缺血后处理对深低温停循环大鼠脑保护作用的实验研究
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摘要
[研究背景]
深低温停循环(DHCA, deep hypothermic circulatory arrest)是心血管外科手术中一项重要的保障技术,在心脏外科尤其是主动脉外科及小儿复杂先天性心脏病手术中有广泛的应用。自从这一技术被应用到临床上,复杂的心血管疾病的手术安全性和疗效显著改善。但是在临床广泛应用的过程中,DHCA手术由于存在手术时间长、损伤大、全脑缺血再灌注损伤或低流量灌注导致的缺血缺氧等因素,术后脑部并发症发生率仍较高,如何有效预防DHCA术后的脑损伤成为近年来的研究热点。
缺血后处理(ischemia postconditioning,Ⅰ-postC)是新近发现的一种内源性保护现象,是指在一个持续较长时间的缺血期后,在再灌注前交替实施短暂的再灌注／缺血处理,其后恢复常规灌注,可以减轻再灌注损伤,从而达到组织器官的保护作用。作为一种全新的对抗缺血再灌注损伤的措施,目前已有研究表明其可以减少心、脑、肝、肾、脊髓等器官或组织的缺血再灌注损伤,进而减少其功能的障碍,起到相应的保护作用。近期许多研究证明缺血后处理可通过多种机制减轻炎症反应和改善氧自由基代谢,有效地减少脑组织神经元凋亡、保护细胞功能。同时,已有大量研究表明：全脑缺血-再灌注损伤主要与氧化应激反应、炎症反应及细胞凋亡等有关。神经细胞凋亡是脑缺血-再灌注损伤的重要环节。
由于缺血后处理具有技术简单、实施方便、可操作性强等优点,因此,将Ⅰ-postC应用于DHCA术后脑保护有良好的应用前景,值得深入研究。
[目的]
为探讨缺血后处理对DHCA大鼠的脑保护作用,通过建立DHCA大鼠脑保护模型,检测大鼠脑组织匀浆相关指标,观察大鼠脑组织超微结构及神经元凋亡的情况,分析缺血后处理在DHCA大鼠脑保护作用中可能机制。
[材料和方法]
健康雄性清洁级SD大鼠60只,随机分为5组：假手术组和DHCA组,缺血后处理(IPost)Ⅰ组、Ⅱ组、Ⅲ组。采用改良的四血管法制作DHCA大鼠全脑缺血模型。造模成功后,假手术组只暴露而不电凝及夹闭血管。DHCA组阻断45min,开放后不做任何处理。IPostⅠ组：在开放时反复进行3个循环的开放15s／阻断15s,然后全面开放。IPostⅡ组：在开放时反复进行3个循环的开放30s／阻断30s,然后全面开放。IPostⅢ组：在开放时反复进行3个循环的开放60s／阻断15s,然后全面开放。
于再灌注24小时,在各组中随机取8只大鼠,麻醉后断头取脑,制作脑组织匀浆,检测炎症因子TNF-α、IL-1、IL-6、SOD活性及脑组织MDA含量；各组中余下4只制作脑组织病理切片,光镜、电镜观察大鼠脑海马组织病理形态及其超微结构变化；TUNEL法检测大鼠脑海马神经细胞的凋亡。
[结果]
早期缺血后处理Ⅰ组、Ⅱ组大鼠脑组织炎症因子TNF-α、IL-1、IL-6较DHCA组明显下降,差别有统计学意义(P<0.05,P<0.01),IPostⅢ组炎症因子与DHCA组相比无统计学差异(P>0.05)。与DHCA组相比,IPostⅠ组脑组织SOD含量升高(P<0.01),IPostⅡ组升高(P<0.05)。IPostⅠ组、Ⅱ组大鼠脑组织MDA含量较DHCA组明显下降,IPostⅢ组再灌注24小时大鼠鼠脑组织中SOD、MPO、MDA的表达与DHCA组相比无统计学差异(P>0.05)。病理学结果显示：早期缺血后处理的IPostⅠ组、Ⅱ组大鼠脑组织破坏和细胞损伤程度明显低于DHCA组,IPostⅢ组与DHCA组相比则无明显差异。海马以及皮层神经细胞的凋亡检测发现：与DHCA组比较,IPostⅠ组、Ⅱ组凋亡指数明显降低。
[主要结论]
早期缺血后处理(15s、30s)可以减轻炎症反应和改善氧自由基代谢,减少了脑组织细胞的凋亡,对深低温停循环大鼠具有脑保护作用。
[Background]
Deep hypothermic circulatory arrest (DHCA) is an important protective technology in cardiovascular surgery. It is widely used in cardiac surgery and aortic surgery in children with complex congenital heart surgery. Effect of complex surgical treatment of cardiovascular disease was improved significantly since this technology was applied to clinical. But because of long operative time, cerebral ischemia and low flow perfusion, ischemia and hypoxia and other factors, postoperative complication of the brain after DHCA is very high. How to prevent the brain injury after DHCA becomes focused in recent years.
Ischemia Postconditioning (I-postC) is a newly discovered endogenous protection. After a longer period of ischemia, carrying out a short reperfusion/ ischemia cycle before reperfusion can reduce the reperfusion injury and achieve the protective effect of tissues and organs. As a new measures fighting against ischemia-reperfusion injury, the current studies have shown that I-postC can decrease the heart, brain, liver, kidney, spinal cord and other organs or tissue ischemia, thereby reducing its barrier function. Many recent studies showed that I-postC could reduce inflammation and improve the oxygen free radicals, reduced neuronal apoptosis in brain tissue effectively, protect cell function through a variety of mechanisms. Meanwhile, numerous studies have indicated that cerebral ischemia-reperfusion injury related mainly with oxidative stress, inflammation and cell apoptosis. Neuronal apoptosis is the important part in brain ischemia-reperfusion injury.
Ⅰ-postC technique is simple and easy to operate, therefore,Ⅰ-postC applied to brain protection after DHCA has a good prospect and worthy of further research.
[ Objective]
To observe the protective effects of theⅠ-postC for brain tissue of DHCA rats. To explore the possible mechanism of this protective effect through the establishment of DHCA model, testing brain tissue related indicators, the ultrastructure of rat brain and neuronal apoptosis.
[Materials and Methods]
60 adult male clean SD rats were randomly divided into 5 groups:the sham group, DHCA group and IPostⅠgroup, IPostⅡgroup and IPostⅢgroup. Use the improved "four-vessel method" to produce global cerebral ischemia model of DHCA rates. After the modeling, exposed the sham operation group without vascular coagulation and clipping. Rats in DHCA group opened up without any treatments after blocking for 45 minutes later. IPostⅠgroup:before the opening hours repeated the cycle "open 15s/block 15s" for three times and then fully opened. IPostⅡgroup:before the opening hours repeated the cycle "open 30s/ block 30s" for three times and then fully opened. IPostⅢgroup:before the opening hours repeated the cycle "open 60s/block 15s" for three times and then fully opened. Animals were killed at reperfusion 24 hours, then make the rats'brain tissue homogenate, inflammatory cytokines TNF-α, IL-1βand IL-6 were detected; and then SOD activity and MDA of this homogenate were detected; ultrastructural changes in brain tissue was observed under electron microscopy; hippocampal and cortical neurons apoptosis were detected by TUNEL.
[Results]
Compared with DHCA group, inflammatory cytokines TNF-α, IL-1βand IL-6 of IPostⅠgroup and IPostⅠgroup and IPostⅡgroup decreased significantly (P<0.05, P<0.01), inflammatory cytokines TNF-α, IL-1βand IL-6 of IPostⅢgroup showed no significant difference (P>0.05). Compared with DHCA group, SOD activity of IPostⅠgroup increased significantly (P<0.01), SOD activity of IPostⅡgroup increased significantly (P<0.05). MDA in IPostⅠgroup and IPostⅡgroup decreased significantly (P<0.05). After 24 hours of reperfusion, SOD、MDA expression of IPostⅢwas not statistically significant compared with DHCA group. The brain tissue pathology display, Compared with DHCA group, ischemic postconditioning group brain histoclasia and cell injury is obviously lighten.
Pathology results showed that:the brain tissue damage and cell injury of IPostⅠgroup and IPostⅡgroup were significantly lower than DHCA groups, the brain tissue damage and cell injury of IPostⅢgroup was similar with the DHCA group Hippocampus and cerebral cortex neural cell apoptosis detected:apoptotic index in IPostⅠgroup and IPostⅡgroup significantly reduced compared to the DHCA group.
[Conclusions]
Eraly ischemic postconditioning(15s、30s) can reduce the inflammatory response, improve the metabolism of oxygen free radicals and reduce the apoptosis of brain cell, can protect the brain of DHCA rats.
深低温停循环(DHCA, deep hypothermic circulatory arrest)是心血管外科手术中一项重要的保障技术,在心脏外科尤其是主动脉外科及小儿复杂先天性心脏病手术中有广泛的应用。自从这一技术被应用到临床上,复杂的心血管疾病的手术安全性和疗效显著改善。但是在临床广泛应用的过程中,DHCA手术由于存在手术时间长、损伤大、全脑缺血再灌注损伤或低流量灌注导致的缺血缺氧等因素,术后脑部并发症发生率仍较高,如何有效预防DHCA术后的脑损伤成为近年来的研究热点。
缺血后处理(ischemia postconditioning,Ⅰ-postC)是新近发现的一种内源性保护现象,是指在一个持续较长时间的缺血期后,在再灌注前交替实施短暂的再灌注／缺血处理,其后恢复常规灌注,可以减轻再灌注损伤,从而达到组织器官的保护作用。作为一种全新的对抗缺血再灌注损伤的措施,目前已有研究表明其可以减少心、脑、肝、肾、脊髓等器官或组织的缺血再灌注损伤,进而减少其功能的障碍,起到相应的保护作用。近期许多研究证明缺血后处理可通过多种机制减轻炎症反应和改善氧自由基代谢,有效地减少脑组织神经元凋亡、保护细胞功能。同时,已有大量研究表明：全脑缺血-再灌注损伤主要与氧化应激反应、炎症反应及细胞凋亡等有关。神经细胞凋亡是脑缺血-再灌注损伤的重要环节。
由于缺血后处理具有技术简单、实施方便、可操作性强等优点,因此,将Ⅰ-postC应用于DHCA术后脑保护有良好的应用前景,值得深入研究。
[目的]
为探讨缺血后处理对DHCA大鼠的脑保护作用,通过建立DHCA大鼠脑保护模型,检测大鼠脑组织匀浆相关指标,观察大鼠脑组织超微结构及神经元凋亡的情况,分析缺血后处理在DHCA大鼠脑保护作用中可能机制。
[材料和方法]
健康雄性清洁级SD大鼠60只,随机分为5组：假手术组和DHCA组,缺血后处理(IPost)Ⅰ组、Ⅱ组、Ⅲ组。采用改良的四血管法制作DHCA大鼠全脑缺血模型。造模成功后,假手术组只暴露而不电凝及夹闭血管。DHCA组阻断45min,开放后不做任何处理。IPostⅠ组：在开放时反复进行3个循环的开放15s／阻断15s,然后全面开放。IPostⅡ组：在开放时反复进行3个循环的开放30s／阻断30s,然后全面开放。IPostⅢ组：在开放时反复进行3个循环的开放60s／阻断15s,然后全面开放。
于再灌注24小时,在各组中随机取8只大鼠,麻醉后断头取脑,制作脑组织匀浆,检测炎症因子TNF-α、IL-1、IL-6、SOD活性及脑组织MDA含量；各组中余下4只制作脑组织病理切片,光镜、电镜观察大鼠脑海马组织病理形态及其超微结构变化；TUNEL法检测大鼠脑海马神经细胞的凋亡。
[结果]
早期缺血后处理Ⅰ组、Ⅱ组大鼠脑组织炎症因子TNF-α、IL-1、IL-6较DHCA组明显下降,差别有统计学意义(P<0.05,P<0.01),IPostⅢ组炎症因子与DHCA组相比无统计学差异(P>0.05)。与DHCA组相比,IPostⅠ组脑组织SOD含量升高(P<0.01),IPostⅡ组升高(P<0.05)。IPostⅠ组、Ⅱ组大鼠脑组织MDA含量较DHCA组明显下降,IPostⅢ组再灌注24小时大鼠鼠脑组织中SOD、MPO、MDA的表达与DHCA组相比无统计学差异(P>0.05)。病理学结果显示：早期缺血后处理的IPostⅠ组、Ⅱ组大鼠脑组织破坏和细胞损伤程度明显低于DHCA组,IPostⅢ组与DHCA组相比则无明显差异。海马以及皮层神经细胞的凋亡检测发现：与DHCA组比较,IPostⅠ组、Ⅱ组凋亡指数明显降低。
[主要结论]
早期缺血后处理(15s、30s)可以减轻炎症反应和改善氧自由基代谢,减少了脑组织细胞的凋亡,对深低温停循环大鼠具有脑保护作用。
[Background]
Deep hypothermic circulatory arrest (DHCA) is an important protective technology in cardiovascular surgery. It is widely used in cardiac surgery and aortic surgery in children with complex congenital heart surgery. Effect of complex surgical treatment of cardiovascular disease was improved significantly since this technology was applied to clinical. But because of long operative time, cerebral ischemia and low flow perfusion, ischemia and hypoxia and other factors, postoperative complication of the brain after DHCA is very high. How to prevent the brain injury after DHCA becomes focused in recent years.
Ischemia Postconditioning (I-postC) is a newly discovered endogenous protection. After a longer period of ischemia, carrying out a short reperfusion/ ischemia cycle before reperfusion can reduce the reperfusion injury and achieve the protective effect of tissues and organs. As a new measures fighting against ischemia-reperfusion injury, the current studies have shown that I-postC can decrease the heart, brain, liver, kidney, spinal cord and other organs or tissue ischemia, thereby reducing its barrier function. Many recent studies showed that I-postC could reduce inflammation and improve the oxygen free radicals, reduced neuronal apoptosis in brain tissue effectively, protect cell function through a variety of mechanisms. Meanwhile, numerous studies have indicated that cerebral ischemia-reperfusion injury related mainly with oxidative stress, inflammation and cell apoptosis. Neuronal apoptosis is the important part in brain ischemia-reperfusion injury.
Ⅰ-postC technique is simple and easy to operate, therefore,Ⅰ-postC applied to brain protection after DHCA has a good prospect and worthy of further research.
[ Objective]
To observe the protective effects of theⅠ-postC for brain tissue of DHCA rats. To explore the possible mechanism of this protective effect through the establishment of DHCA model, testing brain tissue related indicators, the ultrastructure of rat brain and neuronal apoptosis.
[Materials and Methods]
60 adult male clean SD rats were randomly divided into 5 groups:the sham group, DHCA group and IPostⅠgroup, IPostⅡgroup and IPostⅢgroup. Use the improved "four-vessel method" to produce global cerebral ischemia model of DHCA rates. After the modeling, exposed the sham operation group without vascular coagulation and clipping. Rats in DHCA group opened up without any treatments after blocking for 45 minutes later. IPostⅠgroup:before the opening hours repeated the cycle "open 15s/block 15s" for three times and then fully opened. IPostⅡgroup:before the opening hours repeated the cycle "open 30s/ block 30s" for three times and then fully opened. IPostⅢgroup:before the opening hours repeated the cycle "open 60s/block 15s" for three times and then fully opened. Animals were killed at reperfusion 24 hours, then make the rats'brain tissue homogenate, inflammatory cytokines TNF-α, IL-1βand IL-6 were detected; and then SOD activity and MDA of this homogenate were detected; ultrastructural changes in brain tissue was observed under electron microscopy; hippocampal and cortical neurons apoptosis were detected by TUNEL.
[Results]
Compared with DHCA group, inflammatory cytokines TNF-α, IL-1βand IL-6 of IPostⅠgroup and IPostⅠgroup and IPostⅡgroup decreased significantly (P<0.05, P<0.01), inflammatory cytokines TNF-α, IL-1βand IL-6 of IPostⅢgroup showed no significant difference (P>0.05). Compared with DHCA group, SOD activity of IPostⅠgroup increased significantly (P<0.01), SOD activity of IPostⅡgroup increased significantly (P<0.05). MDA in IPostⅠgroup and IPostⅡgroup decreased significantly (P<0.05). After 24 hours of reperfusion, SOD、MDA expression of IPostⅢwas not statistically significant compared with DHCA group. The brain tissue pathology display, Compared with DHCA group, ischemic postconditioning group brain histoclasia and cell injury is obviously lighten.
Pathology results showed that:the brain tissue damage and cell injury of IPostⅠgroup and IPostⅡgroup were significantly lower than DHCA groups, the brain tissue damage and cell injury of IPostⅢgroup was similar with the DHCA group Hippocampus and cerebral cortex neural cell apoptosis detected:apoptotic index in IPostⅠgroup and IPostⅡgroup significantly reduced compared to the DHCA group.
[Conclusions]
Eraly ischemic postconditioning(15s、30s) can reduce the inflammatory response, improve the metabolism of oxygen free radicals and reduce the apoptosis of brain cell, can protect the brain of DHCA rats.
引文
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9.高敏,董秀兰.肿瘤坏死因子与脑缺血再灌注损伤.辽宁医学院学报.2009；30(2)：190-191.
10.沈丽华,叶民,丁新生.大鼠脑缺血再灌注后全脑及血清炎性因子水平研究.J Apoplexy and Nervous Diseases.2009;26(4):456-458.
11.薛晶,冯加纯,杨艺敏等.脑缺血再灌注后炎症和自由基作用的实验研究.中国老年学杂志.2009；3(29)：644-646.
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1. Fox HM, Risso ND, Gifford S. Paychological observations of patients undergoing mitral surgery. Psychosomat Med,1954,16:186-268.
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4. Lee JM, Zipfel GJ, Choi DW. The changing landscape of ischaemic brain injury mechamisms. Nature,1999,399:A7-14.
5. Nikolov R, Rami A, Krieglstein J. Endothelin-1 exacerbates focal cerebral ischemia without exerting neurotoxic action in vitro. Eur J Pharmacol,1993,248:205-208.
6. Palmer AM, Marion DW, Botscheller ML, et al. Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J Neurochem,1993,61:2015-2024.
7.黎红华,郑彩梅.兴奋性氨基酸在缺血性海马神经元损害中的作用研究[J].中华神经精病杂志,1993：19(4)：197.
8. WangY, KawamuraN, Schmelzer JD, et al. Decreased peripheral nerve damage after ischemia-reperfusion injury in mice lackingTNF- α1pha·[J]·Neurol Sc.2008; 267 (1-2):107-111.
9.高敏,董秀兰.肿瘤坏死因子与脑缺血再灌注损伤.辽宁医学院学报.2009；30(2)：190-191.
10.沈丽华,叶民,丁新生.大鼠脑缺血再灌注后全脑及血清炎性因子水平研究.J Apoplexy and Nervous Diseases.2009;26(4):456-458.
11.薛晶,冯加纯,杨艺敏等.脑缺血再灌注后炎症和自由基作用的实验研究.中国老年学杂志.2009；3(29)：644-646.
12. Yun Y; Duan WG; Chen P, et al. Ischemic postconditioning modified renal oxidative stress and lipid peroxidation caused by ischemic reperfusion injury in rats. Transplant Proc.2009; 41(9):3597-3602.
13. Domorakova I; Mechirova E; Dankova M, et al. Effect of antioxidant treatment in global ischemia and ischemic postconditioning in the rat hippocampus. Cell Mol Neurobiol.2009;29(6-7):837-844.
14. Shigeno T, Yamaski Y, Kato G, et al. Reduction of delayed neuronal death by inhibition protein synthesis[J]. Neurosci Cell,1990,120:117-123.
15. Sugawara T, Fujimura M, Noshita N, et al. Neuronal death/survival signaling pathways in cerebral ischemia [J] Neuro Rx.2004; 1:17-25.
16.李桂香,董林森,王运良.全脑缺血后脑损伤的机制.Chinese Journal of Practical Nervous Diseases.2008;11 (11):126-129.
17.江基尧.亚低温脑保护基础与临床[M].上海：第二军医大学出版社,1998：54-57.
18. Busto R,Dietrich WD,Globws MY,et al. Small differences in intraischemic brain temperature critically determine the extent of ischemic neurocal injury[J]. J Cereb Blood Flow Metab.1987;7:729-738.
19. Huh PW,Belayev L,Zhao W,et al. Comparative neuroprotective efficacy of prolonged moderate intraischemic and postischemic hypothermia in focal cerebral ischemia[J]. J Neurosurg.2000;92:91-99.
20.徐庆怀,何学松,刘冈祝等.亚低温对大鼠脑缺血再灌注损伤的保护研究.Nervous Diseases and Mental Health.2008;8(5):358-361.
21. LI JC, LUO J, WANG JL. Experience with cardiopulmonary bypass in non-cardiac surgery [J]. Chinese Journal of Extracorporeal Circulation, 2004,2(4):222-224.
22. YAMADA M, NISHIKAWA K, KAWAHARA F, et al. Anesthetic management for clipping a giant basilar artery aneurysm with moderate hypothermia, extracorporeal circulation assistance, and propofol infusion [J]. J Neurosurg Anesthesiol,2003,15 (3):274-277.
23. Greeley WJ, Kern FH, Ungerleider RM, et al. The effect of hypothermic cardiopulmonary bypass and total circulatory arrest on cerebral metabolism in neonates, infants, and children[J]. J Thorac Cardiovasc Surg,1991,101(5):783-94.
24. Young WL, Lawton MT, Gupta DK, et al. Anesthetic management of deep hypothermic circulatory arrest for cerebral aneurysm clipping[J]. Anesthesiology,2002,96(2):497-503.
25.高勇,邹小明,王武军.深低温停循环中不同脑灌注方式脑保护的实验研究.南方医科大学学报(J South Med Univ) 2006;26(5)644-647.
26. Murry CE,J ennings RB,Reimer KA. Preconditioning wit h ischemia:a delay of let hal cell injury in ischemic myocardium [J]. Circulation. 1986;74 (5):1124-1136.
27. Zhao ZQ,Corvera J S, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion :comparison it h ischemic preconditioning [J]. Am J Physiol Heart Circ Physiol.2003;285 (2):579-588.
28. Zhao ZQ,Vinten Johansen J. Postconditioning:reduction of reperfusion-induced injury [J]. Cardiovasc Res.2006;70:200-211.
29. Zhao H, Sapolsky RM, Steinberg GK, et al. Interrupting reperfusion as a st roke t herapy:ischemic postconditioning reduces infarctize after focal ischemia in rat s [J]. J Cereb Blood Flow Metab.2006; 26 (9):1121-1142.
30. Jiang XJ, Shi E, Nakajima Y, et al. Postconditioning, a series of rief Interruptions of early reperfusion,prevent s neurologic injury after spinal cord ischemia [J]. Ann Surg.2006;244 (1):148-153.
31. Huang H, Zhang L,Wang Y, et al. Effect of ischemic postconditioning on spinal cord ischemic-reperfusion injury in rabbit s [J].Can J Anest h. 2007;54 (1):42-48.
32. Gao X,Ren C,Zhao H,et al. Protective effect s of ischemic postconditioning compared with gradual reperfusion or preconditioning[J]. Neurosci Res.2008;86 (11):2505-2511.
33. Jiang XJ, Shi E, Li LW, et al. Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbit s [J]. Life Sci.2008;82:608-614.
34.张瑜,贺民.缺血预处理、缺血后处理的脑保护进展.华西医学.2009；24(2)：491-493.
35. Bolli R, Bhatti ZA, Tang XL, et al. Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxded[J].Circ Res,1997,81(1):42-52.
36. Riepe MW,Esclaire F,Kasischke K,et al. Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation:"chemical preconditioning" [J]. J Cereb Blood Flow Metab.1997; 17:257-264.
37. Luo WJ, Li B, Chen R,et al. Effect of ischemic postconditioning in adult valve replacement [J]. Eur J Cardiot horac Surg.2008;33 (2):203-208.
38. Staat P, Rioufol G, Piot C, et al. postconditioning t he human heart [J]. Circulation.2005;112 (14):2143-2148.