建立类似于绝经期妇女帕金森病大鼠模型的实验研究

目的探讨利用6-羟基多巴(6-OHDA)制备模拟绝经期妇女帕金森病的大鼠模型。方法应用6-OHDA制备OVX PD模型大鼠,应用免疫组织化学染色、高效液相色谱法等技术对大鼠黑质(SN)的酪氨酸羟化酶(TH)阳性神经元数目、纹状体多巴胺(DA)及其代谢物二羟基苯乙酸(DOPAC)和高香草酸(HVA)含量进行考察并评价。结果阿朴吗啡可诱导出明显的PD大鼠旋转行为;大鼠损伤侧黑质TH阳性神经元数量较健侧显著减少(P<0.01),纹状体DA及其代谢物DOPAC和HVA含量也较健侧明显减少(P<0.01)。结论本模型具有绝经期妇女帕金森病的基本的病理特点。     

姜黄素对帕金森病模型小鼠多巴胺能神经元作用的研究

目的探讨姜黄素（Curcumin,Cur）对1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）制备的帕金森病（PD）模型小鼠多巴胺能神经元的保护作用。方法将C57BL6雄性小鼠随机分为正常对照组、MPTP模型组及姜黄素预处理组。用逆转录-聚合酶链式反应（RT-PCR）技术检测黑质酪氨酸羟化酶（TH）及多巴胺转运体（DAT）基因的表达水平。应用高效液相色谱法检测纹状体（Str）内DA及其代谢物二羟基苯乙酸（DOPAC）含量。结果MPTP模型组黑质TH和DAT基因的表达较正常对照组明显降低,姜黄素预处理组可逆转上述改变（P〈0.01）。MPTP模型组Str内DA及其代谢产物DOPAC的含量较对照组明显减少,应用姜黄素后可逆转上述改变（P〈0.01）。结论姜黄素对MPTP制备的PD模型小鼠黑质纹状体DA能神经元有明显的保护作用。     

Therapeutic effect of human amniotic epithelial cell transplantation into the lateral ventricle of hemiparkinsonian rats

Background Human amniotic epithelial cells （HAECs） are able to secrete biologically active neurotrophins such as brain-derived neurotrophic factor and neurotrophin-3, both of which exhibit trophic activities on dopamine neurons. Previous study showed that when human amniotic epithelial cells were transplanted into the striatum of 6-hydroxydopamine （6-OHDA）-induced Parkinson disease rats, the cells could survive and exert functional effects. The purpose of this study was to investigate the survival and the differentiation of human amniotic epithelial cells after being transplanted into the lateral ventricle of Parkinson＇s disease （PD） rats, and to investigate the effects of grafts on healing PD in models. Methods The Parkinson＇s model was made with stereotactic microinjection of 6-hydroxydopamine （6-OHDA） into the striatum of a rat. The PD models were divided into two groups： the HAECs group and the normal saline （NS） group. Some untreated rats were taken as the control. The rotational asymmetry induced by apomorphine of the HAECs group and the NS group were measured post cell transplantation. The expression of nestin and vimentin in grafts were determined by immunohistology. Ten weeks after transplantation the density of tyrosine hydroxylase positive cells in the substantia nigra of the HAECs group, NS group and the untreated group was determined. The differentiation of grafts was determined by TH immunohistology. High performance liquid chromatography （HPLC） was used to determine monoamine neurotransmitter levels in the striatum. Results The rotational asymmetry induced by apomorphine of the HAECs group was ameliorated significantly compared to the NS group two weeks after transplantation （P 〈0.01）. The grafts expressed nestin and vimentin five weeks after transplantation. TH immunohistochemistry indicated that the TH positive cells in the substantia nigra of the HAECs group increased significantly compared to the NS group （P 〈0.01）. Tyrosine hydroxylase （TH） positive cells in the substantia nigra of the HAEC group and the NS group were decreased compared to the untreated group （P 〈0.01）. Dopamine and DOPAC levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.05）. Homovanillic acid （HVA） levels in the striatum of the HAECs group increased significantly compared to the NS group （P 〈0.01）. In addition dopamine, DOPAC, and HVA levels in the striatum and dopamine levels in the cerebrospinal fluid of the HAECs group and the NS group were decreased compared to the untreated group （P 〈0.05）. Conclusions Human amniotic epithelial cells could be used to ameliorate the rotational asymmetry induced by apomorphine of the PD models. This could have been due to the increased content of dopamine and its metabolic products, DOPAC and HVA, in the striatum in the PD models.     

Dynamic changes of striatal monoamines with in vivo microdialysis in ischemic rats]

Using intracerebral microdialysis, high performance liquid chromatography and electrochemical detection (HPLC-ECD), we determined the contents of extracellular dopamine (DA), serotonin (5-HT) and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid(HVA) and 5-hydroxy-indole acetic acid (5-HIAA) in the striatum of rats after the occlusion of middle cerebral artery (MCAO). The dialysis tube was implanted stereotaxically in the lateral 2/3 of the striatum. No significant effects of stressors were noted on the concentration of neurotransmitter mentioned above. A step rise of extracellular DA and 5-HT accompanied by significant reduction of DOPAC, HVA and 5-HIAA was found 10 minutes after MCAO. From then on, the enhanced concentrations of DA and 5-HT decreased gradually. At 3 hours after MCAO, however, DA and 5-HT were still high with respect to the basal levels. Their metabolites were kept at low levels at 3 hours of MCAO. In conclusion, the massive leakage of DA and its over production of oxygen free radicals are important for the development of ischemic cell damage in the striatum.     

乌鸡白凤丸有效成分对帕金森病模型小鼠的神经保护作用

目的探讨乌鸡白凤丸有效成分（BFP）对1-甲基-4-苯基-1，2，3，6-四氢吡啶（MPTP）诱致C57BL小鼠黑质神经元凋亡的保护作用及其可能机制。方法C57BL小鼠连续5d皮下注射MPTP（30mg／kg）制备帕金森病小鼠模型。给予BFP预处理后，用酪氨酸羟化酶（TH）免疫组织化学法观察黑质神经元的变化；同时应用免疫组织化学法检测Bax蛋白表达; 应用高效液相-电化学方法检测纹状体（Str）多巴胺（DA）及其代谢产物二羟基苯乙酸（DOPAC）和高香草酸（HVA）的含量变化。结果BFP预处理能使黑质致密带（SNc）TH免疫阳性神经元数目较MPTP组明显增多，而Bax蛋白表达较MPTP组明显减少；可使Str区DA及其代谢产物DOPAC、HVA含量明显增加。结论BFP有效成分可以拮抗神经毒性物质MPTP对C57BL小鼠黑质多巴胺能神经元的损伤，其作用机制可能与其抗凋亡作用有关，Bax表达的降低可能是BFP抗凋亡作用的途径之一。     

葛根素对急性乙醇中毒大鼠脑内阿片肽和多巴胺的调控作用

目的： 检测葛根素对急性乙醇中毒大鼠皮层、小脑、海马和纹状体内阿片肽和多巴胺（DA）表达水平的调控作用,为临床防治乙醇中毒和开发中药葛根提供依据。方法： 雄性SD大鼠24只随机分为对照组、乙醇中毒组和葛根素组,每组8只,采用放射免疫法检测各组大鼠皮层、小脑、海马和纹状体内β-内啡肽（β-EP）、强啡肽（DnyA）和亮脑啡肽（L-EK）表达水平,采用高效液相色谱法检测各组大鼠各脑区DA、多巴克（DOPAC）和高香草酸（HVA）的表达水平,并计算（DOPAC+HVA）/DA的比值。结果：与对照组比较,乙醇中毒组和葛根素组大鼠皮层内β-EP、DnyA和L-EK表达水平降低(P<0.01),皮层、小脑、海马和纹状体内 DA、DOPAC和HVA表达水平均升高 (P<0.01);与乙醇中毒组比较,葛根素组大鼠皮层内β-EP、DnyA和L-EK表达水平升高(P<0.05,P<0.01),DA(P<0.01)、DOPAC(P<0.01)和HVA表达水平均降低,小脑、海马和纹状体内所有指标表达水平均降低(P<0.05,P<0.01),皮层、小脑和海马内（DOPAC+HVA）/DA 比值升高,纹状体内该比值降低（P>0.05）。结论：葛根素对急性乙醇中毒造成的脑组织损伤有保护作用,其机制可能与葛根素对脑内阿片肽和DA的调控有关。     

蛋白酶体抑制剂对小鼠黑质多巴胺神经元的毒性作用

目的:观察蛋白酶体抑制剂对小鼠黑质多巴胺神经元的毒性作用.方法:8周龄C57BL/6小鼠随机分组:磷酸盐缓冲液(PBS组)和lactacystin组,二种制剂分别单侧立体定向注射于中脑前脑束(Middle forebrain bundle);12周龄时灌注取脑分离纹状体,采用HPLC法测多巴胺、5-HT及代谢产物,取中脑观察黑质区多巴胺能神经元变性及包涵体样物质形成情况.结果:Lactacystin组同侧黑质区多巴胺细胞数减少51%,并可见泛素染色阳性的类包涵体样物质形成,同侧纹状体多巴胺(DA)及其代谢产物二羟苯乙酸(DOPAC)、高香草酸(HVA)浓度分别减少52%、46%和51%,而5-羟色胺(5-HT)及5-羟吲哚乙酸(5-HIAA)浓度无改变.结论:蛋白酶体抑制剂可引起选择性黑质区多巴胺能神经元变性,并可能导致细胞内异常蛋白质聚集.     

神经降压素对帕金森病模型大鼠纹状体DA含量影响

目的观察神经降压素(NT)对正常和帕金森病模型大鼠纹状体多巴胺(DA)及其代谢产物含量的影响。方法采用6-羟基多巴胺(6-OHDA)单侧损毁内侧前脑束制备帕金森病大鼠模型。将5μL 1 mmol/L NT或生理盐水双侧缓慢注入正常及帕金森病模型大鼠侧脑室30 min后,利用高效液相色谱分析(HPLC)法检测纹状体DA及其代谢产物高香草酸(HVA)和3,4-二羟基苯乙酸(DOPAC)含量的变化。结果正常大鼠NT组纹状体DA含量与生理盐水组相比均明显升高(t=5.32,P<0.01)。帕金森病模型大鼠生理盐水组损毁侧纹状体DA含量与未损毁侧和正常大鼠生理盐水组相比明显降低(F=61.23,P<0.001);损毁侧纹状体DA更新率(DOPAC+HVA)/DA、DOPAC/DA、HVA/DA均明显高于未损毁侧和正常大鼠生理盐水组(F=27.52、21.12、28.43,P<0.001)。帕金森病模型大鼠侧脑室注射NT可明显提高两侧纹状体DA的含量(t=3.87、2.26,P<0.05)。结论 NT可增加正常及帕金森病模型大鼠纹状体DA的含量,提示NT具有抗帕金森病效应。     

自噬诱导剂海藻糖对帕金森症小鼠的治疗作用

目的：探讨自噬诱导剂海藻糖对帕金森症小鼠的治疗作用。方法：使用1-甲基-4-苯基-1,2,3,6-四氢吡啶盐酸盐（MPTP）建立小鼠帕金森症模型,通过观察小鼠中脑黑质区多巴胺（DA）神经元数量、纹状体内神经递质多巴胺及代谢产物变化,判断海藻糖治疗帕金森症的作用。结果：（1）免疫组化染色发现MPTP使小鼠黑质区DA神经元数量明显减少。海藻糖干预后可增加DA神经元数量。（2）HPLC检测发现MPTP造成小鼠纹状体中多巴胺明显减少,海藻糖组可增加神经递质及代谢物。结论：海藻糖能减少MPTP造成的黑质区DA神经元丢失,增加纹状体中多巴胺的含量,海藻糖能有效治疗帕金森症。     

Effects of Echinacoside on Histio-central Levels of Active Mass in Middle Cerebral Artery Occlusion Rats*

Objective To investigate the effects of echinacoside on the extracellular striatal levels of norepinephrine (NE),dopamine (DA),homovanillic acid (HVA),3,4-dihydroxyphenylethanoid acid (DOPAC),5-hydroxyindoleacetic acid (HIAA),and 5-hydroxytryptamine(5-HT) in middle cerebral artery occlusion (MCAO rats.Methods The middle cerebral artery was occluded in male Sprague-Dawley rats.Three days later microdialysis probes were placed into the right striatum of MCAO rat brains and the brains were perfused with Ringer's solution at a rate of 1.5 μL/min.Cerebral microdialysates were collected every 30 minutes from awake and freely moving rats before assaying for NE,DA,HVA,DOPAC,HIAA,and 5-HT levels by reverse phase HPLC with electrochemistry.Results Three days after MCAO,the extracellular striatal levels of NE,DA,DOPAC,HIAA,HVA,and 5-HT of the MCAO rats increased significantly (at least P＜0.05 vs.control).However,simultaneous treatment with echinacoside (30.0 or 15.0 mg/kg) attenuated these increases (at least P＜0.05 vs.non-treated model rats).Conclusion These results imply that echinacoside may protect striatal dopa minergic neurons from the injury induced by MCAO and may help prevent and treat cerebral ischemic diseases.     

3 种不同脑区定点注射 6-OHDA 单侧损伤大鼠帕金森病模型的比较研究

[摘要] 目 的 从 神 经 行 为 学、组 织 病 理 和 生 化 方 面 对 3 种 不 同 脑 区 单 侧 定 点 注 射 6- 羟 基 多 巴 胺 ( 6- hydroxydopamine , 6-OHDA )损伤大鼠帕金森病( Parkinsondisease , PD )模型进行比较研究。方法 健康雄性 SD 大 鼠随机分为 3 组( n =10 ):纹状体组、黑质组、黑质 + 中脑腹侧被盖区组。根据脑立体定位图谱,将微量 6-OHDA 单点定位注入大鼠中脑黑质区、纹状体区和双点定位注入黑质区与中脑腹侧被盖区。观察阿朴吗啡诱发大鼠旋转 行为,免疫组织化学检测大鼠黑质区酪氨酸( tyrosinehydroxy-lase , TH + )阳性神经元数目,电化学高效液相色谱法 检测纹状体中多巴胺( dopamine , DA )及其代谢产物 3 , 4- 二羟苯乙酸( dihydroxy-phenylaceticacid , DOPAC )和高香 草酸( homovanillicacid , HVA )含量。结果 纹状体组 3 周后模型稳定,黑质组与黑质 + 中脑腹侧被盖区组大鼠 2 周后稳定, 3 组 30min 内向健侧转圈数均呈升高趋势,黑质 + 中脑腹侧被盖区组高于纹状体组和黑质组,组间· 时点间交互作用差异有统计学意义( P <0.05 )。纹状体组和黑质 + 中脑腹侧被盖区组造模成功率高于黑质组 ( P <0.05 )。 3 组大鼠注射 6-OHDA 损伤侧纹状体中 DA 、 DOPAC 、 HVA 含量和 TH + 神经元数目均明显低于非 损伤侧( P <0.05 )。结论 纹状体单点注射 6-OHDA 损伤建立 PD 模型成功率高,可作为研究 PD 的一种稳定可靠 的动物模型。     

MPTP对小鼠纹状体多巴胺含量的影响

报道1—甲基—4—苯基—1,2,3,6—四氢吡啶(MPTP)给C_(57)-BL黑色小鼠腹腔注射后,应用高效液相色谱—电化学检测法,测定纹状体多巴胺(DA)、去甲肾上腺素(NA)、5—羟色胺(5-HT)及其代谢产物3,4—二羟基苯乙酸(DOPAC)、高香草酸(HVA),5—羟基吲哚乙酸(5-HIAA)的含量。结果与对照组相比,用药组小鼠纹状体DA、5-HT及其代谢产物显著降低,其中DA减少94%(P<0.001),DOPAC减少61%(P<0.05),HVA减少65%(P<0.001)。实验结果表明用MPTP给予小鼠所产生的动物模型,对研究帕金森病是一种较为有用的方法。     

Comparative study of D2 receptors and dopamine content in striatum before and after electro-acupuncture treatment in rats

Objective To evaluate the change in D(2) receptors and their relationship with dopamine (DA) content in experimental hemi-parkinsonism rats before and after electro-acupuncture (EA) treatment. Methods (125) Ⅰ-IBZM D(2) receptor cerebral autoradio-graphic analysis, HPLC-ECD detection of DA and its metabolites, homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC) were used to study their levels in striatum in pre-EA, EA and EA control group. Results The DA, HVA and DOPAC levels in striatum of the lesioned side in the EA group were elevated compared to the pre-EA and EA control group (P&lt;0.05). For the EA group, the striatum/cerebellum (125) Ⅰ-IBZM uptake ratio of the lesioned side was 8.04±0.71, (29.34%±4.83%) more than that of the contralateral side (P&lt;0.05), while no significant difference was observed as compared with that in the pre-EA group (8.09±0.52, 30.12%±4.53%, higher than that of the intact side P&gt;0.05). It was lower than the EA control group (8.61±0.63, P&lt;0.05), and the latter was (38.63%±3.71%) higher than that in its contralateral side (P&lt;0.05). Conclusion 6-OH-DA lesions in the substantia nigra and ventral tegmental areas induce an up-regulation of striatal D(2) binding sites. EA treatment could elevate the DA level of the lesioned side striatum and prevent D(2) receptor up-regulation in rats with experimental hemi-parkinsonism.     

大鼠尾核多巴胺代谢与运动强度的关系

采用在活体内微透析技术，观察大鼠在不同强度运动时及运动后尾核细胞外液中多巴胺（ＤＡ）及其代谢产物３．４一二羟笨乙酸（ＤＯＰＡＣ）和高香草酸（ＨＶＡ）含量的变化。结果表明，运动时尾核细胞外液中以ＤＯＰＡＣ含量增高；运动后细胞外液中ＤＡ、ＤＯＰＡＣ和ＨＶＡ含量均增高，其中ＤＯＰＡＣ和ＮＶＡ的增高程度取决于运动速度。提示尾核多巴胺代谢与运动强度有关。     

染料木黄酮对帕金森病模型小鼠多巴胺能神经元的保护作用

目的 探讨染料木黄酮对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)制备的帕金森病(PD)模型小鼠多巴胺能神经元的保护作用.方法 将C57BL6雄性小鼠随机分为正常对照组、MPTP模型组及染料木黄酮预处理组.用逆转录-聚合酶链式反应(RT-PCR)技术检测黑质酪氨酸羟化酶(TH)及多巴胺转运体(DAT) 基因的表达水平.应用高效液相色谱法检测纹状体(Str)内DA及其代谢物二羟基苯乙酸(DOPAC)含量.结果 MPTP模型组黑质TH和DAT基因的表达较正常对照组明显降低,染料木黄酮预处理组可逆转上述改变(F=20.31、13.19,P<0.01).MPTP模型组Str内DA及其代谢产物DOPAC的含量较对照组明显减少,应用染料木黄酮后可逆转上述改变(F=63.58、16.12,P<0.01).结论 染料木黄酮对MPTP制备的PD模型小鼠黑质纹状体DA能神经元有明显的保护作用.     

PROTECTIVE ACTION OF BUDIPINE AGAINST MPTP NEUROTOXICITY

Mice treated with large dose of MPTP showed a reduction in the levels of DA, 5-HT and their metabolites in the striatum. The average levels as compared with the control were reduced by 94% for DA (P<0.001), 61% for DOPAC (P<0.05), 65% for HVA (P<0.01). 5-HT and 5-HIAA were lower than detection limits. In mice pretreated with budipine, although the striatal dopamine level was also reduced, mean DA and 5-HT levels were significantly higher than those in mice given MPTP alone. This result suggested that budipine can partially prevent the MPTP neurotoxicity.     

实验性溃疡大鼠胃肠道单胺类物质含量的变化

应用高效液相色谱电化学检测法（ＨＰＬＣ－ＥＣＤ）测定正常及ＭＰＴＰ（１甲基２苯基１，２，３，６四氢吡啶）溃疡大鼠胃体、胃窦和十二指肠粘膜组织中单胺类神经递质，包括肾上腺素（Ｅ）、去甲肾上腺素（ＮＥ）、３，４－双羟苯乙酸（ＤＯＰＡＣ）、多巴胺（ＤＡ）、５羟吲哚乙酸（５ＨＩＡＡ）、高香草酸（ＨＶＡ）和５羟色胺（５ＨＴ）含量的变化。正常大鼠胃体、胃窦和十二指肠粘膜含有丰富的ＤＡ和ＤＯＰＡＣ。侧脑室注射ＭＰＴＰ后大鼠胃体、十二指肠粘膜ＤＡ减少并持续较长时间，减少程度同用药时间、剂量平行。胃体ＤＡ减少的同时伴有５ＨＴ减少、十二指肠５ＨＴ升高。提示ＤＡ在溃疡病的发生机理中具有重要作用，５ＨＴ亦参与溃疡形成。     

Rapid determination of dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection

Objective To determine dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection.Methods Microdialysis probes were placed into the right striatum of Wistar rat brains and perfused with Ringer's solution at a rate of 1.5 μL/min.A reverse phase HPLC with electrochemistry was used to assay DA,DOPAC,and HVA after cerebral microdialysates were collected every 20 minutes from awake and freely moving rats.In order to identify the reliability of this method,its selectivity,linear range,precision and accuracy were tested and the contents of DA,DOPAC,and HVA in rat microdialysates were determined.Results The standard curve was in good linear at the concentration ranging from 74 nmol/L to 1.5 μmol/L for DOPAC(r2= 0.9996),from 66 nmol/L to 1.3 μmol/L for DA(r2=1.0000)and from 69 nmol/L to 1.4 μmol/L for HVA(r2=0.9992).The recovery of DOPAC(0.30,0.77,1.49 μmol/L),DA(0.26,0.69,1.32 μmol/L),and HVA(0.27,0.71,1.37 μmol/L)was 82.00±1.70%,104.00±4.00%,98.70±3.10%;92.30±1.50%,105.30±2.30%,108.00±2.00%;80.00±7.80%,107.69±8.00%,and 108.66±3.10%,respectively at each concentration.Their intra-day RSD was 3.3%,3.4%,and 2.5%,and inter-day RSD was 4.2%,2.3%,and 5.6%,respectively.The mean extracellular concentrations of DOPAC,DA,and HVA in rat brain microdialysates were 10.7,2.4,and 9.2 μmol/L(n=6),respectively.Conclusion The findings of our study suggested that the simple,accurate and stable method can be applied to basic researches of diseases related to monoamines neurotransmitters by cerebral microdialysis in rats.     

多巴胺自身受体通过抑制依赖cAMP的磷酸化调控TH活性

应用ＨＰＬＣ－ＥＣＤ法测定大鼠纹状体突触体ＴＨ的活性，研究ＤＡ自身受体介导的ＤＡ生物合成负反馈调控的机理。研究发现，ＡＣ激活剂ＦＳＫ和ＰＫＡ激活剂ｄｂｃＡＭＰ均以浓度依赖的方式激活突触体ＴＨ的活性，增加ｌ－ｄｐｏａ的生成。ＤＡ自身受体激动剂ＬＹ１７１５５５能抑制ＦＳＫ对ＴＨ的激活效应，但不影响ｄｂｃＡＭＰ对ＴＨ的激活。实验结果表明，ＤＡ自身受体介导的负反馈调控的机理是通过抑制依赖ｃＡＭＰ的ＰＫＡ对ＴＨ的磷酸化激活，其作用环节在于抑制ＡＣ，而不是对ＰＫＡ的直接抑制。