葡萄糖调节蛋白78在PTSD样情感行为异常大鼠大脑皮质中的表达及意义

目的观察创伤后应激障碍(PTSD)大鼠大脑皮质内质网应激标志物葡萄糖调节蛋白78(GRP78)的表达变化,探讨内质网分子伴侣在PTSD发病机制中的作用。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60只,随机分为SPS模型的1d、4d、7d组及正常对照组,采用免疫荧光法、免疫印迹和逆转录--聚合酶链式反应检测大鼠前额皮质GRP 78的表达变化。结果 SPS刺激后大鼠前额皮质神经元细胞内GRP78于1d开始逐渐升高,7d时表达最多;GRP 78mRNA的变化与之相一致。结论大脑皮质GRP 78的表达变化,可能是PTSD大鼠情感行为异常的重要病理生理基础之一。     

创伤后应激障碍大鼠前额内侧皮质MR表达的变化

目的观察创伤后应激障碍(PTSD)样大鼠前额内侧皮质(medial prefrontal cortex,mPFC)神经元核受体-盐皮质激素受体(Mineralocorticoid receptors,MR)表达的变化。方法采用国际认定的单一连续应激(single prolonged stress,SPS)方法建立PTSD大鼠模型,取成年健康雄性Wistar大鼠90只,随机分为PTSD模型1d、7d、14d、28d和正常对照组。采用免疫组化、免疫印迹和RT-PCR方法分别进行各组mPFC神经元MR表达变化的观察及检测,进行图像分析和统计学处理。结果 PTSD大鼠mPFC神经元MR的表达在SPS-1d时高于对照组,随后下降,SPS-14d最低,SPS-28d恢复性上调,但仍然低于对照组(P<0.05)。结论 PTSD模型大鼠经SPS处理后,mPFC中出现MR表达的变化,该变化可能参与PTSD的下丘脑-垂体-肾上腺(hypothalamic pituitary adren axis,HPA)轴的变化机制。     

细胞色素C和BAX在创伤后应激障碍大鼠海马神经元的表达及意义

观察细胞色素C（Cyt C）和凋亡相关基因BAX在创伤后应激障碍（PTSD）大鼠海马神经元中的表达,探讨其在PTSD大鼠海马神经元凋亡中的作用及相互关系。采用国际认定的SPS方法刺激大鼠建立PTSD大鼠模型,取SPS刺激后1、4、7、14、28d组和正常对照组。应用免疫组化、免疫荧光双标、激光共聚焦显微镜技术和免疫印迹法检测CytC和BAX蛋白的表达;     

PTSD样行为异常大鼠杏仁核Ca^2＋/CaM的改变

目的观察创伤后应激障碍（PTSD）样行为异常大鼠杏仁核细胞内Ca^2＋信号及Ca M（钙调蛋白）表达变化,有望揭示PTSD的部分发病机制。方法成年健康雄性Wistar大鼠60只,随机分为连续单一刺激（single prolonged stress,SPS）模型的12h、1d、4d、7d、14d组及正常对照组,采用荧光探针标记法、免疫组化、Western blott等方法,检测PTSD样行为异常大鼠杏仁核神经元游离Ca^2＋含量和钙调蛋白（Ca M）的表达变化。结果SPS刺激后大鼠杏仁核神经元游离Ca^2＋浓度（nmol/L）于12h内升高,24h增至顶峰,4d开始下降,14d恢复正常。CaM的表达于SPS刺激后4d表达最多,之后渐趋下降。结论杏仁核Ca^2＋信号调控与Ca M表达变化,可能与PTSD样大鼠恐惧增强的发病机制相关。     

PTSD样大鼠蓝斑核盐皮质激素受体表达变化的研究

目的探讨PTSD样大鼠蓝斑（locus ceruleus,LC）神经元盐皮质激素受体（mineralocorticoid receptors,MR）表达的变化。方法使用连续单一应激（SPS）方法建立PTSD大鼠模型,随机分为SPS处理后24h、4d、7d、14d和28d组,非SPS刺激大鼠作为对照,应用免疫组化、免疫印迹方法分别进行各组蓝斑神经元MR表达变化的观察及检测,进行图像分析和统计学处理。结果蓝斑神经元MR的表达呈现24h急剧下调,4d、7d,14d和28d恢复性上调。结论PTSD样大鼠蓝斑神经元MR的表达变化可能直接参与了PTSD持续性精神行为障碍的发生发展过程。     

大鼠PTSD后蓝斑核 β-catenin的表达下降伴随细胞凋亡

目的 探讨创伤后应激障碍( PTSD) 大鼠蓝斑神经元β-catenin(β-连环蛋白)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( single prolonged stress,SPS) 模型1 d、4 d、7 d、14 d 组和对照组,应用免疫组化、免疫印迹方法检测PTSD 大鼠蓝斑神经元β-catenin的表达变化;透射电镜观察PTSD大鼠蓝斑神经元的超微结构变化.结果 经SPS 刺激后大鼠蓝斑神经元细胞内β-catenin于1d开始逐渐减少,14d表达最少;蓝斑神经元出现细胞凋亡改变.结论 蓝斑神经元细胞凋亡可能是导致PTSD 患者蓝斑功能失调的重要原因之一.     

创伤后应激障碍大鼠蓝斑核神经元细胞凋亡的实验研究

目的探讨创伤后应激障碍(PTSD)大鼠蓝斑核神经元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(single prolonged 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大鼠蓝斑核神经元Caspase-3和Caspase-9的表达变化。结果SPS刺激后1d,4d,Caspase-9的表达逐渐增强,7d和14d逐渐下降,Caspase-3于SPS刺激后7d表达最多,14d出现下降。结论蓝斑神经元细胞凋亡可能是导致PTSD患者蓝斑功能失调的重要原因之一。     

PTSD样情感行为异常大鼠杏仁核Caspase 9的改变

目的观察创伤后应激障碍(PTSD)样行为异常大鼠杏仁核神经元Caspase 9表达变化,有望揭示PTSD的部分发病机制。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60只,随机分为SPS模型的1d、4d、7d组及正常对照组,采用免疫荧光法、免疫印迹和逆转录-聚合酶链式反应检测大鼠杏仁核Caspase 9的表达变化。结果SPS刺激后大鼠杏仁核神经元细胞内Caspase 9于1d开始逐渐升高,7d时表达最多;Caspase 9 mRNA的变化与之相一致。结论海马Caspase 9的表达变化,可能是PTSD大鼠情感行为异常的重要病理生理基础之一。     

PTSD促进大鼠中缝背核细胞色素c表达

目的研究创伤后应激障碍(PTSD)大鼠中缝背核神经元细胞色素C(Cyt-c)的表达变化。方法应用无连续单一刺激(SPS)方法建立PTSD大鼠模型,随机分为SPS刺激后1d、4d、7d和对照组,应用酶组织化学法和RT-PCR方法观察中缝背核神经元Cyt-c的表达变化。结果光镜酶细胞化学法和RT-PCR法显示中缝背核神经元Cyt-c染色阳性细胞于SPS刺激后1d明显高于对照组,4d逐渐增高,并于7d达到高峰。电镜下显示Cyt-c阳性反应产物主要分布在中缝背核神经元线粒体膜,SPS刺激后可见Cyt-c释放到胞浆中。结论 SPS刺激引起Cyt-c在PTSD大鼠中缝背核神经元呈过表达。     

PTSD与杏仁核神经元细胞凋亡的关系

目的观察创伤后应激障碍（PTSD）大鼠杏仁核神经元细胞凋亡相关基因的表达与细胞凋亡的发生,从杏仁核神经元细胞凋亡揭示PTSD的部分发病机制。方法成年健康雄性Wister大鼠50只,随机分为连续单一刺激（single prolonged stress,SPS）模型的1d、4d、7d、14d组及正常对照组。应用免疫组化和Western Blotting技术检测凋亡相关基因Bax、Bcl-2在PTSD杏仁核神经元的表达;采用TUNEL法检测PTSD大鼠杏仁核神经元细胞凋亡。结果PTSD大鼠杏仁核神经元凋亡相关基因Bax于4d达高峰,Bcl-2于1d表达最高,Bax／Bcl-2比值逐渐升高,于4d达到峰值,之后渐趋下降。TUNEL阳性细胞在SPS各组模型均出现,4d达最高峰。结论PTSD大鼠杏仁核神经元细胞凋亡中,凋亡相关基因Bax、Bcl-2各自发挥促进凋亡和抑制凋亡的作用,Bax／Bcl-2比值升高促进细胞发生凋亡,可能与杏仁核调节的PTSD恐惧异常的发病机制相关。     

The Unfolded Protein Response is Triggered in Rat Neurons of the Dorsal Raphe Nucleus After Single-Prolonged Stress

The dorsal raphe nucleus (DRN) has been suggested playing an important role in the pathophysiology of post-traumatic stress disorder (PTSD), however the underlying cellular mechanisms are not fully understood. The endoplasmic reticulum (ER) is a critical organelle for synthesis of membrane and secretory proteins, and perturbations in ER lead to the unfolded protein response (UPR). In the present experiment, we hypothesized UPR may be associated with the PTSD, and there is an induction of UPR in the DRN neurons of the PTSD-like rats. We first observed the morphological changes of ER in the DRN neurons of the rats exposed to single-prolonged stress (SPS), a model of PTSD, and then we also detected the expression of ER chaperones glucose regulated protein 78 (GRP78) and glucose regulated protein (GRP94) which are two key sensors and mediators of the UPR and are considered an ER stress-specific inducible proteins using methods of western blot and immunohistochemical analysis. Our results demonstrated there were abnormal expansion of ER and up-regulation expression of GRP78 and GRP94 after SPS, which indicated that the UPR was triggered in the DRN neurons of the PTSD-like rats. These results are consistent with our speculation that UPR may be associated with the PTSD, and suggest us the UPR may be a new critical cellular mechanisms of PTSD.     

PTSD大鼠中缝背核神经元TMP酶表达变化的实验研究

目的研究创伤后应激障碍大鼠中缝背核神经元细胞TMP(三偏磷酸酶)活性分布及其表达变化。方法采用SPS刺激方法,建立PTSD样大鼠SPS模型,随机分为SPS刺激后1d、7d、14d和正常对照组,应用光、电镜酶组化技术方法,分别对各组中缝背核神经元细胞TMP活性分布及其变化进行观察和定量检测。结果光镜下TMP酶反应阳性产物为棕褐色颗粒分布于细胞质中;电镜下TMP酶反应阳性产物为高电子密度的黑色颗粒沉淀,分布于各组中缝背核神经元细胞内溶酶体上。SPS刺激后1d、7d、14d中缝背核神经元TMP活性表达比正常对照组明显增强,并于7d达到高峰。结论创伤后应激障碍大鼠中缝背核神经元细胞TMP活性增强,提示TMP参与了中缝背核神经元细胞凋亡产物的降解和处理过程。     

Single-Prolonged Stress Induces Endoplasmic Reticulum - Dependent Apoptosis in the Hippocampus in a Rat Model of Post-Traumatic Stress Disorder

Background

Our previous research indicated that apoptosis induced atrophy in the hippocampus of post-traumatic stress disorder (PTSD) rats. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of several disorder diseases. The aim of this study was to investigate whether endoplasmic reticulum-related pathway is involved in single-prolonged stress (SPS) induces apoptosis in the hippocampus of PTSD rats by examining the expression levels of three important indicators in the ER-related apoptotic pathway: Glucose-regulated protein (GRP) 78, caspase-12 and Ca²⁺/CaM/CaMkinaseIIα (CaMkIIα).

Methods

Wistar rats were sacrificed at 1, 4 and 7 days after SPS. SPS is a reliable animal model of PTSD. The apoptotic cells in the hippocampus were assessed by TUNEL method and transmission electron microscopy (TEM). Free intracellular Ca²⁺ concentration was measured. GRP78 expression was examined by immunohistochemistry, western blotting and RT-PCR. mRNA of caspase-12 and CaM/CaMkIIα were determined by RT-PCR.

Results

Our results showed that apoptotic cells were increased in the SPS rats. TEM analysis revealed characteristic morphological changes of apoptosis in these cells. We observed that GRP78 was significantly up-regulated during early PTSD, and then recovered at 7 days after SPS. By RT-PCR, we observed that the change in caspase-12 expression level was similar to that in GRP78. Moreover, the free intracellular Ca²⁺ concentration was significantly higher at 1 day after SPS and decreased in 7 days. CaM expression increased significantly, while CaMKIIα expression decreased significantly in the hippocampus at 1 day after SPS.

Conclusion

SPS induced change in the expression levels of GRP78, caspase-12 and Ca²⁺/CaM/CaMkIIα in the hippocampus of PTSD rats indicated that the endoplasmic reticulum pathway may be involved in PTSD-induced apoptosis.     

Stress Affects a Gastrin-Releasing Peptide System in the Spinal Cord That Mediates Sexual Function: Implications for Psychogenic Erectile Dysfunction

Background

Many men suffering from stress, including post-traumatic stress disorder (PTSD), report sexual dysfunction, which is traditionally treated via psychological counseling. Recently, we identified a gastrin-releasing peptide (GRP) system in the lumbar spinal cord that is a primary mediator for male reproductive functions.

Methodology/Principal Findings

To ask whether an acute severe stress could alter the male specific GRP system, we used a single-prolonged stress (SPS), a putative rat model for PTSD in the present study. Exposure of SPS to male rats decreases both the local content and axonal distribution of GRP in the lower lumbar spinal cord and results in an attenuation of penile reflexes in vivo. Remarkably, pharmacological stimulation of GRP receptors restores penile reflexes in SPS-exposed males, and induces spontaneous ejaculation in a dose-dependent manner. Furthermore, although the level of plasma testosterone is normal 7 days after SPS exposure, we found a significant decrease in the expression of androgen receptor protein in this spinal center.

Conclusions/Significance

We conclude that the spinal GRP system appears to be a stress-vulnerable center for male reproductive functions, which may provide new insight into a clinical target for the treatment of erectile dysfunction triggered by stress and psychiatric disorders.