急性和慢性束缚应激对大鼠内脏敏感性的影响

目的了解急性和慢性求缚应激对大鼠内脏敏感性的影响和持续时间。方法成年SD大鼠随机分为3组：对照组（没有束缚应激），急性部分束缚应激组（单次2h的束缚应激）和慢性部分束缚应激组（连续束缚应激3天，每天2h），通过腹壁回撤反应（AWR）评分评估应激前后不同时间点大鼠对结直肠扩张（CRD）的内脏敏感性。结果①在扩张压力20mmHg和40mmHg时，急慢性束缚应激组的AWR评分均显著高于应激前基础水平（Od）（P〈0．05），但在60，80mmHg压力时各组的AWR评分均无显著差异；②急性束缚组在应激后24h和应激后第7天，AWR评分显著下降（P〈0．05）；③慢性束缚组在末次应激后24h，AWR评分仍维持增高水平，但在第7天时AWR评分回到基础水平。结论急性和慢性束缚应激郜可以导致大鼠内脏敏感性增高，但其作用可能是短暂的。     

急性束缚应激对大鼠结肠敏感性的影响

背景:内脏高敏感性是肠易激综合征(IBS)重要的病理生理特征,而应激事件能诱发或加重IBS患者的症状。目的:了解急性束缚应激对大鼠结肠敏感性的影响和持续时间,以及对单个平滑肌细胞收缩活动的影响。方法:建立急性部分束缚应激大鼠动物模型,于0天(应激前1天)、1天(应激当天)、2天(应激后1天)行结直肠气囊扩张．并行腹壁回撤反射(AWR)评分;分离平滑肌细胞,测定结肠平滑肌细胞的收缩百分率。结果:20 mm Hg和40 mm Hg扩张压力下,束缚组1天的AWR评分显著高于对照组(1．68±0．79对0．81±0．42、2．45±0．72对1．58±0．60,P<0．05);束缚组大鼠1天时AWR评分在20 mm Hg(P1=0．001;P3=0．006)和40 mm Hg(P2=0．012,P4=0．033)扩张压力下与0天、 2天相比显著增加,但在60 mm Hg和80 mm Hg扩张压力下以及对照组在每天的评分均无显著差异;束缚组结肠平滑肌细胞的收缩百分率与对照组无显著差异(P=0．523)。结论:急性束缚应激使大鼠结肠对低压力球囊扩张的敏感性呈一过性增加,对大鼠单个平滑肌细胞的收缩活动没有影响。     

传统束缚应激动物模型内脏感觉的新评价

目的检测与探讨传统束缚应激动物模型的内脏感觉的变化。方法建立束缚应激动物模型并在大鼠腹壁埋置电极后,经肛门直肠内气囊插入,依次给予不同容量( 0m1、0 .4ml、0 .8m1、1.2m1) ,通过大鼠腹壁外斜肌收缩次数改变来反映其内脏感觉变化。结果束缚应激后,各个容量级别( 0ml、0 .4m1、0 .8m1、1.2m1)大鼠腹部收缩次数分别为3 .75±1.17、5 .0 0±1.2 0、6.2 5±1.49、5 .2 5±1.83 ,均较对照组( 0 .13±0 .64、0 .13±0 .64、0 .2 5±0 .89、0 .13±0 .83 )明显增多,P <0 .0 1;对照组和应激组大鼠腹部收缩次数均随气囊容量增加而增加。结论束缚应激模型除可用于动力研究外,还可进行内脏敏感性研究。     

长期慢性束缚水浸应激对大鼠胃窦Cajal间质细胞数量的影响

目的:研究不同时程低强度束缚水浸应激对大鼠Cajal间质细胞(interstitial cells of Cajal,ICC)数量的影响,探讨慢性应激导致胃动力改变与ICC的相关性.方法:雄性SPF级SD大鼠48只被随机分为6组,即实验3 d、7 d、28 d组和对照3 d、7 d、28 d组,每组8只.实验组23℃水域箱内束缚水浸1h/d,对照组自由摄食饮水.4 d、8d、29d晨序贯脱颈处死.取膜胃小弯侧(ICC-1)、腺胃小弯侧(ICC-3)、腺胃大弯侧上1/3处(ICC-5)、腺胃幽门旁0.5 cm处(ICC-7)组织各一块放入固定液中,制成石蜡切片:抗c-kit免疫组织化学染色,观察不同层次ICC计数.结果:正常大鼠胃内ICC主要分布在肌内(ICC-MY)和肌间(ICC-IM),而黏膜下(ICC-SM)和深肌丛(ICC-DMP)较少.实验3 d组的各层ICC数量与对照组相比无明显差异,实验7 d和28 d组大鼠不同部位、不同层次及同一部位不同层次ICC数量明显异于同期对照组.应激时间长短对不同部位不同层次及同一部位不同层次ICC总数有明显影响,其大致规律是先正常后增加再减少,即先升高后降低.应激主要引起ICC-MY及ICC-IM数量发生变化,而本来较少的ICC-SM和ICC-DMP受影响较轻.结论:长时间低强度的慢性束缚水浸应激可以使大鼠胃窦ICC数量发生变化.     

不同时程慢性束缚水浸应激对大鼠胃窦Cajal间质细胞超微结构的影响

目的:研究不同时程慢性束缚水浸应激大鼠胃窦Cajal间质细胞(ICC)的超微结构改变.方法:♂ SD大鼠48只随机分为6组、即实验3、7、28 d组和对照3、7、28 d组,每组8只.实验组每日束缚水浸1 h,对照组自由摄食饮水;于实验第4、8、29天晨禁食12 h后脱颈处死.取胃窦组织2块放入3%戊二醛中固定并电镜下观察ICC超微结构.结果:所有对照组ICC的超微结构均无异常改变,试验3、7、28 d各组ICC的超微结构与同期对照组比较均有明显的损害,主要表现为ICC的缝隙连接减少、细胞器减少等,以肌内ICC(ICC-MY)和肌间ICC(ICC-IM)为主:随着应激时间的延长,ICC的超微结构受损逐渐加重.结论:慢性束缚水浸应激可以损伤大鼠胃窦ICC的超微结构.     

早期生活应激所致功能性慢性内脏痛对成年后大鼠抑郁样行为和下丘脑室旁核促肾上腺皮质激素释放激素表达的影响

目的:观察大鼠新生期经历直结肠扩张所致功能性慢性内脏痛对成年后大鼠抑郁样行为及下丘脑室旁核(corticotropin releasing hormone,PVN)内促肾上腺皮质激素释放激素(corticotropin releasing hormone,CRH)表达的影响,探讨早期生活应激所致的功能性慢性内脏痛大鼠伴发抑郁样行为的可能作用机制.方法:将新生期♂SD大鼠20只,随机分成2组(n=10):假手术(Sham)组,新生期结直肠扩张(colorectal distension,CRD)组.CRD组大鼠出生后第8、10、12天,每天给予两次结直肠扩张,成年后即出生后第8-10周,检测腹壁撤退反射(abdominal withdrawal reflex,AWR)评分、痛阈以及腹外斜肌放电幅度,出生后第10-12周检测旷场实验、糖水偏好及强迫游泳等抑郁样行为变化,行为学检测后取结肠进行HE染色观察组织病理变化,取血浆检测皮质酮(cortisol,CORT)水平,取脑PVN进行荧光检测CRH表达水平.结果:(1)大鼠新生期经历直结肠扩张,成年后痛阈值下降,AWR评分、腹外斜肌放电幅度增高(P<0.05,P<0.05),结直肠组织均未见明显病理改变;(2)与Sham组相比,CRD组大鼠在5 min旷场实验中的穿格子线数、直立次数和总运动距离明显减少(均P<0.05),总休息时间明显增多(P<0.05),糖水消耗率下降(P<0.05)强迫游泳过程中不动时间增加(P<0.05);(3)与Sham组相比较,CRD组大鼠血浆中CORT的水平增高(P<0.05),PVN内CRH表达也增加(P<0.05).结论:早期生活应激致功能性慢性内脏痛大鼠有抑郁样行为改变,其机制可能与PVN内CRH表达增加,HPA轴功能失调有关.     

慢性束缚应激对小鼠胃食管的影响

目的探讨心理应激在胃食管反流病(GERD)发生、发展中的作用,并为束缚应激模型的成功建立提供参考指标。 方法20只雄性SPF级昆明小鼠随机分2组(每组10只),即慢性束缚应激(CRS)组和正常对照(NC)组。CRS组小鼠每天在自制式束缚器中限制活动2 h,其余时间2组小鼠在相同环境中自由饮水摄食,实验持续14 d。实验结束后通过葡萄糖耐量试验(GTT)、胰岛素耐受试验(ITT)、小鼠胃食管组织及血清,检测相关指标确认该模型建立成功与否,同时在光学显微镜下观察胃食管黏膜组织学改变。 结果CRS组小鼠体重增加量为(8.6±3.1)g,明显低于NC组小鼠(12.5±3.0)g,差异有统计学意义(P<0.05);胰岛素耐受试验(ITT)结果显示,CRS组小鼠出现胰岛素抵抗现象;ELISA实验显示CRS组小鼠HPA轴相关激素水平明显高于NC组,差异有统计学意义(P<0.05)。CRS组小鼠胃食管黏膜可见充血、水肿及炎症细胞的浸润。 结论CRS可影响机体,引起GERD相关组织病理学变化。为阐述心理因素在GERD中的作用机制研究提供简单有效并且具有借鉴意义的实验基础。     

慢性束缚应激对健康大鼠血糖及血脂的影响

目的 观察慢性束缚应激模型大鼠血清甘油三酯、总胆固醇、低密度脂蛋白-胆固醇及血糖的变化规律.方法 健康雄性Wistar大鼠120只,随机分为束缚应激组及对照组,各60只,两组分别于1周、2周、4周、6周、8周时检测血清甘油三酯、总胆固醇、低密度脂蛋白-胆固醇、葡萄糖水平.结果 束缚应激组1周时,大鼠血清甘油三酯、总胆固醇、低密度脂蛋白.胆固醇、血糖水平与对照组相比均显著升高(P<0.05).束缚应激组大鼠除血糖与对照组相比表现为持续升高趋势外,甘油三酯、总胆固醇、低密度脂蛋白一胆固醇水平在慢性束缚应激第4周后均出现下降趋势,甚至低于对照组(P>0.05).结论 慢性束缚应激所引起的血清脂质水平改变呈各自不同的变化趋势,不同于急性应激.     

慢性应激和增龄对糖尿病易感小鼠胰岛素敏感性的影响

目的研究慢性应激和增龄对小剂量链脲佐菌素诱导的糖尿病易感小鼠胰岛素敏感性的影响,并从胰岛素受体水平探讨其机理。方法取15月龄(幼年)、15月龄(成年)和20月龄(老年)昆明小鼠,用小剂量链脲佐菌素多次腹腔内注射,诱导其糖尿病易感状态后,分为应激组和对照组共6组,各20只。应激组分别给予6周实验性应激源刺激(限制、旋转、拥挤),检测空腹血糖(FBG)和空腹胰岛素(FINS)水平,计算胰岛素敏感指数(ISI),用Scatchard图分析肝细胞膜胰岛素受体高、低和平均亲和常数(K1、K2和K),高、低亲和力(R1、R2),以及每mg膜蛋白胰岛素受体数目(R),用ELISA方法测定10-7mol/L胰岛素刺激的肝细胞磷酸化胰岛素受体数(PINSR)。结果经6周实验性应激,FBG水平随增龄增高,幼年组(103±62)mol/L,成年组(152±36)mol/L,老年组(189±29)mol/L(P<005);FINS水平随增龄下降,幼年组(20±6)mIU/L,成年组(13±5)mIU/L,老年组(10±7)mIU/L(P<005);应激组ISI分别较同龄对照组降低〔幼年组(-51±07)对(-46±06),成年(-50±04)对(-48±08),老年组(-56±09)对(-45±12),P<005〕;成年和老年应激组肝细胞膜R1、R2和R均较同龄对照组上升,且老年应激组较成年应激组明显升高(P均<005);各应激组PINSR均较同龄对照组下降,老年应激组下降更明显,幼年组13±     

5-羟色胺4受体在调节应激大鼠内脏敏感性中的作用

目的研究束缚应激大鼠内脏敏感性及血浆5-羟色胺(5-HT)水平的改变,探讨5-HT4受体激动剂和拮抗剂对束缚应激大鼠内脏高敏感性的影响及对血浆5-HT水平的调节作用.方法雄性Wistar大鼠32只,分为对照组、应激组、5-HT4受体激动剂(1 mg/kg替加色罗,腹腔注射)组和5-HT4受体拮抗剂(3 mg/kg GRI13808,腹腔注射)组.以直肠内球囊扩张(0.4～1.2 ml)时腹壁收缩情况代表其内脏敏感性,分别观察各组大鼠腹壁收缩情况;荧光法测定各组大鼠血浆5-HT水平.结果束缚应激2 h后,应激组大鼠在各个容量直肠扩张时的腹壁收缩次数均较对照组显著增多(0.4 ml10.00±3.74比6.57±1.40;0.8 ml16.75±2.92比11.86±3.44;1.2 ml19.50±4.24比14.86±3.19;P<0.05);应激组大鼠血浆5-HT水平的增幅较对照组显著增高(154.60±19.43比97.75±17.95,P<0.001).在0.4～1.2 ml不同容量的直肠扩张时,5-HT4受体激动剂组的腹壁收缩次数依次为5.86±2.34、10.57±3.26、12.14±2.91;5-HT4受体拮抗剂组依次为3.80±1.48、11.40±3.29、12.40±2.07,均较应激组显著减少(P<0.05,P<0.01).两者均明显降低了应激组大鼠血浆5-HT水平的增加(113.74±13.18比154.60±19.43,P<0.01;47.00±17.17比154.60±19.43,P<0.001).结论应激引起的内脏高敏感性与外周5-HT水平相关;5-HT4受体激动剂及拮抗剂可部分通过影响外周5-HT的释放而改善内脏高敏感性.     

大麻素1型受体在应激大鼠内脏敏感性调节中的作用

目的 研究大麻素1型受体(CB1R)对急性部分束缚应激大鼠内脏敏感性的调节作用.方法 将30只Sprague-Dawley大鼠均分成空白对照组(假性应激)、应激对照组和激动剂组.于实验第1、2、5、8天以测定结直肠气囊扩张(CRD)后腹壁肌电图(EMG)放电频率表示大鼠内脏感觉功能的变化.通过RT-PCR方法 检测实验第8天各组肠道CBIR mRNA表达水平.结果 实验第1天,三组大鼠腹壁肌电放电频率基础值差异均无统计学意义(P值均>0.05).实验第2天,激动剂组大鼠在40、60、80 mm Hg(1 mm Hg=0.133 kPa)压力下的腹壁肌电放电频率[分别为(22.37±1.49)、(42.24±3.03)、(69.09±5.54)次/min]较应激对照组大鼠[分别为(39.71±1.84)、(84.45±8.85)、(112.56±11.66)次/min]显著降低(P<0.05).实验第5天,应激对照组大鼠在40、60、80 mm Hg压力下的腹壁肌电放电频率[分别为(104.12±6.77)、(158.07±18.68)、(193.58±25.69)次/min]较空白对照组[分别为(36.33±5.42)、(74.07±8.25)、(102.94±7.95)次/min]和激动剂组[分别为(74.66±6.44)、(140.10±4.68)、(160.39±5.60)次/min]增多,差异均有统计学意义(P值均<0.05).实验第8天各组大鼠同一压力下的腹壁肌电放电频率差异均无统计学意义(P值均>0.05).应激对照组大鼠回盲部、近端结肠和远端结肠CB1R mRNA表达水平(分别为2.53±0.52、2.29±0.42、2.54±0.29)均显著高于空白对照组(分别为0.56±0.15、0.73±0.12、0.82±0.09),差异均有统计学意义(P值均<0.05).CB1R激动剂干预对大鼠肠道CB1RmRNA表达无影响.结论 CB1R在束缚应激所致大鼠内脏高敏感中起重要的保护性调节作用.     

Effects of electroacupuncture on corticotropin-releasing hormone in rats with chronic visceral hypersensitivity

AIM: To investigate the effect of electroacupuncture on corticotropin-releasing hormone (CRH) in the colon, spinal cord, and hypothalamus of rats with chronic visceral hypersensitivity.METHODS: A rat model of chronic visceral hypersensitivity was generated according to the internationally accepted method of colorectal balloon dilatation. In the 7^th week after the procedure, rats were randomly divided into a model group (MG), electroacupuncture group (EA), and sham electroacupuncture group (S-EA). After treatment, the abdominal withdrawal reflex (AWR) score was used to assess the behavioral response of visceral hyperalgesia. Immunohistochemistry (EnVision method), ELISA, and fluorescence quantitative PCR methods were applied to detect the expression of CRH protein and mRNA in the colon, spinal cord, and hypothalamus.RESULTS: The sensitivity of the rats to the colorectal distension stimulus applied at different strengths (20-80 mmHg) increased with increasing stimulus strength, resulting in increasing AWR scores in each group. Compared with NG, the AWR score of MG was significantly increased (P < 0.01). After conducting EA, the AWR scores of the rats were decreased compared with MG rats. The relative expression of CRH mRNA in the colon, spinal cord, and hypothalamus of MG rats was significantly increased compared with NG rats (P < 0.01). CRH mRNA in the colon and spinal cord of EA and S-EA rats was decreased to varying degrees (P > 0.05) compared with normal rats (NG). However, the decrease in EA compared with MG rats was statistically significant (P < 0.01). The average optical density of CRH expression in the colon of the MG rats was significantly enhanced compared with NG (P < 0.05), while the average optical density of CRH expression in the EA and S-EA rats was significantly decreased compared with MG rats (P < 0.01, P < 0.05, respectively). Compared with MG rats, the CRH concentration in the spinal cord of EA rats was significantly reduced (P < 0.01), but there was no significant change in S-EA rats (P > 0.05).CONCLUSION: Electroacupuncture at the Shangjuxu acupoint was able to significantly reduce the visceral hypersensitivity in rats, and regulated the expression of CRH protein and mRNA in the colon, spinal cord and hypothalamus at different levels, playing a therapeutic role in this model of irritable bowel syndrome.     

Fecal pellet output does not always correlate with colonic transit in response to restraint stress and corticotropin-releasing factor in rats

Background Fecal pellet output has been assessed as a colonic motor activity because of its simplicity. However, it remains unclear whether an acceleration of colonic transit correlates well with an increase in fecal pellet output. We examined the causal relationship between colonic transit and fecal pellet output stimulated by the central application of corticotropin-releasing factor (CRF) and restraint stress. Methods Immediately after intracisternal injection of CRF, ⁵¹Cr was injected via a catheter positioned in the proximal colon. Ninety minutes after ⁵¹Cr injection, the total number of excreted feces was counted, and then the rats were killed. The radioactivity of each colonic segment was evaluated, and the geometric center (GC) of the distribution of ⁵¹Cr was calculated. For the restraint stress study, after administration of ⁵¹Cr into the proximal colon, rats were submitted to wrapping restraint stress for 90 min. Then they were killed, and GC was calculated. Results Both restraint stress and CRF significantly accelerated colonic transit. There was a positive correlation observed between fecal pellet output and GC of colonic transit in response to restraint stress, but not CRF, when the number of excreted feces was more than three. In contrast, there was no significant correlation observed between the two in stress and CRF when the number of excreted feces was less than two. Conclusions The acceleration of colonic transit in response to restraint stress and central administration of CRF does not always correlate with an increase in fecal pellet output.     

先天抑郁大鼠内脏敏感性研究

目的 比较先天抑郁Fawn-Hooded(FH/Wjd)大鼠与Sprague-Dawley(SD)大鼠的内脏敏感性,探讨FH/Wjd大鼠作为精神障碍与肠易激综合征共病模型的可行性.方法 采用强迫游泳实验(FST)和糖水实验(SPT)验证FH/Wjd大鼠的抑郁特性.通过腹壁回撤反射(AWR)评估大鼠对不同压力结直肠扩张(CRD)的敏感性.免疫组织化学法检测大鼠结肠5-HT表达水平及结肠、大脑前额叶下边缘皮质(IL)、前边缘皮质(PrL)、前喙扣带回皮质(rACC)区域的c-fos表达水平.结果 FST中FH/Wjd大鼠水中静止时间显著长于SD大鼠(t＝8.931,P＝0.000).SPT中FH/Wjd大鼠糖水饮用量占总饮水量的比例显著低于SD大鼠(t＝4.155,P＝0.001).FH/Wjd大鼠在各CRD压力梯度(20、40、60、80 mm Hg,1 mm Hg=0.133 kPa)下的AWR评分均显著高于SD大鼠(t值分别＝-2.697、-3.464、-6.822、-3.976,P值分别＝0.022、0.006、0.000、0.003).FH/Wjd大鼠对照组结肠5-HT表达水平高于SD大鼠对照组(t=-11.371,P=0.000).FH/Wjd大鼠扩张组和SD大鼠扩张组结肠5-HT表达水平均较各自的对照组上升,且FH/Wjd大鼠表达水平高于SD大鼠(t＝-3.364,P＝0.007).FH/Wjd大鼠对照组和SD大鼠对照组结肠、前额叶各脑区c-fos表达水平差异无统计学意义(结肠:t＝-0.129、P＝0.900;IL:t＝-1.316、P＝0.218;PrL:t＝1.241、P＝0.243;rACC:t＝2.151、P＝0.057).FH/Wjd大鼠扩张组和SD大鼠扩张组结肠与前额叶脑区c-fos 表达水平均较各自的对照组显著上升,且FH/Wjd大鼠表达水平高于SD大鼠(结肠:t＝-5.864、P＝0.000;IL:t＝-2.530、P＝0.030;PrL:t＝-7.039,P＝0.000;rACC:t＝-6.489、P＝0.000).结论 先天抑郁FH/Wjd大鼠存在内脏高敏感性,其肠道高表达5-HT及结肠和大脑IL、PrL、rACC 区域对内脏伤害性刺激的高反应性.有可能作为精神障碍和肠易激综合征共病的动物模型.

Abstract：

Objective To explore the feasibility of inherent depressive Fawn-Hooded (FH/Wjd)rats as a comorbidity model of mental disorder and irritable bowel syndrome (IBS) by comparing visceral sensitivity of FH/Wjd rats and Sprague-Dawley (SD) rats. Methods Depression trait of FH/Wjd rats was validated through forced swimming test (FST) and sucrose preference test (SPT).Visceral sensitivity to colorectal distention (CRD) under various pressures was assessed by abdominal withdrawal reflex (AWR). The expression of 5-HT in rats' colon,c-fos expression in colon and brain areas of infralimbic cortex (IL),prelimbic cortex (PrL) and rostral anterior cingulated cortex (rACC)was tested with immunohistochemistry. Results FST indicated that the immobility time of FH/Wjd rats was significantly longer than that in SD rats (t=-8. 931,P＜0. 01). SPT showed that the ratio of sucrose water in total liquid consumed was significantly lower in FH/Wjd rats than that of SD rats(t=4. 155,P=0. 01). At each CRD pressure gradient (20,40,60 and 80 mm Hg,1 mm Hg=0. 133kPa),AWR score was all significantly higher in FH/Wjd rats than that of SD rats (t=-2. 697,-3.464,-6.822 and -3. 976,P=0. 022,0.006,0.000 and 0.003). The expression level of 5-HT in FH/Wjd rats' control group was significantly higher than that of SD rats' control group (t=-11.371,P=0. 000). Compared with their own control group,5-HT expression increased both in FH/Wjd rats dilated groups and SD rats dilated groups,and the expression level in FH/Wjd rats was higher than that of SD rats (t= -3. 364,P= 0. 007). There was no significant difference of c-fos expression in colon and prefrontal brain areas between FH/Wjd rats' control group and SD rats'control group (colon:t=-0. 129,P= 0. 900;IL:t=-1. 316,P= 0. 218;PrL:t=1. 241,P = 0. 243;rACC:t=2. 151,P = 0. 057). Compared with their own control group,the expression of c-fos in colon and prefrontal brain areas significantly increased both in FH/Wjd rats dilated groups and SD rats dilated groups,and the expression level in FH/Wjd rats was higher than that of SD rats (colon: t=- 5.864,P= 0.000;IL:t=-2. 530,P = 0. 030;PrL:t=-7. 039,P= 0. 000;rACC:t=-6. 489,P=0. 000). Conclusions Inherent depressive FH/Wjd rats present visceral hypersensitivity. Meanwhile,the expression of 5-HT in colon is high,and with hyperreactivity to visceral noxious stimuli in colon and brain IL,PrL,and rACC areas. FH/Wjd rats might be a comorbidity animal model of mental disorder and IBS.