Neuronal Protective Effect Of Ceftriaxone Against Global Brain Ischemia Through Modulating Glial Glutamate Transporter-1

Objective:Glutamate is the major excitatory neuron- transmitter in the mammalian central nervous system. It is involved in brain plasticity and many higher brain functions such as learning and memory. However, its excessive accumulation could result in neuronal death. Excitatory amino acid transporters are main mechanism regulating the concentration of extracellular glutamate. Glial glutamate transporter-1 (GLT-1) plays an essential role in terminating neuro-transmission and maintaining the extracellular glutamate in normal level.Rothstein et al reported in Nature in 2005 that beta-lactam antibiotics such as Ceftriaxone (Cef) could increase the expression of GLT-1 and its glutamate uptake especially. Soon afterwards, Chu et al in 2007 reported that administration of Cef could diminish infarct volumes in rat middle cerebral artery occlusion model by up-regulating the expression of GLT-1. Recently, a research found that Cef can improve neurological performance and survival in rat middle cerebral artery occlusion model.We found that preventive,rather than therapeutic,admini- stration of Cef has the neuronal protective effect against delayed neuronal death (DND of pyramidal neurons normally induced by global brain ischemia in the CA1 hippocampus. Meanwhile, the expression of GLT-1 was up-regulated. However, there is still no direct evidence to show that the up-regulation of GLT-1 after administration of Cef is responsible to the neuronal protective effect of Cef in global brain ischemic insult.Therefore, the present study was undertaken to investigate the neuronal protective effect against global brain ischemia, and changes in expression and glutamate uptake of GLT-1 after preventive administration of Cef in rat global brain ischemic model. Especially the effect of GLT-1 antisense oligodeoxy- nucleotides (AS-ODNs) on them was observed in the same model. The results obtained provided new and more direct evidence for illustrating the role of GLT-1 in the neuronal protection of Cef and provided new clues for study in the prevention and therapy of cerebrovascular disease.1 GLT-1 AS-ODNs attenuated Cef-induced up-regulation of GLT-1 and neuronal protection against global brain ischemic insult1.1 GLT-1 AS-ODNs inhibited GLT-1 up-regulation in the CA1 hippocampus induced by CefGrouping and method: One hundred and twenty male Wistar rats (270-320 g in weight) with permanently occluded bilateral vertebral arteries were randomly assigned to the two parts consisted of six groups.1.1.1 The effect of GLT-1 AS-ODNs on the Cef-induced GLT -1 up-regulation in sham rats(1) Sham group (n=10): The rats were subjected to sham operation for global brain ischemia, in which all procedures for global brain ischemia was performed except for occluding the bilateral common carotid arteries. The animals were sacrificed by decapitation on 12 h and 24 h after the sham operation to observe the expression of GLT-1.(2) Cef+sham group (n=10): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and subjected to the sham operation on 1 d after the last time of the injection. Other procedures were the same as those in sham group.(3) AS-ODNs+Cef+sham group (n=40): On the basis of the Cef+sham group, 10μl AS-ODNs solution was injected into the right lateral ventricle at 36 h and 72 h after the first time of Cef injection, and at 12 h before the sham operations. Two doses in 9 nmol and 18 nmol were used to observe the dose-dependency. Other procedures were the same as those in sham group. At the same time, distilled water (solvent of the AS-ODNs) control group (n=10) and R-ODNs (18 nmol) control group (n=10) were designed.1.1.2 The effect of GLT-1 AS-ODNs on the Cef-induced up-regulation of GLT-1 in brain ischemic ratsBased on the above sham group, the following 3 groups were designed.(4) Global brain ischemia group (n=10): The rats were subjected to a global brain ischemia for 8 min, and then were reperfused. The animals were sacrificed by decapitation on 12 h and 24 h after the global brain ischemia to observe the expression of GLT-1.(5) Cef+global brain ischemia group (n=10): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and were subjected to global brain ischemia for 8 min on 1 d after the last time of the injection with Cef. Other procedures were the same as those in global brain ischemia group.(6) AS-ODNs+Cef+global brain ischemia group (n=40): On the basis of the Cef+global brain ischemia group, 10μl GLT-1 AS-ODNs solution was injected into the right lateral ventricle at 36 h and 72 h after the first time of Cef injection, and at 12 h before the global brain ischemia. Two doses of 9 nmol and 18 nmol were used. Other procedures were the same as those in brain ischemia group. Meanwhile, distilled water (solvent of AS-ODNs) control group (n=10) and R-ODNs (18 nmol) control group (n=10) were designed.The expression of GLT-1 was assayed using Western blot analysis. The relative expression of GLT-1 was represented with the ratio of integral optical density (IOD) of GLT-1 immunoblot band to that ofβ-actin. The more the ratio is, the more the expression of GLT-1 is.Result: Basal expression of GLT-1 in the CA1 hippocampus could be observed in sham group. Compared with sham group, the GLT-1 expressions in both sham and global brain ischemia rats were significantly up-regulated by administrating Cef (P<0.05). Compared with the Cef+sham or Cef+global brain ischemia group, the GLT-1 expression significantly decreased in the AS-ODNs (18 nmol)+Cef+sham group and AS-ODNs(18 nmol)+Cef+global brain ischemia group (P<0.05), respectively. While there was no influence on the up-regulation of GLT-1 induced by Cef after administrating any one of distilled water, AS-ODNs 9 nmol or R-ODNs (18 nmol) (P>0.05). The results indicated that administration of GLT-1 AS-ODNs in a dose of 18 nmol significantly inhibited the up-regulation of GLT-1 induced by Cef in both sham and global brain ischemic rats.1.1 GLT-1 AS-ODNs inhibited the neuronal protection of Cef against DND of pyramidal neurons in the CA1 hippocampus normally induced by global brain ischemiaGrouping and method: Forty male Wistar rats (270-320 g in weight) with permanently occluded bilateral vertebral arteries were randomly assigned to six groups.(1) Sham group (n=5): The rats were subjected to sham operation for global brain ischemia, in which all procedures for global brain ischemia was performed except for occluding the bilateral common carotid arteries. The animals were sacrificed by decapitation on 7 d after the sham operation to determine DND in the CA1 hippocampus.(2) Cef control group (n=5): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and subjected to the sham operation on 1 d after the last time of the injection. Other procedures were the same as those in sham group.(3) AS-ODNs control group(n=5): GLT-1 AS-ODNs solution of 10μl (18 nmol) was injected into the right lateral ventricle 3.5 d, 2 d and 12 h before the sham operations. The time points above were corresponding to those in the group of"AS-ODNs+Cef+global brain ischemia group". Other procedures were the same as those in sham group.(4) Global brain ischemia group (n=5): The rats were subjected to a global brain ischemia for 8 min, and then were reperfused. The animals were sacrificed by decapitation on 7 d after the global brain ischemia operation to determine delayed neuronal death(DND)in the CA1 hippocampus.(5) Cef+global brain ischemia group (n=5): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and were subjected to global brain ischemia for 8 min on 1 d after the last injection with Cef. Other procedures were the same as those in global brain ischemia group.(6) AS-ODNs+Cef+global brain ischemia group (n=15): On the basis of the Cef+global brain ischemia group, 10μl GLT-1 AS-ODNs solution was injected into the right lateral ventricle at 36 h and 72 h after the first time of Cef injection, and at 12 h before the global brain ischemia. Two doses of 9 nmol and 18 nmol were used. Other procedures were the same as those in global brain ischemia group. Meanwhile, R-ODNs (18 nmol) control group (n=5) were designed.The profile of delayed neuronal death (DND) of pyramidal neurons in the CA1 hippocampus was evaluated using thionin staining under light microscope by determining the neuronal density (ND) and histological grade (HG).Result: It was showed that the outline of the pyramidal neurons was kept intact,and no neuronal loss was found in the CA1 hippocampus in sham group. The histological features of the region in Cef control and GLT-1 AS-ODNs control (18 nmol) groups were similar with those in the sham group. Obvious destruction of the CA1 hippocampus was found in global brain ischemia group, the value of ND was decreased, and HG was increased compared with that in the sham group (P<0.05). Compared with global brain ischemia group, the Cef+global brain ischemia group showed a significant protective effect against the DND induced by global brain ischemia, which was represented with the increase in ND value and decrease in HG (P<0.05). Compared with the Cef+global brain ischemia group, obvious destruction of the CA1 hippocampus was observed in AS-ODNs(18 nmol)+Cef+global brain ischemia group, which was represented with the decrease in the value of ND, and increase in HG(P<0.05). The AS-ODNs in lower dose of 9 nmol and R-ODNs (18 nmol) had no effect on the neuronal protection of Cef. The results indicated that the GLT-1 AS-ODNs used in 18 nmol effectively inhibited the neuronal protective effect of Cef. 2 Cef increased the glutamate uptake of GLT-1 in the CA1 hippocampusGrouping and method: Eighty male Wistar (280–320 g in weight) with permanently occluded bilateral vertebral arteries rats were randomly assigned to five groups.(1) Sham group (n=10):The rats were subjected to sham operation for global brain ischemia, in which all procedures for the global brain ischemia was performed except for occluding the bilateral common carotid arteries. The animals were sacrificed by decapitation on 12 h and 24 h after the sham operation to observe the glutamate uptake.(2) Cef control group (n=10): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and subjected to the sham operation on 1 d after the last time of the injection. Other procedures were the same as those in sham group.(3) Global brain ischemia group (n=10): The rats were subjected to a global brain ischemia for 8 min, and then were reperfused. The animals were sacrificed by decapitation on 12 h and 24 h after the global brain ischemia operation to observe the glutamate uptake.(4) Cef+global brain ischemia group (n=10): The rats were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and were subjected to global brain ischemia for 8 min on 1 d after the last time of the injection with Cef. Other procedures were the same as those in global brain ischemia group. (5) AS-ODNs+Cef+global brain ischemia group (n=40):On the basis of the Cef+global brain ischemia group, 10μl GLT-1 AS-ODNs solution was injected into the right lateral ventricle at 36 h and 72 h after the first time of Cef injection, and at 12 h before the global brain ischemia. Two doses of 9 nmol and 18 nmol were used. Other procedures were the same as those in global brain ischemia group. Meanwhile, distilled water (solvent of AS-ODNs) control group (n=10) and R-ODNs (18 nmol) control group (n=10) were designed.[~3H]-glutamate was used to observe the glutamate uptake of GLT-1 in the CA1 hippocampus.Result: Compared with the sham group, glutamate uptake in the CA1 hippocampus significantly increased in the Cef control group (P<0.05), and no significant change in the uptake was found in global brain ischemia group. Compared with the global brain ischemia group, the glutamate uptake significantly increased in the Cef+global brain ischemia group (P<0.05). Compared with the Cef+global brain ischemia group, the glutamate uptake significantly decreased in AS-ODNs+Cef+ global brain ischemia group (P<0.05), especially in the larger dose of the AS-ODNs. While there was no difference in the glutamate uptake of GLT-1 in distilled water control group and R-ODNs control group.Conclusion:The preventive administration of Cef up-regulated the expression and glutamate uptake of GLT-1, and enhanced the tolerance of pyramidal neurons in the CA1 hippocampus to global brain ischemic insult. Administration of GLT-1 AS-ODNs significantly inhibited the above effects of Cef. The results above indicated that Cef enhanced the tolerance of pyramidal neurons in the CA1 hippocampus to ischemia through up-regulating the expression and glutamate uptake of GLT-1.