Background: Brain injury is the leading cause of death and disability in survivors of cardiac arrest, where neuroinflammation is believed to play a pivotal role, but the underlying mechanism remains unclear. Pyroptosis is a pro-inflammatory form of programmed cell death that triggers inflammatory response upon infection or other stimuli. This study aims to understand the role of microglial pyroptosis in post-cardiac arrest brain injury. Methods: Sprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or shamoperation. Flow cytometry analysis, Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), coimmunoprecipitation, and immunofluorescence were used to evaluate activated microglia and CD11b-positive leukocytes after cardiac arrest and assess inflammasome activation and pyroptosis of specific cellular populations. To further explore the underlying mechanism, MCC950 or Ac-YVAD-cmk was administered to block nod-like receptor family protein 3 (NLRP3) or caspase-1, respectively. Results: Our results showed that, in a rat model, successful resuscitation from cardiac arrest resulted in microglial pyroptosis and consequential inflammatory infiltration which was mediated by the activation of NLRP3 inflammasome. Targeting NLRP3 and caspase-1, the executor of pyroptosis, with selective inhibitors MCC950 and Ac-YVAD-cmk treatment significantly prevented microglial pyroptosis, reduced infiltration of leukocytes, improved neurologic outcome, and alleviated neuropathological damages after cardiac arrest in modeling rats. Conclusions: This study demonstrates that microglial pyroptosis mediated by NLRP3 inflammasome is critically involved in the pathogenesis of post-cardiac arrest brain injury and provides a new therapeutic strategy.

The breakdown of the blood-brain barrier (BBB) is related to the occurrence and deterioration of neurological dysfunction in ischemic stroke, which leads to the extravasation of blood-borne substances, resulting in vasogenic edema and increased mortality. However, a limited understanding of the molecular mechanisms that control the restrictive properties of the BBB hinders the manipulation of the BBB in disease and treatment. Here, we found that the glycocalyx (GCX) is a critical factor in the regulation of brain endothelial barrier integrity.First, endothelial GCX displayed a biphasic change pattern, of which the timescale matched well with the biphasic evolution of BBB permeability to tracers within the first week after t-MCAO. Moreover, GCX destruction with hyaluronidase increased BBB permeability in healthy mice and aggravated BBB leakage in transient middle cerebral artery occlusion (t-MCAO) mice. Surprisingly, ultrastructural observation showed that GCX destruction was accompanied by increased endothelial transcytosis at the ischemic BBB, while the tight junctions remained morphologically and functionally intact. Knockdown of ca-veolin1 (Cav1) suppressed endothelial transcytosis, leading to reduced BBB permeability, and brain edema. Lastly, a coimmunoprecipitation assay showed that GCX degradation enhanced the interaction between syndecan1 and Src by promoting the binding of phosphorylated syndecan1 to the Src SH2 domain, which led to rapid modulation of cytoskeletal proteins to promote caveolaemediated endocytosis. Overall, these findings demonstrate that the dynamic degradation and reconstruction of GCX may account for the biphasic changes in BBB permeability in ischemic stroke, and reveal an essential role of GCX in suppressing transcellular transport in brain endothelial cells to maintain BBB integrity. Targeting GCX may provide a novel strategy for managing BBB dysfunction and central nervous system drug delivery. | M AT ER I A L S A N D M ET HOD S | AnimalsMale C57BL/6J background mice were obtained from the Experimental Animal Center of Southern Medical University and housed under a 12-h light/dark cycle in a pathogen-free facility with free access to water and blood-brain barrier, brain edema, caveolin1, glycocalyx, ischemia stroke, transcytosis

The involvement of melanocortin 4 receptor gene (MC4R) in food intake and body weight regulation is well characterized. MC4R mutations are the most frequent monogenic cause of human obesity. Significant associations have been revealed between MC4R mutations and productive traits in pigs, cattle and poultry. Herein, fluorescence-based conformation sensitive gel electrophoresis was used to identify two single nucleotide polymorphisms (SNPs) in the coding region (93G>A and 292G>A) and two SNPs in the 3'-UTR area (1016G>A and 1240T>C) of MC4R gene in 132 German Merino sheep. We found that the 1016G>A mutation in the 3'-UTR was significantly associated with body weight at 120 and 180 days, average daily gain, back fat thickness and loin-eye area. Allele A located at the 292th position of MC4R gene representing Arg98 was associated with significantly higher loin-eye area in sheep. For the synonymous 93G>A mutation, A allele carrier animals had higher back fat thickness. Our results provide evidence that the MC4R gene may be a candidate gene for growth and meat quality traits with MC4R SNPs being potentially valuable as genetic markers for economic traits in German Merino sheep.

BackgroundGlibenclamide (GLB) reduces brain edema and improves neurological outcome in animal experiments and preliminary clinical studies. Recent studies also suggested a strong anti-in ammatory effect of GLB, via inhibiting Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) in ammasome activation. However, it remains unknown whether the anti-in ammatory effect of GLB is independent of its role in preventing brain edema, and how GLB inhibits the NLRP3 in ammasome is not fully understood. MethodsSprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or sham-operation. Wild type and Trpm4 −/− C57BL/6 male mice underwent radiation-induced brain injury or sham-operation. The Trpm4 siRNA and GLB were injected to block sulfonylurea receptor 1-transient receptor potential M4 (SUR1-TRPM4) channel in rats and mice. Western blotting, quantitative real-time polymerase chain reaction, behavioral analysis, histological examination, and MRI Scanning were used to evaluate the role of GLB in preventing NLRP3-mediated neuroin ammation through inhibiting SUR1-TRPM4, and corresponding neuroprotective effect. To further explore the underlying mechanism, BV2 cells were subjected to lipopolysaccharides, oxygen-glucose deprivation/reperfusion, or radiation. ResultsHere, in mice model of radiation-induced brain injury with minimal brain edema, GLB signi cantly alleviated neurocognitive de cit and neuropathological damage, via the inhibition of radiation-induced microglial NLRP3 in ammasome activation by blocking SUR1-TRPM4. Likewise, above neuroprotective effects were also con rmed in rat model of cardiac arrest with brain edema combined with neuroin ammation, through preventing SUR1-TRPM4-mediated NLRP3 activation. Of note, the above effects of GLB could be achieved by gene silencing or knockdown of Trpm4. In vitro, SUR1-TRPM4 and NLRP3 in ammasome were also activated in BV2 cells subjected to lipopolysaccharides, oxygen-glucose deprivation/reperfusion, or radiation, which could be blocked by GLB or 9-phenanthrol, a TRPM4 inhibitor.Importantly, activation of SUR1-TRPM4 in BV2 cells required the P2X7 receptor-mediated Ca 2+ in ux, which in turn magni ed the K + e ux via the Na + in ux-driven opening of K + channels, leading to the NLRP3 in ammasome activation.