Selective activation and proliferation of a quiescent stem cell population in the neuroepithelial body microenvironment.

BACKGROUND: The microenvironment (ME) of neuroepithelial bodies (NEBs) harbors densely innervated groups of pulmonary neuroendocrine cells that are covered by Clara-like cells (CLCs) and is believed to be important during development and for adult airway epithelial repair after severe injury. Yet, little is known about its potential stem cell characteristics in healthy postnatal lungs. METHODS: Transient mild lung inflammation was induced in mice via a single low-dose intratracheal instillation of lipopolysaccharide (LPS). Bronchoalveolar lavage fluid (BALF), collected 16 h after LPS instillation, was used to challenge the NEB ME in ex vivo lung slices of control mice. Proliferating cells in the NEB ME were identified and quantified following simultaneous LPS instillation and BrdU injection. RESULTS: The applied LPS protocol induced very mild and transient lung injury. Challenge of lung slices with BALF of LPS-treated mice resulted in selective Ca2+-mediated activation of CLCs in the NEB ME of control mice. Forty-eight hours after LPS challenge, a remarkably selective and significant increase in the number of divided (BrdU-labeled) cells surrounding NEBs was observed in lung sections of LPS-challenged mice. Proliferating cells were identified as CLCs. CONCLUSIONS: A highly reproducible and minimally invasive lung inflammation model was validated for inducing selective activation of a quiescent stem cell population in the NEB ME. The model creates new opportunities for unraveling the cellular mechanisms/pathways regulating silencing, activation, proliferation and differentiation of this unique postnatal airway epithelial stem cell population.

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

BACKGROUND: The 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. METHODS: A 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. RESULTS: Careful 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. CONCLUSIONS: This 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.

Functional expression of the multimodal extracellular calcium-sensing receptor in pulmonary neuroendocrine cells.

The Ca(2+)-sensing receptor (CaSR) is the master regulator of whole-body extracellular free ionized [Ca(2+)]o. In addition to sensing [Ca(2+)]o, CaSR integrates inputs from a variety of different physiological stimuli. The CaSR is also expressed in many regions outside the [Ca(2+)]o homeostatic system, including the fetal lung where it plays a crucial role in lung development. Here, we show that neuroepithelial bodies (NEBs) of the postnatal mouse lung express a functional CaSR. NEBs are densely innervated groups of neuroendocrine epithelial cells in the lung representing complex sensory receptors in the airways and exhibiting stem cell characteristics. qRT-PCR performed on laser microdissected samples from GAD67-GFP mouse lung cryosections revealed exclusive expression of the CaSR in the NEB microenvironment. CaSR immunoreactivity was present at NEB cells from postnatal day 14 onwards. Confocal imaging of lung slices revealed that NEB cells responded to an increase of [Ca(2+)]o with a rise in intracellular Ca(2+) ([Ca(2+)]i); an effect mimicked by several membrane-impermeant CaSR agonists (e.g. the calcimimetic R-568) and that was blocked by the calcilytic Calhex-231. Block of TRPC channels attenuated the CaSR-dependent increases in [Ca(2+)]i, suggesting that Ca(2+) influx through TRPC channels contributes to the total [Ca(2+)]i signal evoked by the CaSR in NEBs. CaSR also regulated baseline [Ca(2+)]i in NEBs and, through paracrine signaling from Clara-like cells, coordinated intercellular communication in the NEB microenvironment. These data suggest that the NEB CaSR integrates multiple signals converging on this complex chemosensory unit, and is a key regulator of this intrapulmonary airway stem cell niche.

Purinergic signaling in the airways.

Evidence for a significant role and impact of purinergic signaling in normal and diseased airways is now beyond dispute. The present review intends to provide the current state of knowledge of the involvement of purinergic pathways in the upper and lower airways and lungs, thereby differentiating the involvement of different tissues, such as the epithelial lining, immune cells, airway smooth muscle, vasculature, peripheral and central innervation, and neuroendocrine system. In addition to the vast number of well illustrated functions for purinergic signaling in the healthy respiratory tract, increasing data pointing to enhanced levels of ATP and/or adenosine in airway secretions of patients with airway damage and respiratory diseases corroborates the emerging view that purines act as clinically important mediators resulting in either proinflammatory or protective responses. Purinergic signaling has been implicated in lung injury and in the pathogenesis of a wide range of respiratory disorders and diseases, including asthma, chronic obstructive pulmonary disease, inflammation, cystic fibrosis, lung cancer, and pulmonary hypertension. These ostensibly enigmatic actions are based on widely different mechanisms, which are influenced by the cellular microenvironment, but especially the subtypes of purine receptors involved and the activity of distinct members of the ectonucleotidase family, the latter being potential protein targets for therapeutic implementation.

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 GABAA and GABAB (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.

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.

Neuroepithelial bodies as mechanotransducers in the intrapulmonary airway epithelium: involvement of TRPC5.

In rodent lungs, a major part of the myelinated vagal airway afferents selectively contacts pulmonary neuroepithelial bodies (NEBs). Because most myelinated vagal airway afferents concern physiologically characterized mechanoreceptors, the present study aimed at unraveling the potential involvement of NEB cells in transducing mechanosensory information from the airways to the central nervous system. Physiological studies were performed using confocal Ca(2+) imaging of airway epithelium in murine lung slices. Mechanical stimulation by short-term application of a mild hypoosmotic solution (230 mosmol) resulted in a selective, fast, reversible, and reproducible Ca(2+) rise in NEB cells. Other airway epithelial cells could only be activated using more severe hypoosmotic stimuli (< 200 mosmol). NEB cells selectively expressed the Ca(2+)-permeable osmo- and mechanosensitive transient receptor potential canonical channel 5 (TRPC5) in their apical membranes, whereas immunoreactivity for TRP vanilloid-4 and TRP melastatin-3 was abundant in virtually all other airway epithelial cells. Hypoosmotic activation of NEB cells was prevented by GsMTx-4, an inhibitor of mechanosensitive ion channels, and by SKF96365, an inhibitor of TRPC channels. Short application of gadolinium, reported to activate TRPC5 channels, evoked a transient Ca(2+) rise in NEB cells. Osmomechanical activation of NEB cells gave rise to a typical delayed activation of Clara-like cells due to the release of ATP from NEB cells. Because ATP may activate the NEB-associated P2X(2/3) ATP receptor expressing myelinated vagal afferents, the current observations strongly suggest that pulmonary NEB cells are fully equipped to initiate mechanosensory signal transduction to the central nervous system via a purinergic signaling pathway.

Novel insights in the neurochemistry and function of pulmonary sensory receptors.

Afferent nerves in the airways and lungs contribute to optimisation of the breathing pattern, by providing local pulmonary information to the central nervous system. Airway sensory nerve terminals are consequently tailored to detect changes readily in the physical and chemical environment, thereby leading to a variety of respiratory sensations and reflex responses. Most intrapulmonary nerve terminals arise from fibres travelling in the vagal nerve, allowing a classification of "sensory airway receptors", based on their electrophysiologically registered action potential characteristics. Nowadays, at least six subtypes of electrophysiologically characterised vagal sensory airway receptors have been described, including the classical slowly and rapidly adapting (stretch) receptors and C-fibre receptors. The architecture of airways and lungs makes it, however, almost impossible to locate functionally the exact nerve terminals that are responsible for transduction of a particular intrapulmonary stimulus. With the advances in immunohistochemistry in combination with confocal microscopy, airway sensory receptor end organs can now be examined and evaluated objectively. Based on their "neurochemical coding", morphology, location and origin, three sensory receptor end organs are currently morphologically well characterised: smooth muscle-associated airway receptors (SMARs), neuroepithelial bodies (NEBs) and visceral pleura receptors (VPRs). The present information on the functional, morphological and neurochemical characteristics of these sensory receptors leads to important conclusions about their (possible) function. Currently, ex vivo lung models are developed that allow the selective visualisation of SMARs, NEBs and VPRs by vital staining. The described ex vivo models will certainly facilitate direct physiological studies of the morphologically and neurochemically identified airway receptors, thereby linking morphology to physiology by identifying in situ functional properties of a given receptor end organ.

Effects of aging and exercise training on the histological and mechanical properties of articular structures in knee joints of male rat.

The impact of aging on joints can have a profound effect on an individual's functioning. Our objectives were to assess the histological and mechanical properties of the knee joint capsule and articular cartilage with aging, and to examine the effects of exercise on age-related changes in the knee joint. 2-year-old Wistar rats were divided into a sedentary control group and an exercise-trained group. 10-week-old animals were used to investigate the changes with aging. The joint capsule and cartilage were evaluated with histological, histomorphometric, immunohistochemical, and mechanical analyses. Severe degenerative changes in articular cartilage were observed with aging, whereas exercise apparently did not have a significant effect. The articular cartilage of aged rats was characterized by damage to the cartilage surface, cell clustering, and an abnormal cartilage matrix. Histomorphometric analysis further revealed changes in cartilage thickness as well as a decreased number of chondrocytes. Aging led to stiffness of the articular cartilage and reduced the ability to dissipate the load and distribute the strain generated within the joint. Joint stiffness with aging was independent of capsular stiffness and synovitis was not a characteristic feature of the aging joint. This study confirms that aging alone eventually leads to joint degeneration in a rat model. The lack of recovery in aging joint changes may be due to several factors, such as the duration of the intervention and the regeneration ability of the cartilage.

Precision-cut vibratome slices allow functional live cell imaging of the pulmonary neuroepithelial body microenvironment in fetal mice.

We recently developed an ex vivo lung slice model that allows for confocal live cell imaging (LCI) of neuroepithelial bodies (NEBs) in postnatal mouse lungs (postnatal days 1-21 and adult). NEBs are morphologically well-characterized, extensively innervated groups of neuroendocrine cells in the airway epithelium, which are shielded from the airway lumen by 'Clara-like' cells. The prominent presence of differentiated NEBs from early embryonic development onwards, strongly suggests that NEBs may exert important functions during late fetal and neonatal life. The main goal of the present study was to adapt the current postnatal LCI lung slice model to enable functional studies of fetal mouse lungs (gestational days 17-20).In vibratome lung slices of prenatal mice, NEBs could be unequivocally identified with the fluorescent stryryl pyridinium dye 4-Di-2-ASP. Changes in the intracellular free calcium concentration and in mitochondrial membrane potential could be monitored using appropriate functional fluorescent indicators (e.g. Fluo-4).It is clear that the described fetal mouse lung slice model is suited for LCI studies of Clara cells, ciliated cells, and the NEB microenvironment, and offers excellent possibilities to further unravel the significance of NEBs during the prenatal and perinatal period.

Expression of mechanogated two-pore domain potassium channels in mouse lungs: special reference to mechanosensory airway receptors.

Afferent activities arising from sensory nerve terminals located in lungs and airways are carried almost exclusively by fibres travelling through the vagus nerve. Based on electrophysiological investigations, intrapulmonary airway-related vagal afferent receptors have been classified into three main subtypes, two of which are myelinated and mechanosensitive, i.e., rapidly and slowly adapting receptors. To allow for a full functional identification of the distinct populations of airway receptors, morphological and neurochemical characteristics still need to be determined. Nerve terminals visualised using markers for myelinated vagal afferents seem to be almost uniquely associated with two morphologically well-formed airway receptor end organs, smooth muscle-associated airway receptors (SMARs) and neuroepithelial bodies (NEBs), localised in airway smooth muscle and epithelium, respectively. Due to the lack of a selective marker for SMARs in mice, no further neurochemical coding is available today. NEBs are extensively innervated diffusely spread groups of neuroendocrine cells in the airway epithelium, and are known to receive at least two separate populations of myelinated vagal afferent nerve terminals. So far, however, no evidence has been reported for the expression of channels that may underlie direct sensing and transduction of mechanical stimuli by the receptor terminals in NEBs and SMARs. This study focused on the expression of mechanogated two-pore domain K(+) (K(2P)) channels, TREK-1 and TRAAK, in mouse airways and more particular in the NEB micro-environment and in SMARs by multiple immunostaining. TREK-1 could be detected on smooth muscle cells surrounding intrapulmonary airways and blood vessels, while TRAAK was expressed on myelinated vagal afferents terminating both in SMARs and in the NEB micro-environment. Co-stainings with known markers for subpopulations of myelinated vagal afferents and general neuronal markers revealed that all identified SMARs exhibit TRAAK immunoreactivity, and that at least three subpopulations exist in mouse airways. Also, the intraepithelial terminals of both subpopulations of NEB-associated myelinated vagal sensory nerve fibres were shown to express TRAAK. In conclusion, the present study finally characterised an intrinsically mechanosensitive ion channel, the K(2P) channel TRAAK, on the terminals of identified myelinated vagal nodose airway afferents, organised as SMARs and as components of the innervation of NEBs. These data support the hypothesis that both SMARs and NEBs harbour the morphological counterparts of electrophysiologically identified myelinated vagal airway mechanoreceptors. TRAAK appears to be strongly involved in regulating airway mechanosensing since it was found to be expressed on the terminals of all subpopulations of potential vagal mechanosensors.

Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging.

Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of complex sensory airway receptors involved in the regulation of breathing. Together with their surrounding Clara-like cells, they exhibit stem cell potential through their capacity to regenerate depopulated areas of the epithelium following lung injury. We have employed confocal live cell imaging microscopy and novel electrophysiological techniques in a new ex vivo lung slice model to unravel potential purinergic signaling pathways within the NEB microenvironment. Quinacrine histochemistry indicated high amounts of vesicular ATP in NEB cells. Using a "reporter-patching" method adapted to create a uniquely sensitive and selective biosensor for the direct detection of ATP release from NEBs ex vivo, we demonstrated quantal ATP release from NEBs following their depolarization. Enhancing enzymatic extracellular ATP hydrolysis or inhibiting P2 receptors confirmed the central role of ATP in paracrine interactions between NEB cells and Clara-like cells. Combined calcium imaging, pharmacology, and immunohistochemistry showed that ligand-binding to functional P2Y(2) receptors underpins the activation of Clara-like cells. Hence, NEB cells communicate with their cellular neighbors in the NEB microenvironment by releasing ATP, which rapidly evokes purinergic activation of surrounding Clara-like cells. Besides ATP acting on the P2X(3) receptor expressing vagal sensory nerve terminals between NEB cells, local paracrine purinergic signaling within this potential stem cell niche may be important to both normal airway function, airway epithelial regeneration after injury, and/or the pathogenesis of small cell lung carcinomas.

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 P2X(2) and P2X(3) 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.

Experimentally Induced Biliary Atresia by Means of Rotavirus-Infection Is Directly Linked to Severe Damage of the Microvasculature in the Extrahepatic Bile Duct.

Vascular damage has been reported to contribute to atresia formation in several diseases including biliary atresia. This study focused on the extrahepatic biliary plexus in experimental biliary atresia. Newborn BALB/cAnNCrl-pups were infected with rhesus rotavirus within 24 hr after birth to induce experimental biliary atresia. The extrahepatic biliary plexus was examined by confocal microscopy on whole-mount preparations, scored by three independent researchers, and further evaluated at the subcellular level with transmission electron microscopy. Imaging results revealed a progressive destruction of the extrahepatic biliary vascular plexus in the course of experimental biliary atresia induced by rotavirus infection. Endothelial cell damage was already visible as cell swelling and necrosis in the first days after infection and a damaged microcirculation that rapidly deteriorated with progression of obliterative cholangiopathy, was observed in the infected mice as early as 72 hr after birth. In experimental biliary atresia, the destruction of the extrahepatic biliary vascular plexus starts already in the first days postinfection and clearly precedes the morphological symptoms of atresia. The deterioration of the vascular bed architecture continues with disease progression. Therefore, we conclude that the (ultra)structural changes in the extrahepatic biliary microvasculature occurring before the visible onset of atresia has a predictive diagnostic value and this impairment in blood supply to the extrahepatic bile duct may be an important contributing factor to the pathogenesis of acquired biliary atresia. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:818-824, 2019. © 2018 Wiley Periodicals, Inc.