Endovascular Perforation Model Of Subarachnoid Hemorrhage Research Articles

Therapeutic utility of Perfluorocarbon Oxygent in limiting the severity of subarachnoid hemorrhage in mice

Subarachnoid hemorrhage (SAH) is the deadliest form of hemorrhagic stroke; however, effective therapies are still lacking. Perfluorocarbons (PFCs) are lipid emulsion particles with great flexibility and their much smaller size as compared to red blood cells (RBCs) allows them to flow more efficiently within the blood circulation. Due to their ability to carry oxygen, a specific PFC-based emulsion, PFC-Oxygent, has been used as a blood substitute; however, its role in cerebral blood flow regulation is unknown. Adult C57BL/6 wildtype male mice were subjected to an endovascular perforation model of SAH followed by an intravenous (i.v.) injection of 9 ml/kg PFC-Oxygent or no treatment at 5 h after SAH. At 48 h after SAH, functional and anatomical outcomes were assessed. We found that SAH resulted in significant neurologic and motor deficits which were prevented by PFC-Oxygent treatment. We found that SAH-induced vasospasm, reduced RBC deformability, and augmented endothelial dysfunction were also restricted by PFC-Oxygent treatment. Moreover, mitochondrial activity and fusion proteins were also markedly decreased as assessed by oxidative phosphorylation (OXPHOS) after SAH. Interestingly, PFC-Oxygent treatment brought the mitochondrial activity close to the basal level. Moreover, SAH attenuated the level of phosphorylated AMP-activated protein kinase (pAMPK), whereas PFC treatment improved pAMPK levels. These data show the beneficial effects of PFC-Oxygent in limiting the severity of SAH. Further studies are needed to fully understand the mechanism through which PFC-Oxygent exerts its beneficial effects in limiting SAH severity.

PEDF-34 attenuates neurological deficit and suppresses astrocyte-dependent neuroinflammation by modulating astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after subarachnoid hemorrhage in rats

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

Diffuse microglial responses and persistent EEG changes correlate with poor neurological outcome in a model of subarachnoid hemorrhage

The mechanism by which subarachnoid hemorrhage (SAH) leads to chronic neurologic deficits is unclear. One possibility is that blood activates microglia to drive inflammation that leads to synaptic loss and impaired brain function. Using the endovascular perforation model of SAH in rats, we investigated short-term effects on microglia together with long-term effects on EEG and neurologic function for up to 3 months. Within the first week, microglia were increased both at the site of injury and diffusely across the cortex (2.5-fold increase in SAH compared to controls, p = 0.012). Concomitantly, EEGs from SAH animals showed focal increases in slow wave activity and diffuse reduction in fast activity. When expressed as a fast-slow spectral ratio, there were significant interactions between group and time (p < 0.001) with less ipsilateral recovery over time. EEG changes were most pronounced during the first week and correlated with neurobehavioral impairment. In vitro, the blood product hemin was sufficient to increase microglia phagocytosis nearly six-fold (p = 0.032). Immunomodulatory treatment with fingolimod after SAH reduced microglia, improved neurological function, and increased survival. These findings, which parallel many of the EEG changes seen in patients, suggest that targeting neuroinflammation could reduce long-term neurologic dysfunction following SAH.

Abstract TP169: Cannabidiol as a Therapy for Experimental Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH), commonly caused by a ruptured aneurysm, carries a high rate of disability and death. Patients who survive the initial bleed continue to be at risk of secondary insults. Preclinical studies demonstrate SAH induces dysregulation of the cerebrovasculature and increases neuroinflammation, which contributes to early brain injury and delayed cerebral ischemia. Few treatment options currently exist for SAH patients, making it imperative to identify novel therapies to improve outcomes following SAH. Cannabidiol (CBD) has gained attention for its potential therapeutic properties and widespread mechanistic actions. First utilized for the treatment of pain, preclinical studies demonstrate CBD has anti-inflammatory, neuroprotective, and vascular stabilization properties in experimental models of injury. Our objective was to determine the novel therapeutic potential of CBD for SAH injury. Male and female C57Bl/6J mice were subjected to the endovascular perforation model of SAH and treated daily with CBD (10 mg/kg; intraperitoneal (i.p.)) or vehicle control. We demonstrate that fewer mice developed ischemic cortical infarcts following CBD treatment compared to controls, indicating CBD reduces the potential development of delayed cerebral ischemia following SAH. Consistently, we demonstrate a significantly lower mortality rate in animals treated with CBD following SAH. We also detected a CBD-mediated decrease in astrocyte and microglia activation 1-7 days post-injury, as well as reduced levels of iron (Prussian blue), which contributes to apoptosis and cognitive dysfunction. Overall, treatment with CBD following experimental SAH may act to normalize vascular and inflammatory responses to decrease mortality, risk of delayed infarcts, and neurodegeneration, though further investigation is required.

Abstract TP318: Evaluating Hypothalamic Injury in a Rat Model of Subarachnoid Hemorrhage

Background and purpose: Hypothalamus is one of the most affected parts of the brain by subarachnoid hemorrhage (SAH). There are some reports that hypothalamic disorders are important determinants of poor prognosis after SAH from acute to chronic phase. However, there have never been any reports on the development of therapeutic strategies targeting the hypothalamus for SAH, and few reports have even demonstrated immunohistological evidence of hypothalamic injury after SAH. Here, we demonstrated on the investigation of temporal changes in hypothalamic injury from the hyperacute phase to the chronic phase after SAH in a rat model of SAH. Methods: Moderate to severe endovascular perforation model of SAH was created using 300g - 450g Sprague-Dawley rats, and they were divided into two groups: Sham operated and SAH group.The endpoints were 3, 6, 12, 24, and 72 hours after SAH, and 7 days and 4 weeks after SAH, and physiological findings and neurological symptoms were measured and hypothalamic injury and related factors in the hypothalamus were studied immunohistochemically. Results: The SAH group showed significant neurological deterioration, and the expression of FJC, indicative of cell death, and TUNEL-positive cells, indicative of apoptosis, in the hypothalamus was significantly elevated in the early phase and decreased over time compared to the sham SAH group. On the other hand, oxytocin, a hypothalamic hormone associated with neuronal protection, was significantly decreased in the SAH group during the acute phase. Conclusions: The present study shows that hypothalamic injury appears hyper acute phase of SAH and persists at least during the acute phase of SAH. These findings suggest that the hypothalamus may be an important therapeutic target in the acute phase of SAH.

Abstract TP319: Time Course of Novel Hypothalamic Protein Sushi Repeat-Containing Protein X- Linked 2 Expression in a Rat Model of Subarachnoid Hemorrhage

Background: Hypothalamic injury after subarachnoid hemorrhage (SAH) is known to be associated with short- and long-term prognosis. Recently, a novel hypothalamic protein, Sushi repeat-containing protein X-linked 2 (SRPX2), was reported as a potential biomarker for hypothalamic damage after traumatic brain injury. However, there are no reports on SRPX2 in SAH, and its role is unknown. In this study, we investigated the expression of SRPX2 in the hypothalamus after SAH using a rat model of SAH. Methods: We made endovascular perforation model of SAH using 300g - 450g SD rats, and they were divided into two groups: Sham operated and SAH group. The endpoints were 3, 6, 12, 24, and 72 hours after SAH, and 7 days and 4 weeks after SAH, and physiological findings and neurological symptoms were measured. In addition, changes in SRPX-2 in the hypothalamic injury and related factors were examined immunohistochemically. Results: The SAH group showed significant neurological deterioration, and a significant decrease in body temperature after 3 h of SAH. Expression of SRPX2 in the hypothalamus was also significantly decreased at 3 h after SAH. However, it was not associated with changes in FJC- and TUNEL-positive cells suggestive of cell death or DHE expression suggestive of oxidative stress. On the other hand, OXT showed a significant decrease after 3 hours of SAH as measured by ELISA in serum from the jugular vein. Conclusions: This is the first study to investigate the expression of SRPX2 in the hypothalamus after SAH time course. Although our results suggest that SRPX2 may not be directly associated with hypothalamic injury after SAH, the fact that SRPX2 expression was decreased during the hyperacute phase and OXT and body temperature were also decreased at the same time suggests that SRPX2 may be indirectly associated with hypothalamic injury after SAH through regulation of endocrine and autonomic function in the hypothalamic-pituitary axis. Further studies are warranted to investigate the possibility that SRPX2 may be indirectly related to hypothalamic disturbances after SAH through endocrine and autonomic regulation of the hypothalamic-pituitary axis.

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

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

Sestrin2 provides cerebral protection through activation of Nrf2 signaling in microglia following subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a neurological emergency characterized by dysfunctional inflammatory response. However, no effective therapeutic options have been reported so far. Microglia polarization has been proposed to exert an essential role in modulating inflammatory response after SAH. Sestrin2 is a stress response protein. Growing evidence has reported that sestrin2 could inhibit M1 microglia and promote M2 microglia polarization. The current study investigated the effects of sestrin2 on microglia phenotype switching and the subsequent brain injury and sought to elucidate the underlying mechanism. We conducted an endovascular perforation SAH model in mice. It was found that sestrin2 was significantly increased after SAH and was mainly distributed in neurons and microglia. Exogenous recombinant human sestrin2 (rh-sestrin2) evidently alleviated inflammatory insults and oxidative stress, and improved neurofunction after SAH. Moreover, rh-sestrin2 increased M2-like microglia polarization and suppressed the number of M1-like microglia after SAH. The protection by rh-sestrin2 was correlated with the activation of Nrf2 signaling. Nrf2 inhibition by ML385 abated the cerebroprotective effects of rh-sestrin2 against SAH and further manifested M1 microglia polarization. In conclusion, promoting microglia polarization from the M1 to M2 phenotype and inducing Nrf2 signaling might be the major mechanism by which sestrin2 protects against SAH insults. Sestrin2 might be a new molecular target for treating SAH.

Hemoglobin Derived from Subarachnoid Hemorrhage-Induced Pyroptosis of Neural Stem Cells via ROS/NLRP3/GSDMD Pathway.

Accumulating evidence has demonstrated that neural stem cells (NSCs) have regenerative capacity after brain injuries, such as in aneurysmal subarachnoid hemorrhage (SAH). The reactive oxygen species (ROS)-induced NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome triggers inflammatory responses and pyroptosis of cells; however, whether ROS-induced neuroinflammation modulates the fate of endogenous NSCs after SAH remains largely unknown. In this study, the level of IL-1β was increased in the cerebrospinal fluid (CSF) of patients with SAH. In an endovascular perforation model of SAH in mice, the secretion of IL-1β increased to a peak at 24 h following SAH, and the expression of Caspase1 and NLRP3 was elevated in the hippocampus. Primary cultured NSCs were incubated with hemoglobin (Hb) to mimic SAH in vitro. The cell viability, LDH release, intracellular ROS levels, scanning electron microscopy (SEM), and the expression of NLRP3 and pyroptosis indicators (GSDMD, ASC, and Caspase-1) in NSCs after SAH were examined to investigate the process of pyroptosis. We found that pyroptotic death featuring cellular swelling, cell membrane pore formation and elevated IL-1β was increased in cultured primary NSCs after Hb treatment, as was the expression of NLRP3, ASC, Caspase-1, and GSDMD. In addition, we found that ROS-induced pyroptosis of NSCs by activating the NLRP3/GSDMD pathway. These findings suggest that pyroptosis of NSCs induced by Hb can impede neural regeneration after SAH.

EphA4/EphrinB2 signaling mediates pericyte-induced transient glia limitans formation as a secondary protective barrier after subarachnoid hemorrhage in mice

BackgroundMost patients with subarachnoid hemorrhage (SAH) do not exhibit brain parenchymal injury upon imaging but present significant blood–brain barrier (BBB) disruption and secondary neurological deficits. The aim of this study was to investigate whether stressed astrocytes act as a secondary barrier to exert a protective effect after SAH and to investigate the mechanism of glial limitan formation. MethodsA total of 204 adult male C57BL/6 mice and an endovascular perforation SAH model were employed. The spatiotemporal characteristics of glial limitan formation after SAH were determined by immunofluorescence staining and transmission electron microscopy. The molecular mechanisms by which pericytes regulate glia limitans formation were analyzed using polymerase chain reaction, Western blotting, immunofluorescence staining and ELISA in a pericyte-astrocyte contact coculture system. The findings were validated ex vivo and in vivo using lentiviruses and inhibitors. Finally, pericytes were targeted to regulate glial limitan formation, and the effect of the glia limitans on secondary brain injury after SAH was evaluated by flow cytometry and analysis of neurological function. ResultsStress-induced glial limitan formation occurred 1 day after SAH and markedly subsided 3 days after ictus. Pericytes regulated astrocyte glia limitan formation via EphA4/EphrinB2 signaling, inhibited inflammatory cell infiltration and altered neurological function. ConclusionsAstrocyte-derived glia limitans serve as a secondary protective barrier following BBB disruption after SAH in mice, and pericytes can regulate glial limitan formation and alter neurological function via EphA4/EphrinB2 signaling. Strategies for maintaining this secondary protective barrier may be novel treatment approaches for alleviating early brain injury after SAH.

Inhibition of Mer exacerbates early brain injury by regulating microglia/macrophage phenotype after subarachnoid hemorrhage in mice

BackgroundPolarization of microglia/macrophages toward the pro-inflammatory phenotype is a crucial contributor to neuroinflammation after subarachnoid hemorrhage (SAH). Mer belongs to the TAM receptor tyrosine kinases family, which is known to play a significant role in the resolution of inflammation. However, the effect and mechanism of Mer after SAH remain unclear. In this study, we explored the effect of Mer on modulating the microglia/macrophage phenotype and neuroinflammation and possible potential mechanism after SAH. Method: Endovascular perforation model of SAH was performed. There are 3 parts in this study. Firstly, the time course of Mer expression was determined within 72 hours after SAH. Secondly, the effect of Mer downregulation on brain water content, neurological function, and microglial polarization was evaluated at 24 h after SAH. Thirdly, the neuroprotective effects of pharmacological Mer agonist were assessed. Result: The expression of Mer increased after SAH, and was prominently localized in microglia/macrophages. Treatment with Mer siRNA increased pro-inflammatory phenotype and decreased anti-inflammatory phenotype of microglia/macrophage, thus resulted in exacerbation of neurological deficits and brain edema after SAH. Mechanistically, the downregulation of Mer inhibited the downstream anti-inflammatory signals, SOCS1/SOCS3, by decreasing phosphorylated STATs. Conclusion: Mer is involved in the microglia/macrophage polarization and inflammation resolution after SAH, and that mechanism, at least in part, may contribute to the involvement of the STATs/SOCSs pathway.

Dynamic Measurements of Cerebral Blood Flow Responses to Cortical Spreading Depolarization in the Murine Endovascular Perforation Subarachnoid Hemorrhage Model.

Delayed cerebral ischemia (DCI) is the most severe complication after subarachnoid hemorrhage (SAH), and cortical spreading depolarization (CSD) is believed to play a vital role in it. However, the dynamic changes in cerebral blood flow (CBF) in response to CSD in typical SAH models have not been well investigated. Here, SAH was established in mice with endovascular perforation. Subsequently, the spontaneous CBF dropped instantly and then returned to baseline rapidly. After KCl application to the cortex, subsequent hypoperfusion waves occurred across the groups, while a lower average perfusion level was found in the SAH groups (days 1-7). Moreover, in the SAH groups, the number of CSD decreased within day 7, and the duration and spreading velocity of the CSD increased within day 3 and day 14, respectively. Next, we continuously monitored the local field potential (LFP) in the prefrontal cortex. The results showed that the decrease in the percentage of gamma oscillations lasted throughout the whole process in the SAH group. In the chronic phase after SAH, we found that the mice still had cognitive deficits but experienced no obvious tissue damage. In summary, SAH negatively affects the CBF responses to CSD and the spontaneous LFP activity and causes long-term cognitive deficits in mice. Based on these findings, in the specific phase after SAH, DCI is induced or exacerbated more easily by potential causers of CSD in clinical practice (edema, erythrocytolysis, inflammation), which may lead to neurological deterioration.

Exendin-4 Preserves Blood-Brain Barrier Integrity via Glucagon-Like Peptide 1 Receptor/Activated Protein Kinase-Dependent Nuclear Factor-Kappa B/Matrix Metalloproteinase-9 Inhibition After Subarachnoid Hemorrhage in Rat.

In this study, we investigated the role of Exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, in blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH) in rats. The endovascular perforation model of SAH was performed in Sprague-Dawley rats. Ex-4 was intraperitoneally injected 1 h after SAH induction. To elucidate the underlying molecular mechanism, small interfering ribonucleic acid (siRNA) for GLP-1R and Dorsomorphin, a specific inhibitor of adenosine monophosphate-activated protein kinase (AMPK), were intracerebroventricularly injected 48 h before induction of SAH correspondingly. Immunofluorescence results supported GLP-1R expressed on the endothelial cells of microvessels in the brain after SAH. Administration of Ex-4 significantly reduced brain water content and Evans blue extravasation in both hemispheres, which improved neurological scores at 24 h after SAH. In the mechanism study, Ex-4 treatment significantly increased the expression of GLP-1R, p-AMPK, IκB-α, Occludin, and Claudin-5, while the expression of p-nuclear factor-kappa B (NF-κB) p65, matrix metalloproteinase-9 (MMP-9), and albumin was significantly decreased. The effects of Ex-4 were reversed by the intervention of GLP-1R siRNA or Dorsomorphin, respectively. In conclusion, Ex-4 could preserve the BBB integrity through GLP-1R/AMPK-dependent NF-κB/MMP-9 inhibition after SAH, which should be further investigated as a potential therapeutic target in SAH.

Sirtuin 5-Mediated Lysine Desuccinylation Protects Mitochondrial Metabolism Following Subarachnoid Hemorrhage in Mice.

Sirt5 (Sirtuin 5) desuccinylates multiple metabolic enzymes and plays an important role in maintaining energy homeostasis. The goal of this study was to determine whether Sirt5-mediated desuccinylation restores the energy metabolism and protects brain against subarachnoid hemorrhage (SAH). Male C57BL/6 or Sirt5-/- mice were used. The endovascular perforation SAH model was applied. Protein lysine succinylation in the brain cortex was examined using liquid chromatography-tandem mass spectrometry analysis. The brain metabolism was evaluated by measurement of brain pH as well as ATP and reactive oxygen species level. Neuronal cell death and neurobehavioral deficits were assessed 24 hours after SAH. The expression and desuccinylation activity of Sirt5, lysine succinylation of citrate synthase and ATP synthase subunits were investigated by Western blot, immunohistochemistry, and ELISA in SAH mice and patients. Furthermore, the benefits of resveratrol-mediated Sirt5 activation were investigated. A total of 211 lysine succinylation sites were differentially expressed on 170 proteins in mice brain after SAH. Thirty-nine percent of these succinylated proteins were localized in mitochondria and they are related to energy metabolism. SAH caused a decrease of Sirt5 expression and succinylated citrate synthase as well as the subunits of ATP synthase, subsequently lowered brain pH, reduced ATP and increased reactive oxygen species production, leading to neuronal cell death, and neurological deficits. Knockdown of Sirt5 aggravated SAH-induced effects, mentioned above. Administration of resveratrol resulted in activation of Sirt5. The activation was accompanied both with restoration of the mitochondrial metabolism and alleviation of early brain injury as well as with desuccinylating citrate synthase and ATP synthase. Protein lysine succinylation is a biochemical hallmark of metabolic crisis after SAH, and disruption of lysine succinylation through activation of Sirt5 might be a promising therapeutic strategy for the treatment of SAH.

TAK1 mediates neuronal pyroptosis in early brain injury after subarachnoid hemorrhage

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.

Acute T2*-Weighted Magnetic Resonance Imaging Detectable Cerebral Thrombosis in a Rat Model of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is associated with a high incidence of morbidity and mortality, particularly within the first 72h after aneurysm rupture. We recently found ultra-early cerebral thrombosis, detectable on T2* magnetic resonance imaging (MRI), in a mouse SAH model at 4h after onset. The current study examined whether such changes also occur in rat at 24h after SAH, the vessels involved, whether the degree of thrombosis varied with SAH severity and brain injury, and if it differed between male and female rats. Adult Sprague Dawley rats were subjected to an endovascular perforation SAH model or sham surgery and underwent T2 and T2* MRI 24h later. Following SAH, increased numbers of T2* hypointense vessels were detected on MRI. The number of such vessels correlated with SAH severity, as assessed by MRI-based grading of bleeding. Histologically, thrombotic vessels were found on hematoxylin and eosin staining, had a single layer of smooth muscle cells on alpha-smooth muscle actin immunostaining, and had laminin 2α/fibrinogen double labeling, suggesting venule thrombosis underlies the T2*-positive vessels on MRI. Capillary thrombosis was also detected which may follow the venous thrombosis. In both male and female rats, the number of T2*-positive thrombotic vessels correlated with T2 lesion volume and neurological function, and the number of such vessels was significantly greater in female rats. In summary, this study identified cerebral venous thrombosis 24h following SAH in rats that could be detected with T2* MRI imaging and may contribute to SAH-induced brain injury.

Importance of CBF measurement to exclude concomitant cerebral infarction in the murine endovascular perforation SAH model

ObjectiveConcomitant cerebral infarction (CI) is could be a potential concern in experimental subarachnoid hemorrhage (SAH) induced by endovascular perforation. We propose a noninvasive method for excluding CI in a murine SAH model by using Laser speckle flow imaging (LSFI). MethodsAn SAH was induced with endovascular perforation (EVP) in male ddY mice. The cerebral blood flow (CBF) was quantitatively measured in the bilateral cerebral cortex was performed by using LSFI at five timepoints (preprocedure, immediately after, and 3 hours, 6 hours, and 24 hours after the procedure). The mice were then euthanized, and the SAH grade and volume of the CI were evaluated. The mice were divided into the SAH group and the SAH + CI group. Differences between the groups were assessed. ResultsForty-eight mice were used in this study. Six were the sham control group. Five SAH mice died within 24 hours after the procedure. A large CI on the ipsilateral side occurred in 15 (40.5%) mice (i.e., SAH + CI group). The remaining 22 (59.5%) mice were classified as the SAH group. The SAH grading score was not significantly different between the groups. The neurological score and CBF of the ipsilateral hemisphere were significantly higher in the SAH group than in the SAH + CI group (neurological score: 12.3 vs. 8, p < 0.01; CBF: 343.1 vs. 205.5; p < 0.01). The cut-off modified neurological score for excluding CI was 8 (area under the curve [AUC]: 0.77) and CBF at 24 hours after the procedure was 279.2 (AUC:0.856). ConclusionsUsing LSFI is less invasive and effectively excludes concomitant CI in experimental SAH. This methodological protocol may ad in improving the quality of the EVP-SAH model.

Negative Allosteric Modulator of mGluR1 Improves Long-Term Neurologic Deficits after Experimental Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) causes permanent neurological sequelae, but the underlying mechanism needs to be further clarified. Here, we show that inhibition of metabotropic glutamate receptor 1 (mGluR1) with negative allosteric modulator JNJ16259685 improves long-term neurobehavioral outcomes in an endovascular perforation model of SAH. JNJ16259685 improves cerebrovascular dysfunction through attenuation of cerebral blood flow (CBF) reduction, cerebral vasoconstrictio, and microthrombosis formation in a rat SAH model. Moreover, JNJ16259685 reduces experimental SAH-induced long-term neuronal damage through alleviation of neuronal death and degeneration. Mechanically, JNJ16259685 maintains phosphorylation of endothelial NO synthase (eNOS) and vasodilator-stimulated phosphoprotein (VASP) and decreases apoptosis-related factors Bax, active caspase-9, and active caspase-3 following experimental SAH. Altogether, our results suggest JNJ16259685 improves long-term functional impairment through neurovascular protection.

TT01001 attenuates oxidative stress and neuronal apoptosis by preventing mitoNEET-mediated mitochondrial dysfunction after subarachnoid hemorrhage in rats.

Oxidative stress and neuronal apoptosis are considered crucial therapeutic targets against early brain injury (EBI) after subarachnoid hemorrhage (SAH). Emerging evidence indicates that mitochondrial dysfunction is the main reason for oxidative stress and neuronal apoptosis. MitoNEET, an outer mitochondrial membrane protein, has been shown to regulate mitochondrial function. However, whether mitoNEET activation attenuates oxidative stress and neuronal apoptosis after SAH remains unknown. This study was therefore conducted to verify the neuroprotective role of mitoNEET in EBI after SAH in rats. A total of 93 rats were subjected to an endovascular perforation model of SAH. TT01001, a selective agonist of mitoNEET, was administered intraperitoneally 1 h after SAH induction. Neurological tests, immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, dihydroergotamine (DHE) staining, and western blot experiments were performed. The results showed that MitoNEET is expressed in neurons, but significantly decreased at 24 h after SAH induction. Activating mitoNEET with TT01001 significantly improved the neurological deficits, and reduced oxidative stress and neuronal apoptosis as measured by DHE and TUNEL staining, when compared with the SAH+vehicle group. Furthermore, TT01001 treatment decreased the expression of the proapoptotic marker, Bax, while increasing the expression of the antiapoptotic marker, Bcl-2. Together, our results suggested that mitoNEET activation with TT01001 reduced oxidative stress injury and neuronal apoptosis by improving mitochondrial dysfunction in EBI after SAH.

Naringenin reduces early brain injury in subarachnoid hemorrhage (SAH) mice: The role of the AMPK/SIRT3 signaling pathway

• Naringenin improved neurological function and alleviated brain edema after SAH. • Naringenin attenuate neuroinflammation after SAH. • Naringenin attenuated cell apoptosis after SAH. • AMPK/SIRT3 signaling pathway may be the underlying mechanisms. Naringenin exhibits multiple pharmaceutical effects, while the effects of naringenin on early brain injury (EBI) after subarachnoid hemorrhage (SAH) and its underlying mechanism have not been well-studied yet. Here, male C57BL/6 mice were utilized to the endovascular perforation model of SAH. Naringenin, 3-TYP (a selective SIRT3 inhibitor), Compound C (an AMPK inhibitor) were administered. Naringenin can attenuated neurological deficits and cerebral edema after SAH. Besides, naringenin reduced the number of Iba-1-positive and apoptotic cells. Moreover, naringenin decreased the expression of inflammatory cytokines. And naringenin upregulated the expression of phosphorylated AMPK (pAMPK) and SIRT3, which were abolished in mice with combined administration of naringenin and Compound C. However, administration of naringenin combined with 3-TYP did not enhance the expression of pAMPK, implying that AMPK is an upstream protein of SIRT3 in SAH under naringenin treatment. In summary, naringenin attenuated neuroinflammation and apoptosis after SAH. The potential mechanisms involved AMPK/SIRT3 signaling pathway.