CAJ | 학술논문

目的：应用大鼠大脑中动脉缺血再灌注模型研究无创性远端肢体缺血预处理（RLIP）对大鼠脑缺血再灌注损伤的作用，探讨RLIP是否通过降低p38MAPK信号通路的活性抑制神经细胞凋亡发挥脑保护的作用。方法36只健康雄性SD大鼠，体重200～250 g，随机分成3组（n=12）：假手术组（sham组）、缺血再灌注组（I/R组）、无创远端肢体缺血预处理组（RLIP组）。（1）Sham组：分离右侧的颈总动脉、颈内动脉、颈外动脉，但不阻断血流。（2）I/R组：参照Longa的线栓法制作大脑中动脉缺血再灌注（MCAO）模型，阻断大鼠右侧大脑中动脉2 h后再灌注24 h。（3）RLIP组：在大鼠右后肢根部用改良的自制血压袖带施行远端缺血预处理，缺血5 min，再灌注5 min，共3个循环，RLIP后即刻行大鼠右侧大脑中动脉阻塞再灌注，步骤同前。三组大鼠均再灌注24 h后行神经功能缺陷评分（NDS）；用TTC法测定脑梗死容积；HE染色观察海马CA1区椎体细胞的形态；免疫组织化学法测定海马CA1区P-p38MAPK蛋白、Caspase8蛋白及Caspase3蛋白的表达。结果（1）神经功能缺陷评分：sham组无神经功能缺陷；RLIP组比I/R组评分降低，但两组24 h NDS评分中位数相同。（2）TTC染色：sham组无梗死体积，与I/R组相比，RLIP组脑梗死体积显著减小，差异有统计学意义（P＜0.05）。（3）HE染色：sham组海马CA1区椎体细胞形态正常，排列整齐、致密，为2～3层，细胞核圆而大，染色呈浅蓝色。I/R组椎体细胞数明显减少，细胞体积缩小，细胞核碎裂、溶解、染色加深，间质水肿明显。与I/R组比较，RLIP组海马CA1区椎体细胞层次较清楚，数目多且细胞形态较完整，间质水肿较轻。（4）免疫组织化学结果：与 I/R 组比较， RLIP组P-p38MAPK蛋白、Caspase8蛋白和Caspase3蛋白表达显著减少（P＜0.05），但两组蛋白的表达均明显高于sham组（P＜0.05）。结论无创性RLIP对大鼠脑缺血再灌注损伤有保护作用，其机制可能与降低p38MAPK信号通路活性，减少Caspase8蛋白和Caspase3蛋白表达抑制神经细胞凋亡有关。目的：應用大鼠大腦中動脈缺血再灌註模型研究無創性遠耑肢體缺血預處理（RLIP）對大鼠腦缺血再灌註損傷的作用，探討RLIP是否通過降低p38MAPK信號通路的活性抑製神經細胞凋亡髮揮腦保護的作用。方法36隻健康雄性SD大鼠，體重200～250 g，隨機分成3組（n=12）：假手術組（sham組）、缺血再灌註組（I/R組）、無創遠耑肢體缺血預處理組（RLIP組）。（1）Sham組：分離右側的頸總動脈、頸內動脈、頸外動脈，但不阻斷血流。（2）I/R組：參照Longa的線栓法製作大腦中動脈缺血再灌註（MCAO）模型，阻斷大鼠右側大腦中動脈2 h後再灌註24 h。（3）RLIP組：在大鼠右後肢根部用改良的自製血壓袖帶施行遠耑缺血預處理，缺血5 min，再灌註5 min，共3箇循環，RLIP後即刻行大鼠右側大腦中動脈阻塞再灌註，步驟同前。三組大鼠均再灌註24 h後行神經功能缺陷評分（NDS）；用TTC法測定腦梗死容積；HE染色觀察海馬CA1區椎體細胞的形態；免疫組織化學法測定海馬CA1區P-p38MAPK蛋白、Caspase8蛋白及Caspase3蛋白的錶達。結果（1）神經功能缺陷評分：sham組無神經功能缺陷；RLIP組比I/R組評分降低，但兩組24 h NDS評分中位數相同。（2）TTC染色：sham組無梗死體積，與I/R組相比，RLIP組腦梗死體積顯著減小，差異有統計學意義（P＜0.05）。（3）HE染色：sham組海馬CA1區椎體細胞形態正常，排列整齊、緻密，為2～3層，細胞覈圓而大，染色呈淺藍色。I/R組椎體細胞數明顯減少，細胞體積縮小，細胞覈碎裂、溶解、染色加深，間質水腫明顯。與I/R組比較，RLIP組海馬CA1區椎體細胞層次較清楚，數目多且細胞形態較完整，間質水腫較輕。（4）免疫組織化學結果：與 I/R 組比較， RLIP組P-p38MAPK蛋白、Caspase8蛋白和Caspase3蛋白錶達顯著減少（P＜0.05），但兩組蛋白的錶達均明顯高于sham組（P＜0.05）。結論無創性RLIP對大鼠腦缺血再灌註損傷有保護作用，其機製可能與降低p38MAPK信號通路活性，減少Caspase8蛋白和Caspase3蛋白錶達抑製神經細胞凋亡有關。
목적：응용대서대뇌중동맥결혈재관주모형연구무창성원단지체결혈예처리（RLIP）대대서뇌결혈재관주손상적작용，탐토RLIP시부통과강저p38MAPK신호통로적활성억제신경세포조망발휘뇌보호적작용。방법36지건강웅성SD대서，체중200～250 g，수궤분성3조（n=12）：가수술조（sham조）、결혈재관주조（I/R조）、무창원단지체결혈예처리조（RLIP조）。（1）Sham조：분리우측적경총동맥、경내동맥、경외동맥，단불조단혈류。（2）I/R조：삼조Longa적선전법제작대뇌중동맥결혈재관주（MCAO）모형，조단대서우측대뇌중동맥2 h후재관주24 h。（3）RLIP조：재대서우후지근부용개량적자제혈압수대시행원단결혈예처리，결혈5 min，재관주5 min，공3개순배，RLIP후즉각행대서우측대뇌중동맥조새재관주，보취동전。삼조대서균재관주24 h후행신경공능결함평분（NDS）；용TTC법측정뇌경사용적；HE염색관찰해마CA1구추체세포적형태；면역조직화학법측정해마CA1구P-p38MAPK단백、Caspase8단백급Caspase3단백적표체。결과（1）신경공능결함평분：sham조무신경공능결함；RLIP조비I/R조평분강저，단량조24 h NDS평분중위수상동。（2）TTC염색：sham조무경사체적，여I/R조상비，RLIP조뇌경사체적현저감소，차이유통계학의의（P＜0.05）。（3）HE염색：sham조해마CA1구추체세포형태정상，배렬정제、치밀，위2～3층，세포핵원이대，염색정천람색。I/R조추체세포수명현감소，세포체적축소，세포핵쇄렬、용해、염색가심，간질수종명현。여I/R조비교，RLIP조해마CA1구추체세포층차교청초，수목다차세포형태교완정，간질수종교경。（4）면역조직화학결과：여 I/R 조비교， RLIP조P-p38MAPK단백、Caspase8단백화Caspase3단백표체현저감소（P＜0.05），단량조단백적표체균명현고우sham조（P＜0.05）。결론무창성RLIP대대서뇌결혈재관주손상유보호작용，기궤제가능여강저p38MAPK신호통로활성，감소Caspase8단백화Caspase3단백표체억제신경세포조망유관。
Objective To study the cerebral protective effects of noninvasive remote limb ischemic preconditioning (RLIP) against ischemia-reperfusion injury using the middle cerebral artery occlusion in rats and to investigate whether through lowering p38MAPK signal transduction pathway activity to inhibit the apoptosis of nerve cells contributes to cerebral protection. Methods Thirty-six healthy male Sprague-Dawley rats (weight 200-250 g) were randomly divided three groups (n=12, each group): sham-operated group (sham group), I/R group, RLIP group. In the sham group, only separate common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA), but don’t block the blood flow. In the I/R group, rats were subjected to 2 h of right middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. In the RLIP group, rats were accepted 3 cycles of 5 minutes ischemia followed by 5 minutes reperfusion on right hind limb at the beginning of MCAO. Evaluation of neurologic deficit scores was performed on 24 h after reperfusion by the method of Longa's. Six rats were randomly selected to measure the cerebral infarction volume by TTC staining and another rats were observed tissue morphology of hippocampal CA1 region by HE staining. The expression of P-p38MAPK, Caspase8 and Caspase3 in the hippocampal CA1 region were determined by immunohistochemical analysis. Results There was no neurologic deficit in sham group. The NDS in RLIP group was lower than that in I/R, but there was no significant difference between them. TTC staining:There was no infarction volume in sham group, the cerebral infarction volume in RLIP group decreased significantly comparing with that in I/R group (P<0.05). HE staining showed that in the sham group, the pyramidal neuron of hippocampal CA1 region were aligned, dense, for 2-3 layers, its shape was normal and its cell nucleus was round and big, dyed in light blue. But the pyramidal neuron in the I/R group were dyed deepen, its number reduced significantly, its volume shrinked and its cell nucleus were fragmentized and dissolved. And compared with that in I/R group, the hippocampal pyramidal neuron in RLIP group were more integrated and clear. Immunohistochemical analysis:the expression of P-p38MAPK, Caspase8 and Caspase3 protein obviously down-regulated than that in I/R group (P<0.05), but they were up-regulated significantly than that in sham group (P<0.05). Conclusion Nonin-vasive remote limb ischemic preconditioning has protective effects against cerebral ischemia-reperfusion injury which may related to inhibiting apoptosis of neurons by p38MAPK signal transduction pathway.