Gomez-Frittelli Julieta, Hamnett Ryan, Kaltschmidt Julia A
Department of Chemical Engineering, Stanford University, Stanford, CA 94305 USA.
Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA 94305 USA.
bioRxiv. 2023 Jan 20:2023.01.17.524014. doi: 10.1101/2023.01.17.524014.
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 tract 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 as well as a rod-mounted peeling technique. 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, neuronal nitric oxide synthase (nNOS), or somatostatin (SST) in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased marker labeling for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in ileum 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, demonstrating a critical variability in wholemount muscularis dissection methods.
准确报告沿胃肠道（GI）全长的肠神经系统（ENS）神经元亚型的身份和表现，对于增进我们对ENS控制胃肠道功能的理解至关重要。关于亚型标志物表达比例各异的报告采用了不同的解剖技术来获得肌间神经丛的全层肌层标本。在本研究中，我们探讨了胃肠道解剖方法的差异是否会在标志物表达的定量分析中引入变异性。
我们比较了三种常用的ENS全层解剖方法：两种在显微解剖和固定顺序上不同的平板制备方法以及一种杆装剥离技术。我们使用免疫组织化学、基因报告系、共聚焦显微镜和自动图像分析来评估标志物表达。
我们发现两种平板制备方法在回肠肌间神经丛中钙视网膜蛋白、神经元型一氧化氮合酶（nNOS）或生长抑素（SST）的表达上没有显著差异。然而，杆装剥离方法导致钙视网膜蛋白和nNOS的标志物标记减少。该方法还导致回肠中P物质的转基因报告荧光蛋白（tdTomato）以及回肠和远端结肠中胆碱乙酰转移酶（ChAT）减少。这些结果表明，杆装机械剥离方法会使一些标志物（包括天然蛋白和转基因荧光报告物）的标记减少，这表明全层肌层解剖方法存在关键的变异性。
