Abstract Body: Introduction: Post-stroke brain stimulation is a promising neurorestorative approach, yet the molecular mechanisms driving recovery remain unclear. Our previous work demonstrated that post-stroke optogenetic stimulations of the ipsilesional primary motor cortex (iM1) promotes functional recovery. To understand the mechanisms driving post-stroke recovery, we investigated the transcriptome of iM1 in non-stimulated and stimulated mice using RNA sequencing.

Methods: C57Bl6 male mice underwent stereotaxic surgery to express Channelrhodopsin in iM1 excitatory neurons, with optical fiber implanted in the same location. After 5-6 weeks, mice underwent transient middle cerebral artery occlusion (30 minutes). Stimulated mice received optogenetic stimulations from post-stroke days (PD) 5–14. Rotating beam test was performed at pre-stroke baseline, PD4, 7 and 14. iM1 from stimulated, non-stimulated stroke mice at PD 7 & 15, and sham mice were processed for RNA sequencing (n=4-5/group). Expression of cholesterol enzymes such as HMGCS1 was examined using quantitative PCR and immunohistochemistry. Cholesterol levels were visualized using filipin or BODIPY.

Results: iM1 stimulations enhanced recovery at PD14, with longer distance traveled and faster speed on the rotating beam test (p<0.05). RNA sequencing revealed a distinct transcriptomic landscape between stimulated and non-stimulated stroke mice. In non-stimulated mice, 4526 differentially expressed genes (DEGs) were identified in iM1 at PD7, and number of DEGs dropped to 95 by PD15, compared to sham controls. In contrast, stimulated mice exhibited 1446 DEGs at PD7, which remained elevated at 1090 DEGs by PD15. Ingenuity pathway analysis highlighted downregulation of multiple cholesterol biosynthesis and metabolism pathways in iM1-stimulated mice. qPCR confirmed that iM1 stimulation reduced the expression of cholesterol enzyme HMGCS1 in iM1. Preliminary BODIPY and HMGCS1 staining data indicated noticeable cholesterol level changes in iM1 post-stroke.

Conclusions: Our transcriptome data revealed important insights into the molecular signaling of optogenetic stimulation-induced recovery, particularly cholesterol metabolism. Ongoing studies include immunostaining of cholesterol metabolism related genes and co-staining of cholesterol levels with cell type specific markers (neurons, glia, microglia). These data suggest that reduction in cholesterol accumulation may be a key mechanism in post-stroke recovery.