Cerebral Ischemic Preconditioning Enhanced Binding Properties Of Hippocampal Ca1 Region Of Glial Glutamate Transporter-1 On Glutamate Uptake

Objective: L-glutamate serves as the primary excitatory neurotransmitter in the mammalian central nervous system, where it can contribute to either neuronal communication or neuropathological damage through activating a wide variety of excitatory amino acid receptors. Excitatory amino acid transporters are the most important mechanism in controlling the extracellular glutamate homeostasis. Among them, the glial glutamate transporter-1 (GLT-1) plays an essential role in breaking off the transmission of glutamate and maintaining the extracellular glutamate at a normal level.We found that cerebral ischemic preconditioning (CIP) could protect pyramidal neurons of CA1 hippocampus against delayed neuronal death (DND) and increase in extracellular glutamate concentration normally induced by lethal global cerebral ischemia. During the process, the expression of GLT-1 was up-regulated. In addition, dihydrokainate (DHK), a selective inhibitor of GLT-1, and GLT-1 antisense oligodeoxy- nucleotides could dose-dependently prevent the protective effect of the preconditioning to neurons. The results suggested that GLT-1 plays an important role in the mechanism of CIP.However, there is no report about weather the function of GLT-1 including the binding characteristics and uptake capacity to glutamate changes during the process. Therefore, the present study was undertaken to observe changes in GLT-1 binding characteristics including amount and affinity to glutamate, and in uptake capacity of GLT-1 for glutamate during the induction of brain ischemic tolerance using radio-ligand binding and the 3H-glutamate uptake assays. The results obtained provided further experimental evidence for clarifying the role of GLT-1 in the induction of brain ischemic tolerance and provided new clues for study in prevention and treatment of cerebrovascular disease.Methods: Two hundred Wistar rats (280-300g) with permanently occluded bilateral vertebral arteries except for rats in control group were randomly assigned to 5 groups:(1) Control group (n=12): The rats were subjected to without any treatment.(2) Sham group (n=47): The rats were subjected to the sham operation of global cerebral ischemia, in which all procedures for global brain ischemia was performed except for occluding the bilateral common carotid arteries.(3) CIP group (n=47): The rats were subjected to a global cerebral ischemia for 3 min as CIP, and then let the blood reperfused.(4) Cerebral ischemic insult group (n=47): The rats were subjected to a global cerebral ischemia for 8 min, which is lethal for the pyramidal neurons of the CA1 hippocampus, and then let the blood reperfused.(5) CIP+Cerebral ischemic insult group (n=47): The rats were subjected to a CIP for 3 min first, and the followed by a global cerebral ischemia for 8 min after an interval of reperfusion for 2 days, and then let the blood flow.The animals except for control group were sacrificed with decapitation at 0h,1h,1d,2d,3d,5d,7d after the sham operation or the last time of cerebral ischemia. The animals in group of 7 days (n=5) were prepared to observe histological changes in the CA1 hippocampus. The left groups were (n=7) used to observe changes in the function of GLT-1, in which 4 of them were used for changes in the amount and affinity of GLT-1, and the other 3 of them for the glutamate uptake of GLT-1.The 4 vessels occluding method was used to make the global cerebral ischemia model. The bilateral vertebral arteries were first electrocauterized and then the bilateral common carotid arteries were clamped to induce global cerebral ischemia after an interval for 2 days. An occlusion in a short period of 3 min was normally used as CIP, or a relative long one for 8 min was used as cerebral ischemic insult, which is lethal for CA1 pyramidal neurons and usually results in DND. When the CIP was followed by the 8 min ischemic insult, the interval between them was 2 days. The sham operation of the cerebral ischemic included all of the surgery except for clamping the bilateral common carotid arteries.The profile of DND in the CA1 hippocampus were determined by routine histological slice and thionin staining under light microscope and represented with histological grade (HG) and neuronal density (ND). HG was determined by the following standards: Grade 0, no neuron death; GradeⅠ, scattered single neuron death; GradeⅡ, mass neurons death; GradeⅢ, almost complete neurons death. The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The average of the number in 3 areas of the CA1 hippocampus was calculated as value of ND.The amount and affinity of GLT-1 were determined using radio-ligand binding assay and represented with values of Bmax and Kd. The Bmax and Kd value were calculated with total binding minus non-specific binding. The larger Bmax is more amount of GLT-1, and the lower Kd is the greater affinity of GLT-1 for glutamate. Glutamate uptake rate was determined by ~3H-glutamate method, and use the total uptake minus non-specific uptake as the uptake of GLT-1 for glutamate.Results:1 CIP reduced DND of pyramidal neurons in the CA1 hippocampus normally induced by global cerebral ischemia. There were no DND of pyramidal neurons in the CA1 hippocampus in control and sham groups. HG of all the two groups was grade 0 and ND was 208±11.31 and 198.67±9.35, respectively. The histological feature in the CA1 hippocampus in the rats of CIP group was similar to that in control and sham groups, and HG was grade 0~I and ND was 192±18.93. Obvious DND represented with decrease in ND (29.33±13.06) and increase in HG (Ⅱ～Ⅲ) was observed in cerebral ischemic insult group (P<0.01 vs the above groups). When the cerebral ischemic insult was preceded with the CIP, the DND induced by the ischemic insult was effectively prevented, which was represented with the decrease in HG (0～Ⅰ) and increase in ND (166.67±10.98) in the CIP+cerebral ischemic insult group (P<0.01 vs ischemic insult group).2 CIP increased the amount and affinity of GLT-1 in the CA1 hippocampus.2.1 Changes of BmaxThe value of Bmax in sham group had a trend to increase especially from 1d to 3d after the sham operation compared to control group(P<0.05), which indicated that the sham operation had some effects on the amount of GLT-1. The value of Bmax in CIP group were obviously increased and reached to peak on 2 day after CIP(P<0.05 vs 0h,1h,24h). The increase lasted to 5 day after CIP. In addition, compared to the Sham group, the value of Bmax in CIP group significantly increased at all time points (P<0.05). The value of Bmax in cerebral ischemic insult group had a trend to decrease and reached to a lowest point on 3 day after the insult (P<0.05 vs 0h and 1h). Compared to sham group, the values of Bmax on time points of 2d,3d,5d were significant lower than those in sham group at the corresponding time points (P<0.05). The value of Bmax in CIP+cerebral ischemic insult group had the same trend as that in CIP group except for the increase began at earlier time point of 24h. Compared to cerebral ischemic insult group, the value of Bmax increased significantly at all time points(P<0.05)except for the immediate time point. In addition, the values in this group were also increased compared to CIP group at all time points (P<0.05) except for time point 0h,1h and 5d.2.2 Changes of Kd.The value of Kd in the sham group had no significant differences at all time points compared with Control group. Compared with sham group, the value of Kd was obviously decreased in CIP group at time point of 2d(P<0.05), while the increased in cerebral ischemic insult group especially at the time point of 2d(P<0.05). The increased Kd values induced by cerebral ischemic could significantly prevented by CIP given before the cerebral ischemia which was represented with the decrease in the value of Kd in CIP+cerebral ischemic insult group compared with cerebral ischemic insult group (P<0.05)at all time points except time points of 3d and 5d.3 CIP increased the glutamate uptake of GLT-1 in the CA1 hippocampus. Compared to Control group, the uptake of glutamate in sham group had no significant differences at all time points. However, the CIP for 3 min could obviously increase the uptake of glutamate, especially at the early stage after the CIP. The uptake of glutamate gradually recovered, but still higher at time point of 5d than that in sham group. The glutamate uptake in the cerebral ischemia group had a trend to decrease,especially at time point 3d. The glutamate uptake in CIP+cerebral ischemic insult group was the similar as that in CIP group, but the magnitude in the increase of the uptake was more apparent at each time point observed.Conclusion:CIP could increase the amount and affinity and glutamate uptake of GLT-1, and then plays neuronal protective effect on the brain against cerebral ischemia.