Improved understanding of the pathophysiology of Hirschsprung Disease (HD) may allow development of surgical and non-surgical approaches. Whilst abnormal motility in HD is associated with Enteric Nervous System defects, non-neuronal Interstitial Cells (ICs) identified as positive for cKit or platelet derived growth factor receptor alpha (PDGFRa) are also implicated. However, the contribution of these IC types to HD pathology is unclear, typically due to different methods in tissue processing between studies. Having optimised our methodology, this project aims to allow clarification of the contribution of IC subtypes to HD pathology, and by comparing IC in the presence and absence of ganglia, to allow further clarification of IC biology.
Methods and results
Resected bowel specimens from 7 patients with HD were compared to tissue matched samples from stoma closures (n=10) using immunofluorescence. Ten minute immersion fixation in acetic ethanol allowed rapid tissue processing and robust immunostaining revealed by staining intensity and number of visible cells in comparison to formalin and zinc fixation.
ICs were reliably labelled and differentiated with antibodies to the ion channels Ano-1 (c-kit) and SK3 (PDGFRa) Imaris and Image J software based quantification revealed IC is significantly reduced,expressed as mean ± standard-deviation cells/mm3,in the distal section of the colon (4.7±1) in comparison to the proximal region(85.9±3). Reduction in protein was mirrored by significant reductions in mRNA for cKit (P 1.56±0.16,D 0.45±0.092),ANo-1(P 1.77±0.14,D 0.32± 0.03), PDGFRα(P 1.33±0.14,D 0.48± 0.4)and SK3 (P 1.86± 0.4,D 0.33±0.35).
the study IC can be applied to compare IC distribution at the proximal and distal margins from HD. Extension of these approaches will enhance our understanding of HD pathology.