Early Stage Of Subarachnoid Hemorrhage Research Articles

Protective effects of histone deacetylase 6 specific inhibitor tubastatin A on subarachnoid hemorrhage in rats and the underlying mechanisms.

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

Dynamic Evolution of the Glymphatic System at the Early Stages of Subarachnoid Hemorrhage.

The early stages of subarachnoid hemorrhage (SAH) are extremely important for the progression and prognosis of this disease. The glymphatic system (GS) has positive implications for the nervous system due to its ability to clearance tau and amyloid-β (Aβ) protein. Previous studies have shown that GS dysfunction will appear after SAH. However, there is no systematic evaluation of the degree of damage and development process of GS function in the early stage after SAH. In this study, we evaluated the GS function and neurobehavioral in the sham, 6 h, 1, 3, and 7 days after SAH, respectively. Our results showed that the function of GS was severely attenuated in mice after SAH with a decreased polarity of Aquaporin-4 (AQP4), increased expression of AQP4, a linear correlation with the dystrophin-associated complex (DAC), the proliferation of reactive astrocytes, increased tau protein accumulation, and decreased neurological function. Collectively, these findings provide a comprehensive understanding of the functional changes of GS after SAH, provide references for subsequent scholars studying SAH, and suggest some potential mechanistic insight that affects AQP4 polarity and GS function.

Acetyl CoA synthase 2 potentiates ATG5-induced autophagy against neuronal apoptosis after subarachnoid hemorrhage.

ATG5-induced autophagy is triggered in the early stages after SAH, which plays a vital role in subarachnoid hemorrhage (SAH). Acyl-CoA synthetase short-chain family 2 (ACSS2) is not just involved in energy metabolism but also binds to TEFB to form a complex translocated to related autophagy genes to regulate the expression of autophagy-related genes. However, the contribution of ACSS2 to the activation of autophagy in early brain injury (EBI) after SAH has barely been discussed. The purpose of this study was to investigate the alterations of ACSS2 and its neuroprotective effects following SAH. We first evaluated the expression of ACSS2 at different time points (6, 12, 24, and 72 h after SAH) in vivo and primary cortical neurons stimulated by oxyhemoglobin (OxyHb). Subsequently, adeno-associated virus and lentivirus were used to regulate ACSS2 expression to investigate the effect of ACSS2 after SAH. The results showed that the ACSS2 level decreased significantly in the early stages of SAH and was minimized at 24 h post-SAH. After artificial intervention to overexpress ACSS2, ATG5-induced autophagy was further enhanced in EBI after SAH, and neuronal apoptosis was alleviated to protect brain injury. In addition, brain edema and neurological function scores were improved. These results suggest that ACSS2 plays an important role in the neuroprotection against EBI after SAH by increasing ATG5-induce autophagy and inhibiting apoptosis.

Inhibition of the myeloid differentiation primary response protein 88 reducres neuron injury in the early stages of subarachnoid hemorrhage in an in vitro experimental model.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease with high rates of morbidity and mortality and a paucity of effective therapies. The development of early brain injury (EBI) is closely related to prognosis in SAH, and inflammation plays an important role in its pathophysiology. A previous experiment showed that ST2825, a selective inhibitor of MyD88, could alleviate EBI in vivo. However, this protective effect in vivo is affected by a variety of pathophysiology processes making the result to some extent uncertain. whether there is a coincident result in vitro ruling out the effect of other factors remains unknown, and further investigation using cultured neurons is necessary. Primary neuronal cells were cultured to construct an in vitro model of SAH. The cells were cultured and then divided into three groups: (1) a blank control group, (2) an oxygenated hemoglobin + vehicle group, and (3) an oxygenated hemoglobin + ST2825 group. In each group, apoptosis of neuronal cells along with changes in the expression of proteins including MyD88, p-JNK, p-Erk, p-p38, NFκB, Bcl-2, and P53 were measured. Results showed that after stimulating neurons with oxygenated hemoglobin, the expression of the MyD88 protein in the vehicle group increased significantly. The quantity of p-JNK, p-p38, and p-Erk also increased significantly, as did the quantity of p65 in the nucleus. Expression of the anti-apoptotic protein Bcl-2 was markedly reduced, while that of the cleaved caspase-3 protein was significantly increased. In addition, in this group, the apoptosis rate of neurons was significantly increased. In the ST2825 group, the expression of p-JNK, p-p38, p-Erk, cleaved caspase-3, and p65 in the nucleus was significantly decreased, the expression of Bcl-2 was significantly increased, and the apoptosis rate of neurons was significantly reduced. The results of this study suggest that in an experimental in vitro SAH model, ST2825, a selective inhibitor of MyD88, can have a neuroprotective effect by inhibiting neuronal apoptosis mediated by the MAPK and NFκB signaling pathways, and this has a certain protective effect on EBI after SAH.

Early Microcirculatory Hemodynamic Changes Are Correlated With Functional Outcomes at Discharge in Patients With Aneurysmal SAH.

PurposeThe technique of color-coding blood flow analysis was used to explore the correlation between the microcirculatory hemodynamic changes on digital subtraction angiography (DSA) images in patients with aneurysmal subarachnoid hemorrhage (SAH) at the early stage and functional outcomes at discharge.MethodsData of 119 patients who underwent DSA examination due to SAH were retrospectively analyzed. The following hemodynamic parameters of the four region of interests (ROIs) [an ophthalmic segment of the internal carotid artery (ICA), frontal and parietal lobe, and superior sagittal sinus] were analyzed: the time-to-peak (TTP), the area under the curve (AUC), the full width at half maximum (FWHM), mean transit time (MTT), and circulation time. Multifactor regression analysis was performed to explore the correlation between the hemodynamic parameters and functional outcomes in patients at discharge.ResultsOf 119 patients with SAH, good and poor outcomes were found in 83 (69.7%) and 36 (30.3%) patients, respectively. The hemodynamic parameters including the FWHM, relative TTP (rTTP), and circulation time were significantly correlated with the Hunt–Hess grade (p < 0.005, p = 0.03, and p < 0.005) and the World Federation of Neurological Societies Scale grade (p < 0.005, p = 0.02, and p = 0.01). The FWHM was significantly prolonged with the increase of modified Fisher grade (p = 0.02). The multifactor analysis showed that the FWHM [odds ratio (OR) 17.56, 95% CI: 1.13–272.03, p = 0.04] was an independent risk factor predicting the functional outcomes in patients at discharge.ConclusionThe technique of color-coding blood flow analysis could be suitable for the qualified evaluation of disease conditions at an early stage of SAH as well as the prediction of outcomes.

Transcriptome Analysis of Microglia Reveals That the TLR2/IRF7 Signaling Axis Mediates Neuroinflammation After Subarachnoid Hemorrhage.

Microglia-mediated neuroinflammatory response in the early brain injury after subarachnoid hemorrhage (SAH) has been reported to have an impact on progress, and the mechanism is not completely understood. Here, we performed genome-wide transcriptome analysis of microglia purified from damaged hemisphere of adult mice at 3 days after SAH or sham operation. Robust transcriptional changes were observed between SAH-induced and healthy microglia, indicating rapid activation of microglia after suffering from SAH. We identified 1576 differentially expressed genes (DEGs; 928 upregulated and 648 downregulated) in SAH-induced microglia compared with sham microglia, representing a strong alteration of the genome (6.85% of total ∼23,000 genes). Functional enrichment of these DEGs indicated that cell division, inflammatory response, cytokine production, and leukocyte chemotaxis were strongly activated in SAH-induced microglia. Moreover, we identified and proved that the TLR2/IRF7 signaling axis was involved in the regulation of this microglia-mediated inflammation in SAH mice by performing flow cytometry and immunofluorescence. Together, these results provided a perspective of microglia-mediated neuroinflammatory response in the early stage of SAH and might give a new therapeutic target for SAH.

Peroxisomal Dysfunction Contributes to White Matter Injury Following Subarachnoid Hemorrhage in Rats via Thioredoxin-Interacting Protein-Dependent Manner.

Background and PurposeWhite matter injury (WMI) exists in the early stage of subarachnoid hemorrhage (SAH) and has not been well addressed so far.MethodsWe utilized short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR) to verify the role of peroxisomes in WMI following SAH. We evaluated short- and long-term neurobehavior after SAH. Western blotting, immunofluorescence, and Golgi staining techniques were performed to assess the changes in protein levels.ResultsCatalase (CAT) CRISPR treatment significantly attenuated neurological deficits and reduced long-term spatial learning and memory impairments after SAH by increasing the level of myelin basic protein (MBP) while decreasing the levels of amyloid precursor protein (APP), interleukin 6 (IL-6), and tumor necrosis factor (TNF)-α. The use of thioredoxin-interacting protein (TXNIP) shRNA significantly offset the effects of CAT shRNA, and the use of glycerone phosphate acyl transferase (GNPAT) shRNA significantly reversed the effects of CAT CRISPR by decreasing the levels of plasmalogens and reactive oxidative species (ROS).ConclusionPeroxisomal dysfunction induced by SAH reversely exacerbated cerebral WMI following SAH, which was at least partly mediated by TXNIP and GNPAT pathways.

A Case Series of Double Bypass Technique Used for the Treatment of Internal Carotid Blood Blister-like Aneurysms in Patients in Poor Initial Neurological Condition at the Early Stage of Subarachnoid Hemorrhage.

Optimal surgical treatment of blood-blister aneurysms (BBA) remains controversial. Some surgeons prefer clipping reconstruction while others favor primary bypass with trapping. To analyze of benefit of double bypass technique to surgical outcomes in patients with ruptured BBA in poor initial neurological condition (PINC). Retrospective analysis of clinical, radiological, and surgical data in 9 patients treated between January 2009 and December 2015. Intraoperative middle cerebral artery blood pressure (MCABP) measurement was used for selection of bypass procedure. Seven patients presented with World Federation of Neurosurgical Societies (WFNS) subarachnoid hemorrhage (SAH) score 4 or 5. No pulse pressure in the MCA after internal carotid artery (ICA) clamping was found in 3 patients, although their ACoA or PCoA were well visualized on preoperative angiograms. By contrast, only a minimal drop in MCABP following ICA clamping was detected in two cases, although their collaterals were slim/nonvisualized on imaging. Although angiographic vasospasms were not observed in our patients, two of them experienced cerebral infarction, attributable more to the mass effect and postoperative ICA thrombosis than to SAH-induced vasospasm. There were 2 premature intraoperative ruptures, but no perioperative mortality, aneurysm recurrence, or rebleeding. Five patients with WFNS SAH score 4 or 5 achieved favorable outcomes. Early double bypass technique guided by MCABP measurement and combined with trapping (or rarely clipping) seems to be a safe method with excellent long-term outcomes in patients in PINC. This study may thus contribute to the debate on the optimal treatment strategy for BBA.

Mitoquinone attenuates blood-brain barrier disruption through Nrf2/PHB2/OPA1 pathway after subarachnoid hemorrhage in rats

Background and purposeMitochondrial dysfunction is involved in the mechanism of early brain injury (EBI) following subarachnoid hemorrhage (SAH). Blood-brain barrier disruption is a devastating outcome in the early stage of SAH. In this study, we aimed to investigate the role of a mitochondria-related drug Mitoquinone (MitoQ) in blood-brain barrier disruption after SAH in rats. MethodsA total of 181 male Sprague–Dawley SAH rats with the endovascular perforation model were utilized. Intraperitoneal MitoQ was given 1 h (h) post-SAH. Cerebroventricular ML385, an inhibitor of NF-E2-related factor 2 (Nrf2) and Small interfering ribonucleic acid (siRNA) for Prohibitin 2 (PHB2) were injected respectively 24 h and 48 h before SAH. Neurological function evaluation was performed before sacrifice. SAH grade was measured during the sacrifice of each animal. Brain water content was performed at 24 h. Co-immunoprecipitation was used to demonstrate the relationship of proteins Nrf2 and PHB2. Mitochondrial and cytoplasmic fractions were gathered using mitochondria isolation kits. Pathway related proteins were investigated with Western blot and immunofluorescence staining. Transmission electron microscopy was performed for mitochondrial morphology. ResultsExpression of Nrf2 levels peaked at the 3 h time point following SAH and then decreased to normal levels at 24 h, while PHB2 and Optic Atrophy 1 (OPA1) decreased at 24 h and 72 h after SAH compared with the Sham group. MitoQ treatment attenuated neurological deficits and brain edema, thereby resulting in a decreased expression of Albumin, while an increase of Nrf2, PHB2, OPA1 and Claudin-5 proteins compared with SAH + vehicle group. With co-immunoprecipitation, Nrf2 and PHB2 were further demonstrated to show their interaction. And MitoQ administration lead to more binding of the two proteins. ML385 abolished the effects of MitoQ on neurobehavior and protein levels post-SAH. Similarly, PHB2 siRNA reversed the neuroprotection of MitoQ administration with the decreased expression of PHB2 and OPA1 after SAH. Further, MitoQ treatment improved mitochondrial morphology after SAH with an increase of PHB2 and OPA1 in mitochondrial extraction. ConclusionsMitoQ attenuates blood-brain barrier disruption via Nrf2/PHB2/OPA1 pathway after SAH in rats. MitoQ may serve as a potential therapeutic strategy for SAH patients.

Vascular endothelial growth factor A promotes platelet adhesion to collagen IV and causes early brain injury after subarachnoid hemorrhage.

The role of vascular endothelial growth factor A in platelet adhesion in cerebral microvessels in the early stage of subarachnoid hemorrhage remains unclear. In this study, the endovascular puncture method was used to produce a rat model of subarachnoid hemorrhage. Then, 30 minutes later, vascular endothelial growth factor A antagonist anti-vascular endothelial growth factor receptor 2 antibody, 10 μg, was injected into the right ventricle. Immunohistochemistry and western blot assay were used to assess expression of vascular endothelial growth factor A, occludin and claudin-5. Immunohistochemical double labeling was conducted to examine co-expression of GP Ia-II integrin and type IV collagen. TUNEL was used to detect apoptosis in the hippocampus. Neurological score was used to assess behavioral performance. After subarachnoid hemorrhage, the expression of vascular endothelial growth factor A increased in the hippocampus, while occludin and claudin-5 expression levels decreased. Co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells increased, whereas behavioral performance was markedly impaired. After treatment with anti-vascular endothelial growth factor receptor 2 antibody, occludin and claudin-5 expression recovered, while co-expression of GP Ia-II integrin and type IV collagen and the number of apoptotic cells decreased. Furthermore, behavioral performance improved notably. Our findings suggest that increased vascular endothelial growth factor A levels promote platelet adhesion and contribute to early brain injury after subarachnoid hemorrhage. This study was approved by the Biomedical Ethics Committee, Medical College of Xi'an Jiaotong University, China in December 2015.

Early Administration of Hypertonic-Hyperoncotic Hydroxyethyl Starch (HyperHES) Improves Cerebral Blood Flow and Outcome After Experimental Subarachnoid Hemorrhage in Rats

Early cytotoxic brain edema may be a decisive factor that maintains cerebral malperfusion after subarachnoid hemorrhage (SAH). In addition, endothelial cell swelling may be an independent factor restricting cerebral blood flow (CBF) in a very early stage after SAH. Immediate and aggressive treatment may be able to restore CBF in this critical period. Male Sprague-Dawley rats were subjected to SAH by the endovascular filament model and treated by a bolus of hyperoncotic-hypertonic hydroxyethyl starch (4mL/kg body weight) immediately after vessel perforation and 150 minutes later (n= 12) or by the same amount of normal saline (n= 9). Mean arterial blood pressure, intracranial pressure, and local CBF over both hemispheres were continuously measured by laser-Doppler flowmetry. Neurologic assessment was performed 24 hours later. Hippocampal damage was assessed by hematoxylin-eosin and Caspase-3 staining. Arterial blood gases and mean arterial blood pressure were not significantly different between the 2 groups. Intracranial pressure was significantly reduced in the treatment group (P < 0.05). Local CBF was significantly improved in the treatment group over both hemispheres (P< 0.05; 180 minutes after treatment, P < 0.01). There was a trend to better neurologic performance in the treatment group. The rate of injured neurons was significantly reduced in animals of the treatment group compared with controls (P < 0.01). The number of Caspase-3-positive neurons in the hippocampal CA1 field was not reduced. In this study, the effects of very early and repeated treatment with a high-dose hyperoncotic-hypertonic hydroxyethyl starch were investigated. The results of this series show that this therapy can be highly effective to improve CBF and attenuate hippocampal cell damage in the early stage of SAH. Whether delayed cell death could be treated by longer therapy cannot be answered by this study. Because no differential diagnosis of the clinical suspicion of SAH prohibits the administration of hypertonic-hyperoncotic solutions, it may be useful as a first-tier preclinical therapy in suspected SAH and could even be used by emergency rescue services before the patient is admitted to a hospital.

In vivo observation of cerebral microcirculation after experimental subarachnoid hemorrhage in mice

Acute brain injury caused by subarachnoid hemorrhage is the major cause of poor prognosis. The pathology of subarachnoid hemorrhage likely involves major morphological changes in the microcirculation. However, previous studies primarily used fixed tissue or delayed injury models. Therefore, in the present study, we used in vivo imaging to observe the dynamic changes in cerebral microcirculation after subarachnoid hemorrhage. Subarachnoid hemorrhage was induced by perforation of the bifurcation of the middle cerebral and anterior cerebral arteries in male C57/BL6 mice. The diameter of pial arterioles and venules was measured by in vivo fluorescence microscopy at different time points within 180 minutes after subarachnoid hemorrhage. Cerebral blood flow was examined and leukocyte adhesion/albumin extravasation was determined at different time points before and after subarachnoid hemorrhage. Cerebral pial microcirculation was abnormal and cerebral blood flow was reduced after subarachnoid hemorrhage. Acute vasoconstriction occurred predominantly in the arterioles instead of the venules. A progressive increase in the number of adherent leukocytes in venules and substantial albumin extravasation were observed between 10 and 180 minutes after subarachnoid hemorrhage. These results show that major changes in microcirculation occur in the early stage of subarachnoid hemorrhage. Our findings may promote the development of novel therapeutic strategies for the early treatment of subarachnoid hemorrhage.

Preclinical efficacy of human Albumin in subarachnoid hemorrhage

Human Albumin is a unique pleiotropic protein with multiple properties. Previous clinical and laboratory studies have indicated a possible beneficial effect of Albumin in subarachnoid hemorrhage (SAH). The present study aimed to further define the preclinical characteristics of Albumin. SAH was induced by endovascular perforation in Sprague–Dawley rats. In the dose-escalation study, Albumin ranging from 0.02g/kg to 1.0g/kg was intravenously infused immediately after SAH. In the therapeutic window study, 1.0g/kg Albumin was administered at 0h, 2h, 4h or 8h after SAH. Physiologic variables were monitored in different Albumin treatment regimens. One day after SAH, neurological scores, SAH scores, blood–brain barrier permeability, neural degeneration and apoptosis were examined. The efficacy of Albumin for SAH was also determined in female rats and in spontaneously hypertensive rats. We found that 0.2g/kg and 1.0g/kg Albumin significantly attenuated sensorimotor deficits, brain edema, IgG leakage, and neuronal degeneration after SAH. The benefits of Albumin existed even when the administration was delayed to 4h after SAH onset. No significant difference was found between 0.2g/kg and 1.0g/kg Albumin groups. In female rats and spontaneously hypertensive rats, Albumin likewise improved neurological outcomes and early brain injury. In conclusion, Albumin could reduce both cerebral lesions and functional deficits in the early stage of SAH. The beneficial regimen occurs within a favorable therapeutic window and is reproducible in different high-risk subjects.

Sirtuin 1 activation protects against early brain injury after experimental subarachnoid hemorrhage in rats.

Increasing evidence indicates that sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions, such as oxidative stress, inflammation and apoptosis. The aim of this study was to investigate the change of SIRT1 in the brain after subarachnoid hemorrhage (SAH) and its role on SAH-induced early brain injury (EBI). In the first set of experiments, rats were randomly divided into sham group and SAH groups at 2, 6, 12, 24, 48 and 72 h. The expression of SIRT1 was evaluated by western blot analysis, immunohistochemistry and immunofluorescence. In another set of experiments, SIRT1-specific inhibitor (sirtinol) and activator (activator 3) were exploited to study the role of SIRT1 in SAH-induced EBI. It showed that the protein level of SIRT1 was markedly elevated at the early stage of SAH and peaked at 24 h after SAH. The expression of SIRT1 could be observed in neurons and microglia, and the enhanced SIRT1 was mainly located in neurons after SAH. Administration of sirtinol inhibited the expression and activation of SIRT1 pathways after SAH, while activator 3 enhanced the expression and activation of SIRT1 pathways after SAH. In addition, inhibition of SIRT1 could exacerbate forkhead transcription factors of the O class-, nuclear factor-kappa B- and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis, leading to aggravated brain injury after SAH. In contrast, activator 3 treatment could reduce forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative damage, neuroinflammation and neuronal apoptosis to protect against EBI. These results suggest that SIRT1 plays an important role in neuroprotection against EBI after SAH by deacetylation and subsequent inhibition of forkhead transcription factors of the O class-, nuclear factor-kappa B-, and p53-induced oxidative, inflammatory and apoptotic pathways. SIRT1 might be a new promising molecular target for SAH.

Hyperbaric Oxygen Intervention Modulates Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats: Possible Involvement of TLR4/NF-κ B-Mediated Signaling Pathway

Background/Aims: Previous studies have proved that the activation of TLR4/NF-κ B signaling pathway is involved in inflammatory processes in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Hyperbaric oxygen (HBO) intervention has successfully been used to treat several animal models of tissue injury via its anti-inflammation property. This study was undertaken to investigate the influence of HBO administration on the TLR4/NF-κ B signaling pathway in rats at the early stage of SAH. Methods: Male Sprague-Dawley rats (n = 150) were randomly divided into 5 groups: the sham, the sham + 2.8 atmospheres absolute (ATA) HBO group, the SAH group, the SAH + 2.0ATA HBO group, the SAH + 2.8ATA HBO group. Each group (n = 30) was randomly subdivided into three subgroups that were examined at the following time points: 24 h, 48 h and 72 h post-injury. HBO (100% O2, 2.0ATA or 2.8ATA for 90mins) was initiated 12 h after injury. Neurological deficit, brain edema and blood-brain barrier (BBB) permeability were assessed to evaluate the development of EBI. The expressions of TLR4, NF-κ B and pro-inflammatory cytokines in the cortical were determined by real time polymerase chain reaction (RT-PCR), western blot, immunohistochemistry, or enzyme-linked immunosorbent assay (ELISA). Results: Our study showed that treatment with HBO significantly decreased the expressions of TLR4, NF-κ B and the downstream inflammatory agents, such as TNF-α, IL-6, IL-1β and ICAM-1, and also improved brain edema, blood-brain barrier permeability and neurologic function. Conclusions: These findings indicate that HBO treatment may result in abatement of the development of EBI after SAH, possibly through suppression of TLR4/NF-κ B signaling pathway.

Expression of Cytoplasmic Gelsolin in Rat Brain After Experimental Subarachnoid Hemorrhage.

Convincing evidence indicates that apoptosis contributes to the unfavorable prognosis of subarachnoid hemorrhage (SAH), a significant cause of morbidity and case fatality throughout the world. Gelsolin (GSN) is a Ca(2+)-dependent actin filament severing, capping, and nucleating protein, as well as multifunctional regulator of cell structure and metabolism, including apoptosis. In the present study, we intended to investigate the expression pattern and cell distribution of GSN in rat brain after experimental SAH. GSN expression was examined in sham group and at 3, 6, 12 h, day 1 (1 day), 2, 3, 5, and 7 days after SAH by Western blot analysis as well as real-time polymerase chain reaction. Immunohistochemistry and immunofluorescence were performed to detect the localization of GSN. The level of GSN protein expression was significantly decreased in SAH group and reached a bottoming point on 1 day after SAH. GSN mRNA level was significantly decreased in SAH groups in comparison with the sham group, and reached a minimum value at 12 h after SAH. Immunohistochemistry showed that GSN was constitutively and obviously expressed in the cortex of the normal rat brain and significantly decreased in the rat cortex after SAH. In addition, immunofluorescence results revealed that GSN expression could be found in both neurons and microglias, as well as in glialfibrillary acidic protein-positive astrocytes. The decreased expression of GSN could mainly be found in neurons and astrocytes as well, and GSN-positive microglias showed different cell morphological characteristics. Interestingly, the protein and gene levels of GSN seemed to be constant in the rat hippocampus of sham and SAH groups. These findings suggested a potential role of GSN in the pathophysiology of the brain at the early stage of SAH.

Role of the AMPK signaling pathway in early brain injury after subarachnoid hemorrhage in rats.

AMP-activated protein kinase (AMPK) is a key metabolic and stress sensor/effector. Few investigations have been performed to study the role of AMPK in subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was undertaken to investigate the time course of AMPK activation in the early stage of SAH and to evaluate the influence of AICAR (which is known to mimic AMP and activates AMPK) and compound C (a commonly used AMPK inhibitor) on EBI in rats following SAH. Adult male rats were divided into six groups: control, sham, SAH, SAH + vehicle, SAH + AICAR and SAH + compound C. SAHs were induced by a modified endovascular perforation method. Immunohistochemistry, real-time PCR and Western blot were used to detect the spatial and dynamic expression of AMPK after SAH. Cortical apoptosis and the expressions of apoptosis-related proteins such as FOXO3a (forkhead box, class O, 3a) and Bim (Bcl-2-interacting mediator of cell death) were detected after different drug interventions. We found SAH induced prolonged activation of AMPK. Treatment with AICAR markedly induced overactivation of AMPK and upregulation of FOXO3a and Bim. AICAR also significantly exacerbated cerebral apoptosis and neurological impairment following SAH. On the other hand, pre-administration of compound C attenuated EBI in this SAH model by modulating cerebral apoptosis by inhibiting FOXO3a and Bim. Our findings suggest that the AMPK pathway may play an important role in SAH-induced neuronal apoptosis, and the use of AMPK inhibitors can provide neuroprotection in EBI after SAH.

Alterations of Voltage-Dependent Calcium Channel Currents in Basilar Artery Smooth Muscle Cells at Early Stage of Subarachnoid Hemorrhage in a Rabbit Model

ObjectiveTo investigate the changes in the currents of voltage-dependent calcium channels (VDCCs) in smooth muscle cells of basilar artery in a rabbit model of subarachnoid hemorrhage (SAH).MethodsNew Zealand white rabbits were randomly divided into five groups: sham (C), normal (N), 24 hours (S1), 48 hours (S2) and 72 hours (S3) after SAH. Non-heparinized autologous arterial blood (1ml/kg) was injected into the cisterna magna to create SAH after intravenous anesthesia, and 1 ml/kg of saline was injected into cisterna magna in the sham group. Rabbits in group N received no injections. Basilar artery in S1, S2, S3 group were isolated at 24, 48, 72 hours after SAH. Basilar artery in group C was isolated at 72 hours after physiological saline injection. Basilar artery smooth muscle cells were isolated for all groups. Whole-cell patch-clamp technique was utilized to record cell membrane capacitance and VDCCs currents. The VDCCs antagonist nifedipine was added to the bath solution to block the Ca++ channels currents.ResultsThere were no significant differences in the number of cells isolated, the cell size and membrane capacitance among all the five groups. VDCC currents in the S1–S3 groups had higher amplitudes than those in control and sham groups. The significant change of current amplitude was observed at 72 hours after SAH, which was higher than those of 24 and 48 hours. The VDCCs were shown to expression in human artery smooth muscle cells.ConclusionsThe changes of activation characteristics and voltage-current relationship at 72 hours after SAH might be an important event which leads to a series of molecular events in the microenvironment of the basilar artery smooth muscle cells. This may be the key time point for potential therapeutic intervention against subarachnoid hemorrhage.

The role of the sonic hedgehog signaling pathway in early brain injury after experimental subarachnoid hemorrhage in rats

Previous studies have demonstrated that the sonic hedgehog (Shh) pathway plays a neuro-protective role. However, whether the Shh pathway is induced by subarachnoid hemorrhage (SAH) has not been investigated. We sought to investigate Shh activation in the cortex in the early stage of SAH, and assessed the effect of cyclopamine (a specific inhibitor of the Shh pathway) on Shh pathway regulation and evaluated the impact of cyclopamine on SAH. We found that the Shh pathway was up-regulated in the cortex after SAH, and that blocking the Shh pathway increased cell apoptosis. Early brain damages, including brain edema, blood-brain barrier impairment, and cortical apoptosis were significantly aggravated following with cyclopamine treatment compared with vehicle treatment. Our results suggest that the Shh pathway should be activated in the brain after SAH, and plays a beneficial role in SAH development, possibly by inhibiting cerebral oxidative stress through induction of antioxidant and detoxifying enzymes.

Multicenter Prospective Cohort Study on Volume Management After Subarachnoid Hemorrhage

Systemic circulation management has not been established for patients with poor grade aneurysmal subarachnoid hemorrhage (SAH) or delayed cerebral ischemia (DCI) after SAH. The aims of the study were to examine hemodynamic variables in these patients and to establish treatment strategies. A multicenter prospective cohort study of hemodynamic variables from days 1 to 14 was performed using a transpulmonary thermodilution system (PiCCO Plus). Parameters were analyzed by Mann-Whitney test. Multivariate analysis was performed to identify parameters involved in onset of DCI. The subjects were 204 patients, including 138 with poor grade SAH (World Federation of Neurological Surgeons grades IV and V) and 52 who developed DCI. The extravascular lung water index, pulmonary vascular permeability index, and systemic vascular resistance index were significantly greater in patients with poor grade SAH compared with those with good grade SAH (World Federation of Neurological Surgeons I-III) on day 2 (P=0.049, P=0.039, and P=0.038). Cardiac index was significantly lower in patients with poor grade SAH on days 1 and 2 (P=0.027 and P=0.011). In patients with DCI, the global end-diastolic volume index was significantly lower than in those without DCI on days 3 to 5 (P=0.0053; P=0.048; and P=0.048). In multivariate analysis, median global end-diastolic volume index, cardiac index, and systemic vascular resistance index at an early stage of SAH (days 3-6) were independently related to onset of DCI (P=0.023, P=0.013, and P=0.003). Patients with poor grade SAH developed heart failure-like afterload mismatch at an early stage, and those with DCI had decreased global end-diastolic volume index (hypovolemia) in the early stage of SAH. http://www.clinicaltrials.gov. Unique identifier: UMIN000003794.