脑淋巴引流阻滞加重蛛网膜下腔出血后大鼠皮层神经细胞的凋亡

本文旨在探讨在体脑淋巴引流阻滞(cerebral lymphatic blockage,CLB)对蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后大鼠皮层神经细胞凋亡的影响及机制。选用健康成年Wistar大鼠,随机分为正常对照组、SAH组、SAH+CLB组。采用枕大池二次注血法建立SAH模型。于第二次注血3d后,采用HE染色法及碘化丙啶(PI)染色法观察各组大鼠皮层神经细胞形态结构变化;TUNEL荧光标记法检测原位凋亡情况;免疫组织化学激光共聚焦检测大鼠皮层神经细胞Caspase-3和Bcl-2的蛋白表达。结果显示:(1)HE染色和PI染色可见SAH组大鼠部分皮层神经细胞皱缩,细胞核呈波纹状或折缝样,部分呈新月形;SAH+CLB组神经细胞分布稀疏,核碎裂,可见凋亡小体,周围有空泡形成;(2)SAH组和SAH+CLB组TUNEL阳性细胞的数量均高于正常对照组,而SAH+CLB组又显著高于SAH组;(3)SAH组和SAH+CLB组Caspase-3的表达量均高于正常对照组,而SAH+CLB组又显著高于SAH组;(4)SAH组和SAH+CLB组Bcl-2蛋白表达量均高于正常对照组,而SAH+CLB组显著低于SAH组。以上结果表明,CLB可以通过Caspase-3高表达和Bcl-2低表达加重SAH后大鼠皮层神经细胞的凋亡。     

Cerebral angiogenesis after subarachnoid hemorrhage (SAH) and endothelin receptor blockage with BQ-123 antagonist in rats.

The aim of the study was to determine the effect of chronic vasospasm after SAH on angiogenesis and the effect of endothelin-1, the main causative factor in vasospasm, on this process. Male Wistar rats, 220-250 g, were examined. Seven days after cannulation of the cisterna magna (CM), a 100 microl dose of non-heparinized blood was administered to induce SAH. Sham SAH (aSAH) was induced by intracisternal injection of 100 microl of artificial cerebrospinal fluid. Endothelin receptor antagonist BQ-123 in a dose of 40 nmol in 50 microl of cerebrospinal fluid was given three times: 20 min. before SAH and aSAH, 60 min and 24 hours after SAH and aSAH. The same pattern of BQ-123 administration was used in the nonSAH group. The brains were removed 48 hours later for histological evaluation. Vascular surface density was measured in cerebral hemisphere sections (at the level of the dorsal part of the hippocampus) and brain stem sections (1/2 of the pons). An increase in angiogenesis was observed after SAH, compared to control values. The administration of BQ-123, a specific endothelin receptor blocker inhibits angiogenesis in cerebral hemispheres after SAH.     

Cerebral aneurysm exclusion by CT angiography based on subarachnoid hemorrhage pattern: a retrospective study

Background  

To identify patients with spontaneous subarachnoid hemorrhage for whom CT angiography alone can exclude ruptured aneurysms.     

Role of autophagy in early brain injury after subarachnoid hemorrhage in rats

Early brain injury (EBI) occurred after aneurismal subarachnoid hemorrhage (SAH) strongly determined the patients’ prognosis. Autophagy was activated in neurons in the acute phase after SAH, while its role in EBI has not been examined. This study was designed to explore the effects of autophagy on EBI post-SAH in rats. A modified endovascular perforating SAH model was established under monitoring of intracranial pressure. Extent of autophagy was regulated by injecting autophagy-regulating drugs (3-methyladenine, wortmannin and rapamycin) 30 min pre-SAH intraventricularly. Simvastatin (20 mg/kg) was prophylactically orally given 14 days before SAH induction. Mortality, neurological scores, brain water content and blood–brain barrier (BBB) permeability were evaluated at 24 h post-SAH. Microtubule-associated protein light chain-3 (LC3 II/I) and beclin-1 were detected for monitoring of autophagy flux. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, expression of cleaved caspase-3 and cytoplasmic histone-associated DNA fragments were used to detect apoptosis. The results showed that mortality was reduced in rapamycin and simvastatin treated animals. When autophagy was inhibited by 3-methyladenine and wortmannin, the neurological scores were decreased, brain water content and BBB permeability were further aggravated and neuronal apoptosis was increased when compared with the SAH animals. Autophagy was further activated by rapamycin and simvastatin, and apoptosis was inhibited and EBI was ameliorated. The present results indicated that activation of autophagy decreased neuronal apoptosis and ameliorated EBI after SAH. Aiming at autophagy may be a potential effective target for preventing EBI after SAH.     

Impaired vascular responses of insulin-resistant rats after mild subarachnoid hemorrhage

Insulin resistance (IR) impairs cerebrovascular responses to several stimuli in Zucker obese (ZO) rats. However, cerebral artery responses after subarachnoid hemorrhage (SAH) have not been described in IR. We hypothesized that IR worsens vascular reactions after a mild SAH. Hemolyzed blood (300 μl) or saline was infused (10 μl/min) into the cisterna magna of 11-13-wk-old ZO (n = 25) and Zucker lean (ZL) rats (n = 25). One day later, dilator responses of the basilar artery (BA) and its side branch (BA-Br) to acetylcholine (ACh, 10(-6) M), cromakalim (10(-7) M, 10(-6) M), and sodium nitroprusside (10(-7) M) were recorded with intravital videomicroscopy. The baseline diameter of the BA was increased both in the ZO and ZL rats 24 h after the hemolysate injection. Saline-injected ZO animals showed reduced dilation to ACh (BA = 9 ± 3 vs. 22 ± 4%; and BA-Br = 23 ± 5 vs. 37 ± 7%) compared with ZL rats. Hemolysate injection blunted the response to ACh in both the ZO (BA = 4 ± 2%; and BA-Br = 12 ± 3%) and ZL (BA = 7 ± 2%; and BA-Br = 11 ± 3%) rats. Cromakalim (10(-6) M)-induced dilation was significantly reduced in the hemolysate-injected ZO animals compared with the saline control (BA = 13 ± 3 vs. 26 ± 5%; and BA-Br = 28 ± 8 vs. 44 ± 9%) and in the hemolysate-injected ZL rats compared with their saline control (BA = 24 ± 4 vs. 32 ± 4%; but not BA-Br = 39 ± 6 vs. 59 ± 9%). No significant difference in sodium nitroprusside reactivity was observed. Western blot analysis of the BA showed a lower baseline level of neuronal nitric oxide synthase expression and an enhanced cyclooxygenase-2 level in the hemolysate-injected ZO animals. In summary, cerebrovascular reactivity to both endothelium-dependent and -independent stimuli is severely compromised by SAH in IR animals.     

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, L-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas L-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.     

Potential role of CD34 in cerebral vasospasm after experimental subarachnoid hemorrhage in rats

Inflammatory responses have been implicated in the elaboration of several forms of central nervous system injury, including cerebral vasospasm after subarachnoid hemorrhage (SAH). A critical event participating in such responses is the recruitment of circulating leukocytes into the inflammatory site. CD34 is a key adhesion molecule responsible for recruitment of monocytes/macrophages and the attachment of leukocytes to endothelial cells. However, it has not been investigated whether, and to what degree, CD34 is induced by SAH and also the role of CD34 in the pathogenesis of cerebral vasospasm following SAH remains unknown. Experiment 1 aimed to investigate the timecourse of the CD34 expression in the basilar artery after SAH. In experiment 2, we chose the maximum time point of vasospasm (day 3) and assessed the effect of monoclonal antibody against CD34 on regulation of cerebral vasospasm. As a result, the elevated expression of CD34 was detected in the basilar artery after SAH and peaked on day 3. After intracisternal administration of CD34 monoclonal antibody, the vasospasm was markedly attenuated after blood injection on day 3. Our results suggest that CD34 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in a rat experimental model of SAH and administration of the specific CD34 antibody could prevent or reduce cerebral vasospasm caused by SAH.     

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.     

Enhanced myogenic tone in cerebral arteries from a rabbit model of subarachnoid hemorrhage

Cerebral artery vasospasm is a major cause of death and disability in patients experiencing subarachnoid hemorrhage (SAH). Currently, little is known regarding the impact of SAH on small diameter (100-200 microm) cerebral arteries, which play an important role in the autoregulation of cerebral blood flow. With the use of a rabbit SAH model and in vitro video microscopy, cerebral artery diameter was measured in response to elevations in intravascular pressure. Cerebral arteries from SAH animals constricted more (approximately twofold) to pressure within the physiological range of 60-100 mmHg compared with control or sham-operated animals. Pressure-induced constriction (myogenic tone) was also enhanced in arteries from control animals organ cultured in the presence of oxyhemoglobin, an effect independent of the vascular endothelium or nitric oxide synthesis. Finally, arteries from both control and SAH animals dilated as intravascular pressure was elevated above 140 mmHg. This study provides evidence for a role of oxyhemoglobin in impaired autoregulation (i.e., enhanced myogenic tone) in small diameter cerebral arteries during SAH. Furthermore, therapeutic strategies that improve clinical outcome in SAH patients (e.g., supraphysiological intravascular pressure) are effective in dilating small diameter cerebral arteries isolated from SAH animals.     

Inhibition of Rho kinase by hydroxyfasudil attenuates brain edema after subarachnoid hemorrhage in rats

The blood-brain barrier (BBB) disruption and brain edema are important pathophysiologies of early brain injury after subarachnoid hemorrhage (SAH). This study is to evaluate whether Rho kinase (Rock) enhances BBB permeability via disruption of tight junction proteins during early brain injury. Adult male rats were assigned to five groups; Sham-operated, SAH treated with saline, a Rock inhibitor hydroxyfasudil (HF) (10 mg/kg) treatment at 0.5 h after SAH, HF treatment at 0.5 and 6 h (10 mg/kg, each) after SAH, and another Rock inhibitor Y27632 (10 mg/kg) treatment at 0.5 h after SAH. The perforation model of SAH was performed and neurological score and brain water content were evaluated 24 and 72 h after surgery. Evans blue extravasation, Rock activity assay, and western blotting analyses were evaluated 24 h after surgery. Treatment of HF significantly improved neurological scores 24 h after SAH. Single treatment with HF and Y27632, and two treatments with HF reduced brain water content in the ipsilateral hemisphere. HF reduced Evans blue extravasation in the ipsilateral hemisphere after SAH. Rock activity increased 24 h after SAH, and HF reversed the activity. SAH significantly decreased the levels of tight junction proteins, occludin and zonula occludens-1 (ZO-1), and HF preserved the levels of occluding and ZO-1 in ipsilateral hemisphere. In conclusion, HF attenuated BBB permeability after SAH, possibly by protection of tight junction proteins.     

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

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

Role of hepcidin and its downstream proteins in early brain injury after experimental subarachnoid hemorrhage in rats

Early brain injury (EBI) is a major cause of mortality from subarachnoid hemorrhage (SAH). We aimed to study the pathophysiology of EBI and explore the role of hepcidin, a protein involved in iron homeostatic regulation, and its downstream proteins. One hundred and thirty-two male Sprague–Dawley rats were assigned into groups (n = 24/group): sham, SAH, SAH + hepcidin, SAH + hepcidin-targeting small interfering ribonucleic acid (siRNA), and SAH + scramble siRNA. Three hepcidin-targeting siRNAs and one scramble siRNA for hepcidin were injected 24 h before hemorrhage induction, and hepcidin protein was injected 30 min before induction. The rats were neurologically evaluated at 24 h and euthanized at 72 h. Hepcidin, ferroportin-1, and ceruloplasmin protein expression were measured by immunohistochemistry and Western blotting. Brain water content, blood–brain barrier (BBB) leakage, non-heme tissue iron and Garcia scale were evaluated. Hepcidin expression increased in the cerebral cortex and hippocampus after experimental SAH (P < 0.05 compared to sham), while ferroportin-1 and ceruloplasmin decreased (P < 0.05). Hepcidin injection lowered the expression of ferroportin-1 and ceruloplasmin further but siRNA reduced the levels of hepcidin (P < 0.05 compared to SAH) resulting in recovery of ferroportin-1 and ceruloplasmin levels. Apoptosis was increased in SAH rats compared to sham (P < 0.05) and increased slightly more by hepcidin, but decreased by siRNA (P < 0.05 compared to SAH). SAH rats had lower neurological scores, high brain water content, BBB permeability, and non-heme tissue iron (P < 0.05). In conclusion, downregulation of ferroportin-1 and ceruloplasmin caused by hepcidin enhanced iron-dependent oxidative damage and may be the potential mechanism of SAH.

     

Plasma visfatin,a possible prognostic marker in aneurysmal subarachnoid hemorrhage

Visfatin is linked to inflammation and associated with clinical outcomes of intracerebral hemorrhage. This study was designed to investigate whether visfatin might serve as a marker of severity and prognosis in aneurysmal subarachnoid hemorrhage. In this study, plasma visfatin levels of 172 consecutive patients and 172 sex and age-matched healthy subjects were determined using enzyme-linked immunosorbent assay. The recorded clinical outcomes included in-hospital mortality and 6-month mortality and unfavorable outcome (Glasgow Outcome Scale score of 1–3). Plasma visfatin level was substantially higher in patients than in healthy controls (92.1 ± 20.5 ng/mL vs. 12.4 ± 3.2 ng/mL; P < 0.001), was significantly associated with the World Federation of Neurological Surgeons (WFNS) score (r = 0.569, P < 0.001) and Fisher score (r = 0.657, P < 0.001), was an independent predictor of in-hospital mortality [odds ratio (OR), 1.378; 95% confidence interval (CI), 1.036–1.866; P = 0.002] and 6-month mortality (OR, 1.261; 95% CI, 1.018–1.745; P = 0.004) and unfavorable outcome (OR, 1.207; 95% CI, 1.012–1.682; P = 0.008) in multivariate logistic regression analysis and had high predictive value for in-hospital mortality [area under curve (AUC), 0.849; 95% CI, 0.787–0.899; P < 0.001] and 6-month mortality (AUC, 0.868; 95% CI, 0.808–0.915; P < 0.001) and unfavorable outcome (AUC, 0.859; 95% CI, 0.797–0.907; P < 0.001) using receiver operating characteristic curves. AUCs of visfatin were similar to those of WFNS score and Fisher score (all P > 0.05), but visfatin did not improve the predictive values of WFNS score and Fisher score (all P > 0.05). Thus, visfatin may be associated with clinical severity of aneurysmal subarachnoid hemorrhage and also have prognostic value for clinical outcomes.