Launay Pierre-Serge, Godefroy David, Khabou Hanen, Rostene William, Sahel Jose-Alain, Baudouin Christophe, Melik Parsadaniantz Stéphane, Reaux-Le Goazigo Annabelle
INSERM, U968, Paris, F-75012, France; Sorbonne Universités, Université UPMC, Paris 06, UM 80, Institut de la Vision, 75012, Paris, France; CNRS, UMR 7210, Paris, F-75012, France.
INSERM, U968, Paris, F-75012, France; Sorbonne Universités, Université UPMC, Paris 06, UM 80, Institut de la Vision, 75012, Paris, France; CNRS, UMR 7210, Paris, F-75012, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, F-75012, France.
Exp Eye Res. 2015 Oct;139:136-43. doi: 10.1016/j.exer.2015.06.008. Epub 2015 Jun 11.
Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.
组织透明化以及随后对完整透明组织进行成像，为分析神经系统中的解剖通路提供了一种创新方法。在本研究中，我们将最近的溶剂清除器官三维成像（3DISCO）程序、光片显微镜、荧光逆行示踪剂和Imaris软件相结合，对成年小鼠整个三叉神经节（TG）内的角膜感觉神经元进行三维映射。我们首先建立了轻松快速清除固定TG的优化步骤。我们发现3DISCO程序效果极佳，清除TG所需时间不到3小时。在第二组实验中，将逆行示踪剂（霍乱毒素B Alexa 594偶联物）应用于去上皮的角膜，以逆行标记角膜感觉神经元。两天后，通过3DISCO方法清除TG，并使用光片超显微技术进行连续成像。从三维重建的整个小鼠TG中可以轻松快速地获得标记神经元的高分辨率图像。然后，我们对角膜传入神经元进行了三维重建，并分析了它们在TG中的精确定位。因此，我们表明，提供角膜感觉神经支配的神经元在TG内表现出高度特异性的有限背内侧定位。我们报告说，我们的联合方法提供了在清除的小鼠TG中对标记细胞进行手动（在20μm切片上）和自动（在三维重建的TG上）计数的可能性。总之，我们证明3DISCO清除方法与光片显微镜、逆行示踪剂和自动计数相结合，是一种分析外周和中枢神经系统内神经元亚群的快速可靠方法。
