Impairment in biochemical level of arterial dilative capability of a cyclic nucleotides-dependent pathway by induced vasospasm in the canine basilar artery

The authors investigated the changes and the potential of cyclic nucleotide-dependent signal transduction, which induces smooth muscle relaxation, in the basilar artery with severe vasospasm in dogs with double experimental subarachnoid hemorrhage (SAH) to explore at which biochemical level the arterial dilative capability was impaired. The amount of cyclic adenosine and guanosine monophosphates (cAMP and cGMP) decreased significantly in the basilar artery after SAH. The activities of adenylate and guanylate cyclases also were decreased significantly in the smooth muscle cells of the basilar artery 4 days after SAH. In addition to the failure of the pathways to produce cyclic nucleotides, the activities of cAMP- and cGMP-dependent protein kinases, which are representative actual enzymes that amplify the signal for vascular dilation, also significantly decreased together with the almost total loss of activation by cyclic nucleotides in the same basilar artery after SAH. It was revealed that the system for smooth muscle relaxation was impaired severely in the cerebral arteries with severe vasospasm after SAH, on the biochemical basis of significantly less vasodilative capability and in several of the steps to produce the cyclic nucleotides of intracellular signal transduction.     

Specificity and sensitivity of distinctive chest radiographic and/or 67Ga images in the noninvasive diagnosis of sarcoidosis

1. Experimental chronic cerebral vasospasm induced by subarachnoid haemorrhage (SAH) in rabbits was studied in order to evaluate the efficacy of repeated i.v. infusion of Iloprost (ILO). 2. The mean diameter of the basilar artery of intact animals was calculated as 737.5 +/- 52.8 microns while this value was reduced to 237.5 +/- 22.96 microns after SAH. 3. In the ILO treated group the mean diameter of the basilar artery was significantly increased and found to be 593.75 +/- 64.0 microns. 4. No change was observed in intracranial pressure (ICP) except a slight decrease in mean arterial pressure when relatively higher doses of ILO were used. 5. These results were taken as evidence of the high therapeutic value and low side effects of ILO in the treatment of persisting cerebral vasospasm due to SAH.     

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.     

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli can prime and boost immune responses against heterologous CFAs

Calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) are intrinsic vasodilatory substances contained in perivascular nerve fibers innervating large intracranial arteries. Effects of these substances on delayed cerebral vasospasm were examined using a rabbit model of experimental subarachnoid hemorrhage (SAH). Sixty-one anesthetized rabbits received intrathecal fresh arterial blood on day-1 and intrathecal administration of different doses of CGRP, VIP or distilled water on day-4. Prior to the treatment, caliber of the spastic basilar artery was 73.4 +/- 0.9% of pre-SAH values. Serial angiograms after treatment demonstrated that 10(-10)mol/kg of CGRP dilated the spastic artery to 117.1% of pre-SAH levels and that dilatory effect of CGRP continued up to 6 hours after treatment. VIP injection also brought arterial dilatation up to 114.9% of pre-SAH levels, although the duration of the effect was less than 3 hours. Intrathecal administration of CGRP or VIP showed no adverse effect on the systemic and neurological state of the animals. These results indicate that intrathecal CGRP and VIP have therapeutic potential in treating delayed cerebral vasospasm after subarachnoid hemorrhage. Further investigations are expected to extend the effect of CGRP and VIP.     

Transcranial Doppler in vertebrobasilar vasospasm after subarachnoid hemorrhage

OBJECTIVE: The primary objective of this study was to assess the incidence of vertebrobasilar vasospasm after subarachnoid hemorrhage (SAH) by means of transcranial Doppler ultrasonography and to evaluate the clinical significance of this phenomenon. The secondary objective was to analyze the different factors influencing the development, the severity, and the duration of vertebrobasilar vasospasm. METHODS: Fifty-seven patients with traumatic SAH and 44 patients with spontaneous SAH were evaluated and monitored by means of transcranial Doppler ultrasonography. Vasospasm of the anterior and middle cerebral arteries was defined by mean flow velocities (FVs) exceeding 120 cm per second and at least three times the FV of the internal carotid artery. Vasospasm of the basilar and vertebral arteries was defined by a mean FV exceeding 60 cm per second. RESULTS: Vasospasm of the anterior or middle cerebral arteries was found in 27 patients and was associated with vertebrobasilar spasm in 20 patients. FVs in anterior circulation vessels were neither related to the cause of the SAH nor did they correlate with the outcome. Forty-six patients (45.5%) had FVs exceeding 60 cm per second and 25 (24.8%) had FVs exceeding 85 cm per second. In 10 of these patients, direct or computed tomographic angiography showed arterial narrowing involving the vertebrobasilar system, whereas in 21 more patients, computed tomography disclosed a cerebral infarction involving the vertebrobasilar vascular territory. Vertebral artery FVs in this group were twice that of the ipsilateral carotid artery. Vertebrobasilar vasospasm was significantly more frequent after head injury, although it was not related to the type of intracranial lesion or the Glasgow Coma Scale score at admission. It did correlate, however, with outcome (P < 0.0001) and with the intensity of SAH (P < 0.0001). Delayed neurological deterioration occurred in 14 patients and was significantly more frequent in patients with basilar artery FVs above 85 cm per second (P < 0.001). Prognosis, however, could not be reliably predicted by FVs in the basilar artery, even when an FV of 110 cm per second was chosen for prediction criterion. CONCLUSION: These results suggest that vertebrobasilar vasospasm is more common than previously thought, especially in association with head injury, with which it may significantly contribute to brain stem ischemic lesions and therefore justify specific therapeutic measures.     

Dysfunction of nitric oxide induces protein kinase C activation resulting in vasospasm after subarachnoid hemorrhage

We hypothesize that the interaction between protein kinase C (PKC) and nitric oxide (NO) plays a role in the modulation of cerebral vascular tone, and the disturbance of this interaction following subarachnoid hemorrhage (SAH) results in vasospasm. To prove this hypothesis with direct evidence, PKC activities of smooth muscle cells of canine basilar arteries in the control and in the SAH groups were measured by an enzyme immunoassay method. N omega-nitro-L arginine (L-NA), an inhibitor of NO production, enhanced PKC activity. This enhancement was inhibited neither by 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) nor SIN-1, a NO releasing agent. PKC activity in the SAH was significantly higher than in the control; however, no further enhancement was produced with L-NA. In the SAH, PKC activity was not inhibited either by 8-bromo-cGMP or SIN-1. We conclude that NO maintains an appropriate vascular tone through inactivation of PKC, and that this effect is disturbed following SAH, resulting in PKC-dependent vascular contraction, such as vasospasm. On the other hand, once PKC has been activated, NO precursors do not inhibit PKC. These facts indicate NO inactivates PKC through the inhibition of phosphatidylinositol breakdown.     

Adenosine triphosphate causes vasospasm of the rat femoral artery

OBJECTIVE: Adenosine 5'-triphosphate (ATP) causes vasoconstriction by activation of P2-purinoceptors on vascular smooth muscle cells. Erythrocytes contain ATP at a concentration (1.6 mmol/L) that contracts smooth muscle. Previous studies of hemoglobin solutions did not assess whether the vasoactivity was caused by ATP rather than or in addition to hemoglobin. It was hypothesized that the hemolysis of erythrocytes that occurs after subarachnoid hemorrhage releases ATP in concentrations that cause vasospasm. METHODS: Thirty-eight rats were randomly assigned to undergo placement of one of the following compounds in a silastic elastomer cuff around each femoral artery: 1) agarose gel (n = 8); 2) dog erythrocyte hemolysate (n = 8); 3) purified human hemoglobin (Hemolink; Hemosol, Inc., Toronto, Canada; n = 8); 4) ATP (n = 8); or 5) clotted autologous blood (n = 6). The amounts of hemoglobins and adenine nucleotides in the compounds were measured by spectrophotometry and high pressure liquid chromatography. Hemolysate, purified hemoglobin, and ATP were mixed with agarose gel to create an artificial clot. Rats were killed and fixed by perfusion at physiological blood pressure 7 days after perivascular cuff and spasmogen placement. Vasospasm was assessed by image analysis of cross sections of fixed femoral arteries. Arteries were assessed for histopathological changes on 3-point scales. RESULTS: There was significant variance in arterial diameters among groups (mean diameter +/- standard deviation: agarose gel, 0.29 +/- 0.06; purified hemoglobin, 0.28 +/- 0.04; hemolysate, 0.24 +/- 0.05; ATP, 0.25 +/- 0.05; clotted blood, 0.24 +/- 0.01; P < 0.05, analysis of variance, n = 11-20). Animals exposed to clotted blood, hemolysate that contained ATP, or ATP, developed vasospasm, whereas purified hemoglobin and agarose did not cause vasospasm. Endothelial proliferation and perivascular inflammation were more severe (P < 0.05) in arteries exposed to clotted blood, purified hemoglobin, and hemolysate. CONCLUSION: These results suggest that ATP may be a vasospastic substance released by erythrocyte hemolysis. The concentration of ATP in impure solutions of hemoglobin is too low to account for the vasoactivity of these solutions. The discrepancy between arterial narrowing and histopathological changes suggests that either histopathological changes may not be an important correlate of arterial vasospasm or that other substances are important in vasospasm.     

Interleukin-6 and development of vasospasm after subarachnoid haemorrhage

The authors characterized the role of interleukins in the cerebrospinal fluid (CSF) in the development of vasospasm after subarachnoid haemorrhage (SAH), particularly interleukin-6 (IL-6). Concentrations of interleukin-1 beta (IL-1 beta), IL-6, and interleukin-8 (IL-8) were measured serially in CSF of 24 patients and in serum of 9 patients with SAH and correlated clinically. Additionally, the effects of the same cytokines on the cerebral arteries of dogs were analyzed on angiograms after intracisternal injection. Changes in levels of eicosanoids, angiogenic factors, and soluble cell adhesion molecules were investigated in the CSF of injected dogs. CSF concentrations of IL-6 and IL-8 were elevated significantly above control levels from the acute stage of SAH until the chronic stage. Patients with symptomatic vasospasm had significantly higher levels of IL-6 as well as IL-8 in CSF on days 5 and 7. Intracisternal injection of IL-6 induced long-lasting vasoconstriction in five out of eight dogs, while IL-8 did not. The diameter of canine basilar artery after IL-6 was reduced 29 +/- 5% from pretreatment diameter at 8 hours. Prostaglandins E2 and I2 were elevated in CSF for the first 4.5 hour of this IL-6-induced vasospasm. Neither angiogenic factors such as platelet-derived growth factor-AB and vascular endothelial growth factor nor soluble cell adhesion molecules were significantly elevated in CSF. IL-6, which increases to very high concentrations in CSF after SAH, may be important in inducing vasospasm, as IL-6 produced long-lasting vasoconstriction in the canine cerebral artery, which may be partly related to activation of the prostaglandin cascade.     

Subarachnoid haemorrhage: what happens to the cerebral arteries?

1. Subarachnoid haemorrhage (SAH) is a unique disorder and a major clinical problem that most commonly occurs when an aneurysm in a cerebral artery ruptures, leading to bleeding and clot formation. Subarachnoid haemorrhage results in death or severe disability of 50-70% of victims and is the cause of up to 10% of all strokes. Delayed cerebral vasospasm, which is the most critical clinical complication that occurs after SAH, seems to be associated with both impaired dilator and increased constrictor mechanisms in cerebral arteries. Mechanisms contributing to development of vasospasm and abnormal reactivity of cerebral arteries after SAH have been intensively investigated in recent years. In the present review we focus on recent advances in our knowledge of the roles of nitric oxide (NO) and cGMP, endothelin (ET), protein kinase C (PKC) and potassium channels as they relate to SAH. 2. Nitric oxide is produced by the endothelium and is an important regulator of cerebral vascular tone by tonically maintaining the vasculature in a dilated state. Endothelial injury after SAH may interfere with NO production and lead to vasoconstriction and impaired responses to endothelium-dependent vasodilators. Inactivation of NO by oxyhaemoglobin or superoxide from erythrocytes may also occur in the subarachnoid space after SAH. 3. Nitric oxide stimulates activity of soluble guanylate cyclase in vascular muscle, leading to intracellular generation of cGMP and relaxation. Subarachnoid haemorrhage appears to cause impaired activity of soluble guanylate cyclase, resulting in reduced basal levels of cGMP in cerebral vessels and often decreased responsiveness of cerebral arteries to NO. 4. Endothelin is a potent, long-lasting vasoconstrictor that may contribute to the spasm of cerebral arteries after SAH. Endothelin is present in increased levels in the cerebrospinal fluid of SAH patients. Pharmacological inhibition of ET synthesis or of ET receptors has been reported to attenuate cerebral vasospasm. Production of and vasoconstriction by ET may be due, in part, to the decreased activity of NO and formation of cGMP. 5. Protein kinase C is an important enzyme involved in the contraction of vascular muscle in response to several agonists, including ET. Activity of PKC appears to be increased in cerebral arteries after SAH, indicating that PKC may be critical in the development of cerebral vasospasm. Recent evidence suggests that PKC activation may occur in cerebral arteries after SAH as a result of decreased negative feedback influence of NO/cGMP. 6. Cerebral arteries are depolarized after SAH, possibly due to decreased activity of potassium channels in vascular muscle. Decreased basal activation of potassium channels may be due to several mechanisms, including impaired activity of NO (and/or cGMP) or increased activity of PKC. Vasodilator drugs that produce hyperpolarization, such as potassium channel openers, appear to be unusually effective in cerebral arteries after SAH. 7. Thus, endothelial damage and reduced activity of NO may contribute to cerebral vascular dysfunction after SAH. Potassium channels may represent an important therapeutic target for the treatment of cerebral vasospasm after SAH.     

Modulatory role of endothelial and nonendothelial nitric oxide in 5-hydroxytryptamine-induced contraction in cerebral arteries after subarachnoid hemorrhage

OBJECTIVE: Endothelial dysfunction is claimed to play a role in the pathogenesis of delayed cerebral vasospasm after subarachnoid hemorrhage (SAH). We have examined the effect of experimental SAH on the modulatory action of endothelial and nonendothelial nitric oxide (NO) in the contractile response of goat middle cerebral artery to 5-hydroxytryptamine (5-HT). METHODS: We compared the 5-HT-induced contractile responses of cerebral arteries from control goats and from goats with SAH that had been experimentally induced 3 days earlier by delivery of autologous arterial blood into the subarachnoid space. Contractile responses were examined by recording the isometric tension in isolated cerebral arteries. To assess the influence of endothelium, this cell layer was mechanically removed in some of the arteria, segments (rubbed arteries) from both control goats and goats with SAH. RESULTS: In arteries from control goats, contractile responses to 5-HT were significantly higher in rubbed arteries than in arteries with intact endothelium; 5-HT-induced contractions were significantly enhanced by a competitive inhibitor of NO synthesis, NG-nitro-l-arginine, in arteries both with and without endothelium. In arteries from goats with SAH, 5-HT contracted cerebral arteries without showing significant differences between segments with endothelium and those that had been rubbed; in both cases, 5-HT-induced contractions were significantly higher than those obtained in arteries from control goats. NG-Nitro-l-arginine significantly enhanced the contractile response to 5-HT of cerebral arteries from goats with SAH. CONCLUSION: These results suggest that cerebral arteries after SAH exhibit hyperreactivity to 5-HT via a mechanism that involves the absence of the modulatory role of endothelial NO, that SAH does not modify the modulatory role of nonendothelial NO, and that impairment of the modulatory action of endothelial NO on vascular responses to 5-HT could contribute to the pathogenesis of cerebral vasospasm after SAH.     

Monoclonal antibodies against ICAM-1 and CD18 attenuate cerebral vasospasm after experimental subarachnoid hemorrhage in rabbits

BACKGROUND AND PURPOSE: 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. Two of the key adhesion molecules responsible for the attachment of leukocytes to endothelial cells are intercellular adhesion molecule-1 (ICAM-1) and the common beta chain of the integrin superfamily (CD18). This study examined the effects of monoclonal antibodies on ICAM-1 and the effects of CD18 on cerebral vasospasm after SAH. METHODS: A rabbit model of SAH was utilized to test the influence of intracisternally administered antibodies to ICAM-1 and CD18 on cerebral vasospasm. Antibodies were administered alone or in combination, and the cross-sectional area of basilar arteries was assessed histologically on day 2 post-SAH. RESULTS: Treatment with antibodies to ICAM-1 or CD18 inhibited vasospasm by 22% and 27%, respectively. When administered together, the attenuation of vasospasm increased to 56%. All of these effects achieved statistical significance. CONCLUSIONS: These findings provide the first evidence that the severity of cerebral vasospasm can be attenuated using monoclonal antibodies against ICAM-1 and CD18. The results reinforce the concept that cell-mediated inflammation plays an important role in cerebral vasospasm after SAH and suggest that therapeutic targeting of cellular adhesion molecules can be of benefit in treating cerebral vasospasm.     

Comparison of spontaneously released endothelium-derived relaxing factor in cerebral and extracerebral arteries in rabbits

To investigate regional differences in spontaneously released endothelium-derived relaxing factor (EDRF), a bioassay of spontaneously released EDRF was performed on rabbit basilar, ear, common carotid and thoracic arteries using an isometric tension measurement technique and a measurement of cyclic guanosine monophosphate (cGMP) content in the vascular smooth muscle. The amount of spontaneously released EDRF was higher in the basilar artery than in any other arteries examined (p < 0.01). The levels of cGMP were 57.3 +/- 4.4 (n = 7) in basilar, 26.5 +/- 4.3 (n = 6) in ear, 24.5 +/- 2.3 (n = 11) in common carotid, and 30.3 +/- 3.8 pmol/g tissue (n = 8) in thoracic artery with endothelium, while endothelium-denuded arteries showed 24.2 +/- 6.6 (n = 5), 17.5 +/- 5.1 (n = 6), 20.1 +/- 2.9 (n = 7) and 14.4 +/- 2.3 pmol/g tissue (n = 8) in the same order. Haemoglobin (10(-5) M, incubated with the artery for 5 min, significantly reduced the level of cGMP in all vessels with endothelium: 35.3 +/- 4.4 (basilar), 16.0 +/- 2.1 (ear), 14.0 +/- 1.9 (common carotid) and 8.7 +/- 1.2 pmol/g tissue (thoracic artery). Since endothelium-dependent relaxation is associated with a rise in the cGMP content of the smooth muscle cell, the data of cGMP measurement in addition to the bioassay of spontaneously released EDRF in tension measurement suggests that the spontaneous release of EDRF is much greater in the basilar artery than in extracerebral arteries. It is concluded that the intensity of the spontaneously released EDRF is relatively higher in the intracerebral artery than in the extracerebral artery.     

Prevention of cerebrovasospasm following subarachnoid hemorrhage in rabbits by the platelet-activating factor antagonist, E5880

Recently, an important role of platelet-activating factor (PAF), an inflammation mediator, has been demonstrated in the genesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). In the current study, the authors examined whether intravenous administration of the novel PAF antagonist, E5880, can prevent vasospasm following SAH in rabbits. A vasospasm model was produced in three groups of rabbits using two subarachnoid injections of autologous arterial blood, followed by intravenous administration of distilled water (control), a low dose of E5880 (0.1 mg/kg in distilled water), or a high dose of E5880 (0.5 mg/kg in distilled water). Neurological deterioration was largely prevented in the rabbits that received E5880. Basilar artery constriction was also reduced by both doses of E5880. Histological examination at autopsy predominantly showed ischemic changes in the brain. Animals in each E5880-treated group exhibited ischemic changes less frequently than those in the control group. Plasma thromboxane B2 concentrations were reduced in rabbits treated with E5880. Platelet-activating factor was immunolocalized in the intima and media of the basilar artery in the control group. The PAF immunoreactivity demonstrated in the basilar artery was decreased in the E5880 groups in a dose-dependent manner. Thus, this study provides evidence that PAF may play a role in the pathogenesis of vasospasm after SAH and that intravenous administration of E5880 is a promising approach in preventing vasospasm.     

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.     

Oxidative stress in the human brain after subarachnoid hemorrhage

OBJECT: The aim of this study was to verify the patterns of antioxidant enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the human brain after subarachnoid hemorrhage (SAH) to verify whether an "oxidative stress situation" characterizes the brain response to subarachnoid bleeding. METHODS: Forty samples of gyrus rectus or temporal operculum that were obtained during a surgical approach to anterior circulation aneurysms were used for this study. The activity of total SOD, GSH-Px, and the SOD/GSH/Px ratio (which expresses the balance between the production of hydrogen peroxides by dismutation of superoxide radicals and the scavenging potential) were calculated in each case. Twelve samples were obtained from patients who underwent surgery for unruptured aneurysms (control group); 13 samples were obtained during surgical procedures performed within 72 hours of SAH; and 15 samples were obtained during delayed surgical procedures (> 10 days post-SAH). Ten patients presented with clinical deterioration caused by arterial vasospasm. In both SAH groups, the mean total SOD activity was significantly higher than in the control group (p=0.029). The mean activity of GSH-Px did not differ significantly between the SAH and control groups (p=0.731). There was a significant increase in the SOD/GSH-Px ratio in both SAH groups, as compared with controls (p < 0.05). There was a significant correlation between the enzymatic activity and the clinical severity of the hemorrhage, with findings of lower values of SOD and, mainly, of the SOD/GSH-Px ratio in the poor-grade patients. The SOD/GSH-Px ratio was 2.14+/-0.44 in patients who presented with clinical vasospasm and 1.24+/-0.2 in cases without vasospasm. CONCLUSIONS: The results of this study show an imbalance of the antioxidant enzymatic activities in the human brain after SAH. which is linked to the severity of the initial bleeding and possibly modified by the development of arterial vasospasm.     

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

PURPOSE: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. METHODS: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and 3H-thymidine incorporation in SMCs in culture. RESULTS: Arterial HSPGs (680 microg) reduced neointimal formation by 35% at 14 days after injury (P=.029), whereas 2000 microg of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 microg/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1%+/-13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1%+/-15.1%). In contrast, 100 microg/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9%+/-14.6%, controls 35.9%+/-12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 microg/mL compared with a value of 14 microg/mL for Enoxaparin (P< .01). CONCLUSION: When used periadventitially in the rabbit arterial injury model, natural arterial HSPGs are effective inhibitors of neointimal formation. In vitro, the HSPGs maintain SMCs in a quiescent state by inhibiting phenotypic change and DNA synthesis. This study suggests that HSPGs may be a natural agent for the treatment of clinical restenosis.     

Novel mechanism by which hemoglobin induces constriction of cerebral arteries

Since oxyhemoglobin (OxyHb) is implicated in the pathogenesis of cerebral vasospasm, we have investigated the role of protein tyrosine phosphorylation in OxyHb-mediated signalling in canine cerebral arteries and cultured canine cerebrovascular smooth muscle cells. OxyHb produced a contraction of basilar artery preparations, which was reversed by genistein, an inhibitor of tyrosine kinases, and PD098059, an inhibitor of mitogen-activated protein kinase. In cerebrovascular smooth muscle cells, OxyHb induced tyrosine phosphorylation of 42, 46, 54-60 and 80-100 kDa proteins with a time-course which paralleled the contractile action of OxyHb, suggesting that these events might be functionally linked. The 42 and 60 kDa proteins were immunologically related to the mitogen-activated protein kinase, extracellular signal regulated protein kinase (ERK2), and to p60c-Src (c-Src), respectively. The increase in protein tyrosine phosphorylation was attenuated by genistein, and the phosphorylation of the 42 kDa protein (ERK2) was inhibited by PD098059. These results suggest that OxyHb-mediated signalling utilizes a protein tyrosine kinase-based mechanism.     

Expression of heat shock protein 27 in developing and adult human kidney

Clot removal at early surgery has been reported to be clinically effective for the prevention of cerebral vasospasm following subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm. We examined the most efficacious timing of mechanical clot removal on pharmacological responses in a monkey SAH model. Cynomolgus monkeys (Macaca fascicularis) were randomized into five groups: sham-operated, clot removal in which the clot was removed 48, 72, or 96 h after SAH, and clot groups. An autologous blood clot was placed around the bilateral major cerebral arteries after craniectomy to mimic the hemorrhage. Seven days after the SAH, proximal and successively distal parts of the middle cerebral arteries were cut into rings for isometric tension measurement. The contractile responses to potassium chloride, 5-hydroxytryptamine, norepinephrine, adenosine triphosphate, prostaglandin F20, and hemoglobin were greater in the proximal parts than in the distal parts in each group. Compared with the sham-operated group, the responses of the clot-removal and clot groups to the drugs were progressively attenuated. The maximum responses to 5-hydroxytryptamine in the proximal parts and to adenosine triphosphate in the distal parts started to decrease, significantly, in the clot-removal group 48 h after SAH, while most of the responses to the other agonists began to decrease in the clot-removal groups later than 72 h after SAH. These results suggest that the attenuation of cerebrovascular contractile responses 7 days after SAH is pharmacologically inevitable, even if the clot is removed as early as 48 h after the SAH. Clot removal may thus be recommended within 48h after SAH to ameliorate the severity of cerebral vasospasm following SAH.     

Role of the posterior cruciate ligament in total knee arthroplasty

Accumulating evidence indicates that protein kinase C (PKC)-dependent, Ca2+-independent smooth muscle contraction plays the central role in the occurrence of chronic vasospasm following aneurysmal subarachnoid hemorrhage. As far as we know, the nitric oxide/ cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) system comprises the most efficacious inhibitory mechanism against the PKC-dependent contractile mechanism, and the myogenic tonus of normal cerebral arteries is thought to be maintained on the balance between these systems. Recent studies indicate that in spastic cerebral arteries, the rise in the intracellular diacylglycerol level causes PKC activation presumably owing to the overexpression of endothelin (ET)-1 as well as the generation of free radicals, whereas the cGMP level is inversely reduced owing to the inactivation of soluble guanylate cyclase through some as yet unknown mechanism. The resultant loss of balance between the two systems is considered to culminate in the occurrence of chronic vasospasm lasting for nearly 2 weeks. Based on the above concept, recent papers concerning the effects of reactive oxygen species on the arterial smooth muscle, alterations of various membrane ion channels, particularly of adenosine triphospate (ATP)-activated potassium channels in spastic arteries, the preventive effects of ET antagonists on vasospasm, and the causative role of ET-1 were reviewed in the present article. The roles of the above spasmogenic factors or mechanisms may be more clearly understood on the basis of the antagonistic interrelation between the PKC and the PKG systems, which exert diverse influences on the force-generating system as well as on its multifarious regulatory mechanisms in smooth muscle cells.