Springer Basel AG

Methods in Pulmonary Research

Edited by s. Uhlig A. E. Taylor

Editors:

Dr. Stefan Uhlig

Department of Pulmonary Pharmacology

Forschungszentrum Borstel

Parkallee 22

D-23845 Borstel

Germany

Library of Congress Cataloging-in-Publication Data

Professor Aubrey E. Taylor

Department of Physiology

Col lege of Medicine

University of Southern Alabama

Mobile, AL 36688

USA

Methods in pulmonary research/edited by S. Uhlig, A. E. Taylor.

p.cm.

Includes bibliographical references and index.

ISBN 978-3-0348-9803-4 ISBN 978-3-0348-8855-4 (eBook) DOI 10.1007/978-3-0348-8855-4

1. Respiratory organs--Research--Methodology. 2. Respiratory organs--Cytology--Research-­

Methodology. 1. Uhlig, S. (Stefan), 1957- .11. Taylor, Aubrey E.

OP121.M573 1998 98-14358

612.2'07'24--dc21 CIP

Deutsche Bibliothek Cataloging-in-Publication Data

Methods in pulmonary research/edited by S. Uhlig, A. E. Taylor.

Springer Basel AG, 1998

ISBN 978-3-0348-9803-4

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© 1998 Springer Basel AG Originally published by Birkhăuser Verlag in 1998 Softcover reprint of the hardcover 1 st edition 1998

Printed on acid-free paper produced from chlorine-free pulp. TCF 00

Cover illustration: Taken from «Physiologie der Atmung», with the friendly permission of © 1980

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ISBN 978-3-0348-9803-4

987654321

Contents

• List of corresponding authors . . . . . . . . . . . . . . . . . . . . . . . . XIX

• Preface ..................................... XXIII

Airways

II Measurement of lung function in rodents in vivo H.G Hoymann and U. Heinrich

Spontaneous respiration Pulmonary manoeuvres

Material and equipment ... Lung function laboratory

Methods ........... . Preparation and calibration Pulmonary function testing

Examples for applications Discussion ... Troubleshooting

References

The isolated perfused lung s. Uhlig

Advantages and disadvantages of perfused lungs Theoretical background . .

Vascular resistance Respiratory mechanics

Material and equipment Artificial thorax chamber and ventilation Perfusion

............. 1

.2

.3

.4

.5 10

· 10 · 12 · 21 · 23 .27

· 28

· 29

.29

· 31 · 31 · 31 .32

34 · 37

V

VI

Weight measurement . 39 Gas exchange . 39

Methods ......... . 40 Surgery and setting up the lung 40 Criteria for viability . 43 Cleaning the apparatus 43 An example application 44

Discussion ......... 46 Interpretation of the results 46 Constant flow (CFP) versus constant pressure perfusion (CPP) 47 Negative or positive pressure ventilation .... 49 Choice of perfusate ............... 49 Recirculating versus non-recirculating perfusion 50 Additional experimental options ........ 51

Troubleshooting ................... 52 Final comments .................................. 52

References .................................. 53

Lung explants ......... . E.A. Cowley and D.H. Eidelman

Material and equipment Preparation of culture media Preparation of agarose Preparation of animals Preparation of explants Image acquisition ... Variations on this technique

Applications ......... . Effects of bronchoconstriction Measurements of mucociliary clearance Measurements of pulmonary vasculature Long term explant culture techniques Investigations of protein and gene expression

Troubleshooting Discussion ....

Acknowledgements

References

Tracheal preparations M. Eltze

Methods Guinea pig tracheal preparations

Immersion techniques

.57

57 57 59 59 59 61 63 63 63 65 66 66 67 67 68

70 70

. 71

.72 72

.72

Tracheal chain Spirally cut trachea . Zig-zag tracheal strip Tracheal tube preparations

Superfusion techniques Electrically stimulated trachea Epithelium-denuded trachea .

Conclusion

References

Vessels

Intravital microscopy: Airway circulation M.R. Corboz and S. T. Ballard

Materials and equipment Microscope Video equipment Peripheral equipment Ventilation Solutions

Methods Surgery . Experimental procedure Species differences

Discussion

References ...... .

The bronchial circulation R.P. Michel

.72

.74 · 76 · 81 · 84 · 84 · 85 .87

· 87

· 93

· 94 · 94 · 94 · 97 · 97 · 98 · 99 · 99 100 104 104

106

107

Importance and role of the bronchial circulation . . . . . . . . . . . . . . . 107 Postobstructive pulmonary vasculopathy (POPV) and principles of the techniques ................................ 111

Material and equipment .......................... 112 Production of POPV in dogs, rats and guinea pigs: Ligation of the left main pulmonary artery . . . . . . . . . . . . . . . . . . . . . . . . . 112 In situ perfused LLL preparation .................... 113 Morphological assessment of the bronchial and pulmonary vasculature using light microscopy and morphometry ............... 115

Methods ................................ 116 Surgical ligation of the left main pulmonary artery in dogs, rats and guinea pigs 116

Canine model ......................... 116

VII

Rat and guinea pig model ....................... 117 In situ perfused LLL preparation to measure pulmonary and bronchial vascular flows, pressures and resistances using modified AO and VO and bronchial vascular micropuncture . . . . . . . . . 118

Procedure for the in situ perfused LLL preparation ....... 118 AO and VO measurements ................... 119 Modified in situ perfused LLL preparation for bronchial collateral vascular pressure measurements by micropuncture ....... 122

Morphological assessment of the bronchial and pulmonary vasculature, using light microscopy and morphometry ................ 123

Measurement of pulmonary vascular medial thickness and muscu-larization in lungs injected with pigmented gelatin-barium mixtures 123

Fixation and preparation . . . . . . . . . . . . . . . 123 Morphometry .................... 123

Assessment of proliferation in the bronchial vasculature 126 Bronchial vessel number per airway ........ 126 Assessment of bronchial vascular endothelial proliferation using bromodeoxyuridine (BrdU) labeling 126

Discussion and troubleshooting ........ 129 Production of POPV ............... 129 In situ perfused left lower lobar preparation ... 129 Morphological assessment of the bronchial and pulmonary vasculature 130

Acknowledgements 132

References .................................. 132

Segmental vascular resistance and compliance from vascular occlusion ............................ . G.A. Dawson, S.H. Audi, J.H. Linehan

Methods The lumped parameter RCR model . The continuous RC distribution More distributed lumped parameter models

The 3C4R model The 3C2R model

Arterial occlusion in vivo

Acknowledgements

References ........ .

135

139 139 140 144 144 145 152

156

157

Edema

Experimental and clinical measurement of pulmonary edema 161 S. 8ayat and F. Grimbert

Definitions ............................. 163

VIII

Lung water filtration and clearance Lung water filtration ..... . Lung water clearance .... . Edema formation = filtration - clearance

Lung protein filtration and clearance Protein filtration ............. . Protein clearance ............ . Protein accumulation = filtration - clearance

Mechanisms of pulmonary edema ...... . Material and methods .................. .

Quantifying pulmonary edema formation and clearance in the experimental setting ........ .

Lung microvascular filtration rate Isolated lung Intact lung . . . .

Lung water clearance Lymph flow Airway fluid clearance Pleural fluid clearance

Lung water = filtration-clearance Lung weight (isolated lung) . Indicator dilution Gravimetry ....... . Pathology ........ .

Starling equation components Microvascular pressure . Indirect measurements .. Interstitial liquid pressure . Plasma protein osmotic pressure Interstitial colloid osmotic pressure Filtration coefficient (Kt,e) PS ............ . Sigma (0) ,., ..... . The filtered volume method

Quantifying pulmonary edema formation and clearance in the

163 163 164 165 166 166 169 170 171 171

171 171 172 172 173 173 174 175 176 176 176 177 177 178 178 179 181 182 183 184 187 193 195

clinical setting ......................... 197 Lung water and edema ...............,.. 197

Indicator dilution technique: Extravascular thermal lung volume 197 Imaging techniques ................. 198 Transthoracic bioimpedance ............ . 202

Solute filtration: Capillary-alveolar macro-molecule transport . 203 External radioflux detection . . . . . . . . 203

Positron emission tomography (PET) ... . . . . . . 205 Magnetic resonance imaging (MRI) ,....... . 205 Edema fluid protein and Bronchoalveolar Lavage fluid . 205

Starling equation components .............. . 207 Microvascular pressure ................ . 207 Small solute PS .......,',................. 211

Lung water clearance . . . . . . . . . . . . . . . . . . . . . . . . . . 212

IX

Epithelial permeability: DTPA clearance ............. . 212 Identifying pulmonary edema and its mechanism in the clinical setting . 213

Present ......................... . 214 Diagnosis and quantification of pulmonary edema . 214 Identification of mechanisms ............ . 214 Identification of an imbalance in Starling forces . . . . 215 Identification of an altered transvascular permeability . 216

Future . . . . . . . . . . . . . . . . 219 Role of edema clearance ... . 219

Conclusion

Role of exchange surface area Macromolecule transport Anatomic distribution

References ........... .

.220

.220

.222

.222

.223

II Neurogenic inflammation in the airways: Measurement of microvascular leakage ................ . ....... . 231

x

M.G. Belvisi and D.F. Rogers

Material and equipment Methods

Anaesthesia ... Surgery Experimental procedure

Direct electrical stimulation of the vagus nerve Chemical stimulants

Capsaicin ... Bradykinin . . . Cigarette smoke Sodium metabisulphite

Other stimulants Quantification ....... .

Evans blue dye technique Monastral dyes as tracers [1251]-albumin

Application .. . . . Species differences

Discussion ...... . Troubleshooting

Difficulty in cannulating veins Difficulty in cannulating arteries Poor blood pressure trace

Acknowledgements

References ......... .

.233

.235

.235

.236

.238

.239

.242

.242

.242

.242

.244

.244

.244

.244

.246

.246

.247

.247

.248

.248

.248

.249

.249

.249

.250

nil Intravital microscopy: Surface lung vessels and interstitial pressure 251

- D. Negrini

III

Material and equipment Pipette preparation

Methods ...... . Surgery

Cannulation of left atrium and pulmonary artery Preparation of the intact parietal pleural window

Video imaging analysis Experimental procedure

Application ........ . Physiological conditions Transition to edema Lung fluid balance in the newborn Mechanical behavior of interstitial matrix . Video image analysis of the superficial lung structures

Species differences Discussion ..... . Troubleshooting

Technical problems General drawbacks of the micropuncture technique Micropuncture through the intact parietal pleural window

References ......................... .

Lymphatics ................ . RE Drake and J.e. Gabel

Basic physiology of the lymphatic system Methods ............ .

Common technical problems Lymph flow rate measurement Lymph protein concentration

Discussion

Acknowledgements References ....

Airway liquid

Evaluation of secretory and transport processes which determine the composition of airway surface liquid R.H. Scott, M. Acevedo and A. Griffin

Methods Studies using isolated trachea

· 253 · 253 · 256 · 256 · 258 .259 · 260 · 261 · 263 · 264 · 267 · 267 · 268 · 268 · 269 .270 .272 · 272 · 272 .273

.273

· 275

· 275 .277 · 279 · 280 · 282 · 285

· 287

· 287

291

292 292

XI

XII

The ferret isolated whole trachea in vitro preparation Protocol for stimulating secretions Assay for lysozyme ................ . Albumin transport ................. . Measurement of potential difference across the tracheal wall

Ion transport across the airways ................ . Measurement of the ionic composition of periciliary fluid . . . Electrophysiological methods used in the investigation of ion transport in the airways. Measurement of short-circuit current {lsd and transepithelial resistance (RT) •.•••.•.•••.••.•

Acid/base transport .. . . . . . . . . . . . . . . . . . . . . . . Acidlbase transport across cell membranes of isolated tracheocytes

Culture of ovine tracheal submucosal gland cells ............ . Preparation and characterisation .................. .

292 293 295 296 296 297 297

298 299 303 304 304

Methods for studying the effects of secretagogues on lysozyme release from cultured ovine trachea submucosal gland cells 305

307 310 310

Electrophysiology Troubleshooting . . Conclusions

Acknowledgements

References

Bronchoalveolar lavage U. Wagner and P. von Wichert Methods

Endoscopic techniques of SAL in man Premedication Local anaesthesia Site of lavage .. Fluid used to perform lavage Methods to instil and recover the fluid Volumes of fluid to be used . . . . .

Recovery ........... . Should the first aliquot of lavage be processed separately?

Handling of the harvested lavage material Mucus filtration Conventional stains . . . . . .

Membrane filters Cytocentrifuge preparations Romanovsky stain Papanicolaou stain Grocott methenamine silver stain-microwave method Gram stain Iron stain ...... .

In situ DNA hybridization Immunocytochemical stains

311 . 311

313

313 313

. 314 315 315 315 316 316 318 318 319 319 320 320 321 321 322 323 323 324 325 326

Procedure (as one example out of a variety of techniques) . 327 Flow cytometry to quantify lymphocyte subsets . 328

Principle of flow cytometry . 328 Preparation of samples ..... . 330 Analysis by immunofluorescence . 330

Electron microscopy . 332 Differential cell counts . 332 Cultures from BAL 334

Microbial culture . 334 Routine culture . 335 Fungal culture . . 335 Mycobacterial culture 337 Viral culture ..... . 337

Analysis of soluble components of the epithelial lining fluid . 337 Attempt to quantify lavage material . 340

Complications of lavage ..... . 341 Preparation techniques in animals . 344

References .............. . 345

Assessment of surfactant function B. Robertson and S. SchOrch

. 349

In vitro methods for assessment of surfactant function ........... 350 The Langmuir-Wilhelmy balance ................. 350 Bubbles on a tube: The pulsating bubble surfactometer according to Enhorning 354 Captive bubbles . . 356 Microbubble stability 358

Adsorption ...... . 360 The rate of adsorption . 361 Adsorption characteristics of pulmonary surfactants . 362

Spreading . . . . . . . . . . . . . . . . . . . . . 363 The measurements of adsorption and spreading . 364

Adsorption .......... . 364 Spreading ........... . 365

Measurement of surface tension in situ 366 Choosing a test fluid ....... 366 Calibration ............ 368 Alveolar miropuncture and surface tension in situ 369

Influence of surfactant on static lung pressure-volume characteristics 369 Animal models for in vivo evaluation of exogenous surfactants 372

Preterm newborn animals 372 In vivo lung lavage . 376

Comments . 378

Acknowledgements . 379

References .... . 380

XIII

Cell culture

XIV

Isolation of type II alveolar epithelial cells J.S. Lwebuga-Mukasa

Why do we need to isolate cells for in vitro studies? Strategies for isolation of type II cells

Methods ....................... . Steps in type II cell isolation ........... .

Removal of blood products and alveolar macrophages Dissociating lung tissues with digestive enzymes

Selecting the enzyme combinations Recovery of dissociated cells . . . .

Strategies for selective isolation Characterization of isolated type II cells

Morphology ......... . Surfactant phospholipid profile ... . Immunochemical techniques .... .

Adhesion and culture on different substrata Selection of CO2 atmospheric conditions Selection of substrata for culture ....

Mn++-enhanced technique for pneumocyte isolation Reagents ..... Type II Cell Isolation

Discussion ........ . Limitation of studies of isolated cells Alternatives to cell isolation

Acknowledgments

References .......... .

.387

· 387 388 389 389 389 389 389

.390 · 390 · 392 · 392 · 393 · 393 · 394 · 394 · 394 · 395 · 395

396 · 399 · 399 .400

.400

.400

Endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 403 T. Stevens, G.H. Brough, T.M. Moore, P. Baba/ and W,J. Thompson

Material and methods .............................. 411 Tissue culture of pulmonary artery endothelial cells

Equipmentfmedia and chemicals ........ . ........ 411

· 412 Solutions/media preparation ............. . · 412 Macrovascular endothelial cell isolation procedure · 413

Tissue culture of pulmonary microvascular endothelial cells: Method 1 415 Equipmentfmedia and chemicals ........ . · 415 Solutions/media preparation .......... . · 416 Microvascular endothelial cell isolation procedure · 416 Endothelial cell procurement .......... . · 417

Tissue culture of pulmonary microvascular endothelial cells: Method 2 · 418 Solutions/media preparation ................. . · 418 Microvascular endothelial cell isolation procedure. lung isolation · 419

Endothelial cell procurement .. Establishment of primary cultures

Verification of endothelial cells Discussion Troubleshooting

References ..

Histology

Studying lung ultrastructure H. Fehrenbach and M. Ochs

Material and equipment List of equipment

Methods

Solutions Primary fixative for conventional TEM and SEM Primary fixative for immunocytochemistry Cryoprotectant for infiltration of specimens ... Postosmication for SEMITEM ... Postiixation for TEM .. . Postfixation for SEM .. .

Modes of fixation

· 420 .420 · 421 · 422 .423

· 426

· 429

· 431 · 431 · 431 · 431 · 433 · 433 .434 · 434 · 434 · 435 · 435

Fixation by airway instillation . 435 Fixation by vascular perfusion . 436 Combined chemical/physical fixation for immunocytochemistry . 438 Fixation by immersion into a chemical fixative . 438

Sampling of tissue blocks ......... . 439 Processing and embedding of tissue blocks . 442

For conventional LM ......... . 442 For conventional TEM . . . . . . . . . . 443 For TEM based immunocytochemistry . 444 For SEM modified according to an OTOTO method . 445

Sampling of micrographs . 446 Discussion .............. . 448

Selecting the mode of fixation .. . 448 Selecting the mode of application . 449 Selecting the fixing agent . 450 Selecting the mode of tissue processing . 450 Stereological analysis . 451 Interpretation . 452

Acknowledgements . 452

References .... . 452

XV

Autoradiography in the lung R.G. Goldie and P.J. Rigby

Materials and equipment Methods ......... .

Slide preparation Slide cleaning and subbing Preparation of chrome alum/gelatin solution

Application ............. . Preparation of emulsion-coated coverslips

Safelight illumination Emulsion coating of coverslips .

Tissue preparation . . . . . . . . Tracheal and bronchial tissue Peripheral lung tissue

Cutting frozen sections Radioligand binding procedures . . . Attachment of emulsion-coated covers lips Emulsion-dipped auto radiograms Film auto radiograms . . . . . . . . . . . Exposure of autoradiograms ..... . Development and staining of auto radiograms .

Preparation for development and tissue staining Processing solutions Photographic development

Tissue staining and mounting Materials Procedure

Chemography controls Discussion ....... .

Controls ...... . Choosing the appropriate autoradiographic method

Emulsion-dipped preparations Emulsion-coated coverslip preparations Film imaging

Microscopy Photography . Image analysis

References ....

Further methods

XVI

Application of aerosols W. Koch Methods

.455

.458

.458

.462

.462

.462

.463

.463 · 463 · 464 · 465 · 465 .465 · 466 · 466 .467 .468 .469 · 469 .470 .470 · 471 · 471 .472 .472 .473 .474 .474 .475 .475 .475 .477 .478 .478 .478 .480

.480

.485

.487

Physical characterization of aerosols Methods of aerosol generation Methods of aerosol measurement Exposure methodology

References ............. .

rUt1 Cryopreservation of human pulmonary tissues - E. MOller-Schweinitzer

.487 · 491 · 499 · 504

· 507

· 509

Mechanisms of freezing injury . . . . . . . . . . . . . . . . . . . . . . . . 509 Cryoprotective agents and cryomedia .................... 510 Freezing procedure and storage temperature ................ 511 Thawing procedure ............................. 511

Methods, material and equipment . 513 Cryopreservation ................... . 513

Freezing procedure .. .. .. .. .. .. .. .. . 513 Temperature and sample handling during storage . 515 Thawing procedure ..... . 516 Inventory control . 516

Post-thaw functional recovery . 517 Conclusion ....... . 519 Safety recommendations · 520 References ...... . · 520

II Appendix I. Physiological data of various mammalian species · 523

References ................................ . 524

• Appendix II. List of suppliers ........................ 527

• Subject index ................................. 531

XVII

List of corresponding authors

Dr. Sam Bayat TIM C-PRETA, UMR CNRS 5525,

Dept. de Physiologie, Facult6 de M6decine de Grenoble

F-38700 La Tronche, France

Tel. 0033 476 6371-38, Fax 00334766371-86, E-mail: Sam.Bayat@imag.fr

Dr. Maria Belvisi Rhone-Poulenc Rorer Research & Development

Dept. of Pharmacology, Dagenham Research Centre

Rainham Road South, Dagenham, Essex, RMlO 7XS, UK

Tel. 0044 181 919-2075, Fax 0044181919-2497, E-mail: m.belvisi@ic.ac.uk

Dr. Michel R. Corboz Dept. of Physiology, MSB 3024, Univ. of South Alabama, College of Medicine

Mobile, AL 36688-0002, USA

Tel. 001334460-7826, Fax 001334460-6464

E-mail: mcorboz@jaguar1.usouthal.edu

Dr. Christopher Dawson Dept. of Physiology, Medical College of Wisconsin, Research Service 151

5000 West National Avenue, Milwaukee, WI 53295-1000, USA

Tel. 001 414384-2000 ext. 1440, Fax 001 414 384-0115

E-mail: dawson@vms.csd.mu.edu

Dr. Robert E. Drake Dept. of Anesthesiology, The University of Texas,

XIX

Health Science Center at Houston

6431 Fannin, TX 77030, USA

Tel. 001 713 792-5566, Fax 001 713 794-4157

Prof. David Eidelman

Meakins Christie Laboratories, McGill University

Montreal, Canada H2X 2P2

Tel. 001514398-3864, Fax: 001514398-7483

E-mail: david@meakins.lan.mcgill.ca

Dr. Manfrid Eltze

BYK Gulden Lomberg Chemische Fabrik

Byk-Gulden-Str. 2 - FP2, Postfach 10 03 10, D-78403 Konstanz, Deutschland

Tel. 0049-7531-84-2617, Fax 0049-7531-84-2474

Dr. Heinz Fehrenbach

Institut fUr Pathologie, Universitats-Klinikum Gustav Carus

Fetscherstr. 34, D-01307 Dresden, Germany

Tel. 0049351458-5277, Fax 0049351458-4328

Prof. Roy G. Goldie, Ph.D. Dept. of Pharmacology, University of Western Australia

Nedlands, WA, 6907

Tel. 0061 9 346-2812, Fax: 0061 9346-3469

E-mail: rgoldie@receptor.pharm.uwa.edu.au

Dr. Gerd Hoymann, Prof. Dr. Uwe Heinrich

Frauenhofer-Institut fur Aerosolforschung

Nicholai-Fuchs Str. 1, D-30625 Hannover, Germany

Tel. 0049 511 5350-404, Fax: 0049511 5350-155

E-mail: hoymann@ita.thg.de

xx

Dr. Wolfgang Koch Frauenhofer-Institut fiir Aerosolforschung Nicholai-Fuchs Str. 1, D-30625 Hannover, Germany

Tel. 0049 511 5350-117, Fax: 0049 511 5350-155

Dr. Jamson Lwebuga-Mukasa Lung Biology Research Program, Pulmonary and Critical Care Division Dept. of Internal Medicine, SUNY at Buffalo School of Medicine Buffalo General Hospital 100 High Street, Buffalo, New York 14203, USA

Tel: 001 716 859-3682, Fax: 001 716859-3620 E-mail: jlwebuga@ubvm.cc.buffalo.edu

Dr. Rene P. Michel Dept. of Pathology, McGill University, Lyman Duff Medical Sciences Bldg. 3775 University St., Montreal, PQ, Canada H3A 2B4

Tel. 001514398-7194, Fax 001514398-7446 E-mail: michel@pathology.lan.mcgill.ca

Dr. Else Miiller-Schweinitzer Dept. of Internal Medicine, Clinical Pharmacology, University of Basel Peters graben 4, CH-4031 Basel, Switzerland

E-mail: muellere@ubaclu.ch

Dr. Daniela Negrini Instituto di Fisiologica Umana 1, Univesrita di Milano Via Magiagalli 32, 1-20133 Milano, Italy

Tel. 0039 2 7063-8768, Fax 0039 2 7063-0032

Dr. Bengt Robertson Div. for Experimental Perinatal Pathology, Karolinska Hospital S-l71 76 Stockholm, Sweden

Tel. 0046 8 729-6160/6161, Fax 00468729-6165

XXI

Dr. Rod Scott Dept. of Biomedical Sciences, Marischal College, Aberdeen University Aberdeen AB9 lAS, Scotland, UK

Tel. 0044 12242730-51, Fax 0044 12242730-19, E-mail: rod.scott@abdn.ac.uk

Dr. Troy Stevens Dept. of Pharmacology MSB 3130, University of South Alabama, College of Medicine Mobile, AL 36688-0002, USA

Tel. 001334460-6497, Fax 001334460-6798 E-mail: tstevens@jaguar1.usouthal.edu

Dr. Stefan Uhlig Division of Pulmonary Pharmacology, Research Center Borstel Parkallee 22, D-23845 Borstel, Germany

Tel. 00494537 188-478, Fax 0049 4537 188-778 E-mail: Suhlig@fz-borstel.de

Dr. Peter von Wichert Klinikum der Philippis-Universitat Marburg Baldingerstrasse D-35043 Marburg, Germany

Tel. 0049 642128-6451, Fax 0049 642128-8987

XXII

Preface

Pulmonary diseases have increased in incidence over the last 40 years and it is

obvious that basic and clinical pulmonary research must be intensified in order

to understand the underlying mechanisms that cause each particular disease. This

book affords renowned experts the opportunity to present a broad range of meth­

ods now used in pulmonary research. The techniques span the range from eva­

luating cell mechanisms in endothelial and epithelial cell cultures to those stu­

dying the inflammatory responses of in situ lungs. The theoretical background of

each technique is presented in some depth whenever possible. The major em­

phasis of this book lies in the practical application of the methods and it consti­

tutes a valuable guide for investigators not familiar with basic pulmonary meas­

urements that can be incorporated into experiments within their laboratories.

The fIrst part of the book describes techniques used to study lung mechanics

and airway smooth muscle. The fIrst four chapters describe in situ lung prepara­

tions, isolated perfused lungs, lung explants and classical tracheal preparations.

The techniques described in these chapters using perfused lungs and lung ex­

plants can easily be extrapolated to study pulmonary vessels. Chapters 5 to 7 pro­

vide important new methods for studying the pulmonary vasculature in great de­

tail, such as intravital microscopy, pulmonary vascular occlusion techniques and

bronchial circulation studies. Since edema formation is a broad and important

fIeld in pulmonary research, the edema section begins with a review on the me­

thodology in this fIeld. Chapters 9 to 11 describe in detail how microvascular

leakage can be assessed, how intravital microscopy can be applied to interstitial

studies and what techniques are now available to study the lymphatic system.

The next three chapters then present methods to evaluate airway secretion and

their contents in vitro, using bronchoalveolar lavage and surfactant activities.

The lung consists of more than 40 different cell types, some of which are isolat­

ed into cultures and monolayers in many laboratories. With regard to harvesting

XXIII

and culturing techniques, only type II alveolar epithelial cells and endothelial

cells are considered in chapters 15 and 16. Basic histological techniques are pre­

sented in chapters 17 and 18. Chapter 19 explains how to generate aerosols and

how to introduce them into the respiratory tract. Finally, chapter 20 explains how

lung tissue can be preserved over long periods of time to maintain its structure

and even function.

Obviously, many other techniques, such as those used in biochemistry or mo­

lecular biology, are now extensively used in lung studies, and are described in

detail in many other excellent books. In this work we have presented only those

techniques useful to evaluating the lung function at the organ level in order to

stimulate continued pulmonary research in organs and relate these measures to

data collected at the cellular level.

Stefan Uhlig

Borstel, Germany

XXIV

Aubrey E. Taylor

Mobile, AL, USA

October 1997