Degeneration of ganglion cells in the peripheral retina of transgenic animals containingthe mutated mouse myocilin gene.

Abstract

Abstract: : Purpose: To examine changes in the retina of transgenic (TG) animals containing the mouse myocilin gene with the Tyr423His point mutation. This mutation in the mouse gene corresponds to the Tyr437His mutation in the human myocilin gene which may be the cause of severe juvenile open–angle glaucoma. Methods: This mutation was introduced into a BAC clone containing the full length mouse myocilin gene. TG mice containing the mutated BAC DNA were generated using a standard protocol. Whole mount retinas and paraffin sections were quantitatively analyzed after immunostaining with antibodies against specific cell markers. Apoptosis was assessed using a TUNEL technique. Levels of Thy–1 mRNA in the retina were estimated by in situ hybridization and real–time PCR. Results: Since the loss of ganglion cells (GC) in the retina is a hallmark of glaucoma, we focused specifically on changes in the ganglion cell layer. In the central retina, no significant differences were observed between TG animals and their wild–type (WT) littermates at 6–8 months of age. However, degenerative changes were consistently found in the ganglion cell layer in peripheral retinas of TG animals. These changes included the appearance of TUNEL–positive cells. TUNEL–positive cells were not detected in the WT retinas. Cell counts of hematoxylin and eosin stained sections of TG eyes showed a 5–10% decrease in the number of cells in the ganglion cell layer, compared with WT. A similar (5–15%) decrease in cell number was observed in eye sections and whole mount retinas stained with an antibody against the neuronal marker NeuN. However, the most significant changes (up to 20% decrease) were observed in sections or whole mount retinas stained with antibodies against Brn3b or NF68, which specifically label GC. Real–time PCR analysis of mRNA levels of Thy–1, a specific marker to assess RGC degeneration, showed a 2.0–2.9 fold decrease in the quantity of Thy1 mRNA in TG as compared with WT retinas. In situ hybridization analysis demonstrated selective reduction in Thy–1 mRNA expression in RGC on the periphery of TG retina. Conclusions: Comparative characterization of the TG phenotype suggests that the presence of the mouse myocilin gene with the Tyr423His point mutation induces selective degeneration of RGC in peripheral retina. The degenerative changes in the retina of TG animals resemble those observed in human glaucoma patients.

Keywords: retinal degenerations: cell biology • cell death/apoptosis • gene/expression