Presentation
# 7
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6th Internet World Congress for Biomedical Sciences |
Satoshi Iwabuchi(1), Yasushi Sakai(2), Hitoshi Kimura(3), Shinya Yoshii(4), Tetsuya Yokouchi(5), Morikazu Ueda(6), Hirotsugu Samejima(7)
(1)(3)(4)(5)(6)(7)Department of Neurosurgery. Toho University - Tokyo. Japan
(2)Department of Physiology. Showa University College of Medical Sciences - Japan
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Cerebral vasospasm is a major cause of morbidity and mortality in patients who have suffered an aneurysmal SAH (1,2,3). Although the pathogenesis of cerebral vasospasm is still unclear, it is generally considered that the blood component or erythrocyte products in bloody CSF, such as oxyhemoglobin, are closely related to cerebral vasospasm (4,5).
It has been established that adenosine 5'-triphosphate (ATP) regulates smooth muscle tone by binding to P2 purinoceptors (6,7). ATP is abundant in human erythrocytes as well as in other animal red blood cells (8). Therefore, it has been reported that ATP may contribute to the vasoconstriction that occurs in vasospasm (9,10,11).
The purposes of the present study were to determine whether CSF obtained from patients with SAH on successive days following the hemorrhage causes contraction of cerebral smooth-muscle cells, and to investigate intracellular Ca2+ mobilization in smooth-muscle cells during contraction. Furthermore, we examined the involvement of nucleotides in CSF-induced contraction of smooth-muscle cells.
Source and collection of CSF
CSF samples were collected from two men and 5 women with SAH due to ruptured cerebral aneurysm. The mean age of the patients was 55 years. Clinical assessment was performed according to the grading system of Hunt and Hess. Four patients underwent surgical clipping and establishment of cistern drainage. Another 3 patients underwent endovascular embolization using coils and establishment of spinal drainage (Table 1). All patients had undergone one of these two interventions within 48 hours after onset of SAH. CSF samples were aspirated through drainage catheters on successive days until the removal of the catheter. Samples were centrifuged at 3000 G for 10 minutes. The supernatant was collected, frozen and stored at -80șC until analysis. The day of aneurysm rupture was designated on g Day 0h.
Cell isolation
The method used for isolation of rat basilar artery smooth-muscle cells has been described previously (10,11,12,13,14). Briefly, Wistar rats, weighing 250g each, were anesthetized with ether and decapitated. The basilar arteries were removed and placed in medium consisting of (mal/L): 130 NaCl, 5 KCl, 0.8 CaCl2, 1.3 MgCl2, 5 glucose, and 10 N-2-hydroxyethylpiperazine-Nf-2-etane-sulfonic acid (HEPES), pH 7.4. The arteries were cut into 0.2-mm rings and incubated in medium containing 0.2 mM CaCl2 and collagenase (type II, 0.5 g/L), elastase (0.5 g/L), hyaluronidase (type IV-S, 0.5 g/L), and deoxyribonuclease I (0.1 g/L) for 1 hour at room temperature. The isolated smooth-muscle cells were placed on glass coverslips and stored at room temperature in buffer solution containing 2 mM CaCl2. The cells were used within 4 hours after isolation.
Contraction of smooth-muscle cells
The morphological changes of smooth-muscle cells were recorded after application of CSF. Contraction was calculated as a percentage of the maximum contraction induced by 60 mM KCl.
Intracellular Ca2+ mobilization
The changes of intracellular Ca2+ concentration in smooth-muscle cells were determined using the fluorescent Ca2+ indicator fluo-3/AM and a confocal laser scanning fluorescence microscope.
Statistical Analysis
Data are expressed as the mean +/- standard deviation of the mean (SD). Statistical differences between the control and other groups were compared using unpaired t-tests. A probability value of less than 0.05 was considered to be statistically significant.
Contraction of smooth-muscle cells
Isolated smooth muscle cells contracted within 3 minutes after application of CSF (Figure 1). The application of CSF obtained on Day 3 following SAH induced greater contraction than that induced by CSF obtained on Day 7 (Figure 2). To test the involvement of protein in CSF with SAH, CSF pretreated with heating at 80 was also applied in the same fashion. There was no significant difference between the contraction induced by application of CSF after heating and that induced by untreated CSF (Figure 3).
Intracellular Ca2+ mobilization
While CSF produced contraction of isolate smooth-muscle cells, the fluorescence signals of intracellular Ca2+ increased for 20-30 seconds after application of CSF, and then gradually decreased (Figure 4).
Effect of P2-purinoceptor antagonist on CSF-induced contraction
To test the involvement of nucleotides, suramin, an antagonist of P2x and P2y purinoceptors, was applied. Pretreatment of isolated smooth-muscle cells with suramin (100 ÊM) for 5 minutes caused a significant inhibition of the contraction (Figure 5).
Bloody CSF collected from patients with SAH has been reported to produce sustained contractions of canine cerebral arteries (15) and human cerebral arteries (16,17,18,19). Takenaka, et al., (20) reported that CSF from patients with SAH induced cytosolic free Ca2+ elevations in single smooth-muscle cells which were prepared from explants of rat thoracic aorta. In the present study, CSF from all patients with aneurysmal SAH induced contraction of cerebrovascular smooth-muscle cells freshly isolated from rat basilar artery, and an increase of intracellular free Ca2+ concentration ([Ca2+]i) correlated with the contraction induced by CSF. Previously we showed that erythrocyte lysate produced [Ca2+]i elevation in cerebral smooth-muscle cells, and that tyrosine phosphorylation might be involved in erythrocyte lysate-induced [Ca2+]i elevation (14). The results of the present study suggest that some spasmogenic compound, possibly erythrocyte component or its breakdown products, is released into CSF after SAH. The contraction may involve an increase of [Ca2+]i that may be mediated by the activation of tyrosine kinase, protein kinase C and/or other kinases. In this study, CSF obtained on Day 3 following SAH induced greater contraction than that induced by CSF obtained on Day 7. There was a discrepancy between this observation and the fact that the peak of clinical vasospasm occurs about 7 days after SAH. Therefore, some spasmogens released into CSF after SAH may take a few days to affect smooth-muscle cells in the media via the adventitia. Takenaka, et al., (20) demonstrated that the changes in [Ca2+]i plotted over time after SAH in a vasospasm patient group (clinical and radiological evidence of vasospasm) showed a biphasic pattern, with the highest concentrations on Day 2 and 11, and that on Day 2, the [Ca2+]i elevation induced by CSF in the vasospasm group was significantly higher than that in the non-vasospasm group. In our study, there was no difference between the contraction caused by CSF from patients with clinical or radiological vasospasm, and that caused by CSF from patients without clinical or radiological vasospasm. There was no significant difference in the contractions caused by application of CSF after heating and by application of untreated CSF. These results indicate that some substances other than proteins or peptides are related to vasospasm following SAH. Preincubation of cells with suramin, an antagonist of P2x and P2y purinoceptors, attenuated contraction caused by application of CSF. Purine nucleotides may be involved in the contraction of cerebrovascular smooth-muscle cells after SAH. We are presently determining the level of adenine nucleotides such as ATP, ADP, AMP, adenosine and adenine in CSF samples, by high-performance liquid chromatography (HPLC) analysis.
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