Abstract
Abstract: :
Purpose:An interesting finding of corneal vascularization was originally discovered by our group in the Florida manatee (Trichechus manatus latirostris). In every cornea that has been examined, blood vessels have been consistently observed in the anterior portion of the corneas without any signs of infection, injury or other pathological conditions. As an important determinant in blood vessel formation, vascular endothelial growth factor, VEGF and two high affinity tyrosine kinase receptors, VEGF receptor–1 (VEGFR–1/Flt) and VEGF receptor–2 (VEGFR–2/ KDR), have been often examined in association with corneal angiogenesis. The objective of this study is to investigate the presence or absence of VEGF receptors in the corneas of the Florida manatee through the use of immunohistochemistry. Methods:Within 24 hours after death, Florida manatee eyes from a neonate and juvenile were collected from Sea World of Florida and the eyes were placed in 10% BNF (buffered neutral formalin) for 24 hrs, sectioned at 5 microns, quenched with 3% hydrogen peroxide, blocked in 5% normal goat serum and treated with a polyclonal rabbit primary antibody (Flt–1) used to detect VEGFR–1 or a monoclonal mouse primary antibody (KDR) used to detect VEGFR–2 at concentrations of 1:50, 1:100, and 1:200. Streptavidin–HRP was applied, followed by a DAB chromagen substrate. Results:Corneal blood vessels in both the juvenile and neonate animals reacted positively for VEGFR–1. The juvenile had positive reactions in both right and left corneas at 1:50 and 1:100 concentrations. The neonate also showed positive reactions in both corneas at concentrations of 1:50 and 1:100, being most evident in the 1:100 concentration. With both the juvenile and neonate samples, the positive reaction was most apparent in the stromal vessels and anterior epithelial vessels. The control samples demonstrated no positive reaction at all. At concentrations of 1:50, 1:100, and 1:200 of monoclonal mouse primary antibody, positive staining of VEGFR–2 in the corneal specimens was neither observed in the juvenile nor the neonate. Conclusions:We postulate that the process of angiogenesis occurring in this tissue is a non–pathological and non–inflammatory response to currently unknown factors, but most likely due to developmental or evolutionary influences. VEGF receptors detected in the Florida manatee corneas examined are believed occur normally and not under conditions brought on by pathologic factors such as trauma, injury or infection.
Keywords: growth factors/growth factor receptors • immunohistochemistry • cornea: epithelium