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

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Abstract

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.

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References

  • Adriaensen D, Scheuermann DW (1993) Neuroendocrine cells and nerves of the lung. Anat Rec 236:70–85

    Article  CAS  PubMed  Google Scholar 

  • Adriaensen D, Timmermans J-P (2004) Purinergic signalling in the lung: important in asthma and COPD? Curr Opin Pharmacol 4:207–214

    Article  CAS  PubMed  Google Scholar 

  • Adriaensen D, Brouns I, Van Genechten J, Timmermans J-P (2003) Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anat Rec 270A:25–40

    Article  Google Scholar 

  • Bergua A, Neuhuber WL, Naumann GOH (1994) Visualization of human choroidal ganglion cells with the supravital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridium iodide. Ophthalmic Res 26:290–295

    CAS  PubMed  Google Scholar 

  • Brouns I, Adriaensen D, Burnstock G, Timmermans J-P (2000) Intraepithelial vagal sensory nerve terminals in rat pulmonary neuroepithelial bodies express P2X3 receptors. Am J Respir Cell Mol Biol 23:52–61

    CAS  PubMed  Google Scholar 

  • Brouns I, Van Genechten J, Scheuermann DW, Timmermans J-P, Adriaensen D (2002) Neuroepithelial bodies: a morphologic substrate for the link between neuronal nitric oxide and sensitivity to airway hypoxia? J Comp Neurol 449:343–354

    Article  CAS  PubMed  Google Scholar 

  • Brouns I, Van Genechten J, Burnstock G, Timmermans J-P, Adriaensen D (2003a) Ontogenesis of P2X3 receptor-expressing nerve fibres in the rat lung, with special reference to neuroepithelial bodies. Biomed Res 14:80–86

    CAS  Google Scholar 

  • Brouns I, Van Genechten J, Hayashi H, Gajda M, Gomi T, Burnstock G, Timmermans J-P, Adriaensen D (2003b) Dual sensory innervation of pulmonary neuroepithelial bodies. Am J Respir Cell Mol Biol 28:275–285

    Article  CAS  PubMed  Google Scholar 

  • Brouns I, Pintelon I, Van Genechten J, De Proost I, Timmermans J-P, Adriaensen D (2004) Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies. Histochem Cell Biol 121:1–12

    Article  CAS  PubMed  Google Scholar 

  • Carabba VH, Sorokin SP, Hoyt RFJ (1985) Development of neuroepithelial bodies in intact and cultured lungs of fetal rats. Am J Anat 173:1–27

    Article  CAS  PubMed  Google Scholar 

  • Cho T, Chan W, Cutz E (1989) Distribution and frequency of neuro-epithelial bodies in post-natal rabbit lung: quantitative study with monoclonal antibody against serotonin. Cell Tissue Res 255:353–362

    Article  CAS  PubMed  Google Scholar 

  • Cornelissen W, Timmermans J-P, Van Bogaert P-P, Scheuermann DW (1996) Electrophysiology of porcine myenteric neurons revealed after vital staining of their cell bodies. A preliminary report. Neurogastroenterol Mot 8:101–109

    CAS  Google Scholar 

  • Cutz E, Jackson A (1999) Neuroepithelial bodies as airway oxygen sensors. Respir Physiol 115:201–214

    Article  CAS  PubMed  Google Scholar 

  • Cutz E, Chan W, Wong V, Conen PE (1974) Endocrine cells in rat fetal lungs. Ultrastructural and histochemical study. Lab Invest 30:458–464

    CAS  PubMed  Google Scholar 

  • Cutz E, Yeger H, Wong V, Bienkowski E, Chan W (1985) In vitro characteristics of pulmonary neuroendocrine cells isolated from rabbit fetal lung. I. Effects of culture media and nerve growth factor. Lab Invest 53:672–683

    CAS  PubMed  Google Scholar 

  • Fu XW, Nurse CA, Wang YT, Cutz E (1999) Selective modulation of membrane currents by hypoxia in intact airway chemoreceptors from neonatal rabbit. J Physiol (Lond) 514:139–150

    Article  CAS  Google Scholar 

  • Fu XW, Wang D, Nurse CA, Dinauer MC, Cutz E (2000) NADPH oxidase is an O2 sensor in airway chemoreceptors: evidence from K+ current modulation in wild-type and oxidase-deficient mice. Proc Natl Acad Sci USA 97:4374–4379

    Article  CAS  PubMed  Google Scholar 

  • Fu XW, Wang D, Pan J, Farragher SM, Wong V, Cutz E (2001) Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor. Am J Physiol Lung Cell Mol Physiol 281:L931–L940

    CAS  PubMed  Google Scholar 

  • Fu XW, Nurse CA, Wong V, Cutz E (2002) Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit. J Physiol (Lond) 539:503–510

    Article  CAS  Google Scholar 

  • Hage E (1976) Endocrine-like cells of the pulmonary epithelium. In: Coupland RE, Fujita T (eds) Chromaffin, enterochromaffin and related cells. Elsevier, Amsterdam, pp 317–332

    Google Scholar 

  • Hanani M (1992) Visualization of enteric and gallbladder ganglia with a vital fluorescent dye. J Auton Nerv Syst 38:77–84

    Article  CAS  PubMed  Google Scholar 

  • Herrera AA, Banner LR (1990) The use and effects of vital fluorescent dyes: observation of motor nerve terminals and satellite cells in living frog muscles. J Neurocytol 19:67–83

    Article  CAS  PubMed  Google Scholar 

  • Hillsley K, Jennings LJ, Mawe GM (1998) Neural control of the gallbladder: an intracellular study of human gallbladder neurons. Digestion 59:125–129

    Article  CAS  PubMed  Google Scholar 

  • Kelly SS, Anis N, Robbins N (1985) Fluorescent staining of living mouse neuromuscular junctions. Pflügers Arch 404:97–99

    Article  CAS  PubMed  Google Scholar 

  • Kemp PJ, Lewis A, Hartness M, Searle GJ, Miller P, O’Kelly I, Peers C (2002) Airway chemotransduction: from oxygen sensor to cellular effector. Am J Respir Crit Care Med 166:S17–S24

    Article  PubMed  Google Scholar 

  • Lauweryns JM, Peuskens JC (1972) Neuro-epithelial bodies (neuroreceptor or secretory organs?) in human infant bronchial and bronchiolar epithelium. Anat Rec 172:471–481

    Article  CAS  PubMed  Google Scholar 

  • Lauweryns JM, Van Lommel A (1986) Effect of various vagotomy procedures on the reaction to hypoxia of rabbit neuroepithelial bodies: modulation by intrapulmonary axon reflexes. Exp Lung Res 11:319–339

    CAS  PubMed  Google Scholar 

  • Lichtman JW, Magrassi L, Purves D (1987) Visualization of neuromuscular junctions over periods of several months in living mice. J Neurosci 7:1215–1222

    CAS  PubMed  Google Scholar 

  • Loew LM, Cohen LB, Salzberg BM, Obaid AL, Bezanilla F (1985) Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon. Biophys J 47:71–77

    CAS  PubMed  Google Scholar 

  • Magrassi L, Purves D, Lichtman JW (1987) Fluorescent probes that stain living nerve terminals. J Neurosci 7:1207–1214

    CAS  PubMed  Google Scholar 

  • Nurse CA, Farraway L (1989) Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes. Cell Tissue Res 255:125–128

    Article  CAS  PubMed  Google Scholar 

  • Peers C, Kemp PJ (2001) Acute oxygen sensing: diverse but convergent mechanisms in airway and arterial chemoreceptors. Respir Res 2:145–149

    Article  CAS  PubMed  Google Scholar 

  • Rafael J, Nicholls DG (1984) Mitochondrial membrane potential monitored in situ within isolated guinea pig brown adipocytes by a styryl pyridinium fluorescent indicator. FEBS Lett 170:181–185

    Article  CAS  PubMed  Google Scholar 

  • Scheuermann DW (1987) Morphology and cytochemistry of the endocrine epithelial system in the lung. Int Rev Cytol 106:35–88

    CAS  PubMed  Google Scholar 

  • Schrödl F, De Laet A, Tassignon MJ, Van Bogaert P-P, Brehmer A, Neuhuber WL, Timmermans J-P (2003) Intrinsic choroidal neurons in the human eye: projections, targets and basic electrophysiological data. Invest Ophthalmol Vis Sci 44:3705–3712

    Article  PubMed  Google Scholar 

  • Sorokin SP, Hoyt RF (1989) Neuroepithelial bodies and solitary small-granule cells. In: Massaro D (ed) Lung cell Biology. Dekker, New York, pp 191–344

    Google Scholar 

  • Speirs V, Cutz E (1993) An overview of culture and isolation methods suitable for in vitro studies on pulmonary neuroendocrine cells. Anat Rec 236:35–40

    Article  CAS  PubMed  Google Scholar 

  • Speirs V, Wang YV, Yeger H, Cutz E (1992) Isolation and culture of neuroendocrine cells from fetal rabbit lung using immunomagnetic techniques. Am J Respir Cell Mol Biol 6:63–67

    CAS  PubMed  Google Scholar 

  • Stuart AE, Hudspeth AJ, Hall ZW (1974) Vital staining of specific monoamine-containing cells in the leech nervous system. Cell Tissue Res 153:55–61

    Article  CAS  PubMed  Google Scholar 

  • Van Genechten J, Brouns I, Burnstock G, Timmermans J-P, Adriaensen D (2004) Quantification of neuroepithelial bodies and their innervation in fawn–hooded and Wistar rat lungs. Am J Respir Cell Mol Biol 30:20–30

    Article  PubMed  Google Scholar 

  • Van Lommel A, Lauweryns JM (1993) Neuroepithelial bodies in the fawn hooded rat lung: morphological and neuroanatomical evidence for a sensory innervation. J Anat 183:553–566

    PubMed  Google Scholar 

  • Wasano K, Yamamoto T (1978) Monoamine-containing granulated cells in the frog lung. Cell Tissue Res 193:201–209

    Article  CAS  PubMed  Google Scholar 

  • Widdicombe JG (2001) Airway receptors. Respir Physiol 125:3–15

    Article  CAS  PubMed  Google Scholar 

  • Youngson C, Nurse C, Yeger H, Cutz E (1993) Oxygen sensing in airway chemoreceptors. Nature 365:153–155

    Article  CAS  PubMed  Google Scholar 

  • Youngson C, Nurse C, Yeger H, Curnutte JT, Vollmer C, Wong V, Cutz E (1997a) Immunocytochemical localization on O2-sensing protein (NADPH oxidase) in chemoreceptor cells. Microsc Res Tech 37:101–106

    Article  CAS  PubMed  Google Scholar 

  • Youngson C, Nurse CA, Wang D, Cutz E (1997b) Ionic currents and oxygen-sensing mechanism in neuroepithelial body cells. In: Cutz E (ed) Cellular and molecular biology of airway chemoreceptors. Landes Bioscience, Austin, pp 71–108

    Google Scholar 

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Acknowledgements

We are grateful to Prof. G. Burnstock (Director of the Autonomic Neuroscience Institute, Royal Free and University College Medical School, London, UK) for his invaluable input in the ATP receptor studies. We thank H. De Pauw, R. Spillemaeckers, G. Svensson, F. Terloo and G. Vermeiren for technical assistance, J. Van Daele and D. De Rijck for help with microscopy, imaging and illustrations, D. Vindevogel for aid with the manuscript, and S. Kockelberg for secretarial help.

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Authors and Affiliations

  1. Department of Biomedical Sciences, Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, 2020, Antwerp, Belgium

    I. Pintelon, I. De Proost, I. Brouns, H. Van Herck, J. Van Genechten, F. Van Meir, J.-P. Timmermans & D. Adriaensen

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  1. I. Pintelon
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  2. I. De Proost
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  3. I. Brouns
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  4. H. Van Herck
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  5. J. Van Genechten
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  6. F. Van Meir
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  7. J.-P. Timmermans
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  8. D. Adriaensen
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Correspondence to D. Adriaensen.

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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.

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Pintelon, I., De Proost, I., Brouns, I. et al. Selective visualisation of neuroepithelial bodies in vibratome slices of living lung by 4-Di-2-ASP in various animal species. Cell Tissue Res 321, 21–33 (2005). https://doi.org/10.1007/s00441-005-1111-y

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