| Objective：This experiment takes brain ischemia reperfusion guinea pigsas the research object, we use the method of immunohistochemical to evaluatethe IRE1, XBP-1positive expression in the auditory cortex. And use theELISA method to detect the change of CRP, IgM in blood, so as to researchthe effect of endoplasmic reticulum stress and the immunology reaction in theauditory cortex neuron apoptosis after brain ischemia reperfusion.Methods：1Seleceted20healthy male red eye guinea pigs,made sure the auditorybrainstem response (ABR) were under10dB sound pressure level (SPL), anddivided them randomly into5groups: normal control group,ischemia in15minutes group, reperfution for6hours group, reperfution for24hours group,reperfution for7days group,with4guinea pigs in each group.Cerebralischemia reperfusion injury model was produced by occlusion of bilateralcommon carotid arteries under the same conditions, while the control groupwithout treatment. The guinea pigs in the second group received ABR testafter operation,the last three group received ABR test until the correspondingreperfusion time, then determine the hearing was impaired.After that takeblood from the heart for ELISA and take brain tissue forimmunohistochemistry.2ELISA for CRP, IgM: Firstly gave guinea pigs anesthesia byintraperitoneal injection, exposed chest,the blood was sampled by the methodof cardiac puncture by1ml syringe.After that,gave heart perfusion, waitingfor the blood coagulation, then stayed overnight in the4℃refrigerator. Thenext day, centrifuged the blood for separation of serum and put the serum inthe-70℃refrigerator to save. After all serum samples collected,set standard wells and testing sample wells after unified coating,then add standards,samplesto the appropriate well,add HRP-conjugate reagent to each well.Afterincubating and washing,the color in the wells should change by adding thechromogen solution.Measure the Optical Density (OD) at450nm using aspectrophotometer,then produce a standard curve of OD versus theconcentration.The concentration of the samples is determined by comparingOD of the samples to the standard curve.3Immunohistochemistry for IRE1, XBP–1:Perfused saline to hearts afterdrewing blood from heart to douche the systemic blood, then continueperfusing4%paraformaldehyde till the body was stiff, fetching bilateralcerebral hemisphere and preserving it into4%paraformaldehyde for24hoursafter quickly cutting head from nape and carefully dissecting bilateral parietal.Secondly dewatering, embodying in wax, embedding, storing in4℃refrigerator. Till all the wax block were collected, uniformly sectioning,spreading, roasting, dewaxing, hydrating, repairing, closing, plusing firstantibody and keeping overnight, then plused second antibody, usinghorseradish peroxidase (HRP) and diaminobenzidine (DAB) to color undermicroscope. Finally, counted the brown positive cells in the40×10timesoptical microscope with color image processing software.Results:1The result of ABR test:control group was10.0±2.47dB SPL，ischemiaminutes group was20±1.56dB SPL,reperfusion6h group was30±2.03dB SPL，reperfusion24h group was30±1.67dB SPL,reperfusion7d group was15±1.14dB SPL.There were no obvious statistical differences between reperfusion6hgroup and reperfusion24h group（P>0.01）,but there were obvious differencesbetween the other groups（P<0.01）.2ELISA： The result of CRP （umol/ml）:control group was1198.96±50.81，ischemia group was1270.70±34.76，reperfusion6h group was1467.80±73.67，reperfusion24h group was1352.83±175.71，reperfusion7d groupwas1252.56±41.32. There were significant statistical differences between anytwo of the groups (P<0.01).The result of IgM:control group was987.32±39.13, ischemia group was1064.90±39.78，reperfusion6h group was1118.03±57.75,reperfusion24h group was1122.43±55.40， reperfusion7d group was1010.70±55.95. There were no obvious differences between reperfusion6hgroup and reperfusion24h group （P>0.01）,but there were obviousdifferences between the other groups（P<0.01）.3HE staining indicated that the amount of apoptotic cells were differentbetween each group: control group was2.6±1.13, ischemia group was14.1±2.71,reperfusion6h group was24.9±2.20,reperfusion24h group was19.3±1.46,reperfusion7d group was9.4±1.17. There were significant statistical differencesbetween any two of the groups (P<0.01).4Immunohistochemical results showed each experiment group hadpositive color of IRE1, XBP-1. The number of positive IRE1at control groupwas9.4±1.56, ischemia group was25.6±1.58, reperfusion6h group was60.3±1.57,reperfusion24h group was42.2±2.13, reperfusion7d group was18.3±1.59. There were significant statistical differences between any two ofthe last four groups (P<0.01). The number of positive XBP-1at controlgroup was10.6±1.24，ischemia group was24.2±1.52，reperfusion6h group was61.4±1.7， reperfusion24h group was41.1±2.38， reperfusion7d group was18.9±1.59.There were significant statistical differences between any two ofthe last four groups (P<0.01).5The relationships of IREl and cellular apoptotic rate，and the elationshipof XBP-1and cellular apoptotic rate：There were positive correlations betweenIREl and cellular apoptotic rate(r=0．947，P=0.000).There was positivecorrelation between XBP-1and cellular apoptotic rate(r=0．933，P=0.000)．6The relationships of CRP and IRE1,cellular apoptotic rate: There werepositive correlations between CRP and IRE1(r=0．747，P=0.000).There waspositive correlation between CRP and cellular apoptotic rate(r=0．755，P=0.000)．7The relationships of IgM and IRE1,cellular apoptotic rate: There werepositive correlations between IgM and IRE1(r=0．695，P=0.000).There waspositive correlation between IgM and cellular apoptotic rate(r=0．765， P=0.000)．Conclusions:1In brain ischemia reperfusion injury, the expression of IRE1a, XBP-1protein in auditory cortex tissue is increased. IRE1, XBP-1and cellularapoptotic rate have positive correlation. This suggest that endoplasmicreticulum stress is involved in cerebral ischemia reperfusion injury of neuronapoptosis. IRE1, XBP-1mediated signal transduction pathway is the one ofimportant mechanisms of neuronal cell apoptosis caused by endoplasmicreticulum stress.2CRP, IgM and IRE1, cellular apoptotic rate have positive correlation.Prompt hearing impairment caused by cerebral ischemia may causeimmunological changes of blood, endoplasmic reticulum stress response toinspire and strengthen the immune inflammatory reaction. |
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