慢性不可预知性应激与完全弗氏佐剂、福尔马林诱导的妊娠期疼痛-抑郁共病小鼠模型的构建与评价

Establishment and Evaluation of Mouse Model of Pregnancy Pain-depression Comorbidity Induced by Chronic Unpredictable Stress,Complete Freund's Adjuvant and Formalin

目的通过慢性不可预知性应激(chronic unpredictable stress,CUS)联合完全弗氏佐剂(complete Freund's adjuvant,CFA)和福尔马林诱导建立妊娠期疼痛-抑郁共病小鼠动物模型,对其相关表型进行系统评价,并初步探究其共病的病理基础。方法选择8周龄的C57BL/6J雌性小鼠,依据糖水偏好实验数据,采用随机分层方法将其分为对照组(妊娠前不干预)和CUS组(妊娠前予以CUS干预)。CUS造模完成后,将雌雄小鼠合笼配对,妊娠期小鼠分别于右后肢足底皮下注射50%CFA和5%福尔马林溶液,诱导建立妊娠期疼痛-抑郁共病小鼠模型。实验共分8组,即为对照-空白组、CUS-空白组、对照-CFA组、CUS-CFA组、对照-福尔马林组、CUS-福尔马林组、对照-CFA+福尔马林组、CUS-CFA+福尔马林组,每组10只。各组小鼠均在CUS干预前后、妊娠后和分娩后,通过糖水偏好实验、强迫游泳实验、悬尾实验和旷场实验评价行为学变化,并通过机械刺痛实验和热辐射痛实验测定各组小鼠对痛觉的敏感性变化;最后处死小鼠,用酶联免疫吸附法检测各组小鼠海马体中白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α),以及血清皮质醇(cortisol)和促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)的含量。结果与对照-空白组相比,CUS-空白组模型小鼠产生了明显的抑郁样行为,并伴随痛阈明显降低(P<0.001)。与对照-空白组相比,对照-CFA+福尔马林组在CFA注射与福尔马林注射后均出现痛阈下降(P<0.01)。与对照-空白组和对照-福尔马林组相比,CUS-福尔马林组的痛阈明显降低(P<0.01),且三者依次下降。与对照-空白组和对照-CFA组相比,CUS-CFA组的痛阈明显降低(P<0.001),且三者依次下降。与对照-空白组、对照-CFA+福尔马林组相比,CUS-CFA+福尔马林组小鼠的机械痛阈显著降低(P<0.001)且三者依次下降,热辐射耐受时间缩短(P<0.01)且三者依次缩短。与对照-CFA+福尔马林组和CUS-空白组小鼠相比,CUS-CFA+福尔马林组小鼠的糖水偏好百分比显著降低(P<0.001),强迫游泳时不动时间显著延长(P<0.001),悬尾时不动时间也显著延长(P<0.001),在旷场中央探索的时间显著缩短(P<0.001),旷场探索总路程显著缩短(P<0.001),中央探索路程占比减少(P<0.001)。与对照-CFA+福尔马林组和CUS-空白组相比,CUS-CFA+福尔马林组小鼠血清皮质醇和ACTH水平显著升高(P<0.01),海马体中IL-6、TNF-α水平升高(P<0.05)。结论CUS联合CFA和福尔马林注射可作为建立妊娠期疼痛-抑郁共病C57BL/6J小鼠模型的理想方法。该模型小鼠的行为学改变可能是通过调节海马区的炎症反应水平和下丘脑-垂体-肾上腺(hypothalamic–pituitary–adrenal,HPA)轴的激素水平导致的。

Objective To establish a mouse model of pregnancy pain-depression comorbidity induced by chronic unpredictable stress(CUS),complete Freund's adjuvant(CFA),and formalin,and to systematically evaluate the associated phenotypes and preliminarily explore the pathological basis of the comorbidity.Methods Eight-week-old C57BL/6J female mice were randomly strarified divided into a control group(no intervention before pregnancy)and a CUS model group(CUS intervention before pregnancy)based on sucrose preference test(SPT)data.After completing the CUS treatment,female and male mice were paired and mated.Pain was induced by injecting 50%CFA and 5%formalin in the right hind foot during pregnancy to create a model of pregnancy pain-depression comorbidity.The experiment was divided into 8 subgroups:control-blank group,CUS-blank group,control-CFA group,CUS-CFA group,control-formalin group,CUS-formalin group,control-CFA+formalin group,and CUS-CFA+formalin group,with 10 mice in each group.The mice in each group were subject to behavioral tests,including the SPT,forced swimming test,tail suspension test,and open field test before and after CUS intervention,during pregnancy,and after delivery.Pain sensitivity changes were measured using mechanical allodynia and thermal hyperalgesia tests.Mice were then euthanized.Levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in hippocampus,as well as cortisol and adrenocorticotropic hormone(ACTH)in serum,were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with the control-blank group,the CUS-blank group showed a significant depression-like behavior with reduced pain threshold(P<0.001).The control-CFA+formalin group showed a decrease in pain threshold after both CFA injection and formalin injection(P<0.01).Compared with the control-blank and control-formalin groups,the pain threshold was significantly lower in the CUS-formalin group(P<0.01),with a sequential decrease among the three.Compared with the control-blank and control-CFA groups,the pain threshold was significantly lower in the CUS-CFA group(P<0.001),with a sequential decrease among the three.Compared with the control-blank and control-CFA+formalin groups,the mechanical pain threshold of mice in the CUS-CFA+formalin group was significantly lower(P<0.001)and the thermal radiation tolerance time was shorter(P<0.01),both with sequential decreases among the three.Compared with the control-CFA+formalin and the CUS-blank groups,the CUS-CFA+formalin group had a significantly lower percentage of sucrose preference(P<0.001),longer immobility time during the forced swimming test(P<0.001)and tail suspension test(P<0.001),reduced central exploration time in the open field test(P<0.001),reduced total exploration distance(P<0.001),and reduced percentage of distance traveled for central exploration(P<0.001).Compared with the control-CFA+formalin and CUS-blank groups,the serum cortisol and ACTH levels of the CUS-CFA+formalin group were significantly higher(P<0.01),and the levels of IL-6 and TNF-αin the hippocampus were higher(P<0.05).Conclusion The combination of CUS+CFA+formalin injections is an ideal method for establishing a C57BL/6J mouse model of pregnancy pain-depression comorbidity.The behavioral changes in model mice may be attributed to the regulation of inflammatory response in hippocampus and hormone levels in the hypothalamic-pituitary-adrenal(HPA)axis.