帕罗西汀对束缚应激与额叶皮质相关性的影响研究

目的：观察束缚应激对大鼠额叶皮质的影响,探讨帕罗西汀在防治束缚应激引起焦虑的作用机制。方法：复制大鼠束缚应激模型,分为束缚应激组、治疗组和保护组,另设对照组（不束缚应激）,每组均n=10。观察大鼠行为学改变、额叶皮质c-fos基因表达、5-羟色胺（5-HT）和血浆皮质酮含量的变化,观察帕罗西汀干预的影响。结果：与对照组比较,束缚应激组大鼠额叶皮质的c-fos表达显著增加,5-HT表达明显减少,血浆皮质酮含量明显升高;与束缚应激组比较,治疗组和保护组血浆皮质酮含量和额叶皮质c-fos表达明显减少,5-HT表达明显增加。结论：额叶皮质参与了束缚应激反应;帕罗西汀抑制额叶皮质的c-fos基因表达、减少血浆皮质酮含量和增加额叶皮质5-HT含量可能是其抗焦虑作用机制之一。     

慢性束缚应激对大鼠行为及前额叶皮质细胞外信号调节激酶磷酸化的影响

目的 探讨慢性束缚应激模型大鼠的行为及前额叶皮质中磷酸化细胞外信号调节激酶（ERK1／2）表达的变化。方法 将雄性SD大鼠随机分为正常对照组和束缚应激组，每组8只。将束缚应激组大鼠放入特制的束缚器中限制其活动，6k／d，连续21d。比较应激前后大鼠旷场行为和Morris水迷宫行为学的改变，用蛋白免疫印迹技术和免疫组织化学方法观察应激后大鼠前额叶皮质P—ERK1／2表达的变化，并与正常对照组比较。结果 （1）行为学改变：应激后，束缚应激组大鼠的水平活动度[（4265±864）mm]少于正常对照组[（8562±502）mm]，中央停留时间[（39．1±4．3）s]长于正常对照组[（24．6±1．6）s]，均P〈0．01；束缚应激组大鼠在Morris水迷宫实验的目标象限活动时间[（57．2±1．7）s]和穿越站台次数[（2．0±0．8）次]均少于正常对照组[分别为（70．7±3．6）s和（6．2±1．0）次；均P〈0．01]。（2）p-ERK1／2蛋白水平：应激后束缚应激组吸光度（A）值[（0．767±0．006）]低于正常对照组[（0．813±0．015）；P〈0．05]。（3）p-ERK1／2阳性细胞数：应激后束缚应激组[（76±5）个]少于正常对照组[（110±14）个；P〈0．05]，且树突染色浅淡。结论 慢性心理应激明显影响动物的活动度和记忆能力；前额叶皮质中p-ERK1／2表达的减少，提示ERK信号转导通路可能参与了应激发生的机制。     

帕罗西汀对抑郁模型大鼠前额叶皮质鞘磷脂和神经酰胺的影响

目的观察帕罗西汀对慢性不可预见应激(CUS)模型大鼠前额叶皮质(PFC)中鞘磷脂(SM)和神经酰胺(Cer)水平的影响。方法按照随机数字表法将21只SD大鼠随机分为对照组(Sham组)、模型组(CUS组)和帕罗西汀组(Par组),每组7只。CUS组和Par组均接受CUS造模,并且在造模后每天腹腔注射生理盐水(1 ml/kg)或帕罗西汀(10 mg/kg),连续7 d;Sham组在同一时间段每天腹腔注射生理盐水。随后处死大鼠,取PFC进行质谱分析,比较各处理组SM和Cer以及其组成小分子相对丰度的差异。结果CUS组的SM相对丰度倍数为(0.73±0.16),显著低于Sham组的(1.02±0.08)和Par组的(0.89±0.05)(均P<0.05);CUS组的Cer相对丰度倍数为(1.35±0.18),显著高于Sham组的(1.05±0.10)和Par组的(1.14±0.11)(均P<0.05);Sham组和Par组的SM和Cer相对丰度比较,差异均无统计学意义(P>0.05)。与Sham组相比,CUS组的5个SM和2个Cer小分子相对丰度降低,1个SM和5个Cer小分子相对丰度升高(均P<0.05);与Par组相比,CUS组的2个SM小分子相对丰度降低,8个Cer小分子相对丰度升高(均P<0.05);Sham组的1个SM和2个Cer小分子相对丰度降低,1个SM和6个Cer小分子相对丰度升高(均P<0.05)。结论CUS模型大鼠前额叶皮质的SM水平降低而Cer水平升高,帕罗西汀干预可以改善这一现象。     

整合素信号通路可能参与大鼠束缚应激后的恢复过程

目的筛选与束缚应激大鼠应激反应恢复速度相关的基因,探讨应激反应个体恢复的分子机制。方法观察大鼠2小时束缚应激后血浆促肾上腺皮质激素（adrenocorticotropic-hormone,ACTH）及皮质酮的变化规律,根据应激结束后1小时的血浆ACTH及皮质酮下降程度将大鼠分为快速恢复组与慢速恢复组。采用微阵列技术检测快速恢复组与慢速恢复组下丘脑组织基因表达的差异,用实时逆转录-聚合酶链反应验证部分基因的表达差异。结果基因芯片分析结果显示：大部分基因在两组间并无差异表达,在11个差异表达的基因中,快速恢复组中参与整合素信号通路的踝蛋白（talin）、整合素α6和丝/苏氨酸蛋白磷酸酶PP1β催化亚基（serine/threonine pro-tein phosphatase PP1-beta catalytic subunit,PP1B）基因有1.5倍上调,而结合粘附分子1（junctional adhesion mol-ecule 1, F11r）基因有 1.5 倍下调。实时逆转录 - 聚合酶链反应结果与芯片分析结果一致。结论本研究构建了束缚应激大鼠恢复过程的下丘脑基因表达谱,结果提示整合素信号通路可能参与应激的恢复过程。     

慢性应激对大鼠海马CA3区锥体细胞顶树突及血清皮质酮浓度的影响

目的探讨慢性应激对大鼠海马CA3区锥体细胞结构和血清皮质酮浓度的影响。方法将20只雄性Sprague-Dawley大鼠按体质量随机分为应激组和对照组,每组10只。采用高尔基染色法及酶联免疫分析方法,观察慢性强迫游泳应激对大鼠海马CA3区锥体细胞顶树突和血清皮质酮浓度的影响。结果应激组大鼠海马CA3区锥体细胞顶树突的总长度[(112±10)μm]短于对照组[(168±34)μm],差异有统计学意义(P<0.01);一级树突直径[(9.0±1.1)μm]大于对照组[(5.7±0.9)μm],差异有统计学意义(P<0.01);血清皮质酮浓度[(13±14)μg/L]低于对照组[(30±16)μg/L],差异有统计学意义(P<0.05)。结论慢性强迫游泳可引起大鼠海马CA3区锥体细胞顶树突及血清皮质酮浓度的改变。     

整合素信号通路可能参与大鼠束缚应激后的恢复过程(英文)

目的筛选与束缚应激大鼠应激反应恢复速度相关的基因,探讨应激反应个体恢复的分子机制。方法观察大鼠2小时束缚应激后血浆促肾上腺皮质激素(adrenocorticotropic-hormone,ACTH)及皮质酮的变化规律,根据应激结束后1小时的血浆ACTH及皮质酮下降程度将大鼠分为快速恢复组与慢速恢复组。采用微阵列技术检测快速恢复组与慢速恢复组下丘脑组织基因表达的差异,用实时逆转录-聚合酶链反应验证部分基因的表达差异。结果基因芯片分析结果显示:大部分基因在两组间并无差异表达,在11个差异表达的基因中,快速恢复组中参与整合素信号通路的踝蛋白(talin)、整合素α6和丝/苏氨酸蛋白磷酸酶PP1β催化亚基(serine/threonine pro-tein phosphatase PP1-beta catalytic subunit,PP1B)基因有1.5倍上调,而结合粘附分子1(junctional adhesion mol-ecule 1, F11r)基因有 1.5 倍下调。实时逆转录 - 聚合酶链反应结果与芯片分析结果一致。结论本研究构建了束缚应激大鼠恢复过程的下丘脑基因表达谱,结果提示整合素信号通路可能参与应激的恢复过程。     

慢性强迫游泳应激对大鼠情绪和脑细胞外信号调节激酶磷酸化水平的影响

目的探讨慢性强迫游泳应激对大鼠情绪和脑细胞外信号调节激酶磷酸化(P-ERK1/2)水平的影响。方法将30只SD雄性大鼠随机分为游泳应激组、装置对照组和空白对照组,每组10只。游泳应激组每天接受5min的游泳应激,装置对照组每天接受5min的新异场景应激,均连续14 d,空白对照组不进行任何干预,然后观察大鼠行为(体质量增长量、旷场测验和糖精水溶液偏好测验)。采用免疫印迹法测定大鼠海马和前额叶皮质的P-ERK1/2。结果(1)游泳应激组在应激7d和应激14d的体质量增长[分别为(75±22)g和(70±24)g]均低于空白对照组[分别为(101±35)g和(115+47)g],均P＜0.05。(2)装置对照组的粪便排泄量[(1.4±1.9)粒]多于空白对照组[(0.4±1.0)粒]和游泳应激组[(0.1±0.3)粒],均P＜0.05；而游泳应激组的水平活动距离[(2077±1245)cm]少于空白对照组[(2990±1038)cm]和装置对照组[(3110±1462)cm],均P＜0.05。(3)游泳应激组的糖精水溶液摄入量[(11±6)g]和糖精水溶液摄入量占总液体摄入量的比例[(37±16)％]均低于空白对照组[分别为(15±4)g和(47±15)％],均P＜0.05。(4)游泳应激组在海马[(46±95)％]和前额叶皮质[(65±24)％]的P-ERK2水平均低于空白对照组[分别为(76±30)%和(99±42)%],均P＜0．05。结论慢性强迫游泳应激能诱发大鼠的抑郁情绪,降低P-ERK2在海马和前额叶皮质的水平。     

束缚应激血清对免疫细胞功能的影响

应用空斑形成细胞（PFC）方法观察体外免疫脾细胞在应激血清作用下，分泌抗体的能力并与正常血清进行比较，结果表明免疫脾细胞在应激血清作用下分泌抗体的能力与对照组间差异无显著意义；但是应激血清可抑制脂多糖（LPS）刺激的淋巴细胞转化反应，且有量效关系。应用ConA活化的脾细胞制成IL－2反应细胞，在加入应激血清及正常对照血清作用下，观察IL－2反应细胞对重组基因IL－2（rIL－2）的反应性变化发现，当应激血清与rIL－2同时加入培养基时，IL－2反应细胞的增殖效应与对照组间差异无显著性意义；但是预先将应激血清与ConA活化的脾细胞混合培养2h后，再加入rIL－2，在应激血清作用下IL－2反应细胞对rIL－2刺激的反应性则较对照组显著降低；不同稀释度的应激血清的抑制作用也相应不同。说明应激血清可抑制ConA活化的脾细胞对IL－2刺激的反应性。     

文拉法辛对慢性应激抑郁大鼠前额区可塑性相关蛋白mRNA表达的影响

目的：探讨文拉法辛对慢性应激抑郁大鼠前额区3种可塑性相关蛋白mRNA表达的影响。方法：用慢性不可预见应激（CUS）方法建立抑郁大鼠模型,给予2种剂量（5mg／kg和10mg／kg）的抗抑郁药物文拉法辛14d或28d,用逆转录-聚合酶链反应检测大鼠前额区脑源性神经营养因子（BDNF）、转录因子环磷腺苷反应元件结合蛋白（CREB）和神经细胞粘附分子（NCAM）mRNA表达的变化。结果：抑郁模型大鼠体质量增加量,蔗糖水消耗量和行为学测试均较正常组明显下降,提示抑郁模型大鼠在第28天建立成功。CUS28d后前额区BDNF、CREB和NCAM mRNA表达均较正常组明显降低（P〈0．05）,5mg／kg文拉法辛组明显增加抑郁模型大鼠前额区3种可塑性相关蛋白mRNA的表达,10mg／kg文拉法辛组轻度降低抑郁模型大鼠前额区3种可塑性相关蛋白mRNA的表达。结论：文拉法辛在调节前额区神经可塑性时具有剂量依赖性,BDNF、CREB和NCAM在抑郁症病因和治疗中可能发挥着重要作用。     

两种慢性应激诱导的抑郁模型大鼠前额叶认知功能的比较研究

目的本研究旨在比较两种慢性应激诱导的抑郁模型大鼠前额叶介导的认知灵活性损伤特征。方法分别给予应激组大鼠(n=8只/组)两周慢性不可预期性应激(chronic unpredictable stress,CUS)或社会挫败(social defeat,SD)应激。应激结束后采用糖水偏好测试检测快感缺失(一种典型抑郁样行为),采用注意定势转移任务(attentional set-shifting task,AST)检测认知灵活性改变,主要包括逆反学习(reversal learning,REL)和外维度定势转移(extra-dimensional set-shifting,EDS)能力。结果与相应对照组相比,CUS组大鼠的糖水消耗量降低[(7.24±1.64)vs(13.83±1.50),P0.05]。SD组大鼠的糖水消耗量和糖水偏好指数较相应对照组降低[(4.28±1.96)vs(13.17±2.79),P0.01;(27.96±11.64)vs(82.97±16.13),P0.05]。在AST测试中,CUS诱导以EDS损害为特征的认知灵活性缺失,表现在CUS组大鼠在EDS阶段的训练达标次数较相应对照组增加[(15.57±1.53)vs(10.50±1.41),P0.05],而社会挫败应激诱导以REL和EDS损害为特征的认知灵活性缺失,表现在SD组大鼠在REL和EDS阶段的训练达标次数均较相应对照组增加[REL:(17.30±0.76)vs(14.00±0.97),P0.01);EDS:(15.80±1.72)vs(9.33±0.80),P0.01]。结论慢性不可预期性应激和社会挫败应激诱导的抑郁模型大鼠表现出认知灵活性不同成分的损害,为进一步研究抑郁症不同认知表型障碍的神经生物学机理提供了实验基础。     

Repeated restraint stress and corticosterone injections during late pregnancy alter GAP-43 expression in the hippocampus and prefrontal cortex of rat pups

In the offspring of prenatal stress animals, overactivity and impaired negative feedback regulation of the hypothalamic–pituitary–adrenal axis are consistent finding. However, little was known about how prenatal stress can permanently alter developmental trajectories of pup's brain. Growth-associated protein-43 (GAP-43) is a presynaptic membrane phosphoprotein whose expression increases during developmental events such as axonal outgrowth or remodeling and synaptogenesis. Phosphorylation of GAP-43 by protein kinase C was correlated with enhanced axonal growth and transmitter release. In adult animals, increase of GAP-43 correlated with monoaminergic deficit in neuropsychiatric disorders. The present study examines the effects of repeated maternal restraint stress on the level of GAP-43 in the brain of rat pups. The results showed that prenatal stress significantly increased GAP-43 level in the PFC of rat pup during PND 7–14 as compared to control but not significant difference when observed at PND 21. Increased GAP-43 expression was also observed in the pup's hippocampus during the same postnatal periods. However, when observed at PND 60, pups born from stressed mother showed a significant lower (p < 0.001) GAP-43 expression as compare with control group. These changes indicate the direct effect of corticosteroid hormone, since repeated maternal injection with corticosterone (CORT, 40 mg/kg) during GD 14–21 also gave the same results. PND 7–14 is the peak period of synaptogenesis in these brain areas and abnormal axon sprouting and reorganization may lead to a defect in synaptic pruning at later stage of life. The results suggested that maternal stress is harmful to the developing brain and upregulation of GAP-43 indicated a protective mechanism against the toxicity of maternal stress hormone. Prenatal stress alter the normal developmental trajectories in the pup's brain may underlies the mechanism link between early life stress and neuropsychopathology in later life.     

Anxiolysis followed by anxiogenesis relates to coping and corticosterone after medial prefrontal cortical damage in rats

Medial prefrontal cortex (MPFC) damage causes profound behavioral and neuroendocrine alterations. However, many reports have been inconsistent regarding the direction of these effects. We hypothesized that the lesion recovery stage might be a key factor generating discrepancies. To examine changes over time following ibotenic acid lesion in the ventral part of the MPFC, behavioral and endocrine testing was conducted on the second and the fifth week after lesioning. On the second post-lesion week, bilaterally lesioned animals increased social interaction and swimming scores and their corticosterone response to restraint was exaggerated as compared with shams. On the fifth post-lesion week, social interaction and swimming scores were diminished in bilaterally lesioned animals; their basal plasma corticosterone was enhanced, while their corticosterone increase under restraint was blunted relative to shams. These results reveal that the emotional and endocrine responses to stress vary as a function of time following MPFC lesion, which may help to reconcile conflicting reports on effect direction. The role of the MPFC in anxiety, ability to cope with stress and adrenal regulation is also discussed.     

Behavioral and neuroendocrine effects of the exposure to chronic restraint or variable stress in early adolescent rats

Stress events during adolescence may contribute to the expression or exacerbation of physical and behavioral disorders. However, little attention has been given to the physiological and behavioral changes promoted by stress during this period of ontogeny. In the present study we investigated, in adolescent male rats, the effects of repeated exposure to restraint or variable stress on: (a) locomotor activity and corticosterone levels after exposure to a novel environment; (b) corticosterone levels in response to the exposure to restraint stress; and (c) changes in body, thymus and adrenal weights. The results demonstrated that repeated exposure to restraint or variable stress reduced the locomotor response, but did not affect corticosterone secretion, in response to a novel environment. Moreover, both chronic stress procedures did not change corticosterone secretion in response to acute restraint stress. Furthermore, our results showed that repeated restraint, but not variable stress, produced a decrease in body weight along the stress exposure. Finally, we observed that the exposure to variable stress reduced the thymus relative weight. Taken together our results suggest that behavioral and physiological changes induced by exposure to chronic stress during adolescence depend on the stress regimen.     

慢性应激对大鼠行为和体重的影响

目的　观察慢性应激对大鼠行为和体重的影响。方法　采用慢性强迫游泳模型 ,应用行为追踪仪观察大鼠的活动量 ,以电子动物称监测大鼠体重变化。结果　应激组大鼠活动量明显高于对照组 (P <0 0 1) ,排便、排尿增加 ,修饰频繁。应激组大鼠体重增长明显低于对照组 (P <0 0 1)。结论　慢性强迫游泳使大鼠活动量增多 ,抑制体重增长。     

Divergent effects of repeated restraint versus chronic variable stress on prefrontal cortical immune status after LPS injection

Previous work from our group has shown that chronic homotypic stress (repeated restraint – RR) increases microglial morphological activation in the prefrontal cortex (PFC), while chronic heterotypic stress (chronic variable stress – CVS) produces no such effect. Therefore, we hypothesized that stressor modality would also determine the susceptibility of the PFC to a subsequent inflammatory stimulus (low dose lipopolysaccharide (LPS)). We found that RR, but not CVS, increased Iba-1 soma size in the PFC after LPS injection, consistent with microglial activation. In contrast, CVS decreased gene expression of proinflammatory cytokines and Iba-1 in the PFC under baseline conditions, which were not further affected by LPS. Thus, RR appears to promote microglial responses to LPS, whereas CVS is largely immunosuppressive. The results suggest that neuroimmune changes caused by CVS may to some extent protect the PFC from subsequent inflammatory stimuli. These data suggest that modality and/or intensity of stressful experiences will be a major determinant of central inflammation and its effect on prefrontal cortex-mediated functions.     

Region-specific effects of acute and repeated restraint stress on the phosphorylation of mitogen-activated protein kinases

The mitogen-activated protein kinases (MAPKs) are a family of signal transduction mediators that regulate a host of cellular activities, including cell growth and proliferation, and differentiation and survival, via sequential phosphorylation and activation of a cassette of three protein kinases. MAPKs are also recruited when the brain undergoes synaptic plasticity and remodeling (e.g., during induction of long-term potentiation, learning and memory consolidation). The activities of some of these kinases are altered in response to various acute stimuli such as ischemic insult, visceral pain and electroconvulsive shock. In the present study we used immunoblotting techniques to examine the effects of acute and repeated restraint stress on the phosphorylation state of three MAPKs, the extracellular signal-regulated kinase Erk1/2, c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK, in different brain regions. A single exposure to 30 min of restraint stress-elevated phospho-Erk1/2 (P-Erk1/2) levels in all three brain regions examined (hippocampus, medial prefrontal cortex and cingulate cortex), but did not alter the phosphorylation pattern of the other two MAPKs in any region. In marked contrast, exposure to restraint for 11 days (30 min/day) reduced the levels of all three MAPKs, but only in the prefrontal cortex. The results are compared to the reported effects of acute and chronic stress on other biochemical and functional measures.     

Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex

Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1–CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.     

The effects of acute stress and acute corticosterone administration on the immobility response in rats

The immobility response is an innate antipredatory behavior in a broad variety of species. The immobility response varies in its postural components but in general is characterized by an absence of movement and a relative unresponsiveness to stimuli. Experimentally in rats, clamping the neck followed by body inversion and manual restrain elicits a response called “immobility by clamping the neck”. Stress reactions protect animals against predators and are characterized by activation of the sympathetic and hypothalamic–pituitary–adrenal systems. However, in mammals, the role of acute stress as a modulator of immobility response has been less studied. The aim of our study was to assess the effects of acute stress and the injection of corticosterone (5 mg/kg, ip) on immobility by clamping the neck in rats. We observed that either previous acute stress caused by forced exposure to elevated open platform or application of a heat-pain stimulus to the rat's tail during the immobility increased the duration of the immobility response caused by clamping the neck. Also, the corticosterone produced a rapid increase (15 min after injection) in the duration of this immobility response. Our results show that the acute stress, in rats, is a facilitator of the immobility response and suggest a possible nongenomic rapid action of corticosterone over brain structures that control this behavior.     

Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction of long-term potentiation in rats

Objective

To explore the role of the extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway in the induction of long-term potentiation (LTP) in the anterior cingulate cortex (ACC) that may be implicated in pain-related negative emotion.

Methods

LTP of field potential was recorded in ACC slice and the expressions of phospho-ERK (pERK) and phospho-CREB (pCREB) were examined using immunohistochemistry method.

Results

LTP could be induced stably in ACC slice by high frequency stimulation (2-train, 100 Hz, 1 s), while APv (an antagonist of NMDA receptor) could block the induction of LTP in the ACC, indicating that LTP in this experiment was NMDA receptor-dependent. Bath application of PD98059 (50 μmol/L), a selective MEK inhibitor, at 30 min before tetanic stimulation could completely block the induction of LTP. Moreover, the protein level of pERK in the ACC was transiently increased after LTP induction, starting at 5 min and returning to basal at 1 h after tetanic stimulation. The protein level of pCREB was also increased after LTP induction. The up-regulation in pERK and pCREB expressions could be blocked by pretreatment of PD98059. Double immunostaining showed that after LTP induction, most pERK was co-localized with pCREB.

Conclusion

NMDA receptor and ERK-CREB pathway are necessary for the induction of LTP in rat ACC and may play important roles in pain emotion.