Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.     

Pulmonary intraepithelial vagal nodose afferent nerve terminals are confined to neuroepithelial bodies: an anterograde tracing and confocal microscopy study in adult rats

Our present understanding of the morphology of neuroepithelial bodies (NEBs) in mammalian lungs is comprehensive. Several hypotheses have been put forward regarding their function but none has been proven conclusively. Microscopic data on the innervation that appears to affect the reaction of NEBs to stimuli have given rise to conflicting interpretations. The aim of this study has been to check the validity of the hypothesis that pulmonary NEBs receive an extensive vagal sensory innervation. The fluorescent neuronal tracer DiI was injected into the vagal sensory nodose ganglion and NEBs were visualized in toto by using immunocytochemistry and confocal microscopy on 100-μm-thick frozen sections of the lungs of adult rats. The most striking finding was the extensive intraepithelial terminal arborizations of DiI-labelled vagal afferents in intrapulmonary airways, apparently always co-appearing with calcitonin gene-related peptide (CGRP)-immunoreactive NEBs. Not all NEBs received a traced nerve fibre. Intrapulmonary CGRP-containing nerve fibres, including those innervating NEBs, always appeared to belong to a nerve fibre population different from the DiI-traced fibres and hence did not arise from the nodose ganglion. Therefore, at least some of the pulmonary NEBs in adult rats are supplied with sensory nerve fibres that originate from the vagal nodose ganglion and form beaded ramifications between the NEB cells, thus providing support for the hypothesis of a receptor function for NEBs. Received: 13 November 1997 / Accepted: 17 February 1998     

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X₂ and P2X₃ ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.     

Morphology of the apudocytes and neuroepithelial bodies in the lungs of rats

Endocrine cells (apudocytes) and neuroepithelial bodies (NEBs) were revealed in the lungs of rats of different ages by Grimelius' argyrophilic method. Solitary apudocytes were found among the bronchial epitheliocytes, they had the oval, columnar or triangular shape. NEBs comprise groups of argyrophilic cells, in some cases the penetration of neural fibers into the bodies and their branching in the terminals are observed. Apudocytes and NEBs in 1-, 7-, and 15-day-old rats are more numerous in the epithelium of small than big bronchi. In 21- and 30-day-old and in adult animals apudocytes and NEBs are very scarce. The elements under study are likely to take part in the early postnatal development of the rat lungs.     

Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species

Pulmonary neuroepithelial bodies (NEBs) are extensively innervated organoid groups of neuroendocrine cells that lie in the epithelium of intrapulmonary airways. Our present understanding of the morphology of NEBs is comprehensive, but direct physiological studies have so far been challenging because the extremely diffuse distribution of NEBs makes them inaccessible in vivo and because a reliable in vitro model is lacking. Our aim has been to optimise an in vitro method based on vibratome slices of living lungs, a model that includes NEBs, the surrounding tissues and at least part of their complex innervation. This in vitro model offers satisfactory access to pulmonary NEBs, provided that they can be differentiated from other tissue elements. The model was first optimised for living rat lung slices. Neutral red staining, reported to stain rabbit NEBs, proved unsuccessful in rat slices. On the other hand, the styryl pyridinium dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), showed brightly fluorescent cell groups, reminiscent of NEBs, in the airway epithelium of living lung slices from rat. In addition, nerve fibres innervating the NEBs were labelled. The reliable and specific labelling of pulmonary NEBs by 4-Di-2-ASP was corroborated by immunostaining for protein gene-product 9.5. Live cell imaging and propidium iodide staining further established the acceptable viability of 4-Di-2-ASP-labelled NEB cells in lung slices, even over long periods. Importantly, the in vitro model and 4-Di-2-ASP staining procedure for pulmonary NEBs appeared to be equally reproducible in mouse, hamster and rabbit lungs. Diverse immunocytochemical procedures could be applied to the lung slices providing an opportunity to combine physiological and functional morphological studies. Such an integrated approach offers additional possibilities for elucidating the function(s) of pulmonary NEBs in health and disease. This work was supported by the following research grants: Fund for Scientific Research Flanders (G.0155.01 to D.A.), NOI-BOF (to D.A.) and BOF-RUCA Small Projects (KPO2 to D.A., I.B. and F.V.M.) from the University of Antwerp.     

Quantitative characteristics of the Feyrter cells and neuroepithelial bodies of the fetal rabbit lung in normoxia and short term chronic hypoxia

We report here quantitative data on the Feyrter (single) cells (APUD cells) and neuroepithelial bodies (grouped Feyrter cells), in the lungs of rabbit fetuses at 26, 27.5 and 29 days gestational age, during normoxia and short term chronic hypoxia. The apparent number of these cells declines during this period; we suggest that this might be due to increased hypoxemia. Moreover, the number of cells in the lungs of fetuses from short term chronically hypoxic mothers is lower than in the normoxic animals. These findings are in agreement with our previous studies in short term chronically hypoxic neonatal rabbits, and suggest that the increased hypoxemia in the fetus, caused by the induction of hypoxia in the mother, constitutes a stimulus for secretory activity of the Feyrter cells and neuroepithelial bodies (NEBs). This in turn could be part of the mechanism responsible for maintaining the pulmonary vasoconstriction due to hypoxemia. Our results from fetuses of normoxic does provide base line data on the chronological importance of the Feyrter cells and NEBs.     

Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies

The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.     

The organizational characteristics of the APUD system in mammalian lungs at different stages of ontogeny]

The organization peculiarities of APUD-system in the lungs of rabbits, rats and guinea pigs has been studied. The endocrine system in the lungs of rabbits in pre- and postnatal ontogenesis is presented by the adipocytes and neuroepithelial bodies (NEB) containing a considerable number of monoamines. The number of argyrophil adipocytes and NEBs in the lungs of 21 and more day-old adult rats seem to be less than in fetuses and newborns. Monoamines are not revealed in the endocrine rat lung structures by means of the glyoxylic acid. In the lungs of guinea pigs the single argyrophil adipocytes and NEBs are determined in the gestation period.     

Immunoreactivity of neuroendocrine cells in the respiratory tract in rats with experimental uremia after thyroparathyroidectomy

Animals with experimental uremia, which underwent thyroparathyroidectomy, reveal numerous metabolic disorders that can influence morphology and activity of endocrine cells of the scattered neuroendocrine system. The aim of the study was the evaluation of the influence of thyroparathyroidectomy in rats with chronic renal failure on APUD system cells localized in the respiratory tract. The examination was conducted on the group of 20 rats. Thyroparathyroidectomy was performed 30 days after nephrectomy. Fragments of the lungs and trachea were collected 14 days after the operation. Routinely prepared paraffin sections were stained with H+E and with silver method. The immunohistochemical reactions were conducted with the use of antibodies against calcitonin (CT), synaptophysin (SPh), somatostatine (ST), and neuron-specific enolase (NSE) The results were estimated in light microscope on the basis of stain reaction of endocrine cells. Our examination showed that chronic renal failure affects the functioning of endocrine cells. We also observed the increase in APUD system cell number in the trachea and the lungs after thyroparathyroidectomy in uremic rats.     

Preliminary evaluation of endocrine cells in the rat respiratory tract after thyroid and parathyroid gland removal

The complete thyroid and parathyroid gland removal leads to the abrupt reduction of calcitonin, which can be a factor stimulating growth and intensified activity of APUD system cells in the respiratory tract. Thus, neuroendocrine cells in the lungs and trachea in rats after thyroid and parathyroid removal were evaluated. Paraffin specimens of the examined organs were stained with H+E and impregnated with silver. Calcitonin, synaptophysin, somatostatin, and neuronal-specific enolase were detected immunohistochemically by the use of rabbit specific antibodies. Antibodies used in the study immunostained neuroendocrine cells of the examined organs. Rats with removed thyroid and parathyroid glands presented weakened reaction compared to the control group.     

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABA_A and GABA_B (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.     

Immunoreactivity for calcitonin gene-related peptide in neuroepithelial bodies of the newborn cat

Summary Immunoreactivity for calcitonin gene-related peptide is demonstrated for the first time in neuroepithelial bodies in the lung of newborn cats after Bouin fixation and embedding in paraffin. The intense staining clearly identifies these bodies at the level of bronchioli and alveoli. Occasionally, single neuroepithelial endocrine cells, displaying immunoreactivity for calcitonin gene-related peptide are observed. In the kitten lung, identification and localization of neuroepithelial bodies after immunocytochemical staining for calcitonin gene-related peptide are superior to the analysis based on other techniques, i.e., the argyrophilic reaction, periodic acid Schiff-lead hematoxylin method, and immunocytochemical staining for serotonin. The serial-section technique revealed that in neuroepithelial bodies of the newborn kitten lung, immunoreactivity for calcitonin gene-related peptide coexists with immunoreactivity for serotonin in individual cells. The functional significance of the calcitonin gene-related peptide in neuroepithelial bodies remains to be elucidated.     

Hamster pulmonary endocrine cells with positive immunostaining for calbindin-D28K

 We have examined the distribution of calcium-binding proteins (CaBPs) in adult and fetal lungs of Syrian golden hamsters (Mesocricetus auratus) using immunostaining with confocal laser microscopy and electron microscopy. Single and grouped (neuroepithelial body; NEB) endocrine cells were distributed from bronchi to alveolar ducts in the adult lung. Serial frozen sections immunostained for CaBPs in combination with immunostaining for endocrine markers such as calcitonin gene-related peptide, serotonin, PGP9.5, and synaptophysin revealed that positive immunostaining for calbindin-D_28K (CB-D28K) was seen in single endocrine cells and NEBs. However, other so-called EF-hand family CaBPs, parvalbumin and calretinin, were not detected. Electron microscopically, positive immunoreaction for CB-D28K was mainly in the organelle-free cytoplasmic matrix of endocrine cells, and partly in nuclei and associated with secretory granules and endoplasmic reticulum. In fetal developing lungs, endocrine cells appeared first on gestational day 13, and they were positive for all the endocrine markers used. However, pulmonary endocrine cells were positively immunostained for CB-D28K from gestational days 15 and 16 onward. In summary, our observations suggest that CB-D28K is a useful marker for endocrine cells of the lung, and CB-D28K could function as a mediator of endocrine stimulation or calcium homeostasis in pulmonary endocrine cells. Accepted: 17 June 1997     

Evidence for the coexistence of serotonin and calcitonin gene-related peptide at the subcellular level in neuroepithelial bodies in the lung of a marsupial,Isoodon macrourus

Summary The coexistence of serotonin and calcitonin gene-related peptide (CGRP) in neuroepithelial bodies of the bandicoot, Isoodon macrourus, has been examined using immunocytochemistry at the light- and electronmicroscope levels. The avidin-biotin technique of antigen localisation was used initially to identify serotonin-like and CGRP-like immunoreactivity (-LI). Serotonin-LI and CGRP-LI were found in neuroepithelial cells in the lungs of 30-day-old bandicoots. CGRP-LI could also be demonstrated in nerve fibres associated with some neuroepithelial bodies. The protein A-gold technique of antigen localisation was used to label neuroepithelial cells and nerve fibres at the subcellular level. Serotonin-LI and CGRP-LI were observed in the same dense-cored vesicles of most neuroepithelial cells; however, some neuroepithelial cells were shown to possess serotonin-LI without CGRP-LI. Nerve fibres immediately adjacent to neuroepithelial bodies exhibited mainly CGRP-LI. These results show that serotonin-LI and CGRP-LI are present in neuroepithelial cells of the bandicoot in the same secretory vesicles. This pattern of co-localisation may reflect co-ordinated or synergistic actions of these two neuroactive substances.     

Co-occurrence of immunoreactive calcitonin and calcitonin gene-related peptide in neuroendocrine cells of rat lungs

Summary Neuroendocrine cells of the lung, occurring singly or in clusters known as neuroepithelial bodies, contain a variety of biologically active compounds, including several neuropeptides. We have investigated the localization of calcitonin and calcitonin gene-related peptide (CGRP) within single and grouped neuroendocrine cells in the respiratory epithelium of rats by an immunohistochemical double-staining technique which uses specific antisera raised in heterogeneous animal species. Calcitonin- and CGRP-immunoreactivities were nearly totally co-localized in both single neuroendocrine cells and neuroepithelial bodies. CGRP-immunoreactivity was also present in neurons in the jugular, nodose and dorsal root ganglia. The calcitonin-immunoreactivity in neuroendocrine cells, as in thyroid parafollicular (C) cells, was abolished by preincubation of the anticalcitonin serum with synthetic calcitonin. The CGRP-immunoreactivity in neuroendocrine cells and in the neuronal cells was abolished by preincubation of anti-CGRP serum with synthetic CGRP. Thus, while the calcitonin gene is expressed exclusively or predominantly as either calcitonin or CGRP in all other tissues except thyroid C-cells, our results strongly suggest that both peptides are expressed in the rat bronchopulmonary neuroendocrine cells.     

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus)

Summary Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibres containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which form distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P_{CO 2} and so leading to modulation of activity in afferent nerve fibres (type containing agranular vesicles). The presence of NEBs solely with an adrenergic (efferent) innervation poses a problem with this interpretation.     

Immunohistochemical Characterization of the Chemosensory Pulmonary Neuroepithelial Bodies in the Naked Mole-Rat Reveals a Unique Adaptive Phenotype

The pulmonary neuroepithelial bodies (NEBs) constitute polymodal airway chemosensors for monitoring and signaling ambient gas concentrations (pO₂, pCO₂/H⁺) via complex innervation to the brain stem controlling breathing. NEBs produce the bioactive amine, serotonin (5-HT), and a variety of peptides with multiple effects on lung physiology and other organ systems. NEBs in mammals appear prominent and numerous during fetal and neonatal periods, and decline in the post-natal period suggesting an important role during perinatal adaptation. The naked mole-rat (NMR), Heterocephalus glaber, has adapted to the extreme environmental conditions of living in subterranean burrows in large colonies (up to 300 colony mates). The crowded, unventilated burrows are environments of severe hypoxia and hypercapnia. However, NMRs adjust readily to above ground conditions. The chemosensory NEBs of this species were characterized and compared to those of the conventional Wistar rat (WR) to identify similarities and differences that could explain the NMR’s adaptability to environments. A multilabel immunohistochemical analysis combined with confocal microscopy revealed that the expression patterns of amine, peptide, neuroendocrine, innervation markers and chemosensor component proteins in NEBs of NMR were similar to that of WR. However, we found the following differences: 1) NEBs in both neonatal and adult NMR lungs were significantly larger and more numerous as compared to WR; 2) NEBs in NMR had a more variable compact cell organization and exhibited significant differences in the expression of adhesion proteins; 3) NMR NEBs showed a significantly greater ratio of 5-HT positive cells with an abundance of 5-HT; 4) NEBs in NMR expressed the proliferating cell nuclear antigen (PCNA) and the neurogenic gene (MASH1) indicating active proliferation and a state of persistent differentiation. Taken together our findings suggest that NEBs in lungs of NMR are in a hyperactive, functional and developmental state, reminiscent of a persistent fetal state that extends postnatally.     

Breeding and egg survival of a population of a salamander,Hynobius nebulosus tokyoensis Tago

1. A population of a salamander, Hynobius nebulosus tokyoensisTago was studied at Habu, Hinodemachi, in Tokyo during the breeding seasons from 1976 to 1978. Adults appeared in the pond were captured and marked individually by toe-clipping. Egg sacks were counted and tagged with vinyl tapes for the marking.
2. The annual egg production of this population was found to be constant, about 6500 eggs, and total number of breeding adults appeared in the pond also varied little during three years.
3. The appearance of breeding adults in the pond and oviposition seemed to be influenced by the rise of temperature and rainfall.
4. Males appeared earlier in the pond and stayed there for approximately 10 to 20 days. On the other hand, females appeared later in the pond and left there soon after laying eggs.
5. Mean clutch size and body length of breeding adults appeared in the pond showed a tendency to decrease as time proceeded, which seemed to show that the smaller and younger the salamander was, the later it appeared in the pond.
6. Owing to low temperature, the eggs laid earlier took more time to hatch than those laid later.
7. The survival rate of eggs was 67.2% in 1976, 86.4% in 1977 and 81.2% in 1978. The loss rate of eggs laid in the early and late periods of oviposition was higher than that of eggs laid in the middle period.

     

Nisin F in the treatment of respiratory tract infections caused by Staphylococcus aureus

Aims:  To determine the antimicrobial activity of nisin F against Staphylococcus aureus in the respiratory tract.
Methods and Results:  The respiratory tract of nonimmunosuppressed and immunosuppressed Wistar rats were colonized with 4 × 10⁵ viable cells of S. aureus K and then treated by administering 8192 arbitrary units (AU) nisin F intranasal. Symptoms of pneumonia were detected in the trachea and lungs of immunosuppressed rats that had not been treated with nisin F. The trachea and lungs of immunosuppressed rats treated with nisin F were healthy. No significant differences were recorded in blood cell indices. The antimicrobial activity of low concentrations nisin F (80–320 AU ml⁻¹) was slightly stimulated by lysozyme and lactoferrin.
Conclusions:  Nisin F inhibited the growth of S. aureus K in the respiratory tract of immunocompromised rats. Treatment with nisin F at 8192 AU proofed safe, as the trachea, lungs, bronchi and haematology of the rats appeared normal.
Significance and Impact of the Study:  Nisin F is nontoxic and may be used to control respiratory tract infections caused by S. aureus . This is, however, a preliminary study with an animal model and need to be confirmed with studies on humans.