Hibberd Timothy J, Yew Wai Ping, Dodds Kelsi N, Xie Zili, Travis Lee, Brookes Simon J, Costa Marcello, Hu Hongzhen, Spencer Nick J
College of Medicine and Public Health, Flinders Health and Medical Research Institute, Flinders University, Adelaide, South Australia, Australia.
Department of Anesthesiology, The Center for the Study of Itch & Sensory Disorders, Washington University School of Medicine, St. Louis, Missouri, USA.
J Comp Neurol. 2022 Dec;530(18):3209-3225. doi: 10.1002/cne.25403. Epub 2022 Aug 31.
Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene-related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5-35% of myenteric neurons. We aimed to quantify proportions of CGRP-immunopositive (CGRP+) myenteric neurons. Colchicine-treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan-neuronal markers (Hu+/Per+) co-labeled 94% of neurons. Hu+/Per- neurons comprised ∼6%, but Hu-/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225 ± 239 per animal, n = 7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329 ± 13 vs. 261 ± 12 μm , p < .0001). CGRP+ neurons comprised 19% ± 3% of myenteric neurons without significant regional variation. NOS+ neurons comprised 42% ± 2% of myenteric neurons overall, representing a lower proportion in proximal colon, compared to mid and distal colon (38% ± 2%, 44% ± 2%, and 44% ± 3%, respectively). Peripherin immunolabeling revealed cell body and axonal morphology in some myenteric neurons. Whether all CGRP+ neurons were multiaxonal could not be addressed using peripherin immunolabeling. However, of 118 putatively multiaxonal neurons first identified based on peripherin immunoreactivity, all were CGRP+ (n = 4). In conclusion, CGRP+ myenteric neurons in mouse colon were comprehensively quantified, occurring within a range expected of a putative IPAN marker. All Per+ multiaxonal neurons, characteristic of Dogiel type II/IPAN morphology, were CGRP+.
生物系统的定量数据为理解病理学、实验扰动和计算建模提供了有价值的基线信息。在小鼠结肠中，降钙素基因相关肽（CGRP）由具有多轴突（Dogiel II型）形态的肌间神经元表达，这是内在初级传入神经元（IPANs）的特征。其他物种和肠道区域中的类似神经元占肌间神经元的5%-35%。我们旨在量化CGRP免疫阳性（CGRP+）肌间神经元的比例。用秋水仙碱处理的近端、中段和远端结肠整装标本用HuC/D、CGRP、一氧化氮合酶（NOS）和外周蛋白（Per）进行标记。泛神经元标记物（Hu+/Per+）共标记了94%的神经元。Hu+/Per-神经元约占6%，但Hu-/Per+细胞很少见。因此，定量基于Hu+肌间神经元（共8576个；每只动物1225±239个，n = 7）。CGRP+细胞体明显大于所有Hu+神经元的平均值（329±13对261±12μm，p <.0001）。CGRP+神经元占肌间神经元的19%±3%，无明显区域差异。NOS+神经元总体上占肌间神经元的42%±2%，与中段和远端结肠相比，在近端结肠中的比例较低（分别为38%±2%、44%±2%和44%±3%）。外周蛋白免疫标记揭示了一些肌间神经元的细胞体和轴突形态。使用外周蛋白免疫标记无法确定所有CGRP+神经元是否都是多轴突的。然而，在最初基于外周蛋白免疫反应性鉴定的118个假定多轴突神经元中，全部都是CGRP+（n = 4）。总之，对小鼠结肠中CGRP+肌间神经元进行了全面定量，其比例在假定的IPAN标记物预期范围内。所有具有Dogiel II型/IPAN形态特征的Per+多轴突神经元都是CGRP+。
