1单侧损毁6 - 羟基多巴胺帕金森病小鼠模型的发展
Summary
一个6-OHDA单侧病变，内侧前脑束对小鼠进行的协议说明。该方法具有低死亡率与89％的幸存的动物> 95％的损失纹状体多巴胺和90.63率（13.3％）±-4.02％ipsiversive旋转对病灶侧的偏见。
Abstract
单方面损毁6 hyroxydopamine（6 – 羟基多巴胺），损毁帕金森病大鼠模型（PD）的已证实到是在推进我们基本巴金森氏症症状的机制的理解非常宝贵的，因为它概括在基底神经节电路的变化和药理学观察中帕金森患者1-4。然而，在纹状体内的输出途径，这是基底节的主要输入地区的皮质纹状体突触发生的确切的细胞和分子的变化仍然是难以捉摸的，这被认为是网站，在这里出现潜在的帕金森病症状的病理异常5。
在PD，了解基底节电路以下尼格罗 – 纹状体通路变性的变化机制已大大提前过度表达绿色荧光蛋白，通过促进推动发展由细菌人工染色体（BAC）的小鼠RS具体纹状体两个输出途径（EGFP-D1的直接途径，间接的途径：EGFP-D2和EGFP-A2A）让他们在隔离研究。例如，最近的研究表明，有在突触可塑性的病理变化在帕金森病小鼠9,10。然而，这些研究利用幼年小鼠和急性帕金森模型。目前还不清楚是否与稳定的6 – 羟多巴胺损毁大鼠中所描述的变化也发生在这些模型中。其他团体都试图产生一个稳定的单方面的损毁6-OHDA成年PD小鼠模型，损毁内侧前脑束（MFB），不幸的是，在这项研究中的死亡率是非常高，只有14％存活21手术天或更长时间11。最近的研究已产生内黑质病变与低死亡率> 80％的多巴胺能神经元的损失，但表达左旋多巴引起的运动障碍11,12,13,14变量在这些研究中。另一个PD小鼠模型MPTP的损伤小鼠15。虽然这种模式已被证明在评估潜在神经保护剂16有用的，它是不适合理解机制帕金森病的症状，这种模式往往未能引起运动障碍，在18 17病变程度，显示了一个很大的可变性。
在这里，我们已经开发了6-OHDA直接管理到稳定单方面的PD小鼠模型6-OHDA损毁MFB的，一贯导致95％的损失纹状体多巴胺（高效液相色谱法测定），以及生产的行为，不平衡的特点以及单方面的6-OHDA损毁PD大鼠模型中观察到。这个新开发的PD小鼠模型，将被证明是有价值的工具，在了解帕金森病症状的生成机制。
Protocol
1。住房和小鼠的制备这些老鼠是纯FVB小鼠，保持驱动的转基因小鼠8（突变小鼠区域资源中心（MMRRC）的FVB在12:12 h光照黑暗免费获得食物和水的循环细菌人工染色体（BAC）的殖民地。越过这些小鼠与任何其他应变是没有必要的，无论是繁殖的目的，或6-OHDA损伤过程，以确保成功。 为了产生帕金森病模型，出生后一天31-42岁的成年小鼠（P31-42）6-OHDA损伤和假手术的需要。 <…
Discussion
本协议描述了一个稳定的单方面6-OHDA损毁的帕金森氏症，这是非常重复性高病变的成功率，低死亡率，小鼠模型的生成方法。 6-OHDA损伤手术的成功，可以很容易地估计> 70％> 95％，在27的损毁纹状体多巴胺耗竭的指标ipsiversive旋转自发旋转行为的测量。纹状体多巴胺水平的量化纹状体多巴胺耗竭的程度相比，在黑质致密部TH阳性细胞数量quantificiation是最精确的测量，因为它提供了直接?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的外交事务和国际贸易（加拿大政府），多伦多大学，加拿大创新，NSERC，Krembil的基金会和治疗帕金森氏信托基金会诺基金。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| desipramine HCl | Sigma-Aldrich, Oakville, ON, Canada | D125 | 25mg/kg |
| pargyline HCl | Sigma-Aldrich, Oakville, ON, Canada | P8013 | 5mg/kg |
| 6-OHDA HBr | Sigma-Aldrich, Oakville, ON, Canada | H116 | 3mg / mouse |
| stereotaxic Frame | Kopf Instruments, Tujunga, CA, USA | Model 900 | |
| mouse ear cups | Kopf Instruments, Tujunga, CA, USA | Model 921 Zygoma Ear Cups | |
| mouse incisor bar | Kopf Instruments, Tujunga, CA, USA | Model 923B | |
| mouse anaesthesia mask | Kopf Instruments, Tujunga, CA, USA | Model 923B | |
| priming kit (containing 250ml syringe) | Hamilton Company, Reno, NV, USA | PRMKIT 81120 | |
| RN compression fitting kit (1 mm) | Hamilton Company, Reno, NV, USA | 55750-01 | |
| PEEK tubing from RN compression fitting kit< (1/16th inch) | Hamilton Company, Reno, NV, USA | 55751-01 | |
| dual small hub RN Coupler | Hamilton Company, Reno, NV, USA | 55752-01 | |
| luer to small hub RN adaptor | Hamilton Company, Reno, NV, USA | 55753-01 | |
| 1ml 25S syringe model 7001KH | Hamilton Company, Reno, NV, USA | 80100 | |
| *33G removable needle (RN) pack of 6. . Custom 1 inch with 45<° bevel | Hamilton Company, Reno, NV, USA | 7803-05 | |
| Scissors | Fine Science Tools, Vancouver, BC, Canada. | 14084-08 | |
| Scalpel | Fine Science Tools, Vancouver, BC, Canada | 10003-12 | |
| Scalpel blades | Fine Science Tools, Vancouver, BC, Canada | 10035-20 | |
| Forcep | Fine Science Tools, Vancouver, BC, Canada | 11608-15 | |
| Hemostats | Fine Science Tools, Vancouver, BC, Canada. | 13004-14 | |
| Isoflurane | Abbot | 02241315 | 2-3% |
| Suters (Vicryl 4.0) | Syneture | SS-683 | |
| Steriliser | Fine Science Tools, Vancouver, BC, Canada | 18000-45 | |
| Infusion Pump | Harvard Apparatus | PhD 22/2000 | |
| Needles (27G) | Becton Dickinson | 305109 | |
| Needles (25G) | Becton Dickinson | 305127 | |
| Syringes (1ml) | BD syringe | 309692 | |
| Anaesthesia trolley | LEI medical | M2000 | |
| Baytril | CDMV, St. hyacinthe, QC | 102207 | |
| Lidocaine | CDMV, St. hyacinthe, QC | 3914 | |
| Betadine solution | CDMV, St. hyacinthe, QC | 19955 |
Riferimenti
- Costall, B., Naylor, R. J., Pycock, C. Non-specific supersensitivity of striatal dopamine receptors after 6-hydroxydopamine lesion of the nigrostriatal pathway. Eur. J. Pharmacol. 35, 276-283 (1976).
- Maneuf, Y. P., Mitchell, I. J., Crossman, A. R., Brotchie, J. M. On the role of enkephalin cotransmission in the GABAergic striatal efferents to the globus pallidus. Exp. Neurol. 125, 65-71 (1994).
- Robertson, G. S., Robertson, H. A. Evidence that L-dopa-induced rotational behavior is dependent on both striatal and nigral mechanisms. J. Neurosci. 9, 3326-3331 (1989).
- Ungerstedt, U., Arbuthnott, G. W. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res. 24, 485-493 (1970).
- Brotchie, J. M. Novel approaches to the symptomatic treatment of parkinsonian syndromes: alternatives and adjuncts to dopamine-replacement. Curr. Opin. Neurol. 10, 340-345 (1997).
- Besson, M. J., Graybiel, A. M., Nastuk, M. A. [3H]SCH 23390 binding to D1 dopamine receptors in the basal ganglia of the cat and primate: delineation of striosomal compartments and pallidal and nigral subdivisions. Neuroscienze. 26, 101-119 (1988).
- Gerfen, C. R. D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science. 250, 1429-1432 (1990).
- Schiffmann, S. N., Jacobs, O., Vanderhaeghen, J. J. Striatal restricted adenosine A2 receptor (RDC8) is expressed by enkephalin but not by substance P neurons: an in situ hybridization histochemistry study. J. Neurochem. 57, 1062-1067 (1991).
- Hutchison, W. D. Differential neuronal activity in segments of globus pallidus in Parkinson’s disease patients. Neuroreport. 5, 1533-1537 (1994).
- Pan, H. S., Penney, J. B., Young, A. B. Gamma-aminobutyric acid and benzodiazepine receptor changes induced by unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. J. Neurochem. 45, 1396-1404 (1985).
- Pan, H. S., Walters, J. R. Unilateral lesion of the nigrostriatal pathway decreases the firing rate and alters the firing pattern of globus pallidus neurons in the rat. Synapse. 2, 650-656 (1988).
- Gong, S. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature. 425, 917-925 (2003).
- Kreitzer, A. C., Malenka, R. C. Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson’s disease models. Nature. 445, 643-647 (2007).
- Shen, W., Flajolet, M., Greengard, P., Surmeier, D. J. Dichotomous dopaminergic control of striatal synaptic plasticity. Science. 321, 848-851 (2008).
- Lundblad, M., Picconi, B., Lindgren, H., Cenci, M. A. A model of L-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice: relation to motor and cellular parameters of nigrostriatal function. Neurobiol. Dis. 16, 110-123 (2004).
- Grealish, S., Mattsson, B., Draxler, P., Bjorklund, A. Characterisation of behavioural and neurodegenerative changes induced by intranigral 6-hydroxydopamine lesions in a mouse model of Parkinson’s disease. Eur. J. Neurosci. 31, 2266-2278 (2010).
- Dauer, W., Przedborski, S. Parkinson’s disease: mechanisms and models. Neuron. 39, 889-909 (2003).
- Francardo, V. Impact of the lesion procedure on the profiles of motor impairment and molecular responsiveness to L-DOPA in the 6-hydroxydopamine mouse model of Parkinson’s disease. Neurobiol. Dis. 42, 327-340 (2011).
- Jakowec, M. W., Petzinger, G. M. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned model of parkinson’s disease, with emphasis on mice and nonhuman primates. Comp. Med. 54, 497-513 (2004).
- Visanji, N. P., Brotchie, J. M. MPTP-Induced Models of Parkinson’s Disease in Mice and Non-Human Primates. Curr. Protoc. Pharmacol. Chapter 5, 42-42 (2005).
- Sedelis, M., Schwarting, R. K., Huston, J. P. Behavioral phenotyping of the MPTP mouse model of Parkinson’s disease. Behav. Brain Res. 125, 109-125 (2001).
- Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuropharmacology. 39, 777-787 (2000).
- Paul, M. L., Currie, R. W., Robertson, H. A. Priming of a D1 dopamine receptor behavioural response is dissociated from striatal immediate-early gene activity. Neuroscienze. 66, 347-359 (1995).
- Breese, G. R., Traylor, T. D. Effect of 6-hydroxydopamine on brain norepinephrine and dopamine evidence for selective degeneration of catecholamine neurons. J. Pharmacol Exp. Ther. 174, 413-420 (1970).
- Breese, G. R., Chase, T. N., Kopin, I. J. Metabolism of tyramine-3H and octopamine-3H by rat brain. Biochem. Pharmacol. 18, 863-869 (1969).
- Frankin, K. B. J., Paxinos, G. . The Mouse Brain in Stereotaxic Coordinates. , (2008).
- Cornell-Bell, A. H., Finkbeiner, S. M., Cooper, M. S., Smith, S. J. Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. Science. 247, 470-473 (1990).
- Iancu, R., Mohapel, P., Brundin, P., Paul, G. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson’s disease in mice. Behav. Brain Res. 162, 1-10 (2005).
- Tan, Y., Williams, E. A., Lancia, A. J., Zahm, D. S. On the altered expression of tyrosine hydroxylase and calbindin-D 28kD immunoreactivities and viability of neurons in the ventral tegmental area of Tsai following injections of 6-hydroxydopamine in the medial forebrain bundle in the rat. Brain Res. 869, 56-68 (2000).
- Thiele, S. L. Generation of a model of L-DOPA-induced dyskinesia in two different mouse strains. J. Neurosci. Methods. 197, 193-208 (2011).
- Henry, B., Crossman, A. R., Brotchie, J. M. Characterization of a rodent model in which to investigate the molecular and cellular mechanisms underlying the pathophysiology of L-dopa-induced dyskinesia. Adv. Neurol. 78, 53-61 (1998).
Tags
Unilaterally-lesioned 6-OHDA Mouse ModelParkinson’s DiseaseBasal Ganglia CircuitryParkinsonian SymptomsCortico-striatal SynapsesStriatumPathological AbnormalitiesNigro-striatal PathwayBacterial Artificial Chromosome (BAC) MiceGreen Fluorescent ProteinsDirect PathwayIndirect PathwaySynaptic PlasticityJuvenile MiceAcute Models Of Parkinsonism
Citazione di questo articolo
Thiele, S. L., Warre, R., Nash, J. E. Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson’s Disease. J. Vis. Exp. (60), e3234, doi:10.3791/3234 (2012).