SPS模型大鼠前额皮质分子伴侣钙网蛋白的表达变化

目的观察创伤后应激障碍(PTSD)大鼠前额皮质钙网蛋白(calretinculin,CRT)的表达变化,旨在揭示PTSD部分发病机制。方法取健康成年雄性Wistar大鼠75只,随机分为连续单一刺激(singleprolonged stress,SPS)模型1d、4d、7d、14d和正常对照组,用免疫组织化学法、免疫印迹法和RT-PCR法检测PTSD内侧前额皮质(media prefrontal cortex,mPFC)CRT的表达变化。结果在正常大鼠脑mPFC神经细胞中有CRT的表达,与对照组相比SPS刺激后CRT表达有增高趋势,mRNA和蛋白水平从SPS1d开始增多,SPS4d达到高峰,SPS7d组和SPS14d组有降低趋势,但仍高于正常组。结论 CRT在SPS大鼠mPFC过表达提示其可能参与PTSD大鼠记忆情绪改变的病理生理学机制。     

创伤后应激障碍大鼠前额叶皮质PKMζ表达的变化

目的探讨创伤后应激障碍(PTSD)大鼠前额叶皮质PKMζ的表达变化情况。方法采用单程长时应激(SPS)制备PTSD大鼠模型,旷场试验、高架十字迷宫试验检测造模效果。将大鼠分为:SPS1d、SPS3d、SPS7d和SPS14d组,正常饲养大鼠作为对照组。利用Western blot和real-time RT-PCR检测PKMζ蛋白及m RNA在大鼠前额叶皮质表达。结果 SPS模型大鼠表现出明显的焦虑症状,SPS7d、SPS14d组大鼠前额叶皮质PKMζ蛋白和m RNA表达水平显著高于对照组。结论在应激后7d和第14d,PTSD大鼠前额叶皮质PKMζ表达显著增高。     

大鼠下丘脑内糖皮质激素受体在创伤后应激障碍时的表达变化

目的:研究大鼠下丘脑神经元内糖皮质激素受体(GR)在创伤后应激障碍(FTSD)过程中的表达变化.方法:采用国际认定的连续单一应激(SPS)方法刺激大鼠,制作PTSD大鼠模型,SPS处理后分别取24 h、7 d、14 d的大鼠下丘脑;同时以非SPS刺激的下丘脑作为正常对照,应用免疫组织化学、免疫印迹方法分别进行各组下丘脑神经元GR表达变化的观察及定量检测.结果:经SPS处理后,下丘脑神经元GR表达于24 h时降低,7 d时回升且高于正常,14 d时呈现恢复正常趋势.结论:下丘脑神经元内GR在PTSD的表达变化可能是下丘脑与海马反馈调节过程中重要因素之一,为进一步揭示PTSD发病机制奠定理论基础.     

凋亡相关基因在创伤后应激障碍大鼠内侧前额叶皮质神经元的表达

目的探讨创伤后应激障碍(PTSD)大鼠内侧前额叶皮质(medial prefrontal cortex,mPFC)神经元Caspase-3和Caspase-9的表达变化。方法成年健康雄性Wistar大鼠50只,随机分为连续单一刺激(singleprolonged stress,SPS)模型1d、4d、7d、14d组和对照组,应用免疫组化、免疫荧光和RT-PCR方法检测PTSD大鼠mPFC神经元Caspase-3和Caspase-9的表达变化。结果 SPS刺激后Caspase-9的表达于刺激后1d开始升高,4d达到顶峰,7d和14d逐渐下降,Caspase-3于SPS刺激后1d,4d和7d表达逐渐增多,14d出现下降。结论 SPS刺激后PTSD大鼠mPFC神经元Caspase-3和Caspase-9呈规律性过表达。     

PTSD大鼠海马RGMa的表达变化

目的观察RGMa(repulsive guidancemoleculea)在创伤后应激障碍(posttraumatic stress disorder,PTSD)大鼠海马神经元的表达变化。方法建立连续单一刺激(single prolonged stress,SPS)的Wistar大鼠PTSD模型,随机分为SPS刺激后第7天组(SPS7d)、第14天组(SPS14d)及正常对照组,采用免疫组织化学、Western Blot方法检测各组海马神经元RGMa的表达变化。结果免疫组织化学及Western Blot结果均显示SPS7d组海马神经元RGMa表达水平较正常对照组增加(P<0.01),第14天组降低但仍高于正常对照组(P<0.01)。结论 PTSD模型大鼠海马RGMa蛋白水平变化可能是海马神经元遭遇巨大应激源后修复异常并最终导致神经元凋亡、海马体积缩小的重要分子机制之一。     

Caspase-9和caspase-3在创伤后应激障碍大鼠海马神经元的表达及意义

目的:探讨caspase-9和caspase-3在创伤后应激障碍(PTSD)大鼠海马神经元中的表达及意义.方法:采用国际认定的无连续单一应激(SPS)方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1、4、7、14、28 d组和正常对照组.应用免疫组织化学、免疫荧光双标、激光共聚焦显微镜技术和免疫印迹法检测caspase-9和caspase-3蛋白的表达.结果:与正常对照组相比,caspase-9活性于SPS刺激后1 d升高,SPS刺激后7 d再次上调并达到高峰,之后逐渐下降.Caspase-3活性于SPS刺激后7 d达到高峰,之后逐渐下降.结论:caspase-9和caspase-3共同参与了PTSD大鼠海马神经元凋亡的调控.     

创伤后应激障碍模型大鼠行为学改变和杏仁核bcl—2、bax的表达

目的:检测创伤后应激障碍(PTSD)动物行为学改变,初步探讨PTSD大鼠杏仁核神经细胞bax、 bcl-2的表达.方法:将Wistar大鼠分为对照组和模型组.采用无连续单一应激(SPS)建立PTSD模型,采用旷场实验(OFT)、拒俘反应实验、Morris水迷宫实验(MWM)等观察实验动物情感行为改变,采用化学发光法测定血清皮质醇浓度,分别应用免疫组织化学方法和RT-PCR检测杏仁核神经元bcl-2、 bax蛋白和mRNA的表达.结果:对照组水迷宫实验逃避潜伏期、血清皮质醇浓度分别为(5.632±1.065)s、 (1.25±0.12)μg/dL,模型组分别为(20.762±3.236)s、 (0.58±0.09)μg/dL,差异均有统计学意义.杏仁核bcl-2、 bax蛋白和mRNA在PTSD后表达升高,bcl-2/bax上调.结论:PTSD大鼠行为学表现符合PTSD主要临床表现;PTSD大鼠杏仁核神经细胞bcl-2/bax上调,可能与PTSD发病机制有关.     

吗氯贝胺早期干预对创伤后应激障碍大鼠行为及前额皮质pCREB水平的影响

目的探讨吗氯贝胺(Moclobemide,Moc)早期干预对创伤后应激障碍(posttraumatic stress disorder,PTSD)模型大鼠行为及内侧前额皮质pCREB水平的影响。方法采用单一连续刺激(single prolonged stress,SPS)建立大鼠PTSD模型。取成年健康雄性Wistar大鼠48只,随机分为N+sham组(正常对照组),SPS+sham组(SPS刺激后腹腔注射与抑制剂等体积的生理盐水),SPS+Moc组(SPS刺激后腹腔注射吗氯贝胺),N+Moc组(正常大鼠腹腔注射吗氯贝胺)。吗氯贝胺按8mg/kg腹腔注射给药,每日一次,连续14d。采用旷场试验和高架十字迷宫检测大鼠行为表现,并于行为学实验完成后处死大鼠,取前额内侧皮质,采用Western blot检测pCREB的表达水平。结果SPS引起大鼠进入中央区次数减少和总的运动距离缩短,给予Moc后大鼠进入中央区次数增多和总的运动距离增加。SPS引起大鼠进入开臂次数的百分比和开臂停留时间百分比均显著降低,给予Moc后大鼠进入开臂次数的百分比和开臂停留时间百分比明显升高。SPS引起大鼠内侧前额皮质pCREB的表达水平降低,Moc干预后pCREB的表达量增加。结论吗氯贝胺对PTSD大鼠行为改善的作用,可能是与上调前额内侧皮质pCREB水平相关。     

创伤后应激障碍样大鼠杏仁中央核盐皮质激素受体表达的变化

目的:研究创伤后应激障碍(PTSD)样大鼠杏仁中央核(CeA)盐皮质激素受体(MR)表达的变化.方法:使用连续单一应激(SPS)方法建立PTSD样大鼠模型.应用免疫组织化学、免疫印迹法检测各组杏仁中央核的盐皮质激素受体表达.结果:MR分布在CeA神经元的胞体和突起中,SPS刺激后24 h,MR表达明显下调,而7 d时快速回升并可见MR阳性神经元的突起增长、增多,至14 d时趋于稳定,突起减少,但MR表达仍低于正常状态.结论:PTSD样大鼠CeA的神经元突起增长、增多,提供了CeA与PTSD恐怖增强相关的形态学基础;PTSD样大鼠CeA的MR表达变化,可能是HPA轴调节紊乱从而引发PTSD相关症状的重要因素之一.     

创伤后应激障碍大鼠蓝斑神经元整合素的表达变化

目的探讨创伤后应激障碍(PTSD)大鼠蓝斑神经元整合素(Integrin)的表达变化。方法采用国际认定的SPS方法刺激建立大鼠PTSD模型,取成年健康雄性Wistar大鼠100只,随机分为连续单一刺激(singleprolongedstress,SPS)模型1d、4d、7d、14d组和对照组,应用免疫组化、免疫印记和逆转录-聚合酶链式反应(RT-PCR)方法检测PTSD大鼠蓝斑神经元整合素(Integrin)的表达变化。结果大鼠蓝斑神经细胞内整合素蛋白和mRNA于SPS刺激后1d明显降低,4d恢复性增多达到正常对照组水平,之后又下降,14d最低。结论创伤后应激障碍模型大鼠蓝斑整合素的表达变化,可能与PTSD的发病机制相关。     

单一连续应激大鼠内侧前额皮质细胞外信号调节激酶磷酸化增高

目的 探讨单一连续应激(SPS)大鼠内侧前额皮质(mPFC)磷酸化细胞外信号调节激酶(pERK1/2)和c-fos表达的变化.方法将45只雄性Wistar大鼠随机分为对照组、应激组和干预组.应激组和干预组大鼠接受SPS,干预组大鼠接受SPS前30min前额皮质局部注射ERK抑制剂2′-氨基-3′-甲氧黄酮(PD9805...     

Induction of repulsive guidance molecule in neurons following sciatic nerve injury

Repulsive guidance molecule (RGM) is a protein implicated in both axonal guidance and neural tube closure. We examined the expression of RGMa in the spinal cord after the sciatic nerve crush by immunohistochemistry. Although there was no RGMa immunoreactivity under na?ve conditions in the dorsal horn, a weak signal for RGMa was found at 24 h after the nerve crush, and this signal was progressively increased in the NeuN-positive neurons in the ipsilateral dorsal horn from superficial to deep layers at 10 days after surgery. In the neurons of the ipsilateral ventral horn, RGMa was also induced at 10 days after surgery, whereas no RGMa signal could be observed in na?ve conditions or at 24 h after surgery. Thus, RGMa expression is upregulated both in the ipsilateral dorsal and ventral horns in response to the sciatic nerve injury. We next examined the effects of complete Freund's adjuvant (CFA)-induced inflammation on RGMa expression in the spinal cord. However, no RGMa expression was observed at 24 h and 10 days after the CFA injection in the dorsal horn, suggesting that RGMa is not involved in inflammation-induced gyperalgesia. Our present study demonstrates that induction of RGMa is associated with the peripheral nerve injury.     

Glucocorticoid receptor activation is involved in producing abnormal phenotypes of single-prolonged stress rats: a putative post-traumatic stress disorder model

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experience, and presents with characteristic symptoms, such as intrusive memories, a state of hyperarousal, and avoidance, that endure for years. Single-prolonged stress (SPS) is one of the animal models proposed for PTSD. Rats exposed to SPS showed enhanced inhibition of the hypothalamo-pituitary-adrenal (HPA) axis, which has been reliably reproduced in patients with PTSD, and increased expression of glucocorticoid receptor (GR) in the hippocampus. In this study, we characterized further neuroendocrinologic, behavioral and electrophysiological alterations in SPS rats. Plasma corticosterone recovered from an initial increase within a week, and gross histological changes and neuronal cell death were not observed in the hippocampus of the SPS rats. Behavioral analyses revealed that the SPS rats presented enhanced acoustic startle and impaired spatial memory that paralleled the deficits in hippocampal long-term potentiation (LTP) and depression. Contextual fear memory was enhanced in the rats 1 week after SPS exposure, whereas LTP in the amygdala was blunted. Interestingly, blockade of GR activation by administering 17-beta-hydroxy-11-beta-/4-/[methyl]-[1-methylethyl]aminophenyl/-17-alpha-[prop-1-ynyl]estra-4-9-diene-3-one (RU40555), a GR antagonist, prior to SPS exposure prevented potentiation of fear conditioning and impairment of LTP in the CA1 region. Altogether, SPS caused a number of behavioral changes similar to those described in PTSD, which marks SPS as a putative PTSD model. The preventive effects of a GR antagonist suggested that GR activation might play a critical role in producing the altered behavior and neuronal function of SPS rats.     

Expression of repulsive guidance molecule b (RGMb) in the uterus and ovary during the estrous cycle in rats

Repulsive guidance molecule b (RGMb; a.k.a. Dragon), initially identified in the embryonic dorsal root ganglion, is the first member of the RGM family shown to enhance bone morphogenetic protein (BMP) signaling by acting as a BMP co-receptor. BMP signaling has been demonstrated to play an important role in the reproductive organs. Our previous study found that RGMb was expressed in the reproductive axis, but whether RGMb expression in reproductive organs changes across the estrous cycle remains unknown. Here, we show in the rat that RGMb mRNA expression in the uterus was significantly higher during metesterus and diestrus than during proestrus and estrus. Western blotting indicated that RGMb protein was significantly lower during estrus compared with the other three stages. Immunohistochemistry revealed that RGMb protein was mainly localized to the uterine luminal and glandular epithelial cells of the endometrium. RGMb mRNA and protein in the ovary remained unchanged during the estrous cycle. RGMb protein was expressed in the oocytes of all follicles. Weak staining for RGMb protein was also found in corpora lutea. RGMb was not detected in granulosa cells and stromal cells. Taken together, RGMb expression in the uterus and ovary across the estrus cycle demonstrate that RGMb may be involved in the regulation of uterine function, follicular development as well as luteal activity.     

TACE cleaves neogenin to desensitize cortical neurons to the repulsive guidance molecule

Neogenin is a receptor for netrins and proteins of the repulsive guidance molecule (RGM) family. It regulates several key developmental processes within the nervous system. The binding of RGMa to neogenin induces the inhibition of neurite outgrowth and the collapse of the growth cone of neurons. Here, we report that a disintegrin and metalloprotease (ADAM) transmembrane protein regulates the sensitivity of neurons to RGMa, by inducing the shedding of the ectodomain of neogenin. The extracellular domain of neogenin is directly associated with and cleaved off by the tumor necrosis factor-α converting enzyme (TACE), also called ADAM17. TACE is endogenously expressed in embryonic cortical neurons and regulates the cleavage of neogenin, and the inhibition of TACE in turn enhances RGMa-induced inhibition of neurite outgrowth and collapse of the growth cone. Conversely, exogenous expression of TACE abolishes the effect of RGMa. Therefore, TACE may play a role in modulating the RGM-induced repulsive behavior of neurons by regulating the expression of neogenin on the cell surface.     

Conditioned fear stress combined with single-prolonged stress: a new PTSD mouse model

There are still some defects in current single-prolonged stress (SPS) model and conditioned fear (CF) stress model of post-traumatic stress disorder (PTSD). The purpose of this study is to evaluate a novel mouse model of PTSD. Male KM mice suffered the double stresses-SPS and CF. After incubation time, the novel model exhibited the PTSD-like behaviors: sensitive fear and conditioned fear, low activities and defects in novel object recognition abilities. The apoptosis in the hippocampus was significantly increased, which was induced by the double stresses and further caused the synaptic structure damages in the hippocampus. The electron microscopy analysis further proved the synaptic losses and neuronal impairments in the hippocampus. Our results indicated this combined stresses mouse model was better than the SPS model and CF model. In addition, in order to further verify this model, paroxetine was administered after the double stresses. The results showed that paroxetine administration reduced PTSD-like behaviors, hippocampal apoptosis and structure damages. We conclude that this mouse model is novel and more predictably mimicked the clinical characteristics of PTSD, and this model can be further used for investigating the mechanisms of PTSD and screening effective therapeutics agents.     

沉默排斥性导向分子A对脑缺血再灌注损伤大鼠血脑屏障的保护作用

目的 观察沉默排斥性导向分子A（RGMa）对脑缺血再灌注损伤大鼠血脑屏障及紧密连接蛋白的影响。 方法 雄性成年 SD 大鼠立体定位侧脑室注射腺病毒后建立大脑中动脉闭塞(MCAO) /再灌注（I/R）模型。将成年雄性SD大鼠40只,分为空白对照组（sh-con）和RGMa干扰组（sh-RGMa）,分别在注射后1 d和3d观察腺病毒对RGMa的影响。将其余120只SD大鼠随机分为假手术组（sham）、缺血再灌注组（I/R）、空病毒组（I/R+sh-con）及病毒组（I/R+sh-RGMa）;I/R 72 h 后采用神经功能缺损评分评估各组大鼠神经功能恢复情况; 采用2,3,5-氯化三苯基四氮唑(TTC)染色测量脑梗死体积;采用静脉注射伊文思蓝观察血脑屏障通透性的改变;应用Western blotting和免疫组织化学染色检测RGMa的变化;应用Western blotting检测血脑屏障完整性相关紧密连接蛋白5（claudin-5）、基质金属蛋白酶9（MMP-9）和闭锁小带蛋白1（ZO-1）的表达。 结果 缺血再灌注后 72 h,I/R组较 sham 组神经功能评分降低,沉默RGMa能改善血脑屏障通透性,减少脑梗死体积;可下调MMP-9及上调claudin-5和ZO-1的表达。 结论 沉默RGMa对脑缺血再灌注损伤大鼠的血脑屏障有保护作用。     

大鼠脑缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A的表达变化

目的 探讨大鼠大脑中动脉缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A（RGMa）的表达变化。 方法 取雄性SD大鼠30只,随机分成对照组,缺血再灌注损伤7 d、14 d模型组（I/R）,采用线栓法制作大鼠大脑中动脉缺血再灌注损伤模型(tMCAO);Real-time PCR检测M1、M2型小胶质细胞标记分子白细胞介素-1β（IL-1β）、诱导性一氧化氮合酶（iNOS）、精氨酸酶1（Arg-1）及CD206 mRNA的表达;免疫荧光检测缺血区小胶质细胞RGMa的表达;体外培养小胶质细胞,分别用M1或M2型小胶质细胞的诱导物脂多糖（LPS）或IL-4诱导24 h后,利用Real-time PCR检测M1、M2型小胶质细胞标记分子IL-1β、iNOS、Arg-1、CD206 mRNA的表达;Western blotting检测M1、M2型小胶质细胞上RGMa的表达。 结果 缺血再灌注损伤后7 d、14 d,M1、M2型小胶质细胞的标记分子表达增加。缺血后7 d、14 d激活的小胶质细胞上RGMa大量表达。RGMa在体外培养的LPS诱导极化的M1型小胶质细胞和IL-4诱导极化的M2型小胶质细胞上表达均显著增加。 结论 大鼠大脑中动脉缺血再灌注损伤后,RGMa在缺血区M1型和M2型极化的小胶质细胞上均大量表达,RGMa可能在小胶质细胞激活极化过程中发挥重要作用。RGMa可能是缺血性脑卒中治疗的新靶点。     

脑缺血再灌注模型大鼠接受跑台运动锻炼海马反义导向分子A的表达

背景：脑卒中后,运动锻炼对其神经功能的康复具有极其重要的作用,但关于卒中后大鼠运动量的研究鲜有报道。而海马与认知功能密切相关,但大鼠缺血再灌注后海马内反义导向分子A的表达的研究,国内外未见报道。 目的：探索运动锻炼对脑缺血再灌注后大鼠缺血侧海马内反义导向分子A表达的影响。 方法：将120只SD大鼠随机等分为正常组、假手术组、模型7,14,28 d组,模型7,14,28 d组大鼠采用线栓法制作大鼠右侧脑缺血再灌注模型。各组大鼠各等分为`4个亚组：非运动亚组、低、中、高运动量亚组。低运动量组进行5 m/min,5 min;7 m/min,5 min;9 m/min,20 min跑台训练;中运动量组进行8 m/min,5 min;10 m/min,5 min;13 m/min,20 min跑台训练;高运动量组进行8 m/min,5 min;11 m/min,5 min;20 m/min,20 min跑台训练。 结果与结论：非运动时,模型7 d组大鼠缺血侧海马反义导向分子A mRNA和蛋白表达水平最高,且反义导向分子A相对表达水平随时间延长而逐渐降低。与非运动相比,模型14,28 d组大鼠中运动量时缺血侧海马反义导向分子A mRNA和蛋白表达水平明显下降(P < 0.05),而高运动量海马反义导向分子A mRNA和蛋白表达水平表达增加。提示中运动量锻炼可以降低脑缺血再灌注大鼠患侧海马反义导向分子A的表达。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

The single-prolonged stress paradigm alters both the morphology and stress response of magnocellular vasopressin neurons

Vasopressin (AVP) plays an important role in anxiety-related and social behaviors. Single-prolonged stress (SPS) has been established as an animal acute severe stress model and has been shown to induce a lower adrenocorticotropic hormone (ACTH) response upon cortisol challenge. Here, we show results from immunoassays for AVP, ACTH, and corticosterone (CORT), and in situ hybridizations for AVP mRNA performed 7 days after SPS exposure. Immunofluorescence for AVP was also performed during the 7-day period following SPS exposure and after an additional forced swimming stress paradigm. We observed that the plasma concentrations of AVP, ACTH, and CORT were not altered by SPS; ACTH content in the pituitary and AVP mRNA expression in the supraoptic nucleus (SON) were significantly reduced by SPS. During the 7-day period following SPS, the intensity of immunoreactivity, the size of the soma, and the immunoreactive optical density of the dendrites of AVP neurons in the SON all increased. An apparent reduction in the intensity of AVP immunoreactivity was observed in the SON at 4 h after additional stress. Additional forced swimming led to a rapid increase in the dendritic AVP content only in the controls and not in the SPS-treated rats. These findings suggest that AVP is a potential biomarker for past exposure to severe stress and that alterations in AVP may affect the development of pathogenesis in stress-related disorders.