Ferroptosis mechanisms in early brain injury after subarachnoid hemorrhage.

Objectives: Increasing body of experimental and clinical data indicates that early brain injury after initial bleeding largely contributes to unfavorable outcome after subarachnoid hemorrhage (SAH). This review presents molecular mechanisms underlying brain injury at its early stages after SAH.Methods: PubMed was searched using term 'subarachnoid hemorrhage' and key words referring to molecular and cellular pathomechanisms of SAH-induced early brain injury.Results: The authors reviewed intracranial phenomena and molecular agents that contribute to the early development of pathological sequelae of SAH in cerebral and vascular tissues, including cerebral ischemia and its interactions with injurious blood components, blood–brain barrier disruption, brain edema and apoptosis.Discussion: It is believed that detailed knowledge of molecular signaling pathways after SAH will serve to improve therapeutic interventions. The most promising approach is the protection of neurovascular unit including anti-apoptosis therapy.

BackgroundActivated microglia-mediated neuroinflammation has been regarded as an underlying key player in the pathogenesis of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). The therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) transplantation has been demonstrated in several brain injury models and is thought to involve modulation of the inflammatory response. The present study investigated the salutary effects of BMSCs on EBI after SAH and the potential mechanism mediated by Notch1 signaling pathway inhibition.MethodsThe Sprague-Dawley rats SAH model was induced by endovascular perforation method. BMSCs (3 × 106 cells) were transplanted intravenously into rats, and N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a Notch1 activation inhibitor, and Notch1 small interfering RNA (siRNA) were injected intracerebroventricularly. The effects of BMSCs on EBI were assayed by neurological score, brain water content (BWC), blood-brain barrier (BBB) permeability, magnetic resonance imaging, hematoxylin and eosin staining, and Fluoro-Jade C staining. Immunofluorescence and immunohistochemistry staining, Western blotting, and quantitative real-time polymerase chain reaction were used to analyze various proteins and transcript levels. Pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assay.ResultsBMSCs treatment mitigated the neurobehavioral dysfunction, BWC and BBB disruption associated with EBI after SAH, reduced ionized calcium binding adapter molecule 1 and cluster of differentiation 68 staining and interleukin (IL)-1 beta, IL-6 and tumor necrosis factor alpha expression in the left hemisphere but concurrently increased IL-10 expression. DAPT or Notch1 siRNA administration reduced Notch1 signaling pathway activation following SAH, ameliorated neurobehavioral impairments, and BBB disruption; increased BWC and neuronal degeneration; and inhibited activation of microglia and production of pro-inflammatory factors. The augmentation of Notch1 signal pathway agents and phosphorylation of nuclear factor-κB after SAH were suppressed by BMSCs but the levels of Botch were upregulated in the ipsilateral hemisphere. Botch knockdown in BMSCs abrogated the protective effects of BMSCs treatment on EBI and the suppressive effects of BMSCs on Notch1 expression.ConclusionsBMSCs treatment alleviated neurobehavioral impairments and the inflammatory response in EBI after SAH; these effects may be attributed to Botch upregulation in brain tissue, which subsequently inhibited the Notch1 signaling pathway.

Previous studies have demonstrated resveratrol (RSV) has beneficial effects in early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the beneficial effects of RSV and the underlying mechanisms have not been clearly identified. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation plays a crucial role in the EBI pathogenesis. The aim of this study was to investigate the role of RSV on the NLRP3 inflammasome signaling pathway and EBI in rats after SAH. A prechiasmatic cistern injection model was established in rats, and the primary cultured cortical neurons were stimulated with oxyhemoglobin (oxyHb) to induce SAH in vitro. It showed that the NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, mature interleukin-1β (IL-1β), and interleukin-18 (IL-18) were upregulated after SAH, and the enhanced NLRP3 after SAH was mainly located in microglia. Treatment with 60 or 90 mg/kg RSV after SAH dramatically inhibited the expression of NLRP3, but there was no significant difference in the expression of NLRP3 between the SAH + 60 mg/kg RSV and SAH + 90 mg/kg RSV groups. In addition, treatment with 30 mg/kg RSV did not significantly reduced the expression of NLRP3. We next evaluated the neuroprotective effects of RSV against SAH. We determined that SAH-induced NLRP3 inflammasome activation was significantly inhibited in the SAH + 60 mg/kg RSV group. Meanwhile, 60 mg/kg RSV administration could markedly inhibit microglia activation and neutrophils infiltration after SAH. Concomitant with the decreased cerebral inflammation, RSV evidently reduced cortical apoptosis, brain edema, and neurobehavioral impairment after SAH. In vitro experiments, RSV treatment also clearly protected primary cortical neurons against oxyHb insults, including reduced the proportion of neuronal apoptosis, alleviated neuronal degeneration, and improved cell viabilities. These in vitro data further confirm that RSV has an efficient neuroprotection against SAH. Taken together, these in vivo and in vitro findings suggested RSV could protect against EBI after SAH, at least partially via inhibiting NLRP3 inflammasome signaling pathway.

Subarachnoid hemorrhage (SAH) is a kind of severe hemorrhagic stroke, and early brain injury acted as one of the main causes of death and delayed neurological deficit in patients with subarachnoid hemorrhage. In this process, the function and structural integrity of the blood-brain barrier play an important role. In this study, we have observed whether the apolipoprotein E (apoE) mimetic peptide, COG133, can alleviate early brain injury after subarachnoid hemorrhage. For this purpose, an experimental subarachnoid hemorrhage model was constructed in mice and treated by intravenous injection of COG133 at a dosage of 1 mg/kg. Then, the function and integrity of the blood-brain barrier were detected, and the pyroptosis level of the neuron was determined. The results showed that COG133 could protect blood-brain barrier function and structure integrity, reduce early brain injury, and ameliorate neurological function after subarachnoid hemorrhage. In terms of molecular mechanism, COG133 inhibits blood-brain barrier destruction through the proinflammatory CypA-NF-κB-MMP9 pathway and reduces neuronal pyroptosis by inhibiting NLRP3 inflammasome activation. In conclusion, this study demonstrated that apoE-mimetic peptide, COG133, can play a neuroprotective role by protecting blood-brain barrier function and inhibiting brain cell pyroptosis to reduce early brain injury after subarachnoid hemorrhage.

ABSTRACTObjectives: Early brain injury (EBI) is central to the pathological progress of subarachnoid hemorrhage (SAH). In this study, we determined if neuritin protects the brain against EBI in rats and discussed the role of apoptosis pathway mediated by endoplasmic reticulum stress in this neuroprotective route. Methods: A total of 96 male Sprague Dawley rats were divided into control, sham, SAH and SAH + neuritin groups. The rat SAH model was induced by injection 0.3 mL of nonheparinized arterial blood into the prechiasmatic cistern. Mortality assay, neurological scores, brain water content measurement, Evans blue dye assay, TUNEL stain assay and Western blot analysis were performed. Results: Neuritin significantly improved the neurological scores, brain water content, blood-brain barrier (BBB) and apoptosis compared with the control and sham groups within 24 h after SAH. TUNEL staining assay results demonstrated that apoptosis was ameliorated, MMP-9 expression was reduced, whereas GRP78, CHOP, caspase-12 and ASK1 levels were markedly preserved after neuritin application. Conclusions: Our study demonstrated that neuritin plays a neuroprotective role on EBI after SAH by attenuating BBB disruption, brain edema and apoptosis.

Clinical studies have indicated that early brain injury (EBI) following subarachnoid hemorrhage (SAH) is associated with fatal outcomes. Oxidative stress and brain edema are the characteristic pathological events in occurrence EBI following SAH. The present study aimed to examine the effect of 3,4-dihydroxyphenylethanol (DOPET) against SAH-induced EBI, and to demonstrate whether the effect is associated with its potent free radical scavenging property. SAH was induced in rats using an endovascular perforation technique, and 24 h later the rats displayed diminished neurological scores and brain edema. Furthermore, elevated malondialdehyde (an index of lipid peroxidation) and depleted levels of antioxidants were observed in the rat cerebral cortex tissue. Quantitative polymerase chain reaction analysis indicated the upregulated mRNA expression of the apoptotic markers caspase-3 and -9 in the cerebral cortex. Furthermore, the protein expression levels of the proinflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were significantly upregulated in SAH-induced rats. By constrast, treatment with DOPET significantly attenuated EBI by reducing brain edema, elevation of antioxidant status, inhibition of apoptosis and inflammation. In this context, DOPET may be a potent agent in the treatment of EBI following SAH, as a result of its free radical scavenging capacity.

BackgroundInnate immunity can facilitate early brain injury (EBI) following subarachnoid hemorrhage (SAH). Numerous studies suggest that pyroptosis could exacerbate extracellular immune responses by promoting secretion of inflammatory cytokines. Transforming growth factor-β-activated kinase 1 (TAK1) is a quintessential kinase that positively regulates inflammation through NF-κB and MAPK signaling cascades. However, the effects of TAK1 on neuroinflammation in EBI following SAH are largely unknown.MethodsTwo hundred and forty-six male C57BL/6J mice were subjected to the endovascular perforation model of SAH. A selective TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) was administered by intracerebroventricular (i.c.v) injection at 30 min after SAH induction. To genetic knockdown of TAK1, small interfering RNA (siRNA) was i.c.v injected at 48 h before SAH induction. SAH grade, brain water content, BBB permeability, neurological score, western blot, real-time PCR, ELISA, transmission electron microscope, and immunofluorescence staining were performed. Long-term behavioral sequelae were evaluated by the rotarod and Morris water maze tests. Furthermore, OZ was added to the culture medium with oxyhemoglobin (OxyHb) to mimic SAH in vitro. The reactive oxygen species level was detected by DCFH-DA staining. Lysosomal integrity was assessed by Lyso-Tracker Red staining and Acridine Orange staining.ResultsThe neuronal phosphorylated TAK1 expression was upregulated following SAH. Pharmacologic inhibition of TAK1 with OZ could alleviate neurological deficits, brain edema, and brain-blood barrier (BBB) disruption at 24 h after SAH. In addition, OZ administration restored long-term neurobehavioral function. Furthermore, blockade of TAK1 dampened neuronal pyroptosis by downregulating the N-terminal fragment of GSDMD (GSDMD-N) expression and IL-1β/IL-18 production. Mechanistically, both in vivo and in vitro, we demonstrated that TAK1 can induce neuronal pyroptosis through promoting nuclear translocation of NF-κB p65 and activating nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. TAK1 siRNA treatment mitigated SAH-induced neurobehavioral deficits and restrained phosphorylated NF-κB p65 expression and NLRP3 inflammasome activation. TAK1 blockade also ameliorated reactive oxygen species (ROS) production and prevented lysosomal cathepsin B releasing into the cytoplasm.ConclusionsOur findings demonstrate that TAK1 modulates NLRP3-mediated neuronal pyroptosis in EBI following SAH. Inhibition of TAK1 may serve as a potential candidate to relieve neuroinflammatory responses triggered by SAH.

Neuroinflammation is a well-recognized consequence of subarachnoid hemorrhage (SAH), and Toll-like receptor (TLR) 4 may be an important therapeutic target for post-SAH neuroinflammation. Of the TLR family members, TLR4 is expressed in various cell types in the central nervous system, and is unique in that it can signal through both the myeloid differentiation primary-response protein 88-dependent and the toll receptor associated activator of interferon-dependent cascades to coordinate the maximal inflammatory response. TLR4 can be activated by many endogenous ligands having damage-associated molecular patterns including heme and fibrinogen at the rupture of an intracranial aneurysm, and the resultant inflammatory reaction and thereby tissue damages may furthermore activate TLR4. It is widely accepted that the excreted products of TLR4 signaling alter neuronal functions. Previous studies have focused on the pathway through nuclear factor (NF)-κB signaling among TLR4 signaling pathways as to the development of early brain injury (EBI) such as neuronal apoptosis and blood-brain barrier disruption, and cerebral vasospasm. However, many findings suggest that both pathways via NF-κB and mitogen-activated protein kinases may be involved in EBI and cerebral vasospasm development. To overcome EBI and cerebral vasospasm is important to improve outcomes after SAH, because both EBI and vasopasm are responsible for delayed brain injuries or delayed cerebral ischemia, the most important preventable cause of poor outcomes after SAH. Increasing evidence has shown that TLR4 signaling plays an important role in SAH-induced brain injuries. Better understanding of the roles of TLR4 signaling in SAH will facilitate development of new treatments.

Intro: Early Brain Injury (EBI) after non-traumatic subarachnoid hemorrhage (SAH) is a significant mediator of neurological injury, but the injury pathways and markers are not well understood. We hypothesized that the inflammatory mediator soluble ST2 (sST2) is associated with markers of EBI. Methods: We studied two independent cohorts, including 190 SAH patients in a discovery cohort and 50 patients in a replication cohort. Clinical markers of EBI included early loss of consciousness, Glasgow Coma Scale (GCS) score at 24 hours, and neurological deterioration within the first 72 hours (defined as a decrease in GCS ≥ 2 points). Radiographic markers included bicaudate index, clinically significant hydrocephalus requiring external ventricular drainage (EVD), and global cerebral edema. Serial plasma sST2 level was measured at early (3.5 ± 1.2 days post-hemorrhage), intermediate (7.8 ± 1.3 days post-hemorrhage), and late (13 ± 2.3 days post-hemorrhage) time points in the discovery cohort and daily during post-hemorrhage days 1-9 in the replication cohort. In the discovery cohort, the relationship between EBI markers and poor functional outcome (90-day modified Rankin Scale; mRS ≥ 3) was assessed using multivariable logistic regression. The association between sST2 level and EBI markers was evaluated using repeated measures analysis. In the replication cohort, associations were examined using an analysis of response profiles. Results: Clinical and radiographic markers of EBI, except for global edema, were each associated with functional outcome in univariate analysis. Of these EBI markers, independent predictors of poor outcome included 24-hour GCS (OR=5.76, 95% CI 2.31-14.4, p<0.001) and clinically significant hydrocephalus (OR=8.74, 95% CI 2.44-31.3, p=0.001). Elevated sST2 was associated with all markers of EBI except for global edema, and independently predicted poor outcome in a multivariate model that included EBI markers. These findings were further replicated in an independent cohort. Conc: Of the potential clinical and radiographic contributors to EBI, poor 24-hour GCS and clinically significant hydrocephalus remain independent predictors of poor outcome. Furthermore, soluble sST2 level is associated with EBI and poor outcome.

Background: We have developed Xe-containing echogenic liposomes (Xe-ELIP). Using this delivery agent, we have demonstrated neuroprotective effects in an ischemic stroke model. Given the common mediators of cellular injury in ischemic and early-stage hemorrhagic stroke, the goal of this study was to evaluate the neuroprotective effects of Xe-ELIP on early brain injury after subarachnoid hemorrhage (SAH). Methods: Xe-ELIP was prepared by a pressurization-freeze method. The neuroprotective effects of Xe-ELIP on cultured brain astrocytes and microglia were evaluated by a lactate dehydrogenase (LDH) assay. For in vivo evaluation, rats (n=35) were randomly divided into sham, SAH, SAH with Xe-ELIP, ELIP treatment, and Xe saturated saline treatment groups. SAH was induced by an endovascular puncture method. Treatments were administrated intravenously in combination with ultrasound application over the common carotid artery to trigger Xe release from circulating Xe-ELIP at 30 minutes after SAH. Mortality assessments, neurological evaluations, and behavioral tests were conducted for three days following surgery. Brain tissue was harvested for SAH grading, H&E staining, TUNEL staining, and brain water content measurement on the third day after surgery. Results: Treatment with Xe-ELIP protected brain astroacytes and microglia against H 2 O 2 (10 mM) induced cytotoxicity. SAH grading revealed effective reduction of bleeding in association with decreased mortality. Moreover, Xe-ELIP significantly reduced apoptotic neuronal death and improved both general neurological and motor function. Conclusions: Xe-ELIP alleviates early SAH brain injury by inhibiting neuronal death and bleeding. This novel delivery approach, using Xe-ELIP with ultrasound-controlled cerebral drug release, provides a relatively noninvasive strategy for therapeutic gas delivery in SAH.

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) generally causes significant and lasting damage. Pentoxifylline (PTX), a nonselective phosphodiesterase inhibitor, has shown anti-inflammatory and neuroprotective properties in several brain injury models, but the role of PTX with respect to EBI following SAH remains uncertain. The purpose of this study was to investigate the effects of PTX on EBI after SAH in rats. Adult male Sprauge-Dawley rats were randomly assigned to the sham and SAH groups. PTX (30 or 60mg/kg) or an equal volume of the administration vehicle (normal saline) was administrated at 30min intervals following SAH. Neurological scores, brain edema, and neural cell apoptosis were evaluated. In order to explore other mechanisms, changes in the toll-like receptor 4 (TLR4) and the nuclear factor-κB (NF-κB) signaling pathway, in terms of the levels of apoptosis-associated proteins, were also investigated. We found that administration of PTX (60mg/kg) notably improved neurological function and decreased brain edema at both 24 and 72h following SAH. Treatment with PTX (60mg/kg) significantly inhibited the protein expressions of TLR4, NF-κB, MyD88 and the downstream pro-inflammatory cytokines, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). PTX also significantly reduced neural cell death and BBB permeability. Our observations may be the first time that PTX has been shown to play a neuroprotective role in EBI after SAH, potentially by suppressing the TLR4/NF-κB inflammation-related pathway in the rat brain.

Inhibition of mammalian target of rapamycin attenuates early brain injury through modulating microglial polarization after experimental subarachnoid hemorrhage in rats

Recombinant human erythropoietin (rhEPO) alleviates early brain injury following subarachnoid hemorrhage in rats: Possible involvement of Nrf2–ARE pathway

Early brain injury (EBI) after subarachnoid hemorrhage (SAH) is a new therapeutic target. Sulfonylurea receptor 1 (SUR1) is expressed in nerve cells, glial cells, and vascular endothelial cells in EBI. SUR1 promotes intracellular inflow of Na and Ca ions, resulting in cell swelling and depolarization, and finally cell death. Glibenclamide reduced cerebral edema and mortality in a basic study of cerebral ischemia. However, the effects of glibenclamide on EBI have not been fully elucidated. This study examined the inhibitory effect of glibenclamide on EBI. Rats were divided into the sham group, SAH-control group, and SAH-glibenclamide group. The water content of the brain was measured using the dry-wet method. In addition, the brain was divided into the cortex, putamen, and hippocampus, and expression of inflammatory cytokines was evaluated by the polymerase chain reaction method. In addition, microglia in the brain were evaluated immunohistologically. Water content of the brain was significantly decreased in the SAH-glibenclamide group compared to the SAH-control group. Interleukin-1beta (IL-1β), tumor necrosis factor alpha (TNFα), and nuclear factor-kappa B significantly increased in the cerebral cortex after SAH. IL-1β and TNFα in the cortex were significantly decreased in the SAH-glibenclamide group compared to the SAH-control group. Immunohistochemical staining confirmed that SAH causes extensive microglial activation in the brain, which was suppressed by glibenclamide. The present study showed that glibenclamide suppressed cerebral edema and activation of microglia and hypersecretion of inflammatory cytokines. Glibenclamide is a potential therapeutic method which may significantly improve the functional prognosis.

Intranasal administration of insulin to the brain bypasses the blood brain barrier (BBB) and can increase cerebral glucose uptake and prevent energy failure. Intranasal insulin treatment has shown neuroprotective effects in multiple central nervous system (CNS) lesions, but the effects of intranasal insulin on the metabolic and pathological process of subarachnoid hemorrhage (SAH) are not clear. This study is designed to explore the effects of intranasal insulin treatment on metabolic distress and early brain injury (EBI) after experimental SAH. SAH model was built by endovascular filament perforation method in adult male C57BL/6J mice, and then, insulin was administrated via intranasal route at 0, 24, and 48h post-SAH. EBI was assessed according to the neurological performance, BBB damage, brain edema, neuroinflammatory reaction, and neuronal apoptosis at each time point. To evaluate metabolic conditions, microdialysis was used to continuously monitor the real-time levels of glucose, pyruvate, and lactate in interstitial fluid (ISF) in living animals. The mRNA and protein expression of glucose transporter-1 and 3 (GLUT-1 and -3) were also tested by RT-PCR and Western blot in brain after SAH. Compared to vehicle, intranasal insulin treatment promoted the relative mRNA and protein levels of GLUT-1 in SAH brain (0.98 ± 0.020 vs 0.33 ± 0.016 at 24h, 0.91 ± 0.25 vs 0.21 ± 0.013 at 48h and 0.94 ± 0.025 vs 0.28 ± 0.015 at 72h in mRNA/0.96 ± 0.023 vs 0.36 ± 0.015 at 24h, 0.91 ± 0.022 vs 0.22 ± 0.011 at 48h and 0.95 ± 0.024 vs 0.27 ± 0.014 at 72h in protein, n = 8/Group, p < 0.001). Similar results were also observed in GLUT-3. Intranasal insulin reduced the lactate/pyruvate ratio (LPR) and increased ISF glucose level. It also improved neurological dysfunction, BBB damage, and brain edema and attenuated the levels of pro-inflammatory cytokines as well as neuronal apoptosis after SAH. The intranasal insulin treatment protects brain from EBI possibly via improving metabolic distress after SAH.