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Silke Becker, Thalis Charalambous, Hari Jayaram, Karen Eastlake, Angshumonik Angbohang, Peng Tee Khaw, G Astrid Limb; Modulation of Müller glia-derived neurotrophic factors by inflammatory cytokines during hypoxia. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2052.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerative diseases are frequently associated with inflammatory processes and release of inflammatory cytokines. Changes in tissue oxygenation have also been associated with altered neurotrophin production. Müller glia, which maintain retinal health, release various neurotrophic factors, which can mitigate retinal neuronal dysfunction and death during retinal injury and degeneration. We have investigated the production and release of neurotrophic factors by Müller glia in vitro in response to inflammatory cytokines, as well as components of the TGFβ pathway by which neurotrophin release might be affected during hypoxia.
Müller glia were cultured for 7 days with TGFβ1 (50ng/ml), IL-6 (10ng/ml) and TNFα (50ng/ml), normoxia (5% O2) or hypoxia (1% O2). Following extraction of total RNA and reverse transcription, PCR was performed for BDNF, NGF, NT-3, CNTF, GDNF, NRTN, TGFb1, TGFβR1-3 and β-actin. PCR products were quantified by gel electrophoresis followed by densitometric analysis. Protein concentrations of BDNF, NGF and NT-3 in cell culture supernatants were determined by ELISA.
Müller glia expressed mRNA coding for the neurotrophic factors BDNF, NGF, NT-3, CNTF, GDNF and NRTN, as well as TGFβ1 and TGFβR1-3. TGFβ1 supplementation attenuated NGF and BDNF expression, while upregulating NT-3 and GDNF mRNA. IL-6 decreased NGF gene expression and TNFα reduced NGF, BDNF, NT-3 and GDNF mRNA. During hypoxia mRNA expression and protein secretion of TGFβ1, NGF and NT-3 were increased. While BDNF mRNA expression was upregulated, protein release was not altered by hypoxia. CNTF gene expression was decreased during hypoxia, while GDNF and NRTN mRNA remained unaltered. Inhibition of the TGFβ pathway by the SMAD3 inhibitor SIS3 and the Alk5 blocker SB43152 abolished hypoxia-induced upregulation of NT-3, but not NGF and BDNF expression.
Müller glia express a large number of neurotrophic factors, which can be decreased by pro-inflammatory cytokines. Conversely, modulation of neurotrophic factor production by TGFβ1 is complex. Importantly, increased transcription of NT-3, but not NGF and BDNF, during hypoxia was shown to be TGFβ1 dependent. Modulation of pro- and anti-inflammatory factors in Müller glia may therefore represent potential treatment targets in retinal degenerative diseases with changes in tissue oxygenation to promote survival and function of retinal neurons.
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