Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine

Abstract

An important requirement in pathological diagnostics in the human enteric nervous system (ENS) is the estimation of the total numbers of neurons and of proportions of distinct subpopulations. In this study, we compared the suitability of two suggested panneuronal markers, cuprolinic blue (CB) and anti-Hu-protein (HU), for staining and counting human myenteric neurons in wholemounts, derived from small and large intestinal samples. Furthermore, the proportional expression of three cytoskeletal intermediate filaments, a-internexin (IN), neurofilament 200 (NF) and peripherin (PE), was correlated with both CB and HU. In 8 CB- and HUstained wholemounts, 93.3% of all neurons were double labeled, 3.3% of neurons were stained only with CB whereas 3.3% were immuno-stained only for HU. Thus, both markers were comparably reliable in representing the putative total human myenteric neuron population in our material. The wholemounts double stained for IN/CB or IN/HU revealed between 56.2 and 71.5% of neurons to be IN-reactive. Between 42.8 and 50.9% of neurons were immunoreactive for NF whereas 53.9 to 62.4% of neurons were reactive for PE. Although our sample number was too small to allow final conclusions, we suggest that the variations in proportions of intermediate filament expression we observed may be due to individual circumstances rather than to correlation with age or region. The proportions of neurons positive for IN, NF or PE but unstained by CB histochemical or HU immunohistochemical techniques was between 0 and 2.2%. We conclude that both CB and HU techniques are suitable methods for representation of almost all myenteric neurons in the human gut and that the differential expression of the cytoskeletal proteins investigated has to be included in the classification of enteric neurons in pathological diagnostics of human gastrointestinal diseases.