The Effect of Experimental Glaucoma and Optic Nerve Transection on Amacrine Cells in the Rat Retina

A macrine cells of the vertebrate retina are interneurons that modify the visual signal as it moves through the photoreceptor-bipolar-retinal ganglion cell (RGC) chain. They are synaptically active in the inner plexiform layer (IPL), with cell bodies in both the inner portion of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). Amacrine cells integrate, modulate, and interpose a temporal domain in the visual message presented to the RGC.1 Amacrine cells are the most diverse retinal neuronal type, with more than 20 classes, based on size, shape, stratification pattern, and neurotransmitter.2 Most amacrine cells are inhibitory neurons, using the inhibitory neurotransmitters γ-aminobutyric acid (GABA) or glycine. Amacrine cells expressing either GABA or glycine comprise 95% of all amacrine cells in the retina.3 Among human retinal amacrine cells, the GABAergic comprise 55% and the glycinergic 40% of the total. Several studies have evaluated the effect of RGC depletion on amacrine cells, but most research has focused on the neonatal eye. GABAergic and glycinergic amacrine cell distribution was altered by unilateral removal at birth of the superior colliculus, pretectum, and optic tract in rats.4 This experiment found a decrease in the number of INL cells immunoreactive for the GABA isoforms GAD-65 and GAD-67. At the same time, cells expressing the GAD-65 isoform increased in the GCL. In two other investigations, 1-day-old rats underwent unilateral optic nerve transection with survival for 1 to 2 months. There was no change in labeling for tyrosine hydroxylase, choline acetyltransferase, and substance P among amacrine cells.5,6 In the neonatal ferret, early RGC elimination caused a 25% reduction in presumed amacrine cells that were immunoreactive for glycine transporter and a 34% reduction in GABAergic amacrine cells.7 Few studies have specifically quantified a potential change in amacrine cells after optic nerve injury in adult animals or humans. Dijk et al.8 studied the effects of ischemia on amacrine cell subtype-specific transcript levels in the adult rat retina by real-time quantitative polymerase chain reaction. By 4 weeks after ischemic injury, glycinergic amacrine cell markers recovered to 65% to 75% of the normal control level, whereas GABAergic markers completely recovered. There was a substantial reduction 4 weeks after ischemia in cells labeling for parvalbumin, glycine transporter 1, and choline acetyltransferase.9 To our knowledge, there are no studies on the effect of amacrine cells by transection or crush of the nerve. In studies of experimental and human glaucoma, there are qualitative and some quantitative studies of amacrine cells, but the data are modest. Vickers et al.10 and Harwerth et al.11 examined a small number of retinal specimens from monkeys with experimental glaucoma and detected no loss of amacrine cells. A report of another study of four glaucomatous monkey eyes stated that there was a qualitative loss of presumed amacrine cells labeling for NADPH-diaphorase.12 May and Mittag13 studied amacrine cells in DBA/2NNia (DBA) mice with a condition exhibiting some features of glaucoma. Amacrine cell subpopulations had no significant staining differences compared with nonglaucomatous mice when antibodies against tyrosine hydroxylase, GABA, and vesicular acetylcholine transporter (VAChT) were used. Moon et al.14 labeled amacrine cells in normal mice and in DBA/2J mice GABA, glycine and choline acetyltransferase, and nitric oxide synthase. They observed no changes in the population of glycine-immunoreactive cells, but reported increases in some GABA-labeled cells in one layer and decreases in another. No statistical comparisons were presented. In the past, our laboratory has shown preservation of photoreceptor and INL cells in rat,15 monkey,16 and human17 glaucoma eyes, but we did not attempt quantification of amacrine cells. Whether amacrine cells are affected by glaucoma is still a controversial issue. Thus, it is important to study amacrine cells in a mature animal model and to determine quantitatively whether RGC loss results in amacrine cell death. It may be that concomitant loss of amacrine cells would potentiate secondary degeneration of RGC in diseases such as glaucoma and other optic neuropathies.18–20 Amacrine cells frequently synapse with RGCs and may provide some trophic or functional support to them. In addition, there are initial suggestions that progenitor cells may be used to replace lost RGCs in eye diseases, including glaucoma.21 If neuronal replacement is to be contemplated, it is important to establish how many cell types are needed. Our hypothesis is that RGC elimination alters the number or functional status of amacrine cells in the adult rat retina. We sought to identify a large proportion of amacrine cells by immunohistochemically labeling them for GABA and glycine. Investigators in earlier studies, who used antibodies against other molecules, were able to study only particular subpopulations of amacrine cells. We used two rat models known to affect RGCs: surgical optic nerve transection, in which severe ganglion cell loss occurs, and experimental glaucoma, which was produced by translimbal laser photocoagulation of the trabecular meshwork, which produces variable ganglion cell loss.