Multiple endocrine neoplasia 2B: Differential increase in enteric nerve subgroups in muscle and mucosa.

Multiple endocrine neoplasia 2B (MEN2B) is a rare syndrome caused by an activating mutation of the RET gene, leading to enteric gangliomatosis. This child presented with constipation at 1-mo old, was diagnosed with MEN2B by rectal biopsy at 4 mo, had thyroidectomy at 9 mo and a colectomy at 4 years. We studied the extent of neuronal and nerve fibre proliferation and which classes of enteric nerves are affected by examining the colon with multiple neuronal antibodies. Resected transverse colon was fixed, frozen, sectioned and processed for fluorescence immunohistochemistry labelling with antibodies against TUJ1, Hu, ChAT, NOS, VIP, SP and CGRP and cKit. Control transverse colon was from the normal margin of Hirschsprung (HSCR) colon (4-year-old) and a child with familial adenomatous polyposis (FAP, 12 year). Myenteric ganglia were increased in size to as wide as the circular muscle. There was a large increase in nerve cells and nerve fibres. ChAT-, NOS-, VIP- and SP-immunoreactive nerve fibres all increased in the myenteric ganglia. NOS-IR nerves preferentially increased in the muscle, while VIP and SP increased in submucosal ganglia and mucosal nerve fibres. The density of ICC was normal. RET overactivation in MEN2B lead to a large increase in intrinsic nerve fibres in the myenteric and submucosal ganglia, with a relative increase in NOS-IR nerve fibres in the circular muscle and VIP and SP in the submucosal ganglia and mucosa. The changes were associated with severe constipation resulting in colectomy at 4 years.

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system.

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.