疾病小鼠视网膜小胶质动态及突触相互作用的实时成像与表征
January 16th, 2026
本文展示了利用旋转盘共聚焦显微镜观察和量化视网膜中小胶质细胞运动性及与突触后点接触的方法。
我们实验室的研究范围是研究青光眼和小胶质细胞在发育过程中突触重塑中的潜在机制。当前的实验挑战在于小胶质细胞是否主动拆解视网膜神经节细胞突触，还是仅仅被动清除杂质。先将小鼠视网膜放在滤纸上，用实验室擦拭器擦干。
把视网膜倒置到MatTek培养皿上。用金属珠或环压住视网膜。然后，加入三到五滴人工脑脊液。
将样本置于旋转盘共焦显微镜下，调整设置以预览视网膜。沿Z轴采集总深度为10至20微米的图像，步长为0.3微米。将帧间隔保持在20到30秒之间，以便可靠的小区跟踪。
要对小胶质细胞进行表面渲染，请使用文件转换器软件将延时文件转换为IMS格式。根据之前获得的图像属性输入体素尺寸。在分析软件领域，点击观察文件夹打开转换后的文件，确保图像处于三维视图中。
要检测单个小胶质细胞，请点击对象工具栏中的“添加新表面”图标。选择仅切段感兴趣区域，跟踪表面随时间变化，分类曲面，并在算法设置下进行对象-对象统计。然后点击蓝色播放按钮继续。
选择用于表面渲染的通道。可选地，启用 smooth 以平滑表面创建并输入表面细节。在阈值化下，选择机器学习分割来检测细胞。
在训练数据中，使用背景和前景画笔工具。通过选择想要的画笔，使用Shift加左键点击来绘制画笔笔触。确保插值显示已启用，然后选择列车并预测。
用中间的黄色指针在Z轴上导航。在X、Y、Z平面上画多笔，并且跨越几个时间段。每个条目使用三到五次短笔划，反复调整直到形成准确的表面。
用黄色矩形在训练显示和实际生成的表面之间切换，作为最终检查。同时，检查时间流逝时面是否与细胞形态相符。完成后，取消选择拆分对象。
调整体素直方图阈值，去除不属于主表面的小型不连通物体。然后，用剪刀工具手动编辑表面，去除多余的物体或切割边缘。如有需要，设置物体允许的间隙阈值，并选择移动单元的跟踪算法。
过滤追踪与主表面无关联的对象。完成后，将移动单元格显示设置为透明配置文件，以便可视化点，方便下一步操作。要检测单个PSD95点，请点击对象工具栏中的蓝色表面图标。
然后，选择跟踪点，分类点，并统计物体与物体的统计，然后按下播放键。选择突触标记的源通道。关闭切片软件和其他通道，只显示PSD95。
使用控制按钮和指针选择工具来估算点的XY直径。挑几个贯穿帧的明亮点，关闭背景减法。接下来，根据点状质量添加滤镜。
调整直方图，使所有时间段的PSD95点都准确。通过编辑去除假阳性点。在立方体和圆圈光标之间切换，删除只持续一两帧的点。
如有需要，设定允许间隙的阈值，并选择突触点的追踪算法。使用直方图阈值过滤与真实点无关的点轨迹，以排除小物体轨迹。用最短距离定义分类。
被包围时，视为任何小于零微米的点，接触时距离表面约在0至0.5微米之间，未接触时为0.5微米以上。使用上述分类为事件分配标签。一旦表面和斑点恰当地代表小胶质细胞和点，就在统计标签下导出所有统计数据。
与对照组相比，激光诱发眼高血压后小胶质细胞表现出突起位移长度增加。激光治疗眼的微胶质细胞速度显著高于对照组。激光治疗后，小胶质细胞与PSD95点的接触事件数量增加。
延时摄影显示，激光处理视网膜表现出更高的小胶质运动率，且与PSD95点的相互作用增加，相较于对照视网膜。我们的重要发现表明，小鼠在短暂的眼内压升高后，小胶质细胞数量、复杂性和运动能力以及突触共定位均有所增加。我们的方案提供体外成像，有助于避免白内障和角膜不透明度问题，从而简化实验设置和更高效的实验流程和更高的通量。
我们的发现通过利用转基因荧光线实现小胶质细胞-神经元相互作用、细胞通讯和动态的可视化，以及高分辨率延时成像，推动了研究进展。
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Overview
This protocol describes an ex vivo mouse retina explant model to study microglia dynamics and their interactions with synaptic proteins. Using spinning disk confocal microscopy and advanced image analysis, the method enables detailed quantification of microglial motility and synaptic contacts under both homeostatic and injury conditions. The approach is particularly useful for investigating microglia-mediated synaptic pruning in retinal neurodegenerative disease models.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Imaging
Background
- Microglia are the resident macrophages of the CNS, involved in immune responses and synaptic pruning.
- Synaptic pruning by microglia is crucial for neural circuit refinement during development and disease.
- The mechanisms of microglia-mediated synaptic pruning in neuroinflammation and neurodegeneration are not fully understood.
- Retinal explant models allow for controlled ex vivo studies of microglial behavior.
Purpose of Study
- To visualize and quantify microglia motility and their interactions with postsynaptic proteins in the mouse retina.
- To compare microglial dynamics in homeostatic versus injury (laser-induced ocular hypertension) conditions.
- To provide a protocol for high-throughput, high-resolution imaging of microglia-synapse interactions.
Methods Used
- Preparation of mouse retina explants and mounting on filter paper in Petri dishes.
- Labeling of synapses with AAV-PSD95-RFP.
- Time-lapse spinning disk confocal microscopy to capture microglia and synaptic protein dynamics.
- 3D surface and spot reconstruction using image analysis software with machine learning segmentation.
- Quantitative analysis of microglia displacement, process speed, and contact with PSD95 puncta.
Main Results
- Microglia in laser-treated retinas showed increased process displacement length and higher motility compared to controls.
- The number of contact events between microglia and PSD95 puncta increased after laser-induced injury.
- Time-lapse imaging revealed greater microglial complexity and synaptic colocalization following transient intraocular pressure elevation.
- The protocol enabled efficient, high-throughput imaging and analysis, overcoming limitations of in vivo approaches.
Conclusions
- This ex vivo protocol allows detailed visualization and quantification of microglia-synapse interactions in the retina.
- Microglial motility and synaptic contacts are enhanced following neuronal injury.
- The method facilitates studies of microglia-mediated synaptic pruning in neurodegenerative disease models.
Frequently Asked Questions
Executive Industry Relevance
Quantitative live imaging of microglia-synapse interactions in ex vivo murine retina provides a high-throughput, reproducible platform for interrogating neuroimmune mechanisms relevant to neurodegenerative disease. This approach enables precise measurement of microglial motility and synaptic contact dynamics, supporting mechanistic de-risking and target validation in early discovery. The protocol's scalability and imaging fidelity facilitate robust portfolio triage and translational continuity for CNS therapeutic programs.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct visualization of microglia-mediated synaptic remodeling under homeostatic and injury conditions.
- Supports mechanistic de-risking by distinguishing active synapse disassembly from debris clearance.
- Provides quantitative metrics for functional target validation in neuroinflammation and neurodegeneration models.
- Facilitates predictive confidence in pathway interrogation for CNS disease targets.
Screening & Assay Development
- Delivers validated, reproducible imaging outputs for downstream screening workflows.
- Standardizes quantification of microglia motility and synaptic contact events for assay development.
- Enables high-throughput, scalable analysis of cellular interactions using machine learning segmentation.
- Prepares robust biological systems for compound evaluation in neuroimmune modulation studies.
Translational & Preclinical Research
- Aligns ex vivo findings with disease-relevant mechanisms in retinal neurodegeneration models.
- Supports continuity from discovery through preclinical validation by quantifying microglia-synapse dynamics post-injury.
- Informs risk-adjusted advancement decisions for neuroprotective and immunomodulatory strategies.
- Provides translational biomarker candidates based on dynamic cellular interactions.
Pipeline & Workflow Integration
This imaging and analysis protocol integrates from early discovery through preclinical research, bridging mechanistic studies and translational validation in CNS disease models.
- Discovery Biology: Quantifies microglia-synapse interactions to clarify neuroimmune pathways and support hypothesis testing.
- Screening: Establishes reproducible, quantitative imaging assays for evaluating candidate interventions.
- Analytics: Extracts displacement, speed, and contact frequency metrics to enable condition comparison and statistical analysis.
- Translational Research: Connects ex vivo cellular dynamics to disease-relevant endpoints in neurodegeneration.
- Enterprise Reuse: Offers a scalable, adaptable platform for diverse CNS and neuroimmune research pipelines.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neuroimmune target validation.
- Operational Value: Enhances standardization, reproducibility, and throughput in imaging-based assays.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by enabling robust early-stage de-risking.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of CNS therapeutic candidates.
Implementation Considerations
- Requires expertise in confocal imaging, image analysis, and neurobiology.
- Demands access to spinning disk confocal microscopy and advanced analysis software with machine learning segmentation.
- Necessitates cross-team standardization of imaging parameters and data extraction protocols.
- Adaptable to other CNS tissues or disease models with appropriate labeling strategies.
- Throughput and scalability are limited by imaging infrastructure and data processing capacity.
Biopharma FAQs
Chapters in this video
0:00
Introduction
0:24
Time-Lapse Imaging of Mouse Retina for Tracking Analysis of Microglia and Synaptic Puncta
1:14
Microglial Surface Rendering and Tracking Analysis
3:56
Spot Classification for Postsynaptic Density Protein-95 (PSD95) to Detect Synaptic Puncta
5:54
Results
6:35
Conclusion
