| Objectives: With the advent of population aging, the occurrence rate of the Ischemic Cerebrovascular Disease (ICD) tends to ascend in recent years. Its high incidence, mortality and disability rates have seriously damaged people's health and life quality. At the same time, neurons in ICD are very sensitive to ischemic damages. A large number of neurons can die from cerebral ischemia after a certain period of time. At this time, neurons cannot regenerate even if blood supply was restored, which can leave serious sequelae to patients. Therefore, it is very important to develop neurons'resistance and tolerance to decrease damages and survive from serious cerebral ischemia and to make sure these neurons can still have normal functions after restoring blood supply in ICD treatment.Numerous animal experiment shows a large number of neurons in hippocampal CA1 sector died from serious cerebral ischemia. If the experiment animal was pretreated by slight ischemia which cannot cause neuron damage, it's neurons could be protected and tolerate the following serious cerebral ischemia that could bring neuron damages. This phenomenon is called brain ischemic tolerance (BIT). The slight, short-time pretreatment of ischemic is called cerebral ischemic preconditioning (CIP). The method of developing the mechanism of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP) to explore neuron tolerance of ischemia can provide a novel way for ICD clinical treatment.Glutamate is one of the important excitatory neuronal transmitter in the central nervous system(CNS). Ischemia and hypoxia in brain tissue induced by various kinds of diseases can result in dysmetabolism in energy and inhibit the action of Na+-K+-ATPase, which would lead to an obvious increase of extracellular K+ concentration and intracellular Na+ concentration. The changes facilitate depolarization of neurons and release of glutamate in the nerve terminal. In addition, the high concentration of extracellular K+ and intracellular Na+ can reverse the activity of high-affinity glutamate transporter, which would lead to reversal transporting of glutamate from intracellular to extracellular space. These reasons result in an increase in extracellular glutamate concentration. The neurons die of the overload of Na+ and Ca2+. Thus, the glutamate has been referred to as excitotoxin. Excitatory amino acid transports (EAATs) are important mechanism for regulating the extracellular concentration of glutamate. Five kinds of distinct high-affinity, sodium-dependent EAATs are found, which include EAAT1 (glutamate/aspartate transporter,GLAST) , EAAT2 (glial glutamate transport-1,GLT-1), EAAT3(excitatory amino acid carrier 1,EAAC1), EAAT4 and EAAT5. Although both neurons and glia contain EAATs, it is generally accepted that the uptake capacity of astrocytes is much higher than that of neurons. Many studies have shown that GLT-1 plays a principal role in removing the released glutamate from the extracellular space and maintaining the extracellular glutamate below neurotoxic level in the brain. So, the role of GLT-1 in the cerebral ischemia has been becoming a focus of the researches.Our recent study proved that cerebral ischemic preconditioning (CIP) could extend the processes of astrocytes, which surround the pyramidal neurontightly, and increase the expression of GLT-1 and the uptake of glutamate in the CA1 hippocampus. While brain ischemic insult led to down-regulation of the expression of GLT-1 in the CA1 hippocampus, especially in the area where almost complete pyramidal neurons died of ischemic insult. Both inhibiting function of GLT-1 with DHK and reducing the expression of GLT-1 by GLT-1 antisense oligodeoxynucleotides (AS-ODNs) could block brain ischemic tolerance induced by CIP.These results show that GLT-1 of astrocytes participates in the neuronal protection of CIP against brain ischemic insult. Recent studies have reported that there are four kinds of splicing variants of GLT-1, including three kinds of C-terminal splicing variants and one kind of N-terminal splicing variant. Three kinds of C-terminal splicing variants of GLT-1 have been named as GLT-1a, GLT-1b and GLT-1c. The quantities of GLT-1a and GLT-1b in rat brain is plentiful, whereas the quantity of GLT-1c in rat brain is very low. There is no report about which kind of GLT-1 splice variants play a role in the induction of brain ischemic tolerance. Therefore, the present study was undertaken to study whether GLT-1a participates in the induction of brain ischemic tolerance in vivo.Methods: Ninety-six Wistar rats(weighing 260-300 g)were randomly divided into 5 groups. Except for the control group, rats of other groups were permanently occluded bilateral vertebral arteries and recovered for 2 days before brain ischemia or sham operation, the details are as follows:(1) control group (n=3); (2) sham group (n=24): the bilateral common carotid arteries (BCCA)were separated, but without occluding the blood flow; (3) CIP group(n=24): the BCCA were clamped for 3min then reperfused with the blood flow; (4) Ischemic insult (II) group (n=24): the BCCA were clamped for 8 min then reperfused with the blood flow; (5) CIP+II group (n=24): a CIP for 3min was performed then reperfused, 2 day-interval, a lethal ischemic insult for 8min was given then reperfus with the blood flow. Except for the control group, the other groups were further divided into eight time points, 0min, 1h, 3h, 6h , 1d, 3d, 5d and 7d after the sham operation or the last operations(n=3 in each time point). The hippocampal CAL subfield was used to explore the expression of GLT-1a mRNA by RT-PCR.Results:The hippocampal GLT-la mRNA of rats in control group has some expression.The comparison of results in 0 min and 1h: the sham group is much higher than the control group (p<0.05). CIP group is not significantly different from sham group and group I(Ip>0.05). Group CIP+II is remarkably higher than Group II(p<0.05). Comparison in 3h and 6h: sham group is significantly higher than control group (p<0.05). CIP group increased remarkably compared with sham group (p<0.05). Group II decreased slightly compared with sham group but the result is not significantly different (p>0.05). CIP +II group increased significantly than Group II (p<0.05).At the time of 1d: sham group is significantly higher than control group (p<0.05). CIP group increased remarkably than sham group (p<0.05). Group II decreased remarkably compared with sham group, CIP group and CIP+II group separately(p<0.05).At the time of 3d: sham group is slightly increased compared with control group but the result is not significantly differen(tp>0.05).CIP group increased remarkably than that of sham group (p<0.05). Group II decreased remarkably than those of sham group, CIP group and CIP +II group(p<0.05).Comparison of the results in 5d and 7d: sham group is not significantly different from control group( p>0.05). CIP group is not significantly different from sham group (p>0.05). Group II decreased remarkably than those of sham group, CIP group and CIP+II group (p<0.05).Conclusion:Cerebral is chemia for eight minutes can make the experimental rats'hippocampal CA1 pyramidal neurons die remarkably. The expression of GLT-1a mRNA is decreased. The hree-minute cerebral ischemic preconditioning can make the experimental rats tolerate eight-minute ischemic and the expression of GLT-1a mRNA is increased.
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