Calcitonin-gene related peptide and cerebral vasospasm

The pathophysiology of arterial vasospasm following subarachnoid hemorrhage (SAH) is poorly understood and the contribution of endogenous neuropeptides has not been sufficiently elucidated. Recently, we detected an excessive release of vasoconstrictive neuropeptide Y (NPY) in SAH patients and identified a significant correlation of NPY cerebrospinal fluid (CSF) levels with vasospasm-related ischemia. Here, we present the results of an experimental study on the possible role of the potent endogenous vasodilator calcitonin-gene related peptide (CGRP) in the acute stage of SAH. Twelve consecutive patients with SAH were included. Seven patients had severe arterial vasospasm, confirmed by transcranial doppler-sonography (TCD). Prospectively, CSF was collected from day 1 to day 10 after onset of the SAH. The levels of CGRP were determined in a competitive enzyme immunoassay and were correlated with the clinical course and hemodynamic changes. A cohort of 29 patients without CNS disease served as a control. CGRP was significantly higher in SAH patients compared with the control group (p < 0.05). From day 1 to day 4, the CGRP levels in patients without vasospasm were significantly higher than the levels of CGRP in patients with vasospasm (p < 0.05). These patients did not develop cerebral ischemia. The significantly increased levels of the CGRP during the first days after onset of the SAH in the non-vasospasm group indicate a potential protective role of CGRP. CGRP may alleviate arterial vasoconstriction and thus protect the brain from vasospasm and subsequent ischemia.     

Intracisternal administration of SB203580, a p38 mitogen-activated protein kinase inhibitor,attenuates cerebral vasospasm via inhibition of tumor-necrosis factor-α

Tumor-necrosis factor-α (TNF-α) is critical to the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Hence, therapeutic strategies targeting TNF-α can attenuate cerebral vasospasm. This study investigated the effects of SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, on TNF-α concentration in the cerebral arteries and the cerebrospinal fluid (CSF) after SAH and on subsequent cerebral vasospasm. Twenty-three rabbits were divided into four groups: (i) control (without SAH), (ii) SAH (SAH only), (iii) dimethylsulfoxide (DMSO, vehicle), and (iv) SB203580. The severity of vasospasm and the immunoreactivities of TNF-α and phosphorylated p38 MAPK in the brain vessels were determined in all animals, and the concentrations of TNF-α in the CSF were also assessed. Severe vasospasm was observed in the rabbits from the SAH and DMSO groups. SB203580 reversed vasospasm after SAH. Lower immunoreactivities of TNF-α and phosphorylated p38 MAPK were found in the basilar artery in the SB203580 group than in the DMSO group. The concentration of TNF-α in the CSF increased after SAH, but treatment with SB203080 after SAH suppressed this increase. Our data show that SB203580 reversed cerebral vasospasm by inhibiting the phosphorylation of p38 MAPK in the basilar artery and by suppressing the increase in TNF-α in the basilar artery and CSF after SAH. SB203580 could therefore potentially be used for the treatment of cerebral vasospasm after SAH.     

动脉瘤性蛛网膜下腔出血患者脑脊液中一氧化氮浓度的动态变化

目的探讨蛛网膜下腔出血后脑脊液中一氧化氮浓度的动态变化及其与脑血管痉挛的关系。方法采集57例动脉瘤性蛛网膜下腔出血(aSAH)患者脑脊液标本(采集时间为入院后即刻,出血后第3、5、7、10、14天),采用镉粒还原法检测脑脊液中NO浓度。结果出血后第3天脑脊液中NO浓度即有明显降低(P<0.05),在出血后第7～10天达到最低(P<0.01),而后逐渐升高。症状性脑血管痉挛患者NO浓度明显低于未痉挛者及无症状的脑血管痉挛患者。结论症状性脑血管痉挛的发生与脑脊液中NO浓度降低有一定相关性。     

The expression of tissue factor and tissue factor pathway inhibitor in aortic smooth muscle cells is up-regulated in synthetic compared to contractile phenotype

Tissue factor (TF) and its specific inhibitor TF pathway inhibitor (TFPI) are produced by vascular smooth muscle cells (SMCs) in vitro and are increased in vivo in atherosclerotic compared to normal vessels. Besides local regulation of the hemostatic balance, this may be related to non-hemostatic TF/protease dependent functions such as SMC proliferation, adhesion and migration. The aim of the study was to compare the expression of both proteins between the contractile (normal adult) and synthetic (neo-intimal) SMC phenotypes. Primary cultures of SMCs isolated from rat thoracic aorta before and 10 days after balloon injury displayed stable characteristics of the contractile and synthetic phenotype, respectively. Synthetic SMCs expressed more TF mRNA than contractile SMCs, but released excess TF in the conditioned medium, so that the cell-associated TF activity measured by a factor Xa generating assay remained similar in the two subtypes. Accordingly, cell surface thrombogenicity measured under blood flow conditions was also similar. The production and release of functional TFPI was enhanced by a factor 3 to 6 (p < 0.01) in synthetic SMCs. A difference in the quantitative expression of TF and TFPI is a new distinctive feature of SMC phenotypes. Matrix-associated TFPI derived from synthetic SMCs may serve as an anchorage for their migration and regulate protease-activated processes during neo-intima formation.     

亚低温治疗对蛛网膜下腔出血继发性血管痉挛及脑脊液和血浆内皮素、降钙素基因相关肽的影响

目的 探讨亚低温治疗对蛛网膜下腔出血(SAH)继发性血管痉挛及脑脊液和血浆内皮素(ET)、降钙素基因相关肽(CGRP)水平的影响.方法 56例SAH患者随机分成亚低温组和对照组,两组在常规治疗的基础上,亚低温组增加局部亚低温治疗;检测两组入院时及治疗7 d、14 d脑脊液和血浆ET、CGRP水平,并比较两组脑血管痉挛的发病情况.结果 (1)脑脊液、血浆ET水平治疗7 d时亚低温组较对照组显著降低(均P＜0.05);14 d时差异更显著(均P＜0.01);两组CGRP水平治疗第7 d时降至最低,后渐升高,亚低温组较对照组变化幅度小,差异有统计学意义(P＜0.05～0.01).(2)亚低温组脑血管痉挛发病率为6.67%,较对照组的30.77%明显减少(P＜0.05).结论 亚低温治疗减少了SAH患者脑脊液和血浆中ET水平上升幅度及CGRP水平下降幅度,从而降低脑血管痉挛的发生率.     

Bilirubin production and oxidation in CSF of patients with cerebral vasospasm after subarachnoid hemorrhage.

Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) remains a significant cause of mortality and morbidity; however, the etiology is, as yet, unknown, despite intensive research efforts. Research in this laboratory indicates that bilirubin and oxidative stress may be responsible by leading to formation of bilirubin oxidation products (BOXes), so we investigated changes in bilirubin concentration and oxidative stress in vitro, and in cerebral spinal fluid (CSF) from SAH patients. Non-SAH CSF, a source of heme oxygenase I (HO-1), and blood were incubated, and in vitro bilirubin production measured. Cerebrospinal fluid from SAH patients was collected, categorized using stimulation of vascular smooth muscle metabolism in vitro, and information obtained regarding occurrence of vasospasm in the patients. Cerebral spinal fluid was analyzed for hemoglobin, total protein and bilirubin, BOXes, malonyldialdehyde and peroxidized lipids (indicators of an oxidizing environment), and HO-1 concentration. The formation of bilirubin in vitro requires that CSF is present, as well as whole, non-anti-coagulated blood. Bilirubin, BOXes, HO-1, and peroxidized lipid content were significantly higher in CSF from SAH patients with vasospasm, compared with nonvasospasm SAH CSF, and correlated with occurrence of vasospasm. We conclude that vasospasm may be more likely in patients with elevated BOXes. The conditions necessary for the formation of BOXes are indeed present in CSF from SAH patients with vasospasm, but not CSF from SAH patients without vasospasm.     

蛛网膜下腔出血后脑血管痉挛与血管活性物质的关系

目的 探讨蛛网膜下腔出血后脑血管痉挛与血管活性物质的关系。方法 测定52例蛛网膜下腔出血患者血浆血栓素B_2(thromboxane B_2,TXB_2)、6-酮-前列环素F1α(6-keto-prostaglandin,6-Keto)及血浆和脑脊液中血管紧张素转化酶(angiotensin converting enzyme,ACE)活性,同时通过经颅多普勒超声检查法(transcranial Doppler nltrasonography,TCD)观察蛛网膜下腔出血后脑血管平均血流速度(Vm)的变化,并与正常对照组进行比较。结果 蛛网膜下腔出血患者血浆TXB_2水平高于对照组(P<0.01),6-Keto水平低于对照组(P<0.05或P<0.01);发病后血浆ACE活性无明显改变,但脑脊液中ACE活性高于对照组(P<0.01);大脑中动脉平均血流速度于蛛网膜下腔出血后第4～5d的变化最为显著(P<0.01)。结论 蛛网膜下腔出血后患者血浆TXB_2、6-Keto与脑脊液ACE水平均发生明显变化,结合TCD检测结果,推测蛛网膜下腔出血后脑血管痉挛与血管活性物质水平有关。     

蛛网膜下腔出血腰池持续引流前后脑脊液中NO浓度的变化

目的 探讨腰池持续引流防治脑血管痉挛的效果及其对脑脊液中一氧化氮（NO）浓度的影响。方法 50例破裂动脉瘤蛛网膜下腔出血患，随机分为引流组25例。对照组25例。引流组在电解可脱弹簧圈（GDC）栓塞术后立即实施腰池持续引流，对照组行间断腰椎穿刺。脑脊液NO浓度采用镉粒子还原法测定。结果 引流组中发生症状性脑血管痉挛2例。对照组7例。血管痉挛患的NO浓度明显降低，引流组脑脊液中NO浓度在出血后第5d起明显高于对照组。结论 腰池持续引流可有效清除蛛网膜下腔积血，提高脑脊液中NO的浓度。     

Effectiveness of papaverine cisternal irrigation for cerebral vasospasm after aneurysmal subarachnoid hemorrhage and measurement of biomarkers

Cisternal irrigation with thrombolytic agents was used to prevent post-SAH vasospasm, but its role remained inconclusive. To verify effectiveness of papaverine (PPV) in preventing vasospasm, we studied relationship between inflammatory biologic markers and vasospasm. This prospective study included 121 patients with clipped anterior circulation aneurysms that had ruptured, and 372 control patients. Patients were divided into three groups according to cisternal irrigation method: simple drain, papaverine group, and urokinase (UK) group. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in CSF and serum on days 3 and 7 after SAH. The PPV group showed similar incidence of vasospasm with UK group, but lower incidence than the simple drain group. The levels of ICAM-1 and VCAM-1 were significantly higher in the SAH group than in the control group. CSF and serum levels were more elevated on day 7 than day 3, and the degree of elevation were more marked when measured in the CSF than in the serum. However, there was no statistical difference between measured levels of ICAM-1 and VCAM-1, and vasospasm development. PPV cisternal irrigation was similarly effective as UK at preventing vasospasm. Although neither PPV nor UK irrigation could reduce the concentration of adhesion molecules compared with simple drain, we found levels of ICAM-1 and VCAM-1 were specifically elevated in the CSF. Therefore, further research should focus on anti-inflammation as a therapeutic target against cerebral vasospasm and on the CSF as the optimum place where such inflammatory action practically brought about.     

Endothelin and aneurysmal subarachnoid haemorrhage: a study of subarachnoid cisternal cerebrospinal fluid.

Endothelin (ET) is considered one of the most potent vasoconstrictor polypeptides; several experimental studies have suggested its possible role in the pathogenesis of arterial vasospasm after subarachnoid haemorrhage (SAH). Previously reported data on plasma and CSF levels of endothelin in patients with a diagnosis of SAH have been controversial. Cisternal endothelin CSF levels and the possibility that they could be related to vasospasm and other clinical patterns of SAH were investigated. CSF samples were obtained from 55 patients admitted after angiographic diagnosis of intracranial aneurysm. Levels of ET-1 and ET-3 were measured through radio-immunoassay technique. Twelve patients who had operations for unruptured aneurysms were considered control cases; 43 patients with SAH were classified according to: Hunt and Hess grading at admission, vasospasm grading, CT classification and timing of surgery. In all 55 patients ET-1 was measured, while positive levels of ET-3 were found only in 17 cases of 48. No linear correlation was found between cisternal CSF ET-1 levels when considering time of surgery, CT classification, Hunt and Hess grading at admission, and vasospasm grading. The results of ET-3 assay should be considered with great caution because of the low percentage of positive cases. Cisternal CSF levels of ET-1 and ET-3 are not directly related to the occurrence of arterial vasospasm after the aneurysm rupture, or to other major clinical patterns of SAH; however, ET-1 expression occurs either in paraphysiological (unruptured aneurysm) or in pathological conditions (SAH). It is suggested that ET may potentiate, or may be potentiated by, other factors playing a consistent pathophysiological role in the development of vasospasm.     

Cerebrospinal fluid membrane-bound tissue factor and myelin basic protein in the course of vasospasm after subarachnoid hemorrhage.

No marker that predicts accurately the time of occurrence of cerebral vasospasm due to subarachnoid hemorrhage (SAH) has been reported. In the present study, membrane-bound tissue factor (mTF) and myelin basic protein (MBP) concentrations in cerebrospinal fluid (CSF) were evaluated as a predictor of the time of occurrence of cerebral vasospasm. The mTF and MBP concentrations were measured in the CSF from 28 patients with SAH due to ruptured aneurysm. Serial assays were performed from day 4 to day 14 after SAH. CSF mTF and MBP concentrations from days 5 to 9 correlated with the volume of cerebral infarction due to vasospasm and outcome three months after SAH. From the serial assays, CSF mTF measurements predicted the time of occurrence and severity and irreversibility of symptoms due to vasospasm. In conclusion, CSF mTF is predictive of the occurrence and the recovery of cerebral vasospasm, while CSF MBP is only an indicator of severity of brain damage due to vasospasm.     

侧脑室外引流及脑脊液净化治疗蛛网膜下腔出血的疗效观察

目的 探讨蛛网膜下腔出血的治疗方法,评价其治疗效果。方法 治疗组(28例)采用侧脑室外引流及脑脊液净化加常规内科治疗;对照组(30例)采用常规内科治疗。结果 治疗组无1例死亡,对照组死亡4例(13.3％)。治疗组急性脑积水与脑血管痉挛的发生率均为3.6％,对照组急性脑积水与脑血管痉挛的发生率分别为13.3％和20％。治疗组疗效明显优于对照组。结论 侧脑室外引流结合脑脊液净化是治疗蛛网膜下腔出血的有效方法。     

Nonenzymatic derived lipid peroxide, 8-iso-PGF2 alpha, participates in the pathogenesis of delayed cerebral vasospasm in a canine SAH model

We studied whether 8-iso-PGF2alpha, nonenzymatic arachidonyl peroxide, participated in the pathogenesis of delayed vasospasm using a canine subarachnoid hemorrhage (SAH) model. Fourteen adult mongrel dogs were divided into two groups, two-hemorrhage SAH group (n = 8) and control group (n = 6). The contents of 8-iso-PGF2alpha in CSF, the basilar artery segment, and subarachnoid clot were measured by enzyme immunoassay kit. The CSF 8-iso-PGF2alpha content on Day 7 in the SAH group was 67.9+/-29.9 pg ml(-1) (n = 8), which was significantly higher than 27.1+/-13.8 (n = 8) on Day 0 in the SAH group, and 33.2+/-14.4 pg ml(-1) (n = 5) on Day 7 in the control group. The 8-iso-PGF2alpha content in the basilar artery segment with spasm on Day 7 in the SAH group was 13.5+/-1.9 pg mg(-1) wet weight (n = 8), significantly higher than 8.7+/-1.9 (n = 6) in the control group. The 8-iso-PGF2alpha content in subarachnoid clot was 1.7+/-1.4 ng g(-1) wet weight (n = 8). Significant elevation of the 8-iso-PGF2alpha contents in the CSF and the basilar artery segment occurred on Day 7 in the SAH group. The subarachnoid clot enclosed the basilar artery on Day 7, contained a considerable amount of 8-iso-PGF2alpha. These results suggested that 8-iso-PGF2alpha could play a crucial role in the pathogenesis of the delayed cerebral vasospasm.     

Elevated concentrations of sphingosylphosphorylcholine in cerebrospinal fluid after subarachnoid hemorrhage: A possible role as a spasmogen

This study investigates the role of sphingosylphosphorylcholine (SPC) in the mechanisms underlying cerebral vasospasm after subarachnoid hemorrhage (SAH). The levels of SPC were measured in cerebrospinal fluid (CSF) of patients with SAH and also in an experimental canine model. CSF samples were collected from 11 patients with SAH, and from dogs that had received an injection of SPC into the cisterna magna to examine SPC kinetics in the CSF. SPC was assayed using solid-phase extraction and triple quadrupole mass spectrometry. The SPC concentrations in SAH patients on days 3, 8, and 14 after the onset of SAH were significantly higher than those in normal CSF. In the canine model, rapid dilution of SPC in CSF was observed. In combination with data from previous studies, these results suggest that SPC is involved in the development of cerebral vasospasm. Rapid dilution of SPC in CSF suggests that SPC is released into CSF at higher concentrations than those measured in the present study.     

Protease-activated receptor 2 (PAR2) mediates vascular smooth muscle cell migration induced by tissue factor/factor VIIa complex

Protease-activated receptor 2 (PAR2) is one of G-protein-coupled receptors able to be activated by trypsin and coagulation factor VIIa. We previously reported that tissue factor/factor VIIa (TF/FVIIa) complex was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). The migratory response was dependent on a catalytic activity of FVIIa, and did not involve factor Xa and thrombin generation. In this study, we examined TF/FVIIa-induced SMC migration. METHODS: The contribution of PAR2 to TF/FVIIa-induced vascular SMC migration was investigated using a modified Boyden's chamber method, and the distribution of PARs in the human coronary arteries and cultured SMCs was also examined. RESULTS: Trypsin and PAR2-activating peptide (AP; SLIGKV) stimulated SMC migration in a dose-dependent manner, of which abilities were comparable to those of TF/FVIIa complex and platelet-derived growth factor-BB, but PAR1-AP (TFLLR or SFLLR) or PAR4-AP (AYPGOV) did not elicit the migration. The antisera against PAR2-AP significantly inhibited TF/FVIIa-induced SMC migration, but that of PAR1-AP did not. In immunostaining, both intimal SMCs of the human coronary arteries and cultured SMCs showed positive reaction for PAR2-AP. CONCLUSION: These results suggest that PAR2 in SMCs plays a crucial role in the cell migration induced by TF/FVIIa complex.     

L-精氨酸枕大池注射对蛛网膜下腔出血脑血管痉挛的影响

目的:研究L-精氨酸枕大池注射对兔蛛网膜下腔出血后脑血管痉挛的影响及作用机制。方法:采用双侧颈动脉结扎及枕大池二次注血法制造兔蛛网膜下腔出血模型。在蛛血后第4天,以比色法测定血清及脑脊液中NO及脑组织NOS的含量。光镜下测定基底动脉的动脉壁厚度和基底动脉的内径,以其比值作为脑血管痉挛的指标。治疗组分为300μmol及500μmol组,在蛛血后第4天,枕大池持续微泵滴注L-精氨酸,在滴注后再分别测定上述指标。结果:蛛血后第4天,基底动脉的动脉壁厚度和内径的比值明显升高,血清及脑脊液中NO及脑组织NOS的含量降低。在L-精氨酸滴注后,脑血管痉挛缓解。血清及脑脊液中NO及脑组织NOS的含量较对照明显升高。结论:L-精氨酸枕大池注射对兔蛛网膜下腔出血后的血管痉挛具有治疗作用。L-精氨酸可能是通过NO及NOS系统起作用。