An immunocytochemical study on specific amacrine cell subpopulations in the rat retina after ischemia

Transient retinal ischemia leads to the loss of neurons in the inner retina. In an accompanying paper [F. Dijk, S. Van Leeuwen, W. Kamphuis, Differential effects of ischemia/reperfusion on amacrine cell subtype-specific transcript levels in the rat retina, Brain Res., 1026 (2004) 194-204] we present the results of a study on the effects of experimentally induced retinal ischemia on transcript levels of genes expressed by distinct subpopulations of amacrine cells. In response to 60-min ischemia, three different patterns of changes in transcript levels were found, indicating a differential vulnerability of amacrine subtypes: (i) a gradual decrease of transcript level without recovery (parvalbumin; PV); (ii) a gradual decrease, with varying rates and degrees, followed by partial recovery after 72 h of reperfusion (choline acetyltransferase (ChAT), calretinin (CR) and glycine transporter (Glyt1)); (iii) no significant changes (substance P (SP)). In order to verify whether the degree of cell loss can be predicted from the quantified alterations in gene expression level, immunocytochemical stainings were carried out. A 60-min ischemic period was administered to the rat eye by raising the intraocular pressure, followed by a reperfusion period lasting between 2 h and 4 weeks. Cryosections were immunostained for Glyt1, PV, ChAT, CR, and SP. Double-labelling with apoptosis marker TUNEL was used to demonstrate cell type-specific apoptosis. Following ischemia, the numbers of detected PV-, Glyt1, ChAT-, and CR-immunopositive somata showed a substantial, but differential, reduction at 1-4 weeks after ischemia. The total amount of immunoreactivity present in the inner plexiform layer (IPL) also decreased. The extent of alterations derived from immunocytochemical staining was greater than was anticipated from the decrease of transcript levels. Only for SP, no significant decrease in number of cells or in the intensity of immunoreactivity in IPL was observed, which is in agreement with the absence of significant changes in transcript levels. In conclusion, retinal ischemia/reperfusion differentially affects amacrine cell populations. Although both protein and mRNA levels are reduced, transcript levels are less attenuated. Caution must be applied in the use of real-time quantitative PCR (qPCR) screening as a tool to assess the cellular pattern of neurodegeneration in the retina.