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Sheik Pran Babu Sardar Pasha, Münch Robert, Schäfer Patrick, Oertel Peter, Mike O Karl; Retinal cell death dependent reactive proliferative gliosis in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5387.
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© ARVO (1962-2015); The Authors (2016-present)
Neurodegeneration is a common starting point of reactive gliosis, which may have beneficial and detrimental consequences. It remains incompletely understood how distinctive pathologies and neuronal cell death processes differentially regulate glia responses. Müller glia (MG) in the retina are a prime model, which regenerate neurons in some species, whereas in mammals may cause proliferative disorders and scarring. Here, we investigated the relationship of retinal damage and MG cell cycle re-entry in mouse retinal organotypic culture.
Murine retinas after the end of retinogenesis were cultured as explants. Retinal tissue damage was induced by stab wound(SW) injury. Retinal cell death was inhibited by application of selected cell-death signalling pathway inhibitors, e.g. Z-VAD-FMK, Necrostatin-1, and others. Cell death was monitored by TUNEL assay and expression of activated caspase 3. MG proliferation was determined by co expression of SOX2 with EdU and Ki67 immunostaining. Gene expression was analyzed by RT-PCR.
Retinal cell death and MG proliferation occur in a defined the temporal time course in our previously established retina ex vivo explant assay. Hypothermia pre-treatment during eye dissection reduced whereas SW injury increased neuronal cell death and MG proliferation. Combined (but not single) application of apoptotic (Z-VAD-FMK) and non-apoptotic (Necrostatin-1) cell death signalling–pathway inhibitors diminished neuron death(TUNEL+ cells: Necrostatin-1+Z-VAD-FMK 4 ±0.1 vs. CTRL 31 ±1, N=3, P<0.0001) and maintained MG mitotically quiescent (SOX2+KI67+: Necrostatin-1+Z-VAD-FMK 0.2 ±0.05 vs. CTRL 2 ±1 cells, N=4, P<0.05). Gene expression analysis of single and combined inhibitor treatments showed differential changes in cell death related pathways suggesting compensatory mechanisms. The level of neuronal cell death determined MG activity, indicated by extracellular signal-regulated kinase (ERK), and proliferation, which both became diminished by EGFR–inhibition.
Our results suggest that retinal damage depended cues, possibly controlled by both programmed apoptosis or non-apoptotic cell death signaling pathways, empower MG to gain competence to proliferate upon EGF stimulation via EGFR-MAPK signalling. Thus, mouse retina organotypic culture facilitates studies on the mechanism governing MG quiescence and neuron death–dependent MG reactivation and proliferative gliosis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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