Mouse Model Of Subarachnoid Hemorrhage Research Articles

Cortical Spreading Depolarizations in a Mouse Model of Subarachnoid Hemorrhage.

Cortical spreading depolarizations (CSDs) are associated with worse outcomes in patients with aneurysmal subarachnoid hemorrhage (SAH). Animal models are required to assess whether CSDs can worsen outcomes or are an epiphenomenon; however, little is known about the presence of CSDs in existing animal models. Therefore, we designed a study to determine whether CSDs occur in a mouse model of SAH. A total of 36 mice were included in the study. We used the anterior prechiasmatic injection model of SAH under isoflurane anesthesia. A needle was inserted through the mouse olfactory bulb with the point terminating at the base of the skull, and arterial blood or saline (100µl) was injected over 10s. Changes in cerebral blood volume over the entire dorsal cortical surface were assessed with optical intrinsic signal imaging for 5min following needle insertion. CSDs occurred in 100% of mice in the hemisphere ipsilateral to olfactory bulb needle insertion (CSD1). Saline-injected mice had 100% survival (n = 10). Blood-injected mice had 88% survival (n = 23 of 26). A second, delayed, CSD ipsilateral to CSD1 occurred in 31% of blood-injected mice. An increase in the time interval between CSD1 and blood injection was associated with the occurrence of a second CSD in blood-injected mice (mean intervals 26.4 vs. 72.7s, p < 0.0001, n = 18 and 8). We observed one blood-injected animal with a second CSD in the contralateral hemisphere and observed terminal CSDs in mice that died following SAH injection. The prechiasmatic injection model of SAH includes CSDs that occur at the time of needle insertion. The occurrence of subsequent CSDs depends on the timing between CSD1 and blood injection. The mouse prechiasmatic injection model could be considered an SAH plus CSD model of the disease. Further work is needed to determine the effect of multiple CSDs on outcomes following SAH.

Obstructive sleep apnea aggravates neuroinflammation and pyroptosis in early brain injury following subarachnoid hemorrhage via ASC/HIF-1α pathway.

Obstructive sleep apnea can worsen the prognosis of subarachnoid hemorrhage. However, the underlying mechanism remains unclear. In this study, we established a mouse model of subarachnoid hemorrhage using the endovascular perforation method and exposed the mice to intermittent hypoxia for 8 hours daily for 2 consecutive days to simulate sleep apnea. We found that sleep apnea aggravated brain edema, increased hippocampal neuron apoptosis, and worsened neurological function in this mouse model of subarachnoid hemorrhage. Then, we established an in vitro HT-22 cell model of hemin-induced subarachnoid hemorrhage/intermittent hypoxia and found that the cells died, and lactate dehydrogenase release increased, after 48 hours. We further investigated the underlying mechanism and found that sleep apnea increased the expression of hippocampal neuroinflammatory factors interleukin-1β, interleukin-18, interleukin-6, nuclear factor κB, pyroptosis-related protein caspase-1, pro-caspase-1, and NLRP3, promoted the proliferation of astrocytes, and increased the expression of hypoxia-inducible factor 1α and apoptosis-associated speck-like protein containing a CARD, which are the key proteins in the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway. We also found that knockdown of hypoxia-inducible factor 1α expression in vitro greatly reduced the damage to HY22 cells. These findings suggest that sleep apnea aggravates early brain injury after subarachnoid hemorrhage by aggravating neuroinflammation and pyroptosis, at least in part through the hypoxia-inducible factor 1α/apoptosis-associated speck-like protein containing a CARD signaling pathway.

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

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

Suppression of MALAT1 alleviates neurocyte apoptosis and reactive oxygen species production through the miR-499-5p/SOX6 axis in subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH), a common devastating cerebrovascular accident, is a great threat to human health and life. Exploration of the potential therapeutic target of SAH is urgently needed. Previous studies showed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes cell apoptosis in various diseases, while its role in SAH remains unclear. In our study, we established a mouse model of SAH and used the oxyhemoglobin (OxyHb) to induce neuronal injury in vitro. Interestingly, MALAT1 was found upregulated in brain tissues of SAH mice and OxyHb-stimulated neurons. In addition, knockdown of MALAT1 attenuated apoptosis and decreased reactive oxygen species (ROS) production in OxyHb-stimulated neurons. Mechanistically, we demonstrated that MALAT1 bound with miR-499-5p. Furthermore, our findings indicated that miR-499-5p bound to SOX6 3' untranslated region (UTR) and negatively regulated SOX6 mRNA and protein levels. Rescue assays suggested that SOX6 overexpression counteracted the effects of MALAT1 knockdown on neurocyte apoptosis, and ROS production in OxyHb-stimulated neurons. The in vivo assays indicated that knockdown of MALAT1 improved brain injury of SAH mice. Our study demonstrates that silencing of MALAT1 alleviates neurocyte apoptosis and reduces ROS production through the miR-499-5p/SOX6 axis after SAH injury.

ACE2 Rescues Impaired Autophagic Flux Through the PI3K/AKT Pathway After Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is one of the life-threatening neurosurgical diseases in central nervous system. Autophagy has been previously demonstrated to exert vital roles in SAH development. Angiotensin I converting enzyme 2 (ACE2) has been revealed as a regulator of autophagy in neurosurgical diseases. However, effect of ACE2 on autophagy in SAH progression has not been clarified. First, we explored the relationship between autophagy and SAH progression by establishing a mouse model of SAH under the administration of 3-MA (the autophagy inhibitor). Next, we examined ACE2 expression in the cerebral cortex of SAH mice ex vivo with RT-qPCR. Subsequently, we assessed the biological function of ACE2 on brain injury, the autophagic flux pathway and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling ex vivo via neurological scoring, TUNEL assay, western blot analysis and immunofluorescence staining assay. Finally, we carried out rescue assays under chloroquine (CQ, the autophagic flux inhibitor) and LY294002 (the PI3K/AKT signaling inhibitor) administration. 3-MA mitigated brain injury after SAH, and ACE2 was downregulated in cerebral cortex of SAH mice. Moreover, ACE2 elevation alleviated cell apoptosis, cerebral edema, and neurological deficits, ameliorated the autophagic flux pathway and activated the PI3K/AKT signaling in SAH mice. Furthermore, CQ and LY294002 neutralized the effects of overexpressed ACE2 on neuronal apoptosis, cerebral edema, and neurological deficits in SAH mice. Overall, ACE2 lessened neuronal injury via the autophagic flux and PI3K/AKT pathways. This research might provide a potential novel direction for clinical treatment of SAH.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11064-021-03469-w.

Hippocampal Transcriptome Changes After Subarachnoid Hemorrhage in Mice.

After subarachnoid hemorrhage (SAH), up to 95% of surviving patients suffer from post-SAH syndrome, which includes cognitive deficits with impaired memory, executive functions, and emotional disturbances. Although these long-term cognitive deficits are thought to result from damage to temporomesial–hippocampal areas, the underlying mechanisms remain unknown. To fill this gap in knowledge, we performed a systematic RNA sequencing screen of the hippocampus in a mouse model of SAH. SAH was induced by perforation of the circle of Willis in mice. Four days later, hippocampal RNA was obtained from SAH and control (sham perforation) mice. Next-generation RNA sequencing was used to determine differentially expressed genes in the whole bilateral hippocampi remote from the SAH bleeding site. Functional analyses and clustering tools were used to define molecular pathways. Differential gene expression analysis detected 642 upregulated and 398 downregulated genes (false discovery rate <0.10) in SAH compared to Control group. Functional analyses using IPA suite, Gene Ontology terms, REACTOME pathways, and MsigDB Hallmark gene set collections revealed suppression of oligodendrocytes/myelin related genes, and overexpression of genes related to complement system along with genes associated with innate and adaptive immunity, and extracellular matrix reorganization. Interferon regulatory factors, TGF-β1, and BMP were identified as major orchestrating elements in the hippocampal tissue response. The MEME-Suite identified binding motifs of Krüppel-like factors, zinc finger transcription factors, and interferon regulatory factors as overrepresented DNA promoter motifs. This study provides the first systematic gene and pathway database of the hippocampal response after SAH. Our findings suggest that damage of the entorhinal cortex by subarachnoid blood may remotely trigger specific hippocampal responses, which include suppression of oligodendrocyte function. Identification of these novel pathways may allow for development of new therapeutic approaches for post-SAH cognitive deficits.

Sevoflurane and Desflurane Exposures Following Aneurysmal Subarachnoid Hemorrhage Confer Multifaceted Protection against Delayed Cerebral Ischemia.

Numerous studies have demonstrated the ability of isoflurane conditioning to provide multifaceted protection against aneurysmal subarachnoid hemorrhage (SAH)-associated delayed cerebral ischemia (DCI); however, preclinical studies have not yet examined whether other commonly used inhalational anesthetics in neurological patients such as sevoflurane or desflurane are also protective against SAH-induced neurovascular deficits. We therefore sought to identify the potential for sevoflurane and desflurane conditioning to protect against DCI in an endovascular perforation mouse model of SAH. Neurological function was assessed daily via neuroscore. Large artery vasospasm and microvessel thrombosis were assessed three days after SAH or sham surgery. Four groups were examined: Sham, SAH + room air, SAH + 2% Sevoflurane, and SAH + 6% Desflurane. For the SAH groups, one hour after surgery, mice received 2% sevoflurane, 6% desflurane, or room air for one hour. We found that conditioning with sevoflurane or desflurane attenuated large artery vasospasm, reduced microvessel thrombosis, and improved neurologic function. Given their frequent clinical use and strong safety profile in patients (including those with SAH), these data strongly support further studies to validate these findings in preclinical and clinical studies and to elucidate the mechanisms by which these agents might be acting.

Cortical spreading depression aggravates early brain injury in a mouse model of subarachnoid hemorrhage.

Cortical spreading depression (CSD) has been observed during the early phase of subarachnoid hemorrhage (SAH). However, the effect of CSD on the cerebral blood flow (CBF) and cerebral oxyhemoglobin (CHbO) during the early phase of SAH has not yet been assessed directly. We, therefore, used laser speckle imaging and optical intrinsic sinal imaging to record CBF and CHbO during CSD and cerebral cortex perfusion (CCP) at 24 hours after CSD in a mouse model of SAH. SAH was induced by blood injection into the prechiasmatic cistern. When CSD occurred, the change trend of CBF and CHbO in Sham group and SAH group was the same, but ischemia and hypoxia in SAH group was more significant. At 24 hours after SAH, the CCP of CSD group was lower than that of no CSD group, and the neurological function score of CSD group was lower. We conclude that induction of CSD further aggravates cerebral ischemia and worsens neurological dysfunction in the early stage of experimental SAH. Our study underscores the consequence of CSD in the development of early brain injury after SAH.

Anesthetic and subanesthetic doses of isoflurane conditioning provides strong protection against delayed cerebral ischemia in a mouse model of subarachnoid hemorrhage

Delayed cerebral ischemia (DCI) is identified as one of the significant contributors to poor patient outcome after aneurysmal subarachnoid hemorrhage (SAH). We previously reported that a supratherapeutic dose of isoflurane conditioning (2%) provided robust protection against SAH-induced DCI. The aim of our current study is to compare the efficacy of the supratherapeutic dose of isoflurane to that typically used to establish general anesthesia or sedation. After IRB approval for animal studies, ten to fourteen-week-old wild-type male mice (C57BL/6) were divided into five groups – sham, SAH alone, or SAH with isoflurane conditioning (0.5%, 1%, and 2%). Conditioning was performed with one-hour of isoflurane initiated one-hour after induction of SAH via endovascular perforation technique. Vasospasm measurement in the middle cerebral artery was assessed 72 h after SAH. Neurological assessment was performed at baseline and for next three days after SAH. It was identified that all tested doses of isoflurane conditioning (0.5%, 1%, and 2%) significantly attenuated large artery vasospasm and markedly improved neurological deficits following SAH. No significant differences in neurovascular outcome were noted between the three doses of isoflurane conditioning. Our data show that isoflurane dosing typically used for general anesthesia (1%) or sedation (0.5%) provide similar levels of DCI protection in SAH as that provided by a supratherapeutic dose (2%). This result has important implications for future translational studies. Additional studies examining the therapeutic potential of anesthetic conditioning for SAH are therefore warranted.

TREM-1 Exacerbates Neuroinflammatory Injury via NLRP3 Inflammasome-Mediated Pyroptosis in Experimental Subarachnoid Hemorrhage.

Neuroinflammation contributes to the pathogenesis of early brain injury induced by subarachnoid hemorrhage (SAH). Previous reports have demonstrated that triggering receptor expressed on myeloid cells 1 (TREM-1) regulates inflammatory response caused by ischemic stroke or myocardial infarction. However, whether TREM-1 could modulate neuroinflammation after SAH remains largely unknown. Here, using a mouse model of SAH, we found that the expression of TREM-1 was mainly located in microglia cells and increased to peak at 24h following SAH. Then, TREM-1 antagonist or mimic was intranasally administrated to investigate its effect on SAH. TREM-1 inhibition with LP17 improved neurological deficits, mitigated brain water content, and preserved brain-blood barrier integrity 24h after SAH, whereas recombinant TREM-1, a mimic of TREM-1, deteriorated these outcomes. In addition, LP17 administration restored long-term sensorimotor coordination and cognitive deficits. Pharmacological blockade of TREM-1 reduced TUNEL-positive and FJC-positive neurons, and CD68-stained microglia in ipsilateral cerebral cortex. Neutrophil invasion was inhibited as protein level of myeloperoxidase (MPO), and MPO-positive cells were both decreased. Moreover, we found that LP17 treatment ameliorated microglial pyroptosis by diminishing levels of N-terminal fragment of GSDMD (GSDMD-N) and IL-1β production. Mechanistically, both in vivo and in vitro, we depicted that TREM-1 can trigger microglial pyroptosis via activating NLRP3 inflammasome. In conclusion, our results revealed the critical role of TREM-1 in neuroinflammation following SAH, suggesting that TREM-1 inhibition might be a potential therapeutic approach for SAH.

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 °C and positioned in a stereotactic frame with a cervical bend of about 30°. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 µL of blood was injected in the cisterna magna followed by a 30° downward tilt of the animal for 2 minutes. The second infusion of 30 µL of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

Meningeal lymphatics clear erythrocytes that arise from subarachnoid hemorrhage

Extravasated erythrocytes in cerebrospinal fluid (CSF) critically contribute to the pathogenesis of subarachnoid hemorrhage (SAH). Meningeal lymphatics have been reported to drain macromolecules and immune cells from CSF into cervical lymph nodes (CLNs). However, whether meningeal lymphatics are involved in clearing extravasated erythrocytes in CSF after SAH remains unclear. Here we show that a markedly higher number of erythrocytes are accumulated in the lymphatics of CLNs and meningeal lymphatics after SAH. When the meningeal lymphatics are depleted in a mouse model of SAH, the degree of erythrocyte aggregation in CLNs is significantly lower, while the associated neuroinflammation and the neurologic deficits are dramatically exacerbated. In addition, during SAH lymph flow is increased but without significant lymphangiogenesis and lymphangiectasia. Taken together, this work demonstrates that the meningeal lymphatics drain extravasated erythrocytes from CSF into CLNs after SAH, while suggesting that modulating this draining may offer therapeutic approaches to alleviate SAH severity.

Targeting CCL20 inhibits subarachnoid hemorrhage-related neuroinflammation in mice.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1β and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.

Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1.

BackgroundDelayed cognitive impairment (DCI) after subarachnoid hemorrhage (SAH) is one of the most common sequelae in patients. This study aimed to investigate the characteristics of the course and glutamatergic pathogenesis of DCI after SAH in mice.MethodsA SAH mouse model of internal carotid puncture was used. Depressive and cognitive behaviors were detected by forced swimming and sucrose preference tests and Morris water maze test, respectively. Microdialysis and high-performance liquid chromatography (HPLC) were used to detect the interstitial glutamate. The expressions of histone deacetylases (HDACs), glutamate transporters, and glutamate receptors were examined. Primary astrocytes magnetically sorted from adult mice were cultured for glutamate uptake assay and protein and mRNA detection. Selective HDAC2 inhibitor and glutamate transporter-1 (GLT-1) inhibitor administered via were intraperitoneal injection to evaluate their effects on DCI in SAH mice.ResultsDepression and memory impairment lasted for more than 12 weeks and peaked at 8 weeks after SAH. Interstitial glutamate accumulation in the hippocampus and impaired glutamate uptake in astrocytes of the SAH mice were found during DCI, which could be explained by there being a significant decrease in GLT-1 expression but not in glutamate and aspartate transporter (GLAST) in hippocampal astrocytes. Meanwhile, the phosphorylation level of excitatory glutamate receptors (GluN2B and GluA1) in the hippocampus was significantly reduced, although there was no significant change in the expression of the receptors. Importantly, the expression of HDAC2 increased most significantly in astrocytes after SAH compared with that of other subtypes of HDACs. Inhibition of HDAC2 markedly rescued the decrease in GLT-1 expression after SAH through transcriptional regulation. Behavioral results showed that a selective HDAC2 inhibitor effectively improved DCI in SAH mice, but this effect could be weakened by GLT-1 inhibition.ConclusionsIn summary, our study suggests that the dysfunction of GLT-1-mediated glutamate uptake in astrocytes may be a key pathological mechanism of DCI after SAH, and that a specific inhibitor of HDAC2 may exert a potential therapy.

Defining the Mechanism of Subarachnoid Hemorrhage-Induced Pyrexia.

Fever can affect the majority of patients with subarachnoid hemorrhage (SAH) and many times no identifiable source is found for the fever whether infectious or sterile, like deep vein thrombosis. We hypothesized that fever in SAH is mediated by a NON-cyclo-oxygenase-dependent mechanism, which we neologized as subarachnoid hemorrhage-induced pyrexia (SAHiP). This hypothesis was investigated using genetically modified mice, pharmacological manipulation, cerebrospinal fluid from SAH patients, and a large cohort of SAH patients. Mice with deletions of neuronal prostaglandin EP3 receptor, global toll-like receptor 4 (TLR4), myeloid TLR4, and microglial TLR4 were subjected to SAH after being implanted with thermometers. Pathways necessary for SAHiP were identified. In SAH patients, cerebrospinal fluid was examined by flow cytometry and correlated with SAHiP. From a large cohort of SAH patients, independent associations with SAHiP were determined using logistic regression analysis. In our mouse model of SAH, microglial TLR4 is necessary for SAHiP, but independent of the neuronal prostaglandin EP3 receptor, cyclo-oxygenase, and prostaglandins. Macrophages from the cerebrospinal fluid of SAH patients with SAHiP expressed more TLR4-co-receptor than SAH patients without SAHiP. In a large cohort of SAH patients, SAHiP was found to be independently, yet inversely, associated with acetaminophen administration. SAHiP is independent of the neuronal prostaglandin EP3 receptor, cyclo-oxygenase, and prostaglandins, but dependent on microglial/macrophage TLR4 with evidence from both SAH mouse models and SAH patients.

Abstract WP308: Role of Microthrombi and Vasospasm in the Development of Delayed Neurological Deficits After Subarachnoid Hemorrhage in Mice

Rationale: Microthrombosis has been suggested as a major factor contributing to delayed neurological deterioration in patients after subarachnoid hemorrhage (SAH). However, experimental studies on the role of microthrombi in delayed deficits after SAH has not been investigated. Our hypothesis is that, following SAH, mice which develop delayed neurological deficits have a greater number of microthrombi than mice which do not develop delayed neurological deficits. Methods: SAH was induced in adult male and female C57BL/6 mice via endovascular perforation. Mice were randomly assigned into sham (n=6/sex) or SAH groups (n=22-24/sex). Neurobehavior was performed on days 1-3, 5, and 7 post-SAH using a composite neuroscore. Animals were sacrificed on the day of delayed deficits or 7 days post-SAH. Microthrombi count and vessel diameters (for vasospasm) were measured using H&amp;E stained brain slices. All outcomes were performed and all data were analyzed by a blinded investigator. Results: Seventeen percent (4/24) of male mice and thirty-six percent (8/22) of female mice developed delayed deficits on days 3-5 post-SAH (Figures 1A and 1B). Those mice which developed delayed deficits had significantly more microthrombi in their brains than mice which did not develop delayed deficits; vasospasm did not correlate with delayed deficits. Additionally, female SAH mice develop delayed deficits at a higher frequency than males (Figure 1C). Conclusions: This work shows for the first time delayed deficits in a SAH mouse model. Further, microthrombi correlated with delayed deficits, whereas no correlation was between delayed deficits and vasospasm. The data within this study suggests that preventing microthrombi may improve functional recovery and reduce the risk of delayed deficits.

Complement C5 Contributes to Brain Injury After Subarachnoid Hemorrhage

Previous studies showed that complement activation is associated with poor functional outcome after aneurysmal subarachnoid hemorrhage (SAH). We investigated whether complement activation is underlying brain injury after aneurysmal SAH (n = 7) and if it is an appropriate treatment target. We investigated complement expression in brain tissue of aneurysmal SAH patients (n = 930) and studied the role of common genetic variants in C3 and C5 genes in outcome. We analyzed plasma levels (n = 229) to identify the functionality of a single nucleotide polymorphism (SNP) associated with outcome. The time course of C5a levels was measured in plasma (n = 31) and CSF (n = 10). In an SAH mouse model, we studied the extent of microglia activation and cell death in wild-type mice, mice lacking the C5a receptor, and in mice treated with C5-specific antibodies (n = 15 per group). Brain sections from aneurysmal SAH patients showed increased presence of complement components C1q and C3/C3b/iC3B compared to controls. The complement component 5 (C5) SNP correlated with C5a plasma levels and poor disease outcome. Serial measurements in CSF revealed that C5a was > 1400-fold increased 1 day after aneurysmal SAH and then gradually decreased. C5a in plasma was 2-fold increased at days 3–10 after aneurysmal SAH. In the SAH mouse model, we observed a ≈ 40% reduction in both microglia activation and cell death in mice lacking the C5a receptor, and in mice treated with C5-specific antibodies. These data show that C5 contributes to brain injury after experimental SAH, and support further study of C5-specific antibodies as novel treatment option to reduce brain injury and improve prognosis after aneurysmal SAH.

Vitamin D: A New Perspective in Treatment of Cerebral Vasospasm After Subarachnoid Hemorrhage

Abstract INTRODUCTION Cerebral vasospasm (CVS) is a severe complication after subarachnoid hemorrhage (SAH). We explored vitamin D (VitD) as a possible therapeutic option for CVS. METHODS Between 2007 and 2015, 25-vitaminD3 (VitD3) levels and data of SAH patients admitted during the months with a peak vs nadir of VitD3-values were analyzed, retrospectively. Furthermore, VitD3 and vasospasm/outcome data in SAH patients admitted in 2017 were correlated prospectively. A mice SAH model and cell culture studies were used to determine the involved mechanisms of 1,25-dihydroxy-vitaminD3. RESULTS A significant lower vasospasm rate and improved outcome in SAH patients admitted in summer as compared to winter time was identified by the retrospective analysis. In the SAH mouse model, active 1,25-VitD3 attenuated CVS development, as determined by vessel diameter of basilar artery (BA) and reduced neurological deficit. 1,25-VitD3 attenuated the inflammation of the BA in response to blood. Deletion of the myeloid or endothelial VitD-receptor decreased the protective 1,25-VitD3 effect. Co-culture of myeloid and endothelial cells with blood confirmed the anti-inflammatory effect of 1,25-VitD3 and revealed a selective induction of SDF1a, VEGF, and eNOS. In mice, SDF1a mimicked the protective 1,25-VitD3 effect. In patients, high VitD3 levels were associated with a significant higher rate of favorable outcome, prospectively. CVS severity was inversely correlated with the VitD level. Patients with severe CVS exhibited attenuated expression of SDF1a on myeloid cells. CONCLUSION 1,25-VitD3 attenuates CVS after SAH. VitD chould be tested as potential treatment option to prevent CVS after SAH.

Impact of 12/15-Lipoxygenase on Brain Injury After Subarachnoid Hemorrhage.

Background and Purpose- Subarachnoid hemorrhage (SAH) is a devastating form of stroke. Oxidative stress contributes to brain injury, but the mechanisms have been poorly studied. Here, we evaluated the role of 12/15-lipoxygenase (12/15-LOX), an enzyme known to cause cell death in ischemic stroke, on brain injury in a mouse model of SAH. Methods- C57Bl6 wild-type mice and Alox15 knockout mice were subjected to SAH using a direct blood injection technique. In SAH wild-type mice, half received the 12/15-LOX inhibitor ML351 and half received vehicle. Immunohistochemistry, brain edema, blood-brain barrier leakage and functional outcomes were assessed 1 and 3 days after SAH induction. Results- SAH led to increased 12/15-LOX in macrophages of the brain parenchyma, adjacent to the subarachnoid blood. Neuronal cell death after SAH was reduced by ML351 and in Alox15 knockout mice. Similarly, SAH induced brain edema, which was 12/15-LOX dependent. Finally, Alox15 gene knockout and inhibitor treatment in wild-type mice with SAH led to an improved behavioral outcome. Conclusions- 12/15-LOX is overexpressed in macrophages after SAH in mice, and inhibition of the 12/15-LOX pathway decreases brain injury and improves neurological outcome. This study suggests 12/15-LOX as a novel therapeutic target to limit brain injury after SAH.

Central action of rapamycin on early ischemic injury and related cardiac depression following experimental subarachnoid hemorrhage

Early brain injury and related cardiac consequences play a key role in the devastating outcomes after subarachnoid hemorrhage (SAH). We reported that rapamycin exerts neuroprotection against cortical hypoxia early after SAH, but its mechanism is poorly understood. This in vivo study aimed to determine the potential role of the transcription factor STAT3 in the rapamycin-mediated neuroprotection in a mouse model of SAH. Forty C57BL/6 N mice were treated with an intracerebroventricular injection of rapamycin or vehicle (control) given after SAH induction by a filament perforation method, with or without STAT3 (Stattic) or ERK (PD98059) inhibitor pretreatment. Cerebral blood flow signals (%vascularity), brain tissue oxygen saturation (SbtO2), and cardiac output (CO) were analyzed using an ultrasound/photoacoustic imaging system. Clinically relevant neurocardiac depression was notable in severe SAH mice. Rapamycin improved %vascularity, SbtO2, and CO on day 1 after SAH onset. The beneficial effects of rapamycin on cerebral blood flow and oxygenation persisted until day 3, resulting in a significant reduction in post-SAH new cerebral infarctions and survival, as well as improved neurological functions, compared to the control group. All of the effects were attenuated by pretreatment with Stattic or PD98059. These data suggest that ERK and JAK/STAT3 pathways play an important role in the neurocardiac protection by rapamycin after SAH. We propose that rapamycin is a novel pharmacological strategy to target STAT3 activation, with a possible crosstalk through the ERK pathway, for the treatment of post-SAH early brain injury.