Effect of endothelin-1 receptor antagonist BQ-123 on basilar artery diameter after subarachnoid hemorrhage (SAH) in rats.

Aim of the study was to quantify cerebral vasospasm in rats after subarachnoid hemorrhage (SAH) by morphometric examination of basilar artery and to evaluate the influence of endothelin receptor blocker BQ-123 on basilar artery constriction. The rat cisterna magna (CM) was cannulated and after 7 days SAH was developed by administration of 100 microl autologic, non-heparinized blood to the CM. The sham subarachnoid hemorrhage was developed by intracisternal administration of 100 microl of artificial cerebrospinal fluid. Endothelin receptor blocker BQ-123 was injected into the CM in a dose of 40 nmol diluted in 50 microl of cerebrospinal fluid 20 min. before SAH, and 24h and 48 h after SAH. After perfusion fixation the brains were removed from the skull and histological preparations of basilar artery were done. The internal diameter and wall thickness of basilar arteries was measured by interactive morphometric method. The most severe vasospasm was found in rats after SAH. The presence of numerous infiltrations composed of neutrophils and macrophages correlated with advanced vasospasm (index of constriction 5 times lower than in normal), suggesting the role of other factors participating in the late phase of vasospasms after SAH. Administration of BQ-123 in the late phase after SAH caused the dilatation of basilar artery. Following the administration of BQ-123 in the late phase (48 h after SAH) the basilar artery dilated, its wall became thinner, and the number of leukocyte infiltrations in the subarachnoid space decreased compared to the values after SAH alone.     

Time course of production of hydroxyl free radical after subarachnoid hemorrhage in dogs

Vasospasm after subarachnoid hemorrhage (SAH) is associated with lipid peroxidation. However, lipid peroxides increase in a delayed fashion after SAH and may be a byproduct of but not a cause of vasospasm. This study correlated vasospasm with hydroxyl free radical and lipid peroxide levels. 24 dogs had baseline cerebral angiography and induction of SAH by 2 injections of blood into the cisterna magna at baseline and 2 days later. Angiography was repeated 4, 7, 10, 14 or 21 days after the first injection (n = 4 per group) and a microdialysis catheter was inserted into the premedullary cistern. Control dogs (n = 4) underwent angiography and microdialysis but not SAH. Salicylic acid, 100 mg/kg, was administered intravenously, and microdialysis fluid was collected and analyzed by high pressure liquid chromatography for 2,3- and 2,5-dihydroxybenzoic acids (DHBA). Malondialdehyde was measured in subarachnoid clot removed from the prepontine cistern and in the basilar artery itself at the time of euthanasia. Significant vasospasm developed 4 to 14 days after SAH. Malondialdehyde levels were significantly elevated in the basilar artery and subarachnoid clot 4 days after SAH (p < 0.0001, ANOVA) but not at other times. 2,5-DHBA levels were significantly greater than control at 4 to 14 days and they peaked at 4 days (p < 0.05, ANOVA). 2,3-DHBA was significantly increased at 4 days after SAH (p < 0.05, ANOVA). There were significant correlations between basilar artery malondialdehyde levels and vasospasm and cerebrospinal fluid 2,5-DHBA levels and vasospasm. These results suggest the presence of hydroxyl free radical after SAH and demonstrate a correlation between such production, as measured by trapping with salicylate, and the early phase of vasospasm. The correlation with vasospasm implicates free radicals and lipid peroxidation in this phase of vasospasm.     

A novel intravital method to evaluate cerebral vasospasm in rat models of subarachnoid hemorrhage: a study with synchrotron radiation angiography

Precise in vivo evaluation of cerebral vasospasm caused by subarachnoid hemorrhage has remained a critical but unsolved issue in experimental small animal models. In this study, we used synchrotron radiation angiography to study the vasospasm of anterior circulation arteries in two subarachnoid hemorrhage models in rats. Synchrotron radiation angiography, laser Doppler flowmetry-cerebral blood flow measurement, [(125)I]N-isopropyl-p-iodoamphetamine cerebral blood flow measurement and terminal examinations were applied to evaluate the changes of anterior circulation arteries in two subarachnoid hemorrhage models made by blood injection into cisterna magna and prechiasmatic cistern. Using synchrotron radiation angiography technique, we detected cerebral vasospasm in subarachnoid hemorrhage rats compared to the controls (p<0.05). We also identified two interesting findings: 1) both middle cerebral artery and anterior cerebral artery shrunk the most at day 3 after subarachnoid hemorrhage; 2) the diameter of anterior cerebral artery in the prechiasmatic cistern injection group was smaller than that in the cisterna magna injection group (p<0.05), but not for middle cerebral artery. We concluded that synchrotron radiation angiography provided a novel technique, which could directly evaluate cerebral vasospasm in small animal experimental subarachnoid hemorrhage models. The courses of vasospasm in these two injection models are similar; however, the model produced by prechiasmatic cistern injection is more suitable for study of anterior circulation vasospasm.     

Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be due, in part, to altered regulation of arterial smooth muscle contraction. Contraction of cerebral arteries to serotonin is augmented after experimental SAH. We hypothesized that activation of Rho-associated kinase (Rho kinase) contributes to augmented contraction of cerebral arteries to serotonin after SAH. Autologous arterial blood (SAH) or artificial cerebrospinal fluid (control) was injected into the cisterna magna of anesthetized rabbits. At 2 days after injection, the basilar artery was excised and isometric contraction of arterial rings was recorded. Maximum contraction of the basilar artery to serotonin was augmented about fourfold in SAH compared with control rabbits (P < 0.01). Contraction to histamine was similar in the two groups. Fasudil hydrochloride (3 mumol/l), an inhibitor of Rho kinase, markedly attenuated serotonin-induced contraction. Fasudil had little effect on contractions induced by histamine or phorbol 12,13-dibutyrate. In addition, phosphorylation of myosin phosphatase, a major target of Rho kinase in regulation of smooth muscle contraction, in the basilar artery was examined by Western blotting. In basilar arteries of SAH, but not control, rabbits, serotonin increased phosphorylation of myosin phosphatase about twofold at Thr(853) of the myosin-targeting subunit. These results suggest that enhanced activation of Rho kinase contributes to augmented contraction of the basilar artery to serotonin after SAH.     

Expression of Nemo-Like Kinase (NLK) in the Brain in a Rat Experimental Subarachnoid Hemorrhage Model

This study aimed to investigate the expression of the Nemo-like kinase (NLK) in the brain after experimental subarachnoid hemorrhage (SAH) in rats. A total of 90 rats were randomly divided into six groups: control group, day 1, day 3, day 5, day 7, and day 14. Day 1, day 3, day 5, day 7, and day 14 groups were all SAH groups in which the rats were killed on days 1, 3, 5, 7, and 14, respectively. In SAH groups, autologous arterial blood was injected into cisterna magna once on day 0. Cross-sectional area of basilar artery was measured by H&E staining. Immunostaining and immunoblotting experiments were performed to detect the expression of NLK protein. Real-time polymerase chain reaction was used to analyze the presence and quantity of NLK mRNA. The level of oxidative stress in the artery was also measured. The basilar arteries exhibited vasospasm after SAH and became the most severe on day 3. The expressions of NLK protein and mRNA were decreased remarkably in SAH groups compared with the control group. The down-regulated expression of NLK was detected after SAH and the low ebb was on day 3, which was oppositely the peak time of oxidative stress. The expression of NLK was present mainly in the neurons in the brain and smooth muscle cells in the basilar artery. NLK is decreasingly expressed in an opposite time-course to the development of cerebral vasospasm (CVS) and SAH-induced brain injury in this rat experimental model of SAH and these findings might have important implications during the administration of specific NLK agonist to prevent or reduce CVS or neuronal apoptosis caused by SAH.     

Prevention and reversal of vasospasm and ultrastructural changes in basilar artery by continuous infusion of CGS 35066 following subarachnoid hemorrhage

Endothelin-1, a potent vasoconstrictive peptide, has been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). The goal of this study was to evaluate the effect of continuous intravenous infusion of a highly selective endothelin-converting enzyme-1 inhibitor, CGS 35066, on the prevention and reversal of cerebral vasospasm following SAH. New Zealand white rabbits were subjected to SAH by injecting autologous arterial blood into the cisterna magna. Infusion of CGS 35066 at dosages of 1, 3, or 10 mg/kg/ day was initiated either 1 hr and 24 hrs later in the prevention and reversal protocols, respectively. Animals were sacrificed by perfusion-fixation 48 hrs after SAH induction. The cross-sectional areas of basilar arteries were measured using computer-assisted videomicroscopy. Ultrastructural changes in basilar arteries were determined using electron microscopy. CGS 35066 significantly prevented and reversed the arterial narrowing after SAH in all three groups. The mean cross-sectional areas of arteries from animals in both the prevention and reversal protocol groups that received 10 mg/kg/day of CGS 35066 did not differ significantly from those of the healthy controls. Histological studies of the basilar artery in the 10 mg/kg/day treatment group did not show pathomorphological changes, such as corrugation of the endothelium seen at 2 days after SAH induction or vacuole formation in the endothelial cells noted in the vehicle-treated SAH group. These findings suggest that CGS 35066 is a promising therapeutic agent for the prevention and reversal of cerebral vasospasm after SAH. It also prevents the pathological changes in vascular walls due to SAH.     

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

Objective

To 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).

Methods

New 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.

Results

There 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.

Conclusions

The 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.     

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。     

Dynamic Alterations of Cerebral Pial Microcirculation During Experimental Subarachnoid Hemorrhage

The study aimed to investigate the involvement of cerebral microcirculation turbulence after subarachnoid hemorrhage (SAH). Wistar rats were divided into non-SAH and SAH groups. Autologous arterial hemolysate was injected into rat’s cisterna magna to induce SAH. Changes of pial microcirculation within 2 h were observed. It was found that there were no obvious changes of the diameters, flow velocity, and fluid state of microvessels in non-SAH group. With the exception of rare linear-granular flow in A4 arteriole, linear flow was observed in most of the arterioles. There was no blood agglutination in any of the arterioles. After SAH, abnormal cerebral pial microcirculation was found. Spasm of microvessels, decreased blood flow, and agglutination of red blood cells occurred. Five minutes following the induction of SAH, the diameters of the arterioles and venules significantly decreased. The decreased diameters persisted for 2 h after cisternal injection. Decreased flow velocity of venules was found from 5 to 90 min after induction of SAH. Spasm of the basilar artery and increased brain malondialdehyde were also found after SAH. We concluded that cerebral microcirculation turbulence plays an important role in the development of secondary cerebral ischemia following SAH.     

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.     

Participation of Lipoxygenase Products from Arachidonic Acid in the Pathogenesis of Cerebral Vasospasm

To examine the possible involvement of lipoxygenase products from arachidonic acid in the pathogenesis of delayed vasospasm after subarachnoid hemorrhage (SAH), we measured the contents of hydroxyeicosatetraenoic acids (HETEs) in the subarachnoid clot, the cerebrospinal fluid, and the basilar artery, using the canine "two-hemorrhage" model. Lipoxygenase activity in the subarachnoid clot and the basilar artery was measured, ex vivo, using samples obtained 7 days after SAH. For a quantitative analysis of HETEs, each sample was homogenized with either ice-cold saline or methanol. The lipid extract was then submitted to reverse-phase HPLC. The identity of each HETE was further confirmed using straight-phase HPLC and gas chromatography-mass spectrometry. When the basilar artery was homogenized with ice-cold saline, a significant increase in the 5-HETE content was observed on SAH day 8. However, when the artery was homogenized with methanol, HETEs were not detected. In the case of incubation in the presence of arachidonic acid and calcium ionophore A23187, the 5-lipoxygenase activity was remarkably increased in the basilar artery exposed to SAH, compared to that of normal dogs. The subarachnoid clot contained a significant amount of 12-HETE (average 1.8 nmol/g wet weight) from day 2 to day 8. The administration of 1,2-bis(nicotinamido)propane significantly ameliorated vasospasm in the two-hemorrhage model, simultaneously inhibiting the 5-lipoxygenase activity of the basilar artery. Our observations show that the activities of 12- and 5-lipoxygenases are significantly increased after SAH in the subarachnoid clot and the basilar artery, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticotropin releasing hormone (CRH) increases beta-endorphin (beta-end like) concentration in cerebrospinal fluid of rats with vasospasm following subarachnoid hemorrhage.

The chronic stage of vasospasm occurring several days after subarachnoid hemorrhage (SAH) is characterized by the development of histopathologic changes in cerebral arteries causing cerebral ischemia. Numerous experimental data indicate the involvement of immune mechanisms in the angiopathy caused by SAH. Endogenous opioids play also an important role in the ischemic lesions of the brain. Corticotropin releasing hormone (CRH) induces the release of beta-endorphin (beta-END) from hypothalamic neurons and also from mononuclear white blood cells. The function of CRH and beta-END in vasospasm following SAH and the interrelationship between neuroendocrine and immune changes requires further elucidation. In the present study we investigated the influence of CRH injected into cerebral cisterna magna (CM) of rats on beta-END-like level in cerebrospinal fluid (CSF) in acute and chronic phase of cerebral vasospasm following artificial SAH. Acutely CRH induced a significant rise of beta-END-like in CSF both in SAH and sham SAH rats. However, in rats subjected to SAH, a single injection of CRH caused a prolonged rise of 5-END in CSF, which was also seen 2 days after SAH, during the chronic phase of vasospasm. The obtained results indicate that CRH increases neuroendocrine changes induced by SAH, probably by an activation of immune cells involved in the patomechanism of chronic vasospasm.     

Gene transfer of calcitonin gene-related peptide to cerebral arteries

Overexpression of calcitonin gene-related peptide (CGRP), an extremely potent vasodilator, to blood vessels is a possible strategy for prevention of vasospasm. We constructed an adenoviral vector that encodes prepro-CGRP (Adprepro-CGRP) and examined the effects of gene transfer on cultured cells and cerebral arteries. Transfection of Adprepro-CGRP to Cos-7 and NIH-3T3 cells increased CGRP-like immunoreactivity in media and produced an increase in cAMP in recipient cells. Five days after injection of Adprepro-CGRP into the cisterna magna of rabbits, the concentration of CGRP-like immunoreactivity increased by 93-fold in cerebrospinal fluid. In basilar artery, cAMP increased by 2.3-fold after Adprepro-CGRP compared with a control adenovirus. After transfection of Adprepro-CGRP, contraction of basilar artery in vitro to histamine and serotonin was attenuated, and relaxation to an inhibitor of cyclic nucleotide phosphodiesterase 3-isobutyl-1-methylxanthine was augmented compared with nontransduced arteries or arteries transfected with a control gene. Altered vascular responses were restored to normal by pretreatment with a CGRP(1) receptor antagonist CGRP-(8-37). Thus gene transfer of prepro-CGRP in vivo overexpresses CGRP in cerebrospinal fluid and perivascular tissues and modulates vascular tone. We speculate that this approach may be useful in prevention of vasospasm after subarachnoid hemorrhage.     

Phosphoramidon prevents cerebral vasospasm following subarachnoid hemorrhage in dogs: the relationship to endothelin-1 levels in the cerebrospinal fluid

There is increasing evidence that the conversion of big endothelin-1 (big ET-1) to endothelin-1 (ET-1) is specifically inhibited by the metalloproteinase inhibitor phosphoramidon. We investigated the effect of phosphoramidon on delayed cerebral vasospasm from subarachnoid hemorrhage (SAH) using a two-hemorrhage canine model. The magnitude of the vasospasm and the drug effect were determined angiographically. On SAH Day 7, diameter of the basilar artery decreased to about 55% of the control value obtained before SAH (on Day 0). Immunoreactive ET (IR-ET) in the cerebrospinal fluid (CSF) significantly increased after SAH (on Day 7). The intracisternal pretreatment of phosphoramidon potently suppressed the decrease in diameter of the basilar artery after SAH, i.e., observed decrease was only about 20%, compared with the value before SAH. In the phosphoramidon group, IR-ET in CSF markedly increased (on SAH Day 2), but the increased levels of IR-ET significantly declined on SAH Day 7. These results clearly indicate that phosphoramidon effectively prevents delayed cerebral vasospasm. Whether the prevention is due to the inhibition of conversion of big ET-1 to ET-1 is now under study.     

家兔自发性蛛网膜下腔出血早期脑损伤模型的初步研究

目的：建立一种可重复的、有典型神经损伤症状的自发性蛛网膜下腔出血（sAH）动物模型,为研究蛛网膜下腔出血后早期脑损伤（EBI）的机制提供可靠的动物模型。方法：选用新西兰大耳白兔60只,随机分为实验组、对照组、空白组。采用枕大池穿刺一次注入自体动脉血法建立SAH模型。在4h、8h、12h、24h、48h、72h时间点,观察比较行为学与脑组织形态学的变化。结果：（1）实验组枕大池和蛛网膜下腔内发现大量的血凝块和血液。（2）实验组光镜下可观察到蛛网膜下腔大量红细胞,神经元细胞水肿,电镜下可见胶质细胞空泡样改变,神经元细胞线粒体肿胀,髓鞘内存在空泡和板层分离现象。（3）实验组造模后均出现明显的神经系统损伤,对照组4h出现典型神经系统损伤表现,12h后恢复正常,空白组未见神经系统损伤表现。结论：枕大池一次注血法是一种简便、可重复的症状性自发性蛛网膜下腔出血早期脑损伤模型,适宜于研究早期脑损伤的致病机制。     

Reversal of delayed vasospasm by an inhibitor of the synthesis of 20-HETE

This study characterized the time course of changes in cerebral blood flow (CBF) and vascular diameter in a dual-hemorrhage model of subarachnoid hemorrhage (SAH) in rats and examined whether acute blockade of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. Rats received an intracisternal injection of blood (0.4 ml) on day 0 and a second injection 2 days later. CBF was sequentially measured using laser-Doppler flowmetry, and the diameters of the cerebral arteries were determined after filling the cerebral vasculature with a casting compound. CBF fell to 67% of control after the first intracisternal injection of blood but returned to a value near control 24 h later. CBF again fell to 63% of control after a second intracisternal injection of blood and remained 30% below control for 5 days. The fall in CBF after the second intracisternal injection of blood was associated with a sustained 30% reduction in the diameters of the middle cerebral, posterior communicating, and basilar arteries. Acute blockade of the synthesis of 20-HETE with TS-011 (0.1 mg/kg i.v.), 5 days after the second SAH, increased the diameters of the cerebral arteries, and CBF returned to control. These results indicate that the rats develop delayed vasospasm after induction of the dual-hemorrhage model of SAH and that blockade of the synthesis of 20-HETE fully reverses cerebral vasospasm in this model. They also implicate 20-HETE in the development and maintenance of delayed cerebral vasospasm.     

大鼠脑血管痉挛时NO和ET—1变化及尼莫地平的影响

目的探讨蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)时脑组织一氧化氮(NO)和内皮素-1(ET-1)含量变化及尼莫地平(ND)对其影响。方法将135只Wistar大鼠随机均分为SAH组、ND处理组和假手术组,观察手术前后基底动脉管径,及24h内局部脑血流量(rCBF)、脑组织NO和ET-1含量动态改变,并行海马病理检查。结果SAH后rCBF明显而持续降低,基底动脉管径显著缩小;海马CAl区锥体细胞严重受损;脑组织NO和ET-1含量均在SAH后1～24h显著增加(P＜0．05～0．01)。ND处理后使上述异常变化均减轻。结论SAH后脑组织NO、ET-1增多可能参与了CVS所致脑损害过程,ND通过减轻CVS和拮抗脑组织NO及ET-1的病理性改变而发挥脑保护作用。     

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.     

Melatonin Ameliorates Cerebral Vasospasm After Experimental Subarachnoidal Haemorrhage Correcting Imbalance of Nitric Oxide Levels in Rats

In the present study, we investigated the in vivo effects of melatonin on SAH-induced cerebral vasospasm and oxidative stress, resulting from SAH in an experimental rat model. Twenty-eight rats (225–250 g) were divided into four groups equally: group 1; control, group 2; SAH, group 3; SAH plus placebo, and group 4; SAH plus melatonin. We used double haemorrhage method for SAH groups. Beginning 6 h after SAH, 20 mg/kg melatonin or equal volume of 0.9% saline was administered intraperitoneally twice daily for 5 days to groups 3 and 4, respectively. Melatonin or 0.9% saline injections were continued up to fifth day after SAH and rats were sacrificed at the end of this period. Brain sections at the level of the pons were examined by light microscopy. The lumen diameter and the vessel wall thickness of basilar artery were measured using a micrometer. The serum levels of cerebral vasodilator nitric oxide (NO), the brain levels of an intrinsic antioxidant superoxide dismutase (SOD) and a NO regulator arginase activities were measured. The brain levels of inducible nitric oxide (iNOS) and nitrotyrosine, a nitrosative stress parameter immunohistochemiacally determined. In conclusion, melatonin administration ameliorated cerebral vasospasm by increasing serum NO level and decreasing the brain the levels of arginase and oxidative stress. It is therefore possible that increased brain arginase activity after SAH may also have a significant role in the pathogenesis of vasospasm by limiting the availability of arginine for NO production.