June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Electric stimulation reduces microglia proinflammatory activity in vitro
Author Affiliations & Notes
  • Anton Lennikov
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
    Medical Biochemistry, Oslo University Hospital, Oslo, Norway
  • Menglu Yang
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Karen Chang
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
    Medical Biochemistry, Oslo University Hospital, Oslo, Norway
  • Li Pan
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Daisy Y Shu
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Kin-Sang Cho
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Magali Saint-Geniez
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Darlene A Dartt
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Tor Utheim
    Ophthalmology, Oslo University Hospital, Oslo, Norway
    Medical Biochemistry, Oslo University Hospital, Oslo, Norway
  • Dongfeng Chen
    Ophthalmology, Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Anton Lennikov None; Menglu Yang None; Karen Chang None; Li Pan None; Daisy Shu None; Kin-Sang Cho None; Magali Saint-Geniez None; Darlene Dartt None; Tor Utheim None; Dongfeng Chen None
  • Footnotes
    Support  This study supported by The Norwegian Research Council; Department of Ophthalmology, Oslo University Hospital, Oslo, Norway; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway (T.P.U); The Norwegian Association for the Blind and Partially Sighted (T.P.U); NEI 5R01EY029269 grant and The Iraty Award (M.S.G); Fight for Sight Leonard & Robert Weintraub Postdoctoral Fellowship (D.Y.S); BrightFocus Foundation Postdoctoral Fellowship Program in Macular Degeneration Research (D.Y.S); NIH grant EY031696 and Harvard NeuroDiscovery Center grant (D.F.C); Core Grant for Vision Research from NIH/NEI to the Schepens Eye Research Institute (P30EY003790)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3177 – F0451. doi:
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    • Get Citation

      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)

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Abstract

Purpose : 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.

Methods : 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.

Results : 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.

Conclusions : 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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