Microglia repair the disrupted blood-brain barrier following low-intensity pulsed ultrasound through p2ry12 signaling

Abstract In a phase I clinical trial for patients with recurrent glioblastoma, we used an implantable ultrasound device for low-intensity pulsed ultrasound and microbubbles (LIPU/MB) to open the blood-brain barrier (BBB). This strategy increased parenchymal drug concentrations by 3.7- to 5.9-fold. Magnetic resonance imaging analysis with gadolinium injections at different timepoints post-sonication revealed that the BBB integrity began to restore within an hour post-sonication. To investigate the effect of LIPU/MB, we biopsied sonicated and non-sonicated peritumoral brain regions 45 minutes post-sonication before tumor resection in 7 patients. Single-cell RNA sequencing analysis revealed that sonicated microglia clustered separately and had enrichment of gene ontology terms related to the positive regulation of cell adhesion (p < 2x10-6), wound healing (p < 3x10-5), and regulation of vascular development and angiogenesis (p < 4x10-5). Upregulated genes included IL1B (log2fold-change 2.9, p < 1x10-140), PLAUR (log2fold-change 1.9, p < 2x10-177), and VEGFA (log2fold-change 1.8, p < 1x10-125). We also evaluated the spatial distribution of microglia in sonicated and non-sonicated samples by multiplex immunofluorescence. Analysis of 20 vessels per sample demonstrated that sonicated microglia accumulated around blood vessels, averaging at 14 μm from CD31+ cells, compared to 45 μm in non-sonicated biopsies (p < 0.01). Based on previous reports of P2RY12-dependent microglial migration and repair of the BBB, we evaluated the effect of P2RY12 inhibition with clopidogrel on BBB repair post-sonication in mice. Permeability assay with fluorescein revealed that the BBB was 80% (p < 0.001) and 50% (p < 0.05) more permeable at 10 and 45 minutes post-sonication in clopidogrel-treated mice, respectively. This work provides human data supporting the role of microglia in the repair of the BBB, previously described in animals, and provides preclinical and clinical evidence for the role of these cells in the rapid restoration of vascular integrity post-LIPU/MB in humans.