Non-invasive electric stimulation suppresses microglia activation and laser-induced choroidal neovascularization

Abstract

Purpose : Age-related macular degeneration (AMD) manifests with microglia activation and abnormal blood vessel growth leading to rapid loss of central vision. Electrical stimulation (ES) employing a low-intensity current presents a potential therapeutic modality that can be applied non-invasively to the skin of the eyelid. In this study, we investigated the effects of ES on laser CNV in vivo and microglial activation and endothelial cell (EC) tube formation in vitro.

Methods : Laser CNV was induced in adult C57BL/6 mice with a 532-nm laser (150 mW, spot size: 50 μm, pulse: 50 ms). The mice were then subjected to the daily ES (biphasic ramp, 300 µA, 20 Hz, transpalpebral, 4 min) or sham treatment. In vivo evaluations were recorded 7 days post-laser CNV using fluorescein angiography (FA), optic coherence tomography (OCT), and immunolabeling of retinal pigment epithelia/choroidal/scleral complexes (RCSC). In vitro primary microglia and primary human retinal endothelial cells (HREC) were subjected to ES (biphasic, 300 µA, 20 Hz, 1 h). Calcium flux in response to ATP (10^-7 M) stimulation (120 sec) was measured by Fura-2. HREC angiogenic potential was evaluated by tube formation and choroidal explant assays. HREC expression of VEGFa was detected by western blotting.

Results : ES in vivo reduced FA leakage area (p<0.01) and the size of the laser CNV lessons measured by OCT (p<0.01) compared to sham-treated mice (Fig 1). RCSC immunolabeling indicated significant reductions of lectin-positive area (P<0.01), the number of infiltrating IBA-1 (p<0.05) and COX-2 positive cells (p<0.05), and VEGFa signal (p<0.05). ES in vitro reduced ATP-induced Ca²⁺ flux in cultured microglia (p<0.05) and HREC cells (p<0.01). Results of seahorse analysis indicated reduced basal (p<0.05) and maximum (p<0.05) respiration and ATP production (p<0.01) in microglia The tube formation (p<0.01), choroidal explants outgrowth (p<0.05), and VEGF expression (p<0.01) were suppressed by the ES application in vitro.

Conclusions : ES inhibits microglia activation and EC angiogenic responses by reversibly depolarising mitochondrial potential and ATP production, reducing Ca²⁺ flux in response to proinflammatory and pro-angiogenic stimuli. The ES is a promising non-invasive therapeutic strategy for ocular inflammation and angiogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Fig 1. Therapeutic transpalbabral ES attenuates laser-induced choroidal neovascularization in mice.