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。     

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。     

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.     

PTSD样大鼠海马MR和GR变化的研究

目的研究PTSD大鼠海马神经元核受体MR(mineralocorticoid receptor,盐皮质激素受体)和GR(glu-cocorticoid receptor糖皮质激素受体)表达的变化。方法采用国际认定的SPS方法刺激大鼠建立PTSD大鼠模型,分别取SPS处理后24h、7d、14d大鼠脑组织;同时取正常脑组织(非SPS刺激)作为对照,应用免疫组化、Western Blot方法分别进行各组海马神经元GR和MR表达变化的观察及定量检测。结果(1)经SPS处理后,免疫组化和Western Blot结果显示海马神经元MR表达呈现随着24h、7d、14d逐渐下调的趋势;(2)经SPS处理后,免疫组化和Western Blot结果显示海马神经元GR表达于24h时下调,而7d、14d时呈现逐渐回升趋势。结论大鼠经SPS处理后,海马MR表现为持续下调状态;而GR表达为短暂下调,随后回调,揭示PTSD大鼠海马神经元核受体—MR和GR的表达变化可能是引发海马功能降低的重要因素之一。     

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。     

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。     

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。     

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;     

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。     

分子伴侣GRP94在创伤后应激障碍大鼠前额内侧皮质表达变化及其意义

目的检测创伤后应激障碍（Post-traumatic stressdisorder,PTSD）连续单一刺激（single prolonged stress,SPS）模型大鼠前额内侧皮质（medial prefrontal cortex,mPFC）分子伴侣葡萄糖调节蛋白94（Glucose-regulated protein,GRP94）的表达变化,探讨PTSD发病机制过程中存在未折叠蛋白反应（unfolded protein reaction,UPR）的激活及GRP94在UPR中的作用机制及意义。方法健康,雄性,成年Wistar大鼠60只,建立国际认定的PTSD-SPS模型,随机分为正常对照组和模型组,模型组大鼠分别于1d、4d、7d取材。应用免疫组化、蛋白印迹和RT-PCR方法检测PTSD大鼠mPFC神经元GRP94表达变化。结果免疫组化、蛋白印迹和RT-PCR方法均显示,给予SPS刺激后大鼠GRP94的蛋白表达及GRP94mRNA表达均高于正常组（P〈O．05）,7d达到顶峰。结论分子伴侣GRP94表达发生变化,提示SPS刺激后大鼠mPFC神经元出现未折叠蛋白反应的激活,PTSD的发生过程中GRP94参与了未折叠蛋白反应,对揭示PTSD致脑损伤的发病机制具有重要意义。     

疼痛刺激对大鼠脊髓上水平神经元NADPH—d和Fos表达的影响

目的：研究一氧化氮合酶（NOS）阳性神经元在中脑导水管周围灰质（APG）和中缝背核（DR）内的分布,探讨脊髓上水平一氧化氮（NO）与外周痛的关系。方法：SD大鼠20只,随机分为2组,每组10只。实验组大鼠单侧后肢足底皮下注射5％福尔马林0.1ml,对照组大鼠于相同部位注射生理盐水0.1ml。2h后常规灌注、固定、取材大鼠中脑所在段、切片。切片按序分为Ⅰ、Ⅱ、Ⅲ三套。全部切片先作NADPH－d组织化学反应,镜检阳性切片,Ⅰ套继续作中性红染色,Ⅱ、Ⅲ套作c-fos免疫细胞化学反应,其中Ⅲ套用0.01mol/LPBS代替c-fos抗体。封片后镜下观察。结果：NADPH－d阳性神经元主要分布于PAG腹外侧核（CGLV）、背外侧核（CGLD）和DR;大鼠足底注射福尔马林后可在上述部位发现NADPH－d、Fos和NADPH－d/Fos三种标记的阳性神经元。结论：脊髓上水平NO可能在PAG和DR的痛觉调制中起作用。     

The Distribution of Serotonergic Nerves in Microencephalic Rats Treated Prenatally with Methylazoxymethanol

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) induces microencephaly in the offspring. In the present study of these microencephalic rats (MAM rats) we used quantitative autoradiography to investigate [³H] paroxetine binding sites, which are a selective marker of serotonin (5-HT) transporters (5-HTT). The binding in the accumbens, cortex, hippocampus, and dorsolateral thalamus was significantly increased in MAM rats, compared to the control rats, while there was a significant decrease in the dorsal raphe nucleus of the MAM rats. The levels of 5-HTT mRNA in the dorsal raphe nuclei were analyzed by in situ hybridization, which revealed a significant decrease in 5-HTT mRNA-positive neurons in the MAM rats compared to the control rats. The results imply serotonergic hyperinnervation in the cerebral hemispheres of MAM rats, while a target-dependent secondary degeneration of 5-HT neurons might be induced in the dorsal raphe nuclei of MAM rats.     

Spontaneous unit activity of neurons within the dorsal raphe nucleus of the neonatal rat

Extracellular recordings were made of spontaneously active neurons in the dorsal raphe nucleus (DRN) of neonatal rats. The firing pattern and rate of these neurons were similar to those characterized for 5-HT-containing cells in the DRN of adult rats. Neonatal DRN cells were also inhibiteby small systematic doses of LSD, as previously described for 5-HT-containing DRN neurons in adult rats. These results indicate that DRN neurons in neonatal rats are physiologically active and display many characteristics similar to mature 5-HT-containing DRN neurons.     

甘丙肽及其受体2在抑郁症动物模型脑内的表达研究

运用慢性非预见性应激刺激建立抑郁症动物模型,用开野实验（open—field）检测大鼠建模前后行为学变化,HPLC—EC法测定血清皮质醇变化,由此对模型进行初步评价。运用原位杂交、RT—PCR方法检测大鼠海马脑区和中缝背核甘丙肽及其受体2的表达。结果显示．慢性应激后模型组大鼠活动性明显降低,血清皮质醇含量显著升高,海马脑区和中缝背核甘丙肽及其受体2的表达显著上调。在慢性应激大鼠抑郁症模型中,甘丙肽及其受体2在部分脑区的高表达提示甘丙肽很有可能参与了应激过程中神经元功能的调节。     

Bee venom acupoint stimulation increases Fos expression in catecholaminergic neurons in the rat brain

Fos immunocytochemistry was combined with tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH) immunolabeling to examine brainstem catecholaminergic neuronal activation resulting from bee venom (BV) stimulation of the Zusanli acupoint (ST36) in Sprague-Dawley rats. BV injection into the Zusanli acupoint caused increased Fos expression in catecholaminergic neurons located in the hypothalamic arcuate nucleus (Arc), the dorsal raphe (DR), the A5 cell group (A5) and the locus coeruleus (LC). BV acupoint stimulation significantly increased Fos-TH double-labeled neurons in the Arc, LC and DR. Fos-DBH positive neurons were also increased by BV acupoint stimulation in the LC and A5. In contrast BV stimulation of a non-acupoint only increased Fos expression and Fos-TH double-labeled neurons in the Arc. These data indicate that BV acupoint stimulation activates brainstem catecholaminergic neurons and that this activation underlies BV acupoint-induced antinociception.     

The effect of raphe nuclei lesions on the rat ovarian cycle

The effects of lesions of the median raphe or dorsal raphe nuclei on ovarian cycle were studied in rats. Lesions involving raphe nuclei decreased forebrain 5-HT and 5-hydroxyindole acid (5-HIAA) concentrations. Rats with lesions of the raphe showed prolonged estrous phase as well as an increase in both the eosinophilic index and karyopycnotic index of the vaginal smears. Histological examinations revealed that lesions of both the dorsal and median raphe produced marked increase in the number of maturing and mature follicles as well as an increase in corpora lutea. The increase in uterine weight was also observed. Present results indicate that lesions of the ascending 5-HT neurons stimulate ovulation and cause an increase in the estrogenic activity. Thus, the 5-HT neurons of the raphe nuclei seem to inhibit ovulation probably due to inhibiting of the hypothalamic releasing hormones.     

毁损大鼠中缝背核内触液神经元对吗啡依赖和戒断的影响

目的:探索脑内远位触液神经元在吗啡依赖和戒断形成过程中的作用。方法:化学性神经元毁损、侧脑室引入霍乱毒素亚单位B与辣根过氧化物酶复合物(CB-HRP)神经示踪、TMB-ST呈色反应,Western blot、nNOS免疫组织化学。结果:毁损大鼠中缝背核内远位触液神经元后,纳洛酮催促的戒断症状明显减弱,戒断症状评分较戒断未毁损组降低约38%(P<0.05);给予溶媒和毁损触液神经元旁侧的大鼠戒断症状与戒断组比较未见明显变化(P>0.05)。毁损组脑片触液神经元密集区局部细胞损坏明显,仅在其毁损区边缘观测到少量CB-HRP阳性细胞。未毁损组CB-HRP标记细胞位置及数量恒定,形态清晰。毁损触液神经元后,脊髓背角nNOS阳性神经元计数及nNOS蛋白表达较戒断未毁损组减少明显(P<0.05),而较正常组和依赖组增加仍显著(P<0.01)。结论:毁损大鼠中缝背核内部分远位触液神经元可减弱吗啡戒断症状和脊髓背角神经元型一氧化氮合酶的表达,提示中缝背核内部分远位触液神经元可能参与了吗啡依赖和戒断的形成,NO介导脑内触液神经元与脊髓水平对吗啡依赖和戒断的调节。     

Tryptamine Concentrations in Areas of 5-Hydroxytryptamine Terminal Innervation After Electrolytic Lesions of Midbrain Raphe Nuclei

The possible existence of tryptamine-containing neurons originating in the midbrain raphe is suggested by several reports of tryptamine-mediated responses to electrical stimulation of the raphe nuclei. To assess this hypothesis, we have investigated the effects of electrolytic lesions of the median and dorsal raphe nuclei on striatal, hypothalamic, and hippocampal concentrations of tryptamine, 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid. In addition, the rat striatal tryptophan concentrations were also determined. No changes in the concentrations of tryptamine were observed at 1 or 2 weeks after lesioning the dorsal and median raphe nuclei, at which time the other 5-hydroxyindoles were markedly reduced; furthermore, no reductions were observed in tryptamine concentrations in the striatum, hypothalamus, or hippocampus of rats pretreated with a monoamine oxidase inhibitor. The only change observed in these rats was a limited increase in striatal tryptamine and tryptophan observed at 1 day after lesioning. The results indicate that tryptamine concentration is independent of the integrity of 5-HT-containing neurons of the midbrain raphe nuclei. Furthermore, if tryptamine-containing neurons that have terminal projections to the striatum, hypothalamus, and hippocampus exist, their cell bodies are located in regions outside the dorsal and median raphe nuclei. Another possibility could be that tryptamine is located in glial cells.