Effect of Panaxtriol Saponins on synaptophysin and postsynaptic density-95 expression at different periods of cerebral infarction*☆

BACKGROUND： The change in expression of synaptophysin （Syp） and postsynaptic density-95 （PSD-95） alters after cerebral infarction, and the plasticity of synapses contributes greatly to nerve function recovery. Chinese medicinal substances may play an important role in the expression of Syp and PSD-95. OBJECTIVE： To observe the effect of Panaxtriol Saponins （PTS）, an active component in Sanqi tongshu capsules, on the expression of Syp and PSD-95 after cerebral infarction at different time points in rats, so as to examine the cerebral function remodeling mechanism. DESIGN, TIME AND SETTING： A randomized and controlled observation which was performed in Dongzhimen Hospital, Beijing University of Traditional Chinese Medicine from January to March, 2007. MATERIALS： Twenty-six healthy male Sprague Dawley rats were used to establish middle cerebral artery occlusion based on the Longa method. Sanqi tongshu capsules （containing 100 mg PTS per tablet） were provided by the Chengdu Huashen Group and nimodipine tablets （30 mg） by Tianjin Zhongyang Pharmaceutical Co., Ltd. METHODS： Twenty-six rats were randomly divided into an operation group （n = 21 ） and a control group （n = 5）. The operation group underwent the EZ Longa procedure to make the middle cerebral artery occlusion model. After surgery rats were randomly divided into a model group, a PTS group and a nimodipine group, with seven rats in each group. Rats were intragastrically administrated with saline （2 mL/d） in the model group, with Sanqi tongshu capsule （5.4 mg/100 g/d） in the PTS group, and with nimodipine （1.73 mg/100 g/d） in the nimodipine group. Rats in the control group did not undergo model establishment and drug administration. MAIN OUTCOME MEASURES： The expressions of Syp and PSD-95 were measured by immunohistochemical and image analysis at days 3, 7 and 28 after the operation. RESULTS： The expression of Syp and PSD-95 in the operation group was significantly lower than in the control group at days 3, 7     

Effects of genistein and 17 beta-estradiol on hippocampal synaptophysin expression in ovariectomized rats

BACKGROUND:Phytoestrogen,derived from plants,is an estrogen-like element,and is effective and safe for estrogen replacement.OBJECTIVE:To compare the interventional effects of genistein and 17 β-estradiol on learning and memory and synaptophysin(SYN)expression in the hippocampus of ovariectomized rats.DESIGN:Randomized controlled animal study.SETTING:Department of Neurology,the Third Affiliated Hospital,Xiangya Medical College,Central South University. MATERIALS:130 healthy female Sprague Dawley(SD)rats,6 months old and weighing(293.1±10.2)g,were provided by the Second Xiangya Hospital of Central South University.This animal experiment received confirmed consent from the local ethics committee.All rats were randomly divided into 5 groups,including baseline group(n＝10),sham operation group(n＝30),ovariectomlzed group(n＝30),genistein group(n＝ 30),and 17 β-estradiol group(n＝30).Rats in the latter four groups were observed for 3 weeks(n＝10)and for 15 weeks(n＝20)after model establishment.METHODS:This study was performed at the Department of Endocrinology,the Second Affiliated Hospital,Xiangya Medical College,Central South University from August 2005 to January 2006.Animals were not submitted to any treatment in the baseline group,but anesthetized and sacrificed at the 7 months of age.After anesthesia in the ovariectomized,genistein,and 17 β-estradiol groups,both ovaries were separated and resected to establish an ovariectomized model.The same volume of fat was resected in the sham operation group.After surgery,rats were intraperitoneally injected with 5 mg/kg genistein in the genistein group,10 μ g/kg 17 β-estradiol in the 17 β-estradiol group,and 0.1 mL/100 g dimethyl sulfoxide (DMSO)/polyethylene glycol(PEG)-200 stock solution in the sham peration and ovariectomized groups once a day until one day before sacrifice.MAIN OUTCOME MEASURES:①Learning and memory changes of SD rats were detected using water maze behavioral testing 3 and 15 weeks after surgery.②SYN expression in the hippocampus was measured using immunohistochemistry. RESULTS:A total of 16 out of 130 rats died due to infection,and 114 rats were included in the final analysis.①Comparison of water maze results from the five groups:by 3 and 15 weeks after surgery, escape latency was prolonged and platform-crossing times decreased in the ovariectomized group compared to the baseline,genistein,17 β-estradiol,and sham operation groups(t＝4.17--14.64,P＜0.05).However, there were no significant differences in escape latency and platform-crossing times among the sham operation,genistein,and 17 β-estradiol groups(P＜0.05).②Distribution and quantity of SYN immunoreactive products in hippocampus:SYN-immunoreactive cells stained darkly in the baseline and sham operation groups,but were lightly stained in the genistein,17 β-estradiol,and ovariectomized groups.In particular,SYN-immunoreactive cells stained lightly in the ovariectomized group 15 weeks after surgery. SYN correction gray values in hippocampal sub-regions,especially in the mossy fiber layer of the CA3 region,of the ovariectomized group was lower compared to the baseline,sham operation,17 β-estradiol,and genistein groups(t＝12.57-23.92,P＜0.05)15 weeks after surgery.However,there were no significant differences in SYN correction gray values among the baseline,sham operation,17 β-estradiol and genistein groups(P＜0.05).CONCLUSION:Genistein or 17 β-estradiol supplemental therapy antagonizes memory deterioration,due to endogenous estrogen deficiency and blocks the decrease of SYN expression in the hippocampus.The effect of genistein is similar to 17 β-estradiol.     

Long-term synaptic alteration in the rat hippocampal CA3 field following an entorhinal cortex lesion

The entorhinal cortex is a key initial relay for cortical input to the hippocampus. To better understand hippocampal dysfunction resulting from early entorhinal cortex involvement in Alzheimer's disease, we stereotaxically injected ibotenic acid to produce unilateral entorhinal cortex lesions in rats. We then serially examined the CA3 hippocampal region by neuronal counts, histochemistry for acetylcholinesterase, and synaptophysin immunohistochemistry. Over 12 months, the neuronal counts did not change. Acetylcholinesterase-positive fibers were persistently but non-progressively beginning at 3 months. Synaptophysin immunoreactivity progressively declined over 12 months. Since much of the entorhinal cortex output proceeds to CA3 via the dentate gyrus, transsynaptic degeneration is suspected.     

慢性应激刺激成年小鼠对其老年期认知功能的影响

目的:观察慢性应激刺激成年小鼠对其老年期认知功能及海马区神经结构的影响。方法:将8周龄成年雄性小鼠予以慢性束缚性刺激6周(Stress,S组),束缚结束后成年S组小鼠即刻进行水迷宫行为学测试并断头以免疫组化学及硫堇染色方法观察海马区突触素及尼氏小体的变化,并利用医学图像分析系统计算突触素免疫组化反应阳性产物数量;而老年S组小鼠于慢性束缚6周后普通环境下继续饲养11周至老年期(25周龄)再进行上述行为学及病理学检测。对照组小鼠正常环境下饲养并分别于14周龄、25周龄进行上述行为学及病理学检测。结果:成年及老年S组小鼠学习记忆能力明显受损(P<0.05);海马区脑组织突触素免疫反应阳性产物和尼氏小体数量较同龄对照组小鼠明显减少(P<0.01)。结论:给予成年期小鼠慢性应激刺激可导致老年期小鼠认知功能损害,其记忆的损害与年龄老化之间可能具有协同作用。     

Does apolipoprotein E (Apo-E) genotype influence nicotinic receptor binding in Alzheimer's disease

Summary. We wished to determine the influence of the apolipoprotein E (Apo-E) genotype on the loss of high affinity nicotinic acetylcholine receptor (nAChR) binding in Alzheimer's disease (AD). The interaction between ε4 allele gene dose and cholinergic loss in AD remains controversial. We have demonstrated that nicotinic binding is significantly lost in AD. Tissue from the midfrontal (MF) cortex of 7 subjects with no ε4 allele copies (ε−/ε−) (mean death age 75.1 ± 10.4 years) was compared to MF cortex of 14 subjects heterozygous for the ε4 allele (ε4/ε−) (mean death age 81.4 ± 7.3 years) and MF cortex of 10 subjects homozygous for the ε4 allele (ε4/ε4) (mean death age 79.6 ± 5.0 years). All subjects were autopsy confirmed AD (using NIA and CERAD criteria) and met NINCDS-ADRDA clinical criteria for probable or possible AD. Nicotine AChR binding was assayed using the high affinity nicotinic agonist ³H-epibatidine ([³H]-Epi). Apo-E genotype was determined in blood samples or in post-mortem tissue. The mean age at death was not significantly different among the groups (p = 0.19). There was no difference in mean [³H]-Epi total binding among the three groups (6.7 ± 4.6, 6.1 ± 2.4, and 6.0 ± 1.0 fmol/mg protein for ε−/ε−, ε4/ε−, and ε4/ε4 respectively. We conclude that the presence or absence of the Apo-E4 genotype does not influence the loss of high affinity nAChR in AD. Received January 16, 2001; accepted April 16, 2001     

不同环境设置对局灶性脑梗死大鼠梗死灶周围Syn表达的影响

目的：观察不同环境对局灶性脑梗死大鼠梗死灶周围突触膜糖蛋白表达的影响。方法：雄性SD大鼠95只,采用电凝法造成右侧大脑中动脉阻断（MCAO）模型后,随机分为独居组（n=20）,社交组（n=30）,探索学习组（n=20）,丰富环境组（n=20）,假手术对照组（n=5）。于不同时点分批处死大鼠,用免疫组化染色观察梗死灶周围皮质Syn阳性表达。结果：MCAO后梗死灶周围皮质Syn阳性表达均高于假手术对照组,并且随时间的推移,其表达进一步增高。探索学习组、丰富环境组于MCAO术后各时间点Syn表达均明显优于独居组和社交组(P<0.05,P<0.01);探索学习组在3、4周表达高于丰富环境组(P<0.05);社会交往组表达高于独居组(P<0.05)。结论：丰富环境;探索学习及社会交往均能促进梗死灶周围Syn的表达。     

Bone marrow stromal cells upregulate expression of bone morphogenetic proteins 2 and 4, gap junction protein connexin-43 and synaptophysin after stroke in rats

Bone morphogenetic proteins play a key role in astrocytic differentiation. Astrocytes express the gap junctional protein connexin-43, which permits exchange of small molecules in brain and enhances synaptic efficacy. Bone marrow stromal cells produce soluble factors including bone morphogenetic protein 2 and bone morphogenetic protein 4 (bone morphogenetic protein 2/4) in ischemic brain. Here, we tested whether intra-carotid infusion of bone marrow stromal cells promotes synaptophysin expression and neurological functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 h of right middle cerebral artery occlusion. Rats were treated with or without bone marrow stromal cells at 24 h after middle cerebral artery occlusion via intra-arterial injection (n=8/group). A battery of functional tests was performed. Immunostaining of 5-bromo-2-deoxyuridine, Ki67, bone morphogenetic protein 2/4, connexin-43, synaptophysin, glial fibrillary acidic protein, neuronal nuclear antigen, and double staining of 5-bromo-2-deoxyuridine/glial fibrillary acidic protein, 5-bromo-2-deoxyuridine/neuronal nuclear antigen, glial fibrillary acidic protein/bone morphogenetic protein 2/4 and glial fibrillary acidic protein/connexin-43 were employed. Rats treated with bone marrow stromal cells significantly (P<0.05) improved functional recovery compared with the controls. 5-Bromo-2-deoxyuridine and Ki67 positive cells in the ipsilateral subventricular zone were significantly (P<0.05) increased in bone marrow stromal cell treatment group compared with the controls, respectively. Administration of bone marrow stromal cells significantly (P<0.05) promoted the proliferating cell astrocytic differentiation, and increased bone morphogenetic protein 2/4, connexin-43 and synaptophysin expression in the ischemic boundary zone compared with the controls, respectively. Bone morphogenetic protein 2/4 expression correlated with the expression of connexin-43 (r=0.84, P<0.05) and connexin-43 expression correlated with the expression of synaptophysin (r=0.73, P<0.05) in the ischemic boundary zone, respectively. Administration of bone marrow stromal cells via an intra-carotid route increases endogenous brain bone morphogenetic protein 2/4 and connexin-43 expression in astrocytes and promotes synaptophysin expression, which may benefit functional recovery after stroke in rats.     

穹窿海马伞切断鼠脑突触素动态变化及Morris水迷宫重复训练对其影响

目的在Morris水迷宫(MWM)重复训练的过程中观察穹窿海马伞切断大鼠的突触素(SYN)动态变化探讨阿尔茨海默病(AD)的发病机制和MWM重复训练学习和记忆能力对AD的影响,为临床应用提供实验依据。方法采用wistar大鼠60只,随机分为对照组、模型组和实验组,建模后连续四周给予Morris水迷宫重复训练定位航行和探索训练,分别评定大鼠空间学习和记忆能力,后进行海马突触素(SYN)免疫组化染色和超微电镜观察并进行统计学处理。结果随着训练次数的增加,对照组、模型组、实验组逃逸时间均逐渐缩短,实验组短于模型组,长于对照组(P0.05),提示空间学习能力得到提高。对照组、模型组和实验组大鼠在靶象限活动时间(s)百分比无明显差异(P0.05),提示空间记忆能力无明显提高。SYN免疫组化实验组SYN表达高于模型组,弱于对照组。结论MWM重复训练增加海马SYN表达可能与提高穹窿海马伞切断大鼠空间学习能力有关。     

肌萎缩侧索硬化小鼠内源性神经干细胞膜突触蛋白表达

背景：成纤维细胞生长因子2是神经系统主要的神经营养因子之一,目前已经被证明有促进内源性神经干细胞分化的作用。 目的：观察成纤维细胞生长因子2干预下,肌萎缩侧索硬化SOD1G93A G1H转基因小鼠运动功能的改变,内源性神经干细胞的增殖情况,突触蛋白的水平改变及内源性神经干细胞增殖数目与突触蛋白水平改变的相关性。 方法：取新出生SOD1G93A G1H转基因小鼠(肌萎缩侧索硬化模型小鼠)60只分为肌萎缩侧索硬化组及成纤维细胞生长因子2组各30只,取新出生野生B6SJL小鼠30只作为正常对照组,成纤维细胞生长因子2组腹腔注入成纤维细胞生长因子2;肌萎缩侧索硬化组和正常对照组腹腔注入安慰剂生理盐水。分别于出生后60,90,120 d采用Rotarod方法评估小鼠运动功能的改变,采用免疫组织化学方法标记内源性神经干细胞和突触蛋白并计数,用spearman方法评估内源性神经干细胞增殖数目与突触蛋白水平的相关性。 结果与结论：与肌萎缩侧索硬化组相比,成纤维细胞生长因子2组小鼠运动功能明显改善,内源性神经干细胞增殖和突触蛋白水平显著增高。小鼠内源性神经干细胞的增加与突触蛋白水平的增呈正相关。提示成纤维细胞生长因子2神经保护机制可能与其促进内源性神经干细胞增殖和提升突触蛋白水平相关。     

NSCs promote hippocampal neurogenesis,metabolic changes and synaptogenesis in APP/PS1 transgenic mice

Adult neurogenesis and synaptic remodeling persist as a unique form of structural and functional plasticity in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) of the lateral ventricles due to the existence of neural stem cells (NSCs). Transplantation of NSCs may represent a promising approach for the recovery of neural circuits. Here, we aimed to examine effects of highly neuronal differentiation of NSCs transplantation on hippocampal neurogenesis, metabolic changes and synaptic formation in APP/PS1 mice. 12‐month‐old APP/PS1 mice were used for behavioral tests, immunohistochemistry, western blot, transmission electron microscopy and proton magnetic resonance spectroscopy (1H‐MRS). The results showed that N‐acetylaspartate (NAA) and Glutamate (Glu) levels were increased in the Tg‐NSC mice compared with the Tg‐PBS and Tg‐AD mice 10 weeks after NSCs transplantation. NSC‐induced an increase in expression of synaptophysin and postsynaptic protein‐95, and the number of neurons with normal synapses was significantly increased in Tg‐NSC mice. More doublecortin‐, BrdU/NeuN‐ and Nestin‐positive neurons were observed in the hippocampal DG and SVZ of the Tg‐NSC mice. This is the first demonstration that engrafted NSCs with a high differentiation rate to neurons can enhance neurogenesis in a mouse model of AD and can be detected by 1H‐MRS in vivo. It is suggested that engraft of NSCs can restore memory and promote endogenous neurogenesis and synaptic remodeling, moreover, 1H‐MRS can detect metabolite changes in AD mice in vivo. The observed changes in NAA/creatine (Cr) and glutamate (Glu)/Cr may be correlated with newborn neurons and new synapse formation.