Reduced glial cell line-derived neurotrophic factor level in aganglionic bowel in Hirschsprung's disease

Glial cell line-derived neurotrophic factor (GDNF) is suggested to be essential for the development of the enteric nervous system. The aim of this study was to investigate GDNF protein expression in human aganglionic (AG) bowel in Hirschsprung's disease (HD) using immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis, to further understand the pathophysiology of HD.

Colonic specimens were obtained from eight patients with HD (aged 7 days to 14 months) at the time of definite pull-through surgery, including both normoganglionic (NG) and aganglionic (AG) segments. Immunofluorescence was performed using anti-GDNF polyclonal antibody and FITC-conjugated secondary antibody on the formalin-fixed and paraffin-embedded specimens. For ELISA analysis, specimens were homogenized by adding 10 volumes of 10-mmol/L phosphate-buffered saline containing proteinase inhibitors. Centrifuged supernatant was used for the quantitative analysis using sandwich-type ELISA for human GDNF.

There was strong GDNF immunoreactivity in the mucosal epithelium, submucosal and myenteric plexuses, and hypertrophic nerve trunks. In the mucosa in AG bowel, the number of GDNF immunoreactive epithelial cells was significantly reduced compared with NG bowel (mean +/- SD, AG/NG = 227.2 +/- 73.1/310.8 +/- 80.5 GDNF immunoreactive cells per 100 crypts, P < .05). Using ELISA, the level of GDNF in full-thickness bowel was reduced significantly in AG bowel compared with NG bowel (mean +/- SD, AG/NG = 860.2 +/- 309.8/1777.5 +/- 271.4 pg/g wet tissue, P < .001).

Because GDNF is essential for the development of the enteric nervous system, our findings of reduced level of GDNF in AG bowel as well as reduced number of GDNF immunoreactive cells in the mucosa of AG bowel suggest that GDNF may play an important role in the pathogenesis of HD.