Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
J Neurosci Methods. 2022 Sep 1;379:109672. doi: 10.1016/j.jneumeth.2022.109672. Epub 2022 Jul 16.
The spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injury (SCI). Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging for SCI researchers.
We present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord.
Corticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading.
The present method does not require special equipment such as vacuum devices, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented.
By using simple neural staining and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord.
中枢神经系统神经元回路的空间复杂性是理解和治疗脑和脊髓损伤 (SCI) 的障碍。尽管最近已经开发出几种方法来使脊髓透明并标记特定的神经回路，但对于 SCI 研究人员来说，具有高分辨率的长脊髓段的三维可视化仍然具有挑战性。
我们提出了一种方法，该方法结合了用生物素化葡聚糖胺 (BDA) 追踪的神经元束的组织染色和改良的被动清晰度清除方案，以描述小鼠脊髓的长段中的单个纤维。
用 BDA 在胸段脊髓损伤的小鼠模型中追踪皮质脊髓束。脊髓用四种溶液染色和清除两周：染色溶液、水凝胶溶液、清除溶液和观察溶液。用光片显微镜观察样本，并使用 ImageJ 软件进行三维重建。获得了与组织切片相当的高分辨率连续和周向图像。通过平铺，可以获得脊髓长段的高分辨率图像。如果出现褪色，可以轻松重新染色组织。
该方法不需要特殊设备，如真空装置，不需要遗传技术即可标记特定的回路，并且可以轻松实现重新染色轮次。
通过使用简单的神经染色和清除方法，可以获得广泛的脊髓高分辨率三维图像。
