| Neurons are very sensitive to the ischemia or hypoxia ,a very short time of which will bring them irreversible injuries, especially for cortical neurons .The undergoing mechanisms are not well known now. The increase of [Ca2+]j is one of the important reasons for cell injury. The abnormal signal transduction of calcium ion in cells can activate some important enzymes, help some genes to express , some neurotransmitter to release or lead to apoptosis. And it is believed to be the last part of the pathway to neurons' death. For the time being, it is believed that there are at least two calcium channels, voltage-depended channels and receptor-depended channels, in neural cells' membrane whose opening will lead to the increasing of inner [Ca"+]i At the same time there are inner-calcium pools (Endoplasmic reticulum) in neurons which could release Ca2+ when activated. These all could increase [Ca2+]j of cytoplasmia after ischemic injuries. It is short of the related studies domestic. Using Fluo-3/AM as marker for Ca2+, referencing from Mitanni A's methods to setup ischemic model, adopting Laser Scanning Confocal Microscopy (LSCM) monitored the change of [Ca2+]j in ischemic model, observed the affection dantrolene sodium, inhibitor of inner calcium pools, imposed to ischemic neurons and wanted to discuss to the role of calcium channels and Ryannodine receptor in the injuries of ischemic neurons, and the effects of dantrolene sodium.Targets and Methods:Isolated neurons from Kunming mice, added aracytidine in the second day of primary culture (10 u M) .Cells in 96-well plate were determined by MTT and in holed-dishes were used to find out the [Ca2+]j in their cytoplasmia. Change cultures very 3 days and divide them 7 days after inoculation. Control group: treated as Mitanni A's method for 10 min, and divided into two group by the difference whether there is calcium in their ASF. Experimental group: incubated in ASF with 30 uM dantrolene sodium and 20uM nicardipine for 10-15 min, ischemic-like treated for 10 min. Detected the change of [Ca2+]j between pre-ischemia and post-ischemia by LSCM. Selected 2-3 fields, 6-10 cells, in every dish and calculated the mean. Detect the change of OD value in 30min, Ih, 2h and 4h after ischemic-like deal by MTT. 12 wells were involved every group.Results:1 The change of [Ca2"1"]! treated by ischemic-like mode/. There was a significant increasing of [Ca2+]j 3-4 min after ischemic-like treat in cultured neurons isolated from fetal mice cortex. And the increasing reached the climax at 10 min after treat. Data from the cells treated by the same methods but in ASF without calcium showed a very smaller increasing of [Ca2+]; than the group with calcium. The increasing was very slow and no significant increasing was found 5 min after treat.2 The effects of dantrolene sodium could imposed to post-ischemic neurons Five minutes after ischemia, the increasing of [Ca2+]; between control group and experimental group in which ASF contained dantrolene sodium was not significantly different. But after 1 Ominutes ischemia and 15 minutes reperfusion, in the two groups of which no calcium contained, significantly difference of increasing of [Ca2+]j was seen between thedantrolene sodium-treat group and control group.3 The effects of nicardipine could imposed to post-ischemic neurons The increasing extent of [Ca2+]j 10 min after ischemic treat is significantly lower than control group. And [Ca2+]j was significantly lower 15 min after reperfusion.4 The effects of dantrolene sodium could imposed to post-ischemic neurons (determined by MTT) Compared with no dentolene sodium , nicardipine, and MK801 groups, the OD value significantly decreased.These results hints:1. The increasing of [Ca2+]; in the early stage of ischemia can be inhibit by nicardipine, but MK801 and dantolene sodium does not, which tells us that the increasing of [Ca2+]j of early stage mainly benefits from the release of L type calcium channel.2. The release of ROC and CICR are both... |
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