Three-dimensional imaging of the enteric nervous system in human pediatric colon reveals new features of Hirschsprung disease

Abstract

Hirschsprung disease is defined by the absence of enteric nervous system (ENS) from distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS with re-anastomosis of "normal" bowel near the anal verge. Problems after pull-through are common and some may be due to retained hypoganglionic bowel (i.e., low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable even for experts. Tissue clearing and 3-dimensional imaging provides better data about ENS structure than sectioning. Regions from 11 human colons and one ileal specimen resected during Hirschsprung disease pull-through surgery were cleared, stained with antibodies to visualize ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. Quantitative analyses suggest age-dependent changes in myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret diameter using human colon ranging from 3-days-old to 60-years-old. Neuron counting using 3D-images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500 x 500 μm2 regions (36-fold smaller than in adults). Hirschsprung samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. Tissue clearing and 3-dimensional imaging provides more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. 3-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung disease.