Abstract Body: Background: Neuronal circuitry remodeling which compromises excitatory and inhibitory neurons is critical to improve neurological outcome after stroke. Cerebral endothelial cell (CEC) generated small extracellular vesicles (CEC-sEVs) have a therapeutic effect on stroke recovery. However, it remains challenge to use sEVs for specifically targeting individual neurons for enhancement of the circuitry after stroke. Using a chemogenetic approach, we tested the hypothesis that Designer Receptors Exclusively Activated by Designer Drugs (DREADD) activated peri-infarct cortical interneurons preferentially take up exogenous CEC-sEVs to enhance neuronal remodeling and functional recovery.
Methods: Adeno-associated viruses (AAVs) carrying Gq-DREADD and Tomato under the control of the transcription factor distal-less homeobox (Dlx) enhancer element (AAV-hDlx-Gq, 500 nL) were injected into the cortex. The mice were then subjected to middle cerebral artery occlusion (MCAO). CEC-sEVs (1x10¹¹ particles, i.v.) were administered following interneuron activation by Clozapine-N-oxide (CNO, 5mg/kg, i.p). The circuitry was assayed by Golgi-Cox staining and biotinylated dextran amine labeled axons in the corticospinal tract (CST). Transmission electron microscope (TEM) was used to measure CEC-sEVs in neurons and neuronal mitochondrial (Mito) integrity.
Results: The adjunct therapy of CEC-sEVs and CNO increased CEC-sEV uptake by peri-infarct cortical neurons and improved somatosensory functional outcome by ~70% vs CEC-sEVs or CNO alone (p<0.05, n=10/group). The adjunct therapy (p<0.05) augmented peri-infarct cortical axonal/dendritic outgrowth and CST axonal remodeling but did not reduce infarct volume. TEM analysis revealed that the neurons with uptake of CEC-sEVs (p<0.05) increased their Mito with intact cristae. Western blot showed that adjunct CEC-sEVs with CNO (p<0.05) decreased ischemia-increased Drp1 and Mff, and elevated ischemia-reduced Mito oxidative phosphorylation (OXPHOS) protein, COX5A, vs CEC-sEVs or CNO alone. Increases of Mito Drp1 and Mff impair Mito crista integrity, which reduces OXPHOS-induced ATP production.
Conclusions: Our data demonstrate that activated cortical interneurons preferentially increased the uptake of CEC-sEVs, leading to the improvement of functional outcome after stroke. The increased CEC-sEV uptake by activated interneurons reduced ischemic neuronal Mito damage, which may contribute to enhanced neuronal circuitry remodeling.