慢性不可预见性温和应激后大鼠行为及海马细胞支架的改变

目的研究慢性不可预见性温和应激所致的动物行为学改变及细胞支架微管系统的动态性改变。方法将大鼠随机分为应激模型组（8只,以下简称模型组）和对照组（8只）,对模型组大鼠进行连续21d的慢性不可预见性应激。进行行为学观察,使用western blotting检测乙酰化微管蛋白（Acet—Tub）,酪氨酸微管蛋白（Tyr—Tub）。结果（1）模型组大鼠慢性应激后糖水偏好及自主活动显著减低,与对照组有显著差异;（2）模型组大鼠慢性应激后海马Acet—Tub表达升高,Tyr—Tub表达减低,与对照组有显著差异。结论慢性应激后微管动态性减低,神经可塑性受损。     

慢性不可预计温和应激大鼠microRNA差异表达谱分析

目的探讨慢性不可预计温和应激大鼠模型microRNA（miRNA）表达谱差异。方法采用慢性不可预计温和应激模型,将16只SD大鼠随机分为对照组和应激组,每组8只,对照组常规饲养,不予任何处理,应激组每天随机予1～2种温和刺激,共6周;造模结束后均采用旷场试验评价大鼠行为,安乐处死,取伏隔核低温保存。采用miRNA测序技术比较两组大鼠miRNA表达差异。结果与对照组比较,慢性不可预计温和应激大鼠有25个miRNA显著下调,2个miRNA显著上调。结论慢性不可预计温和应激可能使大鼠部分miRNA表达发生改变。     

电针对慢性不可预知温和应激小鼠模型抑郁样行为及肠道菌群的调节作用

目的 观察电针对慢性不可预知温和应激（CUMS）模型小鼠抑郁样行为及肠道菌群组 成的影响。方法 将 40 只雄性 C57BL/6 小鼠随机分为对照组、电针组、模型组（CUMS 组）和治疗组 （CUMS+ 电针组）,每组 10 只。CUMS 组和 CUMS+ 电针组均接受 CUMS 造模,造模结束后,给予对照组和 CUMS 组小鼠假刺激 7 d,给予电针组和 CUMS+ 电针组小鼠 2/15 Hz、1.0 mA 的电针刺激 7 d。最后一次 干预结束 24 h 后,收集粪便保存在 -80℃条件下并对小鼠进行糖水偏好实验、旷场实验和强迫游泳实 验。对 4 组小鼠的粪便进行 16S rDNA 测序,采用操作分类单元（OTU）信息进行 α 多样性和 β 多样性 分析、线性判别分析效应量分析（LEfSe）。采用 Spearman 相关性分析小鼠行为学指标与差异菌群富集 水平之间的相关性。结果 CUMS 组小鼠的旷场中心区域探索时间短于对照组［（22.058±4.148）s 比 （37.864±4.407）s］,糖水偏好率低于对照组［（53.427±14.550）% 比（76.514±15.701）%］,强迫游泳不动 时间长于对照组［（82.599±32.369）s 比（47.606±15.344）s］,差异有统计学意义（P＜ 0.01）。CUMS+ 电针 组小鼠的旷场中心区域探索时间长于 CUMS 组［（30.604±6.060）s 比（22.058±4.148）s］,糖水偏好率高于 CUMS组［（72.731±13.933）%比（53.427±14.550）%］,强迫游泳不动时间短于CUMS组［（53.633±17.933）s 比（82.599±32.369）s］,差异有统计学意义（P＜ 0.05）。CUMS 组小鼠肠道菌群的 OTU 数量、ACE 指数和 Chao 指数低于对照组,差异有统计学意义（P＜ 0.05）;但 CUMS+ 电针组与 CUMS 组比较,差异无统计学 意义（P＞ 0.05）。Ruminococcaceae_UCG_002 属、蓝绿藻菌属（Lachnoclostridium）和 Rikenellaceae_RC9_ gut_group 属在 CUMS 组中富集,且与抑郁样行为呈正相关（P＜ 0.05）;放线菌门和理研菌属、杜氏杆菌属、 Ileibacterium 属、双歧杆菌属和异杆菌属在电针和 CUMS+ 电针组中富集,且均与抑郁样行为呈负相关 （P＜ 0.05）。结论 电针干预可以缓解小鼠的抑郁样行为,且对其肠道菌群组成有调节作用。     

慢性应激及氟西汀治疗后大鼠海马细胞支架的改变

目的 探讨慢性不可预见性应激及氟西汀治疗后大鼠细胞支架微管系统的动态性变化及其可能机制.方法 将24只大鼠按随机数字表法分为对照组(空白对照+生理盐水)、慢性不可预见性温和应激(CUMS)组(CUMS+生理盐水)和氟西汀组(CUMS+氟西汀),每组8只.对大鼠进行连续21 d CUMS后,氟西汀组给予氟西汀(10 mg/kg)治疗21 d,对照组和CUMS组给予生理盐水.实验结束后进行行为学观察,并使用免疫印迹法(western blot)检测大鼠海马乙酰化微管蛋白(Acet-Tub),酪氨酸化微管蛋白(Tyr-Tub),微管结合蛋白2(MAP-2)及磷酸化微管结合蛋白2(phospho-MAP-2).结果 (1)CUMS组糖水偏好[(55.13±11.80)%],总行程[(2736.59±511.20)cm],运动平均速度[(5.69±1.08)cm/s]及直立次数[(2.50±2.00)次]均低于对照组,差异有统计学意义(P＜0.01);氟西汀组上述指标与对照组比较差异无统计学意义(P＞0.05).(2)CUMS组与对照组相比,Acet-Tub表达升高[(171.84±10.34)%],Tyr-Tub[(62.06±9.24)%]和phospho-MAP-2[(68.81±8.93)%]的表达降低,差异有统计学意义(P均＜0.01),MAP-2的表达与对照组比较无统计学意义(P＞0.05);经氟西汀治疗后,Acet-Tub的表达降低为[(96.18±8.92)%],Tyr-Tub和phospho-MAP-2的表达分别升高为[(95.06±8.00)%]、[(100.60±7.30)%],与对照组比较均无统计学意义(P＞0.05).结论 慢性应激后微管动态性减低,神经可塑性受损,氟西汀可以逆转海马的这些损伤,上述过程可能与微管相关蛋白磷酸化水平的变化有关.     

大鼠海马注射溶血磷脂酸诱导tau蛋白高度磷酸化和凋亡研究

目的研究溶血磷脂酸(LPA)在大鼠体内对海马神经细胞tau蛋白磷酸化水平的影响和诱导神经细胞凋亡的细胞毒性作用.方法将72只SD大鼠分为实验组(n=32)、实验对照组(n=32)和对照组(n=8),利用脑立体定位技术在大鼠双侧海马微量注射溶血磷脂酸、溶剂,于注射后12、24、48和72 h各不同时间点采用免疫组化方法测定该区域神经细胞中ser202位点磷酸化tau蛋白(PS202-tau)的表达,TUNEL技术检测细胞凋亡.结果实验组LPA注射后24 h海马CA4区神经细胞中PS202-tau阳性表达到达高峰,阳性表达高于对照组和实验对照组(P＜0.05).LPA注射后48 h TUNEL阳性细胞数达高峰,每个视野中阳性细胞数多于对照组和实验对照组(P＜0.05).结论LPA在动物整体水平可诱导大鼠海马神经细胞tau蛋白高度磷酸化,并导致神经细胞发生凋亡.     

老年期痴呆患者脑脊液微管相关蛋白tau的检测

目的探讨老年期痴呆(SD)患者脑脊液(CSF)中微管相关蛋白tau(以下简称tau)的检测方法.方法采用双抗体夹心酶联免疫吸附(ELISA)法检测26例SD患者和30例非神经系统疾病患者的CSF-tau.结果两者浓度分别为473±279、151±60 pg/ml,有极显著差异(P<0.001);本法灵敏度为53.8%,特异性为83.3%,检出限60 pg/ml,组内变异1.2%～5.9%,组间变异1.7%～6.0%.结论采用双抗体夹心ELISA法检测SD患者CSF-tau的浓度可为诊断SD提供有价值的参考依据.     

慢性脑灌注不足对tau蛋白磷酸化及其相关酶表达的影响

神经原纤维缠结(neurofibrillartangles,NFTs)是阿尔茨海默病(Alzheimer’sdisease ,AD)的标志性病理特征之一,tau蛋白的过度磷酸化被认为是NFTs形成的关键[1] 。近年来,脑血管因素在AD中的作用备受关注,但是,它们与AD神经病理之间的关系还不清楚[2 ,3 ] 。我们通过永久性双侧颈总动脉结扎(2VO)制作慢性脑灌注不足(chroniccerebrovascularinsufficiency ,CVI)大鼠模型,观察海马神经元中Ser2 0 2位点磷酸化的tau蛋白(PS2 0 2 tau)、糖原合成酶激酶3β(glycogensynthasekinase 3β,GSK 3β)和蛋白磷酸酯酶2A (proteinphosphatase 2…     

慢性应激模型大鼠脑内5-HT1A受体表达分析

目的 探讨慢性不可预见性轻度应激(CUMS)模型大鼠各脑区(海马、中缝核、前额叶及纹状体)5-HT1A受体表达及与旷场行为变化的关系;西酞普兰对5-HT1A受体表达的影响.方法 将24只雄性SD大鼠随机分为3组(n=8):A组为对照组;B组为CUMS应激组;C组为CUMS应激+西酞普兰用药组(每天腹腔注射西酞普兰水溶液2 ml,10mg/kg).实验为期6周,通过旷场试验评价大鼠行为,6周后处死大鼠获取脑组织,采用Real-Time PCR检测各脑区5-HT1A mRNA表达水平,并分析其与矿场行为的相关性.结果 B组海马5-HT1A mRNA表达较A组有增高趋势(P=0.05),其余各脑区5-HT1A mRNA表达均差异无统计学意义(P＞0.05).海马5-HT1AmRNA表达大鼠移动次数、转身次数呈正相关,与总不动时间呈负相关;中缝核、纹状体5-HT1A mR-NA表达与中心移动距离呈正相关,纹状体5-HT1A mRNA表达与中心停留时间呈正相关(P均＜0.05).结论 慢性应激可能会引起海马5-HT1A受体表达增高;抗抑郁剂西酞普兰对5-HT1A受体表达无影响.5-HT1A mRNA表达与旷场行为变化有相关性.     

慢性应激对大鼠行为学及脑神经颗粒素水平的影响及药物防治

目的探讨慢性应激对大鼠外显行为学变化的影响,检测体内儿茶酚胺类物质及神经颗粒素(Neurogranin,Ng)表达的变化及三七皂苷Rg1的防治效果。方法成年雄性SD大鼠36只,随机分为对照组(CON)、模型组(CUS)、和治疗组(CUS-G),采用慢性不可预见性应激方法建立慢性应激动物模型;采用旷场试验检测行为学变化,运用Morris水迷宫实验进行学习记忆力测试,使用放射免疫法检测血浆、脑组织儿茶酚胺类含量,Western blot法检测皮质、海马、下丘脑Ng含量。结果 6 w慢性应激后,实验组大鼠在水平运动和垂直运动方面得分均明显低于对照组与药物组,均P0.05;水迷宫实验显示慢性应激后动物逃避潜伏期明显延长,而治疗组大鼠逃避潜伏期呈下降趋势(P0.05);血浆、脑组织去甲肾上腺素与多巴胺在对照组、实验组与药物组间无明显变化;慢性应激大鼠皮质、海马、下丘脑Ng含量水平皆与模型大鼠有差异(P0.05、P0.01、P0.05),治疗大鼠皮质、海马、下丘脑Ng含量亦皆与模型大鼠有差异(P0.01、P0.05、P0.05)。结论慢性应激研究领域,神经颗粒素的敏感性优于单胺类神经递质,可作为敏感指标加以观察;100 mg/kg剂量的三七皂苷Rg1能够抑制应激所致学习记忆功能紊乱,对应激所致外显行为有积极的调节作用,对脑内神经颗粒素表达水平降低趋势有较好抑制作用。     

内质网应激与冈田酸诱导tau蛋白异常磷酸化及其神经毒性作用

目的 探讨内质网应激 (ER stress)在冈田酸 (OA)诱导的tau蛋白磷酸化和神经毒性中的作用。方法：采用不同浓度的OA（25,50,100,200 nmol/L）与人神经母细胞瘤SH-SY5Y细胞共培养24小时.以DMSO作为溶媒对照,ER应激诱导剂tunicamycin（2.5μmol/L）作为阳性对照,分别采用MTT比色法和PI染色法观察细胞增殖和死亡情况;免疫荧光双标、Western blot法检测磷酸化tau蛋白,ER应激相关蛋白BiP/GRP78及其促凋亡转录因子CHOP/Gadd153的表达. RT-PCR检测BiP/GRP78和CHOP/Gadd153 mRNA表达水平。 结果：50 nmol/L OA处理后24 h,SH-SY5Y细胞活力下降,死亡率增加,且随OA浓度增加呈现显著上升趋势,免疫荧光染色和Western blotting结果显示ser202/thr205位点磷酸化tau蛋白水平升高;经100 nmol/L OA处理后,BiP/GRP78 mRNA和蛋白表达显著增加,Gadd153/CHOP 表达略有增加;而2.5μmol/L tunicamycin处理细胞后,BiP/GRP78 和Gadd153/CHOP表达均明显上调。 结论：在OA诱导的tau蛋白磷酸化和细胞毒性过程中,ER 应激是诱导细胞损伤的路径之一。     

Cytoskeletal alterations in rat hippocampus following chronic unpredictable mild stress and re-exposure to acute and chronic unpredictable mild stress

The intrinsic dynamic instability of the cytoskeletal microtubular system is essential for neuronal development and organization. The modulation of microtubule dynamics depends on the phosphorylation of neuronal microtubule-associated proteins (MAPs). Chronic unpredicted mild stress (CUMS) affects hippocampal structure and function in the rat. The aim of the present work was to investigate the possible alteration of cytoskeleton in the hippocampus of rats exposed to CUMS and re-exposed to CUMS to mimic depression and the recurrence of depression of human. We investigated the effects of CUMS, fluoxetine and re-exposure to CUMS on α-tubulin isoforms associated with microtubule dynamics, MAP-2 and phospho-MAP-2 in the hippocampus of rats. Our results showed that rats submitted to CUMS once showed a significant reduction in locomotion and sucrose preference which indicate a state of anhedonia. These behavioral alterations were accompanied by specific alterations in hippocampal α-tubulin isoforms and phospho-MAP-2 expression, indicating less microtubule dynamics and the possible mechanism. Treatment of fluoxetine could reverse CUMS-induced impairment. Moreover, there were more dramatically changes in behaviors, α-tubulin isoforms and phospho-MAP-2 of rats re-exposed to CUMS compared to the rats exposed to CUMS once. These findings provide evidence that rats exposed to CUMS and re-exposed to CUMS showed impairment of microtubule dynamics accompanied with the decreased level of phospho-MAP-2, providing insight into the role of cytoskeleton in the depression and recurrent of depression.     

应激及再应激后大鼠大脑皮质BDNF及细胞支架的改变

目的 研究慢性不可预见性温和应激（CUMS）,氟西汀治疗及再应激后大鼠大脑皮质脑源性神经营养因子（BDNF）及细胞支架微管系统的改变.方法 将32只大鼠随机分为4组：对照组（空白对照＋o.9％氯化钠溶液）,CUMS组（CUMS＋0.9％氯化钠溶液）,氟西汀组（CUMS＋氟西汀）,再应激组（CUMS＋氟西汀＋1周药物清洗期＋CUMS）.大鼠进行21 d CUMS后,氟西汀组给予氟西汀治疗21d,再应激组给予氟西汀治疗21d后进行1周药物清洗,再次给予CUMS.其余两组给予0.9％氯化钠溶液.实验结束后检测糖水偏好,并使用Western Blotting检测鼠大脑总皮质及额叶皮质Acet-Tub,Tyr-Tub,BDNF,总tau,磷酸化tau（Ser356及Thr231）的表达.结果 21 d慢性应激后,CUMS组糖水偏好（52.37±16.758）％与对照组（76.37±7.671）％比较差异有统计学意义（P＜0.01）;氟西汀组糖水偏好与对照组比较差异无统计学意义;CUMS再应激组糖水偏好（42.00±8.03）％与对照组及CUMS组比较差异均有统计学意义（P＜0.01）.与对照组比较,CUMS组大鼠额叶皮质Acet-Tub表达升高（174.53±13.7）,Tyr-Tub表达降低（64.00±9.24）,BDNF表达降低（67.94±9.81）,p-tau（Ser356）的表达（165.49±9.68）及p-tau（Thr231）的表达（210.83±23.12）升高,差异有统计学意义（P＜0.01）.经氟西汀治疗后,Acet-Tub表达降低,Tyr-Tub、BDNF的表达升高,p-tau（Ser356）及p-tau（Thr231）的表达降低,与对照组比较差异无统计学意义.再应激组Acet-Tub表达升高（250.46±15.72）,Tyr-Tub表达降低（33.54±10.23）,BDNF表达降低（34.77±6.24）,p-tau （Ser356）表达（237.42±10.24）及p-tau（Thr231）的表达（373.69±34.14）升高,与对照组及CUMS组比较差异均有统计学意义（P＜0.01）.4组之间总tau的表达比较差异无统计学意义.大鼠大脑总皮质各项指标差异无统计学意义.结论 抑郁动物模型大脑额叶皮质微管动态性减低,伴随BDNF表达减低     

Aggregation of tau protein by aluminum

Aluminum has been detected in Alzheimer neurofibrillary tangles, but the significance of its presence is unknown. The principal component of tangles is the paired helical filament (PHF), comprised of tau protein. We investigated whether aluminum could induce tau protein to form filaments or aggregate. When 10 μM bovine tau or non-phosphorylated recombinant human tau was combined with 400 μM or more aluminum, tau protein appeared to aggregate, observed as a dose-dependent decrease in electrophoretic mobility on SDS-PAGE. Tau appeared as a smear above the region of the expected tau bands and, at higher aluminum,doses, failed to enter the gel. A tau fragment encompassing the microtubule binding domains did not show decreased mobility in the presence of aluminum, but did form aggregates that failed to electrophorese. However no fibrillar structures were observed in the aluminum-treated tau samples when observed by electron microscopy. The effect of aluminum on tau mobility was reversed by incubating with 1 mM deferoxamine. In contrast, the morphology of PHF fibrils was unaffected by deferoxamine treatment and the characteristic abnormal mobility of PHF-tau was not reduced by deferoxamine. This suggests that aluminum is not, by itself, a significant factor in maintaining the assembly of PHF-tau as fibrils or in its abnormal mobility on SDS gels. Aluminum treatment of 3T3 fibroblasts transfected with human tau resulted in toxicity, but did not change tau expression levels or induce tau aggregation. In conclusion, aluminum appears to induce isolated tau protein to aggregate in a phosphate-independent way, without the formation of fibrils. This effect was not observed when tau-transfected cells were treated with toxic doses of aluminum.     

Intracortical perfusion of glutamate in vivo induces alterations of tau and microtubule-associated protein 2 immunoreactivity in the rat

Modifications of microtubule-associated proteins (MAP) have been reported in both acute and chronic degenerative conditions, such as cerebral ischaemia and Alzheimer’s disease, and may be associated with cytoskeletal breakdown. Glutamate excitotoxicity has been implicated in the pathogenesis of both of these conditions and has been shown in some in vitro studies to induce changes in tau similar to those occurring in Alzheimer’s disease. This study examines the effects of high extracellular glutamate concentrations on the distribution of tau and MAP2 in vivo in order to determine whether glutamate induces similar changes in tau to those previously reported in vitro in the intact, adult central nervous system. Monosodium glutamate was perfused into the rat parietal cortex for 90 min using in vivo microdialysis and at 4 h after the start of perfusion the distribution of tau and MAP2 was determined by immunohistochemistry. At the core of the glutamate-induced lesion tau immunostaining, as detected with the Tau 1 antibody, was decreased in axons and increased within perikarya compared to controls. Increased immunostaining was not apparent with polyclonal antibodies raised against full-length tau or towards the N or C termini of the protein. In contrast, increased tau immunoreactivity was detected, with all the antibodies used in this study, within oligodendrocytes following either glutamate or sodium chloride perfusion. MAP2 immunoreactivity was increased within perikarya at the core of the glutamate-induced lesion, while dendritic immunoreactivity was reduced. These results suggest that glutamate excitotoxicity in vivo may not be involved in neurofibrillary tangle formation but may be important in the progression of cytoskeletal pathology following cerebral ischaemia. Received: 9 May 1995 / Revised: 12 December 1995 / Accepted: 26 February 1996     

rTMS ameliorates depressive-like behaviors and regulates the gut microbiome and medium- and long-chain fatty acids in mice exposed to chronic unpredictable mild stress

Introduction

Repetitive transcranial magnetic stimulation (rTMS) is a clinically useful therapy for depression. However, the effects of rTMS on the metabolism of fatty acids (FAs) and the composition of gut microbiota in depression are not well established.

Methods

Mice received rTMS (15 Hz, 1.26 T) for seven consecutive days after exposure to chronic unpredictable mild stress (CUMS). The subsequent depressive-like behaviors, the composition of gut microbiota of stool samples, as well as medium- and long-chain fatty acids (MLCFAs) in the plasma, prefrontal cortex (PFC), and hippocampus (HPC) were evaluated.

Results

CUMS induced remarkable changes in gut microbiotas and fatty acids, specifically in community diversity of gut microbiotas and PUFAs in the brain. 15 Hz rTMS treatment alleviates depressive-like behaviors and partially normalized CUMS induced alterations of microbiotas and MLCFAs, especially the abundance of Cyanobacteria, Actinobacteriota, and levels of polyunsaturated fatty acids (PUFAs) in the hippocampus and PFC.

Conclusion

These findings revealed that the modulation of gut microbiotas and PUFAs metabolism might partly contribute to the antidepressant effect of rTMS.     

Antidepressant-like effects of curcumin on serotonergic receptor-coupled AC-cAMP pathway in chronic unpredictable mild stress of rats

Serotonergic receptors take their physiologic effects by affecting adenylyl cyclase (AC) catalytic activity and cyclic adenosine monophosphate (cAMP) concentration. AC-cAMP second messenger pathway has been recently suggested to play an important role in depression. Therefore, the compound that regulates the signal pathway may have potential as antidepressant. Curcumin is the main component of Curcuma longa L, a well-known indigenous herb with comprehensive bioactivities. In the present study, we investigated the effects of chronic unpredictable mild stress (CUMS) and curcumin on behaviours and serotonergic receptor-coupled AC-cAMP signal pathway in rats. Curcumin produced beneficial effects on the stressed rats by effectively improving CUMS-induced low sucrose consumption and reducing serum corticosterone levels in rats. Moreover, curcumin enhanced AC activity and cAMP levels in platelet and various brain regions, and up-regulated mRNA expressions of AC subtypes AC 2, AC 8 and cAMP response element binding protein (CREB) in the hippocampus, cortex and hypothalamus of the CUMS rats. Curcumin also attenuated CUMS-induced reductions of 5-hydroxytryptamine (5-HT) levels and high expressions of central 5-HT_1A/1B/7 receptors in rats. These results suggested that the potent antidepressant property of curcumin might be attributed to its improvement of AC-cAMP pathway as well as CREB via suppressing central 5-HT_1A/1B/7 receptors in the CUMS rats. Our findings provided a basis for examining the interaction of serotonergic receptors and AC-cAMP pathway in depression and curcumin treatment.