Subarachnoid Hemorrhage Model Research Articles

MCC950 ameliorates cognitive function by reducing white matter microstructure damage in rats after SAH

Neuroinflammation and white matter microstructure damage are important causes of cognitive impairment after subarachnoid hemorrhage (SAH). Nod-like receptor protein 3 (NLRP3) plays an important role in neuroinflammation after SAH and may be a potential therapeutic target for treatment of white matter microstructure injury. In this study, we observed whether MCC950, a specific inhibitor of the NLRP3 inflammasome, exerted a therapeutic effect after SAH. The SAH model was induced by endovascular perforation in SpragueDawley rats. MCC950 was injected intraperitoneally 1 h after SAH at a dose of 10 mg/kg. The results showed that MCC950 significantly attenuated white matter microstructure damage in some brain regions, and behavioral experiments confirmed that MCC950 ameliorated cognitive function in rats after SAH, which may provide a new method for the treatment of cognitive dysfunction in SAH patients.

Effect of Ropivacaine Hydrochloride Nanoliposomes on Delayed Cerebral Vasospasm After Subarachnoid Hemorrhage in Rabbits

This research was aimed to investigate the effect of ropivacaine hydrochloride (RP-HCl) nanoliposome (NLP) on delayed cerebral vasospasm (DCVS) after subarachnoid hemorrhage (SAH) in rabbits. RP-HCl liposomes were prepared with RP-HCl, chloroform-ether, soybean phospholipid, dipalmitoyl phosphatidyl glycerol, and L-lysine 5% glucose solution by secondary emulsification, and blank NLP (without RP-HCl) was constructed at the same time. Additionally, an SAH model was also established by secondary blood injection into the large occipital pool. Forty New Zealand rabbits were randomly assigned into sham operation group (the injected liquid was 0.9% sodium chloride injection), control (Ctrl) group (the injected liquid was autologous blood and sodium chloride injection in sequence), no-load group (the injected liquid was autologous blood and blank liposomes in sequence), drug-loaded group (the injected liquid was autologous blood and RP-HCl NLP in sequence), and conventional group (the injected liquid was sodium chloride injection and ropivacaine in sequence), with eight rabbits in each group. The effects of RP-HCl NLP were analyzed through the changes in mean blood flow velocity (Vm), peak systolic blood flow velocity (Vp), neuron-specific enolase (NSE), S100B, diameter and apoptosis of the basilar artery (BA), vascular endothelial cell (EC) (VEC) endothelin-1 (ET-1), and endothelial nitric oxide synthase (eNOS). Results: the average particle size of RP-HCl NLP was 17.73±3.22 μm, and the average encapsulation efficiency was 86.40%. Relative to those before modeling, the Vm, Vp, NSE, and S100B levels in the BA of the five groups were increased on the 1st, 5th, and 10th days after modeling (P <0.05). In contrast to sham group, Vm, Vp, NSE, S100B, ET-1, and the apoptosis index were increased on the 1st, 5th, and 10th days after modeling in the other four groups, while the caliber and eNOS were decreased (P <0.05). The Vm, Vp, NSE, S100B, ET-1, and apoptosis index on the 1st, 5th, and 10th days after modeling in Ctrl and no-load groups were increased versus drug-loaded group, while the pipe diameter and eNOS were decreased (P <0.05). In conclusions, RP-HCl NLP can regulate the balance between ET-1 and eNOS, enhance neurological function, alleviate EC apoptosis, and reduce cerebral blood flow velocity to relieve DCVS after SAH.

Salvianolic acid B ameliorates neuroinflammation and neuronal injury via blocking NLRP3 inflammasome and promoting SIRT1 in experimental subarachnoid hemorrhage.

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated immuno-inflammatory response plays a critical role in exacerbating early brain injury (EBI) after subarachnoid hemorrhage (SAH). Salvianolic acid B (SalB) has previously been shown to suppress neuroinflammatory responses in many disorders. Meanwhile, a previous study has demonstrated that SalB mitigated oxidative damage and neuronal degeneration in a prechiasmatic injection model of SAH. However, the therapeutic potential of SalB on immuno-inflammatory responses after SAH remains unclear. In the present study, we explored the therapeutic effects of SalB on neuroinflammatory responses in an endovascular perforation SAH model. We observed that SalB ameliorated SAH-induced functional deficits. Additionally, SalB significantly mitigated microglial activation, pro-inflammatory cytokines release, and neuronal injury. Mechanistically, SalB inhibited NLRP3 inflammasome activation and increased sirtuin 1 (SIRT1) expression after SAH. Administration of EX527, an inhibitor of SIRT1, abrogated the anti-inflammatory effects of SalB against SAH and further induced NLRP3 inflammasome activation. In contrast, MCC950, a potent and selective NLRP3 inflammasome inhibitor, reversed the detrimental effects of SIRT1 inhibition by EX527 on EBI. These results indicated that SalB effectively repressed neuroinflammatory responses and neuronal damage after SAH. The action of SalB appeared to be mediated by blocking NLRP3 inflammasome and promoting SIRT1 signaling.

Neurobehavioral Impairments Predict Specific Cerebral Damage in Rat Model of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe form of stroke that can cause unpredictable and diffuse cerebral damage, which is difficult to detect until it becomes irreversible. Therefore, there is a need for a reliable method to identify dysfunctional regions and initiate treatment before permanent damage occurs. Neurobehavioral assessments have been suggested as a possible tool to detect and approximately localize dysfunctional cerebral regions. In this study, we hypothesized that a neurobehavioral assessment battery could be a sensitive and specific method for detecting damage in discrete cerebral regions following SAH. To test this hypothesis, a behavioral battery was employed at multiple time points after SAH induced via an endovascular perforation, and brain damage was confirmed via postmortem histopathological analysis. Our results demonstrate that impairment of sensorimotor function accurately predict damage in the cerebral cortex (AUC 0.905; sensitivity 81.8%; specificity 90.9%) and striatum (AUC 0.913; sensitivity 90.1%; specificity 100%), while impaired novel object recognition is a more accurate indicator of damage to the hippocampus (AUC 0.902; sensitivity 74.1%; specificity 83.3%) than impaired reference memory (AUC 0.746; sensitivity 72.2%; specificity 58.0%). Tests for anxiety-like and depression-like behaviors predict damage to the amygdala (AUC 0.900; sensitivity 77.0%; specificity 81.7%) and thalamus (AUC 0.963; sensitivity 86.3%; specificity 87.8%), respectively. This study suggests that recurring behavioral testing can accurately predict damage in specific brain regions, which could be developed into a clinical battery for early detection of SAH damage in humans, potentially improving early treatment and outcomes.

P38-DAPK1 axis regulated LC3-associated phagocytosis (LAP) of microglia in an in vitro subarachnoid hemorrhage model

BackgroundThe phagocytosis and homeostasis of microglia play an important role in promoting blood clearance and improving prognosis after subarachnoid hemorrhage (SAH). LC3-assocaited phagocytosis (LAP) contributes to the microglial phagocytosis and homeostasis via autophagy-related components. With RNA-seq sequencing, we found potential signal pathways and genes which were important for the LAP of microglia.MethodsWe used an in vitro model of oxyhemoglobin exposure as SAH model in the study. RNA-seq sequencing was performed to seek critical signal pathways and genes in regulating LAP. Bioparticles were used to access the phagocytic ability of microglia. Western blot (WB), immunoprecipitation, quantitative polymerase chain reaction (qPCR) and immunofluorescence were performed to detect the expression change of LAP-related components and investigate the potential mechanisms.ResultsIn vitro SAH model, there were increased inflammation and decreased phagocytosis in microglia. At the same time, we found that the LAP of microglia was inhibited in all stages. RNA-seq sequencing revealed the importance of P38 MAPK signal pathway and DAPK1 in regulating microglial LAP. P38 was found to regulate the expression of DAPK1, and P38-DAPK1 axis was identified to regulate the LAP and homeostasis of microglia after SAH. Finally, we found that P38-DAPK1 axis regulated expression of BECN1, which indicated the potential mechanism of P38-DAPK1 axis regulating microglial LAP.ConclusionP38-DAPK1 axis regulated the LAP of microglia via BECN1, affecting the phagocytosis and homeostasis of microglia in vitro SAH model.3q9dhMG-MSCUeK5kURXPjHVideo

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.

WITHDRAWN: Transcription factor EB (TFEB) promotes autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) is responsible for devastating outcomes for patients with subarachnoid hemorrhage (SAH). Autophagy and apoptosis regulate the process of cell death. The transcription factor EB (TFEB) can increase autophagic flux by promoting autophagosome formation and autophagosome-lysosome fusion, and dysregulation of TFEB activity might induce the development of several diseases. However, the biological functions of TFEB in EBI post-SAH remain unknown. We established an animal model of SAH by modified endovascular perforation and a cellular model by treating primary cortical neurons with oxyhemoglobin (oxyHb). A recombinant adenovirus containing a shRNA targeting TFEB (adv-sh-TFEB) was used to knock down TFEB expression. Protein levels of TFEB, Bax, Bcl-2, cleaved caspase-3, LC3, and Beclin-1 were measured by western blotting and immunofluorescence staining. Neuronal apoptosis was assessed by flow cytometry and TUNEL staining. Short-term neurobehavioral functions were examined by modified Garcia and beam balance scores. Brain edema was determined through assessment of brain water content. TFEB was increased at the protein level in cellular and animal models of SAH. TFEB depletion attenuated the oxyHb-induced apoptosis of primary cortical neurons and autophagy. TFEB downregulation improved the short-term neurobehavioral functions and reduced brain edema after SAH and inhibited neuronal apoptosis in SAH rats. TFEB depletion improves short-term neurologic performance and reduces brain edema by preventing autophagy and apoptosis after SAH.

Evaluation of the Cerebroprotective Properties of Ademol-gel in the Analysis of Specific Indicators in the Open Field Test

Experimental subarachnoid hemorrhage (SAH) modeling in rats led to a significant, by 3 times, decrease in the total activity of animals, a decrease in the distance traveled by 2 times, a significant increase in inactivity when moving from the dark field to the illuminated part by 2 times, indicating a decrease in locomotor and search activity. SAH did not affect the number of unsupported rearing, but led to an increase in supported rearing against the wall, and a decrease in the number of short grooming acts against the background of an unchanged amount of long grooming. This behavior also testified to increased anxiety, excitability, irritability of animals, discomfort, and depression. The intranasal introduction of Ademol-gel at a dose of 2 mg/kg (a developed original intranasal form) to animals with SAH contributed to the positive emotional status and behavior of rats in the open field test, as evidenced by a decrease in anxiety, aggressiveness, and depressive behavior against the background of increased comfort and empathy in animals. Against the background of the intranasal introduction of Ademol-gel, the processes of restoring general motor and exploratory activity in animals after SAH were also accelerated. Nimodipine (30 μg/kg) was inferior to Ademol in terms of the degree of influence on the studied parameters of animal behavior. The results are an experimental justification for further study of a new dosage form – Ademol intranasal gel.

MicroRNA-130b May Induce Cerebral Vasospasm after Subarachnoid Hemorrhage via Modulating Kruppel-like Factor 4.

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for invitro experimentation. In vitro and invivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 (KLF4), oe-KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH. Elevated miR-130b and reduced KLF4 were found in SAH patients and rat models of SAH. KLF4 was the target gene of miR-130b. miR-130b promoted the proliferation and migration of cVSMCs through the Inhibition of KLF4. Besides, KLF4 inhibited the proliferation and migration of cVSMCs through blockage of the p38/MAPK pathway. Furthermore, invivo assay confirmed the inhibitory effect of decreased miR-130b in CVS following SAH. In conclusion, miR-130b may activate the p38/MAPK signaling pathway through targeted inhibition of KLF4, thereby contributing to some extent to the development of cerebral vasospasm after SAH.

Vasodilatory effects of tadalafil in an animal model of cerebral vasospasm: Comparative analysis with oral nimodipine

ObjectivesCerebral vasospasm is one of the most fatal complications after spontaneous aneurysmal subarachnoid hemorrhage. Although various treatments have been tried for the treatment of cerebral vasospasm so far, the effect is insignificant or temporary except for oral nimodipine. Phosphodiesterase isozyme type 5 inhibitor, which is used to treat erection dysfunction, recently has been known to have a cerebrovascular vasodilation. It is thought that this will be effective in cerebral vasospasm, and the effect will be compared and analyzed with oral nimodipine through an animal model of cerebral vasospasm. Material and MethodsA total of 40 rabbits were used to make subarachnoid hemorrhage model and were divided into three groups - a control group, nimodipine group, and tadalafil group. The cerebral vessels were angiographically measured before and on the third day of subarachnoid hemorrhage. Then vertebrobasilar arteries were harvested and evaluated. Under the microscope, lumen area and media area were measured for each group and were compared. ResultsAngiographically, tadalafil group showed significant vasodilation compared with the control group (p < 0.01). Histologically, tadalafil showed a similar effect on lumen and on media area to that of nimodipine group compared with the control group. ConclusionsCerebral vasospasm could leave neurologic deficit or sequelae even after proper treatment. Therefore, prevention is important. Tadalafil showed preventive effect against cerebral vasospasm and vasodilative effect similar to that of nimodipine. Therefore, tadalafil could be considered an alternative preventive treatment of cerebral vasospasm.

Ferrostatin-1 attenuates brain injury in animal model of subarachnoid hemorrhage via phospholipase A2 activity of PRDX6.

Subarachnoid hemorrhage (SAH) is an acute catastrophic neurological disorder with high morbidity and mortality. Ferroptosis is one of the pathophysiological processes during secondary brain injury of SAH, which could be inhibited by ferrostatin-1 (Fer-1) effectively. Peroxiredoxin6 (PRDX6) is an antioxidant protein and is currently proven to be associated with lipid peroxidation in ferroptosis except in GSH/GPX4 and FSP1/CoQ10 antioxidant systems. However, the alteration and function of PRDX6 in SAH are still unknown. In addition, whether PRDX6 is involved in the neuroprotection of Fer-1 in SAH is yet to be investigated. Endovascular perforation was employed to induce the SAH model. Fer-1 and in vivo siRNA aiming to knockdown PRDX6 were administrated intracerebroventricularly to investigate relevant regulation and mechanism. We confirmed the inhibition of ferroptosis and neuroprotection from brain injury by Fer-1 in SAH. The induction of SAH reduced the expression of PRDX6, which could be alleviated by Fer-1. Accordingly, dysregulated lipid peroxidation indicated by GSH and MDA was improved by Fer-1, which was counteracted by si-PRDX6. Similarly, the neuroprotection of Fer-1 in SAH was diminished by the knockdown of PRDX6 and the administration of a calcium-independent phospholipase A2 (iPLA2) inhibitor. PRDX6 is involved in ferroptosis induced by SAH and is associated with Fer-1 neuroprotection from brain injury via its iPLA2 activity.

An in vitro study on the stimulatory effects of extracellular glutamate on astrocytes.

In our previous research, it was found that the cerebrospinal fluid had higher levels of glutamate, astrocytes were stimulated and released pro-inflammatory factors in a subarachnoid hemorrhage model. Glutamate is a neurotransmitter produced in abundance by excitatory neurons in the central nervous system, residual glutamate can cause neurotoxicity. Recent studies indicate that most glutamate is absorbed by astrocytes, to optimize neurological functions and prevent excitotoxicity. However, it is still unclear if astrocytes could be stimulated by glutamate, and the concentration range of glutamate transportable by astrocytes. Thus, further research is necessary. This study aimed to clarify these scientific questions by stimulating primary astrocytes at different glutamate concentrations (0, 25, 50, and 100 µM) for 24h. The results showed that glutamate induced an increased response in astrocytes, the protein levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were upregulated in treatment groups with 50 and 100 µM. Additionally, the protein expression of complement component 3 (C3) significantly increased following glutamate stimulation (50 and 100 µM) for 24h. Furthermore, the supernatant of the 100 µM treatment group significantly decreased the viability of HT-22 (an immortalized mouse hippocampal neuronal cell line). In summary, our results indicate that increased extracellular glutamate levels can activate astrocytes and promote pro-inflammatory factor production. Moreover, the concentration range of glutamate transported by astrocytes is approximately less than 50-100 µM. Therefore, our study suggests that experimental antagonization of glutamate excitotoxicity is feasible.

Apolipoprotein E Polymorphism Impacts White Matter Injury Through Microglial Phagocytosis After Experimental Subarachnoid Hemorrhage

Apolipoprotein E (apoE, protein; APOE, gene), divided into three alleles of E2, E3 and E4 in humans, is associated with the progression of white matter lesion load. However, mechanism evidence has not been reported regarding the APOE genotype in early white matter injury (WMI) under subarachnoid hemorrhage (SAH) conditions. In the present study, we investigated the effects of APOE gene polymorphisms, by constructing microglial APOE3 and APOE4-specific overexpression, on WMI and underlying mechanisms of microglia phagocytosis in a mice model of SAH. A total of 167 male C57BL/6J mice (weight 22–26 g) were used. SAH and bleeding environment were induced by endovascular perforation in vivo and oxyHb in vitro, respectively. Multi-technology approaches, including immunohistochemistry, high throughput sequencing, gene editing for adeno-associated viruses, and several molecular biotechnologies were used to validate the effects of APOE polymorphisms on microglial phagocytosis and WMI after SAH. Our results revealed that APOE4 significantly aggravated the WMI and decreased neurobehavioral function by impairing microglial phagocytosis after SAH. Indicators negatively associated with microglial phagocytosis increased like CD16, CD86 and the ratio of CD16/CD206, while the indicators positively associated with microglial phagocytosis decreased like Arg-1 and CD206. The increased ROS and aggravating mitochondrial damage demonstrated that the damaging effects of APOE4 in SAH may be associated with microglial oxidative stress-dependent mitochondrial damage. Inhibiting mitochondrial oxidative stress by Mitoquinone (mitoQ) can enhance the phagocytic function of microglia. In conclusion, anti-oxidative stress and phagocytosis protection may serve as promising treatments in the management of SAH.

Transient cardiac electrophysiological changes in a rat model of subarachnoid haemorrhage: a brain-heart interaction.

Subarachnoid haemorrhage (SAH) is one of the causes of sudden cardiac death (SCD). However, the time course of ventricular arrhythmias and potential mechanisms responsible for this effect after SAH remain unknown. This study aims to investigate the effect of SAH on ventricular electrophysiological changes and its potential mechanisms in long-term phase. We examined the ventricular electrophysiological remodelling and potential mechanisms in a Sprague Dawley rat model of SAH at six time points (baseline, and Days 1, 3, 7, 14 and 28) and explored the potential mechanisms. We measured the ventricular effective refractory period (ERP), ventricular fibrillation threshold (VFT) and left stellate ganglion (LSG) activity at different time points before and after SAH. We also detected neuropeptide Y (NPY) levels in plasma and myocardial tissues by enzyme-linked immunosorbent assay, and quantified NPY 1 receptor (NPY1R) protein and mRNA expression levels by western blotting and quantitative real-time reverse transcription-polymerase chain reaction, respectively. Subarachnoid haemorrhage gradually prolonged QTc intervals, shortened ventricular ERP and reduced VFT during the acute phase, peaking at Day 3. However, no significant changes were observed from Days 14 to 28 compared to Day 0. Subarachnoid haemorrhage gradually increased LSG activity, increased NPY concentrations and up-regulated NPY1R expression in the acute phase of SAH, peaking at Day 3. However, no significant variations were found from Days 14 to 28 compared to Day 0. Subarachnoid haemorrhage increases the transient susceptibility of VAs in the acute phase, and the underlying mechanisms for this response included increased sympathetic activity and up-regulated NPY1R expression.

Ravoxertinib Improves Long-Term Neurologic Deficits after Experimental Subarachnoid Hemorrhage through Early Inhibition of Erk1/2.

Extracellular signal-regulated kinase 1 and 2 (Erk1/2) signaling has been shown to be involved in brain injury after subarachnoid hemorrhage (SAH). A first-in-human phase I study reported that ravoxertinib hydrochloride (RAH), a novel Erk1/2 inhibitor, has an acceptable safety profile and pharmacodynamic effects. Here, we showed that the level of Erk1/2 phosphorylation (p-Erk1/2) was significantly increased in the cerebrospinal fluid (CSF) of aneurysmal subarachnoid hemorrhage (aSAH) patients who developed poor outcomes. In a rat SAH model that was produced by the intracranial endovascular perforation method, western blot observed that the level of p-Erk1/2 was also increased in the CSF and basal cortex, showing a similar trend with aSAH patients. Immunofluorescence and western blot indicated that RAH treatment (i.c.v injection, 30 min post-SAH) attenuates the SAH-induced increase of p-Erk1/2 at 24 h in rats. RAH treatment can improve experimental SAH-induced long-term sensorimotor and spatial learning deficits that are evaluated by the Morris water maze, rotarod test, foot-fault test, and forelimb placing test. Moreover, RAH treatment attenuates neurobehavioral deficits, the blood-brain barrier damage, and cerebral edema at 72 h after SAH in rats. Furthermore, RAH treatment decreases the SAH-elevated apoptosis-related factor active caspase-3 and the necroptosis-related factor RIPK1 expression at 72 h in rats. Immunofluorescence analysis showed that RAH attenuated neuronal apoptosis but not neuronal necroptosis in the basal cortex at 72 h after SAH in rats. Altogether, our results suggest that RAH improves long-term neurologic deficits through early inhibition of Erk1/2 in experimental SAH.

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.

Sympathetic Nervous Hyperactivity Impairs Microcirculation Leading to Early Brain Injury After Subarachnoid Hemorrhage.

Although early brain injury (EBI) is recognized as a critical step following subarachnoid hemorrhage (SAH), its pathophysiology and underlying mechanisms remain poorly understood. Herein, we investigated the role of cerebral circulation in the acute phase using patient data and a mouse SAH model and evaluated its regulation via the sympathetic nervous system. The cerebral circulation time and neurological outcomes in the human body were retrospectively examined in 34 SAH cases with ruptured anterior circulation aneurysms and 85 cases with unruptured anterior circulation cerebral aneurysms at Kanazawa University Hospital from January 2016 to December 2021. In a mouse study, a SAH model was created via endovascular perforation, and India-ink angiography was performed over time. Additionally, bilateral superior cervical ganglionectomy was performed immediately before surgery, and neurological scores and brain water content were evaluated after SAH. Cerebral circulation time was prolonged in the acute phase of SAH compared with that in the unruptured cerebral aneurysm group, especially in those with electrocardiographic changes. Furthermore, it was more prolonged in the poor prognosis group (modified Rankin Scale scores 3-6) than in the good prognosis group (modified Rankin Scale scores 0-2) at discharge. In mice, cerebral perfusion was significantly reduced at 1 and 3 hours after SAH and recovered at 6 hours. superior cervical ganglionectomy improved cerebral perfusion without altering the diameter of the middle cerebral artery at 1 hour and improved neurological outcomes at 48 hours after SAH. Consistently, brain edema, quantified by brain water content, was improved by superior cervical ganglionectomy 24 hours after SAH. Sympathetic hyperactivity may play a critical role in the development of EBI by impairing cerebral microcirculation and edema in the acute phase following SAH.

Sinomenine Protects against Early Brain Injury by Inhibiting Microglial Inflammatory Response via Nrf2-Dependent Pathway after Subarachnoid Hemorrhage.

Microglial activation and sustained inflammation plays an important role in the processes of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Sinomenine (SIN) has been demonstrated to have neuroprotective effects in the traumatic brain injury (TBI) model. However, the role of SIN in SAH-induced EBI and its latent mechanisms remain unclear. This study was carried out to explore the role of SIN on SAH-induced EBI and its effects on the microglial inflammatory response following SAH. In this study, a model of SAH in rats was established. Modified neurological severity scores (mNSS), encephaledema, and Nissl staining were employed to determine the effects of SIN. Western blot and immunofluorescence analysis were performed to evaluate nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Nrf2-related downstream proteins, including heme oxygenase-1 (HO-1) and quinine oxidoreductase-1 (NQO-1), were detected with immunohistochemistry analyses and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Microglia activation and associated inflammatory factors, factor-kappa B (NF-κB), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were assessed after SAH. The results showed that SIN administration improved neurobehavior function, and attenuated neural apoptosis and brain edema after SAH. In addition, SIN inhibited microglial action and the subsequent inflammatory response after SAH through the upregulated expression of HO-1 and NQO-1 via activation of the Nrf2 pathway. These results demonstrated that SIN supplementation provided protection against SAH-induced neuronal apoptosis by microglial inflammatory response regulation and possible involvement of the Nrf2 pathway.

473 Role of Neutrophils and Neutrophil Extracellular Traps in Subarachnoid Hemorrhage in Mice

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (aSAH) continues to be a devastating disease. The role of neutrophils and neutrophil extracellular traps (NETs) have been investigated in other cerebrovascular disorders. Recent evidence suggest that neutrophils might play a role in outcomes after aSAH. METHODS: Subarachnoid hemorrhage model: The endovascular perforation model was utilized to induce SAH in mice. Flow cytometry: Flow cytometry technique was utilized to quantify neutrophils. Neutrophils were identified as CD45+Cd11b+Ly6G+ cells. NETosis assay: Neutrophils were assayed for their NETosis potential using a previously described method. The ratio of PMA (SYTOX+) to DMSO (SYTOX+) defined the NETosis induction. RNAseq: RNA sequencing was performed on neutrophils isolated from skull and femur bone marrow. Immunohistochemistry: NETs were assessed using classical IHC techniques and in vivo microscopy. Neurobehavior score: Mice were assessed daily for their behavior score using previously reported techniques. RESULTS: Neutrophils infiltrate brain within 24 hours and normalize by day 5. RNAseq demonstrates segregation of skull and femur bone marrow neutrophils following SAH. SAH induces a NETosis phenotype in skull neutrophils. NETs form starting day 1 after injury and persist until at least day 7. Neutrophil depletion and NETs inhibition attenuates early brain injury after SAH. Patients developing DCI have significantly higher blood markers of NETs. CONCLUSIONS: Neutrophils infiltrate the brain within the first 24 hours with NETs forming as early as day 1. Depletion of neutrophils and NET inhibition mitigated early brain injury following SAH. Blood markers of NETs are elevated in DCI patients and may be used as predictors of DCI. This highlights NETs as potential therapeutic target for patients with SAH and paves the way for future studies.

Bone-Marrow-Derived Mesenchymal Stem Cells Attenuate Behavioral and Cognitive Dysfunction after Subarachnoid Hemorrhage via HMGB1-RAGE Axis Mediation.

We evaluated the therapeutic effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on behavioral and cognitive function in a mouse model of mild subarachnoid hemorrhage (SAH) and explored the underlying mechanisms in conjunction with the HMGB1-RAGE axis. The SAH models were generated in a total of 126 male C57BL/6J mice via endovascular perforation and evaluated 24 h and 72 h after the intravenous administration of BMSCs (3 × 105 cells). The BMSCs were administered once, at 3 h, or twice, at 3 h and 48 h after the model induction. The therapeutic effects of the BMSCs were compared to those of the saline administration. Compared to saline-treated SAH-model mice, at 3 h, the mice with mild SAH treated with the BMSCs showed significant improvements in their neurological scores and cerebral edema. The administration of the BMSCs decreased the mRNA expression of HMGB1, RAGE, TLR4, and MyD88, as well as the protein expression of HMGB1 and phosphorylated NF-kB p65. Furthermore, the numbers of slips per walking time, impairments in short-term memory, and the recognition of novel objects were improved. There was some improvement in inflammatory-marker levels and cognitive function according to the BMSCs' administration times, but no large differences were seen. The administration of BMSCs improved behavioral and cognitive dysfunction by ameliorating HMGB1-RAGE axis-mediated neuroinflammation after SAH.