Sulbactam Plays Neuronal Protective Effect Against Brain Ischemia By Up-regulating GLT-1in Rats

Glutamate is one of the primary small molecule neurotransmittersresponsible for fast excitatory signaling in the central nervous system. A largebody of data suggested that extracellular accumulation of exciting amino acids,particularly glutamate lead to neuronal death after brain ischemia. Theglutamate transporters distributed in both glial cells and neurons are the onlymechanism for reversal transporting glutamate from extracellular space tointracellular. To date, five types of cell membrane glutamate transporters havebeen cloned, which include EAAT1(GLAST), EAAT2(is also named as glialglutamate transpor-1, GLT-1), EAAT3(EAAC1), EAAT4and EAAT5. It hasbeen shown that GLT-1plays a dominant role for the long-term maintenanceof low and non-toxic concentrations of glutamate in extracellular space. Forexample, transient global ischemia down-regulated glutamate transporterfunction of hippocampal CA1astrocytes by decreasing mRNA and proteinlevels of GLT-1. Transgenic mice lacking GLT-1showed selective neuro-degeneration in the hippocampus, and exacerbation of acute cortical injury.In transient middle cerebral artery occlusion rats, antisense knockdown ofGLT-1exacerbated the ischemic infarct volume and neuronal damage incerebral cortex and striatum. Our recent study found that cerebral ischemicpreconditioning up-regulated the expression and uptake activity of GLT-1andGLT-1a, one of the splice variants of GLT-1, in the CA1hippocampus. Bothinhibiting function of GLT-1with Dihydrokainate (DHK), a non-transportableblocker of GLT-1, and reducing the expression of GLT-1by GLT-1antisenseoligodeoxy-nucleotides (AS-ODNs) could block brain ischemic toleranceinduced by cerebral ischemic preconditioning. These findings indicate thatup-regulating GLT-1expression and its uptake activity might be a new targetin the prevention and treatment of cerebral ischemia diseases. Rothstein et al reported in Nature that β-lactam antibiotics, such asceftriaxone, cephapinin can cross the blood-brain barrier, enter the centralnervous system including the brain and spinal cord, and greatly and selectivelypromote GLT-1expression and function. Ceftriaxone either pre-orpost-administration in middle cerebral artery occlusion animals improved theneurological performance and neuronal survival by up-regulating GLT-1expression. These findings suggested a new possibility to use ceftriaxone forprevention and treatment of brain ischemic diseases.However, it will be faced with many problems such as the excessive doses,dysbacteriosis and bacterial resistance to use ceftriaxone for the preventionand treatment of brain ischemia in clinic. Sulbactam is an atypical β-lactamantibiotic with little anti-bacterial capacity, and is usually used in combinationwith either ampicillin or cefoperazone in clinical medicine to potentiate theanti-bacterial effect of the β-lactam antibiotics by inhibiting serineβ-lactamases. Because of the similarity in the structure with ceftriaxone,sulbactam may have similar effect on GLT-1expression and uptake activitywith ceftriaxone. Especially, it may be promising to use sulbactam in theprevention and treatment of cerebral ischemic diseases because of littleanti-bacterial capability, and then the little side effects resulted fromanti-bacterial effect of sulbactam, if the protective effect of the medicineagainst cerebral ischemia can be elucidated. Therefore, the present study wasundertaken to investigate whether sulbactam is able to against delayedneuronal death (DND) in the CA1hippocampus after brain ischemia andwhether the protective effect of sulbactam is mediated by regulating theexpression and uptake activity of GLT-1.1Sulbactam protects hippocampal neurons against brain ischemiaThe effect of sulbactam on the delayed neuronal death (DND) ofpyramidal neurons in the CA1hippocampus induced by global brain ischemiain rats was investigated in order to provide evidence to elucidate the protectiveeffect of sulbactam against brain ischemia. Methods：Male Wistar rats (280-320g) provided by The Experimental AnimalCenter of Hebei Medical University were used in this study. The rats weredivided into five groups:(1) Sham group: The rats were subjected to the sham operation for globalbrain ischemia.(2) Sulbactam control group: The rats were administered with sulbactam(180nmol) once a day for5days before the sham operation. Other procedureswere the same as those in sham group.(3) Brain ischemia group: The rats were subjected to global brain ischemiafor8min.(4) Sulbactam prevention group: The rats were first administered withsulbactam once a day for5days, and then subjected to the brain ischemia1dafter the last time of the administration. Other procedures were the same asthose in brain ischemia group. This group was further divided into20nmol,60nmol and180nmol subgroups according to the dose of sulbactam. Meanwhile,a normal saline (NS) group was designed as vehicle control.(5) Sulbactam therapy group: The rats were subjected to the brain ischemia,and then administered with sulbactam once a day for5days. The first time ofthe administration was performed immediately after the brain ischemiaoperation. Other procedures were the same as those in sulbactam preventiongroup.Sulbactam was administered by intra-cerebroventricular injection via astainless steel cannula implanted in the right lateral ventricle. Neuro-pathological evaluation under thionin stain was performed7d after the shamoperation or global brain ischemia in each group. The evaluation waspresented by histological grade (HG) and neuronal density (ND) to reflect thecontent of DND of pyramidal neurons in the CA1hippocampus.Results:Neuropathological evaluation indicated that sulbactam reduced DND ofpyramidal neurons in the CA1hippocampus normally induced by global brainischemia. In sham group, the pyramidal neurons were arranged in order with2 to3cell layers, the outline of the neurons was intact, nucleus was full andnucleolus was clear. The histological grade (HG) was0and neuronal density(ND) was216±17.8mm-1. The histological characteristics in vehicle andsulbactam control groups were similar to those in the sham group. Brainischemia for8min induced obvious DND which resulted in large areas absentneurons in the CA1subfield, and the significant increase in HG to grade Ⅱ～Ⅲ and decrease in ND (P<0.05). Preventive administration of sulbactam insulbactam prevention group effectively prevented the DND normally inducedby global brain ischemia in a dose dependent manner, which was representedwith the increased survival of pyramidal neurons, ND values and decreasedHG following the increase in the dose of sulbactam used(P<0.05). Thehistological characteristics of the CA1hippocampi in the180nmol sulbactamprevention group were similar to those in the sham group. Similar neuronalprotective effects were also observed in sulbactam therapy group in which thesulbactam was administered after the brain ischemia, while the magnitude ofthe protective effect was smaller than that in sulbactam pre-treatmentprotocols even in the largest dose of180nmol. Administration of vehicle ofsulbactam either before or after the brain ischemia had no effects on the DNDinduced by brain ischemia.The above findings indicated that sulbactam could protect the pyramidalneurons against global brain ischemia.2Sulbactam inhibits the production of inflammatory factors in thehippocampus induced by global brain ischemiaThe effect of sulbactam on the production of IL-1β and TNF-α in the CA1hippocampus were investigated in global brain ischemia rats to provide furtherevidence to elucidate the neuronal protection of sulbactam against brainischemia because these inflammatory factors are closely associated withcerebral ischemic injury.Methods:Rat grouping was the same as part1. The dynamic changes in the mRNAexpression and protein production of IL-1β and TNF-α in the CA1 hippocampus were investigated by real-time PCR and ELISA, respectively, attime points of0h,3h,6h,12h,1d,2d and3d after the sham operation orbrain ischemia in each group (n=3in each time point).Results:The real-time PCR analysis indicated that there was basal expression ofIL-1β and TNF-α mRNA in the CA1hippocampus in sham rats. The vehiclecontrol and the sulbactam control groups had similar expression with shamgroup. Compared with sham group, the expressions of both IL-1β and TNF-αmRNA were significantly up-regulated in brain ischemia group6h after theischemia insult (P<0.05). This up-regulation lasted to1d after the ischemicinsult and then returned to sham levels. Pre-treatment with sulbactam insulbactam prevention group significantly inhibited the up-regulation in theexpressions of IL-1β and TNF-α mRNA normally induced by brain ischemiain a dose dependent manner (P<0.05). However, post-treatment withsulbactam had little effect on the expressions in any dose. Meanwhile,administration of vehicle of sulbactam either before or after the brain ischemiahad no effects on the sulbactam-induced changes in the expressions of IL-1βand TNF-α mRNA.Consistent with the changes in the mRNA levels, the ELISA analysisindicated that sulbactam pre-treatment significantly decreased the productionof both IL-1β and TNF-α protein. IL-1β and TNF-α protein contents weredetected in a low level in sham group. The vehicle control and the sulbactamcontrol groups had similar reaction with sham group. The levels of both IL-1βand TNF-α protein were increased1day and lasted at high levels to2day aftercerebral ischemia (P<0.05), and then returned to sham levels. When the ratswere preventively administered with sulbactam, the peak value of theproduction of IL-1β and TNF-α protein had a sharp decline (P<0.05),especially in the largest dose of180nmol. In contrast, therapeuticadministration of sulbactam had little effect on the levels in any dose.Meanwhile, administration of vehicle of sulbactam either before or after thebrain ischemia had no effects on the sulbactam-induced changes in the expressions of IL-1β and TNF-α protein.The above findings indicated that pre-treatment with sulbactam down-regulated the production of the inflammatory factors of IL-1β and TNF-α inthe hippocampus, which aggravate cerebral ischemic injury.3Sulbactam up-regulated the expression of GLT-1in the CA1hippocampus in global brain ischemic ratsThe effect of sulbactam on the GLT-1expression in the CA1hippocampusin global brain ischemia rats was investigated in a pre-treatment protocol ofsulbactam in order to elucidate the role of GLT-1in the neuronal protection ofsulbactam against brain ischemia.Methods:Rat grouping was the same as above group (1) to (4) in the part1. TheGLT-1mRNA level was detected with Semi-quantitative RT-PCR andquantitative (real time) RT-PCR analysis at time points of0h,6h,12h,1dand3d after the sham operation or brain ischemia in each group (n=3in eachtime point). The expression of GLT-1protein was assayed with Western blotanalysis at time points of0h,12h,1d,3d,5d, and7d, and withimmunohistochemistry at time point of7d after the sham operation or brainischemia (n=3in each time point).Results:First, semi-quantitative RT-PCR analysis indicated that GLT-1mRNAwas significantly up-regulated in sulbactam control group compared withsham group (P<0.05) at the observed time points from0hour to2day. Theexpression of GLT-1mRNA was significantly down-regulated in brainischemia group compared with sham group. The down-regulation began fromthe time points of1st day, and lasted to2nd day. When pre-treatment withsulbactam especially in large dose of180nmol significantly up-regulated andreversed the down-regulation of the GLT-1mRNA expression (P<0.05). Theabove effect of sulbactam showed a dose dependent manner. The real-timeRT-PCR analysis showed similar results with the semi-quantitative RT-PCRanalysis. Meanwhile, administration of vehicle of sulbactam had no effects on the GLT-1mRNA expressions compared with brain ischemia group in eithersemi-or quantitative RT-PCR analysis.Western blot analysis showed that the GLT-1protein expression wassignificantly up-regulated in sulbactam control group compared with shamgroup at each time point (P<0.05). Brain ischemic insult significantlydown-regulated the expression compared with sham group. Thedown-regulation began from the time points of3d, and lasted to7day, thelatest day observed, after the brain ischemia (P<0.05). The pre-treatment withsulbactam especially in large dose of180nmol significantly up-regulated andreversed the down-regulation of the GLT-1protein expression inducednormally by the brain ischemia in each time points observed (P<0.05).Administration of vehicle of sulbactam had no effects on the GLT-1proteinexpressions either in sham or brain ischemic rats.Immunohistochemistry assay showed that there were brown, weak, anddiffuse immunoparticles distributed in the peri-pyramidal neuronal area in theCA1hippocampus in the Sham group. Compared with sham group, theintensity of the immunoreactivity was significantly increased in the sulbactamcontrol group (P<0.05). It was represented by the results that a lot of GLT-1immunoreactive particles were observed in the area between the pyramidalneurons, which tightly surrounded pyramidal neurons and made the pyramidallayer looked like “shaped grade”. The integral optical density (IOD) wasincreased significantly (P<0.05). The GLT-1expression was markedlydecreased after the lethal brain ischemic insult for8min in the ischemia group,especially in the area where almost all pyramidal neurons died and in theneighboring areas of the pyramidal layers (P<0.05). It was appeared as a sheetabsence of GLT-1immunoreactivity particles. Compared with brain ischemicgroup, pre-treatment with sulbactam especially in the large dose of180nmolsignificantly up-regulated the GLT-1expression, which made theimmunostaining characteristics in the group to be similar with sulbactamcontrol group.The above findings suggested that it might be the up-regulating effect on GLT-1expression that contributed to the neuronal protection of sulbactamagainst brain ischemia.4Inhibition of GLT-1attenuated the neuronal protection of sulbactamagainst brain ischemiaIn order to convincingly elucidate the role of GLT-1up-regulation in theneuronal protection of sulbactam against brain ischemia, the effect ofinhibiting expression and the uptake activity of GLT-1with GLT-1AS-ODNsor DHK on the neuronal protection of sulbactam against brain ischemia wereinvestigated in global brain ischemia rats.Methods:Based on the above (1)-(4) groups in Part1, another four groups weredesignated as follows:(5) AS-ODNs+sulbactam prevention group: The GLT-1AS-ODNs wereadministered once a day for4times from2days before to1day after the brainischemia. Meanwhile, a double distilled water group was designed as a vehiclecontrol. Other procedures were the same as those in sulbactam preventiongroup.(6) AS-ODNs control group: The GLT-1AS-ODNs were administered tosham rats in the same protocols as those in AS-ODNs+sulbactam preventiongroup. Other procedures were the same as those in sham group.(7) DHK+sulbactam prevention group: The rats were administered withDHK solution30min before the brain ischemia. Other procedures were thesame as those in sulbactam prevention group.(8) DHK control group: The DHK solution was administered to shamrats in the same protocols as those in DHK+sulbactam prevention group.Other procedures were the same as those in sham group.Western blot analysis were first performed at time points of3and7daysin (1)-(6)(n=3in each time point) in order to identify the inhibitory effect ofthe GLT-1AS-ODNs on the expression of GLT-1. Neuropathologicalevaluation was performed on7d after the sham operation or brain ischemia todetermine DND in the CA1hippocampus in all groups (n=5). Results:Western blot analysis showed that the administration of AS-ODNsinhibited the expression of GLT-1in the CA1hippocampus in sham group.Brain ischemia insult significantly down-regulated the expression of GLT-1.The pre-treatment with sulbactam significantly up-regulated and reversed thedown-regulation of the GLT-1protein expression induced normally by thebrain ischemia (P<0.05). After administration of GLT-1AS-ODNs, theup-regulation of the GLT-1expression induced by sulbactam was inhibitedmarkedly (P<0.05), which made the upregulation of the GLT-1expressioninduced by sulbactam pretreatment fallen to brain ischemic level inAS-ODNs+sulbactam+prevention group. However, the vehicle of AS-ODNshad no effect on the up-regulation induced by sulbactam in ischemic rats.Based on the above confirmation for the inhibitory effect of the GLT-1AS-ODNs on the GLT-1expression, we performed neuropathologicalevaluation to investigate the effect of GLT-1AS-ODNs on the neuronalprotective effect of sulbactam against brain ischemia. It was shown that thebrain ischemia induced obvious DND of pyramidal neurons in the CA1hippocampus, and the DND could significantly prevented by the pretreatmentwith sulbactam in sulbactam prevention group. When the animals werepre-administered with AS-ODNs, the neuronal protective effect of sulbactamagainst DND was significantly inhibited, which was represented with theheavy loss of pyramidal neurons appeared as large area absent of pyramidalneurons, the increase in HG and decrease in ND in AS-ODNs+sulbactamprevention group compared with sulbactam+prevention group (P<0.05). Thevehicle of AS-ODNs had no effect on the neuronal protective effect ofsulbactam against DND induced by brain ischemia, and AS-ODNs alone didnot induce DND in sham rats. DHK showed similar results with AS-ODNs inDHK+sulbactam prevention group.The above findings indicated that it was the up-regulating effect on GLT-1that contributed to the neuronal protective effects of sulbactam against brainischemia. Conclusions:1. The administration of sulbactam protected pyramidal neurons againstDND and down-regulated the expression of IL-1β and TNF-α in the CA1hippocampus normally induced by global brain ischemia.2. The administration of sulbactam up-regulated the expression of GLT-1in the CA1hippocampus companied with the neuronal protective effect.3. Inhibiting the expression and uptake activity of GLT-1with GLT-1AS-ODNs or DHK attenuated the neuronal protection of sulbactam againstDND of pyramidal neurons in the CA1hippocampus normally induced byglobal brain ischemia.4. The above findings indicated that sulbactam plays neuronal protectiveeffect against brain ischemia by up-regulating GLT-1.