Mouse Brain Vascular Pericytes from C57BL-6

Background While the role of pericytes in blood-brain barrier (BBB) disruption and neuroinflammation is well-established in adult neurological disorders, their contribut... More

Background While the role of pericytes in blood-brain barrier (BBB) disruption and neuroinflammation is well-established in adult neurological disorders, their contribution to neonatal brain injury is largely unexplored. Here, we investigated the role of brain pericytes in hypoxic-ischemic (HI) brain injury in the developing brain, with a particular focus on the regulatory role of pericyte-derived microRNA210 (miR210) in pericyte dysfunction. Methods HI brain injury was induced on postnatal day 9 transgenic mice, including Atp13a5-tdTomato brain pericyte reporter mice, pericyte-specific diphtheria toxin receptor mice, miR210 knockout mice, and wild-type controls. Post-injury assessments include brain infarct, brain edema, BBB permeability, ELISA, western blotting, immunostaining, and neurological function test. BBB-associated cells, including pericytes and endothelial cells, were isolated from mouse brain using an immunomagnetic approach. RNA sequencing analysis was conducted to examine transcriptomic changes in pericytes after HI. To investigate the regulatory role of miR210 in pericyte dysfunction and its underlying mechanisms, primary pericytes were transfected with miR210 mimic or negative control, followed by oxygen-glucose deprivation. Transfected cells were also treated with either interleukin 1 type 1 receptor neutralizing antibody or recombinant interleukin 1 type 2 receptor chimera protein. Post-assays included RT-qPCR, immunostaining and cell viability assay. Student’s t test or one-way ANOVA followed by Bonferroni test was used, as appropriate. Results HI resulted in a time-dependent loss of pericytes in pericyte reporter mouse pups. Ablation of brain pericytes exacerbated BBB disruption and HI brain injury in neonatal brain. miR210 deletion mitigated brain pericyte loss and BBB leakage post-HI. Transcriptomic analysis revealed that HI-induced pericyte dysfunction was associated with upregulated genes enriched in biological processes such as “cellular response to interleukin 1”. miR210 knockout suppressed the expression of proinflammatory markers such as Il1r1. Mechanistically, miR210 overexpression increased proinflammatory cytokine levels and promoted pericyte cell death under oxygen-glucose deprivation, effects that were reversed by IL1R1 blockade. Importantly, brain pericyte-specific miR210 deletion preserved pericyte viability and BBB integrity, and provided neuroprotection after HI. Conclusions These findings underscore the critical role of brain pericytes in BBB function in the developing brain and identify miR210 as a central regulator of pericyte dysfunction following neonatal HI brain injury. Less

Pericytes line the microvasculature throughout the body and play a key role in regulating blood flow by constricting and dilating vessels. However, the biophysical mechan... More

Pericytes line the microvasculature throughout the body and play a key role in regulating blood flow by constricting and dilating vessels. However, the biophysical mechanisms through which pericytes transduce microenvironmental chemical and mechanical cues to mediate vessel diameter, thereby impacting oxygen and nutrient delivery, remain largely unknown. This knowledge gap is clinically relevant as numerous diseases are associated with the aberrant contraction of pericytes, which are unusually susceptible to injury. Here, we report the development of a high-throughput hydrogel-based pericyte contraction cytometer that quantifies single-cell contraction forces from murine and human pericytes in different microvascular microenvironments and in the presence of competing vasoconstricting and vasodilating stimuli. We further show that murine pericyte survival in hypoxia is mediated by the mechanical microenvironment and that, paradoxically, pre-treating pericytes to reduce contraction increases hypoxic cell death. Moreover, using the contraction cytometer as a drug-screening tool, we found that cofilin-1 could be applied extracellularly to release murine pericytes from hypoxia-induced contractile rigor mortis and, therefore, may represent a novel approach for mitigating the long-lasting decrease in blood flow that occurs after hypoxic injury. Less