Purpose:
Glaucoma damages axons of retinal ganglion cells. Cytoskeleton of axons is expected to change in the disease. The axonal cytoskeleton is made up of three major protein filaments: microtubules (MTs), actin filaments (F-actin) and neurofilaments (NFs). In this study, we used simultaneous staining of F-actin, MTs and NFs to investigate change of these components in glaucomatous retina.
Methods:
High intraocular pressure (IOP) was induced unilaterally in Wistar rats by laser photocoagulation of trabecular meshwork. Isolated retinas were immunohistochemically stained to label F-actin, MTs, and NFs. Distributions of these components in flat-mounted retinas were simultaneously imaged by confocal microscopy. En face images were collected through the retinal nerve fiber layer (RNFL). Cross-sectional images were synthesized from the reconstruction of the en face images.
Results:
In normal retinas, confocal images showed uniform and dense staining of F-actin, MTs and NFs within bundles (Fig.). In eyes with elevated IOP, distortion of cytoskeleton staining was found in all three components. Appearance of distortion varied from loss of staining density, decrease of staining uniformity to decrease of RNFL thickness or even total loss of the structure. Different degrees of distortion could occur in the same retina. Among 20 studied retinas with cumulative IOP ranging from 40 to 350 mmHg-days, 12 retinas showed local distortion, four retinas were found to have thinner RNFL over the whole retinas, and four retinas did not show apparent change of structure.
Conclusions:
Elevation of IOP causes non-uniform alteration of the cytoskeletal components in axons. The degree of change varies from distortion of structural distribution to the total disappearance of the structures, suggesting progressive change of axonal cytoskeleton in glaucoma. Different stages of progressive change found in the same retina indicate that distortion of cytoskeleton precedes loss of the structure and, therefore, can be an early sign of glaucomatous damage.
Keywords: cytoskeleton • nerve fiber layer • immunohistochemistry