The Expression Of GLT-1 MRNA In The Hippocampal CA1 Subfield During The Induction Of Brain Ischemic Tolerance In Rats

Objective Glutamate is one of the important excitatory neuro- transmitters in the central nervous system (CNS). Many studies have proved that extracellular glutamate is increased after hypoxic-ischemic insults in the brain. The over increased glutamate activates glutamate receptors, then induces influx of Na⁺ and Ca²⁺, and further leads to release of Ca²⁺ from intracellular to cytoplasm. The overload of Na⁺ and Ca²⁺ leads to delayed neuronal death (DND). Accordingly, the over increased glutamate is usually referred to as excitotoxin. Therefore, in order to prevent the excitotoxicity of glutamate, it is an impotant strategy to decrease the concentration of extracellular glutamate or to reduce the combination of glutamate with its specific receptor during brain ischemia.It has been well-known that the glial glutamate transporter-1 (GLT-1) is the most plentiful and the most important glutamate transporter in the CNS, which plays a crucial role in terminating glutamate neurotransmission, and maintaining the extracellular glutamate below neurotoxic levels. So, the role of GLT-1 in the cerebral ischemia has been becoming a focus of the researches. As has been proved by our previous study, global cerebral ischemic preconditioning (CIP) obviously upregulated the expression of GLT-1 protein that protected pyramidal neurons in the hippocampal CA1 subfield surviving from the lethal ischemic insult which usually induces severe DND. So, the present study is mainly aimed to observe the expression of GLT-1 mRNA in the hippocampal CA1 subfield during the induction of brain ischemic tolerance (BIT) in rats. And moreover, it is our another aim to observe that GLT-1 mRNA was expressed by which kind of cells, astrocytes and/or neurons? Because GLT-1 is mainly expressed in astrocytes, glial fibrillary acidic protein (GFAP), a specific marker for astrocytes, was also observed by us. The results to be obtained would provide experimental evidence for clarifying the role of GLT-1 in the induction of BIT. The study may help us further understand how to improve the therapeutic effect of ischemia by modulating GLT-1.Method One hundred and 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:â‘ control group (n=5);â‘¡sham group (n=25): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow;â‘¢CIP group (n=25): the BCCA were clamped for 3 min then reperfused with the blood flow; â‘£Ischemic insult (II) group (n=25): the BCCA were clamped for 8 min then reperfused with the blood flow (n=25);⑤CIP+II group (n=25): 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 five time points, 6h, 1d, 3d, 5d and 7d after the sham operation or the last operations (n=5 in each time point). At the determined time point, the rats were sacrificed by decapitation.The brain was sectioned in a thickness of 5μm. Four sets of paraffin sections were made, one set for neuropathological evaluation by thionine staining, another set for the expression of GLT-1 mRNA by in site hybridization (ISH), another set for the expression of GFAP by immunohistochemical staining, and another set for double-staining of both GLT-1 mRNA and GFAP.Neuropathological evaluation of the CA1 hippocampus was examined under light microscope to determine DND by neuronal density (ND) and histological grade (HG) of the CA1 hippocampus. 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. HG was divided into the following 4 grades: grade 0, no neuron death; gradeâ… , scattered single neuron death; gradeâ…¡, death of many neurons; gradeâ…¢, death of almost complete neurons. The average HG of the bilateral hippocampus was counted as statistical data.Result1 Neuropathological evaluationIn the hippocampal CA1 subfield of the control rats, pyramidal neurons were arranged in order with 2³ cell layers; the outline of the neurons was intact, the nucleus was full, and the nucleolus was clear. At each time points observed in the sham and CIP groups, no significant neuronal damage was observed in the hippocampal CA1 subfield by thionin staining. At 7d of the sham or CIP groups, the HG was 0～Ⅰ, and the values of ND were 202.13±12.17 and 196.8±15.09, respectively. Obvious DND in the hippocampal CA1 subfield was observed in the II group from the third day after 8min ischemia. The damage deteriorated with time. Almost complete neurons died at the fifth and seventh days after the lethal ischemic insult. At 7d of the II group, HG was gradeⅡ～Ⅲ, which was much higher than that of the sham group. At the same time, the value of ND was 20.93±9.63, which was much lower than that of the sham group (P<0.01). When the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the above injured changes were prevented clearly. At 7d time point of the CIP+II group, HG (grade 0～Ⅰ) was significantly lower than that of the II group (P<0.01), and the value of ND, 187.67±12.35, was much higher than that of the II group (P<0.01). All the above indicates 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-1 mRNA2.1 The expression of GLT-1 mRNA in the neuronal layer of hippocampal CA1 subfieldIn the hippocampal CA1 subfield of the control rats, the cytoplasm of pyramidal neurons was positive for GLT-1 mRNA and stained as blue; whereas, the nucleus of pyramidal neurons was nearly negative for GLT-1 mRNA. Compared with the control group, the average optical density and the total area of GLT-1 mRNA were upregulated significantly at 6h and 1d in the sham group (P<0.01), both of them return to normal level at 3d. The intensity of GLT-1 mRNA was further increased after CIP for 3 min when compared with that of the sham group. The peak value was at 6h. Compared with the sham group, the average optical density of GLT-1 mRNA upregulated significantly at 6h, 1d and 3d (P<0.01), and the total area upregulated significantly at 1d and 3d (P<0.01), respectively. In the II group, 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 and accompanied by considerable glial cells emerging in the pyramidal neuronal layer. The glial cells were smaller than the pyramidal neurons, which stained as blue and shaped round or stick with different size. The quantity of glial cells increased with time. Compared with the sham group, the average optical density of GLT-1 mRNA downregulated significantly at 6h (P<0.01), and the total area downregulated significantly at 1d (P<0.01). Whereas with the glial cells increasing, the total area upregulated significantly at 3d, 5d and 7d (P<0.01). Compared with the II group, the average optical density of GLT-1 mRNA upregulated significantly at 6h, 1d and 3d in the CIP+II group (P<0.01), the total area upregulated significantly at 1d and 3d (P<0.01). At 5d and 7d time points, the total area decreased gradually in the CIP+II group, whereas the total area increased in the II group with the lots of the glial cells. Therefore, at the 7d time point, the total area in the CIP+II group was significantly lower than that in the II group (P<0.01).2.2 The expression of GLT-1 mRNA in the molecular layer of hippocampal CA1 subfieldIn the control group, the glial cells, which were GLT-1 mRNA positive, scattered in the molecular layer of CA1 hippocampus. Compared with the control group, the average optical density and total area of GLT-1 mRNA upregulated significantly at 6h and 1d in the sham group (P<0.01), which was most predominant at 6h and return to basal level at 3d. Compared with the sham group, the average optical density of GLT-1 mRNA upregulated significant at 6h, 1d and 3d (P<0.01, P<0.05), and total area upregulated significantly at 6h in the CIP group (P<0.01). In the II group, some of the glial cells hypertrophied in soma without quantitative change at 6h and 1d. From the third day, the quantity of glial cells significantly increased with time, the soma of the glial cells, in the shape of round or stick, were still hypertrophic. Compared with the sham group, the average optical density of GLT-1 mRNA downregulated significantly at 6h (P<0.05). However, the total area of GLT-1 mRNA was upregulated significantly at 3d, 5d and 7d (P<0.01), and the average optical density upregulated significantly at 3d and 5d (P<0.01) because of the considerable glial cells. Compared with the II group, the average optical density and the total area of GLT-1 mRNA upregulated significantly at 6h and 1d in the CIP+II group (P<0.01), while the total area was significantly lower than that of the II group at 3d, 5d and 7d (P<0.01).3 The expression of GFAPIn the control group, the astrocytes scattered in the CA1 hippocampus, and the processes of the astrocytes didn't surround the pyramidal neurons. Compared with the control group, the total area of GFAP immunoreactivity downregulated significantly at all time points in the sham group(P<0.01, P<0.05), in spite of no significant changes of average optical density was observed, and few GFAP immunoreactive particles were observed in the area that tightly surrounded the pyramidal neurons. After a CIP for 3 min, the GFAP expression was significantly upregulated at almost all time points compared with that of the sham group. Very interestingly, some GFAP immunoreactive particles were observed in the area between the pyramidal neurons, which tightly surrounded the pyramidal neurons and made the pyramidal layer look like a"shaped grade". Moreover, both the total area and average optical density of GFAP immunostaining after the CIP were significantly upregulated compared with those of the sham group. In the II group, after the lethal ischemic insult for 8 min, astrocytes hypertrophied in soma with thickened and prolonged processes at the early stage. However, no GFAP immunoreactive particles were observed in the area between the pyramidal neurons or the neighboring area of the pyramidal layer at all. From the fifth day after the lethal ischemic insult, the body of the astrocytes became more hypertrophic, whereas the processes of the hypertrophic astrocytes began to collapse and fragment. Although the total area, and average optical density of GFAP immunoreactive cells were increased significantly after the lethal ischemic insult compared with those of the sham group, they were accompanied by severe DND as showed by thionin staining. All the above indicated that the morphologic changes of the astrocytes did not protect pyramidal neurons against DND when the brain suffered from lethal ischemic insult. When the rats were pretreated with a CIP 2 days before the lethal ischemic insult, there were many GFAP immunoreactive particles that tightly surrounded the pyramidal neurons thoroughly, which made the"shaped grid"observed in the CIP group clearer. The phenomenon reached peak at 3d and constantly lasted to 7d (the end of the study). However, the average optical density of GFAP immunoreactivity downregulated obviously at 5d and 7d compared with the II group (P<0.05, P<0.01).4 The quantity of double-positive cells of both GLT-1 mRNA and GFAPCompared with the control group, the number of double-positive cells didn't change significantly at all time points in the sham group. Compared with the sham group, the number of double-positive cells didn't change significantly at all time points in the CIP group. In the II group, the number of double-positive cells didn't change significantly at early stage, from the third day, the astrocytes increased obviously, and hypertrophied in soma with thickened processes. Compared with the sham group, the number of double-positive cells increased obviously at 3d, 5d and 7d (P<0.01). In addition to the above, many small blue glial cells emerged in the CA1 hippocampus from the third day after the lethal ischemic insult for 8 min. The glial cells were smaller than the astrocytes and stained as blue, which indicated the glial cells were positive for GLT-1 mRNA. However, the glial cells were negative for GFAP. This kind of cells increased with time, and reached peak at 5d and 7d. We presume that the glial cells maybe microglial cells. In the CIP+II group, the number of double-positive cells didn't change significantly at all time points compared with the sham group. However, the quantity of double-positive cells at 3d, 5d and 7d of CIP+II group were significantly lower than those of the II group (P<0.01).Conclusion 1 Lethal ischemic insult induces severe DND of the pyramidal neurons in the hippocampal CA1 subfield. 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-1 mRNA upregulated significantly in the pyramidal neurons in the CA1 hippocampus during the induction of BIT. We presume that the upregulated expression of GLT-1 may contribute to the cerebral protection induced by CIP.3 The expression of GLT-1 mRNA upregulated significantly in astrocytes in both pyramidal neuronal layer and molecular layer in the hippocampal CA1 subfield during the induction of BIT. We presume that the upregulated expression of GLT-1 in astrocytes may take part in the cerebral protection induced by CIP.