| Glutamate?Glu?represents the neurotransmitter distributed most widely in the central nervous system and is involved in numerous physiological functions of the central nervous system.When glutamatergic neurons are depolarized upon stimulation,the glutamate is released into the synaptic cleft.The concentration of glutamate in synaptic cleft transiently elevates from several micromolar to several millimolar and activates glutamate receptors,thereby exerting physiological functions.When the glutamate concentration in the synaptic cleft increases aberrantly in certain pathological conditions,the ionic and metabotropic glutamate receptors in the postsynaptic membrane will be excessively activated,which causes influx of a large number of Ca2+and Na+ions,then triggering activation of the intracellular signal cascade.The disorder of the internal environment will induce a series of pathological processes,including the destruction of neuronal membrane structure,the release of intracellular components,local inflammatory reactive edema and even necrosis or apoptosis,which belongs to the excitatory toxicity of glutamate.Because there is no glutamate metabolic enzyme in the synaptic cleft,glutamate in the gap must be timely cleared from the synaptic cleft by the glutamate transporter.Among all types of glutamate transporters,glial glutamate transporter-1?GLT-1?clears most of the glutamate released in the cortex and hippocampus.About 80%of the glutamate transporter expressed in the hippocampus is GLT-1.Up-regulating the clearance ability of glutamate transporters under pathological conditions,increasing the expression and function of GLT-1 can thus be used as a target for the prevention and treatment of cerebral ischemic diseases.Studies have demonstrated that?-lactam antibiotics,such as ceftriaxone,can upregulate the expression and uptaking activity of GLT-1.Previous studies in our department have also shown that ceftriaxone exerts an effect of anti-ischemic brain damage by up-regulating the expression of GLT-1.However,because ceftriaxone is a first-line potent antibiotic,administration in long-term and large dose will induce many side effects such as dysbacteriosis,bacterial resistance and other side effects,which severely limit its application as anti-cerebral ischemic medication.Sulbactam belongs to atypical?-lactam antibiotics and is structurally similar to ceftriaxone,but its antibacterial effect is very weak compared with that of ceftriaxone,In the clinic sulbactam is generally used as an antibiotic medicine,but rarely used alone as an antibiotic Given the similarity of the structure of sulbactam with ceftriaxone,sulbactam may exert a similar neuroprotective effect as ceftriaxone by up-regulating the expression and function of GLT-1.More importantly,almost without antibacterial activities,sulbactam has no related side effects such as dysbacteriosis.Therefore,our department conducted a series of studies regarding the anti-ischemic injury effects of sulbactam,showing that sulbactam,like ceftriaxone,exerts an anti-ischemic brain damage effect through up-regulating the expression of GLT-1.However,it is remained to be clarified whether sulbactam improves the function of GLT-1,including its binding characteristics and its uptake activity for glutamate,and whether these changes of GLT-1 can affect the concentration of glutamic acid in the brain extracellular fluid during cerebral ischemia.In these regards,the present study was carried out to examine the GLT-1binding characteristics and glutamate uptake of hippocampus in rats with global cerebral ischemia and reperfusion with radioligand binding assay?L-3H-glutamate labeling?.On this basis,the effect of these changes on extracellular glutamate concentration in brain tissue was further detected using intracerebralmicrodialysisandhigh-performanceliquid-phasemass spectrometry in the same rat model.It aimed to provide further experimental evidence for the role of GLT-1 in the anti-ischemic brain damage of sulbactam,thereby coming up with new clues and ideas for the clinical research on the prevention and treatment of cerebral ischemic diseases.Part one The effect of sulbactam on GLT-1 binding characteristics in thehippocampal CA1 region in global cerebral ischemic ratObjective:To investigate the effect of sulbactam on the binding characteristics of GLT-1,including maximal binding and affinity,during the global cerebral ischemia,and then to elucadate the effect of sulbactam on GLT-1 function during the global cerebral ischemia.Method:The radioligand binding experiments were performed using3H-labeled glutamate?L-3H-glutamate?to investigate the changes in the binding properties of GLT-1 after the treatment of sulbactam in rat global cerebral ischemic model following the confirmation that sulbactam exerts anti-cerebral cerebral ischemic effect using neuropathological evaluation method.The wistar rats were randomly divided into the following 7 groups.11rats were included in each group,and 6 were used for the determination of the binding properties of GLT-1 and 5 were used for neuropathological evaluation.1.Sham control group:The rats were continuously injected with 10?l of normal saline?NS?into the lateral cerebral ventricle once a day for 5 days.Sham operation was performed 24 h after the last injection.2.Brain ischemia group:The rats were injected with 10?l of NS according to the same protocols as those in the sham group.The global cerebral ischemia for 8 min was performed after 24 h after the last injection.3.Sulbactam control group:The rats were injected with 10?l?180 nmol,the same below?of the sulbactam solution into the lateral cerebral ventricle once a day for 5 days,and sham operation for global cerebral ischemia was performed 24 h after the last injection.According to the previous study of Cui Xin et al.from our department,we selected the effective dose of 180 nmol sulbactam.4.Sulbactam prevention group:The rats were injected with 10?l of sulbactam solution in the same protocols as those in the Sulbactam control group,and global cerebral ischemia for 8 min was performed 24 h after the last injection,and then brain was reperfused.5.Sulbactam prevention+GLT-1 AS-ODNs group:On the basis of the sulbactam prevention group,36 h,72 h after the first injection of sulbactam and 12 h before the global cerebral ischemia,the rats was injected with 18nmol of GLT-1 AS-ODNs which were dissolved in double-distilled water,into the lateral cerebral ventricle.At the same time,R-ODNs control group?Sulbactam prevention+R-ODNs group?was set up,and R-ODNs?18 nmol?were used instead of AS-ODNs.The others were same as sulbactam prevention group and GLT-1 AS-ODNs group.6.GLT-1 AS-ODNs control group:On the basis of the sham group,36 h and 72 h after the first injection of NS and 12 h before the sham operation for global cerebral ischemia,10?l?18 nmol?of GLT-1 AS-ODNs solution was injected into the lateral cerebral ventricle,and other treatments were the same with those of sham groups.Rats were decapitated and the hippocampal CA1 region was dissected at the specified time points,i.e.0 h,6 h,12 h,1 d,2 d and 3 d after the global cerebral ischemia for the determination of the binding properties of GLT-1,and on 7 d for pathological evaluation.The maximum binding amount?Bmax value?and affinity?Kd value?of the GLT-1 transporter were measured using radioactive ligand binding method.The larger the Bmax value,the greater the number of GLT-1 transporters;the smaller the Kd value,the higher the affinity of the GLT-1 transporter.Result:1.Changes of Bmax valueCompared with the sham group,the Bmax value of the sulbactam control group was significantly increased?P<0.05?,while was significantly decreased in the GLT-1 AS-ODNs control group?P<0.05?.After 8 min ischemia in the global cerebral ischemia group,the Bmax value showed a down-regulating trend compared with the time point immediately after the ischemia.The decrease during 48 h-72 h was more severe,and most strikingly at 72 h?P<0.05 vs 0 h,6 h??Compared with the sham group,the Bmax values at each time point were significantly diminished?P<0.05?.In sulbactam prevention group,with the extension of time after ischemia,the Bmax lowered gradually at 6 h,12 h,24 h compared with the time point immediately after the ischemia?P<0.05,vs 0 h?,and then there was a gradual recovery process during 48 h-72 h.The Bmax value was significant up-regulated at 72 h compared with the 24 h time point?P<0.05,vs 24 h?.Compared with the Brain ischemia group,the Bmax value in the sulbactam prevention group significantly increased at each time point?P<0.05?.In the sulbactam prevention+GLT-1 AS-ODNs group,Bmax values gradually decreased?P<0.05,vs 0 h?at each time point with prolonged ischemia time compared with that at the time point immediately after the ischemia.Compared with the sulbactam prevention group,Bmax values diminished significantly at each time point?P<0.05?,which indicated the ability of sulbactam to upregulate GLT-1 and Bmax was inhibited by GLT-1AS-ODNs.The Bmax values in the sulbactam prevention+GLT-1 R-ODNs group had no significant changes compared with the those of sulbactam prevention group?P>0.05?.2.Change in KdCompared with the sham group,there was no significant change in the Kd values of the sulbactam control group and the GLT-1 AS-ODNs control group at each time point observed?P>0.05?.In the global cerebral ischemic group,the Kd value at each time point showed a significant enhancement compared with that at the immediate time point after ischemia,and the most significant increase occurred at 24 h-48 h.Compared with the sham group,the Kd values at all time points were significantly up-regulated compared with those of the sham group?P<0.05?.In the sulbactam prevention group,the change trend of Kd value at each time point was the same as that in the global cerebral ischemia group.The Kd values at all time points were significantly decreased compared with the global cerebral ischemia group?P<0.05?.There was no significant difference in Kd values at each time point compared with those at the immediate time point after ischemia in sulbactam prevention+GLT-1 AS-ODNs group.Compared with sulbactam-prevention group,the Kd values were significantly increased at each time point?P<0.05?.There was no difference of Kd value between the sulbactam prevention+GLT-1 R-ODNs group and the sulbactam prevention group.3.Neuropathological evaluationThe pyramidal cells in the hippocampal CA1 region of the sham group had an intact morphology,no cell loss,and were arranged neatly and clearly.The nucleus is large and round,located in the center of the cell,with clear nucleoli and rich Nissl bodies.The morphology of pyramidal cells in hippocampal CA1 region of sulbactam control group was basically the same as that of sham group.In the global cerebral ischemic group,the pyramidal cells in the CA1hippocampus were sparse and disordered,with a large number of cell fragments,and obvious neuron loss.Compared with sham group,histological grade significantly elevated?P<0.05?,and neurnal density significantly diminished?P<0.05?.In the sulbactam prevention group,pyramidal cells in hippocampal CA1 region were not significantly damaged and the morphology of the CA1 was similar to that in sham group.Compared with the global cerebral ischemic group,The histological grade was significantly decreased?P<0.05?and the neuronal density was significantly enhanced?P<0.05?,indicating that sulbactam has the effect of anti-global cerebral ischemia and delaying neuronal death.The morphology of the hippocampal CA1 region in the sulbactam prevention+GLT-1 AS-ODNs group was generally similar as that in the global cerebral ischemia group,and cell swelling,disintegration,fragmentation and nuclear condensation were also observed.Compared with the sulbactam prevention group,histological grade of this group had significantly increased?P<0.05?and neurnal density decreased?P<0.05?,indicating that GLT-1AS-ODNs inhibited the expression of GLT-1 and reduced the protection of sulbactam against pyramidal neurons in hippocampal CA1 region.The morphology of GLT-1 AS-ODNs control group rat hippocampal CA1 tissue were basically the same as the sham group,indicating that lateral ventricle injection of GLT-1 AS-ODNs did not damage the hippocampal CA1 neurons.The histological morphology of hippocampal CA1 in the sulbactam prevention+GLT-1 R-ODNs control group and the sulbactam prevention group were basically the same.The above results therefore demonstrate that the intracerebroventricular injection of GLT-1 AS-ODNs can inhibit the neuroprotective effect of sulbactam against cerebral ischemic injury.Summary:The above results indicate that sulbactam can improve the maximum binding capacity of GLT-1 in rats with global cerebral ischemia and enhance its affinity with glutamate.Part twoThe effect of sulbactam on glutamate uptake of GLT-1 inhippocampal CA1 region in rats with global cerebral ischemiaObjective:To observe the effect of sulbactam on GLT-1 uptake for glutamate in hippocampal CA1 region,and then to investigate the effect of sulbactam on GLT-1 function during the global cerebral ischemia.Method:Experimental animals and groups are the same as the first part.Rats were decapitated at determined time points:0 h,6 h,12 h,1 d,2 d and3 d.A cell suspension of the hippacampal CA1 region was prepared and the glutamate uptake of the hippocampal CA1 cell was measured using 3H-labeled glutamate?L-3H-glutamate?.Glutamate uptake is equal to total uptake minus nonspecific uptake.Cerebral specimens used for neuropathological evaluation were collected on 7 day after the global brain ischemia.Result:1.Changes of glutamate uptakeCompared with the sham group,GLT-1 significantly up-regulated glutamate uptake in the sulbactam control group,suggesting that glutamate uptake by GLT-1 was significantly increased by lateral ventricular injection of sulbactam?P<0.05?.In GLT-1 AS-ODNs control group,GLT-1 significantly suppressed glutamate uptake?P<0.05,vs sham?,indicating that GLT-1AS-ODNs significantly inhibited the GLT-1 expression.The uptake of glutamate in the brain ischemia group showed a decreasing trend,which was the lowest in 72 h in this experiment?P<0.05,vs 0 h?.Compared with sham group,the uptake of glutamate gradually diminished with the prolongation of the time after ischemia?P<0.05?.In the sulbactam prevention group,there was a downward trend between0-24 h?P<0.05,vs 0 h?,and the uptake increased significantly from 48 h to 72h,and at 72 h time point,it was significantly higher than the 24 h uptake?P<0.05,vs 24 h?.Compared with the global cerebral ischemia group,glutamate uptake by GLT-1 was significantly up-regulated at each time point in the sulbactam prevention group?P<0.05?.Compared to the sulbactam prevention group,glutamate uptake by GLT-1was significantly diminished in the sulbactam prevention+GLT-1 AS-ODNs group?P<0.05?,and it was still decreasing during the period of 48 h-72h?P<0.05,vs 0 h?,indicating that AS-ODNs inhibited the up-regulation of GLT-1 expression and glutamate uptake caused by sulbactam.The sulbactam prevention+R-ODNs?18 nmol?group was basically consistent with the sulbactam prevention group,indicating that AS-ODNs were effective on inhibiting the expression of GLT-1.2.Neuropathological evaluationSame results as the first part.3.Prophylactic administration of sulbactam can effectively reduce the delayed neuronal death in hippocampal CA1 pyramidal neurons and significantly increase the uptake of glutamate by GLT-1 in the rat hippocampal CA1 region.The application of GLT-1 AS-ODNs can significantly inhibit the protective effect of sulbactam on neurons in rats with global cerebral ischemiaSummary:These results indicate that sulbactam significantly enhance the uptake of glutamate by GLT-1 in hippocampal CA1 region of rats with global cerebral ischemia.Part threeThe effect of sulbactam on glutamate concentration in braindialysate of hippocampal CA1 region in rats with global cerebral ischemiaObjective:To investigate the effect of sulbactam on the concentration of glutamate in brain microdialysate of hippocampal CA1 region,and then to elucidate the protective effect of sulbactam against brain ischemia.Method:Experimental animals and groups are the same as those in the part 1.Brain microdialysis was started 3 h before global cerebral ischemia in each group and samples were collected at the determined time points.The concentration of glutamate in the microdialysates was analyzed by high performance liquid chromatography combined with mass spectrometry.The neuropathological evaluation was performed 7 day after global cerebral ischemia.Result:1.The isolation of amino acids.Under the experimental conditions,glutamate and internal standard were separated within 6 min.The peak time was as follows:Asp 1.41 min,Glu 1.50min,and Nor 5.25 min.The peak shapes of three amino acid were good,no overlap,double Peaks,trailing,etc.?Fig.1?.Other miscellaneous peaks in hippocampal dialysis fluid samples can also be completely separated from the amino acids to be tested.2.Changes of glutamate concentrationThe concentration of glutamate in the brain microdialysate of the sham group was at a low level,approximately 2.25±0.361?mol/L,which had no significant change at any time.In the sulbactam control group,the glutamate concentration did not substantially change.In the global cerebral ischemia group,glutamate concentration was up-regulated significantly.Elevation occurred at the onset of ischemia and rapidly increased to approximately 7.5 times the baseline value.After restoration of blood perfusion,it returned to basal levels around 16 minutes.In the sulbactam prevention group,sulbactam significantly inhibited the increase in glutamate concentration which was induced by global ischemia,and its peak value was reduced to approximately 3 times of the baseline concentration,which was significantly lower than that in the global cerebral ischemia group?P<0.05?.In the sulbactam prevention+GLT-1 AS-ODNs group,the highest concentration of glutamate reached up to 5.7 times of the basal value after ischemia,which was significantly higher than that of the sulbactam prevention group.The effect of sulbactam to reduce the intracellular glutamate concentration during ischemia can be significantly inhibited by GLT-1AS-ODNs.However,there was no such change in the sulbactam prevention+GLT-1 R-ODNs,indicating that the selected GLT-1 AS-ODNs were effective and significantly inhibited the expression of GLT-1.In the GLT-1 AS-ODNs control group,the basal concentration of glutamate increased slightly,which was significantly different from that in the sham group,indicating that 3 consecutive injections could effectively inhibit the expression of GLT-1.3.Neuropathological evaluationSame results as the first part.Summary:The above results indicate that sulbactam can suppress the increase of extracellular glutamate concentration caused by cerebral ischemia,thereby reducing its excitatory neurotoxicity.Conclusion:1.Sulbactam improves the maximum binding capacity of GLT-1 in rats with global cerebral ischemia and enhance its affinity with glutamate.2.Sulbactam significantly increases the GLT-1 uptake activity for glutamate in hippocampal CA1 region of rats with global cerebral ischemia.3.Sulbactaminhibitstheincreaseofextracellularglutamate concentration caused by cerebral ischemia,thereby reducing its excitatory neurotoxic effects.The above results demonstrate that sulbactam can play neuronal protective effect against cerebral ischemic insult by up-regulating the functions of GLT-1 and then reducing the glutamate concentration during global cerebral ischemic insult. |
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