A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery.

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Astrocytes have an important role in synaptic formation and function but how astrocytic processes become associated with synaptic structures during development is not well understood. Here we analyzed the pattern of growth of the processes extending off the main Bergmann glial (BG) shafts during synaptogenesis in the cerebellum. We found that during this period BG process outgrowth was correlated with increased ensheathment of dendritic spines. In addition, two-photon time-lapse imaging revealed that BG processes were highly dynamic, and processes became more stable as the period of spine ensheathment progressed. While process motility was dependent on actin polymerization, activity of cytoskeletal regulators Rac1 and RhoG did not play a role in glial process dynamics or density, but was critical for maintaining process length. We extended this finding to probe the relationship between process morphology and ensheathment, finding that shortened processes result in decreased coverage of the spine. Furthermore, we found that areas in which BG expressed dn-Rac1, and therefore had a lower level of synaptic ensheathment, showed an overall increase in synapse number. These analyses reveal how BG processes grow to surround synaptic structures, elucidate the importance of BG process structure for proper development of synaptic ensheathment, and reveal a role for ensheathment in synapse formation.