Dry eye afflicts millions of Americans, and it is the leading cause of visits to eye care clinicians. Dry eye often results from insufficient tear formation from the lacrimal gland. Lacrimal fluid is initially formed in acini and then modified during passage through the duct system. However, little is known about the ducts and their function. It has been assumed that the decreased lacrimal production in dry eye is caused primarily by decreased acinar secretion. However, our preliminary data suggest that a competing hypothesis also must be considered, i.e., lacrimal hyposecretion may result from inappropriate or excessive reabsorption of fluid in the ducts. In this proposal, the investigators will use normal rabbits and two rabbit models, pregnancy and induced autoimmune dacryoadenitis [IAD], both of which exhibit decreased basal lacrimal secretion and pathological features of lacrimal insufficiency, to address three aims: #1 Determine whether the volume and composition of lacrimal fluid is modified in isolated duct segments and deduce the functional capabilities of duct epithelial cells in normal control rabbits. #2 Determine whether transepithelial ion and fluid transport functions of isolated duct segments are altered in pregnancy and IAD. #3 Identify cellular mechanisms underlying decreased lacrimal fluid production in pregnancy and IAD. These studies will employ a broad repertoire of innovative techniques. Microdissection and microperfusion methods will be used to determine ionic and volumetric changes in fluids perfused through specific duct segments under basal and pilocarpine-stimulated conditions, and to assess the potential influence of periductal lymphocytes on these functions. Ex vivo real time imaging using multi-photon excitation laser scanning fluorescence microscopy will be used to assess changes in cytosolic Ca2+ associated with M3 muscarinic acetylcholine receptor (M3AChR) signaling;changes in cell volume associated with activation of K+ and Cl- efflux mechanisms and regulatory activation of Na+ and Cl- influx mechanisms;and changes in cytosolic electrolyte contents associated with activation or inhibition of ion transporters hypothesized to mediate transepithelial absorption or secretion. Laser capture microdissection, real time RT- PCR, immunohistochemistry and in situ hybridization will be used to determine whether expression of aquaporins, M3AChR, G proteins coupled to M3AChR (i.e., Gq and G11), and various ion transporters associated with epithelial electrolyte and fluid absorption and secretion is altered in duct cells in the two models. Therefore, these studies will define the functional roles of specific duct segments within the lacrimal gland in production of lacrimal fluid. This research is highly relevant to the Program Goals of the National Plan for Eye and Vision Research, and knowledge gained from these studies has considerable potential to enhance the development of more effective treatment strategies.Dry eye is the most prevalent problem in ophthalmology which afflicts millions of Americans, and is the leading cause of visits to eye care clinicians. The proposed studies take advantage of two excellent animal models to study the biology of the lacrimal gland duct system, which has heretofore received little attention. Gaining an understanding of the lacrimal duct system is critical to enhancing our ability to develop more effective treatment strategies ultimately benefiting millions of dry eye patients.
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