Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
Neurogastroenterol Motil. 2022 Feb;34(2):e14292. doi: 10.1111/nmo.14292. Epub 2021 Dec 4.
A fundamental understanding of the enteric nervous system in normal and diseased states is limited by the lack of standard measures of total enteric neuron number. The adult zebrafish is a useful model in this context as it is amenable to in toto imaging of the intestine. We leveraged this to develop a technique to image and quantify all enteric neurons within the adult zebrafish intestine and applied this method to assess the relationship between intestinal length and total enteric neuron number.
Dissected adult zebrafish intestines were immunostained in wholemount, optically cleared with refractive index-matched solution, and then imaged in tiles using light-sheet microscopy. Imaging software was used to stitch the tiles, and the full image underwent automated cell counting. Total enteric neuron number was assessed in relation to intestinal length using linear regression modeling.
Whole gut imaging of the adult zebrafish intestine permits the visualization of endogenous and immunohistochemistry-derived fluorescence throughout the intestine. While enteric neuron distribution is heterogeneous between intestinal segments, total enteric neuron number positively correlates with intestinal length.
CONCLUSIONS & INFERENCES: Imaging of all enteric neurons within the adult vertebrate intestine is possible in models such as the zebrafish. In this study, we apply this to demonstrate a positive correlation between enteric neuron number and intestinal length. Quantifying total enteric numbers will facilitate future studies of enteric neuropathies and ENS structure in animal models and potentially in biopsied tissue samples.
由于缺乏衡量肠道神经元总数的标准方法，人们对正常和患病状态下的肠神经系统的基本认识受到了限制。在这方面，成年斑马鱼是一种有用的模型，因为它可以对肠道进行整体成像。我们利用这一优势，开发了一种对成年斑马鱼肠道内所有肠神经元进行成像和定量的技术，并应用该方法评估了肠道长度与肠神经元总数之间的关系。
对成年斑马鱼的肠道进行解剖后，进行全组织免疫染色，然后用折射率匹配溶液进行光学透明处理，再用光片显微镜进行瓷砖成像。成像软件用于拼接瓷砖，对全图像进行自动细胞计数。使用线性回归模型评估肠道长度与肠神经元总数之间的关系。
成年斑马鱼肠道的全肠道成像可以在肠道内可视化内源性和免疫组织化学衍生荧光。虽然肠神经元在肠段之间的分布不均匀，但肠神经元总数与肠道长度呈正相关。
在斑马鱼等模型中，可以对成年脊椎动物肠道内的所有肠神经元进行成像。在这项研究中，我们应用这一方法证明了肠神经元数量与肠道长度之间存在正相关关系。量化肠神经元总数将有助于未来在动物模型中以及潜在地在活检组织样本中研究肠神经病变和 ENS 结构。
