Abstract 36: Astrocyte-derived Small Extracellular Vesicles Mediate Astrocyte-neuron Communication To Promote Neurite Outgrowth After Stroke In Astrocyte-vesicle Reporter Mice

Abstract

As an integral part of the neuron-glia system, astrocytes directly contact and communicate to neurons. Small extracellular vesicles (sEVs) are essential mediators of intercellular communication for both physiological and pathological processes. Using astrocyte-sEV reporter mice, we investigated how endogenous astrocyte-derived sEVs communicate to neurons after ischemic stroke. The reporter mice were generated by crossing a CD63-green fluorescent protein (GFP) floxed mouse line with a GFAP-Cre ERT2 mouse line. Mice were subjected to permanent middle cerebral artery occlusion (n=10). To activate Cre recombinase, tamoxifen was injected daily for 4 days starting 1 day after stroke. Animals were euthanized 14 days after stroke. Coronal brain and spinal cord slices were used for immunofluorescent staining with antibodies against GFAP for astrocytes and SMI32 for neurons. Confocal microscopic imaging analysis showed that ischemia dramatically (p<0.01) increased GFP signal intensity in the brain peri-infarct area (58.4±3.6 A.U.) and in the stroke-impaired gray matter of the spinal cord (42.9±2.8 A.U.), compared to that in normal brain (31.7±1.8 A.U) and spinal cord (34.3±2.7 A.U). Neuronal internalization of astrocyte-sEVs measured as colocalization of GFP with SMI32 neurons in the brain and spinal cord was concomitantly and significantly increased in the stroke mice compared with that in normal mice (stroke brain 0.14±0.01% vs 0.07±0.01% in normal, and spinal cord 0.10±0.01% vs 0.06±0.01% in normal, p<0.05), which was confirmed by electron microscopy. In addition, GFP positive sEVs isolated from ischemic brain of the reporter mice significantly promoted neurite outgrowth of primary cultured mouse embryonic cortical neurons, compared to sEVs isolated from non-ischemic brain (p<0.001). Collectively, our data for the first time provide in vivo evidence that endogenous astrocyte-derived sEVs communicate with neurons under non-ischemic condition, while stroke robustly augments this communication, and thereby promotes neurite remodeling during stroke recovery.