Background & Aims

Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions.

Methods

Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions.

Results

We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm². Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm³ with 38,706 ± 5763 neuron/mm³ and 129,321 ± 25,356 glia/mm³. Images of large areas provided insight into why published values of ENS density vary up to 150-fold—ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions.

Conclusions

We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

中文翻译：

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无需组织切片的人结肠肠神经系统的鲁棒，三维可视化。

背景与目标

通常用于人体病理分析的二维小切片提供了有关肠神经支配的有限信息。我们开发了一种在3个维度上成像人类肠神经系统（ENS）和其他壁内细胞的技术。

方法

我们使用小鼠和人类结肠组织，开发了一种结合组织清除，免疫组织化学，共聚焦显微镜和全厚度肠的定量分析的方法，无需切片就可以在3维上定量ENS和其他壁内细胞。

结果

我们从没有已知肠蠕动障碍的人群中提供了280个成年人结肠共聚焦Z堆栈。我们的大多数图像是使用20倍物镜捕获的肌间神经节。用10倍物镜观察的全层结肠图像大至4×5 mm ²。使用来自2名患Hirschsprung病的儿科患者的结肠显示没有结肠神经节的远端结肠，以及存在ENS的过渡区和近端穿刺切除切缘。用我们的方法测试了一组抗体后，我们确定了16种与神经元，神经胶质，Cajal间质细胞和肌巨噬细胞中的分子结合的抗体。定量分析表明，在扁平结肠Z堆栈区域的24.5％±2.4％中，有肌层神经丛。肌神经节占肌神经丛的34％±4％。单个肌层神经节体积平均为3,527,678±573,832 mm ³，其中神经元为38,706±5763 / mm ³和129,321±25,356 glia / mm ³。大面积的图像可以帮助您了解为何ENS密度的公布值变化高达150倍-ENS密度在毫米范围内变化很大，因此，对来自同一肠道区域的少量薄片进行分析可以得出不同的结果。神经元亚型分析显示，约56％的神经元一氧化氮合酶抗体染色的肌层神经元和约33％的神经元产生并储存乙酰胆碱。Hirschsprung病患者结肠组织的过渡区区域神经节呈多层，神经纤维束粗而无神经元。粘膜下神经元分布在成像的结肠区域之间变化。

结论

我们开发了一种用于结肠的三维成像方法，该方法可提供比组织切片更多的有关ENS结构的信息。这种方法可以改善对人类肠蠕动障碍的诊断，并且对于其他肠疾病也可能有用。