| Objective:Brain ischema is one kind of diseases which severely impair human health. Since brain neurons are very sensitive to ischemia, long-time brain ischemic insult results in neuronal death, which usually causes severe sequelae even if blood supply recovers. In order to make neuron surviving from the ischemia and hypoxia, it is important to clarify the mechanism of the tolerance of neurons to ischemia and hypoxia.Transient sublethal cerebral ischemia could protect hippocampal neurons against delayed neuronal death (DND) induced normally by lethal ischemic insult. The transient cerebral ischemia is usually referred to as cerebral ischemic preconditioning (CIP), and the protective role is named as brain ischemic tolerance (BIT).Glutamate is one of the important excitatory neuronal transmitter in the central nervous system (CNS). Excitatory amino acid transporters (EAATs) are essential for maintaining normal extracellular level of glutamate. Five kinds of distinct high-affinity, sodium-dependent EAATs are found, which include EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glial glutamate transpor-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.It has been proved by us that ischemic preconditioning can extend the dendrite of the astrocytes, and made them surround the pyramidal neuron tightly, while the expression of GLT-1 and the uptake of glutamate were largely up-regulationed. Brain ischemic insult lead to the expression of GLT-1 down-regulated, especially in the area where almost complete pyramidal neurons died. Both inhibiting function of GLT-1 with DHK and reducing the expression of GLT-1 by GLT-1 AS-ODNs can blocked BIT induced by CIP. These results show that GLT-1 participates in the neuro-protection of CIP in vivo.It is reported recently, there are several kinds of splicing variants of GLT-1. Four kinds of splicing variants of GLT-1, including three kinds of c-terminal splicing variants and one kind of N-terminal splicing variant, have been found in rat. Three kinds of c-terminal splicing variants of GLT-1 have been named as GLT-1 a(GLT-1 a),GLT-1 b(GLT-1 v)and GLT-1 c. The quantities of GLT-1a and GLT-1b in rat brain is plentiful, whereas the quantity of GLT-1 c in rat brain is very low. There is no report about which kind(s) 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-1b participates in the induction of brain ischemic tolerance in vivo.Methods:One hundred and thirty-five adult male Wistar rats were randomly divided into 5 groups. Except for the control group, rats of the other groups were permanently occluded bilateral vertebral arteries and recovered for 2 days before brain ischemia or sham operation, the details are as follows:①ontrol group (n=3);②sham group (n=33):the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow;③CIP group (n=33):the BCCA were clamped for 3 min then reperfused with the blood flow;④schemic insult (II) group (n=33):the BCCA were clamped for 8 min then reperfused with the blood flow3;⑤IP+II group (n=33):a CIP for 3 min was preformed then reperfused,2 day-interval, a lethal ischemic insult for 8 min was given then reperfused with the blood flow. Except for the control group, the other groups were further divided into eleven time points, Omin,30 min, lh,3h,6h, Id,2d,3d,5d and 7d after the sham operation or the last operations (n=3 in each time point). At the determined time point, the rats were sacrificed by decapitation. The left brain including hippocampus was sectioned in the thickness of 5μm. Two sets of paraffin sections were made, one set for neuropathological evaluation by thionine staining, another set for the expression of GLT-1b mRNA by in site hybridization (ISH). The right hippocampal CA1 subfield was used to explore the expression of GLT-1b mRNA by RT-PCR.Results: 1 Neuropathological evaluationNeuropathological evaluation showed that in the control group, pyramidal neurons in the CA1 hippocampus were arranged in order with 2 to 3 cell layers, the outline of the neurons was intact, nucleus was full and nucleolus was clear. The ND was 203±9.7 mm-1. No significant neuronal damage was observed in the CA1 subfield at all time points observed of sham group, the ND was not different from that of the control group. No change in ND was found at all time points after CIP compared with that of the control or sham group. During the first two days after the lethal ischemic insult for 8 min, no significant pyramidal neuronal damage was observed in the hippocampal CA1 subfield. However, obvious DND wasobserved from the third day after the ischemic insult, such as decrease in ND. The damage deteriorated with time manifested as pyknosis of cell bodies, karyopyknosis of the nucleus, disappearance of the nucleolus. Almost complete neurons died on the fifth and seventh day after the lethal ischemic insult, represented by more significant decrease in ND compared with that of the lethal ischemic insult group. When the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the above injured changes were prevented clearly, which indicated that the CIP protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult. 2 The expression of GLT-1b mRNA in the hippocampal CA1 subfield revealed by in site hybridizationIn the hippocampal CA1 subfield of the control rats, the cytoplasm of pyramidal neurons was positive for GLT-1b mRNA and stained as brown; whereas, the nucleus of pyramidal neurons was nearly negative for GLT-1b mRNA.In the sham group, the cytoplasm of pyramidal neurons was positive for GLT-1b mRNA and stained as brown; whereas, the nucleus of pyramidal neurons was slight brown for GLT-1b mRNA at 0min-6 h time points. The nucleus of pyramidal neurons was nearly negative for GLT-1b mRNA at other time points. Compared with the control group, the average optical density and the total area of GLT-1b mRNA were upregulated significantly at 0min,30min, 1h,3h,6h and 12h time points of the sham group (P<0.05). The peak value was at 6h time point. Both of them return to normal level from 1d time point.The intensities of GLT-1b mRNA at 3h,6h and 12h time points were further increased after CIP Compared with the sham group, the average optical density of GLT-1b mRNA upregulated significantly at 1h,3h,6h,12h, 1d and 2d time points (P<0.05), and the total area upregulated significantly at 3h,6h,12h, 1d and 2d time points (P<0.05), respectively.In theⅡgroup, the neurons were still intact at the early stage after lethal ischemic insult for 8 min, then the pyramidal neurons obviously injuried from the third day. At 5d and 7d time points considerable glial cells were observed in the CA1 subfield. The glial cells were smaller, which stained as brown and shaped round or stick with different size. Compared with the sham group, the average optical density of GLT-1b mRNA downregulated significantly at 12h, 1d and 2d time points (P<0.05), and the total area downregulated significantly at 12h, 1d and 2d time points (P<0.05).In the early stage of CIP+Ⅱgroup, the cytoplasm and nucleus of pyramidal neurons was positive for GLT-1b mRNA and stained as brown, and the peak is at 3 h time point. At 2 d time point, the positive GLT-1b mRNA was observed in the cytoplasm. From then on, the intensities of GLT-1b mRNA decreased significantly. Compared with theⅡgroup, the average optical density and the total area of GLT-1b mRNA were upregulated significantly at all time points, respectively (P<0.01). 3 The GLT-1b mRNA expression in the rat CA1 hippocampus revealed by RT-PCR analysisIt could be found that basal expression of GLT-1b mRNA in the CA1 hippocampus of control group.At 0 min and 30 min time points, the GLT-1b mRNA expression in the sham group was significantly upregulated compared with that of the same time point in control group. Compared with the sham group, there is no obvious differnce on the expression of GLT-1b mRNA in the CIP group and II group at the same time point. Compared with the II group, the GLT-1b mRNA expression in the CIP+II group were upregulated significantly at the same time point.At 1 h and 3 h time points, the GLT-1b mRNA expression in the sham group were upregulated significantly compared with that of the control group. Compared with the sham group, the expression of GLT-1 b mRNA was further upregulated significantly in the CIP group, whereas downregulated in the II group but without significance. The downregulation of GLT-1b mRNA induced by the lethal ischemic insult was thoroughly prevented by the CIP 2 days before the lethal ischemic insult.At 6 h time point, the GLT-1b mRNA expression in the sham group was upregulated significantly compared with that of the control group. Compared with the sham group, the expression of GLT-lb mRNA was further upregulated significantly in the CIP group, whereas no difference was observed in the II group. Compared with the II group, the GLT-1b mRNA expression in the CIP+II group were upregulated significantly.At 12 h and Id time points, the GLT-lb mRNA expression in the sham group were upregulated significantly compared with that of the control group. Compared with the sham group, the expression of GLT-1b mRNA was further upregulated in the CIP group, whereas downregulated in the II group. The downregulation of GLT-lb mRNA induced by the lethal ischemic insult was thoroughly prevented by the CIP 2 days before the lethal ischemic insult.At 2 d time point, there is no obvious differnce on the expression of GLT-1b mRNA in the sham group compared with the control group. Compared with the sham group, the expression of GLT-lb mRNA was upregulated significantly in the CIP group, whereas downregulated significantly in the II group. The downregulation of GLT-1b mRNA induced by the lethal ischemic insult was thoroughly prevented by the CIP 2 days before the lethal ischemic insult.At 3 d,5 d and 7 d time points, there is no obvious differnce on the expression of GLT-1b mRNA in the sham group compared with that of the control group. Compared with the sham group, the expression of GLT-1b mRNA was downregulated significantly in the II group, whereas no obvious differnce was observed in the CIP group. The downregulation of GLT-lb mRNA induced by the lethal ischemic insult was thoroughly prevented by the CIP 2 days before the lethal ischemic insult.Conclusion: 1 Lethal ischemic insult induces severe DND of the pyramidal neurons in the hippocampal CA1 sub field. The CIP for 3 min 2 days before the lethal ischemic insult protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult. 2 The expression of GLT-1b mRNA upregulated significantly in the pyramidal neurons in the CA1 hippocampus by in site hybridization analysis after CIP. And the quantity of GLT-1b mRNA in the CA1 hippocampus upregulated significantly after CIP by RT-PCR analysis. We presume that the upregulated expression of GLT-1b may contribute to the cerebral protection induced by CIP. 3 Compared with the II group, the expression of GLT-1b mRNA in CIP+II group upregulated significantly in the pyramidal neurons in the CA1 hippocampus by in site hybridization analysis. And the quantity of GLT-1b mRNA upregulated significantly in the CA1 hippocampus by RT-PCR analysis. All the above indicated that GLT-1b participated in the protection of brain ischemic tolerance induced by CIP. |
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