The Role Of GLT-1in The Intermittent Hypobaric Hypoxia Preconditioning And Cerebral Ischemic Preconditioning

Once or more transient sublethal cerebral ischemia could stimulate thebody to produce endogenous protective mechanism, so as to reduce thedamage induced by subsequent serious ischemia. That is named as brainischemic tolerance (BIT). Recent studies found that not only the ischemia, avariety of other pretreatment methods, such as hypoxia, high temperature,chemical drugs can also induce BIT. Therefore, the present study wasundertaken to clarify whether GLT-1participates in the induction of brainischemic tolerance by intermittent hypobaric hypoxia (IH) preconditioningand cerebral ischemic preconditioning (CIP).IH, repeated episodes of hypobaric hypoxia interspersed with normoxicperiods, has long been used for treatment and prevention of human diseasessuch as hypertension, ischemic coronary artery diseases, Parkinson's disease,and acute myeloid leukemia. Although a few studies involved in themechanisms of the brain ischemic tolerance induced by IH preconditioning,the details are not clear until now. For example, Zhu et al found that IHpromotes hippocampal neurogenesis and produces antidepressant-like effectsin adult rats. Abellan et al observed that serum EPO significantly increasedafter intermittent hypoxia exposure. In addition, Rybnikova et al showed thatIH preconditioning significantly decreased the apoptotic damage of neurons inrat hippocampus and neocortex induced by severe hypobaric hypoxia, andprevented the behavioral abnormalities and detrimental learning and memoryimpairments caused by severe hypoxia. Based on the above, we couldreasonably think that the brain ischemic tolerance could be induced by IHpreconditioning. Previous studies have confirmed that GLT-1participated inthe brain ischemic tolerance induced by cerebral ischemic preconditioning,and the higher expression of GLT-1in the CA3and dentate gyrus contributed to their inherent resistance to ischemia. Considering the importance of GLT-1in the acquisition of brain ischemic tolerance induced by cerebral ischemicpreconditioning in rats, it is reasonable to hypothesize that GLT-1may play animportant role in the acquisition of brain ischemic tolerance induced by IHpreconditioning. Therefore, the present study was undertaken to clarifywhether GLT-1participated in the induction of brain ischemic tolerance by IHpreconditioning.Cerebral ischemic injury reduced the uptake of glutamate (Glu) by GLT-1,leading to significant increase of extracellular glutamate in the hippocampus.At the same time, the concentration of aspartate also increased. The inhibitoryneurotransmitter γ-aminobutyric acid (GABA) has a significantneuroprotective effect. For example, stroke induced GABA increased andsustained in high concentration, which attenuated the ischemic damage tobrain tissue. In the preasent study, combined with microdialysis technique andHPLC, the changes of amino acid neurotransmitters were observed in brain inthe induction of BIT induced by cerebral ischemic preconditioning (CIP).Theinfluence of DHK, a specific inhibitor of GLT-1, on amino acidneurotransmitters in the induction of BIT was observed, which couldeffectively reflect the changes of the function of GLT-1, and further confirmthat GLT-1play a role in the induction of BIT.Peroxisome proliferator-activated receptor (PPARγ) pathway is widelyregarded as the self-defense system in central nervous system, which plays arole in endogenous protection and attenuates the cerebral ischemia-reperfusioninjury. GLT-1is one of the target genes of PPARγ. Rosiglitazone, an agonist ofPPARγ, can increase the expression of GLT-1mRNA and its protein, andreduce severe oxygen glucose deprivation-induced neuronal death andglutamate release. Therefore, we presume that CIP might attenuateischemia-reperfusion injury by up-regulation GLT-1expression via PPARγsignaling pathway. Previous study proved that CIP caused the up-regulating ofGLT-1via p38MAPK during the induction of BIT, and the nucleartranscription factor-kappa B (NF-κB) is one of the downstream signaling molecules of p38MAPK. Several studies have demonstrated that NF-κBinvolved in the regulation of the GLT-1expression and played an importantrole in cerebral ischemia. For example, Ghosh et al found that neuronsregulated astrocyte GLT-1transcription via NF-κB signaling pathway. Tai et alfound that amitriptyline induced the up-regulation of GLT-1and GLASTthrough NF-κB in rat suffered morphine chronic intrathecal injection.Accordingly, we presume that CIP might attenuate ischemia-reperfusion injuryby up-regulation GLT-1expression via NF-κB signaling pathway. Based onthe above considerations, in order to explore whether PPARγ or NF-κBsignaling pathway participates in the up-regulation of GLT-1during theinduction of BIT, the influences of GW9662(a specific inhibiter of PPARγ) aswell as BAY11-7082(a specific inhibiter of NF-κB) on the dynamic changesof the levels of glutamate, aspartate and GABA in the hippocampal CA1region.1IH preconditioning induced brain ischemic tolerance by up-regulatingGLT-1in rats1.1Methods: Fifty-seven male Wistar rats (180-220g) were randomlyassigned to the following parts.1.1.1The basal expression of GLT-1and GFAP after IH preconditioningTwelve Rats were divided randomly into two groups which had beensubjected to permanent occlusion of the bilateral vertebral arteries (n=6ineach group).(1) Sham group: The rats were subjected to the sham operationfor global brain ischemia;(2) IH+sham group: The rats were subjected to thesham operation for global brain ischemia after IH preconditioning for28d. Allrats were decapitated after the sham operation immediately. Three rats in eachgroup were used for the expression of GLT-1and GFAP byimmunohistochemistry. Two sets of sections were made, one set for theexpression of GLT-1, and the other set for GFAP. Another three rats in eachgroup were used for GLT-1western blotting analysis.1.1.2The effect of IH preconditioning on GLT-1expression after global brainischemia Twenty-four Rats were divided randomly into four groups which hadbeen subjected to permanent occlusion of the bilateral vertebral arteries (n=6in each group).(1) sham group;(2) IH+sham group;(3) Ischemia group: Therats were subjected to the8-min global brain ischemia;(4) IH+ischemiagroup: The rats were subjected to the8-min global brain ischemia after IHpreconditioning for28d. All rats were decapitated at7d after brain ischemiaor the sham operation, and the GLT-1expression was assayed byimmunohistochemistry (n=3) and western blot (n=3).1.1.3The role of DHK in brain ischemic tolerance induced by IHpreconditioningTwenty-one Rats were divided randomly into seven groups which hadbeen subjected to permanent occlusion of the bilateral vertebral arteries (n=3in each group).(1) sham group;(2) IH+sham group;(3) Ischemia group;(4)IH+ischemia group;(5) DHK+sham group;(6) Artificial cerebrospinal fluid(aCSF)+IH+ischemia group;(7) DHK+IH+ischemia group. Thetreatments for group (1) to group (4) mentioned above were the same as before.The rats for group (5) were administered DHK solution (15μl of200nmol) byintraventricular injection30min before the sham operation, then subjected tothe sham operation for global brain ischemia. The rats for group (6) and group(7), first undertaken IH preconditioning for28d, then injected with15μlaCSF (vehicle) or DHK solution (15μl of200nmol) into the right lateralcerebral ventricle respectively according to the design, after30min, weresubjected to the8-min global brain ischemia. All rats were decapitated at7dafter the global brain ischemia or the sham operation for neuropathologicalevaluation by thionin staining.1.2Results:1.2.1IH preconditioning up-regulated the basal expression of GLT-1proteinImmunohistochemistry assay: At the immediate time point of the shamgroup, the GLT-1immunoparticles were diffusely distributed in hippocampalCA1subfield. At the immediate time point of IH+sham group, the GLT-1immunoparticles was up-regulated significantly after IH preconditioning in the CA1subfield, especially in the area between the pyramidal neurons.Western blot analysis: At the immediate time point, the level of GLT-1protein in hippocampal CA1subfield of IH+sham group was significantlyhigher than that in the sham group.Taken together, the results of immunohistochemistry and western blotcoincidentally indicated that IH preconditioning up-regulated the basalexpression of GLT-1protein in hippocampal CA1subfield.1.2.2IH preconditioning up-regulated the expression of GFAPImmunohistochemical staining for GFAP showed that astrocytes took ona star or spider-like shape with prominent processes in hippocampal CA1subfield. In the sham group, a few GFAP immunoreactive particles wereobserved in the area between the pyramidal neurons. Compared with the shamgroup, significant up-regulation of GFAP immunoreactivity was observed atthe immediate time point in the IH+sham group, the astrocytes showednormal soma and aplenty slender processes, which tightly surrounded thepyramidal neurons in CA1hippocampus.1.2.3IH preconditioning reversed the down-regulation of GLT-1proteininduced by8-min global brain ischemiaImmunohistochemistry assay: At7d time point of the sham group, therewere some GLT-1immunoparticles diffusely distributed in the hippocampalCA1subfield. Compared with the sham group, the intensity of GLT-1immunoreactivity was increased significantly in the IH+sham group. In theischemia group, the GLT-1expression was decreased significantly after8-minglobal brain ischemia when compared with that of the sham group, especiallyin the area where almost complete pyramidal neurons died, and theneighboring area of the pyramidal layer. When the rats were pretreated withthe IH preconditioning before8-min global brain ischemia, thedown-regulation of the GLT-1immunoreactivity induced by the brainischemia was prevented clearly.Western blot analysis: At7d time point, the GLT-1level in IH+shamgroup was much higher than that of the sham group. In the ischemia group, the GLT-1level was significantly down-regulated at7d time point after8-minglobal brain ischemia when compared with that of the sham group. When therats were pretreated with the IH preconditioning before the global brainischemia, the down-regulation of GLT-1protein induced by the brain ischemiawas prevented significantly.The results indicated that IH preconditioning reversed thedown-regulation of GLT-1protein induced by8-min global brain ischemia.1.2.4DHK attenuated the brain ischemic tolerance induced by IHpreconditioningIn the sham group, the pyramidal neurons in hippocampal CA1subfieldwere untouched, the HG was0, and ND was203.53±11.04. No significantneuronal damage was observed at7d in IH+sham group. However, obviousDND in hippocampal CA1subfield was observed at7d after8-min globalbrain ischemia in the ischemia group. When the rats were pretreated with IHpreconditioning for28d before the global brain ischemia, the DND normallyinduced by the ischemia was prevented clearly. The above data indicated thatIH preconditioning effectively protected pyramidal neurons in the CA1hippocampus against DND normally induced by8-min global brain ischemia.The above indicated that IH preconditioning induced brain ischemic tolerancein hippocampal CA1subfield. In DHK+sham group, DHK in dose of200nmol just caused little DND of pyramidal neurons in the CA1hippocampus.That is to say, DHK in this dose is not lethal to pyramidal neurons inhippocampal CA1subfield. There was no apparent DND in aCSF+IH+ischemia group. Compared with the aCSF+IH+ischemia group, significantDND was observed in DHK+IH+ischemia group, which was represented bysignificantly decreased ND and increased HG.These results indicated that DHK in dose of200nmol significantlyattenuated the brain ischemic tolerance induced by IH preconditioning.2The influences of GLT-1in the level of amino acids in the hippocampalCA1region during the induction of BIT and its molecular mechanism2.1Methods: thirty-five healthy male Wistar rats (280-300g) were randomly divided into the following seven groups (n=5in each group), which werepre-embedded double-barrel in the right head and recovery of5days:(1) Sham group: The rats were subjected to permanent occlusion of thebilateral vertebral arteries under chloral hydrate anesthesia by intraperitonealinjection. And then the bilateral common carotid arteries were exposed andwires were embedded. Two days later, microdialysis was carried out, and thewires were pulled out with no influence on the blood flow of bilateralvertebral arteries.(2) CIP group: The rats were subjected to permanent occlusion of thebilateral vertebral arteries under chloral hydrate anesthesia. And then thebilateral common carotid arteries were exposed and wires were embedded.Two days later, microdialysis was carried out, and the rats were subjected toglobal cerebral ischemia for3min. Then the wires were pulled out and theblood flow reperfused.(3) Ischemic insult (II) group: The experimental procedure was the sameas that of the CIP group, except the rats were subjected to global cerebralischemia for8min.(4) CIP+II group: The rats were first subjected to a CIP. Two days afterthe CIP, microdialysis was carried out, and the rats were subjected to globalcerebral ischemia for8min. Then the wires were pulled out and the bloodflow reperfused.(5) DHK+CIP+II group: DHK solution (200nmol,20μl) wasintracerebroventricularly injected into the right lateral ventricle30min beforeglobal cerebral ischemia for8min, the other process was the same as that ofgroup (4).(6) GW9662+CIP+II group: GW9662solution (2.5nmol,25μl) wasintracerebroventricularly injected into the right lateral ventricle30min beforeCIP and6h after CIP, respectively, and the other process was the same as thatof group (4).(7) BAY11-7082+CIP+II group: BAY11-7082solution (2.5nmol,25μl)was intracerebroventricularly injected30min before CIP and6h after CIP, respectively, the other process was the same as that of group (4).The microdialysate of each group was collected and stored at-80℃. Thechanges of glutamate, aspartate and GABA concentration in dialysate wereobserved by high performance liquid chromatography (HPLC) withfluorescence detector.2.2Result:2.2.1The changes of glutamate concentration among the groupsIn the sham group, there was no obvious change in the concentration ofglutamate pre-, during and post-sham operation. A pulse of glutamateconcentration about1.7fold of the basal level could be observed after the CIPfor3min. The8-min global brain ischemia in the ischemic insult groupinduced a significant increase in the concentration of glutamate, which beganat the onset of the ischemia and rapidly reached peak value of7-fold of thebasal level at the end of ischemia. The increased concentration of glutamatereturned to baseline about4min after reperfusion. In CIP+II group, themaximum of the increase in glutamate concentration was about3.9fold of thebasal level, which indicated that the increase in the glutamate concentrationinduced by global brain ischemia for8min was significantly inhibited by CIP.The peak value of the glutamate concentration in the DHK+CIP+II groupreached of about6-fold of the basal level. The maximum of the increase inglutamate concentration in GW9662+CIP+II group and BAY11-7082+CIP+IIgroup was about5.6and4.8fold of its basal level, respectively.2.2.2The changes of aspartate concentration among the groupsIn the sham group, there was no obvious change in the concentration ofaspartate pre-, during and post-sham operation. The tendency of the changes inthe concentration of aspartate in sham, CIP and II groups was similar to that ofglutamate in the corresponding group. The peak values of concentration ofaspartate in the CIP and II group were2and4.8fold of the basal level,respectively. However, in CIP+II group, DHK+CIP+II group,GW9662+CIP+II and BAY11-7082+CIP+II group, the peak value ofconcentration of aspartate were5,4.5,4.8, and4.6fold of the basal level, respectively, and no significant difference was observed in either of the abovefour groups compared with the II group.2.2.3Changes of GABA concentration in each groupIn the sham group, there was no obvious change in the concentration ofGABA pre-, during and post-sham operation. The tendency of the changesin the concentration of GABA in sham, CIP and II groups was similar to thatof glutamate in the corresponding group. The peak values of concentration ofGABA in the CIP and II group were3.5and8fold of the basal level,respectively. However, in CIP+II group, there was a more significantincrease in the concentration of GABA, which rapidly reached the peak valueof20fold of the basal level. The above indicated that the concentration ofGABA further increased after the8min global brain ischemia preconditionedby CIP2d before. In the DHK+CIP+II group, GW9662+CIP+II group andBAY11-7082+CIP+II group, an equal increase in the concentration of GABAwas observed, and no significant changes were found compared with theCIP+II group.Conclusions1IH preconditioning up-regulated the basal expression of GLT-1and GFAPprotein, and blocked the down-regulation of GLT-1induced by8-min globalbrain ischemia. DHK attenuated the brain ischemic tolerance induced by IHpreconditioning. Therefore, it could be concluded that IH preconditioninginduced brain ischemic tolerance by up-regulating the expression of GLT-1protein in rats.2The increase in the concentration of glutamate induced by ischemic insultfor8min was significantly inhibited by CIP. Administration of DHK blockedthe inhibiting effect of CIP on the increase in glutamate concentration inducedby ischemic insult. Therefore, CIP caused the up-regulation of the function ofGLT-1during the induction of BIT. Administration of GW9662(a specificinhibitor of PPARγ) and BAY11-7082(a specific inhibitor of NF-κB) could also block the effect of CIP on the glutamate concentration induced byischemic insult, indicating that CIP caused the up-regulating of GLT-1proteinvia PPARγ and NF-κB signalling pathway during the induction of BIT.3The tendency of the changes of aspartate and GABA in sham, CIP and IIgroup were similar to that of glutamate in the corresponding group. While,CIP had no effect on the aspararte concentration induced by8min ischemic.However, a more pronounced increase of GABA concentration was observed.Each of DHK, GW9662, or BAY11-7082had no effect on the aspararte andGABA concentration induced by ischemic insult.