An immunohistochemical study of the distribution of nitric oxide synthase-immunoreactive neurons and fibers in the reticular groove of suckling lambs

Abstract

The reticular groove (RG) is a specialized region of ruminant forestomach which, in suckling animals, via a vagovagal reflex, transforms itself into a tube to ensure the direct transport of milk from the esophagus to the abomasum. The nervous mechanism controlling the RG movement is not fully understood; however, at this level, the enteric nervous system (ENS) shows the highest neuronal density when compared with other forestomach compartments. Because nitric oxide is considered the putative major mediator of non-adrenergic non-cholinergic smooth muscle relaxation, the aim of the present study was to investigate the ENS of the RG of suckling lambs, both in the floor and in the lip, with particular regard to nitric oxide synthase (NOS)-immunoreactivity (-IR), by means of double immunohistochemical staining. NOS antiserum was used in association with some neurochemical markers which have been utilized by many authors in ENS. A rich innervation of fibers extended along the entire length of the RG. Proceeding distally, the number of neurons stained with a pan-neuronal marker increased; they were more numerous in the lips and lip-floor junction than in the floor itself. However, the percentage of NOS-IR neurons was the same in the proximal and distal parts. Many NOS-IR neurons often co-expressed galanin and dopamine β-hydroxylase. Neurochemical markers, such as calbindin, calcitonin gene-related peptide, IB4 and neurofilament 200 kDa, usually used to identify primary sensory neurons were not expressed in RG neurons, and the co-localization of NOS with tyrosine hydroxylase and substance P was rarely found. When compared with other districts, the RG showed some peculiar aspects, such as the lack of both neurons in the submucosal plexus and the lack of typical sensory neurons.