structural macromolecule using immunochemistry and biochemical techniques.
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