| ObjectiveIschemic Preconditioning is a phenomenon in which the brain protects itself against future long-time ischemic injury by adapting to sublethal ischemic insults, which is a significant protective mechanism.Ischemic Preconditioning has been acknowledged in various animals and other organs,such as heart,skeletal muscle, spinal cord,lung,liver,kidney,stomach,and intestine.The profoundly research can benefit for organism shielding in medical treatment,and have inviting perspective of clinical application.In current study,according to the priming factors,preconditioning is generally divided into two sorts:ischemic preconditioning and drug preconditioning.The latter is a stimulant ischemia practice that some chemistry substance is added to interfere with energy metabolism so as to induce the protective effect.Nowadays, preconditioning stimuli include hypoxic exposure,low dose of CoCl2,DFO and 3-NPA among others,whereby those toxic and side-effect restrict their clinical application.Recently,Huber reports some novel findings about this protective effect that was triggered by clinical frequently used antibiotics such as erythromycin,which attenuate the nervous system damage by ischemia.Erythromycin can impact mitochondrial energy metabolism so as to induce the protective effect.What's more, the research is also indicated preconditioning can not only shield the brain from ischemic insults,but also can preserve other injury tissue include epilepsy and cerebral edema.With regard to the protective mechanism,abundant agents are verified to participate in this procedure,and the Neuronal nitric oxide sythases(nNOS) and tumor necrosis factor-α(TNF-α)have attached increasing attention in the neuroprotective effect.It has been certified that nNOS generate a marked effect by regulating apoptosis related gene such as Caspase3,Bcl-2,and TNF-αis also a frequent researching factor in hypoxic preconditioning and congenerous Molecular Biology mechanism that various tissue and cell mediate hypoxic tolerance in mammalian in present study, however,there is no related report on whether nNOS and TNF-αis effective or which path in the erythromycin-induced Preconditioning.Therefore,we explore the role of nNOS and TNF-αin erythromycin-induced focal cerebral ischemic preconditioning rats,so as to offer actual proof for further illustrating the neuroprotective mechanism in erythromycin-induced focal cerebral ischemic preconditioning rats.Methods1,Animal and groupingHealthy adult Wistar male rat(270-300g) were randomly divided into 6 groups: normal control group,erythromycin-1(EM-1)group,erythromycin-2(EM-2) group, erythromycin-3(EM-3) group,erythromycin-4(EM-4) group,and erythromycin-5(EM-5) group.TTC staining,western-blot and RT-PCR were applied to determine the expression of nNOS and TNF-α2,Establishment of rat MCAo modelFocal ischemic preconditioning in the brain was accomplished by occlusion of the right internal carotid artery using patching a line.The animals have three phenotypes after palinesthesia:1,Left forelimb show adduction and buckling;2,Drawing a circle to opposite the ipsilateral side while crawling;3,Dumpaging when standing.all the rats that have one of the signs above-mentioned could be selected.3,TTC(2,3,5-triphenyltetrazolium chlorid) stainingPreconditioned and control rats(ischemia for 24 h) were subjected to decapitation and the brains were isolated at once,the brains were kept 20 min at-20 C and frozen brains were sliced into uniform coronal section about 2-mm-thick.the slices were incubated in 1%TTC(2,3,5-triphenyltetrazolium chlorid) at 37 C in 0.2 M Tris buffer for 30 min away from light Immobilization in the formaldehyde after full coloration.Tissue infarction was identified by the lack of 2,3,5-triphenyltetrazolium chloride staining(total 6 slices).Both sides of each stained coronal slice were photographed by digital camera and then evaluated by digital analysis.4,Measurement of infarct volume The brain infarct size was evaluated by image analysis software and calculated according to the formula V=Σ(A1+A2)t/2.T represent slicing depth,A1 and A2 make up infarct size of front and tail,respectively.Similarly,homolateral brain infarct volume and its percentage can be worked out.5,Evaluation of moisture capacityPreconditioned and control rats(ischemia for 48 h) were suffered from decapitation and take out of the brain at once.Weight the brain on electronic balance and put into the constant temperature baker(100℃),take out and weight after 48h,computate the moisture capacity[(wet weight-dry weight)/wet weight]×100%6,Resource of cerebral tissuePreconditioned and control model rats(ischemia for 24 h) were rendered anesthetic, experienced decapitation and dislodged the brain right away.The right ischemia half-silent cortex(7-11mm to the top of olfactory bulb,lateral fissure sagittal split up to 1/3) were selected and assembled in EP tube,frozen at-70℃。7,Assay of n NOS and TNF-αby PCRThe PCR products derived from a serious RT-PCR reaction were treatment via electrophoresis in 5%agarose gel and staining with bromphenol blue.The density of amplified bands was evaluated by electrophoresis gelatin image apparatus.The standard value is the expression of proportionality for n NOS and TNF-αto GAPDH8,Estimation of n NOS and TNF-αprotein by Western blotThe content of n NOS and TNF-αcan be manifest by enhanced chemiluminescence using the ECL system.The result was scanned by Chemi Imager5500 V2.03 and detected by ECL system9,Statistical AnalysisStatistical analysis was performed using SPSS soft ware.date was expressed as mean±SEM The comparison was done using Student's t test or ANOVA with Fisher's post hoc comparison.Anαof P＜0.05 was considered significant.Results1,Measurement of infarct volume The infarct volume in erythromycin-induced Preconditioning was attenuated at different level compared with control,and the preconditioning yield to 35mg/kg had significant decrease2,Evaluation of edemaThe edema to erythromycin-induced preconditioning was diminished at different level compared with that of control,and the preconditioning yield to 35mg/kg had significant difference3,Assay of n NOS and TNF-αby PCR and Western blotThe expression of n NOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.4,AnalysisLocal infarct volume in erythromycin-induced preconditioning rats was blunted compared with that of control after 24h ischemia.The degree of decrease was determined by drug dose,while the dose of 35mg/kg had significant difference(P＜0.05).The edema in erythromycin-induced preconditioning was diminished at different level compared with control,and the preconditioning yield to 35mg/kg had significant difference(P＜0.05).The expression of nNOS displayed an up-regulation in Preconditioning rats,but TNF-αdown-regulation,compared with control.The expression of n NOS and TNF-αwith the dose of 35mg/kg had significant difference compared with that of control(P＜0.05)Conclusion1,erythromycin-induced Preconditioning in rats can not only defend the brain from acute ischemic focal insults,but also lessen the infarct volume and the degree of brain edema,moreover the dose of 35mg/kg was the most suitable for the preconditioning2,erythromycin-induced Preconditioning in rats was also enhanced the expression of nNOS in cerebral ischemic model,and then cut down the expression of TNF-α.These findings demonstrate that the protective effect and mechanism of erythromycin-induced Preconditioning may have close relationship with the expression of n NOS and TNF-α...
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