Department of Chemical Engineering, Stanford University, Stanford, California, USA.
Wu Tsai Neurosciences Institute, Stanford University, Stanford, California, USA.
Neurogastroenterol Motil. 2024 Jan;36(1):e14693. doi: 10.1111/nmo.14693. Epub 2023 Oct 26.
Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression.
We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis.
We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.
准确报告沿胃肠道（GI）长度的肠神经系统（ENS）神经元亚型的身份和代表性对于深入了解 ENS 对 GI 功能的控制至关重要。关于亚型标志物表达的不同比例的报告采用了不同的解剖技术来实现肌间神经丛的全肠系膜准备。在这项研究中，我们询问了 GI 解剖方法的差异是否会导致标志物表达的定量存在差异。
我们比较了 ENS 全肠系膜解剖的三种常用方法：两种平面片准备方法，其在微解剖和固定的顺序上有所不同，以及第三种棒状剥皮技术。我们还测试了平面片剥皮的反向取向变化、棒状剥皮技术的两个逐步变化以及作为管的全肠固定。我们使用免疫组织化学、遗传报告基因系、共聚焦显微镜和自动图像分析来评估标志物表达。
我们发现，在回肠肌间神经丛中，两种平面片准备方法在 calretinin 或神经元一氧化氮合酶（nNOS）作为总神经元比例的表达方面没有显著差异。然而，棒状剥皮方法导致 calretinin 和 nNOS 标记的神经元比例降低。该方法还导致远结肠中物质 P 的转基因报告荧光蛋白（tdTomato）和回肠和远结肠中胆碱乙酰转移酶（ChAT）的荧光蛋白减少。这些结果表明，一些标志物的标记，包括天然蛋白和转基因荧光报告，都被棒状剥皮的机械方法降低了。该方法的逐步变化指向组织的机械处理可能是标记减少的原因。因此，我们的研究证明了全肠系膜解剖方法存在显著的可变性。
