小鼠肌肉和末端施旺细胞线粒体形态的 原位 定量方法
2026年4月10日
在 体内 对施旺细胞线粒体末端进行局部注射，用于共聚焦显微镜。该方法利用健康或病变小鼠骨骼肌组织中展示的概念验证，提供高分辨率线粒体可视化。
我们的研究首次研究了神经肌肉交接处末端施旺细胞的线粒体形态。我们的方法能够量化毛细血管和淋巴床等难以获得的组织中的线粒体形态。首先，先获得一只野生型和一只D2-mdx小鼠。
通过脚趾夹击反射确认麻醉深度后，将针头插入右膝腱远端。在胫骨肌内侧（TA）注射50微升两微摩尔远红荧光线粒体染色剂，同时沿注射路径抽出针头，并在体内培养一小时。在麻醉状态下维持动物时，使用剪刀和产钳在安乐死前解剖并分离TA肌肉。
将分离的TA肌肉置于室温下4%的对甲醛中15分钟。然后，在立体显微镜下，通过轻微的轻微分离纤维。将细胞渗透核酸染色剂加入4%的对甲醛，最终浓度为每毫升5至10微克，用于核标记。
然后用PBS洗三次TA肌肉，每次五分钟，以去除多余染料。将肌肉依次用50%75%和100%乙醇脱水，各五分钟。然后，将样本依次浸泡在组织清除液中，分别一号和二号，各30分钟，确保完全浸没。
把肌肉放在盖板上。用一块2.5乘2×1厘米、重7.8克的玻璃块轻轻压平。购买一只S100 beta鼠标。
通过脚趾夹击反射确认麻醉深度后，在臀大肌（GM）上方做一个小皮肤切口。在GM肌肉下方注射75微升两微摩尔远红荧光线粒体染色剂。闭合伤口，让染料培养45分钟，以选择性标记末端施旺细胞。
在麻醉状态下保持动物的同时，10分钟内解剖转基因肌肉，并立即放入冷藏的0.9%无菌生理盐水中。仅在使用D2-mdx小鼠时，将荧光乙酰胆碱受体染料以1比500稀释度加入显微分离浴，并培养组织30分钟。修剪多余脂肪和结缔组织，将GM肌肉转移到腹侧朝下的盖膜上，以便倒置显微镜检查。
在GM背侧表面加入两微升通用装载介质，并在成像前用玻璃块将组织压平。在软件中，将激发波长设置为646纳米。在滴入两滴清除溶液后，选择63倍物镜拍摄TA肌，并将Z叠层深度设为接近100微米。
然后选择反卷积模式，应用系统优化参数。将步长设置为一微米，然后获取图像。要获得末端施旺细胞的图像，选择20倍放大的物镜，将Z堆栈深度设为约17微米，然后获取图像。
然后，选择63倍放大的物镜，拍摄单个末端施旺细胞的图像。导入单个Z切片以训练软件识别线粒体信号。并利用AI驱动的软件进行定量分析。
开发供调查员审查的图像掩码。在算法优化后，将三维图像堆栈分离为单独的光学部分。每从每张3D图像中选取每五个Z切片，计算每个样本的平均值。
标准化所有样本的成像参数，并量化线粒体形态。进行免疫组化和统计分析，评估群体差异和相关性。与野生型小鼠相比，mdx肌肉的线粒体连通性降低。
在mdx小鼠中观察到较多的小型线粒体片段和较少的大型互联线粒体网络。在野生型小鼠中，末端施旺细胞中的线粒体在分岔处表现出相互连接的环状结构。相比之下，在mdx小鼠中，线粒体在分岔处出现碎片化且缺乏环状结构。
共焦点显微镜和电子显微镜确认了末端施旺细胞中类似的甜甜圈形线粒体结构。突触后分析显示，亚肉管线粒体聚集在乙酰胆碱受体周围，缺乏典型的椒盐卷饼状结构。图像分析显示，mdx肌肉中的末端施旺细胞线粒体面积和碎片化程度均较野生型肌肉增加，颗粒大小无差异。
肌肉力测量显示，mdx肌肉的肌萎缩减少，变异性更大，峰值力量下降。随着刺激频率增加，MDX肌肉的力产产生也显著减少。MDX肌肉的纤维尺寸向左偏移，表明肌纤维比野生型小约60%。
该方案使研究人员能够研究和量化骨骼肌及末端施旺细胞中的线粒体形态。未来的研究可以将这一方法调整为研究微循环和淋巴管中的线粒体形态。
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Overview
This protocol presents a reproducible method for labeling and quantifying three-dimensional mitochondrial network morphology in whole-mount skeletal muscle and terminal Schwann cells (tSCs) in mice. Utilizing in vivo delivery of a membrane potential–sensitive mitochondrial dye and standard confocal microscopy, the approach enables detailed analysis of mitochondrial architecture within intact neuromuscular tissues, overcoming challenges associated with tissue disruption and anatomical complexity.
Key Study Components
Area of Science
- Cellular and molecular neuroscience
- Muscle biology
- Imaging and microscopy
Background
- Mitochondrial network morphology is a key indicator of cellular health.
- Quantifying mitochondrial architecture in intact tissues is technically challenging.
- Terminal Schwann cells (tSCs) are critical for neuromuscular junction maintenance and regeneration.
- tSCs are difficult to analyze in situ due to their location and sensitivity to tissue disruption.
Purpose of Study
- To develop a reproducible protocol for labeling and quantifying mitochondrial morphology in whole-mount skeletal muscle and tSCs.
- To enable analysis of mitochondrial networks in situ without the need for transgenic reporters or specialized imaging platforms.
- To validate the method in both healthy and dystrophic muscle tissue.
Methods Used
- In vivo delivery of a membrane potential–sensitive mitochondrial dye.
- Rapid tissue processing to preserve mitochondrial structure.
- High-resolution confocal microscopy for three-dimensional imaging.
- Image stack analysis to quantify mitochondrial network connectivity, area, and fragmentation.
Main Results
- The protocol successfully labels and quantifies mitochondrial networks in whole-mount skeletal muscle and tSCs.
- Validation in dystrophic and healthy muscle confirms expected differences in mitochondrial morphology.
- The method is adaptable to tSCs using S100β reporter mice.
- Analysis can be performed using equipment commonly available in university core facilities.
Conclusions
- This approach enables detailed analysis of mitochondrial morphology in intact neuromuscular tissues.
- It eliminates the need for transgenic mitochondrial reporters or specialized imaging platforms.
- The protocol is adaptable to other thin or superficially accessible tissues.
Frequently Asked Questions
Executive Industry Relevance
Quantitative in situ analysis of mitochondrial morphology in muscle and terminal Schwann cells addresses a critical gap in early discovery and target validation for neuromuscular and metabolic disease research. This protocol enables high-content, reproducible assessment of mitochondrial network integrity within intact tissues, supporting predictive confidence in disease-relevant models and facilitating risk-adjusted portfolio decisions. Its compatibility with standard imaging infrastructure enhances accessibility and scalability across R&D teams.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of mitochondrial network health in physiologically relevant tissue environments.
- Supports functional target validation by quantifying network connectivity, area, and fragmentation in disease and control models.
- Facilitates mechanistic de-risking by distinguishing mitochondrial phenotypes in healthy versus dystrophic muscle.
- Provides quantitative endpoints for hypothesis-driven pathway analysis.
Screening & Assay Development
- Establishes validated imaging and quantification workflows for mitochondrial morphology in whole-mount tissues.
- Delivers reproducible, quantitative outputs suitable for assay standardization and cross-study comparison.
- Enables screening of interventions affecting mitochondrial dynamics in accessible tissue systems.
- Supports platform reuse across multiple tissue types without reliance on transgenic reporters.
Translational & Preclinical Research
- Aligns mitochondrial morphology endpoints with disease-relevant tissue contexts for translational biomarker development.
- Maintains continuity from discovery through preclinical validation by enabling analysis in both muscle and Schwann cells.
- Provides risk-adjusted data to inform advancement of mitochondrial-targeted therapeutics.
- Facilitates adaptation to additional thin or superficially accessible tissues for broader translational impact.
Pipeline & Workflow Integration
This method integrates into the discovery-to-preclinical continuum by enabling robust mitochondrial morphology quantification in intact tissues, supporting both early target validation and downstream translational research.
- Discovery Biology: Supports hypothesis testing and mechanistic de-risking through direct measurement of mitochondrial network features.
- Screening: Provides standardized, quantitative imaging outputs for assay development and compound evaluation.
- Analytics: Delivers network connectivity, area, and fragmentation metrics for comparative analysis across experimental conditions.
- Translational Research: Bridges discovery and preclinical phases by enabling biomarker alignment in disease-relevant tissues.
- Enterprise Reuse: Offers a broadly adaptable workflow compatible with standard confocal microscopy platforms.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in mitochondrial research.
- Operational Value: Promotes reproducibility and scalability using widely available imaging infrastructure.
- Strategic Value: Enhances go/no-go decision-making and capital efficiency by providing robust, quantitative endpoints.
- Portfolio Impact: Supports risk-adjusted prioritization of mitochondrial-targeted programs across disease areas.
Implementation Considerations
- Requires expertise in confocal microscopy and quantitative image analysis.
- Utilizes standard membrane potential–sensitive dyes and imaging platforms common to core facilities.
- Demands cross-team standardization of tissue processing and imaging protocols for reproducibility.
- Adaptable to other thin or superficially accessible tissues with minimal protocol modification.
- Dependent on tissue accessibility and dye compatibility for optimal results.
