Ceftriaxone Protects Pyramidal Neurons Of The CA1 Hippocampus Against Delayed Neuronal Death Induced By Global Brain Ischemia By Up-regulating GLT-1

Objective: It has been well-known that the glial glutamate transporter-1 (GLT-1) plays a dominant role in maintaining the extracellular glutamate below neurotoxic levels and preventing the excitotoxicity of the increased glutamate. For example, the infusion of GLT-1 antisense oligonucleotides led to a significant increase in infarct volumes in rat middle cerebral artery occlusion model. Recently, we found that cerebral ischemic preconditioning protected pyramidal neurons of CA1 hippocampus against delayed neuronal death induced by global brain ischemia by up-regulating GLT-1 expression and function. The results of all the studies have presented evidence that up-regulating the expression and function of GLT-1 could protect cerebral neurons from ischemia. So modulating GLT-1 might provide a novel approach for therapy of ischemic cerebrovascular disease.Rothstein et al reported in Nature in 2005 that beta-lactam antibiotics such as Ceftriaxone (CTX) could increase the expression of GLT-1 and its glutamate uptake especially. Soon afterwards, Chu et al in 2007 reported that administration of CTX could diminish infarct volumes in rat middle cerebral artery occlusion model by up-regulating the expression of GLT-1. The results provided new evidence and clues for studying the neuronal protective effect of CTX against brain ischemia. However, there is no study to observe whether CTX could exert neuronal protection in model of global brain ischemia.Therefore, the present study was undertaken to observe the protective effect of CTX against delayed neuronal death (DND) in CA1 hippocampus after global brain ischemia using rat 4-vessel occlusion global brain ischemia model. Simultaneously, the expression of GLT-1 in CA1 hippocampus was assayed using methods of immunohistochemistry and Western blotting analysis to show the involvement of GLT-1 in the neuronal protection of CTX. The results to be obtained will provide new experimental evidence for the neuronal protection of CTX by up-regulating GLT-1 and provide new clues for study in the treatments of cerebrovascular disease.Methods: One hundred Wistar rats (270-320g) with permanently occluded bilateral vertebral arteries beforehand were randomly assigned to six groups:sham group (n=15): The sham operation, in which all procedures for brain ischemia were included except for occluding the bilateral common carotid arteries, was performed. The animals were sacrificed by decapitation on 1 d and 3 d after the sham operation (n=5 in each time point) to observe the expression of GLT-1, and on 7 d after the sham operation (n=5) to determine delayed neuronal death (DND) in the CA1 hippocampus.CTX control group (n=15): Intraperitoneal injection with CTX (200 mg/kg) once a day for 5 days before the sham operation, 5 times together. Other procedures were the same as those in sham group.Brain ischemia group (n=15): Global brain ischemia for 8 min was given and then the blood was reperfused. Other procedures were the same as those in sham group.CTX prevention group (n=35): Intraperitoneal injection with CTX once a day for 5 days before the global brain ischemia for 8min, 5 times together. This group was further divided into 50 mg/kg, 100 mg/kg, 200 mg/kg subgroups according to the doses of CTX. The animals were sacrificed by decapitation on 7 d after reperfusion to determine DND in the CA1 hippocampus (n=5 in each subgroup). Moreover, to observe the expression of GLT-1, another twenty animals assigned to 50 mg/kg and 200 mg/kg subgroups were sacrificed by decapitation on 1 d and 3 d after the global brain ischemia.CTX therapy group (n=15): Intraperitoneal injection with CTX once a day for 5 days began immediately after the global brain ischemia for 8 min, 5 times together. This group was further divided into 50 mg/kg, 100 mg/kg, 200 mg/kg subgroups according to the doses of CTX (n=5 in each subgroup). The animals were sacrificed by decapitation on 7 d after reperfusion to determine DND in the CA1 hippocampus.CTX prevention+therapy group (n=5): Intraperitoneal injection with CTX began on 5 days before the global brain ischemia for 8 min, and ended on 5 d after global brain ischemia for 8 min, 10 times together. The animals were sacrificed by decapitation on 7 d to determine DND in the CA1 hippocampus.Results:1 CTX attenuated DND of pyramidal neurons in the CA1 hippocampus normally induced by global brain ischemia It was found by thionin staining that there was no significant neuronal damage in the CA1 hippocampus in the sham and CTX (200 mg/kg) control groups. Obvious destruction of the CA1 hippocampus was found in brain ischemia group, the value of ND was decreased, and HG was increased compared with that in the sham group (P<0.05). The preventive administration of CTX in low dose of 50 mg/kg had little protective effect against the DND induced by global brain ischemia. However, following the increase in the dose of CTX used, especially in large dose of 200 mg/kg, the administration of CTX 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 in CTX prevention group (200 mg/kg) compared with brain ischemia group (P<0.05). Therapeutic administration of CTX in any doses used had no protective effect against the DND after global brain ischemia. The protective effect of combination of preventive and therapeutic administration of CTX was similar with that of preventive administration, which was represented with the similar ND and HG values in the preventive+therapeutic group compared with those in CTX (200 mg/kg) preventive group.2 CTX up-regulates expression of GLT-1 in the CA1 hippocampus in global brain ischemic rats Immunohisto- chemical staining showed that some fine GLT-1 immuno- reactive (IR) particles distributed in the CA1 hippocampus in sham group either 1 d or 3 d after the sham operation. Large amount of GLT-1 IR particles were found in the CA1 hippocampus in CTX (200 mg/kg) control group on 1 d and 3 d after the sham operation. The integral optical density (IOD), average optical density (AOD) and the area of GLT-1 IR staining significantly increased compared with the sham group (P<0.05). In brain ischemia group, especially on 3 d after global brain ischemia, there was large absence sheet of GLT-1 expression in the area where almost complete pyramidal neurons died and the neighboring area of the pyramidal layer. Preventive administration of CTX in low dose of 50 mg/kg had no influence on the expression of GLT-1 either 1 d or 3 d after global brain ischemia compared with sham group. However, in the large dose (200 mg/kg) of the CTX prevention group, large amount of GLT-1 IR particles distributed extensively in the CA1 hippocampus, the IOD, AOD and the area of GLT-1 IR staining significantly increased compared with sham group (P<0.05). Western blotting analysis showed the elemental expression of GLT-1 in the CA1 hippocampus in sham group on 1 d after the last treatment. Compared with the sham group, GLT-1 expression in the CA1 hippocampus significantly increased in the CTX (200 mg/kg) control group, which was represented with the increase in the ratio of the IOD of band for GLT-1 immunoblot to that ofβ-actin (P<0.05). There was no difference in the expression of GLT-1 in brain ischemia and CTX prevention group in the low dose CTX (50 mg/kg) compared with sham group. GLT-1 expression in the CA1 hippocampus conspicuously increased in CTX prevention group in the large dose (200 mg/kg), which was represented with the increase in the ratio of the IOD of band for GLT-1 immunoblot to that ofβ-actin (P<0.05). The expression of GLT-1 on 3 d after the last operation or treatment is similar to that on 1 d.Conclusion:1 The preventive administration of CTX exerted neuronal protective effect against DND of the CA1 hippocampus normally induced by global brain ischemia in the dose dependent manner.2 The preventive administration of CTX up-regulated the expression of GLT-1 in the CA1 hippocampus in rats subjected to global brain ischemia.3 The results of the above suggested that preventive administration of CTX has the neuronal protective effect against global brain ischemia, and that up-regulating the expression of GLT-1 might be one of the mechanisms involved in the neuronal protection of CTX.