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

Materials & Methods

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 -80ºC until analysis. The day of aneurysm rupture was designated on �g Day 0�h.

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-N�f-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.

Discussion Board

Any Comment to this presentation?

[ABSTRACT] [Introduction] [Materials & Methods] [Results] [Tables & Figures] [Discussion & Conclusion] [References] [Discussion Board]

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Satoshi Iwabuchi, Yasushi Sakai, Hitoshi Kimura, Shinya Yoshii, Tetsuya Yokouchi, Morikazu Ueda, Hirotsugu Samejima
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Last update: 19/01/00