Microtubules contribute to the birefringence of the retinal nerve fiber layer

PURPOSE. The retinal nerve fiber layer (RNFL) exhibits birefringence that is due to the oriented cylindrical structure of the ganglion cell axons. Possible birefringent structures include axonal membranes, microtubules (MTs), and neurofilaments. MTs are generally assumed to be a major contributor, but this has not been demonstrated. In this study, the MT depolymerizing agent colchicine was used to evaluate the contribution of MTs to RNFL birefringence. METHODS. Retinal nerve fiber bundles of isolated rat retina were observed through an imaging polarimeter set near extinction. Images were taken over an extended period. During baseline, the tissue was perfused with a physiological solution. During a treatment period, the solution was switched either to a control solution identical with the baseline solution or to a similar solution containing colchicine. The contrast of nerve fiber bundles was used to follow change of RNFL birefringence over time. RESULTS. When imaged by the polarimeter, birefringent retinal nerve fiber bundles appeared as either bright or dark stripes. Bundles displayed as bright stripes were used to follow changes in retardance. The contrast of nerve fiber bundles was stable in control experiments. However, in treatment experiments, bundles were bright during the baseline period, but the contrast of bundles decreased rapidly when the colchicine solution was applied; bundles were barely visible after 30 minutes of treatment. After 70 minutes, the bundle contrast was close to zero at all wavelengths studied (440-780 nm). CONCLUSIONS. MTs make a significant contribution to RNFL birefringence. The decrease of RNFL birefringence in glaucoma may indicate a loss of MTs.