Fig. 4

Characterization of neuronal survival in the ischemia/reperfusion model following treatment with AAV_EGFP, AAV_FH1-4^19-20 and AAV_FH1-7^19-20 (n = 3–6). A Cryosections from the central retina revealed TUNEL-positive apoptotic/necrotic cells (left panel) and calretinine-positive amacrine and ganglion cells (right panel) in both non-ischemic control and ischemic eyes treated with AAVs. B Quantitative analysis of the percentage of TUNEL-positive cells in each retinal layer and treatment at 3 and 14 dpi. C Evaluation of the outer plexiform layer (OPL) and the inner plexiform layer (IPL) thickness to assess the integrity of the synaptic connections representing neuronal processes. Values were normalized to non-ischemic control eyes of the corresponding animals. D. Quantification of nuclei within the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) per scan field. Data were normalized to non-ischemic control eyes of the corresponding animals. E To examine cell-specific neuronal loss in more detail, particularly given the susceptibility of inner retinal neurons to ischemia/reperfusion, we quantified calretinin-positive cells. Calretinin labels ganglion cells as well as displaced amacrine cells in the GCL and amacrine cells in the INL. B–E Each data point per biological replicate (n = 3–4) is represented by a dot in the graph. Unpaired t-test: *P < 0.05. C–E Dashed lines indicate the level of each parameter in the non-ischemic control eye.