In group 1, the serum and cerebrospinal fluid endothelin-1 levels did not change significantly over the 8 days, and histopathological examination of the basilar arteries revealed no abnormalities. In group 2, the serum and cerebrospinal fluid endothelin-1 levels increased abruptly and significantly on day 2, and remained high to the end of the study period (day 8). Histopathological examination revealed marked vasospasm. In group 3, the serum and cerebrospinal fluid endothelin-1 levels followed the same pattern observed in group 2; however, the arteries showed significantly less vasospasm than that observed in group 2.
The study findings did not provide information about the mechanism of action of the platelet-activating factor-antagonist EGb-761, but they clearly show that this agent decreases morphologic vasospasm in the dog basilar artery.
The principal complication of intracranial aneurysm rupture is brain damage due to subarachnoid hemorrhage (SAH) itself, or to a form of vasoconstriction of the cerebral arteries that occurs several days after SAH. The term for this is cerebral vasospasm. 1,2 Despite extensive experimental and clinical investigation, the cause and pathophysiology of cerebral vasospasm remain poorly understood.3–7 As a result, no widely accepted therapeutic approach for preventing vasospasm has been established, and the current treatment methods are palliative rather than curative. Although the mechanism underlying aneurysmal SAH-induced cerebral vasospasm is not entirely clear, recent evidence suggests that endothelin-l (ET-1) may play a pivotal role in the pathophysiology of this condition. 8,9,10 Recent in vivo studies in several animal models have demonstrated that administration of endothelin receptor antagonists significantly reduces the magnitude of SAH-induced vasospasm. 8 Thus, endothelin receptor antagonists may be a selective and novel drug class for treating cerebral vasospasm after SAH.
Generation of free radicals has also been implicated in the etiology of cerebral vasospasm after SAH. Ginkgo biloba extract (EGb-76l) has been shown to have anti-radical, anti-oxidant, and platelet-activating factor-antagonist properties, and is known to have beneficial effects on many vascular abnormalities and cerebral ischemia. 8,11–17 In this study, we investigated the effects of EGb-761 on cerebral vasospasm in a canine double-SAH model.
MATERIALS AND METHODS
The Animal Ethics and Review Committee of the Veterinary Faculty of Ankara University approved the study protocol. Twenty-four healthy, middle-aged, male mongrel dogs weighing 20 to 24 kg were randomly assigned to 3 groups (Table 1).
On day 0, all dogs were anesthetized with intramuscular ketamine hydrochloride (30 mg/kg) and xylazine (6 mg/kg). A cut-down catheter was placed in the right brachial vein of each animal. A Tuohy needle catheter was percutaneously inserted into the cisterna magna for cerebrospinal fluid (CSF) collection, and the external end of the device was fixed to the skin with a suture.
The group 1 (n = 8) dogs were not subjected to SAH and received no therapy. They were immediately recovered from anesthesia, and their serum and CSF ET-1 levels were measured daily for the next 8 days.
Each anesthetized dog in groups 2 and 3 was placed in prone position with its head hanging down over the end of the table. The cisterna magna was punctured with a 22G spinal needle using sterile technique, and 6 to 8 mL of CSF was withdrawn. Then 0.8 mL/kg of non-heparinized autologous arterial blood was injected over 15 minutes. The animal was left with its head down for another 15 minutes, and was then awakened from anesthesia. On day 3, the same procedure was repeated in each dog to create a “double-hemorrhage” model in these 2 groups. For the 8 days following the second hemorrhage procedure, group 2 was treated with 100 mL/d intravenous (IV) NaCl and group 3 was treated with 100 mg/kg/d IV EGb-761. As in group 1, serum and CSF ET-1 levels were measured daily for each of the 8 sampling/treatment days in groups 2 and 3.
After the 8 days of sampling (group 1) or sampling/treatment (groups 2 and 3), all 24 animals were re-anesthetized. The same intramuscular injections described previously were administered, and each dog was intubated and placed on maintenance anesthesia with a mixture of O2 and 1 to 3% isoflurane. End-tidal partial CO2 pressure, heart rate, and blood pressure were continuously assessed using a non-invasive monitor (Criticon Dinamap Research Monitor; Criticon, Tampa, FL). Body temperature was maintained with a water-heated blanket (Gaymar Model TP-200, Gaymar, Orchard Park, NY). Anterior thoracotomy was performed, and the descending aorta and superior vena cava were cross-clamped. A cannula was placed in the ascending aorta, and an incision was made in the wall of the superior vena cava. The cerebral circulation was flushed with an infusion of 2000 mL NaCl, and then 2.5% cacodylate-buffered glutaraldehyde was injected for intravital perfusion-fixation. After fixation, the basilar arteries were dissected from the brainstem and the distal one third of each vessel was removed.
Serum and CSF ET-1 levels were determined using a commercially available competitive radioimmunoassay (RIA) kit (Euro-Diagnostica AB, Malmo, Sweden; Cat. No. RB 304). The radioactivity of standards, controls, and samples was measured for 2 minutes in a gamma counter (Gamma C 12; Products Corporation, Berthold, Germany). The data were statistically analyzed using the Wilcoxon test, and P less than 0.05 was considered to indicate statistical significance.
The distal one third of each basilar artery was prepared for light microscopic study. The luminal diameter and thickness of the artery wall were measured just distal to the basilar bifurcation. Sections of the vessels were prepared with Verhoeff's elastic stain, and were examined under 40× and 200× magnification. Morphometric measurements of luminal diameter, wall thickness, and average arterial diameter were obtained using a Zeiss IBAS computerized image analysis system (Karl Zeiss Company, Oberkochen, West Germany). These data were statistically analyzed using the Mann-Whitney U test, and P less than 0.05 was considered to indicate statistical significance.
The serum ET-1 levels for the 3 groups are shown in Figure 1 and Table 2. In group 1 (controls), the levels remained stable throughout the study period, with no statistically significant differences among the 8 samples (P > 0.05). In contrast, both group 2 (SAH treated with saline) and group 3 (SAH treated with EGb-761) showed a sharp and significant rise in serum ET-1 level on day 2 of sampling/treatment (P < 0.05). These levels remained high in both groups throughout the study period. The serum ET-1 levels in groups 2 and 3 on days 2 through 8 were significantly higher than the corresponding levels in group 1 (P < 0.05).
The CSF ET-1 levels are illustrated in Figure 2 and Table 3. The patterns for the 3 groups were similar to those observed in the serum. The ET-1 levels in the control animals remained steady throughout the 8 days of sampling (P > 0.05). Groups 2 and 3 showed a sharp and significant increase in CSF ET-1 on day 2 (P < 0.05), and the levels remained high throughout the rest of the observation period. Also, from days 2 through 8, the CSF ET-1 levels in groups 2 and 3 were significantly higher than the corresponding levels in group 1 (P < 0.05).
On gross examination, none of the dogs in group 1 had a blood clot in the subarachnoid space surrounding the basilar arteries, whereas all animals in groups 2 and 3 had a clot at this site. Light microscopy examination revealed normal basilar arteries in all the group 1 dogs. The luminal surfaces of the vessels from these animals were covered with clearly defined, flat, uniform endothelial cells oriented along the longitudinal axis (Fig. 3). The vessel sections from group 2 showed constricted vessels with marked corrugation of the endothelium and internal elastic laminae (Fig. 4). In contrast, the sections from group 3 showed only mild corrugation of the endothelium and internal elastic laminae (Fig. 5).
Figure 6 and Table 4 show the luminal-diameter and wall-thickness data for each group. Group 2 exhibited significantly smaller luminal diameter and significantly greater wall thickness than groups 1 and 3 (P < 0.05). The findings for these 2 parameters in groups 1 and 3 were similar (P > 0.05).
Cerebral vasospasm after SAH is a major clinical problem in the field of neurosurgery.1,2 Although much experimental and clinical research has been done, the cause and pathophysiology of cerebral vasospasm remain unclear. A number of previous studies have demonstrated high concentrations of vasoactive and potentially spasmogenic compounds in the CSF and serum of patients with SAH. 2,18 The vascular endothelium contributes to the regulation of vascular tone by synthesizing vasorelaxant stimuli, such as endothelium-derived relaxing factor, acetylcholine, bradykinin, adenosine diphosphate, adenosine triphosphate, histamine, vasopressin, substance P, neurokinin A, neurokinin B, and prostaglandin F2. 10,19 It also affects tone by producing endothelium-derived constricting factors, including serotonin, norepinephrine, prostaglandin E2, thromboxane A2, leukotriene C4 and, endothelins. 10,20–24
Endothelins have very potent and long-lasting constrictive effects in vivo and in vitro, and are thus thought to be key factors in the development of cerebral vasospasm after aneurysmal SAH. 25–27 The endothelin family consists of ET-1, -2 and -3, and ET-1 is a 21-amino-acid polypeptide that contains 2 disulfide bonds. 27–29 This substance is one of the most potent vasoconstrictors found in the supernatant of cultured endothelial cells from the porcine aorta. Although it has been reported that patients with SAH exhibit elevated plasma and CSF levels of ET-1, there is still question as to whether this substance actually plays a role in initiating delayed cerebral vasospasm. 25–29
In our study, the control animals (group 1; no SAH, no treatment) showed no changes in serum and CSF ET-1 levels throughout the 8 days of sampling. However, the dogs in group 2 (SAH treated with saline) and group 3 (SAH treated with EGb-761) all showed sudden marked increases in serum and CSF ET-1 on day 2 of sampling/treatment. These levels remained elevated for the remaining 6 days of the study period. Comparing the 2 SAH groups, microscopic study revealed significantly milder vasospasm in the animals treated with EGb-761 (group 3). Interestingly, however, this was not reflected in the endothelin findings, as the serum and CSF ET-1 levels in group 3 remained at the same high levels observed in group 2.
One theory holds that vasospasm is caused by oxygen-derived free radicals released by auto-oxidation of oxyhemoglobin in the subarachnoid blood clot. 6,29 Hemoglobin, hemin, or the iron contained within these compounds may contribute to the generation of these radical species. 29 It is thought that vasoconstriction develops in response to damage from lipid peroxidase, or from the specific effect of oxygen-derived free radicals. It is also apparent, at least theoretically, that free radicals or lipid peroxidase might participate in a series of reactions that could contribute to vasospasm, such as generation of vasoactive eicosanoids, 30 inhibition of endothelium-dependent relaxation, 31 and promotion of inflammation. 32,33
Inflammation has long been suspected to play a role in the initiation of vasospasm after SAH. 33,34 Macrophages, neutrophils, and other inflammatory cells infiltrate the intima, media and subarachnoid space after SAH. 33,35 Research on a canine model of cerebral vasospasm has shown that injection of foreign bodies into the subarachnoid space may lead to inflammation and induce persistent moderate-to-severe vasoconstriction. 33 Platelet-activating factor (PAF) is synthesized by various inflammatory cells, and is a potent mediator involved in inflammation. 32 This substance is the most potent platelet aggravator known, and it may also regulate lipogenase and cyclo-oxygenase activity directly or via the generation of free radicals. 7,36 One investigation demonstrated that infusion of PAF into the carotid arteries of normal rats produces cerebral hypoperfusion. 4 Continual PAF production from injured neurons may aggravate previous glutamate-induced injury. Studies have also shown that PAF may be an important mediator of neuronal and microvascular dysfunction. 4,8,32,37,38 Hirashima et al 32 showed that introduction of PAF to the cisterna magna aggravates neurologic deficits and increases constriction of the basilar artery in a rabbit SAH model. The same study demonstrated that PAF antagonists have a therapeutic effect on cerebral vasospasm in this model. The findings from these various investigations support the suggestion that PAF may contribute to the pathogenesis of vasospasm.
EGb-761 contains chemical factors that are capable of trapping hydroxyl and diphenylpicrylhydrazyl radicals in a manner that is as effective as that of uric acid, a well-known anti-radical agent. Furthermore, the extract is known to prevent the formation of species that uric acid does not trap, such as the adriamycyl radical. 17 It also inhibits lipid peroxidation of membranes, and its anti-radical properties translate to a stimulating effect on prostanoid biosynthesis. EGb-761 may be considered a membrane stabilizer, and its ability to reduce the progression of edema has already been documented. 15 The effects of EGb-761 in reducing capillary hyperpermeability, protecting the blood-brain barrier, and preventing or reducing edema, particularly in the brain, all undoubtedly contribute to this membrane action. 15 In addition, EGb-761 is one of the most potent inhibitors of PAF that has been discovered to date. 38
In conclusion, although we cannot speculate on the mechanism of action of EGb-761, the results of our study clearly demonstrate that this potent PAF-antagonist decreases chronic morphologic vasospasm in the canine basilar artery.
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