Subarachnoid Hemorrhage Mice Research Articles

Low-dose LPS alleviates early brain injury after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling

BackgroundLow-dose lipopolysaccharide (LPS) protects against early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanism underlying the neuroprotective roles of low-dose LPS remain largely undefined. MethodsA SAH mice model was established and the pathological changes of brain were evaluated by wet-dry weight method, HE and Nissl staining, and blood-brain barrier (BBB) permeability assay. Cell apoptosis and inflammation were monitored by TUNEL, flow cytometry and ELISA assays. qRT-PCR, immunofluorescence and Western blot were used to detect the expression of microglial polarization-related or oxidative stress-associated markers. Bioinformatics analysis, luciferase and ChIP assays were employed to detect the direct association between FOXO1 and IL-10 promoter. The ubiquitination of FOXO1 in the in vitro SAH model was detected by co-IP. ResultsLow-dose LPS alleviated SAH-induced neurological dysfunction, brain edema, BBB disruption, damage in the hippocampus, neuronal apoptosis and inflammation via modulating microglial M1/M2 polarization by IL-10/IL-10R1 signaling. Mechanistic studies showed that FOXO1 acted as a transcriptional activator of IL-10. USP19 mediated the deubiquitination of FOXO1 to activate IL-10/IL-10R1 signaling, thereby regulating microglial M1/M2 polarization. Functional experiments revealed that low-dose LPS upregulated USP19 to modulate microglial M1/M2 polarization via FOXO1/IL-10/IL-10R1 signaling in SAH mice. ConclusionLow-dose LPS protected against EBI after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling.

CXCR2 antagonism attenuates neuroinflammation after subarachnoid hemorrhage

ObjectivesOveractivation of neuroinflammation can worsen the prognosis of subarachnoid hemorrhage (SAH) patients. CXCR2 is a widely expressed G protein-coupled receptor that participates in the regulation of inflammation, indicating a potential role of CXCR2 in SAH. Materials and methodsHerein, we examined the expression pattern of CXCR2 in the ipsilateral brain tissue of SAH mice. Then, we evaluated the effects of CXCR2 antagonist on neuroinflammation and neurological function after SAH. ResultsWestern blotting and immunohistochemistry revealed that CXCR2 expression was upregulated following SAH. Our results demonstrated that treatment with SB225002 inhibited inflammatory cytokine (IL-1β, IL-6, TNF-α, MCP-1) production in the brain and cerebrospinal fluid (CSF) following SAH. Our further findings confirmed that treatment with SB225002 ameliorated astrocytosis and microgliosis after SAH. Interestingly, SB225002 significantly improved neurological impairment after SAH. ConclusionsAltogether, these results suggest that pharmacologically targeting CXCR2 may be an effective disease-modifying treatment for SAH.

Regulatory T Cells Secrete IL10 to Suppress Neuroinflammation in Early Stage after Subarachnoid Hemorrhage.

Objective: Accumulating evidence supports neuroprotective effects of regulatory T cells (Tregs) in response to brain injury. However, the precise mechanisms underlying the beneficial effects of Tregs on suppressing neuroinflammation after subarachnoid hemorrhage (SAH) remain unclear. Methods: We performed flow cytometry to detect the infiltration of Tregs into the brain at different time points after SAH. Behavioral tests, including Adhesive and Rotarod, were performed to assess neurological deficits in mice after SAH. Bulk RNA sequencing was used to investigate the transcriptomic change of Tregs infiltrating into the brain after SAH. qPCR was performed to verify the variation of inflammatory cytokines expression in the brain after Tregs exogenous infusion. FoxP3-DTR mice and Il10 gene KO mice were used to explore the mechanism of Tregs inhibiting neuron apoptosis after infiltrating the brain following SAH onset. Results: Peripheral Tregs infiltrated into the brain one day after SAH and gradually accumulated in the hemorrhagic hemisphere. An exogenous infusion of Tregs significantly improved the neurological function of mice after SAH, while poor recovery of neurological function was observed in Tregs depletion mice. Transcriptome sequencing data suggested that the immunosuppressive function of brain-infiltrated Tregs was significantly upregulated. qPCR showed that the expression of pro-inflammatory cytokines decreased in the brain of SAH mice after exogenous Tregs infusion. Bioinformatic analysis revealed that IL-10 and other cytokines secreted by brain-infiltrated Tregs were upregulated after SAH. Moreover, exogenous infusion of Il10 gene KO Tregs did not totally improve neurological function in SAH mice. Conclusions: Tregs infiltrated into the brain in the early stage after SAH and exerted neuroprotective effect by secreting IL-10 to suppress neuroinflammation and reduce neuron apoptosis.

Isoflurane Conditioning-Induced Delayed Cerebral Ischemia Protection in Subarachnoid Hemorrhage-Role of Inducible Nitric Oxide Synthase.

Background Recent evidence implicates inflammation as a key driver in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (SAH). Inducible nitric oxide synthase (iNOS) is one of the known major mediators of inflammation. We previously showed that an inhalational anesthetic, isoflurane, provides strong protection against delayed cerebral ischemia after SAH. Our current study aims to define the role of iNOS in isoflurane conditioning-induced protection against delayed cerebral ischemia in a mouse model of SAH. Methods and Results The experiments used 10- to 14-week-old male wild-type (C57BL/6) and iNOS global knockout mice. Anesthetic conditioning was initiated 1 hour after SAH with isoflurane 2% for 1 hour. Isoflurane-induced changes in iNOS expression were measured. N-(3-(aminomethyl) benzyl) acetamidine, a highly selective iNOS inhibitor, was injected intraperitoneally immediately after SAH and then daily. Vasospasm, microvessel thrombosis, and neurological assessment was performed. Data were analyzed by 1-way ANOVA and 2-way repeated measures ANOVA followed by Student Newman Keuls comparison test. Statistical significance was set at P<0.05. Isoflurane conditioning downregulated iNOS expression in naïve and SAH mice. N-(3-(aminomethyl) benzyl) acetamidine attenuated large artery vasospasm and microvessel thrombosis and improved neurological deficits in wild-type animals. iNOS knockout mice were significantly resistant to vasospasm, microvessel thrombosis, and neurological deficits induced by SAH. Combining isoflurane with N-(3-(aminomethyl) benzyl) acetamidine did not offer extra protection, nor did treating iNOS knockout mice with isoflurane. Conclusions Isoflurane conditioning-induced delayed cerebral ischemia protection appears to be mediated by downregulating iNOS. iNOS is a potential therapeutic target to improve outcomes after SAH.

Effect of Ginseng Extract Ginsenoside Rg1 on Mice with Intracerebral Injury

Background: Traditional Chinese medicine has a long history of treating intracerebral injury. The aim of this research is to test the hypothesis that Ginsenoside Rg1 can alleviate brain injury in subarachnoid hemorrhage (SAH) mice. Methods: SAH mice were induced by autologous blood injection into cisterna magna, the mortality and neurological function were assessed. Ginsenoside Rg1 was administered continuously for seven days after SAH. Then, Y-maze test was used to detect the neurological deficits. Brains were harvested to evaluate the neuroinflammation of SAH by immunofluorescence staining and flow cytometry. Cerebral vasospasm was evaluated by hematoxylin-eosin (HE) staining, and neuronal apoptosis was evaluated by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining and HE staining. Results: We demonstrate that Ginsenoside Rg1 reduced mortality and improved neurological deficits, reduced inflammatory response, ameliorated cerebral vasospasm and neuronal apoptosis after SAH. Conclusion: Ginsenoside Rg1 can alleviate brain damage after SAH in mice.

Epidermal Growth Factor Receptor Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage in Mice.

Early brain injury including neuronal apoptosis is a main contributor to neurological deterioration after subarachnoid hemorrhage (SAH). This study was aimed to investigate whether EGFR (epidermal growth factor receptor)/NFκB (nuclear factor-kappa B) inducing kinase (NIK)/NFκB (p65 and p50) pathway is involved in the neuronal apoptosis after SAH in mice. C57BL/6 adult male mice underwent endovascular perforation SAH modeling or sham-operation (n=286), and 86 mild SAH mice were excluded. In experiment 1, vehicle or an EGFR inhibitor (632.0 ng AG1478) was administered intraventricularly at 30 minutes postmodeling. At 24 or 72 hours, after neurological score was tested, brain water content, double immunolabeling with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and a neuronal marker antimicrotubule-associated protein-2 antibody, Western blotting using whole tissue lysate or nuclear protein extraction of the left cortex, and immunohistochemistry for cleaved caspase-3, phosphorylated (p-) EGFR, NIK, p-NFκB p65, and NFκB p105/50 were evaluated. In experiment 2, after sham or SAH modeling, AG1478+vehicle or AG1478+4.0 ng EGF was administered intraventricularly. The brain was used for TUNEL staining and immunohistochemistry after 24-hour observation. SAH group showed deteriorated neurological score (P<0.01, Mann-Whitney U test), more TUNEL- and cleaved caspase-3-positive neurons (P<0.01, ANOVA), and higher brain water content (P<0.01, Mann-Whitney U test), and these observations were improved in SAH-AG1478 group. Western blotting showed that expression levels of p-EGFR, p-p65, p50, and nuclear-NIK were increased after SAH (P<0.05, ANOVA), and decreased by AG1478 administration. Immunohistochemistry revealed these molecules localized in degenerating neurons. EGF administration resulted in neurological deterioration, increased TUNEL-positive neurons, and activation of EGFR, NIK, and NFκB. Activated EGFR, nuclear-NIK, and NFκB expressions were observed in cortical degenerating neurons after SAH, and were decreased by administration of AG1478, associated with suppression of TUNEL- and cleaved caspase-3-positive neurons. EGFR/NIK/NFκB pathway is suggested to be involved in neuronal apoptosis after SAH in mice.

Role of galectin-3 in cardiac dysfunction induced by subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) is a severe acute cerebrovascular event that not only impairs the central nervous system but also negatively affects various other organs, including the heart. The underlying mechanisms, however, remain unclear. In this study, we discovered that mice with SAH exhibited significant cardiac injuries, such as extended QT and QTc intervals, cardiac fibrosis, and reduced cardiac ejection fractions. This phenomenon was accompanied by increased galectin-3 expression in the cardiac ventricle and can be reversed by galectin-3 inhibitor TD139. Interestingly, we also observed increased co-expression of galectin-3 in macrophage within the heart tissue of SAH mice. Additionally, when macrophage activation was suppressed using the beta-blocker propranolol, cardiac function improved, and galectin-3 expression in the cardiac tissue decreased. Collectively, our findings offer new insights into the role of galectin-3 in SAH-related cardiac dysfunction and suggest a macrophage-galectin-3 axis as a potential therapeutic strategy.

PHLDA1 modulates microglial response and NLRP3 inflammasome signaling following experimental subarachnoid hemorrhage.

Balancing microglia M1/M2 polarization is an effective therapeutic strategy for neuroinflammation after subarachnoid hemorrhage (SAH). Pleckstrin homology-like domain family A member 1 (PHLDA1) has been demonstrated to play a crucial role in immune response. However, the function roles of PHLDA1 in neuroinflammation and microglial polarization after SAH remain unclear. In this study, SAH mouse models were assigned to treat with scramble or PHLDA1 small interfering RNAs (siRNAs). We observed that PHLDA1 was significantly increased and mainly distributed in microglia after SAH. Concomitant with PHLDA1 activation, nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome expression in microglia was also evidently enhanced after SAH. In addition, PHLDA1 siRNA treatment significantly reduced microglia-mediated neuroinflammation by inhibiting M1 microglia and promoting M2 microglia polarization. Meanwhile, PHLDA1 deficiency reduced neuronal apoptosis and improved neurological outcomes after SAH. Further investigation revealed that PHLDA1 blockade suppressed the NLRP3 inflammasome signaling after SAH. In contrast, NLRP3 inflammasome activator nigericin abated the beneficial effects of PHLDA1 deficiency against SAH by promoting microglial polarization to M1 phenotype. In all, we proposed that PHLDA1 blockade might ameliorate SAH-induced brain injury by balancing microglia M1/M2 polarization via suppression of NLRP3 inflammasome signaling. Targeting PHLDA1 might be a feasible strategy for treating SAH.

Anti-Apoptotic Effects of AMPA Receptor Antagonist Perampanel in Early Brain Injury After Subarachnoid Hemorrhage in Mice.

This study was aimed to investigate if acute neuronal apoptosis is induced by activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors (AMPARs) and inhibited by a clinically available selective AMPAR antagonist and antiepileptic drug perampanel (PER) in subarachnoid hemorrhage (SAH), and if the mechanisms include upregulation of an inflammation-related matricellular protein periostin. Sham-operated and endovascular perforation SAH mice randomly received an administration of 3mg/kg PER or the vehicle intraperitoneally. Post-SAH neurological impairments and increased caspase-dependent neuronal apoptosis were associated with activation of AMPAR subunits GluA1 and GluA2, and upregulation of periostin and proinflammatory cytokines interleukins-1β and -6, all of which were suppressed by PER. PER also inhibited post-SAH convulsion-unrelated increases in the total spectral power on video electroencephalogram (EEG) monitoring. Intracerebroventricularly injected recombinant periostin blocked PER's anti-apoptotic effects on neurons. An intracerebroventricular injection of a selective agonist for GluA1 and GluA2 aggravated neurological impairment, neuronal apoptosis as well as periostin upregulation, but did not increase the EEG total spectral power after SAH. A higher dosage (10mg/kg) of PER had even more anti-apoptotic effects compared with 3mg/kg PER. Thus, this study first showed that AMPAR activation causes post-SAH neuronal apoptosis at least partly via periostin upregulation. A clinically available AMPAR antagonist PER appears to be neuroprotective against post-SAH early brain injury through the anti-inflammatory and anti-apoptotic effects, independent of the antiepileptic action, and deserves further study.

Abstract WP214: Inhibition Of 12/15-Lipoxygenase Attenuates Delayed Vasospasm After Subarachnoid Hemorrhage In Mice

Approximately 30% of subarachnoid hemorrhage (SAH) patients who survive the initial ictus develop delayed cerebral ischemia (DCI) 4 to 10 days following SAH. Multiple clinical studies indicate the cause of DCI is multifactorial and includes vasospasm. 12/15-Lipoxygenase (LOX), enzymes that metabolize arachidonic acid into bioactive lipid metabolites, such as 12-HETE and 15-HETE, are expressed in several cell types of the brain including neurons, and microglia. Previous work has shown that 12/15-LOX has detrimental effects on brain vasculature and neurons, triggers inflammation, and thrombosis. In a previous study, inhibition of 12/15-LOX was beneficial in reducing early brain injury in mice after SAH. We hypothesize that inhibition of 12/15-LOX would attenuate delay vasospasm and reduce DCI in SAH mice. C57BL/6 mice of both sexes were randomized into Sham+Vehicle, SAH+Vehicle, SAH+ML351. Vehicle and ML351 (LOX inhibitor, 25mg/kg) were intravenously injected 15 minutes after SAH. 12/15-LOX -/- mice of both sexes were randomized into sham or SAH. Behavior was assessed daily until euthanasia on day 5 for analysis of vasospasm. Following SAH, there, there was an increased expression of 12/15-LOX in brain endothelial cells. Inhibition of 12/15-LOX in C57BL/6 males reduced large artery vasospasm and microvessel constriction, improved functional outcome, and lowered DCI incidence. In female C57BL/6 mice, inhibition of 12/15-LOX also reduced vasospasm and microvessel constriction, and ameliorated behavioral deficits. 12/15-LOX -/- displayed better behavior and have less delayed vasospasm. The findings of this study suggest that inhibition of 12/15-LOX is therapeutically beneficial for male and female mice after SAH via preventing delay vasospasm and DCI.

Abstract WP220: Platelet Inhibition Prevents Delayed Cerebral Ischemia After Subarachnoid Hemorrhage In Mice

Subarachnoid hemorrhage (SAH) induces delayed cerebral ischemia (DCI) in about 30% of patients which is characterized by functional decline. As microthrombi is a cause of DCI, platelets may be a therapeutic target for preventing microthrombi formation and development of DCI. Clinical trials of anti-platelets have been inconclusive for their effect on outcome. However, clinical trials used different anti-platelets. Since platelets have numerous receptors which can each prevent thrombosis, there is no clear choice to target for SAH. Here, we utilize 6 platelet antagonists to identify if any receptor is a therapeutic target for DCI after SAH. Our hypothesis is that P2Y receptor inhibition will prevent microthrombi and reduce DCI after SAH. Male and female mice were randomly assigned into sham, SAH+vehicle, SAH+P2Y antagonism, SAH+PAR4 antagonism, SAH+PAF antagonism, SAH+TPβ-R antagonism, and SAH+GPIIb/IIIa antagonism. Mice were euthanized 2 (n=10/group) or 7 days (n=15/group) post-SAH. All outcomes were performed and data was analyzed by a blinded investigator. Behavior was assessed daily. Brain microthrombi were counted. Both male and female SAH mice treated with inhibitors of PAF and TPβ-R had significantly better day 1 function than vehicle-treated mice. Significantly better function on day 1 was also observed in only male mice treated the P2Y antagonists or GPIIb/IIIa antagonist. Neither sex had improved behavior for the PAR-4 inhibitor. Mice exhibiting better function had fewer brain microthrombi with the exception of the GPIIb/IIIa antagonist which reduced microthrombi in female mice but did not correspond with improved behavior. This suggests there may be another deleterious mechanism which is more prevalent/detrimental in females that is not prevented by GPIIb/IIIa inhibition, such as platelet degranulation. However, this needs to be further investigated. Additionally, inhibition of TPβ-R reduced the incidence of DCI compared to vehicle-treated mice. In conclusion, an anti-platelet drug for SAH needs to be chosen carefully as not all antagonists have a beneficial effect after SAH; of vital importance is that anti-platelet drugs may not have similar beneficial effects in both sexes.

Inhibition of CCR2 attenuates neuroinflammation and neuronal apoptosis after subarachnoid hemorrhage through the PI3K/Akt pathway

BackgroundNeuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C–C motif chemokine receptor 2 (CCR2) in SAH.MethodsPre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood–brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA.ResultsExpression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood–brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up.ConclusionCCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

Gas6 Promotes Microglia Efferocytosis and Suppresses Inflammation Through Activating Axl/Rac1 Signaling in Subarachnoid Hemorrhage Mice.

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is characterized byrapid development of neuron apoptosis and dysregulated inflammatory response. Microglia efferocytosis plays a critical role in the clearance of apoptotic cells, attenuation of inflammation, and minimizing brain injury in various pathological conditions. Here, using a mouse SAH model, we aim to investigate whether microglia efferocytosis is involved in post-SAH inflammation and to determine the underlying signaling pathway. We hypothesized that TAM receptors and their ligands regulate this process. To prove our hypothesis, the expression and cellular location of TAM (Tyro3, Axl, and Mertk) receptors and their ligands growth arrest-specific 6 (Gas6) and Protein S (ProS1) were examined by PCR, western blots, and fluorescence immunostaining. Thirty minutes after SAH, mice received an intraventricular injection of recombinant Gas6 (rGas6) or recombinant ProS1 (rPros1) and underwent evaluations of inflammatory mediator expression, neurological deficits, and blood-brain barrier integrity at 24h. Microglia efferocytosis of apoptotic neurons was analyzed in vivo and in vitro. The potential mechanism was determined by inhibiting or knocking down TAM receptors and Rac1 by specific inhibitors or siRNA. SAH induced upregulation of Axl and its ligand Gas6. The administration of rGas6 but not rPros1 promoted microglia efferocytosis, alleviated inflammation, and ameliorated SAH-induced BBB breakdown and neurological deficits. The beneficial effects of rGas6 were arrogated by inhibiting or knocking down Axl and Rac1. We concluded that rGas6 attenuated the development of early brain injury in mice after SAH by facilitating microglia efferocytosis and preventing inflammatory response, which is partly dependent on activation of Axl and Rac1.

Investigation and modulation of interleukin-6 following subarachnoid hemorrhage: targeting inflammatory activation for cerebral vasospasm

BackgroundCerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH.MethodsHuman aneurysmal blood and cerebral spinal fluid were collected for evaluation. To confirm mechanism, c57/bl6 wild type and c57/bl6 IL-6 female knockout (KO) mice were utilized with groups: saline injected, SAH, SAH + IL-6 blockade, SAH IL-6 KO, SAH IL-6 KO + IL-6 administration, SAH + p-STAT3 inhibition. Dual-labeled microglia/myeloid mice were used to show myeloid diapedesis. For SAH, 50 μm blood was collected from tail puncture and administered into basal cisterns. IL-6 blockade was given at various time points. Various markers of neuroinflammation were measured with western blot and immunohistochemistry. Cerebral blood flow was also measured. Vasospasm was measured via cardiac injection of India ink/gelatin. Turning test and Garcia’s modified SAH score were utilized. P < 0.05 was considered significant.ResultsIL-6 expression peaked 3 days following SAH (p < 0.05). Human IL-6 was increased in aneurysmal blood (p < 0.05) and in cerebral spinal fluid (p < 0.01). Receptor upregulation was periventricular and perivascular. Microglia activation following SAH resulted in increased caveolin 3 and myeloid diapedesis. A significant increase in BBB markers endothelin 1 and occludin was noted following SAH, but reduced with IL-6 blockade (p < 0.01). CV occurred 5 days post-SAH, but was absent in IL-6 KO mice and mitigated with IL-6 blockade (p < 0.05). IL-6 blockade, and IL-6 KO mitigated effects of SAH on cerebral blood flow (p < 0.05). SAH mice had impaired performance on turn test and poor modified Garcia scores compared to saline and IL-6 blockade. A distinct microglia phenotype was noted day 5 in the SAH group (overlap coefficients r = 0.96 and r = 0.94) for Arg1 and iNOS, which was altered by IL-6 blockade. Day 7, a significant increase in toll-like receptor 4 and Stat3 was noted. This was mitigated by IL-6 blockade and IL-6 KO, which also reduced Caspase 3 (p < 0.05). To confirm the mechanism, we developed a p-STAT3 inhibitor that targets the IL-6 pathway and this reduced NFΚB, TLR4, and nitrotyrosine (p < 0.001). Ventricular dilation and increased Tunel positivity was noted day 9, but resolved by IL-6 blockade (p < 0.05).ConclusionCorrelation between IL-6 and CV has been well documented. We show that a mechanistic connection exists via the p-STAT3 pathway, and IL-6 blockade provides benefit in reducing CV and its consequences mediated by myeloid cell origin diapedesis.Graphical abstract

Microglial TLR4 is Critical for Neuronal Injury and Cognitive Dysfunction in Subarachnoid Hemorrhage

BackgroundToll-like receptor 4 (TLR4) activation causes excessive production of proinflammatory mediators and an increased expression of costimulatory molecules that leads to neuroinflammation after subarachnoid hemorrhage (SAH). Although TLR4-mediated inflammatory pathways have long been studied in neuroinflammation, the specific glia implicated in initiation and propagation of neuroinflammation in SAH have not been well elucidated. In this study, we investigated the involvement of glial TLR4 including microglia and astrocytes in brain damage and poor neurological outcome.MethodsIn this study, global TLR4 knockout, cell-specific TLR4 knockout, and floxxed control male and female mice were used. The mice were injected with 60 μl autologous blood near the mesencephalon to induce SAH; animals were euthanized on postoperative day 7 for immunohistochemistry of glia and apoptotic cells. Microglial morphology was evaluated by using immunofluorescence density quantification to determine correlations between morphology and neuroinflammation. Microglial depletion was accomplished with the intracerebroventricular administration of clodronate liposomes. Cognitive function was assessed with Barnes maze.ResultsOn postoperative day 7 after SAH induction, neuronal apoptosis was markedly reduced in the clodronate liposome group compared with phosphate-buffered saline control liposomes, and cognitive performance in the clodronate group was improved, as well. Differences in microglial activation, assessed by morphometric analysis, and neuronal apoptosis were significantly greater in wildtype knockouts compared with cell-specific and global TLR4 knockouts. The mice lacking TLR4 on astrocytes and neurons showed no differences compared with wildtype mice on any end points.ConclusionsOur data suggest that microglial depletion with the intracerebroventricular administration of clodronate can improve the cognitive function in an SAH mouse model, and TLR4 is critical for microglial activation and neuronal injury. Only microglial TLR4 is necessary for brain damage and poor cognitive outcome rather than astrocyte or neuronal TLR4. Thus, microglial TLR4 could be a potent therapeutic target to treat SAH-associated neuronal injury and protect against cognitive dysfunction.

MiR-452-3p Targets HDAC3 to Inhibit p65 Deacetylation and Activate the NF-κB Signaling Pathway in Early Brain Injury after Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of stroke, and early brain injury (EBI) is a contributor to its unfavorable outcome. microRNA (miRNA) is abundantly expressed in the brain and participates in brain injury. This study investigated the effect of miR-452-3p on EBI after SAH. The murine model of SAH was established. miR-452-3p expression was detected 48h after the model establishment. Neurobehavioral function, blood-brain barrier permeability, brain water content, neuronal apoptosis, and inflammatory factors were evaluated. The cell model of SAH was induced by oxygen hemoglobin. Apoptosis rate, lactate dehydrogenase, and reactive oxygen species were detected. The targeting relationship between miR-452-3p and histone deacetylase 3 (HDAC3) was verified. The acetylation of p65 and the binding of HDAC3 to p65 were detected. The inhibitory protein of the nuclear factor κB pathway (IκBα) was detected. Suberoylanilide hydroxamic acid was injected into the SAH mice treated with miR-452-3p inhibitor. SAH mice showed upregulated miR-452-3p expression; reduced the neurological score; increased blood-brain barrier permeability, brain water content, and neuronal apoptosis; elevated pro-inflammatory factors; and reduced anti-inflammatory factors. SAH increased the apoptosis rate, lactate dehydrogenase release, and reactive oxygen species levels in oxygen-hemoglobin-treated neuron cells. Inhibition of miR-452-3p reversed the above trends. miR-452-3p targeted HDAC3. SAH upregulated p65 acetylation. miR-452-3p inhibitor promoted the binding of HDAC3 to p65, decreased p65 acetylation, and upregulated IκBα. Suberoylanilide hydroxamic acid reversed the protective effect of miR-452-3p inhibitor on SAH mice and aggravated brain injury. miR-452-3p targeted HDAC3 to inhibit the deacetylation of p65 and activate the nuclear factor κB pathway, thus aggravating EBI after SAH.

Subarachnoid Hemorrhage Induces Sub-acute and Early Chronic Impairment in Learning and Memory in Mice.

Subarachnoid hemorrhage (SAH) leads to significant long-term cognitive deficits, so-called the post-SAH syndrome. Existing neurological scales used to assess outcomes of SAH are focused on sensory-motor functions. To better evaluate short-term and chronic consequences of SAH, we explored and validated a battery of neurobehavioral tests to gauge the functional outcomes in mice after the circle of Willis perforation-induced SAH. The 18-point Garcia scale, applied up to 4days, detected impairment only at 24-h time point and showed no significant difference between the Sham and SAH group. A decrease in locomotion was detected at 4-days post-surgery in the open field test but recovered at 30days in Sham and SAH groups. However, an anxiety-like behavior undetected at 4days developed at 30days in SAH mice. At 4-days post-surgery, Y-maze revealed an impairment in working spatial memory in SAH mice, and dyadic social interactions showed a decrease in the sociability in SAH mice, which spent less time interacting with the stimulus mouse. At 30days after ictus, SAH mice displayed mild spatial learning and memory deficits in the Barnes maze as they committed significantly more errors and used more time to find the escape box but still were able to learn the task. We also observed cognitive dysfunction in the SAH mice in the novel object recognition test. Taken together, these data suggest dysfunction of the limbic system and hippocampus in particular. We suggest a battery of 5 basic behavioral tests allowing to detect neurocognitive deficits in a sub-acute and chronic phase following the SAH.

Abstract WMP111: Microvascular Occlusion Precedes Delayed Deficits In Mice With Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) induces delayed cerebral ischemia (DCI) in about 30% of patients which is characterized by functional decline. Microvascular occlusion, via microvessel constriction or microthrombi, may be a cause of DCI. Our hypothesis is that microvascular occlusion and reduced cerebral blood flow (CBF) precede the onset of delayed deficits in mice with SAH. Mice were randomly assigned into sham, SAH+saline, and SAH+A3P5P+Clopidogrel (platelet antagonists). Laser speckle imaging (to assess large and microvessel perfusion) and behavior were assessed daily. All outcomes were performed and data was analyzed by a blinded investigator. Both large artery and microvessel/tissue perfusion recover to baseline in SAH mice which do not get delayed deficits. However, in SAH mice which develop delayed deficits, microvessel/tissue perfusion has a prolonged reduction (Figure 1A). Interestingly, large artery perfusion recovers in these mice. Antiplatelets prevent microthrombi, attenuating microvessel occlusion, and reduce delayed deficits (Figure 2B-C). Our findings suggest that 1) microvessel occlusion and prolonged tissue perfusion are causative factors for DCI and 2) preventing platelet activation may be a therapeutic target to reduce the incidence of DCI. Our data also suggests a sex difference in the response to platelet antagonists (data not shown).

Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviate M1 Microglial Activation in Brain Injury of Mice With Subarachnoid Hemorrhage via microRNA-140-5p Delivery.

BackgroundIt is documented that mesenchymal stem cells (MSCs) secrete extracellular vesicles (EVs) to modulate subarachnoid hemorrhage (SAH) development. miR-140-5p expression has been detected in MSC-derived EVs, while the mechanism of MSC-derived EVs containing miR-140-5p in SAH remains unknown. We aim to fill this void by establishing SAH mouse models and extracting MSCs and MSC-EVs.MethodsAfter ALK5 was silenced in SAH mice, neurological function was evaluated, neuron apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling with NeuN staining, and expression of serum inflammatory factors (interleukin-6, interleukin-1β, and tumor necrosis factor-α) was determined by enzyme-linked immunosorbent assay. The effect of ALK5 on NOX2 expression was assessed by western-blot analysis. Targeting the relationship between miR-140-5p and ALK5 was evaluated by dual luciferase assay. Following extraction of MSCs and MSC-EVs, EVs and miR-140-5p were labeled by PKH67 and Cy3, respectively, to identify the transferring of miR-140-5p by MSC-EVs. SAH mice were treated with EVs from miR-140-5p mimic/inhibitor-transfected MSCs to detect effects of MSC-EV-miR-140-5p on brain injury and microglial polarization.ResultsALK5 silencing increased the neurological score and reduced neuron apoptosis and neuroinflammation in SAH mice. ALK5 silencing inhibited M1 microglia activation by inactivating NOX2. ALK5 was a target gene of miR-140-5p. MSC-derived EVs contained miR-140-5p and transferred miR-140-5p into microglia. MSC-EV-delivered miR-140-3p reduced ALK5 expression to contribute to repression of brain injury and M1 microglia activation in SAH mice.ConclusionsMSC-derived EVs transferred miR-140-5p into microglia to downregulate ALK5 and NOX2, thus inhibiting M1 microglia activation in SAH mice.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI).

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.