Role of adenosine 5'-triphosphate in vasospasm after subarachnoid hemorrhage: human investigations.

OBJECTIVE: Adenosine 5'-triphosphate (ATP) is a vasoactive compound found in high concentrations inside erythrocytes. This compound may contribute to vasospasm after subarachnoid hemorrhage (SAH). We assessed the hypothesis that ATP contributes to vasospasm in humans. METHODS: ATP and hemoglobin concentrations were measured in cerebrospinal fluid (CSF) from humans with SAH and in blood incubated in vitro. The vasoactivity of the human CSF samples and of fractionated (fractions with molecular weight greater than or less than 10 kDa) and unfractionated blood incubated in vitro was assessed by application of samples to canine basilar artery segments under isometric tension. RESULTS: ATP in human CSF declined within 72 hours of SAH to concentrations too low to contract cerebral arteries. Vasoactivity of human CSF correlated with the concentration of hemoglobin. The vasoactivity of incubated erythrocyte hemolysates remained high despite a decline in ATP concentrations. Fractionation of incubated erythrocyte hemolysates showed that for incubation periods up to 7 days, all vasoactivity was in a fraction of molecular weight greater than 10 kDa. CONCLUSION: ATP is unlikely to contribute to vasospasm because the concentrations in CSF after SAH in humans are not high enough to cause vasospasm after 72 hours. The vasoactivity of erythrocyte hemolysate is not related to the ATP or ferrous hemoglobin content but may be related to the total hemoglobin content. Therefore, ATP is unlikely to be a major cause of clinically significant delayed vasospasm.

Restriction of secretory granule motion near the plasma membrane of chromaffin cells.

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.

Effect of adenovirus-mediated nitric oxide synthase gene transfer on vasospasm after experimental subarachnoid hemorrhage.

OBJECTIVE: Evidence indicates that vasospasm after subarachnoid hemorrhage (SAH) is caused in part by a decrease in the vasodilator nitric oxide (NO), which is produced mainly in endothelial cells. This study tested whether intracisternal injection of adenovirus-expressing endothelial NO synthase (eNOS) would decrease vasospasm in dogs. METHODS: In 12 dogs, baseline cerebral angiography was performed, and then SAH was produced by two injections of blood into the cisterna magna. The dogs were randomized (n = 6/group) to intracisternal injection of adenovirus-expressing lacZ (Ad327beta-Gal) or eNOS (AdCD8-NOS), administered immediately after the first blood injection. Angiography was repeated on Day 7, and then L-arginine (50 mg) was administered intracisternally, and angiography was repeated. Cerebrospinal fluid aspirated from the cisterna magna on Days 2 and 7 was analyzed for levels of NO metabolites. The dogs were killed, and their basilar arteries were removed and studied pharmacologically. Four control dogs underwent angiography on Day 0, followed by virus injection (n = 2/group). Angiography was repeated on Day 7, and the control dogs were killed. Transgene expression was detected in tissue removed on Day 7 by histochemical staining for lacZ, by polymerase chain reaction for messenger ribonucleic acid for eNOS, and by measurement of NO metabolites in cerebrospinal fluid. RESULTS: Angiography showed significant vasospasm in each group (Ad327beta-Gal, -54 +/- 7% reduction in basilar artery diameter; AdCD8-NOS, -53 +/- 7%), with no significant difference between groups. Injection of L-arginine caused an insignificant increase in arterial diameter in each group. In dogs without SAH, Ad327beta-Gal caused a reduction in basilar artery diameter (-13 +/- 10%, P = 0.42; paired t test), whereas injection of AdCD8-NOS caused an increase in diameter (14 +/- 16%, P = 0.77; paired t test). Histological examination and beta-galactosidase staining of dogs given injections of Ad327beta-Gal showed staining in inflammatory cells in the subarachnoid space, in the adventitia of the cerebral vessels, and in the liver and lungs. Messenger ribonucleic acid for eNOS was detected in the leptomeninges of dogs given injections of AdCD8-NOS. Under isometric tension, basilar arteries from each group demonstrated similar relaxation to L-arginine, but arteries exposed to eNOS demonstrated significantly greater relaxation to L-arginine plus tetrahydrobiopterin than arteries exposed to lacZ. Cerebrospinal fluid levels of NO and its metabolites were significantly higher in dogs treated with AdCD8-NOS than those treated with Ad327beta-Gal 2 days after SAH. CONCLUSION: These results demonstrate that adenovirus vectors can be used to transfer genes to cells in the subarachnoid space of dogs. Enough NO can be produced in the absence of SAH to dilate the basilar artery. After SAH, however, NO plus a cofactor can dilate arteries in vitro, but not enough NO is generated in the subarachnoid space to prevent vasospasm, perhaps owing to the scavenging of NO by hemoglobin.

Adenosine triphosphate and hemoglobin in vasospastic monkeys.

Adenosine triphosphate (ATP) is a vasoactive compound found in high levels inside erythrocytes that may contribute to vasospasm occurring after subarachnoid hemorrhage (SAH). This study was instituted to test whether ATP causes vasospasm in a monkey model. Thirty-two monkeys were randomized to four groups of eight monkeys each to undergo cerebral angiography at baseline (Day 0) and then at Day 7 after subarachnoid placement of: 1) agarose, 2) ATP in agarose, 3) autologous hemolysate in agarose, or 4) purified human hemoglobin A(0) in agarose. Vasospasm was assessed by comparison of Day 0 and Day 7 angiograms between and within groups and by pathological examination of a subset of perfusion-fixed monkeys. Levels of adenine nucleotides were measured on Day 7 in subarachnoid agarose by high-pressure liquid chromatography. There was significant vasospasm of the right middle cerebral artery in groups given ATP (-28 +/- 7% reduction, paired t-test, p < 0.05), hemolysate (-23 +/- 7%, p < 0.05), or pure hemoglobin (-15 +/- 2%, p < 0.005). Analysis of variance revealed no significant differences between groups in diameters of cerebral arteries on Day 7. Pathological examination showed mild inflammation in the subarachnoid spaces of animals exposed to hemolysate or hemoglobin and less inflammation in those given ATP or agarose. There were no pathological changes in the cerebral arteries of animals in any group. Most of the ATP diffused out of the subarachnoid agarose by Day 7, and levels of adenine nucleotides in subarachnoid agarose were higher on Day 7 in animals exposed to hemoglobin or hemolysate. It is concluded that ATP could contribute to vasospasm occurring after SAH but that further investigations are necessary to determine if levels of ATP adjacent to vasospastic arteries are sufficient to contribute to vasospasm. In addition, no observation was made of severe vasospasm with histopathological changes in the arteries equivalent to that produced by whole blood clot in the subarachnoid space of monkeys. It should be determined whether this is because a single compound, such as ATP or hemoglobin, causes vasospasm, but that placing the compound in agarose alters its delivery and decreases the amount of vasospasm produced, or whether vasospasm is a more complex, multifactorial process.

Effect of BQ-123 and tissue plasminogen activator on vasospasm after subarachnoid hemorrhage in monkeys.

BACKGROUND AND PURPOSE: We aimed to determine the effect of intracisternal administration of an endothelin-A receptor antagonist (BQ-123) against vasospasm in a monkey model and to determine whether this drug would have adverse interactions with intracisternal tissue plasminogen activator (TPA). METHODS: Thirty-three monkeys were randomly allocated to undergo baseline cerebral angiography, creation of right subarachnoid hemorrhage (SAH), and intracisternal delivery of (1) placebo (n = 10); (2) low-dose BQ-123 (5 mg/kg per day, n = 7); (3) high-dose BQ-123 (10 mg/kg per day, n = 9); or (4) BQ-123 10 mg/kg per day plus TPA 1 mg every 12 hours for three doses (n = 7). Angiography was repeated after 7 days, and animals were killed. Vasospasm was assessed by comparisons of angiograms within groups across time by paired t test and by comparisons across groups at each time by ANOVA. RESULTS: Significant clot remained in the basal cisterns in all groups except those receiving TPA, in whom complete clot clearance was noted. Comparisons of angiograms at baseline and after 7 days showed significant vasospasm of the right middle cerebral artery in animals receiving placebo (mean +/- SEM reduction in diameter, 36 +/- 7%; P < .05) and low- and high-dose BQ-123 (16 +/- 4% and 18 +/- 7%, respectively). Animals that received TPA did not develop significant right cerebral artery vasospasm. Comparisons of arterial diameters at day 7 revealed significant variance in right middle cerebral artery diameter, with animals in the placebo group having significantly more and animals in the TPA group having significantly less vasospasm than the BQ-123 groups. Histopathological examination of the brains did not show inflammation or pathological change in animals that received BQ-123 or BQ-123 plus TPA. CONCLUSIONS: Intracisternal TPA was efficacious against vasospasm in monkeys. Combination therapy with TPA and BQ-123 was not associated with reduction in efficacy of either drug or with evidence of toxicity.

Time course of changes in arterial relaxation following subarachnoid hemorrhage in dogs.

This experiment determined if the time course of inhibition of arterial relaxation correlates with angiographic vasospasm following subarachnoid hemorrhage (SAH) in dogs. Twenty-two dogs underwent cerebral angiography followed by SAH by 2 injections of blood into the cisterna magna. Dogs had repeated angiography and were sacrificed 4 (n = 5), 7 (n = 4), 10 (n = 4), 14 (n = 5), or 21 (n = 4) days later. Four dogs served as controls and underwent angiography only, followed by sacrifice. The basilar arteries were removed and studied under isometric tension to determine relaxations to acetylcholine and sodium nitroprusside. There was significant reduction in basilar artery diameter at each time after SAH (day 4: -52% +/- 10%, day 7: -37% +/- 10%, day 10: -54% +/- 10%, day 14: -37% +/- 10%, and day 21: -26% +/- 18%, p < 0.05, analysis of variance), although there were no pairwise differences in the degree of vasospasm over time. Relaxations to acetylcholine were significantly reduced at all times after SAH (p < 0.05, analysis of variance). Relaxations to sodium nitroprusside were significantly reduced 7 days after SAH. There was a significant linear correlation between increasing time after SAH and decreasing relaxation to acetylcholine. There was no correlation in univariate or multivariate analysis, between the time after SAH, the degree of vasospasm and the relaxation to acetylcholine or sodium nitroprusside. It is concluded that inhibition of arterial relaxation depends on the relaxant used and does not correlate with vasospasm. The lack of correlation between angiographic vasospasm and relaxation suggests that while the latter occurs after SAH, contractile processes may be more important in the pathogenesis of vasospasm.

Prospective, randomized, double-blind trial of BQ-123 and bosentan for prevention of vasospasm following subarachnoid hemorrhage in monkeys.

Thirty-one monkeys were randomly divided into three groups to undergo baseline cerebral angiography followed by induction of subarachnoid hemorrhage by placement of autologous blood clot along the right-sided arteries of the anterior circle of Willis (Day 0). The monkeys were then given drug vehicle or one of two endothelin (ET) antagonists, BQ-123 (6 mg/kg/day) or bosentan (5 mg/kg/day) intracisternally. The BQ-123 was administered by continuous infusion from a subcutaneous pump and the bosentan was given by twice-daily injections into an Ommaya reservoir in the subcutaneous space with a catheter along the right middle cerebral artery (MCA). Seven days later (Day 7), angiography was repeated and the animals were killed. Comparison of arterial diameters shown on angiograms between Day 0 and Day 7 groups given placebo and bosentan showed significant reductions in the diameters of the right intradural internal carotid (28% +/- 6% and 30% +/- 6%, respectively, paired t-test, p < 0.05), anterior cerebral artery (29% +/- 8% and 32% +/- 6% respectively +/- 6%, respectively) and MCA (34% +/- 6% and 46% +/- 4%, respectively). Animals injected with BQ-123 had significant narrowing of the right extradural internal carotid artery (7% +/- 6%) and the basilar artery (11% +/- 3%), but not of the right MCA. Comparison of arterial diameters between groups at Day 7 showed significant variance in the right extradural internal carotid, both intradural internal carotid, right middle cerebral, and left anterior cerebral arteries; the animals injected with BQ-123 developed significantly less arterial narrowing these those receiving bosentan and placebo. Bosentan was not detected in the cerebrospinal fluid aspirated from the cisterna magna on Day 7, whereas BQ-123 was detected in two animals. We can infer from these results that BQ-123 prevents vasospasm following subarachnoid hemorrhage in monkeys, that further investigations of ET antagonists are warranted, and that ET may be an important pathophysiological mediator of vasospasm. The lack of efficacy of bosentan may be related to inadequate cerebrospinal fluid levels obtained by administration twice-daily through an Ommaya reservoir.

Effects of dimethyl sulfoxide on systemic and cerebral hemodynamic variables in the ischemic canine myocardium.

The effects of dimethyl sulfoxide (DMSO) infusion on cerebral blood flow (CBF) and systemic hemodynamics were studied in a canine model of myocardial ischemia. Immediately after ligation, cardiac output dropped from 2.09 +/- 0.11 (SEM) in the control group and 1.79 +/- 0.13 in the DMSO group to 1.07 +/- 0.21 and 1.0 +/- 0.08 L/min, respectively; there were no significant differences for 2 h. By the third hour and thereafter, the DMSO group had a significantly (p less than .05) higher cardiac output (1.65 +/- 0.08 L/min) than the control group (1.15 +/- 0.10 L/min). The cardiac output increase at 3 h was accompanied by significantly (p less than .05) lower systemic vascular resistance (SVR) in the DMSO group (5315 +/- 248 dyne X sec/cm5) as compared to the control group (7892 +/- 442 dyne X sec/cm5). There were no significant differences in heart rate, mean arterial pressure, pulmonary artery wedge pressure, or cerebral or pulmonary resistances in the control as compared to the DMSO group. Higher CBF values were noted at one hour and thereafter in the DMSO as compared to the control group (p less than .05). Low-dose DMSO given as an iv bolus improved cardiac output and CBF and lowered SVR in this canine model of experimental myocardial ischemia.

The effect of mannitol on cerebral blood flow.

The objective of this study was to evaluate the effects of mannitol, given over different time periods, on regional cerebral blood flow (rCBF) in patients with intracranial aneurysms. Seven patients with unruptured aneurysms (Group I) and 16 patients with Grade I and II subarachnoid hemorrhage (SAH) (Group II) received 1.5 gm/kg/8 hrs of 20% mannitol intravenously over a 24-hour period. Seven other patients with unruptured aneurysms (Group III) received 1.5 gm/kg of mannitol over 8 hours only. The last seven patients with unruptured aneurysms (Group IV) received the same dose, but as an intravenous bolus. Over a period of 24 hours, the patients underwent serial measurements of rCBF, intracranial pressure (ICP), mean blood pressure (MBP), cardiac output, and cerebral metabolic rate of oxygen consumption (CMRO2). Mannitol, when given as a continuous intravenous infusion, increased rCBF significantly without increasing MBP or decreasing ICP. This increase was more pronounced in SAH patients. The effects of mannitol lasted for 18 hours when given over an 8-hour period only; however, when it was given as a bolus, the increase in rCBF lasted for 24 hours, cardiac output tended to increase, and the effect on CMRO2 was variable.

In vivo characterization of vasocontractile activities in erythrocytes.

Partially purified protein from washed and artificially hemolyzed erythrocytes, known to cause significant contractions of isolated canine cerebral vessels in vitro, was injected into the cisterna magna of intact anesthetized dogs. Cerebral blood flow, measured by the xenon-133 washout technique, decreased from a control value of 49.5 +/- 1.17 ml/100 gm/min to an experimental value of 34.1 +/- 1.65 ml/100 gm/min at 2 hours. Cerebral vascular resistance rose from a control value of 2.05 +/- 0.17 PRU (peripheral resistance units) to an experimental value of 2.91 +/- 0.25 PRU at 2 hours. Mean arterial blood pressure, heart rate, intracranial pressure, and cerebral perfusion pressure remained stable. Cardiac output also fell significantly (in 2-hour control animals it was 2.89 +/- 0.37 liter/min, and in 2-hour experimental animals 1.43 +/- 0.13 liter/min) and peripheral vascular resistance rose. These changes were evident by 10 minutes after the cisternal injection of the hemolysate protein, and remained for the duration of the 2-hour monitoring period. Serial vertebrobasilar angiograms demonstrated marked narrowing of the intracranial basilar artery when compared to control values. The narrowing persisted for several days in most animals, and tended to increase with time. Relaxation occurred by the 10th through the 14th day. The authors conclude that this experimental preparation may be a useful model for both in vitro and in vivo investigation of chronic cerebral vasospasm.

Dimethyl sulfoxide in experimental brain injury, with comparison to mannitol.

The effects of dimethyl sulfoxide therapy were studied in rhesus monkeys following a standardized occipitofrontal missile injury. This therapy resulted in substantially higher blood pressure, cerebral perfusion pressure, blood flow, and oxidative metabolism than those of a group of monkeys that had been treated similarly with mannitol, and than those of an untreated group.

Cardiovascular effects of experimental cerebral missile injury in primates.

Ten rhesus monkeys were subjected to detailed cerebral and cardiac monitoring over a period of 90 minutes following an experimental gunshot wound. Bradycardia, decreased cardiac output, and decreased pulmonary and systemic blood pressures were observed in the presence of normal preloading (right and left atrial pressures) and afterloading (pulmonary and systemic vascular resistances). Defective cardiac output, autoregulation and cerebrovascular resistance were associated with low perfusion pressures, low cerebral blood flow, and low CMRO2.

The effects of sodium nitroprusside and trimethaphan camsylate on cerebral blood flow in rhesus monkeys.

Hemispheric cerebral blood flow was measured in the rhesus monkey before and after infusion of the hypotensive agents sodium nitroprusside and trimethaphan camsylate. The intracarotid injections of 133Xe was utilized, and flow was calculated by the "flow initial" technique. Cerebral blood flow did not change significantly with the administration of trimethaphan camsylate. However, with a small reduction in blood pressure (10.6%) during the administration of sodium nitroprusside, the cerebral blood flow fell significantly (15.4%).

An experimental cerebral missile injury model in primates.

An experimental model of cerebal missile injury in rhesus monkey is described. The main objective was to create a "clean" wound devoid of bleeding from major vessels and complications due to bone fragments. There was a correlation between the wounding energy and the physiological signs, although we underestimated the actual energy level. After the right parietooccipital to right frontal injury, there was bradycardia, changes in blood pressure, and, in high-energy wounds, a marked alternation in resperation. This suggests that the missile's energy produces direct brain-stem damage, the extent of which can be related to the wounding energy.

Physiological consequences of experimental cerebral missile injury and use of data analysis to predict survival.

The authors describe cerebrovascular and cerebral metabolic changes in monkeys, subjected to cerebral missile injury. After injury with BB pellet at 90 m/sec, there is a rapid rise in intracranial pressure (ICP), which reaches a peak 2 to 5 minutes posttrauma, and then falls to about 20 to 30 mm Hg. This, with a fall in mean blood pressure (MBP), results in a 50% reduction in cerebral perfusion pressure (CPP), Cerebral blood flow (CBF) is also reduced, although acutely there is no close relationship with (CPP). Cerebrovascular resistance falls initially and then at 30 minutes rises to very high values. Cerebral metabolic rates (CMR's) for oxygen fall after injury and remain low for the rest of the animal's life; CMR's for lactate rise immediately after injury and persists for 5 hours, then fall. After injury with a faster missile (180 m/sec), the ICP rises higher and faster, and the peak is shorter. The CCP is reduced in this injury to approximately 30 mm Hg, and only one animal survived more than 1 hour. With the conventional forms of data analysis, the length of survival after injury correlates well with MBP, ICP, and CBF, but separately they were completely unsatisfactory for prediction of an individuals prognosis. With the technique of multiple linear regression analysis, the survival of individual animals could be predicted with great accuracy. This is possible also when two postinjury parameters,CBF and MBP, are used.