In this study we have shown that a commercially produced mouse monoclonal antibody, anti-Hu, which binds to neuronal specific Hu antigens, is a reliable, accurate and effective marker in labelling all myenteric neurons both in fresh fixed and cultured specimens of human colon. As a first step towards the full quantification of all classes of cells, we have characterized three major populations of neurones, defined by the presence of one or both functionally important markers, ChAT and NOS. In addition, the first evidence has been obtained for the existence of a population of enteric neurones that appears to lack both ChAT and NOS and which is likely therefore to be neither cholinergic nor nitrergic.
Anti-Hu antiserum labels neuronal somata, with minimal labelling of nerve fibres, and this enabled cell bodies to be easily distinguished and counted. This is of particular importance in human intestine, where the ganglia contain nerve cells in several layers, even when placed under maximal stretch. Improved accuracy in counting was demonstrated with the use of anti-Hu, by a greater number of nerve cell bodies, being counted, when compared with counting, using other general neuronal markers such as NSE. When compared with similar attempts at quantification of the proportions of cholinergic and nitrergic neurons using NSE12 or PGP 9.513 to label cell bodies, we showed a lower proportion of labelling (52% as compared with 63% or 65% for cholinergic neurons) as a result of increased identification of cell bodies. It is likely that in other studies utilizing NSE to count all neurons33,34 proportions of markers have also been overestimated.
Labelling of nerve cell bodies by anti-Hu was effective in both fresh fixed and cultured tissue, thus expanding its potential applications. Furthermore, it can be readily combined with double and triple labelling immunohistochemistry, thus giving added advantages over techniques requiring serial processing or sectioning.13,22,35 The small neuronal cells bodies identified by anti-Hu in our study were not glial cells as demonstrated by lack of staining for the S100 protein and also by the shape of the cells.36,37 Similarly Lin et al. found no cross reactivity for S100 protein and Hu in guinea pig myenteric ganglia,31 although Phillips et al. found a few cells (<1%), which were labelled with anti-Hu, yet which also demonstrated immunoreactivity for S100.22 Cuprolinic blue is an alternative histochemical method of labelling all enteric neurons, which has been shown to be successful in both small animals,22,24,25 and humans,26 and suggested as an ideal pan-neuronal marker. Recent work has shown that neuronal quantification with cuprolinic blue is comparable with quantification with anti-Hu,26, however, this contrasts with earlier work from Phillips et al., that demonstrated anti-Hu to label more neurons than cuprolinic blue.22
Proportions of markers of human myenteric neurones
Markers for cholinergic and nitrergic neurons were chosen for the initial investigation of proportions of markers, as previous work has revealed that these two populations account for the majority of myenteric neurons in a number of species,3,8,11,12,38 and because of their known importance in neurotransmission. Of relevance to the study of pathological tissue was the observation, that there was very little variation in proportions between the specimens,12,13 so comparison of small numbers of specimens will enable conclusions to be made, regarding changes in neuronal populations.
Cholinergic neurons belong to a number of different functional classes. In the guinea pig ileum, excitatory motor neurones to the longitudinal and circular muscle layers, ascending interneurons, several classes of descending interneurons, primary afferent neurones, some vasomotor and secretomotor neurones have all been shown to be immunoreactive for ChAT and thus are likely to be cholinergic.2,3,39 The polymodal distribution of cell area measurements of cholinergic neurons labelled in the present study reflects the existence of multiple different functional classes within this general immunohistochemical type. Retrograde labelling studies in human colon have shown cholinergic neurons project to the circular muscle, and in ascending and descending interneuronal pathways.10,11,40,41 Markers to discriminate these subclasses of cholinergic neurons will be needed to achieve a full quantitative analysis such as, has been achieved in the guinea pig
In our study, 47% of human colonic myenteric neurons were NOS immunoreactive, a proportion similar to the 43%, found by Porter et al.11 The reason for this small discrepancy may be that NOS was not found in the small neurones, not identified readily by NSE in Porter's study, but counted by us, with use of anti-Hu. Retrograde tracing studies in humans reveal NOS in motor neurones and descending interneurons,11,41 thus, the number of classes of nitrergic neurones, is less than the number of cholinergic classes, as is supported by the absence of a polymodal distribution of cell area measurements for NOS immunoreactive neurones.
We have shown that the proportion of cells to be immunoreactive for NOS appears to be higher in the myenteric plexus of the human colon than in most other preparations studied to date.11,35,42 It is possible that this reflects age related changes. The total number of neurons within the myenteric plexus decreases with age,43,44 however, it has been reported that the proportion of cells immunoreactive for NOS increases in frequency, in the gastrointestinal tract of aged patients, compared with adult controls.45 Phillips et al. when examining cell numbers in rats, found that neuronal loss in aged rats was specific to cholinergic neurons,46 and this may also occur in humans, resulting in a higher proportion of nitrergic neurons. This finding may also reflect functional adaptations of the human colon for accommodation, which are important in its role as a reservoir.47 A greater number of specimens from the right colon in this study may also be of significance, as there are increased numbers of NOS immunoreactive neurons in rat proximal colon compared with distal colon,48 which may possibly also occur in humans. As the majority of NOS−immunoreactive neurons are likely to function as inhibitory motor neurons, it is possible that the high proportion of NOS−immunoreactive motor neurons reflects a functional requirement for powerful inhibitory neuronal input to the muscle layers.
We have confirmed the presence of myenteric neurons that are reactive for both ChAT and NOS, as reported previously in both guinea pigs2,49 and humans.41 These are known to be descending interneurones from retrograde tracing studies41 and the current study reveals these to comprise 4% of all human colonic myenteric neurons. A number of recent studies by Brehmer et al. have characterized human myenteric neurons on the basis of morphology and neurochemical staining.42,50–52 These investigators have characterized small intestine Dogiel type II neurons on the basis of cell morphology, neurochemistry and projections, visible in wholemounts.51 Whilst the authors of this paper suggest these neurons are primary afferent neurons, this can only be proven conclusively, with further investigations, such as retrograde labelling from the mucosa, and electrophysiological experiments. The same group has also characterized neurons immunoreactive for enkelphalin,52 and NOS and VIP, using the same methods, in both small and large intestine,42 however, an exclusive chemical code for a particular functional class has not been previously demonstrated in human colon. Descending interneurons with immunoreactivity for both ChAT and NOS, are the first identifiable exclusive code denoting a class of myenteric neurons in human colon.
A class of myenteric neurons (as revealed by Hu immunoreactivity) totalling 5% of all neurons revealed neither ChAT nor NOS immunoreactivity. To the best of our knowledge, this is the first description of enteric neurones lacking both ChAT and NOS. Cells with this coding were typically very small and are likely to form a discrete population. On the basis of cell size these may be longitudinal muscle motor neurons.53 A variant peripheral form of ChAT, peripheral ChAT has been recently described in the myenteric plexus of a number of experimental animals.54–57 In guinea pigs and sheep,55,57 the common form of ChAT, central ChAT, labels more neurons, than peripheral ChAT, however, it is possible that a small number of cholinergic neurons were not labelled with central ChAT, yet are still cholinergic and may account for some of these neurons without ChAT and NOS imunoreactivity.
To date studies of neuropathology in human gastrointestinal disorders have largely relied upon semi-quantitative assessments of the numbers of nerve cells and fibres.58–60 More recent analyses of nerve cell numbers and proportions of markers have provided better quantification, and revealed changes not detected by prior methods.33,61,62 These studies have relied upon NSE and PGP 9.5 to assess all nerve cell bodies in enteric ganglia. The use of anti-Hu antiserum to readily label such nerve cell bodies, as demonstrated in our study, offers the ability to improve quantification further, thus enabling more subtle detection of changes in neuronal populations.