脓毒症相关性脑病小鼠模型的建立及其认知功能障碍的初步研究
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摘要
目的:建立脓毒症相关性脑病(sepsis-associated encephalopathy,SAE)小鼠模型并对其认知功能障碍进行初步研究。方法:选取8～10周龄SPF级雄性C57BL/6J小鼠。第1部分实验将小鼠随机分为4组,即正常对照组,盲肠结扎穿孔术(cecal ligation and puncture,CLP)1组和CLP2组(分别用7号和12号针头行小鼠腹腔CLP)和假手术组(小鼠仅开腹分离盲肠)。第2部分实验将小鼠随机分为3组,即正常对照组,假手术组和CLP组。采用Kaplan-Meier法分析小鼠术后存活率;神经行为学评分评价小鼠的神经行为变化;Morris水迷宫实验和避暗实验检测小鼠的认知记忆功能变化;爬杆实验和悬挂实验测试小鼠的运动协调性;ELISA法检测小鼠血清前列腺素E_2(prostaglandin E_2, PGE_2)水平变化。结果:第1部分实验中,CLP术后小鼠出现明显嗜睡、竖毛、寒颤和厌食等症状,CLP1组和CLP2组小鼠48 h死亡率分别为20%和30%。两部分实验中行小鼠腹腔盲肠结扎穿孔术组小鼠神经行为学评分均显著低于对照组和假手术组(P<0.01);Morris水迷宫中逃避潜伏期时间均显著延长(P<0.01),目标象限停留时间和穿越平台次数减少(P<0.01);悬挂实验和爬杆实验中评分均出现显著降低(P<0.01);在避暗实验开始后大部分小鼠活动量显著减小甚至未曾进入暗室(P<0.05)。第2部分实验中,CLP术后小鼠血清中PGE_2释放量显著增加(P<0.01)。结论:通过12号针行盲肠结扎穿孔术可成功建立稳定的脓毒症相关性脑病小鼠模型,用此法建立的SAE小鼠模型存在学习记忆认知功能障碍。
AIM: To establish the mouse model of sepsis-associated encephalopathy(SAE) and the preliminary research of cognitive dysfunction in this model. METHODS: SPF male C57 BL/6 J mice of 8～10 weeks old were selected. The first part of the experiment divided the mice into 4 groups randomly, namely control group, cecal ligation and puncture(CLP)1 group and CLP2 group(CLP was performed with 7 and 12 syringe needle respectively). The mice in sham operation group were only laparotomy. In the second part of the experiment, the mice were randomly divided into control group, sham operation group and CLP group. The Kaplan-Meier method was used to analyze the postoperative survival rate of the mice in the first part experiment. The neurobehavioral scores were used to evaluate the neurobehavioral changes of the mice. The Morris water maze test and the passive avoidance experiment were used to detect the changes of cognitive memory function in the mice. The pole test and the wire suspension test were used to test the motor coordination of the mice. The serum levels of prostaglandin E2(PGE_2) were measured by ELISA. RESULTS: In the first part of the experiment, the CLP mice showed obvious symptoms such as lethargy, piloerection, chills and anorexia. The 48 h mortality in CLP1 group and CLP2 group were 20% and 30% respectively. In the 2 parts of the experiments, the neurobehavioral scores of the CLP mice were significantly lower than those in control group and the sham operation group(P<0.01). In CLP mice, the escape latency time of the Morris water maze was significantly prolonged(P<0.01), the target quadrant dwell time and the number of crossing platforms were decreased(P<0.01), the scores in the suspension experiment and the pole test were significantly reduced(P<0.01), the activity of the mice was decreased or even did not enter the darkroom in the step-through test(P<0.05). In the second part of the experiment, the serum level of PGE_2 in the mice after CLP was significantly increased(P<0.01). CONCLUSION: A stable mouse model of sepsis-associated encephalopathy is successfully established by cecal ligation and puncture with 12 syringe needle. The SAE mouse model established by this method is useful for investigating the learning and memory cognitive dysfunction.
AIM: To establish the mouse model of sepsis-associated encephalopathy(SAE) and the preliminary research of cognitive dysfunction in this model. METHODS: SPF male C57 BL/6 J mice of 8～10 weeks old were selected. The first part of the experiment divided the mice into 4 groups randomly, namely control group, cecal ligation and puncture(CLP)1 group and CLP2 group(CLP was performed with 7 and 12 syringe needle respectively). The mice in sham operation group were only laparotomy. In the second part of the experiment, the mice were randomly divided into control group, sham operation group and CLP group. The Kaplan-Meier method was used to analyze the postoperative survival rate of the mice in the first part experiment. The neurobehavioral scores were used to evaluate the neurobehavioral changes of the mice. The Morris water maze test and the passive avoidance experiment were used to detect the changes of cognitive memory function in the mice. The pole test and the wire suspension test were used to test the motor coordination of the mice. The serum levels of prostaglandin E2(PGE_2) were measured by ELISA. RESULTS: In the first part of the experiment, the CLP mice showed obvious symptoms such as lethargy, piloerection, chills and anorexia. The 48 h mortality in CLP1 group and CLP2 group were 20% and 30% respectively. In the 2 parts of the experiments, the neurobehavioral scores of the CLP mice were significantly lower than those in control group and the sham operation group(P<0.01). In CLP mice, the escape latency time of the Morris water maze was significantly prolonged(P<0.01), the target quadrant dwell time and the number of crossing platforms were decreased(P<0.01), the scores in the suspension experiment and the pole test were significantly reduced(P<0.01), the activity of the mice was decreased or even did not enter the darkroom in the step-through test(P<0.05). In the second part of the experiment, the serum level of PGE_2 in the mice after CLP was significantly increased(P<0.01). CONCLUSION: A stable mouse model of sepsis-associated encephalopathy is successfully established by cecal ligation and puncture with 12 syringe needle. The SAE mouse model established by this method is useful for investigating the learning and memory cognitive dysfunction.
引文
[1] Chaudhry N,Duggal AK.Sepsis associated encephalopathy[J].Adv Med,2014,2014:762320.
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[4] Schramm P,Klein K,Falkenberg L,et al.Impaired cerebrovascular autoregulation in patients with severe sepsis and sepsis-associated delirium[J].Crit Care,2012,16(5):1-8.
[5] Kuperberg SJ,Wadgaonkar R.Sepsis-associated encephalopathy:The blood-brain barrier and the sphingolipid rheostat[J].Front Immunol,2017,8:597.
[6] Zhang QH,Sheng ZY,Yao YM.Septic encephalopathy:when cytokines interact with acetylcholine in the brain [J].Mil Med Res,2014,1:20.
[7] 王丽敏,刘宇,李贯清,等.脓毒症脑病发病机制研究进展[J].中国全科医学,2011,14(6C):2101-2104.
[8] Wen H.Sepsis induced by cecal ligation and puncture[J].Methods Mol Biol,2013,1031:117-124.
[9] Rittirsch D,Huber-Lang MS,Flierl MA,et al.Immunodesign of experimental sepsis by cecal ligation and puncture[J].Nat Protoc,2009;4(1):31-36.
[10] Siami S,Annane D,Sharshar T.The encephalopathy in sepsis[J].Crit Care Clin,2008,24(1):67-82.
[11] Morris R.Developments of a water-maze procedure for studying spatial learning in the rat[J].J Neurosci Methods,1984,11(1):47-60.
[12] Li Y,Liu WZ,Li L,et al.Neuroprotective effects of a GIP analogue in the MPTP Parkinson’s disease mouse model[J].Neuropharmacology,2016,101:255-263.
[13] Stover KR,Campbell MA,Van Winssen CM,et al.Analysis of motor function in 6-month-old male and female 3xTg-AD mice[J].Behav Brain Res,2015,281:16-23.
[14] 郁正亚,缪鹏.脓毒症实验动物模型CLP与CASP进展[J].中华实验和临床感染病杂志,2010,4(4):464-469.
[15] Toscano MG,Ganea D,Gamero AM.Cecal ligation puncture procedure[J].J Vis Exp,2011,(51):e2860.
[16] Rezende E,Silva JuniorJM,Isola AM,et al.Epidemiology of severe sepsis in the emergency department and difficulties in the initial assistance[J].Clinics,2008,63(4):457-464.
[17] Zenaide PV,Gusmaoflores D.Biomarkers in septic encephalopathy:a systematic review of clinical studies[J].Rev Bras Ter Intensiva.,2013,25(1):56-62.
[18] 肖广辉,王露.脓毒症致脓毒性脑病的相关认识[J].中国当代医药,2014,21(36):194-197.
[19] Granger JI,Ratti PL,Datta SC,et al.Sepsis-induced morbidity in mice:Effects on body temperature,body weight,cage activity,social behavior and cytokines in brain[J].Psychoneuroendocrinology,2013,38(7):1047-1057.
[20] Zampieri FG,Park M,Machado FS,et al.Sepsis-associated encephalopathy:Not just delirium[J].Clinics,2011,66(10):1825-1831.
[21] Hosokawa K,Gaspard N,Su F,et al.Clinical neurophysiological assessment of sepsis-associated brain dysfunction:a systematic review[J].Crit Care,2014,18(6):674.
[22] Vorhees CV,Williams MT.Morris water maze:Procedures for assessing spatial and related forms of learning and memory[J].Nat Protoc,2006,1(2):848-858.
[23] Ertel W,Morrison MH,Wang P,et al.The complex pattern of cytokines in sepsis.Association between prostaglandins,cachectin,and interleukins.[J].Ann Surg,1991,214(2):141-148.
[24] Montrose DC,Nakanishi M,Murphy RC,et al.The role of PGE2 in intestinal inflammation and tumorigenesis[J].Prostaglandins Other Lipid Mediat,2015,116-117:26-36.
[25] Hotchkiss RS,Monneret G,Payen D.Sepsis-induced immunosuppression:From cellular dysfunctions to immunotherapy[J].Nat Rev Immunol,2013,13(12):862-874.
[2] Tsuruta R,Oda Y.A clinical perspective of sepsis-associated delirium[J].J Intensive Care,2016,4:18.
[3] Woznica EA,Inglot M,Woznica RK,et al.Liver dysfunction in sepsis[J].Adv Clin Exp Med,2018,27(4):547-551.
[4] Schramm P,Klein K,Falkenberg L,et al.Impaired cerebrovascular autoregulation in patients with severe sepsis and sepsis-associated delirium[J].Crit Care,2012,16(5):1-8.
[5] Kuperberg SJ,Wadgaonkar R.Sepsis-associated encephalopathy:The blood-brain barrier and the sphingolipid rheostat[J].Front Immunol,2017,8:597.
[6] Zhang QH,Sheng ZY,Yao YM.Septic encephalopathy:when cytokines interact with acetylcholine in the brain [J].Mil Med Res,2014,1:20.
[7] 王丽敏,刘宇,李贯清,等.脓毒症脑病发病机制研究进展[J].中国全科医学,2011,14(6C):2101-2104.
[8] Wen H.Sepsis induced by cecal ligation and puncture[J].Methods Mol Biol,2013,1031:117-124.
[9] Rittirsch D,Huber-Lang MS,Flierl MA,et al.Immunodesign of experimental sepsis by cecal ligation and puncture[J].Nat Protoc,2009;4(1):31-36.
[10] Siami S,Annane D,Sharshar T.The encephalopathy in sepsis[J].Crit Care Clin,2008,24(1):67-82.
[11] Morris R.Developments of a water-maze procedure for studying spatial learning in the rat[J].J Neurosci Methods,1984,11(1):47-60.
[12] Li Y,Liu WZ,Li L,et al.Neuroprotective effects of a GIP analogue in the MPTP Parkinson’s disease mouse model[J].Neuropharmacology,2016,101:255-263.
[13] Stover KR,Campbell MA,Van Winssen CM,et al.Analysis of motor function in 6-month-old male and female 3xTg-AD mice[J].Behav Brain Res,2015,281:16-23.
[14] 郁正亚,缪鹏.脓毒症实验动物模型CLP与CASP进展[J].中华实验和临床感染病杂志,2010,4(4):464-469.
[15] Toscano MG,Ganea D,Gamero AM.Cecal ligation puncture procedure[J].J Vis Exp,2011,(51):e2860.
[16] Rezende E,Silva JuniorJM,Isola AM,et al.Epidemiology of severe sepsis in the emergency department and difficulties in the initial assistance[J].Clinics,2008,63(4):457-464.
[17] Zenaide PV,Gusmaoflores D.Biomarkers in septic encephalopathy:a systematic review of clinical studies[J].Rev Bras Ter Intensiva.,2013,25(1):56-62.
[18] 肖广辉,王露.脓毒症致脓毒性脑病的相关认识[J].中国当代医药,2014,21(36):194-197.
[19] Granger JI,Ratti PL,Datta SC,et al.Sepsis-induced morbidity in mice:Effects on body temperature,body weight,cage activity,social behavior and cytokines in brain[J].Psychoneuroendocrinology,2013,38(7):1047-1057.
[20] Zampieri FG,Park M,Machado FS,et al.Sepsis-associated encephalopathy:Not just delirium[J].Clinics,2011,66(10):1825-1831.
[21] Hosokawa K,Gaspard N,Su F,et al.Clinical neurophysiological assessment of sepsis-associated brain dysfunction:a systematic review[J].Crit Care,2014,18(6):674.
[22] Vorhees CV,Williams MT.Morris water maze:Procedures for assessing spatial and related forms of learning and memory[J].Nat Protoc,2006,1(2):848-858.
[23] Ertel W,Morrison MH,Wang P,et al.The complex pattern of cytokines in sepsis.Association between prostaglandins,cachectin,and interleukins.[J].Ann Surg,1991,214(2):141-148.
[24] Montrose DC,Nakanishi M,Murphy RC,et al.The role of PGE2 in intestinal inflammation and tumorigenesis[J].Prostaglandins Other Lipid Mediat,2015,116-117:26-36.
[25] Hotchkiss RS,Monneret G,Payen D.Sepsis-induced immunosuppression:From cellular dysfunctions to immunotherapy[J].Nat Rev Immunol,2013,13(12):862-874.