偏侧猴帕金森病模型的脑深部电刺激研究

目的 探讨丘脑底核脑深部电刺激(STN DBS)对纹状体多巴胺代谢的影响.方法 2只偏侧帕金森病(PD)模型猴,在电极植入术前及电刺激后1、3个月分别行单光子放射计算机断层扫描(SPECT)测定脑内多巴胺转运体(DAT)及多巴胺D2受体(D2R)的含量;利用高效液相色谱分析(HPLE-ECD)检测脑脊液中多巴胺及其代谢产物的含量和变化.结果 偏侧PD模型猴在深部脑刺激下症状明显缓解,SPECT显示在有效刺激后纹状体区DAT特异性摄取率增高,D2R特异性摄取率下降.脑脊液中多巴胺及其代谢产物的含量与术前相比无明显差异.结论 STN DBS可有效改善偏侧PD模型猴的症状.SPECT提示有效电刺激STN提高了纹状体区多巴胺代谢活性.     

丘脑底核脑深部电刺激治疗帕金森病的功能显像研究

目的通过单光子放射计算机断层扫描(SPECT)功能显像研究探讨丘脑底核脑深部电刺激(STN DBS)对纹状体多巴胺系统代谢的影响。方法对2只偏侧帕金森病(PD)模型猴及4例临床PD患者在施行单侧STN DBS手术前后给予SPECT检查,测定纹状体区域多巴胺转运体(DAT)及多巴胺D2受体(D2R)含量变化。结果STN DBS电刺激后2只偏侧PD模型猴及3例疗效较好的PD患者纹状体区DAT含量明显增加,2只PD模型猴D2R含量逐渐下降,4例患者D2R检测与术前无统计学意义。结论STN DBS可以明显改善PD症状,SPECT检查显示刺激侧纹状体区DAT含量升高,提示STN DBS可能改善了刺激侧纹状体区多巴胺的代谢,这可能是STN DBS的作用机制之一。     

偏侧帕金森病猴模型的脑血流灌注SPECT活体显象研究

给恒河猴右侧颈内动脉注射神经毒药物甲基-苯基-四氢吡啶(MPTP)后,产生右侧黑质神经元脱失,黑质纹状体多巴胺(DA)浓度明显降低,以及左侧帕金森样症状。投予抗帕金森病(PD)药物治疗显著地改善帕金森样症状。采用单光子发射计算机断层扫描(SPECT),以~(99m)Tc-ECD作为显象剂显示偏侧PD猴模型的脑血流灌注,发现猴模型建立3个月内的损毁侧脑的脑血流灌注明显减低,8个月时的损毁侧脑的脑血流灌注转为正常。结果表明脑血流灌注的变化反映了PD的病理生理变化过程。     

帕金森病功能显像实验研究

目的　应用99mTc TRODAT 1SPECT显像观察偏侧帕金森病 (PD)猴模型两侧纹状体多巴胺转运体 (DAT)密度的变化。方法　 4只恒河猴 ,编号为M1～ 4,M1为正常者 ,M2～M4为右侧颈总动脉注射神经毒素MPTP的PD模型者。静脉注射99mTc TRODAT 1(3 70～ 5 5 0MBq) ,1～ 2小时后进行SPECT断层扫描 ,选取清晰图像进行处理分析。结果　M1两侧纹状体感兴趣区比值为 1 0 0 ,M2～ 4左右侧纹状体感兴趣区比值为 1 10～ 1 2 2 ,而且右侧纹状体与各脑叶感兴趣区的比值均低于左侧。结论　99mTc TRODAT 1SPECT可以在体评估偏侧PD模型猴纹状体DAT的变化 ,有助于定量研究PD的诊断     

立体定向下恒河猴偏侧部分损伤性帕金森病模型的制作

目的　制作一种能保留内侧前脑束 (MFB)内的多巴胺 (DA)神经纤维的偏侧部分损伤性帕金森病(PD)猴模型。方法　以 6 羟多巴 (6 OHDA)溶液对 12只恒河猴右侧黑质致密部行多靶点毁损。术后进行行为学观察、MR、PET、SPECT检查及酪氨酸羟化酶 (TH)的免疫组织化学染色。结果　猴术后符合帕金森病表现。MR示靶点准确位于黑质 ,PET示毁损侧黑质纹状体区代谢减低。TH染色示黑质致密部DA能神经元毁损达 80 %以上 ,而中脑腹侧被盖区和MFB区的DA能神经纤维保留较好。结论　用立体定向注射 6 OHDA毁损一侧黑质致密部的方法可制作出偏侧部分损伤性帕金森病恒河猴模型     

偏侧猴PD模型STN慢性高频电刺激对脑内多巴胺系统的影响

目的探讨丘脑底核(STN)慢性高频电刺激对猴偏侧帕金森病(PD)模型脑内多巴胺(DA)系统的影响。方法采用单侧颈内动脉注入1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)建立猴偏侧PD模型2只,体内植入脑深部电刺激(DBS)系统,行右侧STN慢性高频电刺激。在电刺激前后不同时间点采用微透析技术检测纹状体区细胞外液的多巴胺(DA)及其代谢产物高香草酸(HVA)含量,腰穿取脑脊液标本测量脑脊液中DA、HVA含量,单光子放射计算机断层扫描(SPECT)检测脑内纹状体区巴胺转运体(DAT)及多巴胺D2受体(D2R)的变化。结果猴偏侧PD模型在给予单侧STN慢性高频电刺激后纹状体区DA、HVA明显增高。SPECT显示在有效刺激后纹状体区DAT特异性摄取率增高,D2R特异性摄取率下降。脑脊液中多巴胺及其代谢产物的含量与术前相比无明显差异。结论通过微透析检测提示在给予STN有效慢性高频电刺激后提高了纹状体区DA及其代谢产物的升高,DAT特异性摄取率增高,D2R特异性摄取率下降提示纹状体区的代谢活性有明显升高,这可能是STN-DBS治疗帕金森病的重要机制之一。     

偏侧帕金森病模型猴神经干细胞移植后的初步观察

目的：观察人类神经干细胞移植到偏侧帕金森病灵长类动物模型脑内后动物的行为学和纹状体多巴胺代谢的改变。方法;恒河猴3只,戊巴比妥钠全身麻醉,立体定向下以6-羟多巴溶液行右侧黑质致密部的多靶点毁损。以MRI、PET验证靶点部位,3个月后在右侧尾状核头部,壳核多点移植人类神经干细胞,行为学观察指标包括阿朴吗啡诱发的旋转次数、计步器测量的肢体活动次数,肌张力、震颤,趴伏动作、步态等。以SPECT和PET检验毁损侧纹状体内多巴胺转运蛋白（DAT）含量在移植前后的变化。结果：SPECT、PET检验显示移植后毁损侧黑质、纹状体代谢减低有所恢复,行为学观察显示6-羟多巴损伤对侧肢体活动减少、肌张力增高并有静止性震颤,步态异常,趴伏动作增多,移植后上述症状改善,结论：偏侧部分损伤的帕金森病动物模型在进行尾状核头部,壳核的人类神经干细胞多点移植后,症状有改善,多巴胺代谢有恢复。     

丘脑底核脑深部电刺激治疗帕金森病临床SPECT随访

目的探讨丘脑底核脑深部电刺激(STN DBS)治疗帕金森病(PD)患者症状的改善及单光子放射计算机断层扫描(SPECT)的影像学变化。方法4例施行单侧STN DBS患者术前和给予电刺激后进行帕金森病综合评分(UPDRS)和SPECT测定。结果STN DBS术后临床症状明显改善,UPDRS运动评分缓解60%。3例改善良好的患者SPECT检查提示纹状体区域多巴胺转运体(DAT)含量较术前提高,另1例疗效欠佳的患者DAT含量降低,所有的患者多巴胺D2受体(D2R)检测与术前无明显差异。结论STN DBS可以明显改善PD患者的临床症状,SPECT检查显示刺激侧纹状体区DAT含量的升高提示STN DBS可能改善了多巴胺的代谢,而这种改善可能是STN DBS缓解PD症状的作用机制之一。     

偏侧PD猴模型黑质HMW MAP-2表达的变化

目的探讨偏侧帕金森病（Parkinson＇s disease，PD）猴模型黑质高分子量微管相关蛋白-2（HMW MAP-2）表达的变化。方法对4只恒河猴经单侧颈内动脉注射1-甲基4-苯基1，2，3，6-四氢吡啶（MPTP）制备成偏侧PD猴模型后，应用免疫组化染色方法，观察4只偏侧PD猴模型和2只正常猴黑质HMW MAP-2表达的变化。结果偏侧PD模型猴MPTP毁损侧的黑质的HMW MAP-2免疫活性较毁损侧对侧和正常对照猴明显降低，但MPTP毁损侧对侧的黑质的HMW MAP-2免疫活性未发现明显变化。结论偏侧PD猴模型黑质的HMW MAP-2表达减少可能对黑质神经元的变性和死亡起重要作用。     

偏侧PD猴模型黑质和纹状体GFAP表达的变化

目的探讨偏侧帕金森病（PD）猴模型黑质和纹状体胶质原纤维酸性蛋白（GFAP）表达的变化。方法向3只恒河猴经单侧颈内动脉注射1-甲基4-苯基1，2，3，6-四氢吡啶（MPTP）制备偏侧PD猴模型，应用免疫组化染色方法观察3只偏侧PD猴模型和1只正常猴黑质和纹状体GFAP表达变化。结果偏侧PD模型猴MPTP毁损侧的黑质和纹状体GFAP免疫阳性细胞数目较对侧和正常对照猴明显增加，但MPTP毁损侧对侧的黑质和纹状体GFAP免疫阳性细胞数目未发现明显变化。结论偏侧PD猴模型MPTP毁损侧黑质和纹状体GFAP的表达增加，推测星形胶质细胞可能在黑质和纹状体神经元变性和死亡过程中起一定作用。     

菲立磁标记骨髓基质细胞动脉移植治疗帕金森病的实验研究

目的:探讨应用骨髓基质细胞(BMSCs)经颈动脉移植治疗帕金森病(PD)大鼠的机制及疗效,以及菲立磁(Feridex)标记的BMSCs移植入PD大鼠体内后,MRI示踪观察的可行性。方法:建立PD大鼠模型,体外分离培养扩增SD大鼠BMSCs,从右侧颈动脉移植治疗10只PD大鼠,移植后进行行为学观察和MRI示踪观察。结果:移植治疗的PD大鼠阿扑吗啡诱发旋转实验较对照组明显减少;组织学和MRI示踪发现移植的BMSCs在PD大鼠脑内存活、迁移,并向神经细胞方向分化。结论:BMSCs移植能显著改善PD大鼠的生物学行为,利用MRI技术可以对菲立磁标记的BMSCs进行活体追踪。     

外源性EGFP和TH基因修饰骨髓基质细胞源神经干细胞帕金森病猴模型脑内移植后的跟踪观察

目的评价外源性酪氨酸羟化酶(TH)和增强型绿色荧光蛋白(EGFP)基因修饰的骨髓基质细胞源神经干细胞(BMSCs-D-NSCs)在猴帕金森病(PD)模型脑内移植后长期存活情况及其功能评定。方法应用MPTP制成偏侧PD猴模型。将构建的pEGFP-hTH转染培养的自体BMSCs- D-NSCs,而后植入PD猴模型脑内,观察其行为学变化,并进行单光子发射计算机体层摄影术(多巴胺转运蛋白显像)和正电子发射计算机体层摄影术(葡萄精代谢显像)检查。用免疫组化法检测移植细胞的存活情况。结果成功构建pEGFP-hTH和pEGFP-C2转染的BMSCs-D-NSCs中TH和EGFP基因均有效表达;细胞移植后5个月PD猴症状仍有所改善。多巴胺转运蛋白显像见细胞移植组术侧纹状体平均标准摄取值低于对侧及健康猴,而高于PD猴术侧。葡萄糖代谢显像见细胞移植组术侧纹状体平均标准摄取值低于对侧及健康猴,而高于PD猴术侧,而细胞移植组间明显差异。免疫组化发现, 细胞移植术后5个月移植区仍存存大量TH、EGFP阳性细胞。结论 pEGFP-hTH和pEGFP-C2转染的BMSCs-D-NSCs存体外和体内均长期表达外源性TH、EGFP基因,基因工程细胞在PD猴脑内存活并发挥治疗作用。     

Intracerebroventricular transplanted bone marrow stem cells survive and migrate into the brain of rats with Parkinson’s disease

In this study,6-hydroxydopamine was stereotaxically injected into the right substantia nigra compact and ventral tegmental area of rats to establish Parkinson’s disease models.The rats then received a transplantation of bone marrow stromal cells that were previously isolated,cultured and labeled with 5-bromo-2’-deoxyuridine in vitro.Transplantation of the bone marrow stromal cells significantly de-creased apomorphine-induced rotation time and the escape latency in the Morris water maze test as compared with rats with untreated Parkinson’s disease.Immunohistochemical staining showed that,5-bromo-2’-deoxyuridine-immunoreactive cells were present in the lateral ventricular wall and the choroid plexus 1 day after transplantation.These immunoreactive cells migrated to the surrounding areas of the lateral cerebral ventricle along the corpus callosum.The results indicated that bone marrow stromal cells could migrate to tissues surround the cerebral ventricle via the cerebrospinal fluid circulation and fuse with cells in the brain,thus altering the phenotype of cells or forming neuron-like cells or astrocytes capable of expressing neuron-specific proteins.Taken together,the present findings indicate that bone marrow stromal cells transplanted intracerebroventricularly could survive,migrate and significantly improve the rotational behavior and cognitive function of rats with experimentally induced Parkinson’s disease.     

Intrastriatal transplantation of mouse bone marrow-derived stem cells improves motor behavior in a mouse model of Parkinson's disease

Strategies of cell therapy for the treatment of Parkinson's disease (PD) are focused on replacing damaged neurons with cells to restore or improve function that is impaired due to cell population damage. In our studies, we used mesenchymal stromal cells (MSCs) from mouse bone marrow. Following our novel neuronal differentiation method, we found that the basic cellular phenotype changed to cells with neural morphology that express specific markers including those characteristic for dopaminergic neurons, such as tyrosine hydroxylase (TH). Intrastriatal transplantation of the differentiated MSCs in 6-hydroxydopamine-lesioned mice led to marked reduction in the amphetamine-induced rotations. Immunohistological analysis of the mice brains four months post transplantation, demonstrated that most of the transplanted cells survived in the striatum and expressed TH. Some of the TH positive cells migrated toward the substantia nigra. In conclusion, transplantation of bone marrow derived stem cells differentiated to dopaminergic-like cells, successfully improved behavior in an animal model of PD suggesting an accessible source of cells that may be used for autotransplantation in patient with PD.     

骨髓基质细胞成年大鼠脑内移植

目的 研究骨髓基质细胞脑内移植后的分布和移行，为细胞移植治疗疾病奠定基础。方法 常规培养大鼠骨髓基质细胞，应用免疫组织学方法对细胞进行鉴定，Hoechst33258标记细胞，立体定向移植到大鼠的纹状体，经过一段时间后处死大鼠，脑组织切片，直接在荧光显微镜下检查存活的细胞。结果 细胞移植到大鼠脑内能够长时间存活，移植细胞与宿主细胞有很好的相容性，宿主脑组织的结构无破坏，移植细胞能够移行一段距离，说明脑内存在的信号诱导细胞向一定的方向迁徙。结论 骨髓基质细胞脑内移植后，能够与宿主脑组织整合在一起，无细胞过度增生和胶质瘢痕形成，这种细胞可能成为中枢神经系统自体移植的细胞来源。     

Therapeutic effects of differentiated bone marrow stromal cell transplantation on rat models of Parkinson's disease

This study was to explore curative effect of bone marrow stromal cells (BMSCs) differentiated into nestin-positive cells transplantation on rat Parkinson's disease (PD) model. The PD rats were selected and allocated randomly into three groups. BMSCs with differentiation, BMSCs without differentiation and physiological saline were injected into right striatum of PD rat. The rotation test and immunofluorescence double staining were done. Frequency of rotation was significantly less in differentiated or non-differentiated BMSCs groups than that in normal saline group. Brdu/GFAP- and Brdu/NSE-positive cells appeared except BrdU/TH-positive cells. BMSCs differentiated had better effect than that of BMSCs without differentiated and physiological saline group.     

Therapeutic effects of intranigral transplantation of mesenchymal stem cells in rat models of Parkinson's disease

Stem cell transplantation is a promising tool for the treatment of neurodegenerative disorders, including Parkinson's disease (PD); however, the therapeutic routes and mechanisms of mechanical approaches to stem cell transplantation must be explored. This study tests the therapeutic effect of transplantation of rat bone marrow mesenchymal stem cells (MSCs) into the substantia nigra (SN) of the PD rat. 5‐Bromo‐2‐deoxyuridine‐labeled rat MSCs were transplanted into the SN of the 6‐hydroxydopamine‐injected side of PD rat brains. The behavioral changes in PD rats were examined before and 4 and 8 weeks after MSC transplantation. The expression of tyrosine hydroxylase (TH) in the SN and the striatum and the survival and differentiation of MSCs were assessed by immunohistochemical and double immunofluorescence techniques. Abnormal behavior of PD rats was significantly improved by the administration of bone marrow MSCs, and the number of TH‐positive cells in the SN and the optical density of TH‐positive fibers in the striatum were markedly increased. Transplanted MSCs can survive and migrate in the brain and differentiate into nestin‐, neuron‐specific enolase‐, and GFAP‐positive cells. Our findings suggest that transplantation of rat bone marrow MSCs into the SN of PD rats may provide therapeutic effects. © 2016 Wiley Periodicals, Inc.     

GDNF诱导分化骨髓基质细胞对PD大鼠的治疗作用

目的探讨胶质细胞源性神经营养因子（GDNF）诱导分化骨髓基质细胞（BMSCs）脑内移植对帕金森病（PD）模型的治疗作用。方法体外培养、分离和纯化的BMSCs，用5-溴脱氧尿核苷（BrdU）标记和GDNF诱导分化。分别将经GDNF诱导分化（A组）和未经GDNF诱导分化（B组）的BMSCs移植到PD大鼠模型纹状体区。另设生理盐水对照组（C组）和PD模型对照组（D组）。于不同时间点检测大鼠旋转行为变化，运用免疫荧光组织化学方法分析比较各组纹状体内酪氨酸羟化酶（TH）阳性细胞数量。结果①旋转行为检测显示，C组和D组在所有时间点未见明显变化；在移植后7～30d，A组和B组分别与C组和D组比较，旋转行为明显减少（P〈0．05）；A组比B组旋转行为减少更为显著（P〈0．05）。②免疫荧光组织化学检测显示，A组与B组比较，GFAP和TH阳性细胞数量明显增多（P〈0．05）。但各组自身各时程比较数量变化无统计学意义（P〉0．05）。结论A组的BMSCs脑内移植有效地改善了PD大鼠模型的旋转行为，提高了移植后TH阳性细胞的数量。     

Increased host neuronal survival and motor function in BMT Parkinsonian mice: involvement of immunosuppression

We examined the potential of bone marrow transplantation (BMT) to rescue dopaminergic neurons in a mouse model of Parkinson's disease (PD). A BMT from mice transgenic for green fluorescent protein (GFP(+)) given either before or after administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) led to the accumulation of transplanted adult GFP(+) bone-marrow-derived cells (BMDC) in the substantia nigra, where dopaminergic neurodegeneration occurs in PD. Post-BMT, mice exposed to MPTP had substantially greater numbers of endogenous tyrosine hydroxylase-positive neuronal cell bodies in the substantia nigra and increased dopamine transporter-positive projections into the striatum compared to controls. Moreover, motor function was restored to normal within 1 month post-MPTP in BMT-treated mice assayed by a rotarod behavioral test. The effect of BMT on PD was indirect, as no evidence of BMDC fusion with or transdifferentiation into dopaminergic neurons was observed. BMDC activated by BMT or associated factors could play a trophic role in rescuing damaged cells. Alternatively, the beneficial effects of BMT are due to immunosuppression reflected by a reduction in the proportion of T-cells and a reduction of T-cell proliferation in BMT mice. These findings highlight that when immunosuppression is required for transplantation studies, the amelioration of symptoms may not be due to the transplant itself. Further, they suggest that the immune system plays a role in the development of characteristics typical of PD.     

Protective action of recombinant neurturin on dopaminergic neurons in substantia nigra in a rhesus monkey model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease characterized by muscular trembling palsy due to lack of dopamine (DA) in the substantia nigra-striatum (nigrostriatal) system resulting from the degeneration and necrosis of dopaminergic neurons. No effective cure has been found. Neurturin (NTN) has been demonstrated to act specifically on midbrain (mesencephalic) dopaminergic neurons with protective actions specifically. In the present study, we induced rhesus monkey model of Parkinson's disease by injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Rhesus monkeys were randomly divided into a PD model group, NTN treatment group and normal control groups. In the NTN treatment group, 1 mg of E. coli-derived recombinant human NTN was injected into the cerebral ventricles 48 h before the injection of MPTP. Results indicated that Rhesus monkeys in the PD model group acquired PD symptoms that increasingly aggravated over time, while monkeys treated with NTN had less apparent or no symptoms. Using fluorospectrophotometry, the dopamine (DA), 5, 5-hydroxytrytamine (5-HT) and the 5-hydroxyindoleacetic acid (5-HIAA) contents of DA, 5-HT and 5-HIAA in substantia nigra, putamen and caudate nucleus in monkeys from the model group was found to be significantly lower than in the normal control group. While no significant differences were found between monkeys treated with NTN and normal control groups, the contents of DA, 5-HT and 5-HIAA in the NTN treatment group were higher than those observed in the PD model group. A dramatic loss of neurons in the substantia nigra in monkeys in the PD model group was observed by light microscopy, while no obvious loss was observed in the NTN treatment group in which the numbers of neurons were similar to those in normal controls. These results indicate that recombinant human NTN can prevent PD symptoms as well as protect dopaminergic neurons and preserve DA content in midbrain substantia nigra in rhesus monkeys exposed to MPTP.