Endothelial dysfunction in a primate model of cerebral vasospasm

OBJECT: Although abnormalities in the control of endothelial vasomotility have been reported in both experimental and clinical studies, the mechanism of the endothelial dysfunction that occurs following subarachnoid hemorrhage (SAH) remains unclear. Because of the absence of previous in vivo studies of endothelial function in cerebral vessels in response to SAH or cerebral vasospasm, the authors investigated endothelium-dependent responses in an established primate model of vasospasm after SAH. Endothelial function was assessed by examining vascular responses to intracarotid injections of various drugs known to act via the endothelium. Drugs that have a rapid total body clearance were selected so that their pharmacological effects would be limited to the cerebral circulation after an intracarotid infusion. METHODS: Seventeen adult male cynomolgus monkeys were used. Cerebrovascular endothelium-dependent responses were examined in control animals and in animals with SAH 7, 14, and 21 days after placement of a subarachnoid clot around the right middle cerebral artery. Cortical cerebral blood flow (CBF) and cerebrovascular resistance (CVR) were recorded continuously during 5-minute intracarotid infusions of 5% dextrose vehicle, acetylcholine, histamine, bradykinin, or Calcimycin. In control animals the intracarotid infusion of acetylcholine produced a significant (7.8 +/- 9.5%) increase in CBF and a 9.3 +/- 8.7% reduction in CVR in comparison with a control infusion of dextrose vehicle. The responses to acetylcholine disappeared in animals 7 days post-SAH, specifically in the subset of animals in which arteriography confirmed the presence of vasospasm. Infusion of Calcimycin produced no significant changes in CBF or CVR in control animals, but resulted in a significant reduction in CBF and increase in CVR in animals 7 days after SAH and in animals with vasospasm. An infusion of histamine or bradykinin had no significant effect on CBF or CVR. CONCLUSIONS: An intracarotid infusion of acetylcholine, but not one of histamine, bradykinin, or Calcimycin, produced a measurable physiological response in the normal primate cerebrovasculature. Cerebral vasospasm that occurred after SAH produced a pathophysiological effect similar to the endothelial denudation shown in the in vitro experiments of Furchgott and Zawadzki, in which acetylcholine constricted the vessels via activation of receptors on smooth-muscle cells. Changes in vascular responses to acetylcholine and Calcimycin in animals with vasospasm, compared with control animals, provide evidence that endothelial dysfunction plays a key role in the development and/or sustenance of vasospasm after SAH.     

Intrathecal nimodipine therapy in a primate model of chronic cerebral vasospasm

The safety, prevention, and treatment of chronic vasospasm by repeated administration of intrathecally applied nimodipine was evaluated in a primate model of chronic cerebral vasospasm. Twenty-four female cynomolgous monkeys were randomized into three groups of 8: sham, clot, and clot + intrathecal nimodipine. All animals underwent a subarachnoid hemorrhage (SAH) induction operative procedure after base line angiography. Nimodipine was administered postoperatively by subcutaneous injection of 1 ml/0.2 mg tid for 6 days through an Ommaya reservoir with the catheter placed in the subarachnoid basal cisterns. Angiography was performed on Day 7 post-SAH induction. Intrathecally applied nimodipine was not effective in the prevention of angiographic vasospasm. It also did not seem to decrease the degree of pathological change when compared to controls. One animal in the nimodipine group died 2 hours after an intrathecal injection secondary to a respiratory arrest. Transient sedation and hypoventilation were common. No adverse pathological effects were noted. Intrathecal nimodipine did not produce a significant dilation of vessels in moderate or severe spasm when assessed by angiography 2 hours after intrathecal injection. Only 1 animal in 8 showed diffuse dilation of vasospastic cerebral vessels after an intrathecal nimodipine injection. Three other animals in this group developed dilation only of the basilar artery, which was in mild spasm.     

Pharmacodynamic evaluation of human cerebral arteries in the genesis of vasospasm

Experiments were performed on isolated human cerebral arteries to evaluate the role desensitization and tachyphylaxis might play in preventing certain agonists from producing prolonged vasoconstriction after subarachnoid hemorrhage. In addition, the antiproteases leupeptin and pepstatin were studied to ascertain whether these peptides might inhibit contraction as does antithrombin III. The maximal contraction to KCl was used as a standard for comparing the responses elicited by the agonists, the decay of the responses to the agonists over 15 minutes was used as an index of desensitization, and the percentage of decrease in response to a second application of the agonist over the first was a measure of tachyphylaxis. The results showed that desensitization and tachyphylaxis greatly reduced or abolished the contractile responses to norepinephrine, serotonin, angiotensin II, arginine vasopressin, substance P, neuropeptide Y, neurotensin, thrombin, uridine triphosphate, linoleic acid, melittin, and cathepsin D. Moreover, some arteries failed to respond to some of these agonists, and no contractile response was elicited by acetylcholine or bradykinin. In contrast, prostaglandins E2, D2, and F2 alpha, as well as plasmin, produced sustained contractions, without tachyphylaxis, but only prostaglandin E2 and plasmin produced contractions at concentrations of 10(-7) M or less that were comparable to those of KCl. None of the antiprotease peptides inhibited the responses to KCl whereas small concentrations (6 X 10(-8) M) of antithrombin III did. The results support the hypotheses that the phenomenon of desensitization and tachyphylaxis would prevent many diverse agents from acting as spasmogens and that substances like antithrombin III present in the cerebrospinal fluid after hemorrhage could immediately protect patients from cerebral vasospasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

The permeability change of major cerebral arteries in experimental vasospasm.

The influence of vasospasm on the permeability of the major cerebral arteries was studied using horseradish peroxidase (HRP). Experimental vasospasm was produced in canine basilar arteries by successive injections, 2 days apart, of autologous blood into the cisterna magna. HRP was injected intravenously or intracisternally 48 hours after the second injection of autologous blood, and all animals were killed by perfusion fixation 60 minutes after the injection of HRP. The distribution of HRP was observed by transmission electron microscopy. In 10 dogs injected intracisternally with HRP, 5 control dogs demonstrated a moderate amount of HRP in the intermuscular space through the adventitia. Five dogs with subarachnoid hemorrhages (SAHs) showed a moderate amount of HRP in the intermuscular space. In 11 dogs injected intravenously with HRP, 5 control dogs showed no leakage of HRP into vessel walls. Six dogs with SAHs showed HRP in the interendothelial space. These findings suggest that, despite SAHs, HRP seems to be able to circulate in the cerebrospinal fluid and makes contact with the cerebral vessels. This study suggests the possibility that spasmogenic substances may penetrate the vessel wall from the extraluminal side more easily than from the intraluminal side.     

Effect of cyclosporine on the development of cerebral vasospasm in a primate model

The authors studied the effect of the immunosuppressive drug cyclosporine (CS) on the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) using a primate model of vasospasm. Eighteen monkeys were randomly divided into three groups: a sham-operated control group, an SAH group, and a CS-treated group. To induce SAH, the right side of the circle of Willis was dissected free of the arachnoid and an autologous blood clot was placed around the arteries. In the CS group, CS (5 mg/kg/day) was administered intramuscularly for 7 days after the induction of SAH. The vessel caliber was evaluated on angiograms before the induction of SAH (Day 0) and 7 days after SAH (Day 7). Histological changes and the deposition of IgG in the arterial wall were studied in the three groups. The combined values of the average reduction of the right cerebral arteries at Day 7 was significant (P less than 0.05) in the SAH group (-43.3%) and in the CS group (-31.3%) as compared with the Sham group (-0.7%); however, there was no significant difference between the values in the SAH and the CS groups. In the CS group, the average reduction in vessel caliber of the right middle and anterior cerebral arteries was significantly (P less than 0.05) less than in the SAH group; this did not prove true for the internal carotid artery, however. Although the deposition of IgG in the media and an inflammatory reaction were observed in the spastic arterial wall in both the SAH and CS groups, there was no definitive difference in these immune/inflammatory reactions between the two groups. It is suggested that CS may be helpful in reducing the severity of vasospasm, but may not have a major therapeutic effect, considering its systemic toxicity.     

Characterization of contractile responses to endothelin in human cerebral arteries: implications for cerebral vasospasm

Cerebral vascular tone is modulated, at least in part, by the vascular endothelium. This probably results from a balance between the release of the endothelium-derived relaxing factor(s) and the endothelium-derived constricting factor(s) (e.g., endothelin). The time course of the induction and the decay of these mutually antagonizing substances differ considerably. Endothelium-derived relaxing factor is probably involved in rapid changes in vascular tone whereas endothelin may be more important in long-term modulation. We have studied the vasoconstrictor properties of endothelin in human cerebral artery strips. Endothelin typically produced an intense, sustained increae in tone over a dose range similar to that seen with other vasoconstrictor substances such as serotonin and prostaglandin F2 alpha (ED50 = 10(-8) M). The response was resistant to selective antagonists of norephinephrine, serotonin, isoproterenol, histamine, acetycholine, and angiotensin II. Only sodium nitroprusside, verapamil, and a disulfide bond reducing agent (dithiothreitol) inhibited the response. The physiological properties of this response are similar to those of a vasoconstrictor protein found in cerebrospinal fluid from patients with cerebral vasospasm after subarachnoid hemorrhage. The time course of the induction of endothelin production is consistent with the temporal sequence of vasospasm, further supporting the hypothesis that endothelin may be involved in this pathological process.     

A primate model for acute and late cerebral vasospasm: Angiographic findings

Summary A subarachnoid haemorrhage (SAH) in the squirrel monkey was produced by injection of blood via a permanently implanted catheter connected to the cisterna magna and a cannula stereotactically inserted into the interpeduncular cistern. Repeated angiographic examinations of the vertebro-basilar and right internal carotid arteries revealed a biphasic vasospasm with a maximal acute spasm at ten minutes and maximal late spasm at six days after blood injection. The present study has shown that a reproducible biphasic vasospasm can be produced in the squirrel monkey and evaluated by repeated angiographic examinations. The model is suitable in the study of basic mechanisms underlying vasospasm in a primate and, due to the size of the animal, autoradiographic evaluation of the cerebral blood flow and metabolism can be performed at an acceptable cost.     

Safety and efficacy of intrathecal thrombolytic therapy in a primate model of cerebral vasospasm

To test the safety of a large intrathecal dose of human recombinant tissue plasminogen activator (rt-PA), 6 cynomolgous monkeys were given 10 mg of rt-PA (mean, 2.7 mg/kg) through an Ommaya reservoir after craniectomy and dissection of the basal cisterns. Bleeding occurred briefly at the incision in 2 animals; otherwise the clinical condition of all 6 remained normal throughout the postoperative period. Systemic fibrinolysis did not occur, and gross and microscopic examination of the brain and meninges revealed no abnormality. Next, we evaluated the efficacy of unilateral administration of rt-PA suspension (0.5 mg) plus slow-release gel rt-PA (1.25 mg) in lysing a bilateral subarachnoid clot and preventing vasospasm in a randomized, placebo-controlled trial. Sixteen monkeys were divided randomly into 2 equal groups, each of which underwent baseline cerebral angiography, followed by frontotemporal craniectomy and induction of subarachnoid hemorrhage on the left side and then on the right. Before closure on the right side either rt-PA or an equal volume of placebo was injected into the subarachnoid space. On day 7 angiography was repeated, and the animals were killed under anesthesia for necropsy. One of the animals in the placebo group developed a cerebral infarction on day 5. In the placebo group significant vasospasm occurred in all major right and left-sided anterior cerebral vessels (P less than 0.01). No vasospasm occurred in the rt-PA-treated animals. Whereas gross subarachnoid clot was found in all animals in the placebo group (mean clot weight 1.13 g), only a small fragment of clot was found in a single rt-PA-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological studies on relaxation of spastic primate cerebral arteries in subarachnoid hemorrhage

Chronic cerebral vasospasm was induced in 16 monkeys by direct placement of a clot of autologous blood over the arteries of the circle of Willis on the right side. The middle cerebral arteries (MCA's) on the clot side all showed angiographic vasospasm, which was maximal 7 days after subarachnoid hemorrhage. Animals were sacrificed at this time and vascular responses to acetylcholine (ACh), histamine, and the calcium ionophore A23187 were studied in MCA rings from the clot (spastic) side and the non-clot (control) side. In control preparations with an intact endothelium, which had been precontracted by prostaglandin F2 alpha (PGF2 alpha), histamine and A23187 produced significant relaxation. The same concentrations of histamine and A23187 did not relax vascular tissues in which the endothelium had been mechanically removed. Acetylcholine did not produce a significant endothelium-dependent relaxation of primate MCA rings, but did relax rings of primate common carotid artery. Pretreatment with chlorpheniramine (an H1-receptor antagonist) prevented histamine-induced relaxation; however, cimetidine (an H2-receptor antagonist) had no inhibitory action. It thus seems that histamine mediates relaxation of intact MCA's mostly by an H1-receptor-mediated release of endothelium-derived relaxing factor (EDRF). Relaxations induced by histamine and A23187 in MCA's from the clot side were substantially reduced. Moreover, the small component of ACh-induced relaxation was also abolished. Endothelium-independent relaxation induced by glyceryl trinitrate (GTN) occurred in arteries from both the control and the clot sides. Constrictions induced by KC1 and PGF2 alpha were reduced on the clot side of the MCA's. These results suggest that subarachnoid hemorrhage influences both the generation of EDRF and the constriction of affected arteries. The small contraction which was elicited in spastic arteries was fully relaxed by GTN.     

Therapeutic trial of intravenous nimodipine in patients with established cerebral vasospasm after rupture of intracranial aneurysms

The therapeutic efficacy of intravenous nimodipine to treat the syndrome of delayed ischemic deterioration or vasospasm after subarachnoid hemorrhage caused by a ruptured aneurysm was investigated in a randomized, double-blind, placebo-controlled multicenter study. A total of 188 patients (nimodipine (N) = 102, placebo (P) = 86) were enrolled in the study, both pre- and postoperatively, within 24 hours of clinical deterioration connected with vasospasm or within 24 hours of arteriography that identified vasospasm. After 61 patients were excluded for not meeting study criteria or for protocol violation, the results were supported by 127 validated case reports: 73 patients received intravenous treatment with nimodipine, and 54 were given placebo. Analysis of the main criterion of efficacy, the number of deaths and of patients with severe deficit related to vasospasm alone, showed a significant statistical difference (N = 8 (19%), P = 17 (49%), P = 0.01). The risk of death or disability was reduced by 66% in the treated group. Analysis of this criterion by type of inclusion (clinical or angiographic) also showed a significant reduction in the clinical group (P = 0.05), but there was no difference in the angiographic group. The risk of mortality connected with vasospasm was reduced by 82%, but analysis by group showed that there was no significant difference for those patients included on clinical criteria, whereas mortality was reduced to a much greater extent in the angiographic group. These results demonstrate the therapeutic efficacy of nimodipine in reducing secondary ischemic brain damage in patients already suffering from angiographic vasospasm or delayed ischemic deterioration.     

A trial of the 21-aminosteroid U74006F in a primate model of chronic cerebral vasospasm

The efficacy of U74006F in the prophylaxis of chronic cerebral vasospasm (VSP) was evaluated in a randomized, double-blind, placebo-controlled trial. Forty cynomolgus monkeys were divided by restricted randomization into 2 treatment groups of 20. Five animals from each treatment group were randomized into subgroups 1 and 2. The animals of subgroup 1 were studied pathologically. Brain biopsies of the animals in subgroup 2 were performed and studied with high-performance liquid chromatography (HPLC). The remaining 20 animals supplemented the number studied angiographically. Significant VSP (P less than 0.05) was detected in the majority of vessels from the clot site (right) of both treatment groups. Electron microscopy results showed positive correlation with the angiographic data. When comparing the effects of U74006F to those of the placebo at day 7, there was a significant difference (P less than 0.05) in the degree of VSP in the right extradural internal carotid and right middle cerebral arteries. This resulted from a greater degree of VSP in placebo animals. Two animals developed delayed ischemic deficits, one from each group. The infarct of the U74006F animal was smaller than the infarct in the placebo animal. Although overall changes in phosphagen levels did not reach statistical significance, HPLC analysis of the cortical biopsies did show a decrease in the ATP/ADP +/- AMP ratio of 54% in placebo animals and only 7% in animals receiving U74006F. The middle cerebral arteries of 2 animals were also studied with HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Management of cerebral vasospasm

Cerebral vasospasm is delayed narrowing of the large arteries of the circle of Willis occurring 4 to 14 days after aneurysmal subarachnoid hemorrhage (SAH). It is but one cause of delayed deterioration after SAH but, in general, is the most important potentially treatable cause of morbidity and mortality after SAH. Development of vasospasm is best predicted by the volume, location, persistence and density of subarachnoid clot early after SAH. Diagnosis is made by catheter angiography or, with less accuracy, by computed tomographic angiography, transcranial Doppler ultrasound or other methods. Treatment remains problematic because it is expensive, time-consuming, associated with substantial risk and largely ineffective. Treatment includes optimization of factors that affect cerebral blood flow and metabolism, systemic administration of nimodipine, hemodynamic therapy and pharmacologic and mechanical angioplasty.     

Transcranial Doppler in cerebral vasospasm

Transcranial Doppler provides a noninvasive method for recording blood flow velocity (and indirectly, diameter) in the basal cerebral arteries and therefore is especially useful in detecting vasospasm following subarachnoid hemorrhage. Vasospasm most commonly involves the basal arteries, where the changes in vessel diameter will be inversely proportional to the mean velocity measurements. Examination of patients requires that the examiner be experienced and familiar with the vascular anatomy and the various TCD indicators of vasospasm. Normal mean velocity for the MCA is 62 +/- 12 cm/sec. Significant spasm on angiogram of the MCA corresponds to a mean velocity of 120 cm/sec. Mean velocities of the MCA of 200 cm/sec or greater indicate severe spasm and correlate with 50% or greater narrowing on angiogram. Cerebral blood flow changes that can occur after subarachnoid hemorrhage and as a result of vasospasm may affect velocity values. A simultaneous index of CBF with either direct flow measurement techniques or by recording extracranial carotid artery velocity measurements may be helpful in reflecting these changes. Knowledge of the time course of the development and resolution of vasospasm using TCD can help the clinician predict which patients are at higher and lower risk of developing ischemic deficits, thereby guiding treatment. Several features of TCD assessment of vasospasm are similar to angiography. High TCD velocities, like severe angiographic vasospasm, are associated with delayed ischemic deficits and infarction, although some patients can remain asymptomatic despite these changes. Delayed ischemic deficits or infarctions in patients following subarachnoid hemorrhage usually will be preceded by markedly elevated velocity or other indicators of severe vasospasm.     

The effect of timing of intrathecal fibrinolytic therapy on cerebral vasospasm in a primate model of subarachnoid hemorrhage

The effect of intrathecal tissue plasminogen activator administered at times from 0 to 72 hours after subarachnoid hemorrhage on the development of cerebral vasospasm in primates was examined. Thirty monkeys were randomly assigned into one of five equal groups: a control group that underwent subarachnoid hemorrhage alone, and 0-, 24-, 48-, and 72-hour treatment groups that received 0.75 mg of tissue plasminogen activator at those times after baseline cerebral angiography and subarachnoid hemorrhage on the right side. Seven days later angiography was repeated and the animals were killed. One animal in the 72-hour group developed a delayed ischemic deficit on Day 7 after subarachnoid hemorrhage. In the control and 72-hour groups significant vasospasm occurred in most of the major, right cerebral arteries (P less than 0.05), but no significant vasospasm developed in the 0-, 24-, and 48-hour groups. Although a large subarachnoid clot remained in the control animals, most clot had been dissolved in all treatment groups. Lysing of subarachnoid hematoma with intrathecal tissue plasminogen activator within 72 hours of subarachnoid hemorrhage is effective in preventing vasospasm in primates.     

Etiology of cerebral vasospasm in primates

A primate model was used to determine whether oxyhemoglobin (OxyHb), methemoglobin (MetHb), or bilirubin is likely to be responsible for cerebral vasospasm following subarachnoid hemorrhage (SAH). Forty cynomolgus monkeys were randomly assigned to one of five groups. On Day 0, each animal underwent angiography followed by right craniectomy and placement of an Ommaya reservoir with its catheter adjacent to the right middle cerebral artery (MCA). The animals received intrathecal injections twice a day for 6 days of one of the following solutions: mock cerebrospinal fluid (CSF); OxyHb; MetHb; bilirubin; or supernatant fluid from an incubated mixture of autologous blood and mock CSF. On Day 7, angiography was repeated and the animals were killed. Comparison of angiograms obtained on Day 0 and Day 7 of the experiment showed significant vasospasm of the right MCA and the right anterior cerebral and internal carotid arteries in the animal groups that had received OxyHb or supernatant fluid. There was a smaller reduction in diameter of the same vessels in the bilirubin group (not statistically significant), while no effects were observed in the groups receiving MetHb or mock CSF. Electron microscopy of the right MCA's gave results consistent with the angiographic findings. One monkey in the OxyHb group developed a delayed-onset right MCA infarction. These data suggest that OxyHb is the cause of cerebral vasospasm following SAH.