Confocal Microscopy and Digital Imaging of Rabbit: Corneal Nerves after Immunohistochemical Preparation

Abstract

Abstract: : Purpose:To evaluate the feasibility of using immunohistochemical staining and confocal microscopy with digital imaging software to visualize corneal nerves in a rabbit model. Methods:Animal Care and Use Committee (ACUC) approval at Northwestern University was obtained. Eight New Zealand white rabbits underwent bilateral LASIK. Corneas were harvested at either 5 or 10 weeks post–LASIK to evaluate corneal nerve morphology. Initial histologic preparation involved incubation of excised cornea sections in hyaluronidase at 4°C for 24 hours. This allowed for greater penetration of subsequent staining through the corneas with mouse anti–tubulin monoclonal primary antibody which binds to corneal nerves. The cornea pieces were then incubated in secondary antibody labeled with Alexa 488 probe. Confocal microscopy at 10X magnification in 1µm tangential sections was employed to visualize the stained corneal nerves. Digital images of corneal nerves were taken. Total surface area of corneal nerves was quantified using a digital imaging program (Carl Zeiss, Inc.) that detected the intensity of corneal nerve fluorescence. Results:Data was obtained from 16 rabbit corneas. The corneas were studied after sectioning into 3 equal pie–shaped pieces. One section was used for data collection. Adequate visualization of corneal nerves with confocal microscopy and digital imaging software was obtained in all 16 studied sections. The quantity of corneal nerves measured as total surface area could be compared between cornea sections using a digital imaging program. Conclusions:We report a novel technique for visualizing rabbit corneal nerves. Immunohistochemical preparation of corneas with anti–tubulin antibody and secondary antibody labeled with a fluorescent probe allows for adequate analysis of corneal nerve morphology and surface area quantification.

Keywords: laser • microscopy: light/fluorescence/immunohistochemistry • microscopy: confocal/tunneling