Anti‐human Neuronal Protein – A New Tool for Quantification of Neurones in the Human Enteric Nervous System

Abstract

Understanding how the enteric nervous system controls gastrointestinal function requires an account of the different classes of nerve cells present in the gut wall. Accurately quantifying immunohistochemically identified neurones, would make it possible to characterize changes underlying disorders such as slow transit constipation. Until now, quantification of classes of neurones has relied on antisera to Neuron Specific Enolase (NSE) or Protein Gene Product 9.5 (PGP9.5), however, both stain nerve fibres, making accurate counting of cell bodies unreliable. Anti‐Human Neuronal Protein Antibodies (Hu) were originally isolated from patients with paraneoplastic encephalomyelitis and paraneoplastic neuropathies and have been tested here as a marker for all neurons in the human myenteric plexus. Specimens of colon were obtained from the uninvolved margins of specimens from 10 patients undergoing elective surgery for carcinoma and were either fixed fresh, or maintained in organ culture for 3 days. Nerve cell bodies were identified using double labelling techniques to compare Hu (mouse monoclonal anti Hu, Chemicon, CA) and NSE antisera for quantification. In both fresh and cultured tissue, neuronal somata were readily identified using the Hu antibody but few axons or varicosities were labelled. In double‐stained preparations, Hu labelling consistently revealed more nerve cell bodies than NSE. In addition, counting was more reliable due to the lack of staining of nerve fibres which obscured some cell bodies in NSE or PGP9.5‐stained preparations. We used preparations triple labeled with antisera raised against nitric oxide synthase (NOS), choline acetyltransferase (ChAT) and Hu, to determine the size of these major populations. Approximately 47 + 1% (mean + SD, n= 4) of all myenteric nerve cell bodies were immunoreactive for NOS and 52 + 2% were immunoreactive for ChAT. In each preparation, approximately 8 + 2% of NOS cells also contained ChAT. In 5 + 1% of neurons, neither NOS nor ChAT was detectable. Anti‐Hu antiserum makes possible reliable labeling of all enteric neurons in the human colon, allowing accurate quantification of different populations of enteric neurones in normal and diseased tissue.