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Background and Purpose: Subarachnoid hemorrhage (SAH) is a devastating stroke, in which acidosis is one of detrimental complications. The extracellular pH reduction can activate G protein-coupled receptor 4 (GPR4) in the brain. Yet, the extent to which proton-activated GPR4 contributes to the early brain injury (EBI) post-SAH remains largely unexplored. Ferroptosis, iron-dependent programmed cell death, has recently been shown to contribute to EBI. We aimed to investigate the effects of GPR4 inhibition on neurological deficits and neuronal ferroptosis after SAH in rats., Methods: A total 253 Sprague Dawley (SD) male rats (weighing 275-330g) were utilized in this study. SAH was induced by endovascular perforation. NE-52-QQ57 (NE), a selective antagonist of GPR4 was administered intraperitoneally 1-h post-SAH. To explore the mechanisms, RhoA activator U-46619 and YAP activator PY-60 were delivered intracerebroventricularly. Short- and long-term neurobehavior, SAH grading, Western blot assay, ELISA assay, immunofluorescence staining, and transmission electron microscopy was performed post-SAH., Results: Following SAH, there was an upregulation of GPR4 expression in neurons. GPR4 inhibition by NE improved both short-term and long-term neurological outcomes post-SAH. NE also reduced neuronal ferroptosis, as evidenced by decreased lipid peroxidation products 4HNE and MDA levels in brain tissues, and reduced mitochondrial shrinkage, increased mitochondria crista and decreased membrane density. The application of either U-46619 or PY-60 partially offset the neuroprotective effects of NE on neuronal ferroptosis in SAH rats., Conclusions: This study demonstrated that acid-sensing receptor GPR4 contributed to neuronal ferroptosis after SAH via RhoA/YAP pathway, and NE may be a potential therapeutic strategy to attenuate GPR4 mediated neuronal ferroptosis and EBI after SAH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic-lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH., Methods: A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro., Results: Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1β at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines., Conclusion: PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients., (© 2024. The Author(s).)

We evaluated whether JWH133, a selective cannabinoid type 2 receptor (CB2R) agonist, prevented neurogenic pulmonary edema (NPE) after subarachnoid hemorrhage (SAH) by attenuating inflammation. Adult male rats were assigned to six groups: sham-operated, SAH with vehicle, SAH with JWH133 (0.3, 1.0, or 3.0 mg/kg) treatment 1 h after surgery, and SAH with JWH133 (1.0 mg/kg) at 1 h with a selective CB2R antagonist, SR144528 (3.0 mg/kg). The perforation model of SAH was performed and pulmonary wet-to-dry weight ratio was evaluated 24 and 72 h after surgery. Western blot analyses and immunohistochemistry were evaluated 24 h after surgery. JWH133 (1.0 mg/kg) significantly and most strongly improved lung edema 24 h after SAH. SR144528 administration significantly reversed the effects of JWH133 (1.0 mg/kg). SAH-induced increasing levels of myeloperoxidase (MPO) and decreasing levels of a tight junction (TJ) protein, junctional adhesion molecule (JAM)-A, were ameliorated by JWH133 (1.0 mg/kg) administration 24 h after SAH. Immunohistochemical assessment also confirmed substantial leukocyte infiltration in the outside of vessels in SAH, which were attenuated by JWH133 (1.0 mg/kg) injection. CB2R agonist ameliorated lung permeability by inhibiting leukocyte trafficking and protecting tight junction proteins in the lung of NPE after SAH.

Whether hypotension that occurs due to neurogenic stunned myocardium after subarachnoid hemorrhage (SAH) is associated with cardiomyocyte apoptotic cell death remains unknown. In this study, 18 male rats were subjected to sham or the endovascular perforation model of SAH surgery. Based on the mean arterial pressure (MAP) after SAH, rats were separated into SAH with hypotension (SAH hypotension) or SAH with blood pressure preservation (SAH BP preservation) groups. All animals were euthanized 2 h after the surgical procedure. Hearts were removed and separated transversely into base and apex parts, then Western blot analyses and immunohistochemistry were performed only in the apex part. One rat died as a result of severe SAH and two rats with mild SAH were excluded. We analyzed data from 15 rats that were divided into three groups: sham, SAH hypotension, and SAH BP preservation (n = 5, each). There was a significantly higher cleaved caspase-3/caspase-3 ratio in the SAH hypotension group compared with sham and the SAH BP preservation group. Cardiomyocyte apoptosis was demonstrated in the SAH rats. This is the first experimental report that describes SAH-induced neurogenic stunned myocardium with ensuing hypotension may result from the acute apoptotic cardiomyocyte cell death in the left ventricle.

Early brain injury (EBI) which comprises of vasogenic edema and apoptotic cell death is an important component of subarachnoid hemorrhage (SAH) pathophysiology. This study evaluated whether cannabinoid receptor type 2 (CB2R) agonist, JWH133, attenuates EBI after SAH and whether CB2R stimulation reduces pro-apoptotic caspase-3 via up-regulation of cAMP response element-binding protein (CREB)-Bcl-2 signaling pathway. Male Sprague-Dawley rats (n=123) were subjected to SAH by endovascular perforation. Rats received vehicle or JWH133 at 1h after SAH. Neurological deficits and brain water content were evaluated at 24h after SAH. Western blot was performed to quantify phosphorylated CREB (pCREB), Bcl-2, and cleaved caspase-3 levels. Neuronal cell death was evaluated with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Additionally, CREB siRNA was administered to manipulate the proposed pathway. JWH133 (1.0mg/kg) improved neurological deficits and reduced brain water content in left hemisphere 24h after SAH. JWH133 significantly increased activated CREB (pCREB) and Bcl-2 levels and significantly decreased cleaved caspase-3 levels in left hemisphere 24h after SAH. CREB siRNA reversed the effects of treatment. TUNEL positive neurons in the cortex were reduced with JWH133 treatment. Thus, CB2R stimulation attenuated EBI after SAH possibly through activation of pCREB-Bcl-2 pathway., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Early brain injury (EBI), following subarachnoid hemorrhage (SAH), comprises blood-brain barrier (BBB) disruption and consequent edema formation. Peripheral leukocytes can infiltrate the injured brain, thereby aggravating BBB leakage and neuroinflammation. Thus, anti-inflammatory pharmacotherapies may ameliorate EBI and provide neuroprotection after SAH. Cannabinoid type 2 receptor (CB2R) agonism has been shown to reduce neuroinflammation; however, the precise protective mechanisms remain to be elucidated. This study aimed to evaluate whether the selective CB2R agonist, JWH133 can ameliorate EBI by reducing brain-infiltrated leukocytes after SAH. Adult male Sprague-Dawley rats were randomly assigned to the following groups: sham-operated, SAH with vehicle, SAH with JWH133 (1.0mg/kg), or SAH with a co-administration of JWH133 and selective CB2R antagonist SR144528 (3.0mg/kg). SAH was induced by endovascular perforation, and JWH133 was administered 1h after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24h after surgery. JWH133 improved neurological scores and reduced brain water content; however, SR144528 reversed these treatment effects. JWH133 reduced Evans blue dye extravasation after SAH. Furthermore, JWH133 treatment significantly increased TGF-β1 expression and prevented an SAH-induced increase in E-selectin and myeloperoxidase. Lastly, SAH resulted in a decreased expression of the tight junction protein zonula occludens-1 (ZO-1); however, JWH133 treatment increased the ZO-1 expression. We suggest that CB2R stimulation attenuates neurological outcome and brain edema, by suppressing leukocyte infiltration into the brain through TGF-β1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Despite decades of study, subarachnoid hemorrhage (SAH) continues to be a serious and significant health problem in the United States and worldwide. The mechanisms contributing to brain injury after SAH remain unclear. Traditionally, most in vivo research has heavily emphasized the basic mechanisms of SAH over the pathophysiological or morphological changes of delayed cerebral vasospasm after SAH. Unfortunately, the results of clinical trials based on this premise have mostly been disappointing, implicating some other pathophysiological factors, independent of vasospasm, as contributors to poor clinical outcomes. Delayed cerebral vasospasm is no longer the only culprit. In this review, we summarize recent data from both experimental and clinical studies of SAH and discuss the vast array of physiological dysfunctions following SAH that ultimately lead to cell death. Based on the progress in neurobiological understanding of SAH, the terms "early brain injury" and "delayed brain injury" are used according to the temporal progression of SAH-induced brain injury. Additionally, a new concept of the vasculo-neuronal-glia triad model for SAH study is highlighted and presents the challenges and opportunities of this model for future SAH applications., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Neuroinflammation contributes to the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Cytotoxic events following SAH, such as extracellular accumulation of adenosine triphosphate (ATP), may activate the P2X purinoceptor 7 (P2X7R)/cryopyrin inflammasome axis, thus inducing the proinflammatory cytokine IL-1β/IL-18 secretion. We therefore hypothesized that inhibition of P2X7R/cryopyrin inflammasome axis would ameliorate neuroinflammation after SAH. In the present study, SAH was induced by the endovascular perforation in rats. Small interfering RNAs (siRNAs) of P2X7R or cryopyrin were administered intracerebroventricularly 24h before SAH. Brilliant blue G (BBG), a non-competitive antagonist of P2X7R, was administered intraperitoneally 30min following SAH. Post-assessments including SAH severity score, neurobehavioral test, brain water content, Western blot and immunofluorescence, were performed. Administration of P2X7R and cryopyrin siRNA as well as pharmacologic blockade of P2X7R by BBG ameliorated neurological deficits and brain edema at 24h following SAH. Inhibition of P2X7R/cryopyrin inflammasome axis suppressed caspase-1 activation, which subsequently decreased maturation of IL-1β/IL-18. To investigate the link between P2X7R and cryopyrin inflammasome in vivo, Benzoylbenzoyl-ATP (BzATP), a P2X7R agonist, was given to lipopolysaccharide (LPS) primed naive rats with scramble or cryopyrin siRNAs. In LPS-primed naive rats, BzATP induced caspase-1 activation and mature IL-1β release were neutralized by cryopyrin siRNA. Thus, the P2X7R/cryopyrin inflammasome axis may contribute to neuroinflammation via activation of caspase-1 and thereafter mature IL-1β/IL-18 production following SAH. Therapeutic interventions targeting P2X7R/cryopyrin pathway may be a novel approach to ameliorate EBI following SAH., (© 2013.)

Objective: Isoflurane, a volatile anesthetic agent, has been recognized for its potential neuroprotective properties and has antiapoptotic effects. We examined whether isoflurane posttreatment is protective against early brain injury after subarachnoid hemorrhage and determined whether this effect needs sphingosine-related pathway activation., Design: Controlled in vivo laboratory study., Setting: Animal research laboratory., Subjects: One hundred seventy-nine 8-wk-old male CD-1 mice weighing 30-38 g., Interventions: Subarachnoid hemorrhage was induced in mice by endovascular perforation. Animals were randomly assigned to sham-operated, subarachnoid hemorrhage-vehicle, and subarachnoid hemorrhage+2% isoflurane. Neurobehavioral function and brain edema were evaluated at 24 and 72 hrs. The expression of sphingosine kinase, phosphorylated Akt, and cleaved caspase-3 was determined by Western blotting and immunofluorescence. Neuronal cell death was examined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling staining. Effects of a sphingosine kinase inhibitor N, N-dimethylsphingosine or a sphingosine 1 phosphate receptor inhibitor VPC23019 on isoflurane's protective action against postsubarachnoid hemorrhage early brain injury were also examined., Measurements and Main Results: Isoflurane significantly improved neurobehavioral function and brain edema at 24 hrs but not 72 hrs after subarachnoid hemorrhage. At 24 hrs, isoflurane attenuated neuronal cell death in the cortex, associated with an increase in sphingosine kinase 1 and phosphorylated Akt, and a decrease in cleaved caspase-3. The beneficial effects of isoflurane were abolished by N, N-dimethylsphingosine and VPC23019., Conclusions: Isoflurane posttreatment delays the development of postsubarachnoid hemorrhage early brain injury through antiapoptotic mechanisms including sphingosine-related pathway activation, implying its use for anesthesia during acute aneurysm surgery or intervention.

The blood-brain barrier (BBB) disruption and brain edema are important pathophysiologies of early brain injury after subarachnoid hemorrhage (SAH). This study is to evaluate whether Rho kinase (Rock) enhances BBB permeability via disruption of tight junction proteins during early brain injury. Adult male rats were assigned to five groups; Sham-operated, SAH treated with saline, a Rock inhibitor hydroxyfasudil (HF) (10 mg/kg) treatment at 0.5 h after SAH, HF treatment at 0.5 and 6 h (10 mg/kg, each) after SAH, and another Rock inhibitor Y27632 (10 mg/kg) treatment at 0.5 h after SAH. The perforation model of SAH was performed and neurological score and brain water content were evaluated 24 and 72 h after surgery. Evans blue extravasation, Rock activity assay, and western blotting analyses were evaluated 24 h after surgery. Treatment of HF significantly improved neurological scores 24 h after SAH. Single treatment with HF and Y27632, and two treatments with HF reduced brain water content in the ipsilateral hemisphere. HF reduced Evans blue extravasation in the ipsilateral hemisphere after SAH. Rock activity increased 24 h after SAH, and HF reversed the activity. SAH significantly decreased the levels of tight junction proteins, occludin and zonula occludens-1 (ZO-1), and HF preserved the levels of occluding and ZO-1 in ipsilateral hemisphere. In conclusion, HF attenuated BBB permeability after SAH, possibly by protection of tight junction proteins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Subarachnoid hemorrhage (SAH) results in significant long-lasting cognitive dysfunction. Therefore, evaluating acute and long-term outcomes after therapeutic intervention is important for clinical translation. The aim of this study was to use minocycline, a known neuroprotectant agent, to evaluate the long-term benefits in terms of neurobehavior and neuropathology after experimental SAH in rats, and to determine which neurobehavioral test would be effective for long-term evaluation. SAH was induced by endovascular perforation in adult male Sprague-Dawley rats (n=118). The animals were treated with intraperitoneal injection of minocycline (45 mg/kg or 135 mg/kg) or vehicle 1 h after SAH induction. In the short-term, animals were euthanized at 24 and 72 h for evaluation of neurobehavior, brain water content, and matrix metalloproteinase (MMP) activity. In the long-term, neurobehavior was evaluated at days 21-28 post-SAH, and histopathological analysis was done at day 28. High-dose but not low-dose minocycline reduced brain water content at 24 h, and therefore only the high-dose regimen was used for further evaluation, which reduced MMP-9 activity at 24 h. Further, high-dose minocycline improved spatial memory and attenuated neuronal loss in the hippocampus and cortex. The rotarod, T-maze, and water maze tests, but not the inclined plane test, detected neurobehavioral deficits in SAH rats at days 21-28. This study demonstrates that minocycline attenuates long-term functional and morphological outcomes after endovascular perforation-induced SAH. Long-term neurobehavioral assessments using the rotarod, T-maze, and water maze tests could be useful to evaluate the efficacy of therapeutic intervention after experimental SAH.