Reduced number of intrinsic pulmonary nitrergic neurons in Fawn-Hooded rats as compared to control rat strains.

The Fawn-Hooded rat (FHR) strain reveals a congenital predisposition to primary (idiopathic) pulmonary hypertension (PPH), and can therefore be regarded as an animal model in which to study possible mechanisms underlying an inherited susceptibility to pulmonary hypertension. Pulmonary hypertension can be induced in FHRs after a short exposure to mild hypoxia, presumably because of an altered peripheral oxygen sensitivity. Given the presence of pulmonary nitrergic neurons in rat lungs, the observed link between airway hypoxia and the expression of pulmonary neuronal nitric oxide synthase (nNOS), and the fact that nNOS appears to be involved in peripheral chemoreceptor sensitivity, we examined the intrinsic pulmonary nitrergic innervation in the FHR. In the present study the number of intrapulmonary nitrergic nerve cell bodies, detected by NADPH diaphorase (NADPHd) histochemistry, was quantified in the FHR and three control rat strains. Compared to the control rat strains, the FHR lungs revealed a highly significantly lower number of intrinsic nitrergic neurons, while no apparent differences were found in the number of enteric nitrergic neurons in the esophagus. In conclusion, the possible links between neuronal NO, hypersensitivity to airway hypoxia, and the development of PPH clearly deserve further investigation.

Distribution, immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens.

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.

The longitudinal smooth muscle layer of the pig small intestine is innervated by both myenteric and submucous neurons.

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.

Neuroepithelial bodies: a morphologic substrate for the link between neuronal nitric oxide and sensitivity to airway hypoxia?

Currently, the significance of nitric oxide (NO) in the respiratory tract is a matter of great interest because NO is believed to play a major role in the physiological regulation of airway function but also in lung pathology. What is especially intriguing with respect to the present investigation, are reports that the pulmonary expression of neuronal NO synthase (nNOS) is altered as a result of airway hypoxia. We examined the possible relationship between intrapulmonary nitrergic structures and pulmonary neuroepithelial bodies (NEBs), chemoreceptor-like epithelial cell groups that are known to have all necessary components for oxygen perception. Tyramide-enhanced immunostaining for nNOS was combined with known markers for NEBs in an ontogenetic study of rat lungs. From postnatal day 2 onward, nNOS-immunoreactive (-IR) neuronal cell bodies, present mainly in the lamina propria at all levels of intrapulmonary airways, were seen to give rise to remarkable intraepithelial terminal arborizations that invariably colocalized with NEBs. nNOS immunoreactivity was absent from the vagal calbindin D28k(CB) -IR and the spinal calcitonin gene-related peptide(CGRP) -IR extrinsic sensory nerve fiber populations that our group reported earlier to selectively contact NEBs. Quantitative analysis showed that all NEBs receiving nNOS-IR terminals were also contacted by CGRP-IR nerve fibers, whereas approximately 55% were additionally contacted by CB-IR nerves. The reported nitrergic neurons did not express the cholinergic marker vesicular acetylcholine transporter and were always surrounded by a basket of CGRP-IR nerve terminals. In conclusion, part of the pulmonary NEBs selectively receive extensive nitrergic nerve terminals that originate from intrinsic neurons. Together with literature data on lung physiology and pharmacology, some interesting suggestions for the functional significance of the association between pulmonary CGRP-IR NEBs, nNOS-IR neurons, and CGRP-IR afferents described in the present study, are discussed.

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.