神经肌肉疾病小鼠模型行为花型的低成本盖特分析
Summary
足迹分析是数字化步态分析程序的低成本替代方案,用于研究人员量化小鼠的运动异常。由于其速度、简单性和纵向潜力,它是小鼠模型行为型型的理想选择。
Abstract
动物运动测量是一种常见的行为工具,用于描述特定疾病、损伤或药物模型的表型。这里所展示的低成本步态分析方法是一种简单但有效的测量鼠步态异常的方法。通过用无毒的可洗漆涂一个老鼠的脚,让受试者在一张纸上穿过隧道来分析脚印。测试隧道的设计利用了自然小鼠行为及其对小黑暗场所的亲和力。使用尺子和铅笔可以轻松测量每只鼠标的步长、步幅宽度和脚趾分布。这是一种成熟且可靠的方法,它生成多个类似于数字系统的指标。这种方法足够灵敏,可以检测表型表示的早期步幅变化,并且由于其非侵入性方法,它允许在寿命或表型表示中测试组。
Introduction
运动需要复杂的神经和肌肉骨骼协调,在运动路径的单一方面出现缺陷可以产生可观察到的步态异常1,2。盖特分析是研究人员测试小鼠模型的关键工具,因为它提供了可量化的行为数据,了解特定疾病、损伤或药物如何影响动物的运动3。然而,数字化步态分析需要购买跑步机、照相机和相关软件,这对研究人员来说可能非常昂贵。盖特分析经常间歇性地用于跟踪电机功能的纵向变化,因此,如果偶尔使用4,则很难证明支出的合理性。尽管数字化分析可能比简单的足迹分析提供更详细的步态指标,但这些更复杂的度量并不总是必要的,也并不总是与行为表型5的表征相关。
在这里,我们提出了一个低成本的手动足迹分析方法,作为一个快速和敏感的替代数字化步态分析程序6,7。人工足迹分析已被证明,以检测在众多的鼠病模型4,7,8,9,10,11的显著步态差异 ,12,13,14,15,16,17,和至少一个情况下, 这种低成本的方法识别步态的变化,没有检测到一个常见的数字化步态分析程序12。材料的总成本是象征性的,它可以很容易地适应其他啮齿动物研究模型。
虽然可以从中绘制数据,但我们描述的方法侧重于三个特定的指标:步长、步长宽度(又名”轨道宽度”)和脚趾分布。必须指出,应逐个模型确定要评估的参数。这种步态分析方法不是用来测量认知功能的,也不推荐用于需要复杂的步态生物力学测量的研究。
我们提供来自一组症状前和症状后小鼠的行为数据,这些小鼠模拟X链接脊柱和布尔巴肌肉萎缩症(SBMA),这是一种神经肌肉疾病,其特征是运动神经元退化和肌肉萎缩。这些小鼠在步态中出现渐进性缺陷,与其他疾病特异性表型的发病一致。这证明了这种方法的有效性和特异性,并证实它可以可靠地区分受影响的和未受影响的动物。
本研究中的实验鼠为2.5岁(症状前)和9个月大(症状后)BAC fxAR121转基因小鼠,背景为C57BL/6(nexpt=12)。该模型在我们的实验室生成,并已被完全描述为SBMA9的强大鼠标模型。非转基因杂物用作对照(nctrl#8)。SBMA是一种性别限制疾病,只在雄性中完全显现出来,因此雄性小鼠被专门用于这项研究。在规划阶段,研究人员必须考虑到国家卫生研究院对性别作为生物变量的考虑,以确定群体的大小和组成18。
Protocol
Representative Results
Discussion
采用上述低成本步态分析方法,在SBMA的BAC fxAR121小鼠模型中,成功地识别了症状后年龄的步态功能障碍的几个参数。步幅长度的减小与先前SBMA对小鼠模型和人类患者的研究一致。我们还首次表明,与非转基因的杂物对照相比,有症状的SBMA小鼠的后肢脚趾扩散存在显著差异。有趣的是,后脚趾扩张的减少可能是由爪子扩张肌肉的虚弱、爪子屈肌的紧绷或神经内侧不良2,19引起,这也与SBMA的…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢A.M.提供的动物鉴定援助。这项工作得到了美国国家卫生研究院(R01 7 RF1 AG057264 至 A.R.L.S.和 C.J.C.和 R01 NS100023 到 A.R.L.S) 和肌肉萎缩协会(基础研究补助金给 A.R.L.S.S.,发展赠款给 C.J.C.S.)的资助。
Materials
| Caliper | n/a | n/a | must have markings down to 0.1 mm |
| Craft Glue | E6000 | n/a | |
| Footprint Paint (Tempera Paint) | Artmind | n/a | must be non-toxic |
| Round Barrel Paintbrushes | Symply Simmons | n/a | 0.5 cm diameter |
| Ruler | n/a | n/a | must have markings down to millimeters |
| Scoring Paper (Watercolor Pads) | Canson | n/a | cut to size |
| Tunnel and Goal Chamber | Interstate Plastics | n/a | cut to size |
Referências
- Clarke, K. A., Still, J. Development and consistency of gait in the mouse. Physiology & Behavior. 73 (1-2), 159-164 (2001).
- Mendes, C. S., et al. Quantification of gait parameters in freely walking rodents. BMC Biology. 13, 50 (2015).
- Carter, R. J., Morton, J., Dunnett, S. B. Motor coordination and balance in rodents. Current Protocols in Neuroscience. , (2001).
- Tillerson, J. L., Caudle, W. M., Reveron, M. E., Miller, G. W. Exercise induces behavioral recovery and attenuates neurochemical deficits in rodent models of Parkinson’s disease. Neurociência. 119 (3), 899-911 (2003).
- Pallier, P. N., Drew, C. J., Morton, A. J. The detection and measurement of locomotor deficits in a transgenic mouse model of Huntington’s disease are task- and protocol-dependent: influence of non-motor factors on locomotor function. Brain Research Bulletin. 78 (6), 347-355 (2009).
- Sugimoto, H., Kawakami, K. Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress. Journal of Visualized Experiments. (143), (2019).
- Carter, R. J., et al. Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation. Journal of Neuroscience. 19 (8), 3248-3257 (1999).
- Barlow, C., et al. Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell. 86 (1), 159-171 (1996).
- Cortes, C. J., et al. Muscle expression of mutant androgen receptor accounts for systemic and motor neuron disease phenotypes in spinal and bulbar muscular atrophy. Neuron. 82 (2), 295-307 (2014).
- D’Hooge, R., et al. Neuromotor alterations and cerebellar deficits in aged arylsulfatase A-deficient transgenic mice. Neuroscience Letters. 273 (2), 93-96 (1999).
- Fernagut, P. O., Diguet, E., Labattu, B., Tison, F. A simple method to measure stride length as an index of nigrostriatal dysfunction in mice. Journal of Neuroscience Methods. 113 (2), 123-130 (2002).
- Guillot, T. S., Asress, S. A., Richardson, J. R., Glass, J. D., Miller, G. W. Treadmill gait analysis does not detect motor deficits in animal models of Parkinson’s disease or amyotrophic lateral sclerosis. Journal of Motor Behavior. 40 (6), 568-577 (2008).
- Harper, S. Q., et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proceedings of the National Academy of Sciences of the United States of America. 102 (16), 5820-5825 (2005).
- Lin, C. H., et al. Neurological abnormalities in a knock-in mouse model of Huntington’s disease. Human Molecular Genetics. 10 (2), 137-144 (2001).
- Sopher, B. L., et al. Androgen receptor YAC transgenic mice recapitulate SBMA motor neuronopathy and implicate VEGF164 in the motor neuron degeneration. Neuron. 41 (5), 687-699 (2004).
- Tillerson, J. L., Caudle, W. M., Reveron, M. E., Miller, G. W. Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Experimental Neurology. 178 (1), 80-90 (2002).
- Wheeler, V. C., et al. Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice. Human Molecular Genetics. 11 (6), 633-640 (2002).
- Clayton, J. A., Collins, F. S. Policy: NIH to balance sex in cell and animal studies. Nature. 509 (7500), 282-283 (2014).
- Maricelli, J. W., Lu, Q. L., Lin, D. C., Rodgers, B. D. Trendelenburg-Like Gait, Instability and Altered Step Patterns in a Mouse Model for Limb Girdle Muscular Dystrophy 2i. PLoS One. 11 (9), e0161984 (2016).
- Castelhano-Carlos, M. J., Sousa, N., Ohl, F., Baumans, V. Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation. Lab Animals. 44 (2), 88-103 (2010).
- Lakes, E. H., Allen, K. D. Gait analysis methods for rodent models of arthritic disorders: reviews and recommendations. Osteoarthritis Cartilage. 24 (11), 1837-1849 (2016).
