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Anton Lennikov, Menglu Yang, Karen Chang, Li Pan, Daisy Y Shu, Kin-Sang Cho, Magali Saint-Geniez, Darlene A Dartt, Tor Utheim, Dongfeng Chen; Electric stimulation reduces microglia proinflammatory activity in vitro. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3177 – F0451.
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© ARVO (1962-2015); The Authors (2016-present)
Electrical stimulation (ES) employing a low-intensity electric current with minimal tissue heating presents a potential therapeutic modality that can be applied noninvasively to the eye. However, the cellular level effects of the ES on several retinal cell types are largely unexplored. In this study, we evaluated the effects of ES on microglia (MG) and human retinal endothelial cells (HREC) in vitro.
Primary brain MG was isolated from adult C57BL/6 mice. The resulting cultures were subjected to ES (biphasic, 300 µA, 20 Hz) for 1 hour. The activation profile of MG was studied by immunostaining, RT-PCR, and cytokine arrays. Calcium flux in response to ATP 10-7 M stimulation (120 sec) was measured by Fura-2. MG cells oxygen consumption and mitochondria activity were studied through high-resolution respirometry (Seahorse XF24, Agilent), JC-1 mitochondria membrane potential assay, and cellular ATP content quantification. The effect of ES on primary human endothelial cells (HREC) angiogenic activity was investigated using migration and tube formation assays combined with a measure of VEGFA expression. Findings were validated using the choroidal explant sprouting assay.
ES reduced the expression of proinflammatory cytokines, including IL-6, TNFα, COX-2, and ATP-induced Ca2+ flux in the MG cells (p<0.05). Seahorse XF24 analysis of ES MG cells indicated reduced basal (p<0.05) and maximal (p<0.05) respiration and ATP production (p<0.01). Proton leak was not significantly affected compared to control cultures (p>0.05), suggestive of a lack of mitochondria damage. The results were confirmed by reduced cellular ATP content (p<0.01) and reduced JC-1 mitochondrial potential in the ES MG cells. In HREC, endothelial cells migration (p<0.01) and tube formation (p<0.01) were suppressed by ES, vascular outgrowth from choroidal explants was also reduced (p<0.05). Western blotting and immunostaining analysis of ES HREC further confirmed reduced VEGFa expression.
ES inhibits MG activity by reversibly depolarising mitochondrial potential and ATP production. The lack of ATP reduces Ca2+ flux in response to proinflammatory stimuli due to depletion of endoplasmic reticulum (ER) Ca2+ depo. ES also reduces the angiogenic potential of HREC by suppressing VEGF expression and choroidal vessels ex-vivo. These data suggest the therapeutic potential of biphasic ES in inflammatory and neovascular diseases of the eye.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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