【作者】 吴国媛；

【导师】 吴斌；

【作者基本信息】 福建医科大学 ， 儿科学， 2010， 硕士

【摘要】 目的:采用行为学和电生理学评价方法,观察不同新生期母婴分离(NMS)时间对幼鼠内脏痛觉敏感性影响,探讨采用NMS建立幼年动物内脏高敏感模型所需的NMS适宜时间,对进一步认识NMS远期危害,指导临床防治具有重要指导意义。方法: 50只SD新生大鼠随机分成NMS39、NMS60、NMS78、NMS120组和对照组(C组),每组10只;NMS39组出生后第2~14d每天与母鼠分离3h(8:00-11:00am),连续13d,NMS60组出生后第2~21d每天与母鼠分离3h(8:00-11:00am),连续20d,NMS78组生后第2~14d(每天上午、下午分别与母鼠分离3h(8:00-11:00am;14:00-17:00pm),连续13d,NMS120组生后2~21d每天上午、下午分别与母鼠分离3h(8:00-11:00am;14:00-17:00pm),连续20d,对照组不予任何处理,与母鼠同笼。常规饲养到6周龄,通过不同压力结直肠扩张(CRD)刺激后的腹壁撤退反射评分(AWR)、痛阈测定和腹外斜肌放电波幅值测量进行幼鼠肠道痛觉敏感性评价,测定幼鼠排便粪点数和玻璃小球排出时间评估幼鼠结肠动力,处死幼鼠,留取降结肠进行病理学检查。采用SPSS11.0软件进行统计分析,α=0.05为显著性检验标准。结果:随CRD压力增加,5组幼鼠AWR评分和腹外斜肌放电波幅值均逐渐增加;当CRD为20mmHg、40mmHg、60mmHg时,NMS39组、NMS60组、NMS78组、NMS120组幼鼠AWR评分明显高于C组,CRD为80mmHg时,5组幼鼠AWR评分差异无显著意义;当CRD为15mmHg、30mmHg、45 mmHg、60mmHg时,NMS39组、NMS60组、NMS78组、NMS120组幼鼠腹外斜肌放电波幅值明显高于C组;CRD为75mmHg时,5组幼鼠腹外斜肌放电波幅值差异无显著意义;NMS39组、NMS60组、NMS78组、NMS120组及C组幼鼠痛阈值分别为15.83±5.05mmHg、16.50±8.40mmHg、13.50±2.88 mmHg、16.00±5.28 mmHg、33.83±4.31mmHg;NMS39组、NMS60组、NMS78组、NMS120组幼鼠间AWR评分、腹外斜肌放电波幅值及痛阈差异均无显著性意义;5组幼鼠玻璃小球排出时间及粪点数差异无统计学意义;各组降结肠均未见明显病理组织损伤。结论:长时间不同新生期母婴分离均能造成大鼠痛阈下降,出现内脏高敏感性;并且这种高敏感性能持续到幼年期,但幼鼠没有肠道动力改变,也没有排便习惯改变及大便性状改变,同时没有明显的组织病理学改变。采用新生大鼠在出生后第2~14d给予每天母婴分离3h可建立一种理想的幼鼠内脏痛高敏感模型。目的:采用新生期母婴分离(NMS)建立幼鼠内脏高敏感性模型,观察内脏高敏感性模型幼鼠下丘脑室旁核(PVN)及前皮质扣带回(ACC)内Fos蛋白表达变化,探讨其在参与幼鼠内脏痛高敏感形成的机制,为临床上防治NMS所引起的危害及疾病提供理论依据。方法:32只SD新生大鼠按析因设计分成4组,每组8只。A1B1组为NMS组并在6周龄时接受CRD,A1B2组为NMS组,6周龄未给予CRD,A2B1组未给予NMS并在6周龄接受CRD,A2B2组未给予NMS,6周龄未给予CRD。A1B1、A2B1组幼鼠CRD接受刺激后2h和A1B2、A2B2组幼鼠留取PVN和ACC,应用免疫组织化学染色及计算机图像分析系统进行PVN和ACC内Fos蛋白免疫阳性(FLI)细胞数半定量分析。采用SPSS11.0软件包进行统计分析,α=0.05为显著性检验标准。结果:A1B1组、A1B2组、A2B1组及A2B2组幼鼠PVN内FLI细胞数分别为50.61±11.68、31.25±7.28、43.67±16.03和18.13±3.41,ACC内FLI细胞数分别为53.48±15.45、38.42±11.67、35.02±9.80和20.13±2.08。NMS和幼鼠6周龄时CRD均可使幼鼠PVN和ACC内FLI细胞数显著升高,差别均有统计学意义;NMS引起幼鼠PVN和ACC内FLI细胞数增加的主效应分别为10.03个、18.38个,幼鼠6周龄时CRD引起幼鼠PVN和ACC内FLI细胞数增加的主效应分别为22.45个、14.98个;NMS和CRD对PVN和ACC内FLI细胞数影响无交互作用。结论:新生期持续NMS可导致幼鼠中枢神经系统发育异常和敏化,接受伤害性CRD刺激后能显著引起PVN和ACC内神经元Fos蛋白高表达,出现内脏痛觉高敏感性;PVN和ACC是幼鼠大脑中枢内脏伤害性刺激信号传导处理过程和感知觉的重要区域,其激活和致敏在NMS诱导的幼鼠内脏痛高敏性中起到至关重要作用。更多还原

【Abstract】 Objective: Observe the effect of various neonatal maternal separations (NMS) stimulation on visceral sensibility of pain in developing rats by the method of behavior and electrophysiology research. Explore the best time of the visceral hyperalgesia model in developing rats by NMS. This study can deeply help understanding the future dangers of NMS and providing guidance for prevention and cure in clinic.Method: 50 SD neonatal rats were divided randomly into NMS39、NMS60、NMS78、NMS120 group (n=10) and control group (n=10). Rats in the NMS39 group were separated from the dam for 3h/d (8:00-11:00am) on postnatal days 2~14 for 13 consecutive days, NMS60 group were separated on postnatal days 2~21 for 20 consecutive days;NMS78 group were separated from the dam for 3h/d (8:00-11:00am) and 3h/d (14:00-17:00pm) on postnatal days 2~14 for 13 consecutive days, NMS120 group were separated on postnatal days 2~21 for 20 consecutive days; and the control C group were not treated and remained in the home cage with the dam. Conventionally breeding to the young period (6-week age), observing the score of Abdominal Withdrawal Reflex (AWR)、visceral pain threshold and measurement of electrical discharge of external oblique muscle of abdomen (EOMA) under different pressures of colorectal distension irritation (CRD) to evaluate visceral algesthesia in rats. Measure the amount of fecal pellets and the time of glass pellets output in rats to evaluate colon motility. Kill the rats and select descending colon to do pathological examination. SPSS 11.0 software for Windows was used in all statistical tests.α=0.05 was considered significant.Result: The AWR score and the amplitudes of spike of EOMA increased gradually with the rising of the CRD pressure in 5 groups of developing rats. When the CRD pressure were 20mmHg,40mmHg,60mmHg, the AWR score in NMS39 group ,NMS60 group ,NMS78 group and NMS120 group were higher than the control C group. When pressure reached 80mmHg, there were no significant differences of AWR score among five groups. When the CRD pressure were 15mmHg, 30mmHg, 45mmHg, 60mmHg the amplitudes of spike of EOMA score in NMS39 group ,NMS60 group ,NMS78 group and NMS120 group was higher than the control C group. When pressure reached 75mmHg, there were no significant differences of the amplitudes of spike of EOMA score among five groups. The Pain thresholds of the group NMS39, NMS60, NMS78, NMS120 and group C were (15.83±5.05) mmHg, (16.50±8.40) mmHg, (13.50±2.88) mmHg, (16.00±5.28) mmHg, (33.83±4.31) mmHg, but the differences among the group NMS39, NMS60, NMS78 and NMS120 were no statistical significances. There were no significant differences of fecal pellets and the time of glass pellets output among the five groups. There were no obvious histopatholigical changes in descending colon among the five groups.Conclusion: The persistent NMS can result in decreasing of pain threshold in rats, emerging chronic visceral hypersensibility. This hyperalgesia can continue to juvenile stage and there is no altered intestinal motility, no altered bowel function and properties, and no histological change found in colorectal tissues. The visceral hyperalgesia model of developing rat established by maternal separation for 3h/d on postnatal days 2~14 for 13 consecutive days. Objective: Establish the model of visceral hyperalgesia in developing rats by Neonatal maternal separation (NMS). Detect FOS expression in hypothalamus paraventricular nucleus and anterior cingulated cortex of developing rats with visceral hyperalgesia. Explorer the mechanism of visceral hyperalgesia in developing rats, to provide the theoretical basis for prevents and cures the disease of NMS in clinic.Method: According to the factorial design, 32 SD rats were divided into four groups with each 8, Group A1B1 was NMS group and imposed on CRD at 6-week age, yet Group A1B2 was not imposed on CRD as NMS group. Group A2B1 was imposed on CRD at 6-week age while Group A2B2 without treatment both as the control groups. On the 6th week, after the rats of Group A1B1 and A2B1 were imposed on CRD for 2 hours while rats of Group A1B2 and A2B2 not imposed on CRD were sampled hypothalamus paraventricular nucleus and anterior cingulated cortex, the semi-quantity analysis of the cell numbers of Fos-Like Immunoreactivity (FLI) of these sites were made through immunohisrochemical coloration and computer image analyzing system. SPSS 11.0 software for Windows was used in all statistical tests.α=0.05 was considered significant.Result: The number of FLI in PVN in group A1B1、A1B2、A2B1、A2B2 were (50.61±11.68), (31.25±7.28), (43.67±16.03), (18.13±3.41), and (53.48±15.45)、(38.42±11.67)、(35.02±9.80)、(20.13±2.08) in ACC, respectively, NMS and CRD at 6-week age both can make it increase significantly. The main effect of the number of FLI in PVN and ACC caused by NMS were 10.03 and 18.38, while 22.45 and 14.98 by CRD at 6-week age. There was no interaction between NMS and CRD on affecting the number of FLI in PVN and ACC.Conclusion: The persistent stimulation of MS in neonatal period may cause rat’s central nervous system abnormalities and sensitization. Accepting detrimental CRD can significantly cause the high expression of FOS in PVN and ACC, and result in emerging visceral hyperalgesia. PVN and ACC were the important domains which transmit the visceral detrimental stimulation and sense signal in developing rats’central nervous system. The activation and hyperalgesia of PVN and ACC play an important role in developing rats’visceral hyperalgesia induced by NMS.更多还原