NLRP3 inhibition attenuates early brain injury and delayed cerebral vasospasm after subarachnoid hemorrhage

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 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-1β production and stimulate microglia reactivity, both hallmarks of SAH pathology; thus, we hypothesized that inhibition of NLRP3 could alleviate 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 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 inhibitors could be promising tools in the development of therapeutics for EBI and DCI.

Keywords: Inflammation; Microglia; NLRP3 inflammasome; Stroke; Subarachnoid hemorrhage.

Fig. 1

MCC950 treatment inhibits NLRP3 inflammasome activation 24 h post-SAH. A Representative western blot and densitometric analysis for NLRP3 and caspase-1 (p10 subunit). B Representative western blot and densitometric analysis of caspase-1 (p20 subunit). C Representative western blot and densitometric analysis for IL-1β. Data presented as mean ± SEM, * p < 0.05 compared to sham + vehicle group, ** p < 0.01 compared to sham + vehicle group by Kruskal-Wallis test with Dunn’s multiple comparisons test

Fig. 2

NLRP3 inhibition with MCC950 prevents microglia morphology shift after SAH. A–C 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 = 50μm, 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, n = 5–6 per group for all data, ** p < 0.01 compared to sham surgery group by Kruskal-Wallis test with Dunn’s multiple comparisons test

Fig. 3

NLRP3 inhibition reduces early brain injury after SAH. A Brain water content measurements of the cerebral cortex 24 h post-SAH, n = 3 per group. B, C Western blot for transmembrane tight junction protein occluding (B) and ZO-1 (C) 24 h post-SAH, n = 4 per group. D Immunofluorescence staining for infiltrating neutrophils (red) 24 h post-SAH with Iba1 co-stain (green) to distinguish microglia in sham (left), SAH + vehicle (middle), and SAH + MCC950 (right) groups with DAPI nuclear counterstain (blue), scale bars = 50μm, red arrowheads indicate individual neutrophils. E Quantification of neutrophils (Ly6G/C⁺ cells) per HPF, n = 4 per group. F Representative images of fibrinogen-stained (red) cerebral cortex images captured with 40× or 64× (inset) objective lens. G Quantification of microthrombi per high-powered field (40×), n = 5–6 per group. All data presented as mean ± SEM, * p < 0.05, ** p < 0.01, compared to sham surgery group, # p < 0.05 compared to SAH + vehicle group by Kruskal-Wallis test with Dunn’s multiple comparisons test

Fig. 4

NLRP3 inhibition reduces neuronal apoptosis 24 h after SAH. A Representative images of TUNEL assay with NeuN co-stain in sham + vehicle (top), SAH + vehicle (middle), and SAH + MCC950 (bottom). B Representative images of TUNEL assay with Iba1 co-stain. C Quantification of total TUNEL-positive cells per high-powered field (HPF). D Quantification of TUNEL-positive neurons as a proportion of total NeuN-positive cells per HPF. E Quantification of TUNEL-positive microglia as a proportion of total Iba1-positive cells per HPF. Images were captured with 10× or 40× objective lens, scale bars = 200μm for 10× images and 50μm for 40× images, n= 4 in each group, * p < 0.05 compared to sham+ vehicle group, ** p < 0.01 compared to sham + vehicle group by Kruskal-Wallis test with Dunn’s multiple comparisons test

Fig. 5

NLRP3 inhibition decreases delayed cerebral vasospasm after SAH. A Representative images of vessel-casted brains of sham + vehicle (left), SAH + vehicle (middle), and SAH + MCC950 (right) groups 5 days after SAH. The internal carotid artery (ICA), middle cerebral artery (MCA), and anterior cerebral artery (ACA) are identified in sham image. Arrows indicate areas of significant vasospasm, scale bars = 100μm. B Middle cerebral artery measurements normalized to sham group. Data presented as mean ± SEM, n = 7–9 per group, *** p < 0.001 compared to sham surgery group by Kruskal-Wallis test with Dunn’s multiple comparison test. C Composite neurological evaluation score, p > 0.05 at all timepoints and across all groups by two-way ANOVA. D Prevalence of right turns in the corner test, * p < 0.05, ** p < 0.01 vs sham + vehicle group, # p < 0.05 vs SAH + vehicle group by two-way ANOVA with Tukey’s multiple comparison test