Ferroptosis as a Therapeutic Target in Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a cerebrovascular disorder with significant mortality and morbidity. Neural injury in SAH is mediated through a variety of pathophysiological processes. Currently available treatments are either nonspecific in targeting the basic pathophysiological mechanisms that result in neural damage in SAH, or merely focus on vasospasm. Ferroptosis is a type of programmed iron dependent cell death, which has received attention due to its possible role in neural injury in SAH. Herein, we review how intracellular iron overload mediates the production of reactive free radicals and lipid peroxidation through a variety of biochemical pathways in SAH. This in turn results in induction of ferroptosis, as well as exacerbation of vasospasm. We also discuss several therapeutic agents that have been shown to inhibit ferroptosis through targeting different steps of the process. Such agents have proven effective in ameliorating vasospasm, neural damage, and neurobehavioral outcomes in animal models of SAH. Human studies to test the safety and efficacy of intrathecal or parenteral administration of the inhibitors of ferroptosis in improving outcomes of SAH patients are warranted. There are currently a few ongoing clinical trials pursuing this therapeutic concept, the results of which will be critical to determine the value of ferroptosis as a novel therapeutic target in SAH.

Pathogenic mechanisms and therapeutic implications of extracellular matrix remodelling in cerebral vasospasm.

Cerebral vasospasm significantly contributes to poor prognosis and mortality in patients with aneurysmal subarachnoid hemorrhage. Current research indicates that the pathological and physiological mechanisms of cerebral vasospasm may be attributed to the exposure of blood vessels to toxic substances, such as oxyhaemoglobin and inflammation factors. These factors disrupt cerebral vascular homeostasis. Vascular homeostasis is maintained by the extracellular matrix (ECM) and related cell surface receptors, such as integrins, characterised by collagen deposition, collagen crosslinking, and elastin degradation within the vascular ECM. It involves interactions between the ECM and smooth muscle cells as well as endothelial cells. Its biological activities are particularly crucial in the context of cerebral vasospasm. Therefore, regulating ECM homeostasis may represent a novel therapeutic target for cerebral vasospasm. This review explores the potential pathogenic mechanisms of cerebral vasospasm and the impacts of ECM protein metabolism on the vascular wall during ECM remodelling. Additionally, we underscore the significance of an ECM protein imbalance, which can lead to increased ECM stiffness and activation of the YAP pathway, resulting in vascular remodelling. Lastly, we discuss future research directions.

Circulating Extracellular Vesicles in Subarachnoid Hemorrhage Patients: Characterization and Cellular Effects.

Circulating extracellular vesicles (EVs) may play a pathophysiological role in the onset of complications of subarachnoid hemorrhage (SAH), potentially contributing to the development of vasospasm (VP). In this study, we aimed to characterize circulating EVs in SAH patients and examine their effects on endothelial and smooth muscle cells (SMCs). In a total of 18 SAH patients, 10 with VP (VP), 8 without VP (NVP), and 5 healthy controls (HC), clinical variables were recorded at different time points. EVs isolated from plasma samples were characterized and used to stimulate human vascular endothelial cells (HUVECs) and SMCs. We found that EVs from SAH patients expressed markers of T-lymphocytes and platelets and had a larger size and a higher concentration compared to those from HC. Moreover, EVs from VP patients reduced cell viability and mitochondrial membrane potential in HUVECs and increased oxidants and nitric oxide (NO) release. Furthermore, EVs from SAH patients increased intracellular calcium levels in SMCs. Altogether, our findings reveal an altered pattern of circulating EVs in SAH patients, suggesting their pathogenic role in promoting endothelial damage and enhancing smooth muscle reactivity. These results have significant implications for the use of EVs as potential diagnostic/prognostic markers and therapeutic tools in SAH management.

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 in vitro experimentation. In vitro and in vivo 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, in vivo 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.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms. / ç»„è›‹ç™½è„±ä¹™é °é ¶6特异性抑制剂tubastatin Aå¯¹å¤§é¼ è››ç½‘è†œä¸‹è ”å‡ºè¡€çš„ä¿æŠ¤ä½œç”¨åŠå ¶æœºåˆ¶.

OBJECTIVES: Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms. METHODS: Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery. RESULTS: The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) . CONCLUSIONS: HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Subarachnoid hemorrhage (SAH) is a serious cerebrovascular disease. Early brain injury (EBI) and cerebral vasospasm are the main reasons for poor prognosis of SAH patients. The specific inhibitor of histone deacetylase 6 (HDAC6), tubastatin A (TubA), has been proved to have a definite neuroprotective effect on a variety of animal models of acute and chronic central nervous system diseases. However, the neuroprotective effect of TubA on SAH remains unclear. This study aims to investigate the expression and localization of HDAC6 in the early stage of SAH, and to evaluate the protective effects of TubA on EBI and cerebral vasospasm after SAH and the underlying mechanisms.@*METHODS@#Adult male SD rats were treated with modified internal carotid artery puncture to establish SAH model. In the first part of the experiment, rats were randomly divided into 6 groups: a sham group, a SAH-3 h group, a SAH-6 h group, a SAH-12 h group, a SAH-24 h group, and a SAH-48 h group. At 3, 6, 12, and 24 h after SAH modeling, the injured cerebral cortex of rats in each group was taken for Western blotting to detect the expression of HDAC6. In addition, the distribution of HDAC6 in the cerebral cortex of the injured side was measured by immunofluorescence double staining in SAH-24 h group rats. In the second part, rats were randomly divided into 4 groups: a sham group, a SAH group, a SAH+TubAL group (giving 25 mg/kg TubA), and a SAH+TubAH group (giving 40 mg/kg TubA). At 24 h after modeling, the injured cerebral cortex tissue was taken for Western blotting to detect the expression levels of HDAC6, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining to detect apoptosis, and hematoxylin and eosin (HE) staining to detect the diameter of middle cerebral artery.@*RESULTS@#The protein expression of HDAC6 began to increase at 6 h after SAH (P<0.05), peaked at 24 h (P<0.001), and decreased at 48 h, but there was still a difference compared with the sham group (P<0.05). HDAC6 is mainly expressed in the cytoplasm of the neurons. Compared with the sham group, the neurological score was decreased significantly and brain water content was increased significantly in the SAH group (both P<0.01). Compared with the SAH group, the neurological score was increased significantly and brain water content was decreased significantly in the SAH+TubAH group (both P<0.05), while the improvement of the above indexes was not significant in the SAH+TubAL group (both P>0.05). Compared with the sham group, the expression of eNOS was significantly decreased (P<0.01) and the expressions of iNOS and HDAC6 were significantly increased (P<0.05 and P<0.01, respectively) in the SAH group. Compared with the SAH group, the expression of eNOS was significantly increased, and iNOS and HDAC6 were significantly decreased in the SAH+TubA group (all P<0.05). Compared with the SAH group, the number of TUNEL positive cells was significantly decreased and the diameter of middle cerebral artery was significantly increased in the SAH+TubA group (both P<0.05) .@*CONCLUSIONS@#HDAC6 is mainly expressed in neurons and is up-regulated in the cerebral cortex at the early stage of SAH. TubA has protective effects on EBI and cerebral vasospasm in SAH rats by reducing brain edema and cell apoptosis in the early stage of SAH. In addition, its effect of reducing cerebral vasospasm may be related to regulating the expression of eNOS and iNOS.

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

BACKGROUND: Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH. METHODS: Human 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. RESULTS: IL-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). CONCLUSION: Correlation 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.

New Mechanisms and Targets of Subarachnoid Hemorrhage: A Focus on Mitochondria.

Spontaneous subarachnoid hemorrhage (SAH) accounts for 5-10% of all strokes and is a subtype of hemorrhagic stroke that places a heavy burden on health care. Despite great progress in surgical clipping and endovascular treatment for ruptured aneurysms, cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) threaten the long-term outcomes of patients with SAH. Moreover, there are limited drugs available to reduce the risk of DCI and adverse outcomes in SAH patients. New insight suggests that early brain injury (EBI), which occurs within 72 h after the onset of SAH, may lay the foundation for further DCI development and poor outcomes. The mechanisms of EBI mainly include excitotoxicity, oxidative stress, neuroinflammation, blood-brain barrier (BBB) destruction, and cellular death. Mitochondria are a double-membrane organelle, and they play an important role in energy production, cell growth, differentiation, apoptosis, and survival. Mitochondrial dysfunction, which can lead to mitochondrial membrane potential (Δψm) collapse, overproduction of reactive oxygen species (ROS), release of apoptogenic proteins, disorders of mitochondrial dynamics, and activation of mitochondria-related inflammation, is considered a novel mechanism of EBI related to DCI as well as post-SAH outcomes. In addition, mitophagy is activated after SAH. In this review, we discuss the latest perspectives on the role of mitochondria in EBI and DCI after SAH. We emphasize the potential of mitochondria as therapeutic targets and summarize the promising therapeutic strategies targeting mitochondria for SAH.

Attenuation in Proinflammatory Factors and Reduction in Neuronal Cell Apoptosis and Cerebral Vasospasm by Minocycline during Early Phase after Subarachnoid Hemorrhage in the Rat.

BACKGROUND: Subarachnoid hemorrhage (SAH) is an important subcategory of stroke due to its high mortality rate as well as severe complications such as neurological deficit. It has been suggested that cerebral inflammation is a major factor in advanced brain injury after SAH. Microglia and astrocytes are known supporting cells in the development and maintenance of inflammation in central nervous system. However, the role of microglia and astrocytes in the development of inflammation and neuronal cell apoptosis during the early phase after SAH has not been thoroughly investigated. MATERIALS AND METHODS: Sprague-Dawley rats were divided into 4 groups (n = 6/group): sham group, animals subjected to SAH without treatment, SAH animals pretreated with the microglia inhibitor minocycline (50 mg/kg, ip), and SAH animals pretreated with the astrocyte inhibitor fluorocitrate (50 mg/kg, ip). SAH was induced by injecting autologous blood (1 ml/kg) into the cistern magna on day 0. Pretreatment with minocycline or fluorocitrate was given three days prior to the induction of SAH. Rats were sacrificed 6 hr after SAH, and their cerebral spinal fluids were used to measure protein levels of neuroinflammatory cytokines IL-1ß, IL-6, and TNF-α by ELISA. In addition, the cerebral cortex was utilized to determine the levels of caspase-3 by western blot and to evaluate neuronal cell apoptosis by immunohistochemistry staining and detect microglia and astrocyte by immunofluorescence staining for Iba-1 and GFAP. In this study, all SAH animals were given an injection of autologous blood and SAH rats treated with minocycline or fluorocitrate received ip injections on day 1, 2, and 3 before inducing SAH. Neurological outcome was assessed by ambulation and placing/stepping reflex responses on day 7. RESULTS: Immunofluorescence staining showed that SAH induced proliferation of microglia and astrocyte and minocycline inhibited the proliferation of both microglia and astrocyte. However, fluorocitrate inhibited only the proliferation of astrocyte. ELISA analysis showed that SAH upregulated TNF-α and IL-1ß, but not IL-6 at 6 hr after SAH. Minocycline, but not fluorocitrate, attenuated the upregulation of TNF-α and IL-1ß. Western blot analysis and immunohistochemistry staining showed that SAH induced neuronal cell apoptosis. Pretreatment with minocycline, but not fluorocitrate, decreased SAH-induced neuronal death and cerebral vasospasm. Furthermore, significant improvements in neurobehavioral outcome were seen in the minocycline treatment group, but not in animals treated with fluorocitrate. CONCLUSIONS: Microglia may play an important role to regulate neuronal cell apoptosis and cerebral vasospasm through inhibiting inflammation at an early phase after SAH in the rat.

Scavenging Free Iron Reduces Arteriolar Microvasospasms After Experimental Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia resulting in high acute mortality and severe chronic neurological deficits. Spasms of the pial and intraparenchymal microcirculation (microvasospasms) contribute to acute cerebral ischemia after SAH; however, the underlying mechanisms remain unknown. We hypothesize that free iron (Fe3+) released from hemolytic red blood cells into the subarachnoid space may be involved in microvasospasms formation. METHODS: Male C57BL/6 mice (n=8/group) received 200 mg/kg of the iron scavenger deferoxamine or vehicle intravenously and were then subjected to SAH by filament perforation. Microvasospasms of pial and intraparenchymal vessels were imaged three hours after SAH by in vivo 2-photon microscopy. RESULTS: Microvasospasms occurred in all investigated vessel categories down to the capillary level. Deferoxamine significantly reduced the number of microvasospasms after experimental SAH. The effect was almost exclusively observed in larger pial arterioles (>30 µm) covered with blood. CONCLUSIONS: These results provide proof-of-principle evidence that Fe3+ is involved in the formation of arteriolar microvasospasms after SAH and that arteriolar and capillary microvasospasms are triggered by different mechanisms. Deciphering the mechanisms of Fe3+-induced microvasospasms may result in novel therapeutic strategies for SAH patients.

Peroxisome proliferator­activated receptor ß/δ regulates cerebral vasospasm after subarachnoid hemorrhage via modulating vascular smooth muscle cells phenotypic conversion.

Cerebral vasospasm (CVS) is a common complication of subarachnoid hemorrhage (SAH) with high deformity rates and cerebral vascular smooth muscle cells (VSMCs) phenotypic switch is considered to be involved in the regulation of CVS. However, to the best of the authors' knowledge, its underlying molecular mechanism remains to be elucidated. Peroxisome proliferator­activated receptor ß/δ (PPARß/δ) has been demonstrated to be involved in the modulation of vascular cells proliferation and maintains the autoregulation function of blood vessels. The present study investigated the potential effect of PPARß/δ on CVS following SAH. A model of SAH was established by endovascular perforation on male adult Sprague­Dawley rats, and the adenovirus PPARß/δ (Ad­PPARß/δ) was injected via intracerebroventricular administration prior to SAH. The expression levels of phenotypic markers α­smooth muscle actin and embryonic smooth muscle myosin heavy chain were measured via western blotting or immunofluorescence staining. The basilar artery diameter and vessel wall thickness were evaluated under fluorescence microscopy. SAH grade, neurological scores, brain water content and brain swelling were measured to study the mechanisms of PPARß/δ on vascular smooth muscle phenotypic transformation. It was revealed that the expression levels of synthetic proteins were upregulated in rats with SAH and this was accompanied by CVS. Activation of PPARß/δ using Ad­PPARß/δ markedly upregulated the contractile proteins elevation, restrained the synthetic proteins expression and attenuated SAH­induced CVS by regulating the phenotypic switch in VSMCs at 72 h following SAH. Furthermore, the preliminary study demonstrated that PPARß/δ downregulated ERK activity and decreased the expression of phosphorylated (p­)ETS domain­containing protein Elk­1 and p­p90 ribosomal S6 kinase, which have been demonstrated to serve an important role in VSMC phenotypic change. Additionally, it was revealed that Ad­PPARß/δ could positively improve CVS by ameliorating the diameter of the basilar artery and mitigating the thickness of the vascular wall. Furthermore, subsequent experiments demonstrated that Ad­PPARß/δ markedly reduced the brain water content and brain swelling and improved the neurological outcome. Taken together, the present study identified PPARß/δ as a useful regulator for the VSMCs phenotypic switch and attenuating CVS following SAH, thereby providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

Role of SIRT1 in Isoflurane Conditioning-Induced Neurovascular Protection against Delayed Cerebral Ischemia Secondary to Subarachnoid Hemorrhage.

We recently reported that isoflurane conditioning provided multifaceted protection against subarachnoid hemorrhage (SAH)-induced delayed cerebral ischemia (DCI), and this protection was through the upregulation of endothelial nitric oxide synthase (eNOS). SIRT1, an NAD-dependent deacetylase, was shown to be one of the critical regulators of eNOS. The aim of our current study is to examine the role of SIRT1 in isoflurane conditioning-induced neurovascular protection against SAH-induced DCI. Mice were divided into four groups: sham, SAH, or SAH with isoflurane conditioning (with and without EX-527). Experimental SAH via endovascular perforation was performed. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. EX-527, a selective SIRT1 inhibitor, 10 mg/kg was injected intraperitoneally immediately after SAH in the EX-527 group. SIRT1 mRNA expression and activity levels were measured. Vasospasm, microvessel thrombosis, and neurological outcome were assessed. SIRT1 mRNA expression was downregulated, and no difference in SIRT1 activity was noted after isoflurane exposure. Isoflurane conditioning with and without EX-527 attenuated vasospasm, microvessel thrombosis and improved neurological outcomes. Our data validate our previous findings that isoflurane conditioning provides strong protection against both the macro and micro vascular deficits induced by SAH, but this protection is likely not mediated through the SIRT1 pathway.

The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model.

Cerebral vasospasm (CVS) causes mortality and morbidity in patients after subarachnoid hemorrhage (SAH). The mechanism and adequate treatment of CVS are still elusive. R-568 is a calcimimetic agent known to exert a vasodilating effect. However, there is no report on its vasodilator effect against SAH-induced vasospasm. In the present study, we investigated the therapeutic effect of R-568 on the SAH-induced CVS model in rats. Seventy-two adult male Sprague-Dawley rats were divided into 8 groups: sham surgery; SAH only; SAH + Vehicle, SAH + R-568; SAH + R-568 + Wortmannin (the PI3K inhibitor); SAH + Wortmannin; SAH + R-568 + Calhex-231 (a calcilytic agent); SAH + Calhex-231. SAH was induced by blood (0.3 mL) given by intracisternal injection. R-568 (20 µM) was administered intracisternal immediately prior to experimental SAH. Basilar arteries (BAs) were obtained to evaluate PI3K/Akt/eNOS pathway (immunoblotting) and morphological changes 48 h after SAH. Perimeters of BAs were decreased by 24.1% in the SAH group compared to the control group and the wall thickness was increased by 75.3%. With R-568 treatment, those percentages were 9.6% and 29.6%, respectively, indicating that vasospasm was considerably improved when compared with the SAH group (P < 0.001 in both). While p-PI3K/PI3K and p-Akt/Akt ratio and eNOS protein expression were markedly decreased in the SAH rats, treatment with R-568 resulted in a significant increase in these levels. The beneficial effects of R-568 were partially blocked in the presence of Calhex-231 and completely blocked in the presence of Wortmannin. Herein, we found that treatment with R-568 would attenuate SAH-induced CVS through the PI3K/Akt/eNOS pathway and demonstrate therapeutic promise in CVS treatment following SAH.

Glycemic indices predict outcomes after aneurysmal subarachnoid hemorrhage: a retrospective single center comparative analysis.

Although hyperglycemia is associated with worse outcomes after aneurysmal subarachnoid hemorrhage (aSAH), there is no consensus on the optimal glucose control metric, acceptable in-hospital glucose ranges, or suitable insulin regimens in this population. In this single-center retrospective cohort study of aSAH patients, admission glucose, and hospital glucose mean (MHG), minimum (MinG), maximum (MaxG), and variability were compared. Primary endpoints (mortality, complications, and vasospasm) were assessed using multivariate logistic regressions. Of the 217 patients included, complications occurred in 83 (38.2%), 124 (57.1%) had vasospasm, and 41 (18.9%) died. MHG was independently associated with (p < 0.001) mortality, MaxG (p = 0.017) with complications, and lower MinG (p = 0.015) with vasospasm. Patients with MHG ≥ 140 mg/dL had 10 × increased odds of death [odds ratio (OR) = 10.3; 95% CI 4.6-21.5; p < 0.0001] while those with MinG ≤ 90 mg/dL had nearly 2× increased odds of vasospasm (OR = 1.8; 95% CI 1.01-3.21; p = 0.0422). While inpatient insulin was associated with increased complications and provided no mortality benefit, among those with MHG ≥ 140 mg/dL insulin therapy resulted in lower mortality (OR = 0.3; 95% CI 0.1-0.9; p = 0.0358), but no increased complication risk. While elevated MHG and MaxG are highly associated with poorer outcomes after aSAH, lower MinG is associated with increased vasospasm risk. Future trials should consider initiating insulin therapy based on MHG rather than other hyperglycemia measures.

Role of endothelial nitric oxide synthase for early brain injury after subarachnoid hemorrhage in mice.

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS-/-) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS-/- mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS-/- mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.

SIRT1 mediates hypoxic preconditioning induced attenuation of neurovascular dysfunction following subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Vasospasm and delayed cerebral ischemia (DCI) contribute significantly to the morbidity/mortality associated with aneurysmal subarachnoid hemorrhage (SAH). While considerable research effort has focused on preventing or reversing vasospasm, SAH-induced brain injury occurs in response to a multitude of concomitantly acting pathophysiologic mechanisms. In this regard, the pleiotropic epigenetic responses to conditioning-based therapeutics may provide an ideal SAH therapeutic strategy. We previously documented the ability of hypoxic preconditioning (PC) to attenuate vasospasm and neurological deficits after SAH, in a manner that depends on the activity of endothelial nitric oxide synthase. The present study was undertaken to elucidate whether the NAD-dependent protein deacetylase sirtuin isoform SIRT1 is an upstream mediator of hypoxic PC-induced protection, and to assess the efficacy of the SIRT1-activating polyphenol Resveratrol as a pharmacologic preconditioning therapy. METHODS: Wild-type C57BL/6J mice were utilized in the study and subjected to normoxia or hypoxic PC. Surgical procedures included induction of SAH via endovascular perforation or sham surgery. Multiple endpoints were assessed including cerebral vasospasm, neurobehavioral deficits, SIRT1 expression via quantitative real-time PCR for mRNA, and western blot for protein quantification. Pharmacological agents utilized in the study include EX-527 (SIRT1 inhibitor), and Resveratrol (SIRT1 activator). RESULTS: Hypoxic PC leads to rapid and sustained increase in cerebral SIRT1 mRNA and protein expression. SIRT1 inhibition blocks the protective effects of hypoxic PC on vasospasm and neurological deficits. Resveratrol pretreatment dose-dependently abrogates vasospasm and attenuates neurological deficits following SAH - beneficial effects that were similarly blocked by pharmacologic inhibition of SIRT1. CONCLUSION: SIRT1 mediates hypoxic preconditioning-induced protection against neurovascular dysfunction after SAH. Resveratrol mimics this neurovascular protection, at least in part, via SIRT1. Activation of SIRT1 is a promising, novel, pleiotropic therapeutic strategy to combat DCI after SAH.

Preliminary results in the analysis of the immune response after aneurysmal subarachnoid hemorrhage.

Cerebral vasospasm (VSP) is a common phenomenon after aneurysmal subarachnoid hemorrhage (aSAH) and contributes to neurocognitive decline. The natural history of the pro-inflammatory immune response after aSAH has not been prospectively studied in human cerebrospinal fluid (CSF). In this pilot study, we aimed to identify specific immune mediators of VSP after aSAH. Peripheral blood (PB) and CSF samples from patients with aSAH were prospectively collected at different time-points after hemorrhage: days 0-1 (acute); days 2-4 (pre-VSP); days 5-9 (VSP) and days 10 + (post-VSP peak). Presence and severity of VSP was assessed with computed tomography angiography/perfusion imaging and clinical examination. Cytokine and immune mediators' levels were quantified using ELISA. Innate and adaptive immune cells were characterized by flow cytometry, and cell counts at different time-points were compared with ANOVA. Confocal immunostaining was used to determine the presence of specific immune cell populations detected in flow cytometry. Thirteen patients/aneurysms were included. Five (38.5%) patients developed VSP after a mean of 6.8 days from hemorrhage. Flow cytometry demonstrated decreased numbers of CD45+ cells during the acute phase in PB of aSAH patients compared with healthy controls. In CSF of VSP patients, NK cells (CD3-CD161 +) were increased during the acute phase and progressively declined, whereas CD8+CD161+ lymphocytes significantly increased at days 5-9. Microglia cells (CD45dimCD11b +) increased over time after SAH. This increase was particularly significant in patients with VSP. Levels of VEGF and MMP-9 were consistently higher in VSP patients, with the highest difference occurring at the acute phase. Confocal immunostaining demonstrated the presence of CD8+CD161+ lymphocytes in the arterial wall of two unruptured intracranial aneurysms. In this preliminary study, human CSF showed active presence of innate and adaptive immune cells after aSAH. CD8+CD161+ lymphocytes may have an important role in the inflammatory response after aneurysmal rupture and were identified in the aneurysmal wall of unruptured brain aneurysms. Microglia activation occurs 6 + days after aSAH.

Targeting High Mobility Group Box 1 in Subarachnoid Hemorrhage: A Systematic Review.

Aneurysmal subarachnoid hemorrhage (aSAH) is a complex and potentially deadly disease. Neurosurgical clipping or endovascular coiling can successfully obliterate ruptured aneurysms in almost every case. However, despite successful interventions, the clinical outcomes of aSAH patients are often poor. The reasons for poor outcomes are numerous, including cerebral vasospasm (CVS), post-hemorrhagic hydrocephalus, systemic infections and delayed cerebral ischemia. Although CVS with subsequent cerebral ischemia is one of the main contributors to brain damage after aSAH, little is known about the underlying molecular mechanisms of brain damage. This review emphasizes the importance of pharmacological interventions targeting high mobility group box 1 (HMGB1)-mediated brain damage after subarachnoid hemorrhage (SAH) and CVS. We searched Pubmed, Ovid medline and Scopus for "subarachnoid hemorrhage" in combination with "HMGB1". Based on these criteria, a total of 31 articles were retrieved. After excluding duplicates and selecting the relevant references from the retrieved articles, eight publications were selected for the review of the pharmacological interventions targeting HMGB1 in SAH. Damaged central nervous system cells release damage-associated molecular pattern molecules (DAMPs) that are important for initiating, driving and sustaining the inflammatory response following an aSAH. The discussed evidence suggested that HMGB1, an important DAMP, contributes to brain damage during early brain injury and also to the development of CVS during the late phase. Different pharmacological interventions employing natural compounds with HMGB1-antagonizing activity, antibody targeting of HMGB1 or scavenging HMGB1 by soluble receptors for advanced glycation end products (sRAGE), have been shown to dampen the inflammation mediated brain damage and protect against CVS. The experimental data suggest that HMGB1 inhibition is a promising strategy to reduce aSAH-related brain damage and CVS. Clinical studies are needed to validate these findings that may lead to the development of potential treatment options that are much needed in aSAH.

Toll-Like Receptor 4 and Tenascin-C Signaling in Cerebral Vasospasm and Brain Injuries After Subarachnoid Hemorrhage.

Toll-like receptor 4 (TLR4) is expressed in various cell types in the central nervous system and exerts maximal inflammatory responses among the TLR family members. TLR4 can be activated by many endogenous ligands having damage-associated molecular patterns including heme and fibrinogen at the rupture of a cerebral aneurysm, and therefore its activation is reasonable as an initial step of cascades to brain injuries after aneurysmal subarachnoid hemorrhage (SAH). TLR4 activation induces tenascin-C (TNC), a representative of matricellular proteins that are a class of inducible, nonstructural, secreted, and multifunctional extracellular matrix glycoproteins. TNC is also an endogenous activator and inducer of TLR4, forming positive feedback mechanisms leading to more activation of the signaling transduction. Our studies have demonstrated that TLR4 as well as TNC are involved in inflammatory reactions, blood-brain barrier disruption, neuronal apoptosis, and cerebral vasospasm after experimental SAH. This article reviews recent understanding of TLR4 and TNC in SAH to suggest that the TLR4-TNC signaling may be an important therapeutic target for post-SAH brain injuries.

Effect of Intra-arterial Nimodipine on Cerebral Oxygen Saturation and Systemic Hemodynamic Indices in Patients With Cerebral Vasospasm: A Prospective Cohort Study.

BACKGROUND: Intra-arterial nimodipine (IaN) is used in the management of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (aSAH). The impact of IaN therapy on regional cerebral oxygen saturation (rScO2) assessed by near infra-red spectroscopy, and dynamic cardiac indices, is currently unknown. This study assessed the effect of IaN on rScO2 and systemic hemodynamic indices during IaN therapy for cerebral vasospasm after aSAH. METHODS: This prospective cohort study was conducted in 20 patients over sixteen month period after ethics committee approval and informed consent. Patients with angiographic evidence of vasospasm received IaN 3mg over 30 minutes in the spastic vessels. Data regarding rScO2 heart rate (HR), mean blood pressure (MBP) cardiac index (CI), stroke volume index (SVI), stroke volume variation (SVV), and total peripheral resistance index (TPRI) were collected during IaN treatment. The primary outcome measure was change in rScO2 after IaN therapy. RESULTS: There was no significant change from baseline in ipsilateral and contralateral rScO2 after IaN administration (mean difference [MD], 0.2; 95% confidence interval [CI], -2.1 to 1.6; P=0.804, and 1.3; -1.1 to 3.8; P=0.276, respectively). There was a significant decrease in MBP and TPRI (MD, -12.4; 95% CI, -6.6 to -18.2; P<0.001, and -674.3; -374.9 to -973.7; P<0.001, respectively) and increase in SVI and CI (MD, 7.5; 95% CI, 14.4 to 0.6; P=0.035 and 0.7; 0.9 to 0.4; P<0.001, respectively) after IaN therapy. HR and SVV were unchanged. CONCLUSIONS: IaN for aSAH-related cerebral vasospasm did not improve rScO2 but was associated with significant systemic hemodynamic effects, including a decrease in MBP and TPRI. These hemodynamic changes might offset any potential effects of IaN to improve rScO2.