Retinal Ganglion Cell Quantification by Rbpms Immunohistochemistry in Models of Retinal Ganglion Cell Degeneration

Abstract

Purpose: : We previously demonstrated that RNA binding protein with multiple splicing (Rbpms) is a retinal ganglion cell (RGC) marker. This study evaluated RGC survival by Rbpms immunohistochemistry in the retinas after optic nerve crush injury and N-methyl-D-aspartate (NMDA)-induced excitotoxicity.

Methods: : Adult male Wistar rats weighing 300-350g were used. For the optic nerve crush injury model, the unilateral optic nerve was exposed and crushed for 10 seconds while the contralateral eye was served as an untreated control. Retinas were collected 1, 2, and 4 weeks after optic nerve crush (n=4 each). For the retinal excitotoxicity model, solutions of 1.2, 3, 12, 30, and 120 mM NMDA were injected intravitreally (n=4 each) and retinas were obtained 1 week post-injection. Administration of 120 mM NMDA and MK-801 (1mg/mL), an NMDA-receptor antagonist, was performed in control animals (n=4). Immunohistochemistry with antibody against Rbpms was performed on the retinal wholemounts. The numbers of Rbpms-positive cells at locations in the posterior and peripheral retina were quantified.

Results: : The densities of Rbpms-positive cells at 1, 2, 3, and 4 mm from the center of the optic nerve head in the untreated control retinas were 2881±349, 2722±372, 2319±619 and 1762±659 per mm² respectively. The means of Rbpms-positive cell density in the retinas 1, 2 and 4 weeks after optic nerve crush were 1537±147, 306±55, 168±20 respectively which corresponded to 36.5%, 87.4% and 93.1% loss of Rbpms-positive cells. Significant losses of Rbpms-positive cells were observed at all time points after optic nerve crush injury (P<0.05). The densities of Rbpms-positive cells in the retinas 1 week after injection of 1.2, 3, 12, 30 and 120 mM NMDA were 2442±528, 2304±537, 504±127, 200±57 and 233±35 per mm² respectively. There were significant losses of Rbpms-positive cells after injections of 12, 30 and 120 mM NMDA when compared to controls (P<0.05). The co-injection of MK-801 significantly reduced the loss of Rbpms-positive cells after 120 mM NMDA injection from 90% to 41% (P=0.019).

Conclusions: : Our temporal and dose dependent findings after optic nerve crush injury and NMDA-induced retinal excitotoxicity indicate that Rbpms can be effectively used to evaluate RGC survival in animal models of RGC degeneration.

Keywords: ganglion cells • optic nerve • immunohistochemistry