Temporal changes of neurogenesis in the mouse hippocampus after experimental subarachnoid hemorrhage

Abstract

Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain. Increased neurogenesis has been shown following cerebral ischemia and traumatic brain injury; however, the involvement of neurogenesis in subarachnoid hemorrhage (SAH) has not been examined. Adult male CD-1 mice were subjected to SAH by endovascular perforation of the left anterior cerebral artery. Mice received intraperitoneal injections of the cell proliferation-specific marker 5 ′ -bromodeoxyuridine (BrdU) after SAH induction. BrdU incorporation was examined from 1 to 30 days after SAH by immunohistochemistry. The BrdU-positive cells were detected in SVZ and DG of normal control brain, and were significantly decreased in both areas three days after SAH. The number of these cells had recovered to its control level seven days after SAH. Double staining with BrdU and NeuN indicated that the majority of the BrdU-positive cells migrating into the granular cell layer of the DG became NeuN-positive 30 days after SAH. In conclusion, temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functional outcome after SAH. The induction of the neurogenesis can provide therapeutic value against SAH.     

Temporal changes of neurogenesis in the mouse hippocampus after experimental subarachnoid hemorrhage

Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain. Increased neurogenesis has been shown following cerebral ischemia and traumatic brain injury; however, the involvement of neurogenesis in subarachnoid hemorrhage (SAH) has not been examined. Adult male CD-1 mice were subjected to SAH by endovascular perforation of the left anterior cerebral artery. Mice received intraperitoneal injections of the cell proliferation-specific marker 5'-bromodeoxyuridine (BrdU) after SAH induction. BrdU incorporation was examined from 1 to 30 days after SAH by immunohistochemistry. The BrdU-positive cells were detected in SVZ and DG of normal control brain, and were significantly decreased in both areas three days after SAH. The number of these cells had recovered to its control level seven days after SAH. Double staining with BrdU and NeuN indicated that the majority of the BrdU-positive cells migrating into the granular cell layer of the DG became NeuN-positive 30 days after SAH. In conclusion, temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functional outcome after SAH. The induction of the neurogenesis can provide therapeutic value against SAH.     

Type V phosphodiesterase expression in cerebral arteries with vasospasm after subarachnoid hemorrhage in a canine model

Abstract

Cyclic GMP (cGMP) mediates smooth muscle relaxation in the central nervous system. In subarachnoid hemorrhage (SAH), decreases in intrinsic nitric oxide (NO) cause cerebral vasospasms due to the regulation of cGMP formation by NO-mediated pathways. As phosphodiesterase type V (PDE V) selectively hydrolyzes cGMP, we hypothesized that PDE V may function in the initiation of vasospasm. This study sought to identify the altered PDE V expression and activity in the vasospastic artery in a canine SAH model. We also used this system to examine possible therapeutic strategies to prevent vasospasm. Using a canine model of SAH, we induced cerebral vasospasm in the basilar artery (BA). Following angiographic confirmation of vasospasm on day 7, PDE V expression was immunohistochemically identified in smooth muscle cells of the vasospastic BA but not in cells of a control artery. The isolation of PDE enzymes using a sepharose column confirmed increased PDE V activity in the vasospastic artery only through both inhibition studies, using the highly selective PDE V inhibitor, sildenafil citrate, and Western blotting. Preliminary in vivo experiment using an oral PDE V inhibitor at 0.83 mg kg^-1 demonstrated partial relaxation of the spastic BA. PDE V activity was increased from control levels within the BA seven days after SAH. PDE V expression was most prominent in smooth muscle cells following SAH. These results suggest that clinical administration of a PDE V inhibitor may be a useful therapeutic tool in the prevention of vasospasm following SAH. [Neurol Res 2002; 24: 607-612]     

Improved rat model for cerebral vasospasm studies

Abstract

While the rat has been used extensively in subarachnoid hemorrhage (SAH)-cerebral vasospasm studies, concerns exist whether this animal represents a usable model because its time course and pattern of cerebral vasospasm following SAH is not comparable to that observed in man. At present, our knowledge of the rat model is based almost exclusively on studies using a 'single hemorrhage' method. Since there is a positive correlation between severity of cerebral vasospasm, and volume of subarachnoid blood, an obvious question is whether the rat will show modifications in vascular responses when insulted by a second SAH. Here, an SAH was produced in rats using a 'double hemorrhage' method. Following SAH, cerebral arteries showed pathological alterations, significant decreases in luminal perimeter, and increases in arterial wall thickness, over a 7-day post-SAH period. The above vascular features are considered to be indicative of cerebral vasospasm and their presence over a 7-day post-SAH period represents a significant time extension when compared to a single hemorrhage. These modified vascular responses made the double hemorrhaged rat a much-improved animal model. [Neurol Res 2001; 23: 761-766]     

Dopamine D2-Receptor-Mediated Increase in Vascular and Endothelial NOS Activity Ameliorates Cerebral Vasospasm After Subarachnoid Hemorrhage In Vitro

Introduction  Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a serious complication resulting in delayed neurological deficit, increased morbidity, mortality, longer hospital stays, and rehabilitation time. It afflicts approximately 35 per 100,000 Americans per year, and there is currently no effective therapy. We present in vitro data suggesting that increasing intrinsic nitric oxide relaxation pathways in vascular smooth muscle via dopaminergic agonism ameliorates cerebral vasospasm after SAH. Methods  Cerebrospinal fluid (CSF) from patients with cerebral vasospasm after SAH (CSF_V) was used to induce vasospasm in porcine carotid artery in vitro. Dopamine was added to test its ability to reverse spasm, and specific dopamine receptor antagonists were used to determine which receptor mediated the protection. Immunohistochemical techniques confirmed the presence of dopamine receptor subtypes and the involvement of NOS in the mechanism of dopamine protection. Results  Dopamine receptor 1, 2, and 3 subtypes are all present in porcine carotid artery. Dopamine significantly reversed spasm in vitro (67% relaxation), and this relaxation was prevented by Haloperidol, a D₂R antagonist (10% relaxation, P < 0.05), but not by D₁ or D₃-receptor antagonism. Both eNOS and iNOS expression were increased significantly in response to CSF_V alone, and this was significantly enhanced by addition of dopamine, and blocked by Haloperidol. Conclusion  Cerebral vasospasm is significantly reversed in a functional measure of vasospasm in vitro by dopamine, via a D₂R-mediated pathway. The increase in NOS protein seen in both the endothelium and vascular smooth muscle in response to CSF_V is enhanced by dopamine, also in a D₂R-dependent mechanism.     

Imatinib mesylate prevents cerebral vasospasm after subarachnoid hemorrhage via inhibiting tenascin-C expression in rats

Platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the mechanism remains unknown. The purpose of this study was to assess whether imatinib mesylate (imatinib), an inhibitor of the tyrosine kinases of PDGF receptors (PDGFRs), prevents cerebral vasospasm after SAH in rats, and to elucidate if tenascin-C (TNC), a matricellular protein, is involved in the mechanism. Imatinib (10 or 50 mg/kg body weight) was administered intraperitoneally to rats undergoing SAH by endovascular perforation, and the effects were evaluated by neurobehavioral tests and India-ink angiography at 24-72 h post-SAH. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms in cerebral arteries at 24h post-SAH. Recombinant TNC was administered intracisternally to imatinib-treated SAH rats, and the effects were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 h post-SAH. Both dosages of imatinib significantly prevented post-SAH neurological impairments and vasospasm at 24-72 h. SAH caused PDGFR-β upregulation, PDGFR activation, mitogen-activated protein kinase activation, and TNC upregulation in the spastic cerebral arteries, all of which were significantly suppressed by imatinib treatment. Recombinant TNC reversed the anti-vasospastic effects and protein expression changes by imatinib. This study suggests that imatinib prevents cerebral vasospasm at least partly via inhibiting the upregulation of TNC, implying that TNC may be a new therapeutic target for post-SAH vasospasm.     

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.     

Simvastatin attenuation of cerebral vasospasm after subarachnoid hemorrhage in rats via increased phosphorylation of Akt and endothelial nitric oxide synthase

The mechanisms involved in simvastatin-mediated attenuation of cerebral vasospasm after subarachnoid hemorrhage (SAH) are unclear. We investigated the role of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway and endothelial nitric oxide synthase (eNOS) in the cerebral vasculature in statin-mediated attenuation of cerebral vasospasm using wortmannin, an irreversible pharmacological PI3K inhibitor, and a rat SAH endovascular perforation model. Simvastatin was administered intraperitoneally in two dosages (1 mg/kg and 20 mg/kg) at 0.5, 24, and 48 hr after SAH and histological parameters of ipsilateral intracranial carotid artery (ICA) were assessed at 24 and 72 hr. SAH significantly decreased ICA diameter and perimeter while increasing wall thickness at both 24 and 72 hr. High-dosage simvastatin prevented the reduction of ICA diameter and perimeter following SAH, whereas both high and low dosages reduced wall thickness significantly at 24 and 72 hr. The effects of simvastatin were significantly reversed by wortmannin. High-dosage simvastatin increased pAkt and peNOS (phosphorylated forms) levels without increasing Akt and eNOS expression compared with the SAH group and also improved neurological deficits at 24 and 72 hr. Simvastatin did not affect protein levels by itself compared with untreated sham group. The present study elucidates the critical role of the PI3K activation leading to phosphorylation of Akt and eNOS in simvastatin-mediated attenuation of cerebral vasospasm after SAH.     

脑血管痉挛血管壁损害的发病机制研究

迟发性脑血管痉挛是蛛网膜下隙出血的一种常见并发症。目前,其确切发生机制还不完全清楚。近年来,一些学者发现,蛛网膜下隙出血后血管壁发生了器质性病理变化,伴随蛛网膜下隙出血的免疫炎性反应、血管壁细胞增殖与凋亡导致的血管壁的损害可能是脑血管痉挛发生的关键病理途径。本文就其研究的相关进展进行总结。     

Impact of Systemic Inflammatory Response Syndrome on Vasospasm,Cerebral Infarction,and Outcome After Subarachnoid Hemorrhage: Exploratory Analysis of CONSCIOUS-1 Database

Background  

Systemic inflammatory response syndrome (SIRS) may develop after aneurysmal subarachnoid hemorrhage (SAH). We investigated factors associated with SIRS after SAH, whether SIRS was associated with complications of SAH such as vasospasm, cerebral infarction, and clinical outcome, and whether SIRS could contribute to a difference in outcome between patients treated by endovascular coiling or neurosurgical clipping of the ruptured aneurysm.     

Type V phosphodiesterase expression in cerebral arteries with vasospasm after subarachnoid hemorrhage in a canine model

Cyclic GMP (cGMP) mediates smooth muscle relaxation in the central nervous system. In subarachnoid hemorrhage (SAH), decreases in intrinsic nitric oxide (NO) cause cerebral vasospasms due to the regulation of cGMP formation by NO-mediated pathways. As phosphodiesterase type V (PDE V) selectively hydrolyzes cGMP, we hypothesized that PDE V may function in the initiation of vasospasm. This study sought to identify the altered PDE V expression and activity in the vasospastic artery in a canine SAH model. We also used this system to examine possible therapeutic strategies to prevent vasospasm. Using a canine model of SAH, we induced cerebral vasospasm in the basilar artery (BA). Following angiographic confirmation of vasospasm on day 7, PDE V expression was immunohistochemically identified in smooth muscle cells of the vasospastic BA but not in cells of a control artery. The isolation of PDE enzymes using a sepharose column confirmed increased PDE V activity in the vasospastic artery only through both inhibition studies, using the highly selective PDE V inhibitor, sildenafil citrate, and Western blotting. Preliminary in vivo experiment using an oral PDE V inhibitor at 0.83 mg kg(-1) demonstrated partial relaxation of the spastic BA. PDE V activity was increased from control levels within the BA seven days after SAH. PDE V expression was most prominent in smooth muscle cells following SAH. These results suggest that clinical administration of a PDE V inhibitor may be a useful therapeutic tool in the prevention of vasospasm following SAH.     

Expression of vascular endothelial growth factor (VEGF) in rat brain after subarachnoid haemorrhage and endothelin receptor blockage with BQ-123

Cerebral vasospasm is one of the most severe complications of subarachnoid haemorrhage (SAH), leading to pathological changes in the vessel wall itself and in the nervous tissue, due to ischaemia of endothelial cells and neurones. Amongst the known substances inducing vasospasm, the most potent spasmogenic effect is exerted by endothelin-1 (ET1). The constriction of cerebral arteries and obliteration of capillaries highly stimulates the secretion of growth factors by endothelial cells and induces compensatory formation of collateral circulation in response to brain ischaemia. Expression of vascular endothelial growth factor (VEGF), the main factor responsible for angiogenesis and vascular permeability, was found to be increased in hypoxic cells (irrespective of the cause of hypoxia) as well as in neoplastic cells in the brain. The aim of the study was to determine whether chronic vasospasm and hypoxia of endothelial cells stimulate expression of VEGF, and whether blockage of the endothelin receptor ET(A) reduces this expression. The SAH was induced experimentally in male Wistar rats and the ET(A) receptor antagonist--BQ-123 was administered into the cisterna magna. After 48 hours the brain was removed and expression of VEGF studied immunohistochemically on paraffin sections. We found that hypoxia of endothelial cells, induced by chronic vasospasm after SAH, caused increased expression of VEGF in brain vessels and neurones of the cerebral hemispheres, brain stem and cerebellum. After administration of the endothelin receptor antagonist BQ-123, no changes in VEGF expression in the brain were found.     

血管内一氧化氮合酶基因转染预防脑血管痉挛的实验研究

目的采用血管内基因转染的方法,将重组内皮型一氧化氮合酶(eNOS)基因转染入蛛网膜下腔出血(SAH)后迟发性脑血管痉挛大鼠脑动脉,探讨防治SAH后迟发性脑血管痉挛的新方法。方法首先构建携带eNOS基因的重组腺病毒。采用小脑延髓池二次注血法建立大鼠SAH后迟发性脑血管痉挛模型。通过颈动脉微泵持续滴注方法进行基因转染,并设置对照组。结果第7天采用免疫组化证实重组eNOS基因表达,重组eNOS主要表达于内皮层。第7天显微镜下测定血管内eNOS转染组脑动脉环平均直径较单纯SAH组增大,电镜观察血管痉挛较单纯SAH组减轻。结论通过本研究证实采用颈动脉微泵持续滴注方法可在大鼠脑动脉表达重组eNOS,重组基因主要表达于动脉内皮细胞,可达到缓解SAH后迟发性脑血管痉挛的目的。     

Sexual dimorphism in gene expression after aneurysmal subarachnoid hemorrhage

Background and Purpose: Inflammation and compromise in structure and function of cerebral parenchymal microvasculature begins early after subarachnoid hemorrhage (SAH). We recently found greater inflammation and greater vascular compromise in male than in female rats following SAH. In this study, we investigated whether this cross-sexual difference in pathology is reflected in expression levels of genes related to vascular inflammation and structural compromise.

Method: Age-matched male and female rats underwent sham surgery or SAH by endovascular perforation. Early physiology (intracranial pressure (ICP), blood pressure (BP), heart rate, and cerebral blood flow) was monitored. Cerebral RNA was extracted at sacrifice 3 h after surgery and assayed for expression of thrombomodulin (Thbd), endothelial nitric oxide synthase (eNos;Nos3), intracellular adhesion molecule-1 (Icam1), vascular endothelial growth factor (Vegf), interleukin-1beta (Il1β) tumor necrosis factor-alpha (Tnf-α), and arginine vasopressin (Avp).

Results: Increases in ICP and BP at SAH appeared slightly greater in males but the difference did not reach statistical difference, indicating that SAH intensity did not differ significantly between the sexes. Of the seven genes studied two; Tnf-α and Vegf, did not change after injury, while the remainder showed significant responses to SAH. Response of Nos3 and Thbd was markedly different between the sexes, with expression greater in males.

Conclusion: This study finds that sexual dimorphism is present in the response of some but not all genes to SAH. Since products of genes exhibiting sexual dimorphism have anti-inflammatory activities, our results indicate that previously found sex-based differences in vascular pathology are paralleled by sexually dimorphic changes in gene expression following SAH.     

Mechanism of cerebral vasospasm following subarachnoid hemorrhage in monkeys.

This paper reviews our recent studies on the mechanism of cerebral vasospasm following subarachnoid hemorrhage (SAH) in monkeys. Middle cerebral artery (MCA) vasospasm was maximal at 7 days, resolving by 14 days, and absent at 28 days after SAH. Arterial fibrosis was not detected during vasospasm, although there was intimal hyperplasia with fibrosis 28 days after SAH. On scanning electron microscopy, smooth muscle cells from vasospastic arteries had corrugated cell membranes and appeared similar to cells contracted pharmacologically, suggesting that vasospastic smooth muscle is contracted. Morphometric analysis of arteries obtained 7 days after SAH showed no significant increases in arterial wall area of vasospastic arteries compared with normal MCAs. The results suggest vasospasm in monkeys is not due to hypertrophy, hyperplasia, or fibrosis in the arterial wall. Vasospasm may be mainly vascular smooth muscle contraction, which damages the arterial wall, leading to secondary structural changes in the arterial wall which occur after angiographic vasospasm.     

Search for Genetic Variants in the Ryanodine Receptor 1 Gene in Patients with Symptomatic Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage

Background  

Cerebral vasospasm is one of the most serious complications after subarachnoid hemorrhage (SAH). The cerebral artery diameter is regulated by complex physiological mechanisms. Among them the regulation of intracellular calcium homeostasis seems to play a crucial role. Recent data suggest that ryanodine receptors (RYRs) are involved in regulating the luminal calcium concentration in vascular smooth muscle cells. In this gene association investigation, we studied the question as to whether variants in the gene for the ryanodine receptors subtype 1 (RYR1) are associated with symptomatic cerebral vasospasm following SAH.