Pulmonary Neuroepithelial Bodies Research Articles

The pulmonary neuroepithelial body microenvironment represents an underestimated multimodal component in airway sensory pathways.

Exciting new imaging and molecular tools, combined with state-of-the-art genetically modified mouse models, have recently boosted interest in pulmonary (vagal) sensory pathway investigations. In addition to the identification of diverse sensory neuronal subtypes, visualization of intrapulmonary projection patterns attracted renewed attention on morphologically identified sensory receptor end-organs, such as the pulmonary neuroepithelial bodies (NEBs) that have been our area of expertise for the past four decades. The current review aims at providing an overview of the cellular and neuronal components of the pulmonary NEB microenvironment (NEB ME) in mice, underpinning the role of these complexly organized structures in the mechano- and chemosensory potential of airways and lungs. Interestingly, the pulmonary NEB ME additionally harbors different types of stem cells, and emerging evidence suggests that the signal transduction pathways that are active in the NEB ME during lung development and repair also determine the origin of small cell lung carcinoma. Although documented for many years that NEBs appear to be affected in several pulmonary diseases, the current intriguing knowledge on the NEB ME seems to encourage researchers that are new to the field to explore the possibility that these versatile sensor-effector units may be involved in lung pathogenesis or pathobiology.

Microscopic Anatomy of the Pulmonary Neuroepithelial Bodies in Spontaneously Hypertensive Rats

Microscopic Anatomy of the Pulmonary Neuroepithelial Bodies in Spontaneously Hypertensive Rats

Mechanosensitivity of Murine Lung Slowly Adapting Receptors: Minimal Impact of Chemosensory, Serotonergic, and Purinergic Signaling.

Murine slowly adapting receptors (SARs) within airway smooth muscle provide volume-related feedback; however, their mechanosensitivity and morphology are incompletely characterized. We explored two aspects of SAR physiology: their inherent static mechanosensitivity and a potential link to pulmonary neuroepithelial bodies (NEBs). SAR mechanosensitivity displays a rate sensitivity linked to speed of inflation; however, to what extent static SAR mechanosensitivity is tuned for the very rapid breathing frequency (Bf) of small mammals (e.g., mouse) is unclear. NEB-associated, morphologically described smooth muscle-associated receptors (SMARs) may be a structural analog for functionally characterized SARs, suggesting functional linkages between SARs and NEBs. We addressed the hypotheses that: (1) rapid murine Bf is associated with enhanced in vivo SAR static sensitivity; (2) if SARs and NEBs are functionally linked, stimuli reported to impact NEB function would alter SAR mechanosensitivity. We measured SAR action potential discharge frequency (AP f, action potentials/s) during quasi-static inflation [0–20 cmH2O trans-respiratory pressure (PTR)] in NEB-relevant conditions of hypoxia (FIO2 = 0.1), hypercarbia (FICO2 = 0.1), and pharmacologic intervention (serotonergic 5-HT3 receptor antagonist, Tropisetron, 4.5 mg/kg; P2 purinergic receptor antagonist, Suramin, 50 mg/kg). In all protocols, we obtained: (1) AP f vs. PTR; (2) PTR threshold; and (3) AP f onset at PTR threshold. The murine AP f vs. PTR response comprises high AP f (average maximum AP f: 236.1 ± 11.1 AP/s at 20 cmH2O), a low PTR threshold (mean 2.0 ± 0.1 cmH2O), and a plateau in AP f between 15 and 20 cmH2O. Murine SAR mechanosensitivity (AP f vs. PTR) is up to 60% greater than that reported for larger mammals. Even the maximum difference between intervention and control conditions was minimally impacted by NEB-related alterations: Tropisetron −7.6 ± 1.8% (p = 0.005); Suramin −10.6 ± 1.5% (p = 0.01); hypoxia +9.3 ± 1.9% (p < 0.001); and hypercarbia −6.2 ± 0.9% (p < 0.001). We conclude that the high sensitivity of murine SARs to inflation provides enhanced resolution of operating lung volume, which is aligned with the rapid Bf of the mouse. We found minimal evidence supporting a functional link between SARs and NEBs and speculate that the <10% change in SAR mechanosensitivity during altered NEB-related stimuli is not consistent with a meaningful physiologic role.

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

The Pulmonary NEB ME Is a Complex Intraepithelial Unit.

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.Complexly organised pulmonary NEBs are an integral part of the intrapulmonary airway epithelium of all air-breathing vertebrates. For decades, a quest has been going on to unravel the functional significance of these intriguing structures that appear to be modified in the course of many pulmonary diseases.The pulmonary NEB microenvironment (ME) is composed of organoid clusters of pulmonary neuroendocrine cells (PNECs) that are able to store and release neurotransmitters and are closely contacted by extensive (mainly afferent) nerve terminals, emphasising a potential receptor/effector role and probable signalling to the central nervous system. PNECs are largely shielded from the airway lumen by a special type of Clara cells, the Clara-like cells, with potential stem cell characteristics.Since pulmonary NEBs are widely dispersed in the airway epithelium, and represent less than one percent of the cells in the epithelial lining, investigating the NEB ME largely depends on its unequivocal microscopic identification. Nowadays, multidisciplinary approaches allow to combine functional morphological investigations in cryosections and cleared whole lungs, live cell imaging in lung vibratome slices, and selective gene expression analysis after laser microdissection of genetically tagged NEBs.So far, functional studies of the pulmonary NEB ME revealed that PNECs can be activated by various mechanical and chemical stimuli, resulting in a calciummediated release of neurotransmitters. A number of publications in the past decades have exposed NEBs as potential hypoxia sensors.In the past few years, combination of in vivo experiments and gene expression analysis unveiled the pulmonary NEB ME as a quiescent stem cell niche in healthy postnatal mouse lungs. The stem cell population was shown to be activated by transient mild inflammation, and silencing appears to involve bone morphogenetic protein signalling that may be mediated by vagal afferents.Today, it is clear that only an integrated approach that takes all current information into account will be able to explain the full role of the pulmonary NEB ME in health and disease.

Pulmonary Sensory Receptors.

Among the intrapulmonary myelinated vagal sensory airway receptors, pulmonary neuroepithelial bodies (NEBs) definitely reveal the most complex organisation. This updated review aims at delivering the broad and thorough knowledge of the system that is essential for understanding its physiological relevance.

Correction to: The Pulmonary Neuroepithelial Body Microenvironment.

Correction to: The Pulmonary Neuroepithelial Body Microenvironment.

Multiple Immunofluorescence, 3D Visualization and Quantification in Cleared Whole Mouse Lungs: Pulmonary Neuroepithelial Bodies Contacted by Myelinated Vagal Afferents

Neuroepithelial bodies (NEBs) are clusters of pulmonary neuroendocrine cells (PNECs) that represent less than one percent of the airway epithelial cells. PNECs store and release neurotransmitters, are surrounded by a unique population of stem cells, and are contacted by extensive nerve terminals. Today, multidisciplinary approaches allow combining functional morphological investigations in cryosections, live cell imaging in lung vibratome slices and selective gene expression analysis after laser microdissection of genetically tagged NEBs. However, due to their relatively low number and widespread distribution, the information remains highly fragmentary and quantification of NEBs, associated nerve endings and stem cells is problematic.The present study aimed at acquiring the first whole lung visualization and quantification of the total population of NEBs. As a proof‐of‐concept, we used a challenging triple immunolabeling and four channel imaging protocol for simultaneous identification of the selective myelinated vagal sensory innervation of NEBs.Lungs of GAD67‐GFP mice, which harbor GFP fluorescent NEB cells, were subjected to multiple immunostaining and tissue clearing. The list of optical clearing techniques is quickly growing, and several were tested; for our goals the iDisco+ protocol appeared to result in both the most complete clearing of mouse lungs and the best preservation of immunofluorescence in NEB nerve fiber populations. After fixation, methanol treatment, and permeabilization, whole mouse lungs were simultaneously immunostained for GFP (NEB cells), calbindin D‐28k and P2X3 ATP receptors (selective markers for two subpopulations of myelinated vagal afferents in mouse NEBs), and myelin basic protein (marker for all myelinated nerve fibers).Immunostained whole lungs could be imaged using light sheet microscopy (Ultramicroscope II, LaVision Biotec; Olympus MVPLAPO 2× objective; lasers: 488, 561, 640 and 785nm; emission filters: 525/50, 620/60, 680/30 and 845/55nm; Andor Neo sCMOS camera) at a total magnification of 1.26× and a z‐step of 10 μm. Optical sections were recorded in a mosaic of two by two tiles, and images stitched and merged with a linear blending algorithm (ImageJ).It could be shown that the applied staining, clearing and imaging protocols allow for 3D rendering of the complete lungs, with all NEBs and associated myelinated nerve terminals, including possibilities for 3D quantification. Selected individual NEB/nerve terminal complexes can be further analyzed in detail using high resolution confocal imaging and 3D reconstruction (Leica TCS SP8 DLS).The presented methodology opens interesting opportunities for imaging and quantification of total populations of unevenly dispersed microscopic structures in whole lungs, even when visualization requires complex immunostaining. Future goals include dedicated automated 3D quantification and shortening of the protocol time, which for now is about four weeks.Support or Funding InformationSupport: UA grant GOA BOF 2015 (30729 to DA).

Rare Neuroepithelial Stem Cells May Underlie Small-Cell Lung Cancer

In mouse pulmonary neuroepithelial bodies, few stem cells per cluster proliferate upon lung injury.

Spatiotemporal Expression of Three Secretoglobin Proteins, SCGB1A1, SCGB3A1, and SCGB3A2, in Mouse Airway Epithelia.

Secretoglobins (SCGBs) are cytokine-like small molecular weight secreted proteins with largely unknown biological functions. Three SCGB proteins, SCGB1A1, SCGB3A1, and SCGB3A2, are predominantly expressed in lung airways. To gain insight into the possible functional relationships among the SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice, using Scgb3a1-null and Scgb3a2-null mice as negative controls, by immunohistochemistry and by qRT-PCR analysis, respectively. The three SCGBs exhibited unique spatiotemporal expression patterns during embryogenesis. The lack of Scgb3a1 or Scgb3a2 did not affect expression of the other Scgb genes as determined by mRNA measurements. Moreover, the lack of Scgb3a1 or Scgb3a2 did not affect development of the pulmonary neuroepithelial bodies during embryogenesis, while the lack of Scgb3a2 may have resulted in slightly fewer ciliated cells than in the wild-type. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function.

The Neuroepithelial Body Microenvironment as a Postnatal Airway Epithelial Stem Cell Niche: Critical Role for BMP Signaling

Main objective was to develop models that enable exploring the stem cell characteristics of pulmonary neuroepithelial bodies (NEBs) in postnatal mouse lungs. The NEB microenvironment (ME) consists of innervated groups of pulmonary neuroepithelial endocrine cells, covered by socalled Clara‐like cells (CLCs). Epithelial cells of intrapulmonary conductive airways typically show a very low turn‐over rate in healthy postnatal lungs. Own and literature data point out that the NEB ME is important during development and for adult lung epithelial repair after total ablation, and that CLCs may act as stem cells under these circumstances. However, the absence of a model to physiologically induce selective cell proliferation in the healthy NEB ME, has so far hampered the identification of signaling processes to support their characterization as a quiescent postnatal stem cell niche involved in postnatal airway epithelial homeostasis.We developed a laser microdissection (LMD)‐based method that is compatible with high‐end PCR techniques to selectively isolate the NEB ME and compare its gene expression with control airway epithelium (CAE) [1]. To identify genes that may be involved in signaling mechanisms in the NEB ME, as a potential postnatal stem cell niche, pooled NEB and CAE samples were collected using LMD and gene expression was compared using a panel of stem cell related PCR Arrays. Expression of stem cell ‘signature’ genes in the NEB ME was confirmed and quantified by qPCR [1].Since inhibition of bone morphogenetic protein (BMP) signaling has been reported to induce proliferation of basal progenitor cells in the mouse trachea [2], and members of the BMP pathway showed a high expression in the NEB ME in multiple PCR arrays, their possible involvement in the stem cell characteristics of the NEB ME was further evaluated. Localization of BMP2, BMP7 and BMP receptors in the NEB ME could be confirmed by multiple immunofluorescent labeling.Additionally, mild transient acute lung injury (ALI), induced by a single low‐dose intratracheal lipopolysaccharide (LPS) challenge, was shown to induce a remarkable selective proliferation of CLCs in the NEB ME. It also resulted in a strongly decreased expression of BMP ligands in the NEB ME, indicating that stem cell activation by ALI might involve inhibition of the BMP pathway. Treating mice with LDN‐193189, a small molecule inhibitor of BMP type I receptors, recently revealed that compromising BMP signaling on its own appears to cause cell proliferation in the NEB ME that is reminiscent of what was seen after LPS treatment.In conclusion, a highly reproducible mild ALI model was generated for the selective activation of a quiescent postnatal airway stem cell population in the NEB ME, creating novel opportunities for unraveling the mechanisms that are involved. So far, combined LMD, gene and protein expression analysis, and specific inhibition of BMP signaling strongly suggest the involvement of BMP in silencing cell proliferation in the non‐activated stem cell niche of the NEB ME.Support or Funding InformationSupported by UA grant GOA BOF 2015 (30729 to DA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective gene expression analysis of the neuroepithelial body microenvironment in postnatal lungs with special interest for potential stem cell characteristics

BackgroundThe pulmonary neuroepithelial body (NEB) microenvironment (ME) consists of innervated cell clusters that occur sparsely distributed in the airway epithelium, an organization that has so far hampered reliable selective gene expression analysis. Although the NEB ME has been suggested to be important for airway epithelial repair after ablation, little is known about their potential stem cell characteristics in healthy postnatal lungs. Here we report on a large-scale selective gene expression analysis of the NEB ME.MethodsA GAD67-GFP mouse model was used that harbors GFP-fluorescent NEBs, allowing quick selection and pooling by laser microdissection (LMD) without further treatment. A panel of stem cell-related PCR arrays was used to selectively compare mRNA expression in the NEB ME to control airway epithelium (CAE). For genes that showed a higher expression in the NEB ME, a ranking was made based on the relative expression level. Single qPCR and immunohistochemistry were used to validate and quantify the PCR array data.ResultsCareful optimization of all protocols appeared to be essential to finally obtain high-quality RNA from pooled LMD samples of NEB ME. About 30% of the more than 600 analyzed genes showed an at least two-fold higher expression compared to CAE. The gene that showed the highest relative expression in the NEB ME, Delta-like ligand 3 (Dll3), was investigated in more detail. Selective Dll3 gene expression in the NEB ME could be quantified via single qPCR experiments, and Dll3 protein expression could be localized specifically to NEB cell surface membranes.ConclusionsThis study emphasized the importance of good protocols and RNA quality controls because of the, often neglected, fast RNA degradation in postnatal lung samples. It was shown that sufficient amounts of high-quality RNA for reliable complex gene expression analysis can be obtained from pooled LMD-collected NEB ME samples of postnatal lungs. Dll3 expression, which has also been reported to be important in high-grade pulmonary tumor-initiating cells, was used as a proof-of-concept to confirm that the described methodology represents a promising tool for further unraveling the molecular basis of NEB ME physiology in general, and its postnatal stem cell capacities in particular.

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O2 sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)–hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of Phd1−/− mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of Phd1−/−, Phd2+/−, and Phd3−/− mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in Phd1−/− and Phd3−/− mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of Phds is associated with striking hyperplasia and hypertrophy of NEBs. In Phd2+/− mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all Phd-deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.

Selective Activation and Proliferation of a Unique Stem Cell Population in the Neuroepithelial Body Microenvironment by Transient Acute Lung Injury

The microenvironment (ME) of pulmonary neuroepithelial bodies (NEBs) has been suggested to be implicated in airway development and adult airway epithelial repair after severe injury. Regardless the increasing evidence that NEBs act as a stem cell niche under these circumstances, little is known about the potential stem cell characteristics of the NEB ME in healthy postnatal lungs. The NEB ME consists of highly innervated groups of pulmonary neuroepithelial endocrine cells that are specifically covered by a unique population of Clara‐like cells (CLCs) with supposed stem cell characteristics. The relatively low number and widespread distribution of NEBs in the epithelium of intrapulmonary airways of postnatal animals has so far hampered the direct identification of mechanisms and signaling processes to support their characterization as a stem cell niche.A GAD67‐GFP mouse model, in which NEB cells selectively express GFP, permits their straightforward identification in cryostat sections of postnatal mouse lungs. Transient acute lung injury (ALI) was induced via intratracheal instillation of lipopolysaccharide (LPS; 1mg/kg bw). Lung function was monitored using a noninvasive unrestrained whole body plethysmograph. In one experiment, bronchoalveolar lavage fluid (BALF) was collected 24h after LPS instillation, and used to challenge the NEB ME in ex vivo lung slices of control mice. In another experiment, BrdU (10 mg/kg bw), as a marker for cells that divide during the observation window, was injected intraperitoneally at the time of LPS instillation and after 24h, the animals were euthanized after 48h, and the lungs were processed for freeze sectioning and BrdU immunostaining.Challenge of lung slices with BALF of ALI mice was shown to induce a reversible and reproducible selective Ca2+‐mediated activation of CLCs in the NEB ME of control mice. 48h after LPS challenge and BrdU injection, lung sections of ALI mice revealed a remarkable and highly significant increase in the number of BrdU‐labeled cells that surround NEBs, as compared to control lungs. As judged from sham‐treated mice, the instillation procedure itself also resulted in a modest increase in NEB‐associated BrdU‐labeled cells. Preliminary plethysmography data revealed that a single intratracheal LPS instillation results in short and transient ALI. Lung function parameters show the largest changes around six hours after treatment, and have restored to values that resemble those of the control group after 24h.In conclusion, mild transient ALI appears to result in the production of soluble mediators in the airways that selectively activate, and induce proliferation of, a stem cell population in the NEB ME. The changes that occur in the dividing cells will be further explored using gene expression profiling. The described workflow, together with our previously optimized LMD protocol, provides a solid base for unraveling molecular characteristics of the NEB ME.Support or Funding InformationHercules Foundation grant AUAH‐09‐001 (DA); UA grant GOA BOF 2015 (DA).

Sensory input to the central nervous system from the lungs and airways: A prominent role for purinergic signalling via P2X2/3 receptors.

Specific subpopulations of lung-related primary afferent neurons in dorsal root and vagal sensory ganglia have been reported to express P2X2 and P2X3 receptors both in the neuronal cell bodies and in their peripheral terminals. The afferent innervation of airways and lungs is organised as sensory receptor structures, of which at least seven types with a vagal origin and two with a spinal origin have been reported. In view of the recently suggested therapeutic promise of ATP antagonism - specifically at P2X3 receptor expressing nociceptive fibres - in respiratory disorders, the present work focusses on four distinct populations of pulmonary sensory receptors that have so far been reported to express P2X2/3 receptors. Three of them originate from myelinated nerve fibres that display similar mechanosensor-like morphological and neurochemical characteristics. Two of the latter concern vagal nodose sensory fibres, either related to pulmonary neuroepithelial bodies (NEBs), or giving rise to smooth muscle-associated airway receptors (SMARs); the third gives rise to visceral pleura receptors (VPRs) and most likely arises from dorsal root ganglia. The fourth population concerns C-fibre receptors (CFRs) that also derive from neuronal cell bodies located in vagal nodose ganglia. Although the majority of the airway- and lung-related sensory receptors that express P2X2/3 receptors apparently do not belong to accepted nociceptive populations, these data definitely point out that ATP may be an important player in the physiological transduction of different lung-related afferent signals from the periphery to the CNS. The observed variety within the populations of pulmonary sensory receptors that express P2X2/3 receptors argues for a critical and careful interpretation of the functional data.

Pulmonary neuroepithelial bodies are polymodal airway sensors: evidence for CO2/H+ sensing.

Pulmonary neuroepithelial bodies (NEB) in mammalian lungs are thought to function as airway O2 sensors that release serotonin (5-HT) in response to hypoxia. Direct evidence that NEB cells also respond to airway hypercapnia/acidosis (CO2/H(+)) is presently lacking. We tested the effects of CO2/H(+) alone or in combination with hypoxia on 5-HT release from intact NEB cells in a neonatal hamster lung slice model. For the detection of 5-HT release we used carbon fiber amperometry. Fluorescence Ca(2+) imaging method was used to assess CO2/H(+)-evoked changes in intracellular Ca(2+). Exposure to 10 and 20% CO2 or pH 6.8-7.2 evoked significant release of 5-HT with a distinct rise in intracellular Ca(2+) in hamster NEBs. This secretory response was dependent on the voltage-gated entry of extracellular Ca(2+). Moreover, the combined effects of hypercapnia and hypoxia were additive. Critically, an inhibitor of carbonic anhydrase (CA), acetazolamide, suppressed CO2/H(+)-mediated 5-HT release. The expression of mRNAs for various CA isotypes, including CAII, was identified in NEB cells from human lung, and protein expression was confirmed by immunohistochemistry using a specific anti-CAII antibody on sections of human and hamster lung. Taken together our findings provide strong evidence for CO2/H(+) sensing by NEB cells and support their role as polymodal airway sensors with as yet to be defined functions under normal and disease conditions.

Augmented 5-HT Secretion in Pulmonary Neuroepithelial Bodies from PHD1 Null Mice.

Sustained exposure to low oxygen concentration leads to profound changes in gene expression to restore oxygen homeostasis. Hypoxia-inducible factors (HIFs) comprise a group of transcription factors which accumulate under hypoxia and contribute to the complex changes in gene expression. Under normoxic conditions HIFs are degraded by prolyl-hydroxylases (PHD), however during hypoxia this degradation is inhibited causing HIF accumulation and subsequent changes in gene expression. Pulmonary neuroepithelial bodies (NEB) are innervated serotonin (5-HT)-producing cells distributed throughout the airway epithelium. These putative O(2) sensors are hypothesized to contribute to the ventilatory response to hypoxia. NEB dysfunction has been implicated in several paediatric lung diseases including neuroendocrine cell hyperplasia of infancy and sudden infant death syndrome, both characterized by a marked NEB hyperplasia with unknown functional significance. We have previously reported striking NEB hyperplasia in PHD1(-/-) mice making these mice a potential model to study the role of NEBs in paediatric lung diseases. Here we report in vitro studies on 5-HT release from NEB using this model.

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.

Murine airway slowly‐adapting receptor responses to lung inflation: a role for serotonin? (879.19)

The mechanical phenotype of murine airway slowly‐adapting receptors (SARs), which are mechanosensitive afferents physiologically characterized by maintained discharge to sustained transmural pressure, is only partially defined. Adriaensen et al. suggest that densely‐innervated pulmonary neuroepithelial bodies (NEBs) are a morphological equivalent of SARs (Adriaensen et al. 2006, J Appl Physiol). Since NEBs release serotonin (5‐HT) in response to stretch in vitro (Pan et al. 2006, Am J Physiol Lung Cell Mol Biol), we characterized the mechanosensitivity of SARs during quasi‐static inflation to 20 cmH2O in male C57BL/6 mice and tested the impact of 5‐HT3 receptor blockade (i.v. tropisetron, Trop; 4.5mg/kg). SAR (n=8) discharge frequency (f; impulses/s) mechanosensitivity was proportional to tracheal pressure. Trop caused a small (3‐8%) but significant decrease in f vs. control (P=0.005; 2‐way ANOVA‐RM; mean±SEM (imp/s): 5cmH2O ‐ con: 91±13, Trop: 87±13; 10cmH2O ‐ con: 153±25, Trop: 143±22; 15cmH2O ‐ con: 192±29, Trop: 180±28; 20cmH2O ‐ con: 176±14, Trop: 164±15). It remains to be seen whether such a modest reduction in SAR discharge has a physiologic impact on the reflex effects of SAR medullary input.Grant Funding Source: Supported by: CIHR (JTF; MOP81211) &amp; NSERC PostGrad Scholarship (NJD)

Neuroepithelial bodies as airway hypoxia sensors: re‐evaluation of the concept in different mouse live cell imaging models (869.6)

In pulmonary neuroepithelial bodies (NEBs), acute hypoxia has been suggested to close O2‐sensitive K+ channels, resulting in membrane depolarization, voltage‐gated Ca2+ entry and subsequent transmitter release.To visualize this mechanism in NEBs, our established live cell imaging (LCI) model, based on lung vibratome slices, was applied. Real‐time changes in different cellular processes were monitored during hypoxic challenge in NEBs of postnatal, prenatal and GAD67‐GFP mice.As a proof of concept, LCI experiments were performed on isolated mouse carotid body, proving that our LCI set up is indeed capable of visualizing hypoxia‐induced changes. However, in none of the NEBs studied in the lung slice models, severe (2% O2), mild (12% O2) or intermittent hypoxia, resulted in a rise in intracellular Ca2+ ([Ca2+]i) or subsequent neurotransmitter release. Moreover, low O2 levels appeared to decrease NEB cell excitability. The observed hypoxic mitochondrial membrane depolarization and increased ROS production rather seemed to be part of a general cellular defense mechanism, as supported by the non‐selective and non‐functional expression of the key O2 sensing protein NADPH oxidase.All together, our data argue for a reconsideration of the proposed straightforward hypoxic signal transduction pathway in pulmonary NEBs.Grants: FWO (G.0589.11), Hercules Foundation (AUAH‐09‐001); UA (GOA BOF 2007)