K. S. Ng1,2, D. Mahns3, M. A. Gladman1,2 1Sydney Medical School – Concord, University Of Sydney,Academic Colorectal Unit,Sydney, NSW, Australia 2ANZAC Research Institute, University Of Sydney,Enteric Neuroscience & Gastrointestinal Research Group,Sydney, NSW, Australia 3University Of Western Sydney,Department Of Integrative Physiology, School Of Medicine,Sydney, NSW, Australia
Introduction:
Recent appreciation of enteropathies characterised by impaired gut motility secondary to aberrations of intrinsic nerve structure and/or function (GI neuromuscular disorders) has only served to highlight gross inadequacies in our understanding of the human enteric nervous system (ENS). Indeed, most of our current knowledge is derived from animal studies that have used inaccurate techniques such as unpaired tissue sections rather than ‘gold-standard’ paired wholemount preparations. Therefore, this study aimed to quantitatively investigate and neurochemically code the myenteric plexus of the human hindgut using paired wholemount samples of colon and rectum.
Methods:
Paired samples of human colon and rectum were procured from anterior resection specimens. The tissues were pinned flat in both relaxed and stretched states and fixed in Zamboni’s fixative. Wholemounts of colonic and rectal myenteric plexi were prepared by dissecting the mucosa, submucosa, and circular muscle off the longitudinal muscle. Myenteric neuronal immunostaining was performed using anti-Hu, anti-NOS, and anti-ChAT primary antibodies. These antibodies were secondarily labelled with fluorescent dyes. Wholemount images (approximately 100mm2) were acquired using an epifluorescence microscope equipped with a motorised stage to allow accurate assessment of ganglionic density, average ganglionic size, ganglionic area density, and neuronal density. ‘Stretch-corrected’ values were obtained taking account of tissue dimensions in the stretched and relaxed state. Data from paired colonic and rectal tissues were compared using the Wilcoxon signed-rank test.
Results:
Overall, 12 paired samples of colon and rectum were studied, all of which produced high quality immunostains permitting detailed analysis. Ganglia and individual neurons were readily identified and counted, and the fluorescent dyes discriminated according to their spectral wavelengths. Stretch-corrected ganglionic densities were similar between colon and rectum (colon: median 564 ganglia/100mm2 [range 386–921], rectum: 581 [360–923]; P=0.70), as were average ganglionic sizes (colon: 57,047μm2 [42,350–90,363], rectum: 52,021 [38,701–90,210], P=0.43). Whilst ganglionic area density tended to be lower in the rectum (colon: 11.96 mm2 per 100mm2 [7.53–18.64], rectum: 9.76 [5.80–17.19], P=0.12), there was no overall difference in stretch-corrected neuronal densities (colon: 176.3 neurons/mm2 [107.4–357.3], rectum: 174.7/mm2 [94.7–313.3], P=0.58).
Conclusion:
This is the first study to use paired samples of human gut tissue and apply wholemount immunostaining techniques whilst accounting for tissue stretch to quantitatively assess and neurochemically code the myenteric plexus of the human hindgut. This has allowed the development of the first robust normative data set to advance our current understanding of the intrinsic innervation of the human gut.