依托咪酯后处理对大鼠脑缺血再灌注损伤MDA和SOD的影响
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摘要
缺血组织在一定时间后血流恢复可能致进一步的组织损伤和功能障碍，这种恢复血流灌注后引起的进一步损害称为再灌注损伤(reperfusion injury)[1]。
目的
建立大鼠脑缺血再灌注损伤模型，观察不同剂量的依托咪酯后处理对大鼠脑缺血再灌注损伤（Ischemia-reperfusion,I/R）丙二醛(malondialdehyde,MDA)和超氧化物歧化酶（superoxide dismutase,SOD）的影响，探讨依托咪酯对大鼠脑缺血再灌注损伤的脑保护机制及依托咪酯脑保护的适宜剂量。
方法
脑缺血再灌注模型的建立：动物称重→麻醉→固定→暴露大鼠的股静脉，用温盐水纱布覆盖备用→颈正中切口→暴露双侧颈总动脉→夹闭双侧颈总动脉→缺血20min→再灌注前1min的药物后处理→恢复血流。
实验动物：Wistar大鼠40只随机分为5组（每组8只），Sham组（假手术组），I/R组（缺血再灌注组）,Eto1,2，3组（依托咪酯后处理1,2,3组,分别给予依托咪酯0.3mg/kg，0.9mg/kg,2.7mg/kg）。Sham组不结扎颈总动脉，其余同缺血再灌注组。其余四组均夹闭双侧颈总动脉20min，于再灌注前1min,Eto1,2,3组股静脉注射0.3mg/kg，0.9mg/kg,2.7mg/kg依托咪酯注射液，Sham组与I/R组股静脉注射等量生理盐水。
大鼠再灌注24h时处死动物，断头取脑，由视交叉平面冠状切取约4mm组织块，以10%多聚甲醛固定48h。行常规石蜡包埋，作连续冠状切片（厚5μ m）,进行HE染色，光镜下观察组织学变化。
另取0.5g大脑组织在pH值7.4的PBS缓冲液中制成10%组织匀浆，5000转/min离心20min，取上清液根据试剂盒说明，进行生化指标检测。采用硫代巴比妥酸(TBA)法测定MDA含量，羟胺氧化法测定SOD活性。
结果
与sham组比较，I/R组MDA含量显著增高，SOD活性明显降低（P均<0.05）；
与I/R组比较，Eto1,2,3组可降低MDA含量，提高SOD活性（P均<0.05）；
Eto3组与Eto1,2组比较，MDA含量增高、SOD活性降低（P均<0.05）。
Eto1组与Eto2组比较，其MDA含量与SOD活性无显著差异。
结论
1、通过对MDA含量以及SOD活性的测量，发现大鼠脑缺血再灌注后脑组织MDA含量较假手术组有所升高，SOD活性较后者降低。
2、依托咪酯后处理对大鼠脑缺血再灌注损伤有保护作用，其机制可能与抑制氧自由基有关。3、Eto1,2剂量组的脑保护作用优于Eto3剂量组。
The restoration of blood flow after ischemia can also cause further tissue demage anddysfunction in some cases which we called it reperfusion injury.
Objective
To establish the cerebral ischemia reperfusion injury model of rats. To observe the effects ofEtomidate postconditioning on the brain tissue malondialdehyde(MDA)concentration andsuperoxide dismutase(SOD)activity in rats induced by cerebral ischemia-reperfusion(CIR) so asto investigate cerebral protection mechanism of Etomidate.and the dose of Etomidate whosecerebral protection is better.
Methods
The establishment of the cerebral ischemia reperfusion model: Animalweighing→Anesthesia→Fixation of animal→Exposed rats’ femoral vein to standby coveredwith warm saline gauze→Neck incision to exposed the bilateral common carotid artery andligate them→After20mins’ ischemia, give them the drug treatment for1min→Restoration ofblood flow.
Experimental animal:A total of40adult Wistar rats were randomly divided into5groups(n=8each):Sham operation group (Sham),cerebral ischemia reperfusion group (I/R),Etomidatepostconditioning group1,2,3(Eto1,2,3group respectively be given0.3mg/kg，0.9mg/kg,2.7mg/kgdoses of Etomidate).Then,make the cerebral ischemia model by ligating two-sides commoncarotid arteries except the Sham group.After20min, we will give Eto1,2,3group respectively0.3mg/kg，0.9mg/kg,2.7mg/kg doses of Etomidate, in the same time sham group and I/R groupwill be injected an equal volume of saline solution from femoral vein. Restoration of blood flowafter1min’ ischemic postconditioning.
Sacrificed all rats after24hours’ reperfusion. Seperated the Rat’s head to get its brain. To fixthe brain tissue48hours with10%paraformaldehycle after coronary cutting from the opticchiasm plane for about4mm wide. Paraffin embedding conventionally. Then observe the coronalslices (thickness5μm) whichare alreadybe stained by HE under light microscope, and find theirhistological changed.
Put another0.5g brain tissue into PH7.4PBC buffer to make up the10%tissue homogenate.After20minutes’ centrifugal5000/min, take supernatant to test the biochemical indexesaccording to kit. Using thiobarbituric acid (TBA) method to test the content of MDA. Using hydroxylamine oxidation method to test the SOD activity.
Results
Compared with the Sham group, the MDA content in the rat brain tissue significantlyincreased in I/R group(P <0.05), SOD activity significantly decreased (P <0.05);
Compared with the I/R group, the MDA content in the rat brain tissue in Eto1,2,3groupsdecreased (all P <0.05), while the SOD activity increased (all P <0.05).
Meanwhile, the Eto3groups have the higher MDA content and lower SOD activity than Eto1、2group (P<0.05).
And Eto1group compared with Eto2group, the MDA content and SOD activity had nosignificant difference.
Conclusion
1Though the testing of MDA content, SOD activity and the observation under the lightmicroscope from the HE staining, we found that the rat brain tissue under the ischemiareperfusion was really damaged than a sham operation group does.
2Etomidate postconditioning shows an effect against ischemia reperfusion brain injury, themechanism of the protection may be related to inhibiting of the oxygen free radicals.
3Eto1,2group have better cerebral protection than Eto3group.
目的
建立大鼠脑缺血再灌注损伤模型，观察不同剂量的依托咪酯后处理对大鼠脑缺血再灌注损伤（Ischemia-reperfusion,I/R）丙二醛(malondialdehyde,MDA)和超氧化物歧化酶（superoxide dismutase,SOD）的影响，探讨依托咪酯对大鼠脑缺血再灌注损伤的脑保护机制及依托咪酯脑保护的适宜剂量。
方法
脑缺血再灌注模型的建立：动物称重→麻醉→固定→暴露大鼠的股静脉，用温盐水纱布覆盖备用→颈正中切口→暴露双侧颈总动脉→夹闭双侧颈总动脉→缺血20min→再灌注前1min的药物后处理→恢复血流。
实验动物：Wistar大鼠40只随机分为5组（每组8只），Sham组（假手术组），I/R组（缺血再灌注组）,Eto1,2，3组（依托咪酯后处理1,2,3组,分别给予依托咪酯0.3mg/kg，0.9mg/kg,2.7mg/kg）。Sham组不结扎颈总动脉，其余同缺血再灌注组。其余四组均夹闭双侧颈总动脉20min，于再灌注前1min,Eto1,2,3组股静脉注射0.3mg/kg，0.9mg/kg,2.7mg/kg依托咪酯注射液，Sham组与I/R组股静脉注射等量生理盐水。
大鼠再灌注24h时处死动物，断头取脑，由视交叉平面冠状切取约4mm组织块，以10%多聚甲醛固定48h。行常规石蜡包埋，作连续冠状切片（厚5μ m）,进行HE染色，光镜下观察组织学变化。
另取0.5g大脑组织在pH值7.4的PBS缓冲液中制成10%组织匀浆，5000转/min离心20min，取上清液根据试剂盒说明，进行生化指标检测。采用硫代巴比妥酸(TBA)法测定MDA含量，羟胺氧化法测定SOD活性。
结果
与sham组比较，I/R组MDA含量显著增高，SOD活性明显降低（P均<0.05）；
与I/R组比较，Eto1,2,3组可降低MDA含量，提高SOD活性（P均<0.05）；
Eto3组与Eto1,2组比较，MDA含量增高、SOD活性降低（P均<0.05）。
Eto1组与Eto2组比较，其MDA含量与SOD活性无显著差异。
结论
1、通过对MDA含量以及SOD活性的测量，发现大鼠脑缺血再灌注后脑组织MDA含量较假手术组有所升高，SOD活性较后者降低。
2、依托咪酯后处理对大鼠脑缺血再灌注损伤有保护作用，其机制可能与抑制氧自由基有关。3、Eto1,2剂量组的脑保护作用优于Eto3剂量组。
The restoration of blood flow after ischemia can also cause further tissue demage anddysfunction in some cases which we called it reperfusion injury.
Objective
To establish the cerebral ischemia reperfusion injury model of rats. To observe the effects ofEtomidate postconditioning on the brain tissue malondialdehyde(MDA)concentration andsuperoxide dismutase(SOD)activity in rats induced by cerebral ischemia-reperfusion(CIR) so asto investigate cerebral protection mechanism of Etomidate.and the dose of Etomidate whosecerebral protection is better.
Methods
The establishment of the cerebral ischemia reperfusion model: Animalweighing→Anesthesia→Fixation of animal→Exposed rats’ femoral vein to standby coveredwith warm saline gauze→Neck incision to exposed the bilateral common carotid artery andligate them→After20mins’ ischemia, give them the drug treatment for1min→Restoration ofblood flow.
Experimental animal:A total of40adult Wistar rats were randomly divided into5groups(n=8each):Sham operation group (Sham),cerebral ischemia reperfusion group (I/R),Etomidatepostconditioning group1,2,3(Eto1,2,3group respectively be given0.3mg/kg，0.9mg/kg,2.7mg/kgdoses of Etomidate).Then,make the cerebral ischemia model by ligating two-sides commoncarotid arteries except the Sham group.After20min, we will give Eto1,2,3group respectively0.3mg/kg，0.9mg/kg,2.7mg/kg doses of Etomidate, in the same time sham group and I/R groupwill be injected an equal volume of saline solution from femoral vein. Restoration of blood flowafter1min’ ischemic postconditioning.
Sacrificed all rats after24hours’ reperfusion. Seperated the Rat’s head to get its brain. To fixthe brain tissue48hours with10%paraformaldehycle after coronary cutting from the opticchiasm plane for about4mm wide. Paraffin embedding conventionally. Then observe the coronalslices (thickness5μm) whichare alreadybe stained by HE under light microscope, and find theirhistological changed.
Put another0.5g brain tissue into PH7.4PBC buffer to make up the10%tissue homogenate.After20minutes’ centrifugal5000/min, take supernatant to test the biochemical indexesaccording to kit. Using thiobarbituric acid (TBA) method to test the content of MDA. Using hydroxylamine oxidation method to test the SOD activity.
Results
Compared with the Sham group, the MDA content in the rat brain tissue significantlyincreased in I/R group(P <0.05), SOD activity significantly decreased (P <0.05);
Compared with the I/R group, the MDA content in the rat brain tissue in Eto1,2,3groupsdecreased (all P <0.05), while the SOD activity increased (all P <0.05).
Meanwhile, the Eto3groups have the higher MDA content and lower SOD activity than Eto1、2group (P<0.05).
And Eto1group compared with Eto2group, the MDA content and SOD activity had nosignificant difference.
Conclusion
1Though the testing of MDA content, SOD activity and the observation under the lightmicroscope from the HE staining, we found that the rat brain tissue under the ischemiareperfusion was really damaged than a sham operation group does.
2Etomidate postconditioning shows an effect against ischemia reperfusion brain injury, themechanism of the protection may be related to inhibiting of the oxygen free radicals.
3Eto1,2group have better cerebral protection than Eto3group.
引文
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