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S. Saraswathy, N. A. Rao; Tlr4 Upregulation in the Retina Leads to Photoreceptor Mitochondrial Oxidative Stress and DNA Damage. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6256.
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© ARVO (1962-2015); The Authors (2016-present)
Our recent studies in a highly reproducible model of innate immunity showed upregulation of TLR4 in activated retinal microglia. The current study investigates mitochondrial oxidative stress and mitochondrial DNA damage from such TLR4 upregulation.
Innate immunity response in the retina was induced by subcutaneous injection of complete Freund’s adjuvant (CFA). CFA-injected B10RIII, Rag1-/-, MyD88-/- and TLR4-/- mice were killed on day 5 post injection. The controls were non-injected B10.RIII and C57BL/6 mice. RNA isolated from the retinas of B10RIII and MyD88-/-mice was subjected to oxidative stress and apoptosis PCR arrays. Retinas from B10RIII and TLR4-/- mice were subjected to a novel PCR technique for detection of DNA strand break and lesions in the mitochondria. Retinal cryosections from B10RIII, Rag1-/-, TLR4-/- and MyD88-/- mice were immunostained with 8-OHdG to localize oxidative DNA damage.
The oxidative stress genes, including the oxidases, cytokines, MnSOD, and glutathione peroxidases, were upregulated in the CFA-injected B10RIII mice compared to the controls; no such upregulation occurred in the MyD88-/- mice. There was a significant increase in mitochondrial DNA lesions in the CFA-injected B10RIII mice. Positive staining of 8-OHdG colocalizing with the mitochondrial marker was detected in the inner nuclear layer, photoreceptors, and ganglion layer. A similar staining was seen in the CFA-injected RAG1-/- mice. There was a significant reduction in the mtDNA lesions and 8-OHdG staining in the CFA-injected TLR4-/- mice. Staining for 8-OHdG was negative in the CFA-injected MyD88-/- mice. Apoptosis PCR array in the CFA-injected B10RIII mice demonstrated upregulation of apoptotic genes, caspase7, TNF, and TNF receptors.
Upregulation of TLR4 expression in retinal microglia during innate immune response is associated with neuronal cells (photoreceptors, inner nuclear layer) mitochondrial oxidative stress, and mitochondrial DNA damage. This novel finding is associated with upregulation of apoptotic genes. This oxidative stress and mtDNA damage is abrogated in MyD88-/- and TLR4-/- mice, indicating the importance of TLR4 in oxidative stress insult.
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