The Experts below are selected from a list of 39447 Experts worldwide ranked by ideXlab platform
Ian De Proost - One of the best experts on this subject based on the ideXlab platform.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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 P2Y2 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 P2X3 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.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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-...
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functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine Cells invariably accompanied by Clara-like Cells. Together with NEBs, Clara-like Cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem Cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal Live Cell Imaging of physiological reactions in identified NEBs and surrounding epithelial Cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like Cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated Cells, Clara Cells, and Clara-like Cells in Live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB Cells and ciliated Cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intraCellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial Cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extraCellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB Cells, while Clara-like Cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
Paul J Kemp - One of the best experts on this subject based on the ideXlab platform.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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 P2Y2 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 P2X3 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.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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-...
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functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine Cells invariably accompanied by Clara-like Cells. Together with NEBs, Clara-like Cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem Cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal Live Cell Imaging of physiological reactions in identified NEBs and surrounding epithelial Cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like Cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated Cells, Clara Cells, and Clara-like Cells in Live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB Cells and ciliated Cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intraCellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial Cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extraCellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB Cells, while Clara-like Cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
Jeanpierre Timmermans - One of the best experts on this subject based on the ideXlab platform.
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precision cut vibratome slices allow functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment in fetal mice
Advances in Experimental Medicine and Biology, 2012Co-Authors: Kathy Schnorbusch, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract: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).
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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 P2Y2 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 P2X3 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.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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-...
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functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine Cells invariably accompanied by Clara-like Cells. Together with NEBs, Clara-like Cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem Cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal Live Cell Imaging of physiological reactions in identified NEBs and surrounding epithelial Cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like Cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated Cells, Clara Cells, and Clara-like Cells in Live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB Cells and ciliated Cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intraCellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial Cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extraCellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB Cells, while Clara-like Cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
Inge Brouns - One of the best experts on this subject based on the ideXlab platform.
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precision cut vibratome slices allow functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment in fetal mice
Advances in Experimental Medicine and Biology, 2012Co-Authors: Kathy Schnorbusch, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract: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).
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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 P2Y2 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 P2X3 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.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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-...
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functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine Cells invariably accompanied by Clara-like Cells. Together with NEBs, Clara-like Cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem Cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal Live Cell Imaging of physiological reactions in identified NEBs and surrounding epithelial Cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like Cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated Cells, Clara Cells, and Clara-like Cells in Live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB Cells and ciliated Cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intraCellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial Cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extraCellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB Cells, while Clara-like Cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
Isabel Pintelon - One of the best experts on this subject based on the ideXlab platform.
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precision cut vibratome slices allow functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment in fetal mice
Advances in Experimental Medicine and Biology, 2012Co-Authors: Kathy Schnorbusch, Robrecht Lembrechts, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Dirk AdriaensenAbstract: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).
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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 P2Y2 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 P2X3 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.—De Proost, I., Pintelon, I., Wilkinson, W. J., Goethals, S., Brouns, I., Van Nassauw, L., Riccardi, D., Timmermans, J.-P., Kemp, P. J., Adriaensen, D. Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging.
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purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by Live Cell Imaging
The FASEB Journal, 2009Co-Authors: Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Ian De Proost, William J Wilkinson, Sofie Goethals, Luc Van Nassauw, Daniela Riccardi, Paul J KempAbstract: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-...
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functional Live Cell Imaging of the pulmonary neuroepithelial body microenvironment
American Journal of Respiratory Cell and Molecular Biology, 2008Co-Authors: Ian De Proost, Isabel Pintelon, Jeanpierre Timmermans, Inge Brouns, Daniela Riccardi, Paul J Kemp, Alfons B A Kroese, Dirk AdriaensenAbstract:Pulmonary neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine Cells invariably accompanied by Clara-like Cells. Together with NEBs, Clara-like Cells form the so-called "NEB microenvironment," which recently has been assigned a potential pulmonary stem Cell niche. Conclusive data on the nature of physiological stimuli for NEBs are lacking. This study aimed at developing an ex vivo mouse lung vibratome slice model for confocal Live Cell Imaging of physiological reactions in identified NEBs and surrounding epithelial Cells. Immunohistochemistry of fixed slices demonstrated that NEBs are almost completely shielded from the airway lumen by tight junction-linked Clara-like Cells. Besides the unambiguous identification of NEBs, the fluorescent dye 4-Di-2-ASP allowed microscopic identification of ciliated Cells, Clara Cells, and Clara-like Cells in Live lung slices. Using the mitochondrial uncoupler FCCP and a mitochondrial membrane potential indicator, JC-1, increases in 4-Di-2-ASP fluorescence in NEB Cells and ciliated Cells were shown to represent alterations in mitochondrial membrane potential. Changes in the intraCellular free calcium concentration ([Ca2+](i)) in NEBs and surrounding airway epithelial Cells were simultaneously monitored using the calcium indicator Fluo-4. Application (5 s) of 50 mM extraCellular potassium ([K+](o)) evoked a fast and reproducible [Ca2+](i) increase in NEB Cells, while Clara-like Cells displayed a delayed (+/- 4 s) [Ca2+](i) increase, suggestive of an indirect, NEB-mediated activation. The presented approach opens interesting new perspectives for unraveling the functional significance of pulmonary NEBs in control lungs and disease models, and for the first time allows direct visualization of local interactions within the NEB microenvironment.
