Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine

Abstract

The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.