Effects of glutamate and nimodipine on survival rate of embryonic rat neuronal stem cells culturedin vitro

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Abstract

BACKGROUND:

At least three types of calcium ion channel (T, N, and L) have been recognized in nerve cells, but only the L type of channel is sensitive to drugs. Theoretically, nimodipine can lead to L-type channel inactivation and prevent calcium ion inflow, thereby exhibiting protective effects on nerve cells.

OBJECTIVE:

To observe the protective effects of nimodipine on glutamate-induced injury to embryonic rat neural stem cells, and to make a comparison with MK-801, a nonselective glutamate receptor antagonist.

DESIGN, TIME AND SETTING:

The present in vitro experiment pertaining to neural stem cells was performed at the Department of Neurology, First Hospital, Jilin University between January 2005 and December 2006.

MATERIALS:

Glutamate was sourced from the Shanghai Biological Research Institute of the Chinese Academy of Sciences. Nimodipine was provided by Bayer Company, Germany. Brain tissue was taken from Wistar rats on day 15 of gestation for isolation and culture of neural stem cells.

METHODS:

Passage 2 neural stem cell spheres were taken for preparation of single cell suspension. The prepared single cell suspension was divided into 4 groups: (1) Normal control, normally cultured. (2) Glutamate, cultured with 50, 100, 200, 500, and 1 000 μmol/L glutamate. (3) Nimodipine, received a 30-minute nimodipine [1×(10−8-10−2) g/L] culture followed by a glutamate (200 μmol/L) treatment step. (4) MK-801, given as 30- minute MK-801 (100 μmol/L) culture, followed by a glutamate (200 μmol/L) treatment step.

MAIN OUTCOME MEASURES:

Determination of glutamate-induced cell death by methyl thiazolyl tetrazolium (MTT) assay; calculation of neural stem cell survival rate following addition of nimodipine.

RESULTS:

The survival rate of neural stem cells was approximately 25.26% following 24 hour 50 μmol/L glutamate culture and gradually decreased as the glutamate dose increased (P < 0.05–0.01). Only 9.27% of neural stem cells survived when the glutamate dose was 1 000 μmol/L. The survival rate of neural stem cells was significantly higher, in a dose-dependent manner, in the nimodipine [1×(10−7-10−2) g/L] group than in the glutamate group. In addition, the MK-801 group exhibited a higher survival rate after 24 hour treatment than the glutamate group (P < 0.01).

CONCLUSION:

Glutamate (50–1 000 μ mol/L) induces injury to neural stem cells dose-dependently. Nimodipine exhibits protective effects on injury to neural stem cells and presents the effects in a dose-dependent manner at 1×(10−7-10−2) g/L. Nimodipine displays neuroprotective effects equivalent to MK-801.

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