FIGURE 5

FIGURE 5

Distinct responses in Müller glia gliosis and proliferation in the CLL and AL models. Müller glia (MG) intermediate filaments were immunolabelled with anti-GFAP antisera (GFAP; green) and cell cycle re-entry was demonstrated with anti-PCNA antisera (PCNA; red). Nuclei were stained with TO-PRO-3 (blue). (A–H) The CLL model revealed minimal evidence of MG gliosis, localized to the inner retinal end feet. PCNA+ cells were minimal until 14 dpl, which demonstrated a significant increase in PCNA+ cells in the ONL that increased through 28 dpl. (I–P) Classic MG-mediated retinal regeneration in response to AL damage revealed an early increase in GFAP intensity in the ONL MG end feet at 24 hpl, and a second GFAP peak at 5 dpl. PCNA + cells resulting from MG division appeared in the INL at 36 hpl, which was followed by peak MGPC proliferation in the ONL at 72 hpl (Q) PCNA-positive nuclei quantified by hand-count over a 300 µm linear distance demonstrated the robust stem cell proliferation in the AL model (72 hpl-5 dpl; p < 0.0001), and late accumulation of PCNA + cells in the CLL model (14–28 dpl p < 0.0001). (R,S) Gene expression changes in pcna and gfap in CLL vs. AL damage models displayed as transcript pseudocounts from 3′mRNA-seq of individual retinas. (R) The transcriptional response of pcna followed the histological observations, with a strong peak in the AL model at 72 hpl vs. a slow mild increase in expression in the CLL model. (S) The gfap expression profile highlighted the robust gliotic response in the AL model in contrast with the absence of a significant response in the ALL model. (ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer; scale bar in panel P = 5 µm).