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Diving under a Microscope—A New Simple and Versatile In Vitro Diving Device for Fluorescence and Confocal Microscopy Allowing the Controls of Hydrostatic Pressure, Gas Pressures, and Kinetics of Gas Saturation

Published online by Cambridge University Press:  17 April 2013

Qiong Wang ,
Marc Belhomme ,
François Guerrero ,
Aleksandra Mazur ,
Kate Lambrechts  and
Michaël Theron
Qiong Wang
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
Marc Belhomme
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
François Guerrero
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
Aleksandra Mazur
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
Kate Lambrechts
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
Michaël Theron*
Affiliation:
Laboratory ORPHY, Université Européenne de Bretagne, Université de Brest, 6 Avenue Le Gorgeu CS 93837, 29238 Brest-CEDEX 3, France
*
*Corresponding author. E-mail: Michael.theron@univ-brest.fr
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Abstract

How underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Keywords

air divedecompression sicknessactivity of arterial endothelial cellkinetics of gas saturationin vitro real-time monitoringfluorescence and confocal microscopy
Type
Equipment and Techniques Development: Biological
Information
Microscopy and Microanalysis , Volume 19 , Issue 3 , June 2013 , pp. 608 - 616
Copyright
Copyright © Microscopy Society of America 2013 

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References

Besch, S.R. & Hogan, P.M. (1996). A small chamber for making optical measurements on single living cells at elevated hydrostatic pressure. Undersea Hyperb Med 23, 175–184.Google ScholarPubMed
Bosco, G., Yang, Z.J., Savini, F., Nubile, G., Data, P.G., Wang, J.P. & Camporesi, E.M. (2001). Environmental stress on diving-induced platelet activation. Undersea Hyperb Med 28, 207–211.Google ScholarPubMed
Brouns, R. & De Deyn, P.P. (2009). The complexity of neurobiological processes in acute ischemic stroke. Clin Neurol Neurosurg 111, 483–495.CrossRefGoogle ScholarPubMed
Brubakk, A.O. & Møllerløkken, A. (2009). The role of intra-vascular bubbles and the vascular endothelium in decompression sickness. Diving Hyperb Med 39, 162–169.Google ScholarPubMed
Crenshaw, H.C., Allen, J.A., Skeen, V., Harris, A. & Salmon, E.D. (1996). Hydrostatic pressure has different effects on the assembly of tubulin, actin, myosin II, vinculin, talin, vimentin, and cytokeratin in mammalian tissue cells. Exp Cell Res 227, 285–297.CrossRefGoogle ScholarPubMed
Crenshaw, H.C. & Salmon, E.D. (1996). Hydrostatic pressure to 400 atm does not induce changes in the cytosolic concentration of Ca2+ in mouse fibroblasts: Measurements using fura-2 fluorescence. Exp Cell Res 227, 277–284.CrossRefGoogle ScholarPubMed
D'Agostino, D.P., McNally, H.A. & Dean, J.B. (2012). Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications. J Microsc 246, 129–142.CrossRefGoogle ScholarPubMed
Davis, J.S. & Gutfreund, H. (1976). The scope of moderate pressure changes for kinetic and equilibrium studies of biochemical systems. FEBS Lett 72, 199–207.CrossRefGoogle ScholarPubMed
Fichtlscherer, S., Breuer, S. & Zeiher, A.M. (2004). Prognostic value of systemic endothelial dysfunction in patients with acute coronary syndromes: Further evidence for the existence of the “vulnerable” patient. Circulation 110, 1926–1932.CrossRefGoogle ScholarPubMed
Frey, B., Hartmann, M., Herrmann, M., Meyer-Pittroff, R., Sommer, K. & Bluemelhuber, G. (2006). Microscopy under pressure—An optical chamber system for fluorescence microscopic analysis of living cells under high hydrostatic pressure. Microsc Res Tech 69, 65–72.CrossRefGoogle ScholarPubMed
Geeves, M.A. & Ramatunga, K.W. (1990). Effect of hydrostatic pressure on isometric concentration of intact fibre bundles from rat muscles. J Physiol (Lond) 425, 16.Google Scholar
Hartmann, M., Kreuss, M. & Sommer, K. (2004). High pressure microscopy—A powerful tool for monitoring cells and macromolecules under high hydrostatic pressure. Cell Mol Biol (Noisy-le-grand) 50, 479–484.Google ScholarPubMed
Hogan, P.M., Ornhagen, H.C., Doubt, T.J., Laraway, B.S., Morin, R.A. & Zaharkin, J. (1981). Hyperbaric chamber for evaluating hydrostatic pressure effects on tissues and cells. Undersea Biomed Res 8, 51–58.Google ScholarPubMed
Horstman, L.L., Jy, W., Jimenez, J.J. & Ahn, Y.S. (2004). Endothelial microparticles as markers of endothelial dysfunction. Front Biosci 9, 1118–1135.CrossRefGoogle ScholarPubMed
Jimenez, J.J., Jy, W., Mauro, L.M., Soderland, C., Horstman, L.L. & Ahn, Y.S. (2003). Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis. Thromb Res 109, 175–180.CrossRefGoogle ScholarPubMed
Koyama, S. (2007). Cell biology of deep-sea multicellular organisms. Cytotechnology 55, 125–133.CrossRefGoogle ScholarPubMed
Koyama, S., Miwa, T., Sato, T. & Aizawa, M. (2001). Optical chamber system designed for microscopic observation of living cells under extremely high hydrostatic pressure. Extremophiles 5, 409–415.CrossRefGoogle ScholarPubMed
Laden, G., Madden, L. & Purdy, G. (2004). Endothelial damage as a marker of decompression stress. Undersea Hyperb Med 31, 344.Google Scholar
Madden, L.A. & Laden, G. (2007). Endothelial microparticles in vascular disease and as a potential marker of decompression illness. Eur J Underw Hyperb Med 8, 6–10.Google Scholar
Martin, J.D. & Thom, S.R. (2002). Vascular leukocyte sequestration in decompression sickness and prophylactic hyperbaric oxygen therapy in rats. Aviat Space Environ Med 73, 565–569.Google ScholarPubMed
Mazur, A., Wang, Q., Lambrechts, K., Belhomme, M., Theron, M. & Guerrero, F. (2012). Influence of decompression sickness on the endothelium dependent and independent relaxation in isolated rat vessels. In Abstract and Proceedings from EUBS Annual Scientific Meeting 2012, Marroni, A., Medić, M. & Sedlar, M. (Eds.), p. 68. Serbia: European Underwater and Baromedical Society.Google Scholar
Pagliaro, L., Reitz, F. & Wang, J. (1995). An optical pressure chamber designed for high numerical aperture studies on adherent living cells. Undersea Hyperb Med 22, 171–181.Google ScholarPubMed
Peters, S.C., Reis, A. & Noll, T. (2005). Preparation of endothelial cells from micro- and macrovascular origin. In Practical Methods in Cardiovascular Research, Dhein, S., Mohr, F.W. & Delmar, M. (Eds.), pp. 618–620. Berlin, Heidelberg: Springer-Verlag.Google Scholar
Pickles, D.M., Ogston, D. & Macdonald, A.G. (1990). Effects of hydrostatic pressure and inert gases on platelet aggregation in vitro . J Appl Physiol 69, 2239–2247.CrossRefGoogle ScholarPubMed
Pontier, J.M., Vallée, N., Ignatescu, M. & Bourdon, L. (2011). Pharmacological intervention against bubble-induced platelet aggregation in a rat model of decompression sickness. J Appl Physiol 110, 724–729.CrossRefGoogle Scholar
Salmon, E.D. & Ellis, G.W. (1975). A new miniature hydrostatic pressure chamber for microscopy. Strain-free optical glass windows facilitate phase-contrast and polarized-light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature. J Cell Biol 65, 587–602.CrossRefGoogle ScholarPubMed
Wisloff, U., Ellingsen, O. & Kemi, O.J. (2009). High-intensity interval training to maximize cardiac benefits of exercise training? Exerc Sport Sci Rev 37, 136–146.CrossRefGoogle ScholarPubMed