UCLA/Digestive Diseases Research Core Center, Vatche and Tamar Manoukian Digestive Diseases Division, Department of Medicine, UCLA David Geffen School of Medicine, Los Angeles, CA, USA.
VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA.
Neurogastroenterol Motil. 2021 Apr;33(4):e14030. doi: 10.1111/nmo.14030. Epub 2020 Nov 10.
We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus.
We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5.
In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer.
The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.
我们之前报道了一种新型抗人外周胆碱乙酰转移酶（hpChAT）抗血清的特异性，该抗血清可用于免疫染色人类结肠中的胆碱能神经元细胞体和纤维。在这项研究中，我们研究了人类乙状结肠固有胆碱能神经支配的 3D 结构及其与肠神经丛中氮能神经元的关系。
我们开发了一种改良的 CLARITY 组织技术，适用于清除人类乙状结肠标本，并使用 hpChAT 抗血清进行免疫染色，以及与神经元一氧化氮合酶（nNOS）抗体进行共标记。使用 Imaris 9.2 和 9.5 对 Z 堆叠共聚焦图像进行 3D 重建/分割/数字追踪和计算定量。
在黏膜中，由 hpChAT-ir 纤维和少量神经元细胞体组成的局部微神经元网络被数字化组装。3 层黏膜下神经丛的 3D 结构得以显示，它们通过 hpChAT-ir 纤维束相互连接，并且 hpChAT-ir 神经元很少与 nNOS 共标记。在肌间神经丛中，30.1%的 hpChAT-ir 体包括 Dogiel 型 I 和 II 与 nNOS 共标记，并且在 3D 图像和视频中可以观察到 3 类 hpChAT-ir 神经纤维束。3D 结构中 hpChAT-ir 纤维的密度和强度值在环形层中明显高于纵形层。
首次在 3D 微观结构中逐层展示了人类乙状结肠的固有胆碱能神经支配。这可能为评估结肠疾病中的结构-功能关系和病理改变开辟新途径。
