We hypothesized that NLRP3 inhibition via MCC950 would prevent cerebral vasospasm. group, *** p 0.001 compared to sham surgery group by Kruskal-Wallis test with Dunns multiple comparison. 12974_2021_2207_MOESM2_ESM.tif (1.6M) GUID:?C67FDD2E-0CE8-447D-BCFB-DA9228027987 Additional file 3. Raw blot files. 12974_2021_2207_MOESM3_ESM.pdf (589K) GUID:?14A47A02-927E-4E3E-9935-3B8707C67AD5 Data Availability StatementThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Abstract Background The NLRP3 inflammasome is a critical mediator of several vascular diseases through positive regulation of proinflammatory pathways. In this study, we defined the role of NLRP3 in both the acute and delayed phases following subarachnoid hemorrhage (SAH). SAH is associated with devastating early brain injury (EBI) in the acute phase, and those that survive remain at risk for developing delayed cerebral ischemia (DCI) due to NGD-4715 cerebral vasospasm. Current therapies are not effective in preventing the morbidity and mortality associated with EBI and DCI. NLRP3 activation is known to drive IL-1 production and stimulate microglia reactivity, both hallmarks of SAH pathology; thus, we hypothesized that inhibition of NLRP3 could alleviate DDPAC SAH-induced vascular dysfunction and functional deficits. Methods We studied NLRP3 in an anterior circulation autologous blood injection model of SAH in mice. Mice were NGD-4715 randomized to either sham surgery + vehicle, SAH + vehicle, or SAH + MCC950 (a selective NLRP3 inhibitor). The acute phase was studied at 1 day post-SAH and delayed phase at 5 days post-SAH. Results NLRP3 inhibition improved outcomes at both 1 and 5 days post-SAH. In the acute (1 day post-SAH) phase, NLRP3 inhibition attenuated cerebral edema, tight junction disruption, microthrombosis, and microglial reactive morphology shift. Further, we observed a decrease in apoptosis of neurons in mice treated with MCC950. NLRP3 inhibition also prevented middle cerebral artery vasospasm in the delayed (5 days post-SAH) phase and blunted SAH-induced sensorimotor deficits. Conclusions We demonstrate a novel association between NLRP3-mediated neuroinflammation and cerebrovascular dysfunction in both the early and delayed phases after SAH. MCC950 and other NLRP3 NGD-4715 inhibitors could be promising tools in the development of therapeutics for EBI and DCI. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-021-02207-x. 0.05 compared to sham + vehicle group, ** NGD-4715 0.01 compared to sham + vehicle group by Kruskal-Wallis test with Dunns multiple comparisons test NLRP3 inhibition prevents microglia morphology shift after SAH Microglia are well-known to adopt a morphologic shift from ramified to amoeboid upon reacting to stroke injury [35]. We assessed the effect of NLRP3 inhibition on microglia morphology by automated counting of the number of endpoints of Iba1+ cell bodies in the cerebral cortex 24 h post-SAH. SAH surgery caused a significant decrease in endpoints (sham 12.37 1.24 NGD-4715 vs SAH + vehicle 5.05 0.97 endpoints/cell) (Fig. ?(Fig.22 A and B). MCC950 treatment blunted this response (10.33 1.12 endpoints/cell) (Fig. ?(Fig.22 C). Total microglial burden in the ipsilateral cerebral cortex was unchanged in all groups (sham + vehicle 12.53 1.05, SAH + vehicle 11.75 0.76, SAH + MCC950 12.79 0.81 Iba1+ cells/HPF) (Fig. ?(Fig.22 E). These results indicate NLRP3 inhibition prevents microglial morphology shift without affecting the number of microglia present. Open in a separate window Fig. 2 NLRP3 inhibition with MCC950 prevents microglia morphology shift after SAH. ACC Representative images of Iba1-stained (red) cerebral cortex in A sham, B SAH + vehicle, and C SAH + MCC950 groups with DAPI nuclear counterstain (blue). Scale bars = 50m, all images captured with 40 objective. Inset: Enlarged images of individual cell bodies. D Microglia morphology analysis via quantification of ramification endpoints per cell. E Total number of Iba1+ cells per high-powered field as a measurement of microglial burden. Data presented as mean SEM, = 5C6 per group for all data, ** 0.01 compared to sham surgery group by Kruskal-Wallis test with Dunns multiple comparisons test NLRP3 inhibition reduces early brain injury after SAH Cerebral edema, tight junction disruption, and peripheral immune cell infiltration are characteristic components of early brain injury. We assayed these parameters to evaluate the role of NLRP3 inflammasome in the early phase of SAH pathology. MCC950 partially reduced the development of cerebral edema 24 h post-SAH (sham + vehicle 3.20 0.01, SAH + vehicle 3.86 0.04, SAH + MCC950 3.43 0.03 g H2O/g dry weight) (Fig. ?(Fig.33 A). Further, MCC950 preserved the expression of the transmembrane tight junction protein occludin (sham + vehicle 1.00 0.05, SAH + vehicle 0.31 0.04, SAH + MCC950 0.56 0.08 relative expression units) and tight junction-associated protein ZO-1 (sham + vehicle 1.00 0.05, SAH + vehicle 0.62 0.05, SAH + MCC950 0.93 0.06 relative expression units) at the same time point (Fig. ?(Fig.33 B and C). We also found that there is.