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Claire Hippert, Amanda C. Barber, Anastasios Georgiadis, James W. Bainbridge, Alexander J. Smith, Jane C. Sowden, Robin R. Ali, Rachael Pearson; Modulation Of Gliosis Using shGFAP Or/and shvimentin To Determine Its Role In Photoreceptor Transplantation Efficiency. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2010.
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Cell transplantation is a potential therapeutic strategy for the irreversible loss of photoreceptors occurring in retinal degeneration. In the degenerating retina, Müller glia undergo gliosis, a process that includes hypertrophy and increased production of the intermediate filaments GFAP and vimentin. Glial scarring is likely to have a major impact on regenerative strategies, including cell transplantation, by acting as a physical barrier and/or as a reservoir of inhibitory molecules. Here, we studied the role of gliosis in impeding photoreceptor integration success in the rhodopsin knockout (rho-/-) model of retinal degeneration. To examine the impact of GFAP and Vimentin upregulation on cell integration efficacy, we downregulated GFAP and/or Vimentin in the degenerating retina.
Gliosis was examined by western blot and immunostaining. Photoreceptor transplantation was assessed by transplanting FACS-sorted rod precursors into rho-/- recipients at early (4 week), mid (6 week) or late (10 week) degenerative stages. GFAP and vimentin expression was manipulated using AAV2/9-shGFAP, AAV2/9-shVimentin (Vim) or AAV2/9-lshGFAPshVim vectors. Their efficacy was studied by qRT-PCR and immunostaining at 1 week post subretinal injection.
Total GFAP expression was higher in the rho-/- at 4 wks compared to wildtype, and increased significantly between 4 and 10 weeks. The number of GFAP+ve processes extending into the outer nuclear layer (ONL) also increased together with the appearance of lateral glial scars at the outer edge of the ONL. Transplanted photoreceptor integration was significantly lower in the rho-/- mouse than in age-matched wildtype controls and declined markedly with degeneration. Expression of GFAP and vimentin was specifically knocked down by subretinal injection of AAV2/9-shGFAP or AAV2/9-shVim, which led to significant decrease in GFAP (~80%) and Vimentin (~50%) respectively, compared to non-injected control.
There is an inverse correlation between increasing gliosis and transplanted photoreceptor integration in the rho-/- mouse. We have demonstrated the efficacy of our AAV2/9-shGFAP and shVim expression vectors in the retina. We are using these vectors to determine whether or not GFAP and vimentin, the hallmarks of gliosis, impede transplanted photoreceptor integration.
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