IN-VIVO CONFOCAL IMAGING OF CORNEAL NEOVASCULARIZATION

Purpose. To investigate the cellular dynamics of vessel formation duri ng corneal neovascularization in the living eye by confocal microscopy . Methods. Corneal neovascularization was initiated by placing a 7-0 s ilk suture through the corneal stroma 3 mm from the limbus at the 12 o 'clock position in both eyes of 10 New Zealand white rabbits. The corn eas were examined for vessel ingrowth at intervals from 1 to 15 days a fter suture placement using a tandem scanning confocal microscope with a 20X water immersion objective, as well as a slit-lamp biomicroscope . Changes in the limbal vessels were recorded on videotape for later a nalysis. As early vessel growth appeared to be associated with corneal nerves. the total number of sprouts and the number of sprouts along n erves were counted in confocal images, and the results analyzed for st atistical significance. Vessel growth and the structural relationship between vascular buds and the deep stromal nerves were examined by lig ht and transmission electron microscopy. Results. The early events of cell migration from the limbal microvessels were found to be associate d with the deep stromal nerves; although this association was easily v isualized by confocal microscopy, it could not be documented by slit-l amp biomicroscopy. By 18 h after suture placement, the limbal vessels were dilated and the first vascular buds appeared as short, pointed, o r flat-topped protrusions from the deep limbal capillaries. By 96 h, t he capillary buds had increased in density and had begun to form lumen s. Movement of red blood cells was established between 72 and 80 h aft er the first signs of bud formation, at the same time that cells of im mune origin were seen. Confocal microscopy revealed and transmission e lectron microscopy verified that new bud formation began with the form ation of vascular tubes by endothelial migration along the deep stroma l nerves. The total number of sprouts and the number of sprouts associ ated with stromal nerves were similar on days 1 and 2 but differed on days 3-7, suggesting an association between sprouts and nerves in the early stages of neovascularization. Conclusion, Using real-time white light confocal microscopy, we were able, for the first time, to observ e the process of corneal neovascularization in the living eye, from th e earliest stages within hours after initiation to 2 weeks. The deep s tromal nerves appear to serve as a focus for the growth of new vessels , by attracting and supporting vessel growth and/or by providing a pot ential space for movement of the endothelial cells. Confocal microscop y may provide a new approach to achieving a better understanding of th e mechanisms involved in corneal neovascularization.