drug discovery and evaluation || respiratory activity

Chapter DRespiratory Activity

D.1 In Vitro Tests . . . . . . . . . . . . . . . . . . . . . 511D.1.0.1 Histamine (H1) Receptor Binding . . . 511D.1.0.2 Muscarinic Receptor Binding . . . . . . . 512D.2 Effects on Air Ways . . . . . . . . . . . . . . . 514D.2.1 Tests in Isolated Organs . . . . . . . . . . . . 514D.2.1.1 Spasmolytic Activity

in Isolated Guinea Pig Lung Strips . . 514D.2.1.2 Spasmolytic Activity

in Isolated Trachea . . . . . . . . . . . . . . . . . 515D.2.1.3 Reactivity of the Isolated

Perfused Trachea. . . . . . . . . . . . . . . . . . . 518D.2.1.4 Bronchial Perfusion

of Isolated Lung . . . . . . . . . . . . . . . . . . . 519D.2.1.5 Vascular and Airway Responses

in the Isolated Lung . . . . . . . . . . . . . . . . 520D.2.2 In Vivo Tests. . . . . . . . . . . . . . . . . . . . . . . 522D.2.2.1 Bronchospasmolytic Activity

in Anesthetized Guinea Pigs(Konzett–Rössler method) . . . . . . . . . . 522

D.2.2.2 Effect of Arachidonic Acid or PAFon Respiratory Function In Vivo . . . . 524

D.2.2.3 Bronchial Hyperreactivity . . . . . . . . . . 525D.2.2.4 Body Plethysmography and Res-

piratory Parameters After Histamine-Induced Bronchoconstrictionin Anesthetized Guinea Pigs . . . . . . . . 528

D.2.2.5 Pneumotachographyin Anesthetized Guinea Pigs . . . . . . . . 530

D.2.2.6 Airway Microvascular Leakage . . . . . 532D.2.2.7 Isolated Larynx In Situ . . . . . . . . . . . . . 533D.2.2.8 Animal Models of Asthma. . . . . . . . . . 534D.2.2.8.1 Treatment of Asthma . . . . . . . . . . . . . . . 534D.2.2.8.2 Prevention of Allergic

Asthma Reaction . . . . . . . . . . . . . . . . . . . 539D.2.2.9 Bleomycin-Induced

Pulmonary Fibrosis . . . . . . . . . . . . . . . . 541D.2.2.10 Influence of Cytokines

on Lung Fibrosis . . . . . . . . . . . . . . . . . . . 544D.2.2.11 Emphysema Models. . . . . . . . . . . . . . . . 547D.2.2.12 Models of Chronic Obstructive

Pulmonary Disease (COPD) . . . . . . . . 549

D.3 Antitussive Activity . . . . . . . . . . . . . . . 551D.3.0.1 Antitussive Activity After Irritant

Inhalation in Guinea Pig . . . . . . . . . . . . 551D.3.0.2 Cough Induced

by Mechanical Stimulation . . . . . . . . . 553D.3.0.3 Cough Induced by Stimulation

of the Nervus Laryngicus Superior . . 554D.4 Effects on Tracheal Cells

and Bronchial Mucus Secretionand Transport . . . . . . . . . . . . . . . . . . . . 555

D.4.0.1 In Vitro Studies of Mucus Secretion . 555D.4.0.2 Acute Studies of Mucus Secretion . . 556D.4.0.3 Studies of Mucus Secretion

With Chronic Cannulation . . . . . . . . . . 557D.4.0.4 Bronchoalveolar Lavage . . . . . . . . . . . . 558D.4.0.5 Ciliary Activity . . . . . . . . . . . . . . . . . . . . 559D.4.0.6 Studies of Mucociliary Transport . . . 561D.4.0.7 Culture of Tracheal Epithelial Cells . 562D.4.0.8 Alveolar Macrophages . . . . . . . . . . . . . 563D.5 Safety Pharmacology

of the Respiratory System . . . . . . . . . 564

D.1In Vitro Tests

D.1.0.1Histamine (H1) Receptor Binding

PURPOSE AND RATIONALEHistamine receptors have been classified on the ba-sis of pharmacological analysis (Hill et al. 1997).Histamine exerts its action via at least four receptorsubtypes. The H1 receptor couples mainly to Gq/11,thereby stimulating phospholipase C, whereas the H2receptor interacts with Gs to activate adenylyl cy-clase. The histamine H3 and H4 receptors couple to Giproteins to inhibit adenylyl cyclase, and to stimulateMAPK (Hough 2001).

Histamine is considered to play a major role in asth-matic attacks (Bryce et al. 2006). H1-antagonists havebeen used since decades as therapeutic agents. This as-

512 Chapter D · Respiratory Activity

say is used to determine the affinity of test compoundsto the histamine H1 receptor by measuring their in-hibitory activities on the binding of the H1 antago-nist 3H-pyrilamine to a plasma membrane preparationfrom guinea pig brain.

PROCEDUREBrains from guinea-pigs are homogenized in ice-coldTris buffer (pH 7.5) in a Potter homogenizer (1 g brainin 30 ml buffer). The homogenate is centrifuged at 4°Cfor 10 min at 50,000 g. The supernatant is discarded,the pellet resupended in buffer, centrifuged as before,and the final pellets resupended in Tris buffer (1 g freshweight/5 ml). Aliquots of 1 ml are frozen at –70°C.

In the competition experiment, 50 µl 3H-pyrilamine(one constant concentration of 2 × 10−9 M), 50 µl testcompound (>10 concentrations, 10−5–10−10 M) and100 µl membrane suspension from guinea pig wholebrain (approx. 10 mg wet weight/ml) per sample areincubated in a shaking bath at 25°C for 30 min. Incu-bation buffer: 50 mM Tris-HCl buffer, pH 7.5.

Saturation experiments are performed with 11 con-centrations of 3H-pyrilamine (0.1–50 × 10−9 M). Totalbinding is determined in the presence of incubationbuffer, non-specific binding is determined in the pres-ence of mepyramine or doxepin (10−5 M).

The reaction is stopped by rapid vacuum filtra-tion through glass fibre filters. Thereby the membrane-bound is separated from the free radioactivity. Theretained membrane-bound radioactivity on the filteris measured after addition of 3 ml liquid scintillationcocktail per sample in a liquid scintillation counter.

EVALUATION OF RESULTSThe following parameters are calculated:

• total binding of 3H-pyrilamine• non-specific binding: binding of 3H-pyrilamine in

the presence of mepyramine or doxepin• specific binding = total binding–non-specific bind-

ing• % inhibition of 3H-pyrilamine binding: 100–spe-

cific binding as percentage of control value

The dissociation constant (Ki) and the IC50 value ofthe test drug are determined from the competition ex-periment of 3H-pyrilamine versus non-labeled drug bya computer-supported analysis of the binding data.

MODIFICATIONS OF THE METHODDe Backer et al. (1993) reported genomic cloning, het-erologous expression in COS-7 cells and pharmaco-logical characterization of a human H1-receptor.

REFERENCES AND FURTHER READINGBryce PJ, Mathias CB, Harrison KL, Watanabe T, Geha RS,

Oettgen HC (2006) The H1 histaminic receptor regulatesallergic lung responses. J Clin Invest 116(6):1624–1632

Carswell H, Nahorski SR (1982) Distribution and characteristicsof histamine H1-receptors in guinea-pig airways identifiedby [3H]mepyramine. Eur J Pharmacol 81:301–307

Chang RSL, Tran VT, Snyder SH (1979) Heterogeneity ofhistamine H1-receptors: Species variations in [3H]me-pyramine binding of brain membranes. J Neurochem32:1653–1663

De Backer MD, Gommeren W, Moereels H, Nobels G, VanGompel P, Leysen JE, Luyten WH (1993) Genomiccloning, heterologous expression and pharmacologicalcharacterization of a human H1-receptor. Biochem BiophysRes Commun 197:1601–1608

Hill SJ, Emson PC, Young JM (1978) The binding of [3H]me-pyramine to histamine H1 receptors in guinea-pig brain.J Neurochem 31:997–1004

Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP,Young JM, Schunack W, Levi R, Haas HL (1997) Interna-tional Union of Pharmacology. XIII. Classification of his-tamine receptors. Pharmacol Rev 49:253–278

Hough LB (2001) Genomics meets histamine receptors: newsubtype, new receptor. Mol Pharmacol 59:415–419

Ruat M, Schwartz JC (1989) Photoaffinity labeling and elec-trophoretic identification of the H1-receptor: Comparisonof several brain regions and animal species. J Neurochem53:335–339

D.1.0.2Muscarinic Receptor Binding

PURPOSE AND RATIONALEMuscarinic receptors in the airways are important inboth the normal physiology and the pathophysiologyof pulmonary function. Acetylcholine released fromparasympathetic nerve terminals causes contraction ofairway smooth muscle. Animals with asthma or otherchronic inflammation of the airways exhibit hyper-sensitivity of the airways to muscarinic agonists, andmuscarinic antagonists are used therapeutically in pa-tients with asthma and chronic obstructive pulmonarydisease (Nathanson 2000). Muscarinic receptors arepresent in neurons in the central and peripheral ner-vous system, cardiac and smooth muscles, and a vari-ety of exocrine glands. Mammals possess genes encod-ing five different subtypes of mAChR, termed M1–M5,which can be divided into two broad functional cat-egories: the M1, M3, and M5 receptors preferentiallycouple to the Gq family of G-proteins whereas the M2and M4 receptors preferentially couple to the Gi familyof G-proteins.

For involvement of acetylcholine receptors in thegastrointestinal tract see J.3.6.1.2.

Several papers deal with the distribution of mus-carinic receptors in the lung, their role in pulmonarydisease and the use of muscarinic antagonists for the

D.1 · In Vitro Tests 513

treatment of obstructive airways disease (Mak andBarnes 1990; Barnes 1993, 2001, 2004; Disse 2001;Disse et al. 1993, 1999; Haddad et al. 1994; Barneset al. 1995, 1997; Patel et al. 1995; Peták et al. 1996;Alabaster 1997; Matsumoto 1997; Chelala et al. 1998;Hislop et al. 1998; Okazawa et al. 1998; Wale et al.1999; Rees 2002; Sarria et al. 2002; Tohda et al. 2002;Costello et al. 2006).

Hirose et al. (2001) described the pharmacologicalproperties of a muscarinic antagonist with M2-sparingantagonistic activity.

PROCEDUREBinding Affinity for Humanand Rat Muscarinic Receptor SubtypesIn competition studies, specific binding of [3H]N-methylscopolamine (NMS; New England Nuclear,Boston, Mass., USA) was determined using mem-branes from Chinese hamster ovary (CHO) cells ex-pressing cloned human m1, m2, m3, m4, or m5 re-ceptors (Receptor Biology, Baltimore, Md., USA), ratm1 or m3 receptors (American Type Culture Collec-tion, Manassas, Va., USA), and rat heart tissue. TheseCHO cells expressing cloned rat m1 or m3 receptors,and rat heart tissue were homogenized in 3 vols of50 mM Tris-HCl (pH 7.4) and 1 mM EDTA containing20% sucrose with a Polytron PT-10. The homogenateswere centrifuged at 10,000 g for 30 min at 4°C. Thesupernatants were centrifuged at 100,000 g for 60 minat 4°C. The pellets were suspended in 50 mM Tris-HCl (pH 7.4) and 5 mM MgCl2 and centrifuged at100,000 g for 60 min at 4°C. The pellets were re-suspended in the above-mentioned buffer (25 mg/mlfor CHO cells expressing cloned rat m1 or m3, and50 mg/ml for rat heart tissue) and stored at –80°Cas membrane preparations. In the binding assay, themembrane preparations were incubated with 0.19 to0.2 nM [3H]NMS in 50 mM Tris-HCl, 10 mM MgCl2,and 1 mM EDTA (pH 7.4) for 2 h at room tempera-ture. Final protein concentrations were 22 µg/ml (hu-man m1), 70 µg/ml (human m2), 54 µg/ml (humanm3), 20 µg/ml (human m4), 116 µg/ml (human m5),481 µg/ml (rat m1 and m3), and 2500 µg/ml (rat heart).Assays were performed in a total volume of 500 µl.Non-specific binding was measured in the presence of1 µM NMS and it was less than 2% of total binding.Free and membrane-bound [3H]NMS were separatedby filtration over glass filters (UniFilter-GF/C; PackardInstruments, Meriden, Conn., USA) using a cell har-vester (Filtermate 196; Packard Instruments). Radioac-tivity was counted by a liquid scintillation counter(TopCount; Packard Instruments).

EVALUATIONFor saturation studies, membranes from CHO cells ex-pressing human m3 were incubated with an increasedconcentration of [3H]NMS (0.1–3.2 nM) in the pres-ence or absence of 10 nM compound A, and specificbinding of [3H]NMS was determined after incubationfor 2 h.

Competition binding data were analyzed by a non-linear regression fitting program using GraphPadPrism Software (San Diego, Calif., USA). Saturationbinding data were transformed to make a Scatchardplot and analyzed by a linear regression fitting programusing GraphPad Prism Software.

The Ki values were calculated from the IC50 valuesby using the following equation:

Ki = IC50/(1 + [L]/Kd)

where Kd is the dissociation constant of [3H]NMS ineach receptor subtype, and [L] is the concentration of[3H]NMS (Cheng and Prussoff 1973). Kd values of[3H]NMS in each receptor subtype were determinedby Scatchard plot analysis. The Kd and Bmax values be-low were used in this study. Data of human cloned re-ceptors were extracted from Receptor Biology’s Prod-uct Information Sheets (Receptor Biology).

Human m1receptor

Kd = 51 pM Bmax = 1.28 pmol/mgof protein

Human m2receptor

Kd = 290 pM Bmax = 1 pmol/mg ofprotein

Human m3receptor


Human m4receptor


Human m5receptor


Rat m1 receptor Kd = 62 pM Bmax = 0.039 pmol/mg of wet weight

Rat m2 receptor Kd = 210 pM Bmax = 0.0090 pmol/mg of wet weight

Rat m3 receptor Kd = 72pM Bmax = 0.019 pmol/mg of wet weight

In saturation studies, the Ki value was calculated us-ing the following equation:

Ki = Kd/(K′d − Kd) × [C]

where K′d or Kd is the dissociation constant of

[3H]NMS in human m3 receptors in the presence orabsence of an inhibitor, respectively, and [C] is theconcentration of the test drug (Nishikibe et al. 1999).


MODIFICATIONS OF THE METHODStruckmann et al. (2003) investigated the role of mus-carinic receptor subtypes in the constriction of periph-eral airways by studies on receptor-deficient mice.

REFERENCES AND FURTHER READINGAlabaster VA (1997) Discovery and development of selective

M3 antagonists for clinical use. Life Sci 60:1053–1060Barnes PJ (1993) Muscarinic receptor subtypes: implications for

therapy. Agents Actions Suppl 43:243–252Barnes PJ (2001) Tiotropium bromide. Exp Opin Invest Drugs

10:733–740Barnes PJ (2004) Distribution of receptor targets in the lung.

Proc Am Thorac Soc 1:345–351Barnes PJ, Belvisi MG, Mak JCW, Haddad EB, O’Connor B

(1995) Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstruc-tive airway disease. Life Sci 56:853–859

Barnes PJ, Haddad EB, Rousell J (1997) Regulation of mus-carinic M2 receptors. Life Sci 60:1015–1021

Chelala JL, Kilani A, Miller JM, Martin RJ, Ernsberger P (1998)Muscarinic receptor binding sites of the M4 subtype inporcine lung parenchyma. Pharmacol Toxicol 83.200–207

Cheng YC, Prusoff WH (1973) Relationship between the inhibi-tion constant (K1) and the concentration of inhibitor whichcauses 50 percent inhibition (I50) of an enzymatic reaction.Biochem Pharmacol 22:3099–3108

Costello RW, Jacoby DB, Fryer AD (2006) Pulmonary neu-ronal M2 muscarinic receptor function in asthma and an-imal models of hyperreactivity. Thorax 53:613–618

Disse B (2001) Antimuscarinic treatment for lung diseases.From research to clinical practice. Life Sci 68:2557–2564

Disse B, Reichl R, Speck G, Traunecker W, Ludwig-Romming-er KL, Hammer R (1993) Ba 679 BR, a novel long-actinganticholinergic bronchodilator. Life Sci 52:537–544

Disse B, Speck GA, Rominger KL, Witek TJ Jr, Hammer R(1999) Tiotropium (Spiriva™): mechanistical considera-tions and clinical profile in obstructive lung disease. LifeSci 64:457–464

Haddad EB, Mak JC, Barnes PJ (1994) Characterization of[3H]Ba 679 BR, a slowly dissociating muscarinic antago-nist, in human lung: radioligand and autoradiographic map-ping. Mol Pharmacol 45:899–907

Hirose H, Aoki I, Kimura T, Fujikawa T, Numazawa T, Sasaki K,Sato A, Hasegawa T, Nishikibe M, Mitsuya M, Ohtake N,Mase T, Noguchi K (2001) Pharmacological propertiesof (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylace-tamide: a novel muscarinic antagonist with M2-sparingantagonistic activity. J Pharmacol Exp Ther 297:790–797

Hislop AA, Mak JCW, Reader JA, Barnes PJ, Haworth SG(1998) Muscarinic receptor subtypes in the porcinelung during postnatal development. Eur J Pharmacol359:211–221

Mak JC, Barnes PJ (1990) Autoradiographic visualization ofmuscarinic receptor subtypes in human and guinea piglung. Am Rev Respir Dis 141:1559–1568

Matsumoto S (1997) Functional evidence of excitatory M1receptors in the rabbit airway. J Pharmacol Exp Ther281:531–539

Nathanson NM (2000) A multiplicity of muscarinic mecha-nisms: enough signaling pathways to take your breathaway. Proc Natl Acad Sci USA 97:6245–6247

Nishikibe M, Ohta H, Ishikawa K, Hayama T, Fukuroda T,Noguchi K, Saito M, Kanoh T, Ozaki S, Kamei T,

Hara K, William D, Kivlighn S, Krause S, Gabel R,Zingaro G, Nolan N, O’Brien J, Clayton F, Lynch J,Pettibone D, Siegl P (1999) Pharmacological propertiesof J-104132 (L-753,037), a potent orally active, mixedETA/ETB endothelin receptor antagonist. J Pharmacol ExpTher 289:1262–1270

Okazawa A, Cui ZH, Lötvall J, Yoshihara S, Skoogh BE,Kashimoto K, Lindén A (1998) Effect of a novel PACAP-27 analogue on muscarinic airway responsiveness inguinea-pigs in vivo. Eur Respir J 12:1062–1066

Patel HJ, Barnes PJ, Takahashi T, Tadjikarimi S, Yacoub MH,Belvisi MG (1995) Evidence for prejunctional muscarinicautoreceptors in human and guinea pig trachea. Am JRespir Crit Care Med 152:872–878

Peták F, Hantos Z, Adamicza A, Asztalos T, Sly PD (1996)Metacholine-induced bronchoconstriction in rats: effectsof intravenous vs. aerosol delivery. J Appl Physiol80:1841–1849

Rees PJ (2002) Tiotropium in the management of chronic ob-structive pulmonary disease. Eur Respir J 19:205–206

Sarria B, Naline E, Zhang Y, Cortijo J, Molimard M, Moreau J,Therond P, Avenier C, Morcillo EJ (2002) MuscarinicM2 receptors in acetylcholine-isoproterenol functional an-tagonism in human isolated bronchus. Am J PhysiolL11254–L1132

Struckmann N, Schwering S, Wiegand S, Gschnell A, Ya-mada M, Kummer W, Wess J, Haberberger RV (2003) Roleof muscarinic receptor subtypes in the constriction of pe-ripheral airways: studies on receptor deficient mice. MolPharmacol 64:1444–1451

Tohda Y, Haraguchi R, Itoh M, Ohkawa K, Kubo H, Fukuoka M,Nakajima S (2002) Role of muscarinic receptors ina guinea pig model of asthma. Intern Immunopharmacol2:1521–1527

Wale JL, Peták F, Sly PD (1999) Muscarinic blockade of metha-choline induced airway and parenchymal lung responses inanaesthetized rats. Thorax 54:531–537

D.2Effects on Air Ways

D.2.1Tests in Isolated Organs

D.2.1.1Spasmolytic Activity in Isolated Guinea Pig Lung Strips

PURPOSE AND RATIONALESeveral autacoids such as histamine and leukotrienes,induce bronchoconstriction. Histamine is an impor-tant mediator of immediate allergic and inflamma-tory reactions. It causes bronchoconstriction by acti-vating H1-receptors. Calcium ionophores induce therelease of leukotrienes via the 5-lipoxygenase path-way. Leukotrienes are powerful bronchoconstrictorsthat appear to act on smooth muscles via specific re-ceptors. In this method, drugs are tested for their ca-pability of inhibiting bronchospasm induced by his-tamine or calcium ionophore. It is used to detect H1-and leukotriene receptor blocking properties of testcompounds.

D.2 · Effects on Air Ways 515

PROCEDUREAlbino guinea-pigs of either sex weighing 300–450 gare sacrificed with an overdose of ether. The chest cav-ity is opened and the lungs are removed. They arecut into strips of 5 cm and placed into a physiologicalsaline solution. Thereafter, the lung strips are mountedin an organ bath containing a nutritive solution. Thebath is bubbled with carbogen and maintained at 37°C.Under a pre-load of 0.5 g–3 g, the tissue is left to equi-librate for 30–60 min. Prior to testing, carbachol isadded to the bath to test the lung strips’ ability of con-traction. Twenty min later, two prevalues are obtainedby adding the spasmogen

• histamine dihydrochloride 10−6 g/ml for 5 min, or• Ca – ionophore 5 × 10−6 g/ml for 5 min, or• Leukotriene LTC4 10−9–10−8 g/ml for 10 min, or• Leukotriene LTD4 10−9–10−8 g/ml for 10 min

to the bath and recording the contractile force at itsmaximal level. Following a 20 min equilibration pe-riod, the spasmogen is administered again. Five min-utes thereafter, the test compound is added in cumula-tive doses from 10−8 to 10−4 g/ml at 5 or 10 min inter-vals. The contractile response is determined isometri-cally.

Test Modification: Inhibition of Prostaglandin SynthesisThis procedure is identical to the test described abovewith the exception that the prostaglandin synthesis isinhibited by addition of indomethacin at 10−6 g/mlprior to spasmogen administration.

EVALUATIONThe percent inhibition of spasmogen induced contrac-tion is calculated.

MODIFICATIONS OF THE METHODLung parenchyma strips from various species wereused to measure bronchoactivity by Kleinstiver andEyre (1979).

A descriptive model of the events occurring duringan inflation-deflation cycle using excised rat lungs wasproposed by Frazer et al. (1985).

Barrow (1986) measured volume-pressure cycles inair-filled or liquid-filled rabbit lungs ranging from in-tact lungs with the rib cage immobilized to isolatedlungs.

REFERENCES AND FURTHER READINGBarrow RE (1986) Volume-pressure cycles from air and liquid-

filled intact rabbit lungs. Resp Physiol 63:19–30

Foreman JC, Shelly R, Webber SE (1985) Contraction of guinea-pig lung parenchymal strips by substance P and related pep-tides. Arch Int Pharmacodyn 278:193–206

Frazer DG, Weber KC, Franz GN (1985) Evidence of sequentialopening and closing of lung units during inflation-deflationof excised rat lungs. Resp Physiol 61:277–288

Kleinstiver PW, Eyre P (1979) Evaluation of the lungparenchyma strip preparation to measure bronchoactivity.J Pharmacol Meth 2:175–185

Lach E, Haddad EB, Gies JP (1993) Contractile effect ofbombesin on guinea pig lung in vitro: involvement of GRP-preferring receptors. Am J Physiol 264:L80–86

Lach E, Trifilieff A, Muosli M, Landry Y, Gies JP (1994)Bradykinin-induced contraction of guinea pig lung in vitro.Naunyn-Schmiedeberg’s Arch Pharmacol 350:201–208

Lulich KM, Papadimitriou JM, Paterson JW (1979) The isolatedlung strip and single open tracheal ring: a convenient com-bination for characterizing Schultz-Dale anaphylactic con-tractions in the peripheral and central airways. Clin ExpPharmacol Physiol 6:625–629

D.2.1.2Spasmolytic Activity in Isolated Trachea

PURPOSE AND RATIONALEThe isolated tracheal chain of guinea pigs can beused to test for β-blocking activity (see Sect. A.1.2.7).In addition, this model can be used to test com-pounds which inhibit bronchospasms. It is used todetect β-sympathomimetic, H1-receptor blocking andleukotriene receptor blocking properties of test drugs.

Carbachol is a cholinergic agonist that producescontraction of bronchial smooth muscle by muscarinicstimulation.

Histamine is an important mediator of immediateallergic (type 1) and inflammatory reactions. It causesbronchoconstriction by activating H1-receptors.

Calcium ionophores induce the release ofleukotrienes via the 5-lipoxygenase pathway. Leuko-trienes are powerful bronchoconstrictors that appearto act on smooth muscle via specific receptors.

To asses a compound’s ability to inhibit carba-chol induced bronchospasm via β-receptor activation,a β-receptor blocking agent (for example propranolol)must be added. If relaxation of bronchial smooth mus-cle is brought about by β-receptor activation, the spas-molytic effect will decrease following propranolol ad-ministration.

The effect of bradykinin can be abolished bybradykinin antagonists (Hock et al. 1991).

The effects of potassium channel openers can alsobe studied in this test (Englert et al. 1992).

PROCEDUREAlbino guinea pigs of either sex weighing 300–550 gare sacrificed by CO2 narcosis. The entire trachea is


dissected out and cut into individual rings (2–3 car-tilaginous rings wide). Twelve–fifeteen rings are tiedtogether with silk threads and mounted in the organbath containing Krebs-Henseleit solution. The tissue ismaintained at 37°C under a tension of 0.5 g and gassedwith carbogen. Isometric contractions are recorded viaa strain-gauge transduced on a polygraph. Forty-fiveminutes are allowed for equilibration before the addi-tion of the spasmogen.

The following spasmogens are used:

• carbachol (2 × 10−7 g/ml)• histamine (10−7 g/ml)• Ca – ionophore for release of leukotrienes• leukotriene LTC4 (10−9–10−8 g/ml)• leukotriene LTD4 (10−9–10−8 g/ml)

When the contraction has reached its maximum(initial spasm) after 10–12 min, the standard drug, e. g.,isoprenaline (1 ng/ml) or aminophylline (10 ng/ml) isadministered. The bronchial responses are allowed toplateau and are recorded. The tissue is rinsed thor-oughly and control contractions are induced again byadding spasmogen. After obtaining the initial spasmagain, the test drug is added and the contractile forceis recorded at its maximal level.

Determination of mechanism of action (testingfor β-sympathomimetic effect). After obtaining theinitial carbachol induced spasm, propranolol is admin-istered 5 min before the addition of the test drug. Threeminutes later, the tissue is challenged by carbachol ad-ministration.

EVALUATIONThe percent inhibition of carbachol or other spasmo-gen induced contractions is calculated. From dose-response curves ED50 values can be calculated.

CRITICAL ASSESSMENT OF THE METHODThe isolated guinea pig trachea has been proven to bean useful tool for several purposes, e. g., screening pro-cedures and studies on mode of action, e. g., of potas-sium channel openers.

MODIFICATIONS OF THE METHODThe molecular mechanisms of β-adrenergic relaxationof airway smooth muscle were described by Kotlikoffand Kamm (1996).

Farmer et al. (1986) studied the effects of epithe-lium removal on the sensitivity of guinea-pig isolatedtrachealis to bronchodilator drugs.

Wilkens et al. (1992) described a bioassay systemfor a tracheal smooth muscle-constricting factor using

an isolated guinea pig trachea which was cannulatedfrom both sides, mounted in an organ bath and mon-itored by a TV camera attached to a microscope. Thepicture was digitized continuously, and the diameterof the trachea calculated and displayed on a monitorthroughout the experiment.

Coleman and Nials (1989) described a versatile,eight-chamber superfusion system for the evaluationof spasmogenic and spasmolytic agents using guineapig isolated tracheal smooth muscle.

Goldie et al. (1986a) studied the influence of the ep-ithelium on responsiveness of guinea pig isolated tra-chea to contractile and relaxant agonists.

Lee et al. (1997) studied the effects of bupivacaineand its isomers on guinea pig tracheal smooth mus-cle. The trachea from the larynx to the carina was re-moved and cut into single rings. Seven tracheal ringswere tied together with the circular muscle running onthe same side of the chain, placed into an organ bathcontaining Krebs Ringer’s solution and connected toa force placement transducer for measurement of iso-metric tension.

Wong et al. (1997) tested the effects of tyrosinekinase inhibitors on antigen challenge of guinea piglung in vitro. Guinea pigs were passively sensitized bya single i.p. injection of 1 mg/kg rabbit IgG antibodyagainst ovalbumin. Bronchial rings, 3 mm in length,were obtained from the hilar bronchi of sacrificed an-imals and suspended isometrically in an organ bath inKrebs bicarbonate buffer with a resting load of 2 g.To determine maximum antigen-induced contractions,bronchial rings were exposed to increasing concentra-tions of ovalbumin. To evaluate the role of protein ty-rosine kinase in mediating smooth muscle anaphylac-tic contraction, protein tyrosine kinase inhibitors werepreincubated with bronchial rings 30 min before addi-tion of ovalbumin.

Eltze and Galvan (1994) compared the inhibitionof preganglionic and postganglionic contraction of therabbit isolated bronchus/trachea by antagonists withselectivity for different muscarinic receptor subtypeswith their affinities at M1, M2, M3, and M4 receptors(Barnes 1993).

For experiments with vagus nerve stimulation,the vagi were isolated with rings of the proximal mainstem bronchi and a small portion of the distal tra-chea. The tissues were hung on stainless steel hooks,which passed through the lumen, and were placed ina water-jacketed organ bath filled with Krebs solutionplus 2 × 10–5 M choline chloride to promote resynthe-sis of acetylcholine, 10–5 M indomethacin to preventgeneration of cyclo-oxygenase products, and 10–6 M


DL-propranolol to block possible β-adrenoceptor-me-diated effects. The preparation was fixed under a rest-ing tension of 1 g for isometric contraction measure-ment using a force transducer. The vagi were passedaround bipolar platinum electrodes held at the sur-face of the bath. Electrical stimulation was performedwith trains (20 Hz, 0.3 ms at 20 V) elicited every20 min.

For experiments with field stimulation, two-ringpreparations from the distal trachea of the rabbit weresuspended in organ bath in Krebs solution with theabove mentioned additions under a resting tension of1 g. Isometric contractions were elicited at 1 h inter-vals by continuous electrical field stimulation via plat-inum electrodes (20 Hz, 0.3 ms at 20 V) for an average3–7 min to reach a stable plateau.

Vaali et al. (1996) studied in isolated tracheal ringsof rats and guinea pigs the bronchorelaxing effects ofnitric oxide donors. The epithelium of some rings wasremoved by gentle rubbing. Rat mesenteric rings werecut from the same animals as the bronchi and similarlyprepared by removing the endothelium by gently rub-bing of the intimate surface.

Farmer et al. (1994) used the isolated trachea frommale ferrets weighing 1.5–2.5 kg to study the effectsof bradykinin receptor agonists.

Toews et al. (1997) assessed the effects of the phos-pholipid mediator lysophosphatidic acid on the con-tractile responsiveness of isolated tracheal rings fromrabbits and cats.

Tamaoki et al. (1993) used isolated rings of seg-mental bronchi from dogs to study atypical β-adrenoceptor-(β3-adrenoceptor)mediated relaxation.

The preparation of bovine tracheal smooth musclefor measuring airway responsiveness in vitro was de-scribed by Hashjin et al. (1995).

Wali (1987) used tracheal strips from 2- to 4-week-old chicks to study inhibition of cholinergic and non-cholinergic neural and muscular contractions by localanesthetics.

Lulich and Paterson (1980) used human isolatedbronchial muscle preparations and compared the ef-fects of histamine and other drugs with the effects ob-served on the central and peripheral airways of therat.

Goldie et al. (1986b) measured the responses ofhuman bronchial strip preparations to contractile andrelaxant agonists in preparations from non-diseasedand from asthmatic lung obtained 3–15 h post mor-tem.

Hulsman and de Jongste (1993) reviewed the meth-ods to study human airways in vitro.

REFERENCES AND FURTHER READINGBarnes PJ (1993) Muscarinic receptor subtypes in airways. Life

Sci 52:521–527Castillo JC, de Beer EJ (1947) The tracheal chain. I. A prepa-

ration for the study of antispasmodics with particular ref-erence to bronchodilator drugs. J Pharmacol Exp Ther90:104–109

Coleman RA, Nials AT (1989) Novel and versatile superfu-sion system. Its use in the evaluation of some spasmogenicand spasmolytic agents using guinea pig isolated trachealsmooth muscle. J Pharmacol Meth 21:

Da Silva A, Amrani YS, Trifilieff A, Landry Y (1995) Involve-ment of B2 receptors in bradykinin-induced relaxationof guinea-pig isolated trachea. Br J Pharmacol 114:103–108

Eltze M, Galvan M (1994) Involvement of muscarinic M2 andM3, but not of M1 and M4 receptors in vagally stimulatedcontractions of rabbit bronchus/trachea. Pulmon Pharmacol7:109–120

Englert CE, Wirth K, Gehring D, Fürst U, Albus U, Scholz W,Rosenkranz B, Schölkens BA (1992) Airway pharma-cology of the potassium channel opener, HOE 234, inguinea pigs: in vitro and in vivo studies. Eur J Pharmacol210:69–75

Farmer SG, Fedan JS, Hay DWP, Raeburn D (1986) The ef-fects of epithelium removal on the sensitivity of guinea-pigisolated trachealis to bronchodilator drugs. Br J Pharmacol89:407–414

Farmer SG, Broom T, DeSiato MA (1994) Effects of bradykininreceptor agonists, and captopril and thiorphan in ferret iso-lated trachea: evidence for bradykinin generation in vitro.Eur J Pharmacol 259:309–313

Foster RW (1966) The nature of the adrenergic receptors of thetrachea of the guinea-pig. J Pharm Pharmacol 18:1–12

Goldie RG, Papadimitriou JM, Paterson JW, Rigby PJ, Self HM,Spina D (1986a) Influence of the epithelium on responsive-ness of guinea pig isolated trachea to contractile and relax-ant agonists. Br J Pharmacol 87:5–14

Goldie RG, Spina D, Henry PJ, Lulich KM, Paterson JW(1986b) In vitro responsiveness of human asthmaticbronchus to carbachol, histamine, β-adrenoceptor agonistsand theophylline. Br J Clin Pharmacol 22:669–676

Hashjin GS, Henricks PAJ, Folkerts G, Nijkamp FP (1995)Preparation of bovine tracheal smooth muscle for invitro pharmacological studies. J Pharmacol Toxicol Meth34:103–108

Hock JF, Wirth K, Albus U, Linz W, Gerhards HJ, Wiemer G,Henke St, Breipohl G, König W, Knolle J, Schölkens BA(1991) HOE 140 a new potent and long acting bradykinin-antagonist. In vitro studies. Br J Pharmacol 102:769–773

Hulsman AR, de Jongste JC (1993) Studies of human airways invitro: A review of the methodology. J Pharm Toxicol Meth30:117–132

Kotlikoff MI, Kamm KE (1996) Molecular mechanisms of β-adrenergic relaxation of airway smooth muscle. Ann RevPhysiol 58:115–141

Lee T-L, Adaikan PG, Lau L-C, Ratnam SS, Dambisya YM(1997) Effects of bupivacaine and its isomers on guinea pigtracheal smooth muscle. Meth Find Exp Clin Pharmacol19:27–33

Lulich KM, Paterson JW (1980) An in vitro study of variousdrugs on central and peripheral airways of the rat: a com-parison with human airways. Br. J Pharmac 68:633–636

Rhoden KJ, Barnes PJ (1989) Effect of hydrogen peroxideon guinea-pig tracheal smooth muscle in vitro: role ofcyclo-oxygenase and airway epithelium. Br J Pharmacol98:325–330


Sheth UK, Dadkar NK, Kamat UG (1972) Selected Topics inExperimental Pharmacology Published by Kothari BookDepot, India

Tamaoki J, Yamauchi F, Chiyotani A, Yamawaki I, Takeuchi S,Konno K (1993) Atypical β-adrenoceptor-(β3 -adreno-ceptor) mediated relaxation of canine isolated bronchialmuscle. J Appl Physiol 74:297–302

Toews ML, Ustinowa EE, Schultz HD (1997) Lysophosphatidicacid enhances contractility of isolated airway smooth mus-cle. J Appl Physiol 83:1216–1222

Vaali K, Li L, Redemann B, Paakkari I, Vapaatalo H (1996) Invitro bronchorelaxing effect of novel nitric oxide donorsGEA 3268 and GEA 5145 in guinea pigs and rats. J PharmPharmacol 48:1309–1314

Waldeck B, Widmark E (1985) Comparison of the effects offorskolin and isoprenaline on tracheal, cardiac and skeletalmuscles from guinea-pig. Eur J Pharmacol 112:349–353

Wali FA (1987) Local anaesthetics inhibit cholinergic andnon-cholinergic neural and muscular contractions inavian tracheal smooth muscle. Acta Anaesthesiol Scand31:148–153

Wilkens JH, Becker A, Wilkens H, Emura M, Riebe-Imre M,Plien K, Schöber S, Tsikas D, Gutzki FM, Frölich JCl(1992) Bioassay of a tracheal smooth muscle-constrictingfactor released by respiratory epithelial cells. Am J Physiol263 (Lung Cell Mol Physiol 7):L137–L141

Wong WSF, Koh DSK, Koh AHM, Ting WL, Wong PTH(1997) Effects of tyrosine kinase inhibitors on antigen chal-lenge of guinea pig lung in vitro. J Pharmacol Exp Ther283:131–137

D.2.1.3Reactivity of the Isolated Perfused Trachea

PURPOSE AND RATIONALEThe mechanism by which the epithelium affects thereactivity of tracheal musculature can be studied usingthe isolated perfused trachea preparation. Contractileagonists can be added either to the serosal (extralumi-nal) or to the mucosal (intraluminal) surface (Fedanand Frazer 1992).

PROCEDUREA 4-cm segment of the trachea of male guineapigs is removed after sacrifice of the animal andplaced for cleaning in modified Krebs-Henseleit so-lution at 37°C containing (millimolar) NaCl 113.0,KCl 4.8, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2,NaHCO3 25.0, and glucose 5.7 (pH 7.4) being gassedwith 95% O2/5% CO2. The trachea is then attachedto a stainless-steel perfusion holder (Munakata et al.1988), extended to its in situ length and placed in anorgan chamber (the serosal compartment) at 37°C con-taining 25 ml of gassed modified Krebs-Henseleit solu-tion. This solution is also pumped at a constant rate of30 ml/min through the lumen (mucosal compartment).Responses of the tracheal musculature are obtained bymeasuring changes in inlet-outlet �P between the sideholes of indwelling catheters, while the trachea as per-

fused at a constant rate of modified Krebs-Henseleitsolution. The inlet and outlet catheters are connectedto the positive and negative sides, respectively, of a dif-ferential pressure transducer.

Agonists are added in step-wise increasing, cumu-lative concentrations. Two consecutive dose-responsecurves are obtained after the addition either to theserosal or mucosal compartment. The second dose-response curve is obtained 1.5 h after the end of thefirst, the preparation being washed every 15 min dur-ing the intervening period.

EVALUATIONResponses are quantified as �P in centimeters of H2O.Geometric EC50 values are determined from least-square analysis of a logit model and are presentedalong with 95% confidence intervals.

MODIFICATIONS OF THE METHODBaersch and Frölich (1996) measured continuously thechanges of the diameter of isolated guinea pig tra-cheal tubes by a newly developed imaging bioassaysystem. The tracheal tube between larynx and bifur-cation was prepared to a length of 1.8–2.5 cm, can-nulated at both ends and mounted in a 15 ml organbath filled and perfused with oxygenated Krebs solu-tion at 37°C. The lumen of the trachea was perfusedwith warm (37°C) Krebs solution from a reservoir thatwas isolated from the tissue bath. Changes in diameterof the trachea were assessed by computerized videomicroscopy. The tracheal diameter was used as markerfor airway size, thus allowing calculation of musclecontractions under experimental conditions. Follow-ing a 30-min equilibrium period, a contraction wasinduced by intraluminal application of methacholine(0.1 mmol/l) as reference contraction. After washoutand return to a stable baseline, cumulative concentra-tion-response curves were obtained by luminal or ex-traluminal drug application.

Yang et al. (1991) studied the role of epitheliumin airway smooth muscle responses to relaxant agents.The results suggested that the epithelium is a relativelyweak barrier for lipophilic agents but has a major roleas a diffusion barrier to hydrophilic substances.

Munataka et al. (1988, 1989) developed an in vitrosystem to assess the role of epithelium in regulatingairway tone using the intact guinea pig trachea. Theresponses to histamine, acetylcholine and hypertonicKCl when stimulated from the epithelial or serosal sitewere first examined in tracheae with intact epithelium.Then the responses to these agonists were registeredafter epithelial denudation.


Pavlovic et al. (1989) studied the role of airway ep-ithelium in the modulation of bronchomotor tone inthe isolated trachea of rats. An organ bath was con-structed that permitted independent circulation of fluidwithin the lumen or around the exterior of the trachealsegment. In one-half of the preparations the epitheliumwas mechanically removed.

Fernandes et al. (1989) described a co-axial bioas-say system, whereby rat cross-cut aorta strip prepara-tions were set up in co-axial assemblies under 500 mgresting tension within a guinea pig tracheal segmentserving as donor of the smooth muscle relaxant factorfrom guinea-pig tracheal epithelium.

Lewis and Broadley (1995) investigated the influ-ence of spasmogen inhalation by guinea pigs uponsubsequent demonstration of ovalbumin-induced hy-perreactivity in isolated airway tissues. Guinea pigswere sensitized with ovalbumin (i.p.) 14 days be-fore use. In vitro airway hyperreactivity induced byovalbumin inhalation was determined by challengingwith aerosolized spasmogen (5-HT, methacholine, thethromboxane-mimetic U-46619, or adenosine) 24 hbefore and again 18–24 h after the ovalbumin inhala-tion. One h later, the animals were sacrificed and iso-lated airways perfused lung halves and tracheal spiralswere set up for determination of tissue sensitivity tocarbachol, histamine, and adenosine.

Sparrow and Mitchell (1991) used bronchial seg-ments obtained from the lungs of Large-white-/Land-race-cross pigs to study the modulation by the epithe-lium of the extent of bronchial narrowing produced bysubstances perfused through the lumen.

Mitchell et al. (1989) compared the reactions of per-fused bronchial segments and bronchial strips of pigsto histamine and carbachol.

Hulsman et al. (1992) recommended the perfusedhuman bronchiolar tube as a suitable model.

Omari et al. (1993) studied the responsiveness ofhuman isolated bronchial segments and its relationshipto epithelial loss.

REFERENCES AND FURTHER READINGBaersch G, Frölich JC (1996) A new bioassay to study contrac-

tile and relaxant effects of PGE2 on perfused guinea pigtrachea. J Pharmacol Toxicol Meth 36:63–68

Fedan JS, Frazer DG (1992) Influence of epithelium on the reac-tivity of guinea pig isolated, perfused trachea to bronchoac-tive drugs. J Pharm Exp Ther 262:741–750

Fernandes LB, Paternon JW, Goldie RG (1989) Co-axial bioas-say of smooth muscle relaxant factor released from guinea-pig tracheal epithelium. Br J Pharmacol 96:117–124

Hulsman AR, Raatgeep HR, Bonta IL, Stijnen T, Kerrebijn KF,de Jongste JC (1992) The perfused human bronchiolar tube.Characteristics of a new model. J Pharm Toxicol Meth28:29–34

Lewis CA, Broadley KJ (1995) Influence of spasmogen inhala-tion by guinea pigs upon subsequent demonstration of oval-bumin-induced hyperreactivity in isolated airway tissues.J Pharmacol Toxicol Meth 34:187–198

Mitchell HW, Willet KE, Sparrow MP (1989) Perfusedbronchial segment and bronchial strip: narrowing vs. iso-metric force by mediators. J Appl Physiol 66:2704–2709

Munakata M, Mitzner W, Menkes H (1988) Osmotic stimuli in-duce epithelial-dependent relaxation in the guinea pig tra-chea. J Appl Physiol 64:466–471

Munakata M, Huang I, Mitzner W, Menkes H (1989) Protectiverole of epithelium in the guinea pig airway. J Appl Physiol66:1547–1552

Omari TI, Sparrow MP, Mitchell HW (1993) Responsiveness ofhuman isolated bronchial segments and its relationship toepithelial loss. Br J Clin Pharmacol 35:357–365

Pavlovic D, Fournier M, Aubier M, Pariente R (1989) Epithelialvs. serosal stimulation of tracheal muscle: role of epithe-lium J Appl Physiol 67:2522–2526

Sparrow MP, Mitchell HW (1991) Modulation by the epithe-lium of the extent of bronchial narrowing produced bysubstances perfused through the lumen. Br J Pharmacol103:1160–1164

Yang J, Mitzner W, Hirshman C (1991) Role of epithelium inairway smooth muscle responses to relaxant agents. J ApplPhysiol 71:1434–1440

D.2.1.4Bronchial Perfusion of Isolated Lung

PURPOSE AND RATIONALEBronchial perfusion of the isolated lung was describedby Sollmann and von Oettingen (1928) as a sim-ple method for studying pharmacological reactions ofbronchiolar muscle. The method consists in perfusingfluid down the trachea through the bronchi, and allow-ing it to escape from the alveoli through scratches onthe surface of the lungs. Bronchoconstriction resultsin a reduced rate of flow, bronchodilatation is indi-cated by an increased flow. The method has been usedto evaluate sympathomimetic drugs by Tainter et al.(1934) and by Luduena et al. (1957).

PROCEDUREGuinea pigs weighing about 200 g are sacrificed bya head blow. The chest is opened, the trachea cut atthe upper end and removed with the lung. The tracheais attached to the cannula of an perfusion apparatus.Only one lung is perfused, the other being tied off. Thelower part of the lower lobe is cut off and the rest ofthe lung surface is scratched deeply assuring maximalpre-medication flow.

The perfusion fluid has the following compositionin percentage of anhydrous salts: NaCl 0.659,NaHCO3 0.252, KCl 0.046, CaCl2 0.005,MgCl2 0.0135, NaH2PO4 0.01, Na2HPO4 0.008,glucose 5%, pH 8.0. The temperature of the perfu-sion medium is 37.5°C and the lung is enclosed in


a glass cylinder to be protected from variations in theenvironmental temperature.

The trachea is attached to the cannula of a perfu-sion apparatus which pumps the solution at a constantrate into a manometric tube connected with the per-fused organ. Resistance to the flow (bronchoconstric-tion) results in an increase in the height of the col-umn of fluid in the manometer. The intensity of bron-chodilator effect is measured by the fall of the columnin the manometer.

After the lung is attached to a T-shaped cannula, thepump is set in motion and the fluid, after filling thelung, flows out of the system through the third open-ing of the cannula. By gentle pressure air bubbles areforced out of the lung into the overflow. The lung isthen treated in the aforesaid manner, and the upper out-let of the cannula closed. Histamine HCl is added ina concentration of 1:2500000 as soon as the perfusionstarts and the flow is adjusted to obtain a constant pro-gressive increase in pressure.

The drugs are injected near the cannula when theperfusion pressure reaches a level of 500–650 ml ofwater. The volume injected is always 0.1 ml.

Each drug is tested for bronchodilating activityagainst the bronchoconstriction induced by histaminein parallel with l-arterenol following a Latin square,including three doses of each drug and three doses ofl-arterenol graded at 0.5 log intervals.

EVALUATIONActivity ratios of bronchodilating agents versus thestandard can be calculated with a 3 + 3 point assay in-cluding confidence limits.

REFERENCES AND FURTHER READINGLuduena FP, von Euler L, Tullar BF, Lands AM (1957) Effect

of the optical isomers of some sympathomimetic amineson the guinea pig bronchioles. Arch Int Pharmacodyn111:392–400

Sollmann T, von Oettingen WF (1928) Bronchial perfusion ofisolated lung as a method for studying pharmacologic re-actions of bronchiolar muscle. Proc Soc Exp Biol Med25:692–695

Tainter ML, Peddenm JR, James M (1934) Comparative actionsof sympathomimetic compounds: bronchodilator actions inperfused guinea pig lungs. J Pharm Exp Ther 51:371–386

D.2.1.5Vascular and Airway Responses in the Isolated Lung

PURPOSE AND RATIONALEThe isolated perfused rat lung allows the simultane-ous registration of pulmonary vascular and airway re-sponses to various drugs.

PROCEDUREMale Sprague-Dawley rats weighing 300–350 g are in-traperitoneally anesthetized with pentobarbital sodium(50 mg/kg). The trachea is cannulated with a shortsection of polyethylene tubing, connected to a rodentventilator, and ventilated with room air enriched with95% O2/5% CO2, with a tidal volume of 4–5 ml/kg and2 cm H2O positive end-respiratory pressure. The ratsare heparinized with 1000 units of intravenous heparinand are rapidly exsanguinated by withdrawing bloodfrom the carotid artery.

The lung is exposed by median sternotomy, anda ligature is placed around the aorta to prevent sys-temic loss of blood. The main pulmonary artery iscatheterized, and the lung is removed en block and sus-pended in a warmed (39°C), humidified (100%) water-jacketed chamber. An external heat exchanger is usedto maintain the temperature of the perfusate and theisolated lung chamber constant throughout the exper-iment. The perfusate solution (15 ml of heparinizedblood and 5 ml modified Krebs-Henseleit solution) isplaced in a reservoir and mixed constantly by a mag-netic stirrer. The lungs are perfused with a peristalticroller pump at a flow rate of 8–14 ml/min to maintaina physiological baseline pulmonary arterial perfusionpressure of 15 ± 0.5 mmHg. Pulmonary arterial per-fusion pressure, airway pressure, and reservoir bloodlevel are continuously monitored, electronically aver-aged and recorded with a polygraph.

EVALUATIONChanges (increase or decrease) in pulmonary arterialpressure and in airway pressure after injection of testcompounds are measured in mm Hg and comparedwith baseline values.

MODIFICATIONS OF THE METHODBernard et al. (1997) described an isolated perfusedlung model with real time data collection and analy-sis of lung function. Male Sprague Dawley rats wereanesthetized with 130 mg/kg pentobarbital i.p. The tra-chea was cannulated and then ventilated with 5% CO2and 95% air at e rate of 60 breath/min and a tidalvolume of 2.5 ml. An injection of 650–700 units/kgof heparin was made into the right ventricle. A can-nula was placed into the main pulmonary artery. Theleft ventricle was incised and the lungs were washedfree of blood with warmed Krebs-Henseleit bicarbon-ate buffer with 4.5% BSA and 0.1% glucose. The leftatrium was then cannulated to allow outflow of the per-fusate. The lung was then removed and suspended ina chamber for perfusion. The flow rate of the perfusate


was adjusted to 8–10 ml/min/kg. Ventilation was main-tained at 60 breaths/min with humidified and warmedgas. The lung was allowed to recover for 15 min atwhich time the lung mechanic parameters of flow, vol-ume, transpulmonary pressure, pulmonary artery pres-sure, weight, resistance, elastance, and positive enddi-astolic pressure were measured.

Hauge (1968) studied the conditions governing thepressor responses to ventilation hypoxia in isolatedperfused rat lungs.

Uhlig and Heiny (1995) measured the weight ofthe isolated perfused rat lung during negative pressureventilation for quantitating edema formation in the iso-lated lung.

Uhlig and Wollin (1994) described an improvedsetup for the isolated perfused rat lung. Breathing me-chanic, such as tidal volume, pulmonary compliance,and pulmonary resistance, as well as perfusate charac-teristics, such as pulmonary vascular resistance, pul-monary pre- and postcapillary resistance, perfusatepH, PO2, and PCO2, and the capillary filtration coef-ficient were determined.

Byron et al. (1986) used the isolated perfused ratlung preparation for the study of aerosolized drug de-position and absorption.

Hendriks et al. (1999) published a modified tech-nique of isolated left lung perfusion in the rat.

Riley et al. (1981) determined the tissue elasticproperties of saline-filled isolated hamster lungs bymeasuring the pressure-volume relationships and stud-ied the prevention of bleomycin-induced pulmonary fi-brosis by cis-4-hydroxy-L-proline.

Lewis and Broadley (1995) tested the influence ofspasmogen inhalation by guinea pigs upon subsequentdemonstration of albumin-induced hyperreactivity inisolated airway tissues, such as perfused lung halvesand tracheal spirals.

Corboz et al. (2000) used the isolated guinea piglung to study the inhibition of capsaicin-induced bron-choconstriction by nociceptin. The lungs and the heartwere removed en bloc from guinea pigs euthanizedwith an intraperitoneal overdose of sodium pentobar-bital. The trachea and pulmonary artery were rapidlycannulated and half of the heart was cut to facilitatedrainage. The lungs were then placed inside a warmed(37°C) glass chamber and suspended from a forcedisplacement (Grass FT-03). They were mechanicallyventilated with room air using a small animal venti-lator. The respiratory rate was set at 60 strokes/minwith a volume of 2.0 ml/stroke. Pulmonary inflationpressure was continuously monitored with a pressuretransducer connected to a side arm of the tracheal can-

nula. Perfusion pressure was maintained with a peri-staltic pump at a rate of 4.5–5.0 ml/min to producea baseline pulmonary arterial pressure of between 6and 14 cmH2O. The pulmonary artery pressure wascontinuously monitored using a pressure transducerconnected to the side arm of the pulmonary artery can-nula. Transducers were connected to a polygraph forcontinuous monitoring of variables. The lungs wereperfused with Tyrode’s solution maintained at 37°C.Cumulative dose–response curves were constructed byadding increasing doses of capsaicin or the tachykininNK2 receptor agonist neurokinin A directly into thepulmonary artery. Test drugs were perfused 30 min be-fore the addition of increasing doses of capsaicin orneurokinin A.

Anglade et al. (1998) measured the pulmonary cap-illary filtration coefficient in isolated rabbit lungswhich were suspended by a string tied around the tra-cheal cannula from a counterbalanced force transducerto perform continuous weight measurement. To mea-sure the pulmonary capillary filtration coefficient, pul-monary venous pressure was raised stepwise which re-sults in an initial large weight gain for a few secondsfollowed by a slower rate of weight gain. The pul-monary capillary filtration coefficient was calculatedon the slow phase of the weight curve by a time zeroextrapolation or from the slope of the curve.

Nakamura et al. (1987) studied neurogenic pul-monary edema in lung perfusion preparations in situin the dog.

Allen et al. (1993) studied the cardiovascular effectsof a continuous prostacycline administration into anisolated in situ lung preparation in the dog.

Pogrebniak et al. (1994) investigated the influenceof tumor necrosis factor in an isolated lung perfusionmodel in pigs.

The fluid filtration coefficient before and after in-fusion of Escherichia coli endotoxin was measured inexcised goat lungs by Winn et al. (1988).

REFERENCES AND FURTHER READINGAllen DA, Schertel ER, Bailey JE (1993) Reflex cardiovas-

cular effects of continuous prostacycline administrationinto an isolated in situ lung in the dog. J Appl Physiol74:2928–2934

Anglade D, Corboz M, Menaouar A, Parker JC, Sanou S,Bayat S, Benchetrit G, Grimbert FA (1998) Blood flow vs.venous pressure effects on filtration coefficient in oleic-acidinjured lung. J Appl Physiol 84:1011–1023

Bernard CE, Dahlby R, Hoener BA (1997) An isolated perfusedlung model with real time data collection and analysis oflung function. J Pharmacol Toxicol Meth 38:41–46

Byron PR, Roberts NSR, Clark Ar (1986) An isolated perfusedrat lung preparation for the study of aerosolized drug depo-sition and absorption. J Pharm Sci 75:168–172


Corboz MR, Rivelli MA, Egan RW, Tulshian D, Matasi J,Fawzi AB, Benbow L, Smith-Torhan A, Zhang H, Hey JA(2000) Nociceptin inhibits capsaicin-induced bronchocon-striction in isolated guinea pig lung. Eur J Pharmacol402:171–179

Hauge A (1968) Conditions governing the pressor responsesto ventilation hypoxia in isolated perfused rat lungs. ActaPhysiol Scand 72:33–44

Hendriks JHM, van Schil PEY, Eyskens EJM (1999) Modifiedtechnique of isolated left lung perfusion in the rat. Eur SurgRes 31:93–96

Nakamura J, Zhang S, Ishikawa N (1987) Role of pulmonaryinnervation in canine in situ lung-perfusion preparation:a new model of neurogenic pulmonary edema. Clin ExpPharmacol Physiol 14:535–542

Lewis CA, Broadley KJ (1995) Influence of spasmogen inhala-tion by guinea pigs upon subsequent demonstration of al-bumin-induced hyperreactivity in isolated airway tissues.J Pharmacol Toxicol Meth 34:187–198

Nossaman BD, Feng CJ, Kadowith PJ (1994) Analysis of re-sponses to bradykinin and influence of HOE 140 in the iso-lated perfused rat lung. Am J Physiol Heart Circ Physiol266:H2452–2461

Pogrebniak HW, Witt CJ, Terrill R, Kranda K, Travis WD,Rosenberg SA, Pass HI, Graeber GW, Webb WR,Mathisen DJ (1994) Isolated lung perfusion with tumornecrosis factor: A swine model in preparation of human tri-als. Ann Thorac Surg 57:1477–1483

Riley DJ, Kerr JS, Berg RA, Ianni BD, Pietra GG, Edelman NH,Prockop DJ (1981) Prevention of bleomycin-induced pul-monary fibrosis in the hamster by cis-4-hydroxy-L-proline.AM Rev Respir Dis 123:388–392

Uhlig S, Heiny O (1995) Measuring the weight of the iso-lated perfused rat lung during negative pressure ventilation.J Pharmacol Toxicol Meth 33:147–152

Uhlig S, Wollin L (1994) An improved setup for the isolatedperfused rat lung. J Pharmacol Toxicol Meth 31:85–94

Winn R, Nickelson S, Rice CL (1988) Fluid filtration coefficientof isolated goat lungs was unchanged by endotoxin. J ApplPhysiol 64:2463–2467

D.2.2In Vivo Tests

D.2.2.1Bronchospasmolytic Activity in Anesthetized GuineaPigs (Konzett–Rössler method)

PURPOSE AND RATIONALEThe principle war first described by Kiese (1935).Konzett and Rössler (1940) published a method suit-able for screening procedures which found worldwideacceptance. A survey on the history and further modi-fications was given by Döring and Dehnert (1997).

The method is based on registration of air volumechanges of a living animal in a closed system con-sisting of the respiration pump, of the trachea andthe bronchi as well as of a reservoir permitting mea-surement of volume or pressure of excess air. Bron-chospasm decreases the volume of inspired air andincreases the volume of excess air. Thus, the degree

of bronchospasm can be quantified by recording thevolume of excess air. Administration of spasmogenslike acetylcholine, histamine, bradykinin, serotonin,ovalbumin, PAF, substance P, methacholine or leu-kotrienes, results in contraction of bronchial smoothmuscle.

The method permits the evaluation of a drug’s bron-chospasmolytic effect by measuring the volume of air,which is not taken up by the lungs after bronchospasm.

PROCEDUREGuinea-pigs of either sex weighing 250–500 g areanaesthetized with 1.25 g/kg i.p. urethane. Pentobar-bital (60 mg/kg s.c.) and alcuronium chloride (1 mg/kg s.c.) are to be preferred when the bronchospasm iselicited by PAF or substance P. Anesthesia has to bedeep enough in order to prevent influence of sponta-neous respiration. The trachea is cannulated by meansof a two way cannula, one arm of which is connectedto the respiratory pump and the other to a StathamP23 Db transducer. The animal is artificially respiredusing a Starling pump with an inspiratory pressure setat 90–120 mm of water, an adequate tidal volume of3 ml/100 g body weight and a frequency of 60 strokesper minute. Excess air, not taken up by the lungs, ismeasured and recorded on a polygraph. The internaljugular vein is cannulated for the administration ofspasmogens and test compounds. The carotid artery iscannulated for measuring blood pressure.

TestingGuinea-pigs receive the following spasmogens by i.v.administration:

• acetylcholine hydrochloride (20–40 µg/kg), or• methacholine (20–40 µg/kg), or• histamine dihydrochloride (5–20 µg/kg), or• bradykinin triacetate (10–20 µg/kg), or• ovalbumin (1 mg/kg), or• PAF (25–50 ng/ml), or• leukotrienes LTC4, LTD4 (about 1 µg/kg), or• substance P (0.5 µg/kg).

After obtaining two bronchospasms of equal inten-sity, test compounds are administered i.v., p.o., s.c. orintraduodenally.

The spasmogen is given again at the following timeintervals:

• 5, 15 and 30 min after i.v. administration of the drug• 15, 30 and 60 min after intraduodenal administra-

tion of the drug• 30 and 60 (sometimes also 120) min after p.o. ad-

ministration of the drug.


The following standard compounds are used:

• atropine sulfate (0.01 mg/kg, i.v.) to inhibit acetyl-choline or methacholine induced spasms

• aminophylline (6 mg/kg, i.v.) to inhibit bradykinininduced spasms

• tolpropamine-HCl (0.2 mg/kg) to inhibit histamineinduced spasms

• imipramine-HCl (3–5 mg/kg) to inhibit serotonin-kreatinin-sulphate induced spasms.

EVALUATIONResults are expressed as percent inhibition of inducedbronchospasm over the control agonistic responses.The ED50 value is calculated.

CRITICAL ASSESSMENT OF THE METHODThe “Konzett-Roessler”-method has been proven tobe a standard procedure in respiratory pharmacologybeing modified by several authors (Rosenthale andDervinis 1968).

MODIFICATIONS OF THE METHODForced insufflation was proposed as a simple but accu-rate inhalation procedure for investigating the activityof anti-asthmatic drugs in guinea pigs by Schiantarelliet al. (1982).

Lundberg et al. (1983), Belvisi et al. (1989), Miuraet al. (1994) determined airway opening pressure (Pao)as an index for tracheobronchial resistance to air flow.

Orr and Blair (1969) and Riley et al. (1987) sen-sitized rats intravenously with a potent antiserumto ovalbumin, obtained by infecting ovalbumin-sen-sitized donor rats with the parasitic nematode Nip-postrongylus brasiliensis in order to boost IgE anti-body production. The anesthetized animals were chal-lenged with ovalbumin 48 h after sensitization and thesubsequent increase in tracheal pressure was recorded.

Collier et al. (1993), Collier and James (1967) pub-lished a modification of the Konzett-Rössler-methodusing the forced re-inflation to overcome the severebronchoconstriction occurring in sensitized guineapigs.

Further modifications are by Schliep et al. (1986),Marano and Doria (1993).

Groeben and Brown (1996) measured changes inthe cross-sectional area of conducting airways by cu-mulative doses of ipratropium with and without gal-lamine, a selective M2 muscarinic receptor blocker,and after metaproterenol in anesthetized dogs usinghigh-resolution computed tomography. Using a So-matom Plus scanner (Siemens), 50 to 55 contigu-

ous scans were obtained, starting approximately 5 mmabove the origin of the right upper lobe bronchus fromthe trachea and proceeding caudally using 1-mm tablefeed and 2-mm slice thickness. The dogs were anes-thetized with thiopental. After paralysis was inducedby succinylcholine, the trachea was intubated and thelungs ventilated with a volume-cycled ventilator with100% oxygen. During the scans, the dogs were apneicat function residual capacity (approximately 2 min).Images were reconstructed using a high-spatial fre-quency algorithm.

REFERENCES AND FURTHER READINGBelvisi MG, Chung KF, Jackson DM, Barnes PJ (1989a) Opioid

modulation of non-cholinergic neural bronchoconstrictionin guinea-pig in vivo. Br J Pharmacol 97:1225–1231

Belvisi MG, Ichinose M, Barnes PJ (1989b) Modulation of non-adrenergic, non-cholinergic neural bronchoconstriction inguinea-pig airways via GABAB-receptors. Br. J Pharmacol97:1225–1231

Collier HOJ, Hammond AR, Whiteley B (1963) Anti-anaphylactic action of acetylsalicylate in guinea pig lung.Nature 200:176–178

Collier HOJ, James GWL (1967) Humoral factors affecting pul-monary inflation during acute anaphylaxis in the guinea pigin vivo. Br J Pharmacol 30:283–301

De la Motta S (1991) Simultaneous measurement of respiratoryand circulatory parameters on anesthetized guinea pigs.Seventh Freiburg Focus on Biomeasurement (FFB7) Publ.by Biomesstechnik Verlag, 79232 March, Germany. B IV,pp 45–65

Döring HJ, Dehnert H (1997) Methoden zur Untersuchungder Atmungsorgane für die experimentelle Pharmakologieund Physiologie. Biomesstechnik-Verlag March GmbH, D-79323 March, Germany, pp 225–288

Groeben H, Brown RH (1996) Ipratropium decreases airwaysize in dogs by preferential M2 muscarinic receptor block-ade in vivo. Anesthesiology 85:867–873

Kiese M (1935) Pharmakologische Untersuchungen an derglatten Muskulatur der Lunge (insbesondere mit einigenephedrinartigen Substanzen) Naunyn Schmiedeberg’s Archexp Path Pharmakol 178:342–366

Konzett H, Rössler R (1940) Versuchsanordung zu Un-tersuchungen an der Bronchialmuskulatur. Naunyn-Schmiedeberg’s Arch Exp Path Pharmakol 192:71–74

Lau WAK, Rechtman MP, Boura ALA, King RG (1989)Synergistic potentiation by captopril and propranolol ofbradykinin-induced bronchoconstriction in the guinea-pig.Clin Exp Pharmacol Physiol 16:849–857

Lefort J, Vargaftig BB (1978) Role of platelets in aspirin-sensi-tive bronchoconstriction in the guinea pig; interactions withsalicylic acid. Br J Pharmacol 63:35–42

Lundberg JM, Brodin E, Saria A (1983) Effects and distributionof vagal capsaicin-sensitive substance P neurons with spe-cial reference to the trachea and lungs. Acta Physiol Scand119:243–252

Marano G, Doria GP (1993) Lung constant-pressure inflation:fluid dynamic factors are the basis of airway overpressureduring bronchoconstriction. Pharmacol Res 28:185–192

Miura M, Belvisi MG, Barnes PJ (1994) Modulation of non-adrenergic noncholinergic neural bronchoconstriction bybradykinin in anesthetized guinea pigs in vivo. J Pharm ExpTher 268:482–486


Orr TSC, Blair AMJN (1969) Potentiated reagin response toegg albumin and conalbumin in Nippostrongylus brasilien-sis infected rats. Life Sci 8:1073–1077

Riley PA, Mather ME, Keogh RW, Eady RP (1987) Activity ofnedocromil sodium in mast-cell-dependent reactions in therat. Int Arch Allergy Appl Immun 82:108–110

Rosenthale ME, Dervinis A (1968) Improved apparatus for mea-surement of guinea pig lung overflow. Arch Int Pharmaco-dyn 172:91–94

Schiantarelli P, Bongrani S, Papotti M, Cadel S (1982) Investi-gation of the activity of bronchodilators using a simple butaccurate inhalation procedure: forced insufflation. J Phar-macol Meth 8:9–17

Schliep HJ, Schulze E, Harting J, Haeusler G (1986) Antag-onistic effects of bisopropol on several β-adrenoceptor-mediated actions in anesthetized cats. Eur J Pharmacol123:253–261

D.2.2.2Effect of Arachidonic Acid or PAFon Respiratory Function In Vivo

PURPOSE AND RATIONALEBased on the classical method of Konzett and Rössler(1940), Lefort and Vargafting (1978), Vargafting et al.(1979) studied the effects of arachidonic acid and PAFon respiratory function of guinea pigs in vivo.

Arachidonic acid is metabolized into thromboxane(TXA2) and prostacyclin (PGI2). TXA2 produced inthe lung leads to bronchoconstriction, which is inde-pendent from circulating platelets and leukotrienes;TXA2 produced intracellularly in platelets inducesa reversible thrombocytopenia. PGI2 produced in thevessel wall leads to the reduction of systolic and di-astolic blood pressure. All three effects are inhibitedby drugs which block cyclo-oxygenase. In contrast,agents which block thromboxane synthetase inhibitbronchoconstriction and thrombocytopenia, but lead toa potentiation of blood pressure reduction.

In contrast to arachidonic acid, PAF as inducer leadsto bronchoconstriction, which is platelet-dependent.In addition, PAF induces thrombocytopenia, leukocy-topenia, reduction of blood pressure and increase ofhematocrit. These effects are also reversible, but morepersistent than those induced by arachidonic acid, andquickly result in tachyphylaxis. The test allows to eval-uate the sites of action of drugs, which interfere withthe mechanisms of bronchoconstriction and thrombo-cytopenia; in an in vivo-model guinea pigs are chal-lenged with the spasmogens and platelet-aggregatingsubstances arachidonic acid or PAF (platelet activatingfactor).

PROCEDUREMale guinea pigs (Pirbright White) weighing 300–600 g are anesthetized with 60 mg/kg pentobarbital

sodium (i.p.). One of the jugular veins is cannu-lated for the administration of spasmogen and testcompound. Both external carotid arteries are cannu-lated; one is connected to a pressure transducer toregister blood pressure, the other is used for bloodwithdrawal. The trachea is connected to a Starlingpump with an inspiratory pressure set of 80 mm H2O,an adequate tidal volume of approx. 10 ml/kg bodyweight and a frequency of 70–75 strokes/min. Sponta-neous respiration is inhibited by intravenous injectionof pancuronium (4 mg/kg) or gallamine (2 mg/kg) ontime.

In some experiments, pulmonal β-receptors areblocked by intraperitoneal administration of propra-nolol (2 mg/kg).

Excess air, not taken up by the lungs, is conductedto a transducer with bronchotimer (Rhema, Germany)which translates changes in air flow to an electrical sig-nal. Changes in air flow and arterial blood pressure arerecorded continuously.

Animals receive multiple intravenous injections ofthe same dose of arachidonic acid (Sigma, 250–600 µg/kg prepared from a stock solution 10 mg/mlethanol, 1:20 dilution with Na2CO3) until two bron-chospasms of equal intensity are obtained. The testcompound is administered intravenously and the spas-mogen is given again at the following intervals: 2, 10,20 and, if necessary, 30 min after administration of thedrug.

Ordinarily, the lung has to be passively dilated(bronchotimer) after each of the bronchoconstrictions.Immediately before and 30–45 s after each of thearachidonic acid applications, approx. 50 µl blood arecollected into Na-EDTA-coated tubes. The number ofthrombocytes is determined with a platelet analyzer(Becton Dickinson Ultra-Flo-100 or Baker 810) in10 µl samples of whole blood.

PAF (Paf-acether C16, Bachem, 0.03–0.04 µg/kg in0.9% saline+0.1% human serum albumin) as induceris injected intravenously 60 min before, 5 min and, ifnecessary, 60 min after intravenous drug administra-tion. Blood samples are collected 30 s before and 15 safter each of the PAF applications. The number ofleukocytes and hematocrit values are determined au-tomatically (TOA-microcell counter CC 108, ColoraMeßtechnik).

Standard compounds:

• dazoxiben HCl (inhibitor of thromboxane syn-thetase, TSI)

• acetylsalicylic acid (inhibitor of cyclo-oxygenase,COI)


EVALUATIONPercent inhibition or increase of bronchospasm, re-duction of blood pressure, thrombocytopenia, leuko-cytopenia and hematocrit following test drug admin-istration are calculated in comparison to control val-ues before drug treatment. For the reduction of bloodpressure, both the magnitude [mm Hg, systolic and di-astolic] and the duration [min] are determined. Evena sole increase in duration of blood pressure reduc-tion is considered as an increase of the effect. Fromthe pattern profile of the influence on bronchocon-striction, thrombocytopenia and blood pressure reduc-tion, the mechanism of action of a test drug is con-cluded:

• inhibitor of thromboxane synthetase• inhibitor of cyclo-oxygenase• other effect = no profile

In addition, the inherent action of the test substanceon blood pressure is determined before arachidonicacid- or PAF-administration.

MODIFICATIONS OF THE METHODSKagoshima et al. (1997) used a modification of theKonzett-Rössler-method to test the suppressive ef-fects of a PAF-antagonist on asthmatic responses inguinea pigs actively sensitized with ovalbumin. Theimmediate and the late asthmatic response were mea-sured by the oscillation method according to Mead(1960).

Pauluhn (1994, 2004) described inhalation systems,mainly for toxicological studies, by which large num-bers of animals can be studied simultaneously.

REFERENCES AND FURTHER READINGKagoshima M, Tomomatsu N, Iwahisha Y, Yamaguchi S, Mat-

suura M, Kawakami Y, Terasawa M (1997) Suppressive ef-fects of Y-24180, a receptor antagonist to platelet activat-ing factor (PAF), on antigen-induced asthmatic responsesin guinea pigs. Inflamm Res 46:147–153

Lefort J, Vargaftig BB (1978) Role of platelets in aspirin-sensi-tive bronchoconstriction in the guinea pig; interactions withsalicylic acid. Br. J. Pharmac. 63:35–42

Mead J (1960) Control of respiratory frequency. J Appl Physiol15:325–336

Pauluhn J (1994) Validation of an improved nose-only exposuresystem for rodents. J Appl Toxicol 14:55–63

Pauluhn J (2004) Acute inhalation studies with irritant aerosols:technical issues and relevance for risk characterization.Arch Toxicol 78:243–251

Vargaftig BB, Lefort J, Prancan AV, Chignard M, Benveniste J(1979) Platelet-lung in vivo interactions: An artifact ofa multipurpose model?. Haemostasis 8:171–182

D.2.2.3Bronchial Hyperreactivity

PURPOSE AND RATIONALESymptoms like asphyctic convulsions resemblingbronchial asthma in patients can be induced by in-halation of histamine or other bronchospasm inducingagents in guinea pigs. The challenging agents are ap-plied as aerosols produced by an ultra-sound nebulizer.The first symptoms are increased breathing frequency,forced inspiration, and finally asphyctic convulsions.The occurrence of these symptoms can be delayed byantagonistic drugs. Pre-convulsion time, i. e. time untilasphyctic convulsions, can be measured.

PROCEDURETen male albino guinea pigs weighing 300–400 g pergroup are used. The inhalation cages consist of 3 boxeseach ventilated with an air flow of 1.5 l/min. The an-imal is placed into box A to which the test drug orthe standard is applied using an ultra-sound nebulizerLKB NB108 which provides an aerosol of 0.2 ml so-lution of the test drug injected by an infusion pumpwithin 1 min. Alternatively, the animal is treated orallyor subcutaneously with the test drug or the standard.Box B serves as a sluice through which the animal ispassed into box C. There, the guinea pig is exposed toan aerosol of a 0.1% solution of histamine hydrochlo-ride provided by an ultra-sound nebulizer (De Vilbiss,Model 35 A). Time until appearance of asphyctic con-vulsions is measured. Then, the animal is immediatelywithdrawn from the inhalation box. The aerosols areremoved from the back wall of the boxes by applyinglow pressure.

EVALUATIONPercent of increase of pre-convulsion time is calcu-lated versus controls. ED50 values can be found, i. e.50% of increase of pre-convulsion time.

CRITICAL ASSESSMENT OF THE METHODThe “guinea pig asthma” has been applied as usefulmethod in various modifications by many laboratories.

MODIFICATIONS OF THE METHODSSimple methods to test bronchospasmolytic activity inconscious animals, called “thoracography” were de-scribed by Herxheimer (1956) Olsson (1971), Beumeet al. (1985). A silicon tube with a diameter of 1 mmis filled with mercury and serves as strain gauge ap-plied as belt around the thorax of conscious guineapigs. Changes in electrical resistance due to breathing


movements of the thorax are registered. The animalsare exposed in a Plexiglas chamber to acetylcholinenebulized by an ultrasonic device. Time until onset ofcoughing indicated by an increase of signal amplitudeand of severe asthmatic dyspnoea is registered.

Immunological factors are involved in bronchialhyperreactivity (Reynolds 1991).

Harris et al. (1976) immunized rabbits with ther-mophilic actinomyces antigen (Micropolyspora faeni).Lesions resembling hypersensitivity in man werefound, characterized by a mononuclear cell interstitialreaction and a marked increase in the number of intra-alveolar cells.

Ufkes et al. (1983) induced bronchial and cardio-vascular anaphylaxis in Brown-Norway rats, sensi-tized with trinitrophenyl haptenized ovalbumin andAlPO4 as adjuvant 12 days prior to challenge withtrinitrophenyl haptenized bovine serum albumin intra-venously.

Raeburn et al. (1992) gave a survey on techniquesfor drug delivery to the airways and the assessment oflung functions in animal models including parameterssuch as lung compliance and airway resistance.

Elwood et al. (1992) studied the effects of dexam-ethasone and cyclosporin A on the airway hyperre-sponsiveness and the influx of inflammatory cells intobronchoalveolar lavage fluid seen 18 to 24 h after ex-posure to aerosolized ovalbumin in actively ovalbu-min-sensitized Brown-Norway rats.

Schmiedl et al. (2003) found an increase of inactiveintra-alveolar surfactant subtypes in lungs of asthmaticBrown Norway rats. The volume fractions of surfac-tant subtypes and the epithelial surface fraction cov-ered with alveolar edema were determined by pointand intersection counting. The surface activity of sur-factant from broncho-alveolar lavage was determinedas the minimum surface tension at minimal bubble sizewith a pulsating bubble surfactometer.

Pahl et al. (2002) and Kuss et al. (2003) exposedovalbumin-sensitized Brown Norway rats to an oval-bumin-containing aerosol in a nose-only inhalationsystem (TSE, Bad Homburg, Germany) for 1 h toprovoke an influx of inflammatory cells into the air-ways. At the time of maximal influx of eosinophilicgranulocytes into the airways (48 h later), the animalswere sacrificed and a broncho-alveolar lavage was per-formed.

A model of bronchial hyperreactivity after activeanaphylactic shock in conscious guinea pigs has beendescribed by Tarayre et al. (1990). The guinea pigswere sensitized by an intramuscular injection of a largedose of ovalbumin in Freund’s adjuvant. The adminis-

tration of ovalbumin to induce anaphylactic shock wasby aerosol. Bronchial hyperreactivity to histamine wasobserved 3–6 h after the anaphylactic shock.

Bolser et al. (1995) sensitized guinea pigs by in-traperitoneal injection of 200 µg/kg ovalbumin mixedwith 200 mg/kg aluminum hydroxide. The animalswere placed 28 days later in a transparent plastic cham-ber and exposed to aerosols of ovalbumin (0.1%–1%)at an airflow of 4 l/min to elicit coughing. Coughs weredetected by a microphone placed in the chamber andconnected to an audio monitor and chart recorder. Thenumber of coughs elicited during a 4-min exposurewas counted by visual inspection of the chart record.

Pons et al. (2000) described a guinea pig modelof asthma. Animals were sensitized by two intraperi-toneal injections of 20 µg ovalbumin and 100 mg alu-minum hydroxide given 24 h apart. The sensitized an-imals were anesthetized and mechanically ventilated.Air flow was measured with a pneumotachograph,along with transpulmonary pressure and arterial pres-sure. Lung resistance and dynamic compliance werecalculated. Then, 30 min after administration of testdrug, the animals were challenged by inhaled ovalbu-min (5 mg/ml).

The importance of eosinophil activation for the de-velopment of allergen-induced bronchial hyperreactiv-ity was underlined by Santing et al. (1994). A signif-icant increase in bronchoreactivity to histamine wasobserved at 6 h after allergen exposure, which was as-sociated with an increase of eosinophils in the bron-choalveolar lavage and an increase in the eosinophilperoxidase activity.

The increased pulmonary vascular permeabilitymay be related to the adult respiratory distress syn-drome in man (Snapper and Christman 1989).

The infusion of small amounts of Escherichia coliendotoxin into chronically instrumented awake sheepresults in well-characterized pulmonary dysfunction(Brigham and Meyrick 1986).

One animal model associated with both increasedairway responsiveness and pulmonary inflammation isendotoxemia in sheep (Hutchinson et al. 1983).

The effect of a platelet activating factor receptor an-tagonist on the sheep’s response to endotoxin was stud-ied by Christman et al. (1987).

Chiba and Misawa (1995) characterized muscariniccholinoceptors in airways of antigen-induced airwayhyperresponsive rats. The animals were sensitized with2,4-dinitrophenylated Ascaris suum extract togetherwith Bordetella pertussis (2 × 106 as an adjuvant andwere boosted with 2,4-dinitrophenylated Ascaris suumextract 5 days later. Isometric contractions of the circu-


lar muscle of isolated bronchial rings after addition ofincreasing doses of acetylcholine were measured witha force-displacement transducer.

Laboratory infection of primates with Ascarissuum may provide a model of allergic bronchocon-striction (Patterson et al. 1983; Pritchard et al. 1983;Eady 1986).

Richards et al. (1986) used various models of air-way hypersensitivity, such as inhalation of Ascarissuum antigen in monkeys and dogs.

Rylander and Marchat (1988) studied the effect ofa corticosteroid on an acute inflammation in the lungsof guinea pigs exposed to an aerosol of bacterial en-dotoxin. The subsequent inflammatory response wasevaluated counting the number of cells obtained fromairway lavage and in the lung interstitium as well asthe chemotactic effect of alveolar macrophages.

Hatzelmann et al. (1996) reported on automaticleukocyte differentiation in broncho-alveolar lavagefluids of guinea pigs and Brown-Norway rats using anautomatic cell analyzing system (Cobas Helios 5Diff;Hoffmann-La Roche; Grenzach-Wyhlen, Germany).

Minshall et al. (1993) demonstrated that neonatalimmunization of rabbits with Alternia tenuis can leadto the development of persistent airway hyperrespon-siveness.

Okada et al. (1995) studied late asthmatic reactionsin guinea pigs sensitized with Ascaris antigen. Theyevaluated interleukin-1 production by immunostainingwith anti-IL-1β antibody and elucidated the action ofIL-1 in late asthmatic reactions with recombinant IL-1receptor antagonist.

Folkerts et al. (1995) found that intratracheal inoc-ulation of parainfluenza type 3 virus to guinea pigs in-duces a marked increase of airway responsiveness toincreasing doses of intravenous histamine. In sponta-neously anesthetized breathing guinea pigs, inhalationof an aerosol containing the nitric oxide precursor L-arginine completely prevented the virus-induced hy-perresponsiveness to histamine.

Fryer et al. (1994, 1997) measured M2 muscarinicreceptor function in anesthetized and paralyzed guineapigs by electrical stimulation of both vagus nerves pro-ducing bronchoconstriction (measured as pulmonaryinflation pressure) and bradycardia.

REFERENCES AND FURTHER READINGBeume R, Kilian U, Mussler K (1985) Die Prüfung

bronchospasmolytischer Substanzen am wachen Meer-schweinchen. Atemw – Lungenkrh 11:342–345

Bolser DC, DeGennaro FC, O’Reilly S, Hey JA, Chapman RW(1995) Pharmacological studies of allergic cough in theguinea pig. Eur J Pharmacol 277:159–164

Brigham KL, Meyrick B (1986) Endotoxin and lung injury: stateof the art review. Am Rev Respir Dis 133:913–927

Chiba Y, Misawa M (1993) Strain differences in change inairway hyperresponsiveness after repeated antigenic chal-lenge in three strains of rats. Gen Pharmacol 24:1265–1272

Chiba Y, Misawa M (1995) Characteristics of muscariniccholinoceptors in airways of antigen-induced airway hyper-responsive rats. Comp Biochem Physiol 111C:351–357

Christman BW, Lefferts PL, Snapper JR (1987) Effect ofa platelet activating factor receptor antagonist (SRI 63–441) on the sheep’s response to endotoxin. Am Rev RespirDis 135:A82

Eady RP (1986) The pharmacology of nedocromil sodium. Eur JRespir Dis 69: (Suppl 147):112–119

Elwood W, Lötvall JO, Barnes PJ, Chung KF (1992) Effect ofdexamethasone and cyclosporin A on allergen-induced air-way hyperresponsiveness and inflammatory cell responsesin sensitized Brown-Norway rats. Am Rev Respir Dis145:1289–1294

Folkerts G, van der Linde HJ, Nijkamp FP (1995) Virus-inducedairway hyperresponsiveness in guinea pigs is related toa deficiency in nitric oxide. J Clin Invest 95:26–30

Fryer AD, Yarkony KA, Jacoby DB (1994) The effect of leuko-cyte depletion on pulmonary M2 muscarinic receptor func-tion in parainfluenza virus-infected guinea pigs. Br J Phar-macol 112:588–594

Fryer AD, Costello RW, Yost BL, Lobb RR, Tedder TF, Stee-ber DA (1997) Antibody to VLA-4, but not to L-selectin,protects neuronal M2 muscarinic receptors in antigen-challenged guinea pig airways. J Clin Invest 99:2036–2044

Harris JO, Bice D, Salvaggio JE (1976) Cellular and humoralbronchopulmonary immune response of rabbits immunizedwith thermophilic actinomyces antigen. Am Rev Respir Dis114:29–43

Hatzelmann A, Haefner D, Beume R, Schudt C (1996) Auto-matic leukocyte differentiation in bronchoalveolar lavagefluids of guinea pigs and Brown-Norway rats. J PharmacolToxicol Meth 35:91–99

Herxheimer H (1956) Bronchoconstrictor agents and their an-tagonists in the intact guinea pig. Arch Int Pharmacodyn106:371–380

Hutchinson AA, Hinson JM, Brigham KL, Snapper JL (1983)Effect of endotoxin on airway responsiveness to aerosolhistamine in sheep. J Appl Physiol 54:1463–1468

Kallos P, Pagel W (1937) Experimentelle Untersuchungen überAsthma bronchiale. Acta Med Scand 91:292–305

Kuss H, Hoefgen N, Johanssen S, Kronbach T, Rundfeldt C(2003) In vivo efficacy in airway disease models of N-(3,5-dichloro-pyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-glycolic acid amide (AWD 12–281),a selective phosphodiesterase 4 inhibitor for inhaledadministration. J Pharmacol Exp Ther 307:373–385

Minshall EM, Riccio MM, Herd CM, Douglas GJ, Seeds EAM,McKennif MG, Sasaki M, Spina D, Page CP (1993)A novel animal model for investigating persistent air-way hyperresponsiveness. J Pharmacol Toxicol Meth30:177–188

Okada S, Inoue H, Yamauchi K, Iijima H, Okhawara Y, Tak-ishima T, Shirato K (1995) Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guineapigs: Suppressive effect of interleukin-1 receptor antago-nist on late asthmatic reaction. J Allergy Clin Immunol95:1236–1245

Olsson OAT (1971) Histamine-induced bronchospasm inunanaesthetized guinea pigs. Acta Allergol 26:438–447


Pahl A, Zjang M, Kuss H, Szelenyi I, Brune K (2002) Regulationof IL-13 synthesis in human lymphocytes: implications forasthma therapy. Br J Pharmacol 135:1915–1926

Patterson R, Suszko IM, Harris KE (1983) The in vivo transfer ofantigen-induced airway reactions by bronchial lumen cells.J Clin Invest 62:519–524

Pons R, Santamarïa P, Suchankova J, Cortijo J, Morcillo EJ(2000) Effects of inhaled glaucine on pulmonary responsesto antigen in sensitized guinea pigs. Eur J Pharmacol397:187–195

Pritchard DI, Eady PR, Harper ST, Jackson DM, Orr TSC,Richards IM, Trigg S, Wells E (1983) Laboratory infectionof primates with Ascaris suum to provide a model of aller-gic bronchoconstriction. Clin Exp Immunol 54:469–476

Raeburn D, Underwood SL, Villamil ME (1992) Techniquesfor drug delivery to the airways, and the assessment oflung functions in animal models. J Pharmacol Toxicol Meth27:143–159

Reynolds HY (1991) Immunologic system in the respiratorytract. Physiol Rev 71:1117–1133

Richards IM, Dixon M, Jackson DM, Vendy K (1986) Alter-native modes of action of cromoglycate. Agents Actions18:294–300

Rosenthale ME, Dervinis A (1968) Improved apparatus for mea-surement of guinea pig lung overflow. Arch Int Pharmaco-dyn 172:91–94

Rosenthale ME, Dervinis A, Begany AJ, Lapidus M, Gluck-mann MI (1970) Bronchodilator activity of prostaglandinE2 when administered by aerosol to three species. Experi-entia 26:1119–1121

Rylander R, Marchat B (1988) Modulation of acute endotoxinpulmonary inflammation by a corticosteroid. J Clin Lab Im-munol 27:83–86

Santing RE, Hoekstra Y, Pasman Y, Zaagsma J, Meurs H(1994) The importance of eosinophil activation for the de-velopment of allergen-induced bronchial hyperreactivityin conscious, unrestrained guinea pigs. Clin Exp Allergy24:1157–1163

Schmiedl A, Hoymann HG, Ochs M, Menke A, Fehrenbach A,Krug N, Tschernig T, Höhlfeld JM (2003) Increase of inac-tive intra-alveolar surfactant subtypes in lungs of asthmaticBrown Norway rats. Virchows Arch 442:56–65

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Tarayre JP, Aliaga M, Barbara M, Tisseyre N, Vieu S, Tisne-Versailles J (1990) Model of bronchial hyperreactivity afteractive anaphylactic shock in conscious guinea pigs. J Phar-macol Meth 23:13–19

Ufkes JGR, Ottenhof M, Aalberse RC (1983) A new method forinducing fatal, IgE mediated, bronchial and cardiovascularanaphylaxis in the rat. J Pharmacol Meth 9:175–181

D.2.2.4Body Plethysmography and Respiratory ParametersAfter Histamine-Induced Bronchoconstrictionin Anesthetized Guinea Pigs

PURPOSE AND RATIONALEGuinea pigs can be placed in a plethysmograph formeasurement of respiratory parameters. Respiratoryfrequency and respiratory amplitude are recorded. Thedecrease of respiratory amplitude (diminished respira-tory volume due to bronchoconstriction) and the re-

flectory increase of respiratory frequency after his-tamine inhalation are attenuated by bronchodilata-tory drugs. Additional respiratory parameters can berecorded using a Fleisch tube and a catheter insertedinto the pleural cavity (Englert et al. 1992). Themethod can be used for various purposes, e. g., to eval-uate the antagonism against bradykinin-induced bron-choconstriction (Wirth et al. 1991, 1993) or the bron-chodilator effects of potassium channel openers (En-glert et al. 1992) or to measure the effect of morphineon respiration in rats (Kokka et al. 1965).

PROCEDUREGuinea pigs of either sex weighing 400–600 g areanesthetized with 70 mg/kg pentobarbital i.p. The tra-chea, pleural cavity, jugular vein, and carotid arteryare prepared and cannulated. The animals are mechani-cally ventilated with a Starling respiratory pump whichdelivers an inspiration volume that represents a tra-cheal pressure of 8 cm water at a rate of 60 strokes/min.Succinylcholine chloride at a dose of 1 mg/kg is giveni.v. to prevent interference from spontaneous respira-tion. The guinea pigs are placed inside a whole bodyplethysmograph and tracheal, pleural, venous and ar-terial catheters are connected to onset ports in thewall of the plethysmograph box. The tracheal port isthen connected with the respiration pump. Airflowrate into and out of the plethysmograph are mea-sured as pressure difference with a No. 000 Fleischtube and a differential pressure transducer (Fa. Hel-lige, PM 97 TC). Airflow is calibrated by passing com-pressed air through a rotameter. The tidal volume (VT)is calculated from the flow signal. Transpulmonarypressure (PTP) is measured with a differential pres-sure transducer (Hellige, PM 97 TC), with one side at-tached to a catheter inserted into the right pleural cav-ity and the other side connected to a side port of the tra-cheal cannula. PTP is calibrated with a water manome-ter. Signals from airflow, tidal volume and transpul-monary pressure are fed into an on-line computer sys-tem (PO-NE-MAH, Model PF-1, Storrs) for calcula-tion of pulmonary resistance (RL) and dynamic lungcompliance (CDYN). These parameters are calculatedfor each breath with a sampling rate of 100 s−1 foreach circle. Flow and pressure signals for computa-tion are obtained from a PLUGYS measuring system(Fa. Hugo Sachs Elektronik, Freiburg, Germany). Sys-temic arterial pressure is measured using a Stathampressure transducer (P 23 Db). Heart rate is computedfrom pressure pulses (Döring and Dehnert 1997).

Three doses of test compound or standard are in-jected intravenously. Saline injections serve as con-


trols. Intravenous injections of histamine (0.5–2 µg/kg)lead to a short decrease in CDYN and to a short increasein RL by approximately 200% compared with baseline.Challenges are repeated at 5-min intervals, yielding thesame increase in RL during the whole 1-h experimentalperiod. After 3 reproducible responses the test agent isadministered intravenously 1 min before the histamineinjection.

To evaluate test compounds for inhalation route,aerosols are generated with an ultrasonic nebulizer(LKB, model NB 108) and are administered to the ani-mals through a shunt in the afferent limb of the respira-tory pump, allowing the inspired air to pass through thenebulizer chamber before entering the animals lungs.

EVALUATIONInhibition of histamine induced bronchoconstrictionby various doses of test compound and standard isrecorded. ED50 values for inhibition in RL are calcu-lated. Furthermore, the time course of histamine antag-onism can be evaluated. Compounds can be tested ei-ther after iv. injection of histamine (prevention) or dur-ing intravenous infusion of histamine (intervention).

CRITICAL ASSESSMENT OF THE METHODWhole body plethysmography has been proven to bea useful tool in respiratory pharmacology for studieson the antagonism against various bronchoconstric-tors, such as histamine and bradykinin, as well as forairway pharmacology of potassium channel openers.

MODIFICATIONS OF THE METHODSeveral authors use body plethysmography to studyrespiratory functions in animals (Amdur and Mead1958; Blümcke et al. 1967), Pennock et al. 1979; Agar-wal (1981), James and Infiesto (1983), Kisagawa et al.(1984), Griffith-Johnson (1988), Danko and Chapman(1988), Ball et al. (1991), Chand et al. (1993).

The effect of β-blockers on pulmonary function andbronchoconstrictor responsiveness in guinea pigs andrats has been studied by Chapman et al. (1985).

Finney and Forsberg (1994) developed a techniquefor quantification of nasal involvement in a guineapig plethysmograph. Nasal and lower respiratory sys-tem conductance could be measured simultaneously inanesthetized animals.

A whole body plethysmograph for conscious ani-mals has been described by Elliott et al. (1991) andimprovements by Linton (1991).

Studies of bronchospasmolytic agents with aerosolchallenge in conscious guinea pigs using a double

chamber plethysmograph box have been reported bySchlegelmilch (1991).

Ball et al. (1991) described a method for the evalu-ation of bronchoactive agents in the conscious guineapig. The method involves the use of “head out” wholebody plethysmographs from which respiratory rate canbe recorded by monitoring respiration-related changesin pressure within the body chamber.

Hey et al. (1995) used a head-out, whole bodyplethysmograph to examine the effects of GABAB re-ceptor agonists on minute ventilation, tidal volume andrespiratory rate due to room air and carbon dioxide-en-riches gas hyperventilation in conscious guinea pigs.

Murphy et al. (1998) developed a method forchronic measurement of pleural pressure in con-scious rats. Pleural pressures were measured by sur-gically implanting a fluid-filled polyurethane catheterattached to a pressure-sensitive radiotelemetry trans-mitter (Model TA11PA-C40, Data Sciences Int., St.Paul, MN) beneath the pleural surface. A compatiblereceiver (Model RLA0120) and a data acquisition andanalysis software system (LabPRO, Version 3) sam-pling at a rate of 500 Hz were used to analyze the tele-metric signals.

Sinnett et al. (1981) described a fast integrated flowplethysmograph for small animals (mice).

Glaab et al. (2001) used tidal mid-expiratory flowin a head-out body plethysmograph as a measure ofairway hyperresponsiveness in allergic mice.

Simple methods to determine respiratory parame-ters in small animals were described by Schütz (1960),Höbel et al. (1971).

Schlenker (1984) and Schlenker and Metz (1989)evaluated ventilatatory parameters in dystrophic Syr-ian hamsters.

Wasserman and Griffin (1977) studied bronchoac-tivity in the intact anesthetized dog.

Paré et al. (1976) determined pulmonary resistanceand dynamic lung compliance in rhesus monkeys.

Wegner et al. (1984) measured dynamic respiratorymechanics in monkeys by forced oscillations gener-ated by a loudspeaker in an airtight chamber.

REFERENCES AND FURTHER READINGAgrawal KP (1981) Specific airway conductance in guinea pigs:

normal values and histamine induced fall. Resp Physiol43:23–30

Amdur MO, Mead J (1958) Mechanics of respiration in unanes-thetized guinea pigs. Am J Physiol 192:364–368

Arch JRS, Buckle DR, Bumstead J, Clarke GD, Taylor JF, Tay-lor SG (1988) Evaluation of the potassium channel acti-vator cromakalim (BRL 34915) as a bronchodilator in theguinea pig: comparison with nifedipine. Br J Pharmacol95:763–770


Ball DI, Coleman RA, Hartley RW, Newberry A (1991) A novelmethod for the evaluation of bronchoactive agents in theconscious guinea pig. J Pharmacol Meth 26:187–202

Blümcke S, Rode J, Niedorf HR (1967) Eine einfache Meth-ode der Körper-Plethysmographie der Ratte. Naturwiss54:343–344

Chand N, Nolan K, Pillar J, Lomask M, Diamantis W, Sofia RD(1993) Aeroallergen-induced dyspnea in freely movingguinea pigs: quantitative measurement by bias flow ven-tilated whole body plethysmography. Allergy 48:230–235

Chapman RW, Danko G, Siegel MI (1985) Effect of propra-nolol on pulmonary function and bronchoconstrictor re-sponsiveness in guinea pigs and rats. Pharmacol Res Comm17:149–163

Danko G, Chapman RW (1988) Simple, noninvasive method tomeasure antibronchoconstrictor activity of drugs in con-scious guinea pigs. J Pharmacol Meth 19:165–173


Elliott RD, Fitzgerald MF, Clay TP (1991) A whole bodyplethysmograph for small animals. 7th Freiburg Focuson Biomeasurement. Cardiovascular and Respiratory invivo Studies. Biomesstechnik-Verlag March GmbH, 79232March, Germany, pp 66–71

Englert CE, Wirth K, Gehring D, Fürst U, Albus U, Scholz W,Rosenkranz B, Schölkens BA (1992) Airway pharma-cology of the potassium channel opener, HOE 234, inguinea pigs: in vitro and in vivo studies. Eur J Pharmacol210:69–75

Finney MJ, Forsberg KI (1994) Quantification of nasal in-volvement in a guinea pig plethysmograph. J Appl Physiol76:1432–1438

Glaab T, Daser A, Brain A, Neuhaus-Steinmetz U, Fabel H,Alarie Y, Renz H (2001) Tidal midexpiratory flow as a mea-sure of airway hyperresponsiveness in allergic mice. Am JPhysiol 280:L565–L573

Griffith-Johnson DA, Nicholl PJ, McDermott M (1988) Mea-surement of specific airway conductance in guinea pigs.A noninvasive method. J Pharmacol Meth 19:233–242

Hey JA, Mingo G, Bolsner DC, Kreutner W, Krobatsch D, Chap-man RW (1995) Respiratory effects of baclofen and 3-aminopropylphosphinic acid in guinea pigs. Br J Pharmacol114:735–738

Höbel M, Maroske D, Eichler O (1971) Eine einfache Meth-ode zur Bestimmung des Atemminutenvolumens vonRatten und Meerschweinchen. Arch Int Pharmacodyn194:371–374

James JT, Infiesto BP (1983) Concurrent measurement of res-piratory and metabolic parameters in rats during exposureto a test vapor: Respiratory stress test. J Pharmacol Meth10:283–292

Kisagawa K, Saitoh K, Tanizaki A, Ohkubo K, Irino O (1984)A new method for measuring respiration in the consciousmouse. J Pharmacol Meth 12:183–189

Kokka N, Elliott HW, Way L (1965) Some effects of morphineon respiration and metabolism in rats. J Pharmacol ExpTher 148:386–392

Linton P (1991) Improvements incorporated in the animalwhole-body plethysmograph after Elliott et al. 7th FreiburgFocus on Biomeasurement. Cardiovascular and Respira-tory in vivo Studies. Biomesstechnik-Verlag March GmbH,79232 March, Germany, pp 72–76

Murphy DJ, Renninger JP, Gossett KA (1998) A novel methodfor chronic measurement of pleural pressure in consciousrats. J Pharmacol Toxicol Meth 39:137–141

Paré PD, Michoud MC, Hogg JC (1976) Lung mechanics fol-lowing antigen challenge of Ascaris suum-sensitive rhesusmonkeys. J Appl Physiol 41:668–676

Pennock BE, Cox CP, Rogers RM, Cain WA, Wells JH (1979)A noninvasive technique for measurements of changes inspecific airway resistance. J Appl Physiol 46:399–406

Schlegelmilch R (1991) Respiratory measurements on consciousguinea pigs using a double chamber plethysmograph boxwith aerosol challenge. 7th Freiburg Focus on Biomea-surement. Cardiovascular and Respiratory in vivo Studies.Biomesstechnik-Verlag March GmbH, 79232 March, Ger-many, pp 136–140

Schlenker EH (1984) An evaluation of ventilation in dystrophicSyrian hamsters. J Appl Physiol 56:914–921

Schlenker EH, Metz TJ (1989) Ventilatory responses of dys-trophic and control hamsters to naloxone. PharmacolBiochem Behav 34:681–684

Schütz E (1960) Bestimmung der Atemgröße narkotisierter Rat-ten. Arzneim Forsch/Drug Res 10:52–53

Sinnett EE, Jackson AC, Leith DE, Butler JP (1981) Fast in-tegrated flow plethysmograph for small mammals. J ApplPhysiol 50:1104–1110

Wasserman MA, Griffin RL (1977) Thromboxane B2 – compar-ative bronchoactivity in experimental systems. Eur J Phar-macol 46:303–313

Wegner CD, Jackson AC, Berry JD, Gillepsie JR (1984) Dy-namic respiratory mechanics in monkeys measured byforced oscillations. Resp Physiol 55:47–61

Wirth K, Hock FJ, Albus U, Linz W, Alpermann HG, Anagnos-topoulos H, Henke St, Breipohl W, Knolle J, Schölkens BA(1991) HOE 140, a new potent and long acting bradykinin-antagonist: in vivo studies. Br J Pharmacol 102:774–777

Wirth KJ, Gehring D, Schölkens BA (1993) Effect of HOE 140on bradykinin-induced bronchoconstriction in anesthetizedguinea pigs. Am Rev Resp Dis 148:702–706

D.2.2.5Pneumotachography in Anesthetized Guinea Pigs

PURPOSE AND RATIONALEThe use of a pneumotachograph based on the principleof the Fleisch-tube and of additional pressure trans-ducers allows simultaneous measurements of severalrespiratory and circulatory parameters in anesthetizedguinea pigs (de la Motta 1991).

PROCEDUREGuinea pigs (Pirbright white) weighing 300–400 g areanesthetized with 1.5 g/kg urethane i.p. The animalsare shaved ventrally at the neck, placed on a heatedoperating table, and fixed at the upper extremities.A metal cannula with a blunted tip is inserted into thetrachea and secured with a loop within the caudal sec-tion of the trachea. A thin plastic catheter is insertedinto the esophagus and the tip located inside the tho-rax in order to register intrathoracic pressure. Further-more, the cephalic vein at one side and the carotidartery on the opposite side are cannulated. The tra-cheal cannula is connected with pieces of tubing toa Fleisch tube (pneumotachograph), size 0000. In or-


der to avoid water condensation, the Fleisch tube isheated. The Fleisch tube is connected with a sensitivedifferential pressure transducer with a range of 2 cmH2O (Validyne, model MP 45 -xx-871). One side ofanother pressure differential transducer with a range of20 ml H2O is connected with the esophageal catheter,the other side remaining open to room air. Both Vali-dyne pressure transducers are connected to a separatepreamplifier. For recording of the arterial blood pres-sure a Gould pressure transducer, Type P23Gb, is used.The signals for airflow and esophageal pressure aremonitored at the output of the preamplifier with a dig-ital 2-channel oscilloscope. To obtain various respi-ratory and circulatory parameters from the three pri-mary signals they are calculated by certain formulaeby an analogue computer (Buxco Pulmonary Mechan-ics Analyzer, Model 6). The following parameters arepresented at the output of the instrument as analogueelectrical signals:

CirculationSystolic blood pressure, diastolic blood pressure, meanblood pressure

RespirationTidal volume, respiratory volume per minute, respira-tory rate

Pulmonary MechanicsAirway resistance, dynamic compliance, end-respi-ratory work.

A multi-channel recorder (Graphtec LinearcorderMark VII) serves a functional check on the Buxco An-alyzer and as analogue presentation of the calculatedparameters. Data processing is performed by a 12-channel A/D converter (Buxco Data Logger, ModelD/C-12 F/V) which digitizes the analogue output sig-nals of the Buxco Analyzer and sends them througha serial interface (RS232) to an IBM PC. A specialsoftware program (Lomask 1987; Hastings 1990a, b)provides a flexible facility for data reduction and sta-tistical evaluation.

EVALUATIONFor each individual experiment the data of the last5 min before the first substance application are aver-aged and used as controls. The response values aftersubstance application are then expressed as percent-ages of the controls. In this way each animal servesas its own control. For an analysis of the results theresponse values are averaged over certain time inter-vals.

CRITICAL ASSESSMENT OF THE METHODThe method described in great detail by de la Motta(1991) may be modified using different equipment ac-cording to individual needs.

MODIFICATIONS OF THE METHODMeasurement of respiratory parameters is based onearlier studies to be mentioned for historical reasons(Gad 1880; Pflüger 1882; Zwaardemaker and Ouwe-hand 1904; Jaquet 1908; Rohrer 1915; Gildemeister1922; Fleisch 1925; v. Neergaard and Wirz 1927; sur-vey by Döring HJ 1991).

Lorino et al. (1988) assessed respiratory mechanicsof histamine bronchopulmonary reactivity in guineapigs.

O’Neil et al. (1981) published a comparative studyof respiratory responses to bronchoactive agents inrhesus and cynomolgus monkeys.

Rayburn et al. (1989) described a computer-con-trolled pulmonary function system for studies in largeanimals.

Five methods of analyzing respiratory pressure-vol-ume curves have been compared by Lai and Diamond(1986).

A specially designed pneumotachograph that isplaced inside the trachea of guinea pigs was describedby Santing et al. (1992) allowing the evaluation of air-way functions in conscious, unstressed animals.

Lorino et al. (1993) estimated the changes in end-respiratory lung volume accompanied histamine-in-duced bronchoconstriction in anesthetized, paralyzed,and mechanically ventilated guinea pigs from mea-surements of thoracic cross-sectional area, assessedfrom the voltage induced by an external uniform mag-netical field in a pickup coil encircling the rib cage.

Gozzard et al. (1996) evaluated the effects of PDE-inhibitors in New Zealand White rabbits which wereimmunized within 24 h of birth with Alternaria tenuisantigen. Spontaneously breathing rabbits were intu-bated in neuroleptanalgesia with a cuffed endotrachealtube connected to a thermoregulated Fleisch pneumo-tachograph to allow measurement of tidal air flow.An oesophageal balloon catheter was inserted to pro-vide a measure of intra-pleural pressure and transpul-monary pressure. Antigen challenge was performedwith inhaled Alternaria tenuis extract.

An excellent survey on various methods and equip-ment to measure air flow is given by Döring and Dehn-ert (1997).

REFERENCES AND FURTHER READINGDe la Motta S (1991) Simultaneous measurement of respiratory

and circulatory parameters on anesthetized guinea pigs.


Seventh Freiburg Focus on Biomeasurement (FFB7) Publ.by Biomesstechnik Verlag, 79232 March, Germany. B IV,pp 45–65

Döring HJ (1991) Historical review of methods for the measure-ment and evaluation of respiratory parameters, in particu-lar airway resistance. Seventh Freiburg Focus on Biomea-surement (FFB7) Publ. by Biomesstechnik Verlag, 79232March, Germany. B IV, pp 17–29


Fleisch A (1925) Der Pneumotachograph; ein Apparat zurGeschwindigkeitsregistrierung der Atemluft. Pflüger’sArch 209:713–722

Gad J (1880) Die Regulierung der normalen Athmung. ArchAnat Physiol, Physiol Abthlg:1–32

Gildemeister M (1922) Über die Messung der Atmung mitGasuhr und Ventilen. Pflügers Arch 195:96–100

Gozzard N, Herd CM, Blake SM, Holbrook M, Hughes B,Higgs GA, Page CP (1996) Effects of theophylline androlipram on antigen-induced airway responses in neona-tally immunized rabbits. Br J Pharmacol 117:1405–1412

Hastings SG (1990a) An integrated system for data acquisi-tion and analysis. Sixth Freiburg Focus on Biomeasurement(FFB6) Publ. by Biomesstechnik Verlag, 79232 March,Germany, pp 206–209

Hastings SG (1990b) Typical data reduction process. SixthFreiburg Focus on Biomeasurement (FFB6) Publ. byBiomesstechnik Verlag, 79232 March, Germany. G IV1–27

Jaquet A (1908) Zur Mechanik der Atembewegungen. Archexp Path Pharmakol, Suppl, Festschr. O Schmiede-berg:309–316

Lai YL, Diamond L (1986) Comparison of five methods of an-alyzing respiratory pressure-volume curves. Respir Physiol66:147–155

Lomask MR (1987) BUXCO respiratory mechanics ana-lyzer for non invasive measurements in conscious ani-mals. Third Freiburg Focus on Biomeasurement (FFB3)Publ. by Biomesstechnik Verlag, 79232 March, Germany,pp 212–226

Lorino AM, Bénichou M, Macquin-Mavier I, Lorino H, Harf A(1988) Respiratory mechanics for assessment of histaminebronchopulmonary reactivity in guinea pigs. Resp Physiol73:155–162

Lorino AM, Jarreau PH, Sartene R, Mathieu M, Macquin-Mavier I, Harf A (1993) Bronchoconstriction-induced hy-perinflation assessed by thoracic area measurement inguinea pigs. Am Rev Resp Dis 147:392–397

O’Neil RM, Ashack RJ, Goodman FR (1981) A compara-tive study of respiratory responses to bronchoactive agentsin rhesus and cynomolgus monkeys. J Pharmacol Meth5:267–273

Pflüger E (1882) Das Pneumonometer. Pflügers Arch29:244–246

Rayburn DB, Mundie TG, Phillips YY (1989) Computer-con-trolled large-animal pulmonary function system. ComputMeth Progr Biomed 28:1–9

Rohrer F (1915) Der Strömungswiderstand in den men-schlichen Atemwegen und der Einfluss der unregelmäs-sigen Verzweigungen des Bronchialsystems auf den At-mungsverlauf in verschiedenen Lungenbezirken. PflügersArch 162:225–299

Santing RE, Meurs H, van der Mark TW, Remie R, Oost-erom WC, Brouwer F, Zaagsma J (1992) A novel methodto assess airway function parameters in chronically in-

strumented, unrestrained guinea-pigs. Pulmon Pharmacol5:265–272

v. Neergaard K, Wirz K (1927) Über eine Methode zu Messungder Lungenelastizität am lebenden Menschen, insbeson-dere beim Emphysem. Z Klin Med 105:35–50

Zwaardemaker H, Ouwehand CD (1904) Die Geschwindigkeitdes Athemstromes und das Athemvolum des Menschen.Arch Anat Physiol, Physiol Abthlg. Suppl:241–263

D.2.2.6Airway Microvascular Leakage

PURPOSE AND RATIONALEPlasma exudation in guinea-pig airways in vivo can bedetermined by Evans Blue dye and is fairly correlatedwith radiolabelled albumin (Rogers et al. 1989). Thismethod can be used to study the antagonism againstbradykinin- and platelet-activating factor-induced air-way microvascular leakage and vagal stimulation-in-duced airway responses (Sakamoto et al. 1992, 1994).

PROCEDUREFemale Dunkin-Hartley guinea pigs weighing 380–600 g are anesthetized with an initial dose of 1.5 g/kgurethane injected i.p. Additional urethane is given i.v.30 min later to achieve an appropriate level of anesthe-sia. A tracheal cannula is inserted into the lumen ofthe cervical trachea, a polyethylene catheter into theleft carotid artery to monitor blood pressure and heartrate and another polyethylene catheter into the exter-nal jugular vein for administration of drugs. The an-imals are connected to a constant volume mechanicalventilator and then given an injection of 1.0–1.5 mg/kgsuxamethonium i.v. to prevent interference with spon-taneous respiration. A tidal volume of 10 ml/kg anda frequency of 60 strokes/min are used.

Lung resistance is measured as an index of airwayfunction and monitored throughout the experiment.Transpulmonary pressure is measured with a pressuretransducer with one side attached to a catheter insertedinto the right pleural cavity and the other side attachedto the side port of the intratracheal cannula. Airflow ismeasured by a pneumotachograph connected to a pres-sure transducer. The signals of the transducers are usedfor instantaneous calculation of lung resistance by anappropriate computer program.

The test compound (bradykinin receptor antago-nist) is given intravenously. Ten min later, Evans Bluedye (20 mg/ml) is injected i.v. for 1 min. After 1 min,bronchoconstriction and microvascular leakage is in-duced by injection of bradykinin or by inhalation ofbradykinin or PAF or vagal stimulation.

Six min after induction of leakage, the thoraciccavity is opened, and a cannula is inserted into the


aorta through a ventriculotomy. Perfusion is per-formed with 100–150 ml 0.9% saline at a pressure of100–120 mm Hg in order to remove the intravasculardye from the systematic circulation. Blood and perfu-sion liquid are expelled through an incision in the rightand left atrium. Subsequently, the right ventricle isopened, and the pulmonary circulation is perfused with30 ml of 0.9% saline. The lungs are then removed, andthe connective tissue, vasculature, and parenchyma aregently scraped. The airways are divided into 4 com-ponents: lower part of the trachea, main bronchi, theproximal 5 mm portion, and the distal intrapulmonaryairways. The tissues are blotted dry, and then weighed.Evans Blue dye is extracted in 2 ml of formamide at40°C for 24 h, and measured in a spectrophotometer at620 nm.

EVALUATIONEvans Blue dye concentration, expressed as ng/mg tis-sue, as well as lung resistance are compared by statisti-cal means (unpaired Student’s t-test or Mann-WhitneyU test) between treated groups and controls receivingthe challenge only.

MODIFICATIONS OF THE METHODBoschetto et al. (1989) tested the effect of antiasthmadrugs on microvascular leakage in guinea pig airways.Microvascular leakage was induced by intravenous in-jection of platelet-activating factor (50 ng/kg) whichacts directly on venular endothelial cells, and mea-sured by quantifying extravasation of Evans blue dye.

Xu et al. (1998) induced pulmonary edema in ratsby injection of 20 µg/kg angiotensin I and studied thesuppression by ACE-inhibitors, ATII antagonists andα-adrenergic receptor blockers.

Rapidly developing pulmonary edema was inducedby intravenous injection of 1.2 mg/kg of the GABA ag-onist bicuculline in rats and the role of endogenous en-dothelin was examined by Herbst et al. (1995).

REFERENCES AND FURTHER READINGBoschetto P, Roberts NM, Rogers DF, Barnes PJ (1989) Effect

of antiasthma drugs on microvascular leakage in guinea pigairways. Am Rev Resp Dis 139:416–421

Herbst C, Tippler B, Shams H, Simmet T (1995) A role ofendothelin in bicuculline-induced neurogenic pulmonaryoedema in rats. Br Pharmacol 115:753–760

Rogers DF, Boschetto P, Barnes PJ (1989) Plasma exsudation:Correlation between Evans Blue dye and radiolabelled al-bumin in guinea-pig airways in vivo. J Pharmacol Meth21:309–315

Sakamoto T, Elwood W, Barnes PJ, Chung FK (1992) Ef-fect of Hoe 140, a new bradykinin receptor antago-nist, on bradykinin- and platelet-activating factor-induced

bronchoconstriction and airway microvascular leakage inguinea pig. Eur J Pharmacol 213:367–373

Sakamoto T, Sun J, Barbnes PJ, Chung KF (1994) Effectof a bradykinin receptor antagonist, HOE 140, againstbradykinin- and vagal stimulation-induced airway re-sponses in the guinea-pig. Eur J Pharmacol 251:137–142

Xu ZH, Shimakura K, Yamamoto T, Wang LM, Mineshita S(1998) Pulmonary edema induced by angiotensin I in rats

D.2.2.7Isolated Larynx In Situ

PURPOSE AND RATIONALEThe in situ isolated larynx of rats has been rec-ommended by Willette et al. (1987) for evalua-tion peripheral opiate receptor antagonists. Peripheralopioid-induced laryngospasm and central opioid-in-duced respiratory depression can be measured simul-taneously. Fentanyl citrate stimulates both peripheraland central opiate receptors, whereas [D-Ala2-Met5]-enkephalinamide stimulates only peripheral opiate re-ceptors. Compounds that inhibit both laryngeal andrespiratory effects of fentanyl, e. g., naloxone HCl, canbe considered both central and peripheral opiate antag-onists. Compounds that inhibit only the peripheral ef-fects of fentanyl, e. g., naltrexone methylbromide, canbe considered peripheral opiate receptor antagonists.

PROCEDURELaryngeal resistance experiments are carried out inmale Sprague Dawley rats weighing 340–360 g andanesthetized with urethane (900 mg/kg, i.p.) The ani-mals are secured in supine position and the left femoralartery and the left and right femoral veins are can-nulated for recording of arterial blood pressure andadministration of drugs. A right-angle polyethylenecannula (ID 1.67 mm, OD 2.42 mm) is inserted intothe caudal trachea at the level of the manubrium formeasuring tracheal flow via a small animal pneumo-tachograph. Care has to be taken to avoid damagingsurrounding blood vessels or adjacent bilateral laryn-geal nerves. Tracheal air flow is continually sampled(300 ml/min) for the breath to breath analysis of endtidal carbon dioxide with an infrared gas analyzer.

Laryngeal resistance in the rat is determined usinga modification of the methods described by Stranskyet al. (1973), Bartlett et al. (1973), and Willette et al.1982b). The rostral portion of the trachea is cannulatedwith a right-angle polyethylene tube (ID 1.67 mm,OD 2.42 mm). This cannula is carefully advanced to-wards the larynx and secured with a suture (4–0 silk).A constant flow (V = 30 ml/min) of compressed air,maintained with a flow meter, is delivered througha cannula. Pre-laryngeal pressure is measured with


a needle tipped pressure transducer inserted into thelumen of the flow cannula. A 1.75-cm segment ofpolyethylene tubing (OD 8.4 mm, ID 4.6 mm) is placedinto the mouth to retract resistive components in thenasopharyngeal region.

Laryngeal resistance (LR) is calculated by the fol-lowing equation:

LR = (PLPi − PLP0)/V

where PLPi is the laryngeal pressure in the cannula di-recting a constant flow (V) through the larynx. PLP0 isthe pressure in the cannula when it is removed fromthe trachea.

The agonist fentanyl is administered through the leftfemoral vein at a dose of 12 µg/kg which is equiv-alent to 1.5 times the ED99 in the conscious rat tailflick assay. The enkephaline analogue [D-Ala2-Met5]-enkephalinamide is injected at a dose of 250 µg/kgwhich acts peripherally and increases laryngeal resis-tance (Willette 1982a). The opiate receptor antagonistsare administered similarly into the right femoral vein.

At the conclusion of the experiment, the pulmonaryafferent stimulant, phenyldiguanide (25 µg/kg, i.v.) isinjected into the right femoral vein to elicit laryn-gospasm and to determine the viability of the prepa-ration.

EVALUATIONAll summary values are expressed as the mean plus orminus the standard error of the mean (SEM) Compar-isons are made with independent and paired two-tailedt-tests.

MODIFICATIONS OF THE METHODInagi et al. (1998) assessed the effect of botulin toxinin the rat larynx be measurement of the optical densityof PAS-stained laryngeal muscle after electrical stim-ulation, spontaneous laryngeal muscle activity, and la-ryngeal movement.

O’Halloran et al. (1994) studied the effects of upperairway cooling and CO2 on breathing and on laryngealand supraglottic resistance in anesthetized rats.

González-Barón et al. (1989) studied the mod-ifications of larynx resistance changing bronchialtone in cats evoked by intravenous administration of10 µg/kg carbachol as bronchoconstrictor and by fen-terol (10 µg/kg) or isoproterenol (0.1 mg/kg) as bron-chodilators.

Wang et al. (1999) developed an isolated, luminallyperfused laryngeal preparation in anesthetized para-lyzed cats in order to compare the effects of solutions

with varying levels of pH and pCO2 on pressure-sen-sitive laryngeal receptor sensitivity.

REFERENCES AND FURTHER READINGBartlett D, Remmers JE, Gautier H (1973) Laryngeal regulation

of respiratory airflow. Respir Physiol 18:194–202González-Barón S, Dawid-Miner MS, Lara JP, Clavijo E,

Aguirre JA (1989) Changes in laryngeal resistance andbronchial tonus. Rev Esp Fisiol 45, Suppl:191–196

Inagi K, Connor NP, Ford CN, Schultz E, Rodriquez AA,Bless DM, Pasic D, Heisey DM (1998) Physiologic assess-ment of botulinum toxin effects in the rat larynx. Laryngo-scope 108:1048–1054

O’Halloran KD, Curran AK, Bradford A (1994) Ventilatory andupper-airway resistance response to upper airway coolingand CO2 in anesthetized rats. Pflüger’s Arch 429:262–266

Stransky A, Szereda-Przestazewska M, Widdicombe J (1973)The effect of lung reflexes on laryngeal resistance and mo-toneuron discharge. J Physiol 231:517–518

Wang ZH, Bradford A, O’Regan RG (1999) Effects of CO2 andH+ on laryngeal receptor activity in the perfused larynx ofanesthetized cats. J Physiol (Lond) 519:591–600

Willette RN, Krieger AJ, Sapru HN (1982a) Pulmonary opiatereceptor activation evokes a cardiorespiratory effect. Eur JPharmacol 78:61–70

Willette RN, Krieger AJ, Sapru HN (1982b) Opioids increase la-ryngeal resistance and motoneuron activity in the recurrentlaryngeal nerve. Eur J Pharmacol 80:57–63

Willette RN, Evans DY, Dooley BM (1987) The in situ isolatedlarynx for evaluation peripheral opiate receptor antagonists.J Pharmacol Meth 17:15–25

D.2.2.8Animal Models of Asthma

D.2.2.8.1Treatment of Asthma

PURPOSE AND RATIONALESeveral authors described asthma models in mice.Hammad et al. (2004) found in a mouse model ofasthma that activation of peroxisome proliferator-ac-tivated receptor-γ (PPAR-γ ) in dendritic cells inhibitsthe development of eosinophilic airway inflammation.

PROCEDUREBone Marrow Dendritic CellsOVA-TCR transgenic mice (DO10.11) on a BALB/cbackground, bred at the Erasmus University (Rotter-dam) (Murphy et al. 1990), are used. Femurs and tib-iae of female mice are removed and flushed with RPMI1640. Vigorous pipetting disintegrates clusters withinthe marrow suspension. The cells are washed, enumer-ated, and plated in Petri dishes. Cell-culture medium(TCM) is supplemented with gentamicin (60 µg/ml),2-mercaptoethanol (5 × 10−5 mol/l) and 5% fetal calfserum (Lutz et al. 1999). At day 0 of the culture,cells are seeded at a concentration of 2 × 106/dish inmedium containing granulocyte-macrophage colony-


stimulating factor (GM-CSF, 200 IU/ml). At days 6and 8, half of the medium is collected, centrifuged, andthe pellet is resuspended in TCM containing 200 IU/mlof recombinant murine GM-CSF.

At day 9 of the culture, dendritic cells (Banchereauand Steinman 1998) are pulsed overnight with oval-bumin containing the vehicle (dimethylsulfoxide,DMSO) in which the standard PPAR-γ agonist (e. g.,rosiglitazone) is suspended or with medium aloneas control. Other plates are treated with the testdrug or a PPAR-γ antagonist. After antigen puls-ing overnight, non-adherent dendritic cells are col-lected, washed to remove free ovalbumin, and resus-pended in phosphate-buffered saline at a concentrationof 12.5 × 106 cells/ml.

Eosinophilic Airway InflammationFor intratracheal injection of dendritic cells (Lam-brecht et al. 2000), mice are anesthetized with Avertinand 80 µl of the cell suspension (1 × 106 dendriticcells) is instilled through the opening vocal cords.Mice are injected with unpulsed dendritic cells,ovalbulmin-treated dendritic cells or dendritic cellstreated additionally with the PPAR-γ antagonist. Fromdays 10–13, mice are exposed to 30-min ovalbuminaerosols. They are sacrificed 24 h after the last aerosol.Broncho-alveolar lavage is performed with 3 × 1 mlof Ca2+- and Mg2+-free buffer supplemented with 0.1mmol/l sodium EDTA. The broncho-alveolar lavageis centrifuged; the cells are resuspended in buffer andenumerated with a hemocytometer. They are differen-tiated (Vremec and Shortman 1997) by staining for30 min with anti-I-Ad/I-Ed FITC (macrophages), anti-CCR3 PE (eosinophils), antiCD3-cy-chrome, anti-B220 cytochrome (T and B cells, respectively) andanti-CD11c APC (macrophages) in PBS containing0.5 bovine serum albumin and 0.01% sodium azide.Cells are washed and analyzed by flow cytometry (VanRijt et al. 2002).

EVALUATIONThe difference between the groups is calculated usingthe Mann–Whitney U-test for unpaired data.

MODIFICATIONS OF THE METHODIwasaki et al. (2001) recommended atopic NC/Ngamice as a model for allergic asthma: after immuniza-tion with ovalbumin, severe allergic responses wereelicited by a single intranasal challenge.

For testing PPAR agonists, Trifilieff et al. (2003)used a murine model of asthma using lung inflamma-tion induced by ovalbumin or by LPS (Trifilieff et al.

2000). Female BALB/c mice (4 weeks old) were im-munized with ovalbumin on days 0 and 14, exposedto an aerosol challenge of ovalbumin or phosphate-buffered saline (PBS) on day 21 and killed on day 23for measurement of inflammatory cells in the broncho-alveolar lavage. Animals were intranasally treated withcompounds 1 h before the aerosol exposure using PBScontaining 2% DMSAO as vehicle.

For LPS-induced lung inflammation mice werechallenged intranasally with 0.3 mg/kg of LPS(Salmonella typhosa) in 50 ml of sterile PBS or withsterile PBS alone and killed 3 h later for broncho-alveolar lavage. Tumor necrosis factor alpha (TNF-α)and the neutrophil chemokine KC levels were mea-sured using an enzyme-linked immunosorbent assaykit (R&D Systems, Abingdon, UK). Animals wereintranasally treated with compounds 1 h before theaerosol exposure using PBS containing 2% DMSO asvehicle.

Mueller et al. (2003) reported that peroxisome pro-liferators-activated receptor γ ligands attenuate im-munological symptoms of allergic asthma. For asthmainduction, Balb/c mice were injected with 10 µg ofovalbumin in Alum twice on days 1 and 5 (Zhang et al.1999). On day 10, they were challenged intranasallywith 40 µg ovalbumin in sterile saline, every day for3 days. On the day after the last intranasal challenge,the mice were sacrificed. The degree of inflammationin the lung was evaluated by histopathology. Gene ex-pression was tested by microarray analysis, and serumIgE determined by ELISA.

Regal et al. (2001) described trimellitic anhydride-induced eosinophilia in a mouse model of occupa-tional asthma. Trimellitic anhydride was conjugatedto mouse serum albumin. Female BALB/c mice weresensitized on days 1 and 3 intradermally with 0.1 ml of0.3% ovalbumin suspended in corn oil. On day 12, an-imals were additionally sensitized intratracheally with0.04 ml ovalbumin, trimellitic anhydride conjugated tomouse serum albumin, or mouse serum albumin dis-solved in water. For elicitation of the allergic response,mice were challenged intratracheally beginning on day19 with 0.04 ml of aqueous solutions of ovalbumin,trimellitic anhydride conjugated to mouse serum albu-min, or mouse serum albumin under ketamine/xylazineanesthesia. After the last intratracheal instillation, themice were anesthetized with pentobarbital, EDTAplasma was collected by cardiac puncture, the tracheawas cannulated and the lungs were lavaged with two0.9-ml aliquots of PBS to obtain bronchial lavage fluid.The lungs were removed for homogenization and anal-ysis of eosinophil peroxidase and myeloperoxidase.


Churg et al. (2001) studied anti-inflammatory ef-fects of alpha-1 antitrypsin and a metalloprotease in-hibitor in C57 BL/6 mice or macrophage metallo-elastase knockout mice after intratracheal instillationof a single 7-mg dose of crystalline silica (α-quartz,Minusil-5; US Silica Corporation, Clarkstown, W.Va.,USA). This dose produced a rapid and persisting acuteinflammatory infiltrate. Mice were euthanized 2 or24 h after dust exposure by halothane overdose andthe lungs removed from the chest cavity. A 20-gaugecatheter was inserted into the trachea and the lungslavaged six times with 1 ml of ice-cold saline. For in-flammatory cell measurements, the saline lavage wascentrifuged at 200 g at 4°C for 10 min. The super-natants were decanted, and the cell pellets were re-suspended in 200 µl of saline. Total cell counts wereperformed using a hemocytometer and differential cellcounts were performed on a 10-µl drop of the cellsuspension heat-fixed on a slide and stained withhematoxylin-eosin. Lavage samples were analyzed fordesmosine and hydroxyproline.

De Sanctis and Drazen (1997) discussed the genet-ics of native airway responsiveness in mice.

Several animal species have been used such as rats.Misawa et al. (1987) found strain differences among

Wistar, Lewis and Fischer 344 rats in an allergicasthma model.

Misawa and Sugiyama (1993) described an airwayhyperresponsiveness model in rats, inducing allergicasthma with DNP-Ascaris extract.

Uhlig et al. (1998) reported the effects of long-termoral treatment with leflunomide on allergic sensitiza-tion, lymphocyte activation, and airway inflammationin rats.

Birrell et al. (2003) investigated the nitric-oxidesynthase isoform involved in eosinophilic inflamma-tion in a rat model of Sephadex-induced airway inflam-mation. The rat model of Sephadex-induced airway in-flammation was also used by Belvisi et al. (2005) forpreclinical studies on ciclesonide, an inhaled corticos-teroid for the treatment of asthma.

Many authors used sensitized Brown Norway ratsin experimental models of asthma (Elwood et al. 1992;Steerenberg et al. 1999; Nonaka et al. 2000; Xu et al.2000; Blesa et al. 2002; Glaab et al. 2002; Huang et al.2002; Belvisi et al. 2005; Valstar et al. 2006).

Guinea pigs have been used as animal models forasthma by many authors. Most studies were performedwith sensitization either by injection with ovalbuminsuspended in an adjuvant (Santives et al. 1976; Banneret al. 1996; Lawrence et al. 1998; Regal et al. 2000;Li et al. 2001; Santing et al. 2001; Mukaiyama et al.

2004; Boskabady and Zarei 2004; Ikezono et al. 2005;Tang et al. 2005) or by repetitive sensitization with in-haled ovalbumin (Sagara et al. 1996, 1997; Smith et al.1996; Liu et al. 1997; Tohda et al. 1998; Cheng et al.2001; Zhang et al. 2002) and then challenge by inhaledovalbumin.

Fujimura et al. (1997) described a guinea pigmodel of ultrasonically nebulized distilled-water-induced bronchoconstriction. Guinea pigs sensitizedby intradermal injection of ovalbumin in Freund’sadjuvant were treated with aerosols of physiologicalsaline generated by an ultrasonic nebulizer.

Zhou et al. (1998) reported a dose–response rela-tionship between exposure to cockroach allergens andinduction of sensitization in an experimental asthma inHartley guinea pigs.

Larsen and Regal (2002) studied trimelliticanhydride dust-induced airway obstruction andeosinophilia in non-sensitized guinea pigs.

Nishitsuji et al. (2004) described a guinea pig modelof cough variant asthma. Bronchial responsiveness tomethacholine and cough reflex sensitivity to capsaicinwere measured 72 h after ovalbumin inhalation in ac-tively sensitized guinea pigs.

Dogs were used as animal models for asthma byseveral authors. Many studies preferred the BasenjiGreyhound dog, which manifests various characteris-tics of human asthma, including airway hypersensitiv-ity to low concentrations of methacholine (Hirshmanet al. 1980; Hirshman and Downes 1981; Chan et al.1985; Darowski et al. 1989; Emala et al. 1993, 1996).

Redman et al. (2001) studied pulmonary immunityto ragweed in a Beagle dog model of allergic asthma.

Some studies were reported using cats as an ani-mal model for asthma. Norris et al. (2003) and Norris-Reinero et al. (2004) described an experimental modelof allergic asthma in cats sensitized to the house dustmite and Bermuda grass allergen.

Several studies were performed using monkeysas animal models for asthma. Ascaris suum-sensitivemonkeys were used by Mauser et al. (1995) and Zouet al. (2002). Rhesus monkeys (Macaca mulatta) wereused by Patterson et al. (1975) and Patterson andHarris (1981, 1990, 1992). Turner et al. (1996) usedMacaca fascicularis to demonstrate in vitro and in vivoeffects of leukotriene B4 antagonism in primates.

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lowing immunization elicits recruitment of eosinophils but


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Belvisi MG, Bundschuh DS, Stoeck M, Wicks S, Under-wood S, Battram CH, Haddad EB, Webber SE, Foster ML(2005) Preclinical profile of ciclesonide, a novel corticos-teroid for the treatment of asthma. J Pharmacol Exp Ther314:568–574

Birrell MA, McCluskie K, Haddad el B, Battram CH, WebberSE, Foster ML, Yacoub MH, Belvisi MG (2003) Pharma-cological assessment of the nitric-oxide synthase isoforminvolved in eosinophilic inflammation in a rat model ofsephadex-induced airway inflammation. J Pharmacol ExpTher 304:1285–1291

Blesa S, Cortijo J, Martinez-Losa M, Mata M, Seda E, San-tangelo F, Morcillo EJ (2002) Effectiveness of oral N-acetylcysteine in a rat experimental model of asthma. Phar-macol Res 45:135–140

Boskabady MH, Zarei A (2004) Increased tracheal responsive-ness to beta-adrenergic agonist and antagonist in ovalbu-min-sensitized guinea pigs. Pharmacology 71:73–79

Chan SC, Hanifin JM, Holden CA, Thompson WJ, Hirsh-man CA (1985) Elevated leukocyte phosphodiesterase asa basis for depressed cyclic adenosine monophosphate re-sponses in the Basenji greyhound dog model of asthma.J Allergy Clin Immunol 76(2 Pt 1):148–158

Cheng G, Ueda T, Sugiyama K, Toda M, Fukuda T (2001)Compositional and functional changes of pulmonary sur-factant in a guinea pig model of chronic asthma. RespirMed 95:180–186

Churg A, Dai J, Zay K, Karsan A, Hendricks S, Yee C, MartinR, MacKenzie R, Xie C, Zhang L, Shapiro S, Wright JL(2001) Alpha-1 antitrypsin and a broad metalloprotease in-hibitor, RS113456, have similar acute anti-inflammatoryeffects. Lab Invest 81:1119–1131

Darowski MJ, Hannon VM, Hirshman CA (1989) Corti-costeroids decrease airway hyperresponsiveness in theBasenji-Greyhound dog model of asthma. J Appl Physiol66:1120–1126

De Sanctis GT, Drazen JM (1997) Genetics of native air-way responsiveness in mice. Am J Respir Crit Care Med156:S82–S88

Elwood W, Lotvall JO, Barnes PJ, Chung KF (1992) Effectof dexamethasone and cyclosporine A on allergen-inducedairway hyperresponsiveness and inflammatory cell re-sponses in sensitized Brown-Norway rats. Am Rev RespirDis 145:1289–1294

Emala C, Black C, Curry C, Levine MA, Hirshman CA (1993)Impaired beta-adrenergic receptor activation of adenylylcyclase in airway smooth muscle in the basenji-greyhounddog model of airway hyperresponsiveness. Am J RespirCell Mol Biol 8:668–675

Emala CW, Aryana A, Hirshman CA (1996) Impaired activationof adenylyl cyclase in lung of the Basenji-greyhound modelof airway hyperresponsiveness. Decreased numbers of highaffinity beta-receptors. Br J Pharmacol 118:2009–2016

Fujimura M, Amamiya M, Myou S, Mizuguchi M, Matsuda T(1997) A guinea-pig model of ultrasonically nebulizeddistilled water-induced bronchoconstriction. Eur Respir J10:2237–2242

Glaab T, Hoymann HG, Hohlfeld JM, Korolewitz R, Hecht M,Alarie Y, Tschernig T, Braun A, Krug N, Fabel H (2002)Noninvasive measurement of midexpiratory flow indi-cates bronchoconstriction in allergic rats. J Appl Physiol93:1208–1214

Hammad H, Jan de Heer H, Soullié T, Angeli V, Trottein F,Hoogsteden HC, Lambrecht BN (2004) Activation of per-

oxisome proliferator-activated receptor-γ in dendritic cellsinhibits the development of eosinophilic airway inflamma-tion in a mouse model of asthma. Am J Pathol 164:263–271

Hirshman CA, Downes H (1981) Basenji-Greyhound dog modelof asthma: influence of atropine on antigen-induced bron-choconstriction. J Appl Physiol 50:76–765

Hirshman CA, Malley A, Downes H (1980) Basenji-Greyhounddog model of asthma: reactivity to Ascaris suum, citric acid,and methacholine. J Appl Physiol 49:953–957

Huang TJ, Eynott P, Salmon M, Nicklin PL, Chung KF (2002)The effect of topical immunomodulators on acute allergicinflammation and bronchial hyperresponsiveness in sensi-tized rats. Eur J Pharmacol 437:187–194

Ikezono K, Kamata M, Mori T (2005) Adrenal influences on theinhibitory effects of procaterol, a selective β2-adrenoceptoragonist, on antigen-induced airway microvascular leak-age and bronchoconstriction in guinea pigs. Pharmacology73:209–215

Iwasaki T, Tanaka A, Itakura A, Yamashita N, Ohta K, Mat-suda H, Onuma M (2001) Atopic NC/Nga mice as a modelfor allergic asthma: severe allergic responses by single in-tranasal challenge with protein antigen. J Vet Med Sci63:413–419

Lambrecht BN, Pauwels RA, de St. Groth BF (2000) Inductionof rapid T cell activation, division, and recirculation by in-tratracheal injection of dendritic cells in a TCR transgenicmodel. J Immunol 164:2937–2946

Larsen CP, Regal JF (2002) Trimellitic anhydride (TMA)dust induces airway obstruction and eosinophilia in non-sensitized guinea pigs. Toxicology 178:89–99

Lawrence TE, Millecchia LL, Fedan JS (1998) Fluticasone pro-pionate and pentamidine isethionate reduce airway hyper-reactivity, pulmonary eosinophilia and pulmonary dendriticcell response in a guinea pig model of asthma. J PharmacolExp Ther 284:222–227

Li Y, Martin LD, Minnicozzi M, Greenfeder S, Fine J, Pet-tersen CA, Chorley B, Adler KB (2001) Enhanced ex-pression of mucin genes in a guinea pig model of allergicasthma. Am J Respir Cell Mol Biol 25:644–651

Liu M, Wang L, Holm BA, Enhorning G (1997) Dysfunction ofguinea pig pulmonary surfactant and type II pneumocytesafter repetitive challenge with aerosolized ovalbumin. ClinExp Allergy 27:802–807

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Mauser PJ, Pitman AM, Fernandez X, Foran SK, Adams GK3rd Kreutner W, Egan RW, Chapman RW (1995) Effects ofan antibody to interleukin-5 in a monkey model of asthma.Am J Respir Crit Care Med 152:467–472

Misawa M, Sugiyama Y (1993) An airway hyperresponsivenessmodel in rat allergic asthma. Arerugi 42:107–114

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D.2.2.8.2Prevention of Allergic Asthma Reaction

PURPOSE AND RATIONALEType 1 allergy is an abnormal reaction to protein sub-stances that occur naturally. B-lymphocytes producean antibody against the allergen. An allergic reactionoccurs every time the body is exposed to the allergencausing release of histamine. Allergic reactions can beasthma, hay fever or nettle rash. Sensitization to nor-mally harmless environmental antigens (e. g., pollen,house-dust mite) is the prerequisite for initiating theinflammatory cascade in bronchial asthma. Inflamma-tion of the airway mucosa is orchestrated by Th-2 typeT cells, which produce Th-2 cytokines (IL-4, IL-5, IL-9, IL-13, and IL-15), which regulate both IgE produc-tion and airway inflammation. At present, no cure ofasthma disease is available, but primary and secondaryprevention is the therapy of choice (Cieslewicz et al.1999). Specific immunotherapy is performed by in-jection of increasing amounts of allergens to inducehyporesponsiveness to the respective allergen. By mu-cosal application of soluble antigen, mucosal tolerancecan be achieved (van Halteren et al. 1997; Astori et al.2000). Many studies have been performed in spite ofthe fact that no animal model is available that resem-bles all features of human bronchial asthma (Herz et al.1998). In particular, models using recombinant aller-gens were described (Hoyne et al. 1997, 2000; Herzet al. 2004).

These studies concern specific allergens, such asovalbumin (Renz et al. 1992; Marth et al. 2000;Neuhaus-Steinmetz et al. 2000; Raap et al. 2003;Reader et al. 2003; Wegmann et al. 2005), mite dustallergen (Hoyne et al. 1996; Clarke et al. 1999; Yasueet al. 1998a, 1998b, 1998c, 1999; Jarnicki and Thomas2002), pollen allergen (Hirahara et al. 1998; Wieder-mann et al. 1999, 2001; Batanero et al. 2002; Repaet al. 2004; Hufnagl et al. 2005; Winkler et al. 2006),latex protein allergen (Thakker et al. 1999; Woolhiseret al. 2000; Meade and Woolhiser 2002; Hufnagl et al.2003), bee venom allergen (Von Garnier et al. 2000),and cat allergen (Briner et al. 1993; Treter and Luq-man 2000).

Wegmann et al. (2005) studied involvement of distalairways in a chronic model of experimental asthma.

PROCEDUREAnimalsPathogen-free female BALB/c mice (Harlan Winkel-mann, Hannover, Germany), weighing 18–22 g and6–8 weeks of age, were used in all experiments. The

animals were maintained under standard housing con-ditions, fed an ovalbumin- (OVA-) free diet and sup-plied with food and water ad libitum.

Sensitization ProtocolMice were sensitized to OVA by three intraperitonealinjections [10 µg OVA grade VI (Sigma, Deisenhofen,Germany) adsorbed to 1.5 mg Al(OH)3 diluted in200 µl phosphate-buffered saline (PBS)] on days 1, 14,and 21. The mice were challenged with OVA (gradeV) aerosol (1% wt/vol in PBS) via the airways twicea week on two consecutive days over a period of12 weeks (Renz et al. 1992). Sham sensitization andchallenges were carried out with sterile Al(OH)3 inPBS. Animals were analyzed after 1 or 12 weeks ofOVA aerosol challenge. To investigate persistence ofairway inflammation and lung physiological changesmice were analyzed after 6 weeks of OVA aerosolchallenge discontinuation following 12 weeks of OVAaerosol challenge.

Differential Cell Countsin Broncho-Alveolar Lavage FluidAt 48 h after the last allergen challenge broncho-alve-olar lavage (BAL) was performed and analyzed (Renzet al. 1992).

Measurement of Cytokines in BAL FluidIL-4, IL-5 and tumor necrosis factor alpha (TNF-α)were measured in cell-free lavage fluids by cytometricbead array (CBA, BD Biosciences, San Diego, Calif.,USA). The detection limit for each of the cytokineswas 10 pg/ml. Complete BAL transforming growthfactor beta (TGF-β) was measured after acidic acti-vation using chicken anti-human TGF-β in a standardELISA protocol. The detection limit for TGF-β was20 pg/ml.

Lung HistologyLungs were fixed ex situ with 4% (wt/vol) para-formaldehyde via the trachea, removed and stored in4% paraformaldehyde. Lung tissues were embeddedinto paraffin, and 3 µm sections were stained withhaematoxylin and eosin or periodic acid-Schiff stain-ing. For localization of collagen fibrils, Sirius redstaining was performed according to Uhal et al. (1998).

ImmunohistochemistryIndirect immunohistochemistry was used to stain lungsections for smooth muscle cells and myofibrob-lasts (mouse monoclonal IgG against α-SMA, clone1A4, Immunotech, Marseilles, France), and nerve cells


(polyclonal rabbit antiserum against protein gene prod-uct 9.5, Biogenesis, Poole, UK) according to Fehren-bach et al. (2002). To distinguish smooth muscle cellsfrom myofibroblasts a rat monoclonal IgG against fi-broblast-specific peptide (clone ER-TR7; Biogenesis)was utilized.

Quantitative MorphologyParaffin sections stained with Fast Green/Sirius Redand for α-SMA, respectively, were used to quantifychanges in airway epithelial cell, collagen and smoothmuscle cell layers of distal airways according to stan-dard stereological methods. A distal airway was de-fined as the segment of a terminal bronchiolus that,starting at the broncho-alveolar duct transition, ex-tended up to five alveoli along the proximal direc-tion. Using a PC-based Olympus light microscope BX51 equipped with a CAST-Grid System (Visiopharm,Hoersholm, Denmark), all distal airways of a givensection were delineated (at a magnification of × 426),and the fields of view to be analyzed (at a final mag-nification of ×1.700) were automatically defined ac-cording to systematic uniform random sampling. Thearithmetic mean thicknesses were determined as thevolume of the respective component, determined bycounting all points hitting airway epithelium, SiriusRed- and α-SMA-positive components, respectively.Results were referred to the reference surface deter-mined by counting all intersections with the airwayepithelial basal membrane (Reader et al. 2003). Thearithmetic mean thicknesses were calculated.

Electron Microscopy and Definition of Proximaland Distal AirwaysFor analysis of airway wall ultrastructure, lungs ofmice chronically challenged with OVA and controlmice challenged with PBS (n = 3 per group) were fixedby instillation of 4% (wt/vol) paraformaldehydevia thetrachea. Beginning at the lobar bronchus, the airwayswere microdissected along the axial pathway accord-ing to Plopper et al. (2001). For histopathological andultrastructural analysis, proximal airways were definedas the lobar bronchi and the axial pathway down to thefourth intrapulmonary branch point. Mid-level airwayswere defined as the axial pathways between the intra-pulmonary branch points 6–12. Terminal bronchioleswith direct connection to the alveolar ducts were de-fined as distal airways (Postlethwait et al. 2000).

Non-invasive Measurement of Mid-Expiratory Airflowat Baseline and of Bronchial Responsivenessto MethacholineThe mid-expiratory airflow (EF50) was measured 24h after the last OVA aerosol challenge using head-outbody plethysmography (Glaab et al. 2001).

EVALUATIONResults are presented as mean values ±SD unlessstated otherwise. One-way ANOVA test or Student’sunpaired t-test was used to determine the significanceof differences between animal groups.

REFERENCES AND FURTHER READINGAstori M, von Garnier C, Kettner A, Dufour N, Corradin G,

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Briner TJ, Kuo MC, Keating KM, Rogers BL, Greenstein JL(1993) Peripheral T-cell tolerance induced in naive andprimed mice by subcutaneous injection of peptides fromthe cat major allergen Fel d I. Proc Natl Acad Sci USA90:7608–7612

Cieslewicz G, Tomkinson A, Adler A, Duez C, Schwarze J,Takeda K, Larson KA, Lee JJ, Irvin CG, Gelfand EW(1999) The late, but not early, asthmatic response is de-pendent on IL-5 and correlates with eosinophil infiltration.J Clin Invest 104:301–308

Clarke AH, Thomas WR, Rolland JM, Dow C, O’Brien RM(1999) Murine allergic respiratory responses to the majorhouse dust mite allergen Der p 1. Int Arch Allergy Immunol120:126–134

Fehrenbach H, Fehrenbach A, Pan T, Kasper M, Mason RJ(2002) Keratinocyte growth factor-induced proliferation ofrat airway epithelium is restricted to Clara cell in vivo. EurRespir J 20:1185–1197

Glaab T, Daser A, Brain A, Neuhaus-Steinmetz U, Fabel H,Alarie Y, Renz H (2001) Tidal midexpiratory flow as a mea-sure of airway hyperresponsiveness in allergic mice. Am JPhysiol 280:L565–L573

Herz U, Braun A, Rückert H, Renz H (1998) Various immuno-logical phenotypes are associated with increased airwayhyperresponsiveness. Clin Exp Allergy 28:625–634

Herz U, Renz H, Wiedermann U (2004) Animal models of type Iallergy using recombinant allergens. Methods 32:271–280

Hirahara K, Saito S, Serizawa N, Sasaki R, Sakaguchi M, In-ouye S, Taniguchi Y, Kaminogawa S, Shiraishi A (1998)Oral administration of a dominant T-cell determinantpeptide inhibits allergen-specific TH1 and TH2 cell re-sponses in Cry j 2-primed mice. J Allergy Clin Immunol102:961–967

Hoyne GF, Askonas BA, Hetzel C, Thomas WR, Lamb JR(1996) Regulation of house dust mite responses by in-tranasally administered peptide. Transient activation ofCD4+ T cells precedes the development of tolerance invivo. Int Immunol 8:335–342

Hoyne GF, Jarnicki AG, Thomas WR, Lamb JR (1997) Char-acterization of the specificity and duration of T celltolerance to intranasally administered peptides in mice:


a role for intramolecular epitope suppression. Int Immunol9:1165–1173

Hoyne GF, Le Roux I, Corsin-Jimenez M, Tan K, Dunne J,Forsyth LMG, Dallman MJ, Owen MJ, Ish-Horowicz D,Lamb JR (2000) Serrate1-induced Notch signalling regu-lates the decision between immunity and tolerance madeby peripheral CD4+ T cells. Int Immunol 12:177–185

Hufnagl K, Wagner B, Winkler B, Baier K, Hochreiter R, Thal-hammer J, Kraft D, Scheiner O, Breiteneder H, Wieder-mann U (2003) Induction of mucosal tolerance with re-combinant Hev b 1 and recombinant Hev b 3 fpr preven-tion of latex allergy in BALB/c mice. Clin Exp Immunol133:170–176

Hufnagl K, Winkler B, Focke M, Valenta R, Scheiner O,Renz H, Wiedermann U (2005) Intranasal tolerance induc-tion with polypeptides derived from 3 noncross-reactivemajor aeroallergens prevents allergic polysensitization inmice. J Allergy Clin Immunol 116:370–376

Jarnicki AG, Thomas WR (2002) Stimulatory and inhibitory epi-topes in the T cell responses of mice to Der p 1. Clin ExpAllergy 32:942–950

Marth T, Ring S, Schulte D, Klensch N, Strober W, Kelsall BL,Stallmach A, Zeitz M (2000) Antigen-induced mucosal Tcell activation is followed by Th1 T cell suppression in con-tinuously fed ovalbumin TCR-transgenic mice. Eur J Im-munol 30:3478–3486

Meade BJ, Woolhiser M (2002) Murine models for natural rub-ber latex allergy assessment. Methods 27:63–68

Neuhaus-Steinmetz U, Glaab T, Daser A, Braun A, Lom-matzsch M, Herz U, Kips J, Alarie Y, Renz H (2000) Se-quential development of airway hyperresponsiveness andacute airway obstruction in a mouse model of allergic in-flammation. Int Arch Allergy Immunol 121:57–67

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Winkler B, Hufnagl K, Spittler A, Ploder M, Kállay E, Vrtala S,Valenta R, Kundi M, Renz H, Wiedermann U (2006) Therole of Foxp3+ T cells in long-term efficacy of prophylac-tic and therapeutic mucosal tolerance induction in mouse.Allergy 61:173–180

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D.2.2.9Bleomycin-Induced Pulmonary Fibrosis

PURPOSE AND RATIONALEPulmonary fibrosis has been induced by bleomycin invarious species: golden Syrian hamsters (Giri et al.1986; Chen et al. 1997; Gurujeyalakshmi et al. 1999;Iyer et al. 1999, 2000), mice (Taooka et al. 1997; Tam-agawa et al. 2000; Keerthisingam et al. 2001; Terasakiet al. 2000; Terasaki 2001; Atzori et al. 2004) and rats(Howell et al. 2001; Simler et al. 2002; Wang et al.2002; Morcillo and Bulbena 2003).


Iyer et al. (1999) determined the effects of pir-fenidone on procollagen gene expression at the tran-scription level in a bleomycin hamster model of lungfibrosis.

PROCEDURETreatment of AnimalsMale golden Syrian hamsters weighing 90–110 g werehoused in groups of four in facilities with filtered airand constant temperature and humidity. A 12-h/12-hlight/dark cycle was maintained, and the animals hadaccess to water and either pulverized Rodent Labora-tory Chow 5001 or the same pulverized chow contain-ing pirfenidone (0.5% w/w). The hamsters were fedthese diets 2 days before intratracheal instillation andthroughout the study period. Hamsters were instilledintratracheally with saline or bleomycin (7.5 units/kgper 5 ml). The animals were sacrificed at 3, 7, 10,14, and 21 days after the bleomycin or saline instilla-tion by decapitation, and their lungs were removed andfreeze-clamped, dropped in liquid nitrogen, and storedat –80°C. The major portion of the sample was usedfor direct total RNA isolation, and the remainder wasused for other biochemical studies.

Tissue Processing for Biochemical StudyThe frozen lungs were thawed and homogenized in0.1 M KCl/0.02 M Tris·HCl buffer, pH 7.6, witha Polytron homogenizer. After recording the total ho-mogenate volume (5–6 ml), it was mixed, dividedinto aliquots, and stored at –80°C, except for thealiquots for lipid peroxidation and hydroxyproline as-says, which were processed and assayed the same dayon which the lungs were homogenized.

Determination of Lipid PeroxidationThe lung malondialdehyde equivalent (MDAE) level,an index of lipid peroxidation, was determined inthe whole homogenate according to the method ofOhkawa et al. (1979)

Determination of Prolyl Hydroxylase ActivityThe method for prolyl hydroxylase assay is based onthe release of tritiated water from 3,4-[3H]proline-la-beled unhydroxylated procollagen substrate preparedin vitro using 10-day-old embryonic chick tibiae (Giriet al. 1983). During the reaction, tritium is releasedin stoichiometric proportion to prolyl hydroxylationas tritiated water, which is collected and counted asa measure of the prolyl hydroxylase activity (Huttonet al. 1966). The activity was expressed as the totaldpm released/lung per 30 min.

Determination of Prolyl Hydroxylase Activity In VitroControl hamsters not subjected to any treatment werefirst anesthetized with sodium pentobarbital (80–100mg/kg). Their lungs were perfused with ice-cold iso-tonic saline; then, all lung lobes were dissected outand rinsed in saline. They were immediately homog-enized in buffer containing 0.1 M Tris and 0.05% Tri-ton X. The homogenate was spun down at 6000 gfor 20 min at 4°C. The supernatant was gently aspi-rated and used to determine prolyl hydroxylase ac-tivity and its protein content. The procedure to mea-sure the prolyl hydroxylase activity was essentially thesame as described above. Briefly, the reaction mixturein a total volume of 2.2 ml consisted of 200 µl of α-ketoglutarate (0.001 M), 200 µl of ferrous ammoniumsulfate (0.005 M), 250 µl of supernatant as enzymesource, 200 µl of pirfenidone to produce the desired fi-nal concentration, 200 µl of Tris·HCl (1 M), and 20 µlof 3H-unhydroxylated PC substrate (400,000 cpm).The reaction mixture was first preincubated with pir-fenidone at different concentrations for 30 min at 37°Cin a shaking water bath. The reaction was started byadding 200 µl of ascorbic acid (0.005 M); 30 min later,the reaction was terminated by adding 200 µl of 50%trichloroacetic acid. The tritiated water released wascollected by vacuum distillation. Then 1 ml of the triti-ated water was mixed with 10 ml of Ready Safe liquidscintillation cocktail (Beckman), and the radioactivitywas determined at 45% counting efficiency in a scintil-lation counter. The protein content of the supernatantwas determined according to the method of Lowryet al. (1951). The prolyl hydroxylase activity was ex-pressed as total dpm associated with tritiated water re-leased in the reaction mixture/mg protein per 30 min.

Determination of HydroxyprolineFor assay of lung hydroxyproline as a measure ofcollagen content, 1 ml of whole homogenate wasprecipitated with 0.25 ml of ice-cold 50% (w/v)trichloroacetic acid and centrifuged, and the precip-itate was hydrolyzed in 2 ml of 6 N HCl for 18 h at110°C. The hydroxyproline content was measured ac-cording to the method of Woessner (1961).

EVALUATIONAll data are expressed as mean ± SEM. Bleomycintreatment increases the amount of proteins of extrapul-monary origin that can result in the artificial loweringof all values (Karlinski and Goldstein 1980; Goldsteinand Fine 1986). Thus, the in vivo data are expressed ona per-lung basis. The data were compared within thegroups at the corresponding times using the two-way


ANOVA where four groups were involved, and the ttest between the two groups. A value of P ≤0.05 wasconsidered to be the minimum level of statistical sig-nificance.

MODIFICATIONS OF THE METHODHowell et al. (2001) induced pulmonary fibrosis inrats. Male Lewis rats, aged 6 weeks and weighing140–170 g, were anesthetized by intramuscular in-jection of 0.75–1.0 ml/kg Hypnorm (fentanyl citrate,0.315 mg/ml, and fluanisone, 10 mg/ml). Bleomycinsulfate was administered by a single intratracheal in-jection (l.5 mg/kg body weight in 0.3 ml of ster-ile saline). Control animals received 0.3 ml of salinealone. In initial experiments, groups of six rats werekilled by pentobarbitone overdose after 6 days to al-low assessment of thrombin levels in broncho-alveo-lar lavage fluid. Separate groups of two rats were sac-rificed 1, 3, 6, and 14 days after bleomycin instilla-tion for immunohistochemical assessment of throm-bin and protease-activated receptor1. Lungs were fixedby intratracheal instillation of 4% paraformaldehyde,the trachea ligated, and the inflated lungs and heartremoved en bloc. Tissues were fixed and transferredto 15% sucrose in phosphate-buffered saline, beforealcohol dehydration and embedding in paraffin wax.An additional series of animals was killed 6 days af-ter bleomycin or saline instillation for measurementof blood coagulation parameters, total and differentialcell counts in broncho-alveolar lavage fluid, and forNorthern blot analysis of lung tissue connective tissuegrowth factor and α1(I) procollagen mRNA levels. Formeasurement of coagulation parameters, blood wascollected from the inferior vena cava of animals af-ter laparotomy, and was immediately mixed 10:1 witha solution of 3.8% trisodium citrate (w/v). For mea-surement of total lung collagen and connective tissuegrowth factor and procollagen mRNA levels, the vas-culature was perfused with 5 ml of sterile saline con-taining 100 U/ml heparin. The lungs were removed,weighed, and immediately snap-frozen in liquid N2 af-ter removing the trachea and major airways.

Adachi et al. (2003) and Azoulay et al. (2003) stud-ied the effects of granulocyte colony-stimulating fac-tor (G-CSF) on lung injury induced by various dosesof bleomycin in rats.

Sogu et al. (2004) found that endosteine, an antiox-idant, prevents bleomycin-induced pulmonary fibrosisin rats.

Chen et al. (2006) reported that short coursesof low-dose dexamethasone delay bleomycin-inducedlung fibrosis in rats.

Chaudhary et al. (2006) described the time course ofinflammation and fibrosis in the rat bleomycin modeland studied the effect of timing of anti-inflammatoryand antifibrotic treatments on efficacy.

Barrio et al. (2006) studied in vitro tracheal hyper-responsiveness to muscarinic receptor stimulation bycarbachol in the rat model of bleomycin-induced pul-monary fibrosis.

Terasaki et al. (2000) and Terasaki (2001) studiedthe effect of epimorphin in bleomycin-induced pul-monary fibrosis in mice. Pulmonary fibrosis was in-duced in 8-week-old male ICR mice by a single in-tratracheal instillation of bleomycin. On selected daysafter injection (days 0, 3, 7, 14, 21, 28, 35, 42, and 56),the lungs were harvested and investigated. Immuno-histochemical analysis was performed for epimorphinusing light microscopy and confocal microscopy andalso the analysis by immunoelectron microscopy andin situ hybridization.

Avivi-Green et al. (2006) reported that discoidindomain receptor 1-deficient mice are resistant tobleomycin-induced lung fibrosis. Matsuyama et al.(2006) found that suppression of discoidin domainreceptor 1 by RNA interference attenuates lung in-flammation induced by intratracheal instillation ofbleomycin in mice.

Inayama et al. (2006) reported that a Iκ B kinase-β inhibitor ameliorates bleomycin-induced pulmonaryfibrosis in mice.

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Howell DCJ, Goldsack NR, Marshall RP, McAnulty RJ,Starke R, Purdy G, Laurent GJ, Chambers RC (2001) Di-rect thrombin inhibition reduced lung collagen, accumu-lation, and connective tissue growth factor mRNA lev-els in bleomycin-induced pulmonary fibrosis. Am J Pathol159:1383–1395

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Inayama M, Nishioka Y, Azuma M, Muto S, Aono Y, Makino H,Tani K, Uehara H, Izumi K, Itai A, Sone S (2006) A novelIκ B kinase-β inhibitor ameliorates bleomycin-inducedpulmonary fibrosis in mice. Am J Respir Crit Care Med173:1016–1022

Iyer SN, Gurujeyalakshmi G, Giri SN (1999) Effects of pir-fenidone on procollagen gene expression at the transcrip-tion level in bleomycin hamster model of lung fibrosis.J Pharmacol Exp Ther 289:211–218

Iyer SN, Hyde DM, Giri SN (2000) Anti-inflammatory effectof pirfenidone in the bleomycin-hamster model of lung in-flammation. Inflammation 24:477–491

Karlinsky JB, Goldstein RH (1980) Fibrotic lung disease–a per-spective. J Lab Clin Med 96(6):939–942

Keerthisingam CB, Jenkins RG, Harrison NK, Hernandez-Ro-driguez NA, Booth H, Laurent GJ, Hart SL, Foster ML,McAnulty RJ (2001) Cyclogenase-2 deficiency results ina loss of the anti-proliferative response to transforminggrowth factor-b in human fibrotic lung fibroblasts and pro-motes bleomycin-induced pulmonary fibrosis in mice. AmJ Pathol 158:1411–1422

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Matsuyama W, Watanabe M, Shirahama Y, Hirano R, Mit-suyama H, Higashimoto I, Osame M, Arimura K (2006)Suppression of discoidin domain receptor 1 by RNAinterference attenuates lung inflammation. J Immunol176:1928–1936

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D.2.2.10Influence of Cytokines on Lung Fibrosis

PURPOSE AND RATIONALESeveral cytokines (Murphy et al. 2000) are involvedin development of pulmonary fibrosis, such as gran-ulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), and trans-forming growth factor beta (TGFβ) (Kelly et al. 2003;Agostini and Gurrieri 2006).

GM-CSF is involved in fibrotic reactions of thelung (Xing et al. 1996; Adachi et al. 2002, 2003).

TGFβ is a central modulator of pulmonary and air-way inflammation and fibrosis (Sime et al. 1997; Kolbet al. 2002; Gauldie et al. 2003; Xu et al. 2003; Yaoet al. 2004; Hardie et al. 2006; Lee et al. 2006; Shep-pard 2006).

Smad proteins are involved in the TGFβ-mediatedpulmonary fibrosis (Zhao et al. 2000; Bonniaud et al.2004, 2005; Kobayashi et al. 2006).

TNFα induces TGFβ (Sime et al. 1998; War-shamana et al. 2001; Sullivan et al. 2005).

Xing et al. (1996) reported that the transfer of GM-CSF gene to rat lung induces eosinophilia, monocyto-sis, and fibrotic reactions.

PROCEDUREConstruction of Recombinant Adenovirus VectorsAn 800-bp fragment of murine GM-CSF cDNA wasisolated from pCDSRα by digestion with BamHIand DraI. The shuttle plasmid pACCMV contain-


ing 0 to 17 mu human type 5 adenovirus genomewith a CMV promoter (760 bp), multicloning sitesand SV40 splicing junction/polyA signal (430 bp) in-serted in the E1 region of viral genome was first di-gested with SalI, and the ends were repaired usingT4 kinase and dNTPs (New England Biolabs, Bev-erly, Mass., USA), followed by a secondary digestionwith BamHI to generate the 3′ complimentary ends.The GM-CSF fragment was then subcloned into theBamHI/SalI site in PACCMV using T4 ligase (NewEngland Biolabs) to generate the recombinant plas-mid PACCMVmGM-CSF. The presence of the GM-CSF insert was confirmed by restriction digestions.The PACCMVmGM-CSF was co-transfected, follow-ing a standard procedure described previously (Gra-ham and Prevec 1991), into 293 cells along with a plas-mid pAdBHG10 which contained the most rightwardsequences (3.7 to 100 mu) of human type 5 adenovirusgenome with a partial deletion in the E3 region (Bettet al. 1994). The recombinant replication-deficientadenovirus Ad5E1PACCMVmGM-CSF (Ad5E1GM-CSF) was rescued by homologous recombination. Thepresence of GM-CSF cDNA in the viral genome wasverified by analyzing viral genome fragments uponHindIII digestion and by Southern hybridization (Gra-ham and Perec 1991). The control virus Ad5dl70–3was constructed and characterized as previously de-scribed (Bett et al. 1994), and, similar to Ad5E1GM-CSF, this virus had the E1 region crippled, hence inca-pable of replication.

High titers of the above viruses were generated(Graham and Prevec 1991). Briefly, viruses purifiedby two rounds of CsCl gradient centrifugation weresubjected to chromatography using PD-10 Sephadexcolumns (Pharmacia Biotech, Baie d’UrFe,Quebec,Canada) to remove CsCl. The virus fractions werecollected in PBS containing 10% glycerol, measuredfor conductance to ensure complete removal of CsCl,pooled, titered, aliquoted and stored at –70°C until use.

Characterization of Recombinant Adenovirus VectorExpressing GM-CSF In VitroGM-CSF transgene mRNA was examined by North-ern hybridization analysis (Xing et al. 1993) using to-tal RNA from 293 cells infected with 10 plaque-form-ing units (pfu)/cell of Ad5E1GM-CSF for 24 and 48h. The supernatants from these cells and from infectedrat alveolar macrophages were assayed for GM-CSFusing an ELISA kit (Endogene, Cambridge, Mass.,USA). This ELISA was specific for mouse GM-CSFwithout crossreactivity with rat GM-CSF, with a sen-sitivity of 4 pg/ml.

Delivery of Recombinant Adenovirus Vectors to the LungFollowing a standard procedure (Xing et al. 1994),300 µl of Ad5E1GM-CSF or control virus Ad5dl70–3diluted in PBS to a concentration of 1 × 109 pfuwas instilled intratracheally to the lung of SpragueDawley male rats weighing 220–280 g (CharlesRiver Laboratories, Ottawa, Canada). At the end of1, 2, 4, 7, 12, 18, and 24 days after gene transfer, ratswere anesthetized, blood samples were taken from theabdominal aorta, and serum preparation and broncho-alveolar lavage (BAL) were performed.

Transgene and Transgene Protein Expression in the LungThe left lung of rats obtained at each time point wassnap-frozen in liquid nitrogen. Total lung RNA ex-traction and Northern hybridization were performed.Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine transgene mRNA ex-pression with total lung RNA using PCR reagents fromPromega (Madison, Wis., USA) following the standardprotocol. The specific primers for PCR were chosento ensure the amplification of the transgene-specificGM-CSF mRNA but not the endogenous rat GM-CSFmRNA (Miyatake et al. 1985; Smith et al. 1994).The sense and anti-sense primer sequences were5′-GTCTCTAACGAGTTCTCCTTCAAG-3′ and5′-TTCAGAGGGCTATACTGCCTTCCA-3′, respec-tively. The primers for rat GAPDH were designed asdescribed by Rosenfeld et al. (1992). BAL sampleswere collected at various times and assayed fortransgene protein GM-CSF by ELISA as describedabove or for TNFα by ELISA specific for both murineand rat TNFα (Genzyme, Cambridge, Mass., USA).Serum samples collected from the same animals werealso assayed for circulating levels of GM-CSF byELISA.

Cytologic Examination of BAL and Blood SamplesTotal cell numbers in BAL were determined usinga hematocytometer. Differential cell types were de-termined on cytospins stained with Diff-Quik (Bax-ter, McGaw Park, Ill., USA) by randomly counting300–400 cells/cytospin. To analyze the total peripheralblood leukocyte counts and differentials, blood sam-ples were collected into heparin-coated tubes. Totalleukocyte numbers were counted on a hematocytome-ter after lysing red blood cells with a lysis buffer con-taining 94% H2O, 3% acetic acid, and 3% Diff-Quikpurple stain. Differential leukocytes were determinedon blood smears stained with Diff-Quik by counting500–700 cells/blood smear.


Histopathologic Examination of Lung and other TissuesThe right lung and in some instances the whole lungof each animal were fixed by perfusion with 10% for-malin (Fisher Scientific, Fairlawn, N.J., USA). Tissuesfrom heart, liver, spleen, and kidney were also fixed in10% formalin. Multiple sections from different lobesof the lung or from other organs were stained withhematoxylin/eosin for routine histopathology, withCongo Red for identification of tissue eosinophils, orwith Elastic van Gieson for collagen and elastin.

MODIFICATIONS OF THE METHODUnderwood et al. (2000) demonstrated in guinea pigsand rats that a p38 MAPK inhibitor reduces neu-trophilia, inflammatory cytokines, MMP-9, and fibro-sis in lung.

Kolb et al. (2001) reported that the proteoglycansdecorin and biglycan differentially modulate TGF-β-mediated fibrotic responses in the lung of mice.

Uhal et al. (2003) showed that in rats amiodaroneinduced lung fibrosis and alveolitis, which could bepartially inhibited by angiotensin system antagonists.

Jiang et al. (2004) described regulation of pul-monary fibrosis by the chemokine receptor CXCR3,which is the receptor for the interferon-γ -inducible C-X-C chemokines MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11.

Kim et al. (2006) tested the alveolar epithelial-to-mesenchymal cell transition, which develops in vivoduring pulmonary fibrosis, and found regulation by theextracellular matrix.

Shi-Wen et al. (2006) found that constitutive ALK5-independent c-Jun N-terminal kinase activation con-tributes to endothelin-1 overexpression in pulmonaryfibrosis and gave evidence of an autocrine endothelinloop operating through the endothelin A and B recep-tors.

REFERENCES AND FURTHER READINGAdachi K, Suzuki M, Sugimoto T, Suzuki S, Niki R, Oyama A,

Uetsuka K, Nakamaya H, Doi K (2002) Granulocytecolony-stimulating factor exacerbates the acute lung in-jury and pulmonary fibrosis induced by intratracheal ad-ministration of bleomycin in rats. Exp Toxicol Pathol53:501–510

Adachi K, Suzuki M, Sugimoto T, Uetsuka K, Nakamaya H,Doi K (2003) Effects of granulocyte colony-stimulatingfactor on the kinetics of inflammatory cells in the peripheralblood and pulmonary lesions during the development ofbleomycin-induced lung injury in rats. Exp Toxicol Pathol55:21–32

Agostini C, Gurrieri C (2006) Chemokine/cytokine cocktailin idiopathic pulmonary fibrosis. Proc Am Thorac Soc3:357–363

Bett AJ, Haddara W, Prevec L, Graham FL (1994) An efficientand flexible system for construction of adenovirus vectors

with insertions or deletions in early regions 1 and 2. ProcNatl Acad Sci USA 91:8802–8806

Bonniaud P, Kolb M, Galt T, Robertson J, Robbins C,Stampfli M, Lavery C, Margetts PJ, Roberts AB, Gauldie J(2004) Smad3 null mice develop airspace enlargement andare resistant to TGF-β-mediated pulmonary fibrosis. J Im-munol 173:2099–2108

Bonniaud P, Margetts PJ, Ask K, Flanders K, Gauldie J, Kolb M(2005) TGF-β and Smad3 signaling link inflammation tochronic fibrogenesis. J Immunol 175:5390–5395

Gauldie J, Galt T, Bonnioud P, Robbins C, Kelly M, Warbur-ton T (2003) Transfer of the active form of the transforminggrowth factor-β1 gene to newborn rat lung induces changesconsistent with bronchopulmonary dysplasia. Am J Pathol163:2575–2584

Graham FL, Prevec L (1991) Gene transfer and expression pro-tocols. In: Murray EJ, Walker JM (eds) Methods in molec-ular biology.. Humana, Clifton, N.J.. pp 109–127

Hardie WD, Le Cras TD, Jiang K, Tichelaar JW, Azhar M, Ko-rfhagen TR (2006) A conditioned expression of transform-ing growth factor-α in adult mouse causes pulmonary fi-brosis. Am J Physiol 286:L741–L749

Jiang D, Liang J, Hodge J, Lu B, Zhu Z, Yu S, Fan J, Gao Y,Yin Z, Homer R, Gerard C Noble PW (2004) Regulationof pulmonary fibrosis by the chemokine receptor CXCR3.J Clin Invest 114:291–299

Kelly M, Kolb M, Bonnlaud P, Gauldie J (2003) Re-evaluationof fibrinogenic cytokines in lung fibrosis. Curr Pharm Des9:39–49

Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG,Brumwell AN, Sheppard D, Chapman HA (2006) Alve-olar epithelial cell mesenchymal transition develops invivo during pulmonary fibrosis and is regulated by the ex-tracellular matrix. Proc Natl Acad Sci USA 103:13180–13185

Kobayashi T, Liu X, Wen FQ, Kohyama T, Shen L, Wang XQ,Hashimoto M, Mao L, Togo S, Kawasaki S, Sigiura H,Kamio K, Rennard SI (2006) Smad3 mediates TGF-β1-induced collagen gel contraction by human lung fibrob-lasts. Biochem Biophys Res Commun 339:290–295

Kolb M, Margetts PJ, Sime PJ, Gauldie J (2001) Proteogly-cans decorin and biglycan differentially modulate TGF-β-mediated fibrotic responses in the lung. Am J Physiol280:L1327–L1334

Kolb M, Bonnlaud P, Galt T, Sime PJ, Kelly MM, Margetts PJ,Gauldie J (2002) Differences in the fibrogenic response af-ter transfer of active transforming growth factor-β1 geneto lungs of “fibrosis-prone” and “fibrosis-resistant” mousestrains. Am J Respir Cell Mol Biol 27:141–150

Lee CG, Kang HR, Homer RJ, Chupp G, Elias JA (2006) Trans-genic modeling of transforming growth factor-β1. Role ofapoptosis in fibrosis and alveolar remodeling. Proc AmThorac Soc 3:418–423

Miyatake S, Otsuka T, Yokota T, Lee F, Arai K (1985) Struc-ture of the chromosomal gene for granulocyte-macrophagecolony stimulating factor: comparison of the mouse and hu-man genes. EMBO J 4:2561–2568

Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Mat-sushima K, Miller LH, Oppenheim JJ, Power CA (2000)International Union of Pharmacology. XXII. Nomenclatureof chemokine receptors. Pharmacol Rev 52:145–176

Rosenfeld MA, Seigfried W, Yoshimura K, Yoneyama,Fukayama KM, Stier LE, Paakko PK, Gilardi P, Straford-Perricaudet LD, Pericaudet M, Guggino WB, Pavirani A,.Lecocq JP, Crystal RG (1992) In vivo transfer of the humancystic fibrosis transmembrane conductance regulator geneto the airway epithelium. Cell 68:143–155


Sheppard D (2006) Transforming growth factor β: a centralmodulator of pulmonary and airway inflammation and fi-brosis. Proc Am Thoric Soc 3:413–417

Shi-Wen X, Rodrïguez-Pascual F, Lamas S, Holmes A, Howat S,Pearson JD, Dashwood MR, du Bois RM, Denton CP,Black CM, Abraham DJ, Leask A (2006) ConstitutiveALK5-independent c-Jun N-terminal kinase activation con-tributes to endothelin-1 overexpression in pulmonary fi-brosis: evidence of an autocrine endothelin loop operatingthrough the endothelin A and B receptors. Mol Cell Biol26:5518–5527

Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J (1997)Adenovector-mediated gene transfer of active transform-ing growth factor-β1 induces prolonged severe fibrosis inrat lung. J Clin Invest 100:768–776

Sime PJ, Marr RA, Gauldie D, Xing Z, Hewlett BR, Gra-ham FL, Gauldie J (1998) Transfer of tumor necrosisfactor-α to rat lung induces severe pulmonary inflamma-tion and patchy interstitial fibrogenesis with induction oftransforming growth factor-β1 and myofibroblasts. Am JPathol 153:825–832

Smith LR, Lundeen KA, Dively JP, Carlo DJ, Brostoff SW(1994) Nucleotide sequence of the Lewis rat granulo-cyte-macrophage colony-stimulating factor. Immunogenet-ics 39:80

Sullivan DE, Ferris MB, Pociask D, Brody AR (2005) Tumornecrosis factor-α induces transforming growth factor-β1expression in lung fibroblasts through the extracellular sig-nal-regulated kinase pathway. Am J Respir Cell Mol Biol32:342–349

Uhal BD, Wang R, Laukka J, Zhuang J, Soledad-Conrad V, Fil-ippatos G (2003) Inhibition of amiodarone-induced lung fi-brosis but not alveolitis by angiotensin system antagonists.Pharmacol Toxicol 92:81–86

Underwood DC, Osborn RR, Bochnowicz S, Webb EF, Rie-man DJ, Lee JC, Romnic AM, Adams JL, Hay DWP, Gris-wold DE (2000) SB 239063, a p38 MAPK inhibitor, re-duces neutrophilia, inflammatory cytokines, MMP-9, andfibrosis in lung. Am J Physiol 279:L895–L902

Warshamana GS, Corti M, Brody AR (2001) TNF-α, PDGF, andTGF-β1 expression by primary mouse bronchiolar-alveo-lar epithelial and mesenchymal cells: TNF-α induces TGF-β1. Exp Mol Pathol 71:13–33

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D.2.2.11Emphysema Models

PURPOSE AND RATIONALESeveral animal models of genetically determined em-physema are known, such as the tight-skin mouse(Rossi et al. 1984; Martorana et al. 1989; Gayraudet al. 2000), beige mutants of the C57BL6J strain(O’Donnell et al. 1999; Lucattelli et al. 2003), theblotchy mouse (Ranga and Kleinerman 1981; Mc-Cartney et al. 1988), and homozygous mutant klotho(KL--) mice (Suga et al. 2000).

Hoyle et al. (1999) described emphysematous le-sions, inflammation, and fibrosis in the lungs of trans-genic mice overexpressing platelet-derived growth fac-tor. Le Cras et al. (2004) reported that vascular en-dothelial growth factor causes pulmonary hemorrhage,hemosiderosis, and air space enlargement in neonatalmice. Tsa et al. (2004) reported that overexpression ofplacenta growth factor contributes to the pathogenesisof pulmonary emphysema in mice.

Blanco et al. (1989), Whitney et al. (1999), Mas-saro et al. (1995), Massaro and Massaro (2000, 2003),and Dirami et al. (2004) reported that in rats septationof gas-exchange saccules occurs during the first twopostnatal weeks. Treatment with dexamethasone irre-versibly impairs septation. Treatment with all-trans-retinoid acid prevents the dexamethasone-induced in-hibition of septation.

Hind and Maden (2004) and Maden and Hind(2004) found that retinoic acid induces alveolar re-generation in the adult mouse lung after damage bydisulphiram treatment. These data could not be con-firmed in an elastase-induced emphysema model byFujita et al. (2004).

Several authors used elastase instillation to produceemphysema-like lesions in rats (Tepper et al. 2000;March et al. 2004), mice (Inoue et al. 2003; Murakamiet al. 2005), rabbits (Qi et al. 2004), and dogs (Morinoet al. 2005).

Kuraki et al. (2002) described inhibition of humanneutrophil elastase-induced emphysema in rats by anoral neutrophil elastase inhibitor.

PROCEDURERat Emphysema ModelMale Wistar rats weighing 228± 15 g were dividedinto controls (saline treated), low-dose group (treatedwith 200 U human neutrophil elastase), and a high-


dose group (treated with 400 U human neutrophil elas-tase). Human neutrophil elastase was sprayed abovethe carina of the trachea using a microsprayer withouttracheotomy. Eight weeks after human neutrophil elas-tase application, rats were sacrificed and lungs weredissected out to evaluate the morphologic changes.

Effects of Neutrophil Elastase InhibitorRats were divides into four groups: saline control, hu-man neutrophil elastase + CMC, human neutrophilelastase + low dose of inhibitor, and human neutrophilelastase + high dose of inhibitor given orally. Six hoursafter human neutrophil elastase, lung hemorrhage andneutrophil accumulation in the lung were determined.Eight weeks after the application, the functional andmorphologic changes were determined.

Lung Hemorrhage and Neutrophil Accumulationin the LungAfter tracheostomy, broncho-alveolar lavage was per-formed for determination of neutrophil counts andhemoglobin in lavage fluid. In addition, neutrophil ac-cumulation was estimated using the myeloperoxidaseactivity.

Lung Volume and Pulmonary MechanicsEight weeks after human neutrophil elastase applica-tion, rats were anesthetized and tracheotomy was per-formed. Using a whole-body plethysmograph for smallanimals, the functional residual capacity (FRC), totallung capacity and static lung compliance were deter-mined.

EVALUATIONData were presented as mean ± SD. Differences be-tween groups were evaluated for statistical signifi-cance using one-way analysis of variance.

MODIFICATIONS OF THE METHODBoström et al. (1996) showed that platelet-derivedgrowth factor A signaling is a critical event in lungalveolar myofibroblast development and alveogenesis.

Kirschvink et al. (2005) induced production of pul-monary MMP-2 and MMP-9 and emphysema by re-peated cadmium nebulizations in rats.

Corteling et al. (2002) studied the migration and ac-tivation of neutrophils into the airway in pathologicalconditions such as pulmonary emphysema in BALB/cand C57BL/6 mice and in golden hamsters. Theanimals were sequentially treated intranasally with0.3 mg/kg lipopolysaccharide and with 0.5 mg/kg N-formyl-Met-Leu-Phe.

Selman et al. (2003) induced emphysema in guineapigs by exposure to the whole smoke of 20 cigarettesper day, 5 days per week, for 1 month, 2 months,and 4 months through a whole-body exposure cham-ber. Half of the animals received a matrix metallopro-teinase inhibitor. After death, the lungs were lavagedwith saline solution, and matrix metalloproteinases inthe lavage fluid were determined by zymography andimmunoblot. Lungs were fixed for histology, immuno-chemistry, and morphometry.

Lucattelli et al. (2003) described collagen phagocy-tosis by lung alveolar macrophages in animal modelsof emphysema.

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Murakami S, Nagaya N, Itoh T, Iwase T, Fujisato T, Nishioka K,Hamada K, Kangawa K, Kimura H (2005) Adrenomedullinregenerates alveoli and vasculature in elastase-induced pul-monary emphysema in mice. Am J Respir Crit Care Med172:581–589

O’Donnell MD, O’Connor CM, FitzGerald MX, Lungarella G,Cavarra E, Martorana PA (1999) Ultrastructure of lungelastin and collagen in mouse models of spontaneous em-physema. Matrix Biol 18:357–360

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Selman M, Cisneros-Lira J, Gaxiola M, Ramïrez R, Kud-lacz EM, Mitchell PG, Pardo A (2003) Matrix metallopro-teinases inhibition attenuates tobacco smoke-induced em-physema in guinea pigs. Chest 123:1633–1641

Suga T, Kurabayashi M, Sando Y, Ohyama Y, Maeno T,Maeno Y, Aizawa H, Matsumura Y, Kuwaki T, Kuro-o M,Nabeshima Y, Nagai R (2000) Disruption of the klotho genecauses pulmonary emphysema in mice. Defect in mainte-nance of pulmonary integrity during postnatal life. Am JRespir Cell Mol Biol 22:26–33

Tepper J, Pfeiffer J, Aldrich M, Tumas D, Kern J, Hoffman E,McLennan G, Hyde D (2000) Can retinoic acid amelioratethe physiologic and morphologic effects of elastase instil-lation in the rat? Chest 117:242–244

Tsa PN, Su YN, Li H, Huang PH, Chien CT, Lai YL, Lee CN,Chen CA, Cheng WF, Wei SC, Yo CJ, Hsieh FJ, Hsu SM

(2004) Overexpression of placenta growth factor con-tributes to the pathogenesis of pulmonary emphysema. AmJ Respir Crit Care Med 169:505–511

Whitney D, Massaro GD, Massaro D, Clerch LB (1999) Geneexpression of cellular retinoid-binding proteins: modula-tion by retinoic acid and dexamethasone in postnatal ratlung. Pediatr Res 45:2–7

D.2.2.12Models of Chronic Obstructive Pulmonary Disease(COPD)

PURPOSE AND RATIONALEChronic obstructive pulmonary disease (COPD) isa severe respiratory condition that is increasing inprevalence worldwide. The disease is characterized byairflow limitation that is not fully reversible. The air-flow limitation is usually progressive and associatedwith abnormal inflammatory response of the lungs tonoxious particles and gases. Three conditions com-prise COPD, namely mucus hypersecretion, emphy-sema and bronchiolitis. Cigarette smoking is the ma-jor risk factor for development of COPD and accountsfor the majority of cases (Donnelly and Rogers 2003).The relevance of the present animal models for COPDhas been questioned (Canning 2003). Several authorsused chronic cigarette-smoke exposure in mice (Hau-tamaki et al. 1997; Cavarra et al. 2001a, 2001b; Wrightand Churg 2002; Bartalesi et al. 2005; Martorana et al.2005), in rats (Escolar et al. 1995; Lee et al. 2005),in guinea pigs (Wright 2001; Meshi et al. 2002), or indogs (Frasca et al. 1983).

Lee et al. (2005) reported inhibition of cigarette-smoking-induced emphysema and pulmonary hyper-tension in rat lungs by a HMG-CoA reductase in-hibitor.

Martorana et al. (2005) found that a selective phos-phodiesterase-4 (PDE4) inhibitor fully prevents em-physema in mice chronically exposed to cigarettesmoke.

PROCEDURESix-week-old C57Bl/6J male mice were, in acute stud-ies, exposed either to room air or the smoke of fivecigarettes (Virginia filter cigarettes: 12 mg of tar and0.9 mg of nicotine) for 20 min. In chronic studies, themice were exposed to either room air or to the smokeof three cigarettes/day for 5 days/week for 7 months.Antioxidant capacity was assessed at the end of expo-sure in broncho-alveolar lavage fluid. Cytokines andchemokines were determined.

In acute studies, mice were divided in three groupsof 40 animals each. These groups were then divided


into four subgroups of 10 mice: no treatment/air ex-posed; no treatment/smoke exposed; low dose of testcompound/smoke exposed; high dose of test com-pound/smoke exposed.

In chronic studies, five groups of animals wereused: no treatment/air exposed; drug treatment/air ex-posed; no treatment/smoke exposed; low dose of testcompound/smoke exposed; high dose of test com-pound/smoke exposed. After 7 months, animals weresacrificed and the lungs fixed intratracheally with 5%formalin at a pressure of 20 cmH2O. Lung volumewas measured by water displacement. Assessment ofemphysema included mean linear intercept and inter-nal surface area. The volume density of macrophages,marked immunohistochemically with antimouse Mac-3 monoclonal antibodies, was determined by pointcounting.

EVALUATIONThe significance of the differences was calculated us-ing one-way analysis of variance.

MODIFICATIONS OF THE METHODKumar et al. (2003) compared a selective PDE4 in-hibitor with pentoxifylline (a non-selective phospho-diesterase inhibitor) and dexamethasone in ameliorat-ing the lesions of chronic asthma in BALB/c mice sen-sitized to ovalbumin and chronically challenged withaerosolized antigen for 6 weeks.

Kodavanti et al. (2000) reported that the combina-tion of elastase and sulfur dioxide exposure causesCOPD-like lesions in the rat.

The potential of tachykinin receptor antagonists inairways diseases was discussed by Joos and Pauwels(2001).

Sturton and Fitzgerald (2002) reviewed PDE4 in-hibitors for the treatment of COPD.

Billah et al. (2002) described the pharmacology ofan orally active PDE4 inhibitor.

Pitfalls and opportunities for modeling allergicasthma in mice were discussed by Kumar and Foster(2002).

Inoue et al. (2003) described an impaired pul-monary inflammation response as a prominent featureof streptococcal pneumonia in mice with experimentalemphysema.

Dual dopamine D2 receptor and β2-adrenoreceptoragonists were proposed for the treatment of COPD(Dougall et al. 2003; Ind et al. 2003).

Lessons from transgenic mice for the relationshipbetween asthma and COPD were discussed by Elias(2004).

Lappalainen et al. (2005) found that interleukin-1β

causes pulmonary inflammation, emphysema, and air-way remodeling in the adult murine lung.

Romano (2005) discussed selectin antagonists fortheir therapeutic potential against asthma and COPD.

Jones et al. (2002) described a model for the con-tinuous monitoring of polymorphonuclear leukocytetrapping in the pulmonary vasculature of the rabbit.

REFERENCES AND FURTHER READINGBartalesi B, Cavarra E, Fineschi S, Lucattelli M, Lunghi B, Mar-

torana PA, Lungarella G (2005) Different lung responses tocigarette smoke in two strains of mice sensitive to antioxi-dants. Eur Resp J 25:15–22

Billah MM, Cooper N, Minnicozzi M, Warneck J, Wang P,Hey JA, Kreutner W, Rizzo CA, Smith SR, Young S,Chapman RW, Dyke H, Shih NY, Piwinski JJ, Cuss FM,Montana, Ganguly AK, Egan RW (2002) Pharmacol-ogy of N-(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinoline carboxamide (SCH 351591)a novel, orally active phosphodiesterase 4 inhibitor. J Phar-macol Exp Ther 302:127–137

Canning BJ (2003) Modeling asthma and COPD in animals:a pointless exercise? Curr Opin Pharmacol 3:244–250

Cavarra E, Lucattelli M, Gambelli F, Bartalesi B, Fineschi S,Szarka A, Giannerini F, Martorana PA, Lungarella G(2001a) Human SLPI inactivation after cigarette smoke ex-posure in a new in vivo model of pulmonary oxidativestress. Am J Physiol 281:L412–L417

Cavarra E, Bartalesi B, Lucattelli M, Fineschi S, Lunghi B,Gambelli F, Ortiz LA, Martorana PA, Lungarella G (2001b)Effects of cigarette smoke in mice with different levels ofα1-proteinase inhibitor and sensitivity to oxidants. Am JRespir Crit Care Med 164:886–890

Donnelly LE, Rogers DF (2003) Therapy of chronic ob-structive pulmonary disease in the 21st century. Drugs63:1973–1998

Dougall IG, Young A, Ince F, Jackson DM (2003) Dualdopamine D2 receptor and β2-adrenoreceptor agonists forthe treatment of chronic obstructive pulmonary disease: thepre-clinical rationale. Respir Med 97 Suppl A:S3–S/

Elias J (2004) The relationship between asthma and COPD.Lessons from transgenic mice. Chest 126:111S–116S

Escolar JD, Martinez MN, Rodriguez FJ, Gonzalo G, Esco-lar MA, Roche PA (1995) Emphysema as a result of in-voluntary exposure to tobacco smoke: morphometric studyin the rat. Exp Lung Res 21:255–273

Frasca JM, Auerbach O, Carter HW, Parks VR (1983) Morpho-logic alterations induced by short term cigarette smoking.Am J Pathol 111:11–20

Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD (1997)Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277:2002–2004

Ind PW, Laitinen L, Laursen L, Wenzel S, Wouters E, Deamer L,Nystrom P (2003) Early clinical investigations of Viozan(sibenadel HCl), a novel D2 dopamine receptor, β2-adrenoreceptor agonist for the treatment of chronic obstruc-tive pulmonary disease symptoms. Respir Med 97 SupplA:S0–S21

Inoue S, Nakamura H, Otake K, Saito H, Terashita K, Sato J,Takeda H, Tomoike H (2003) Impaired pulmonary inflam-mation responses are a prominent feature of streptococcalpneumonia in mice with experimental emphysema. Am JResp Crit Care Med 167:764–770

D.3 · Antitussive Activity 551

Jones H, Paul W, Page CP (2002) A new model for the continu-ous monitoring of polymorphonuclear leukocyte trappingin the pulmonary vasculature of the rabbit. J PharmacolToxicol Meth 48:21–29

Joos GF, Pauwels RA (2001) Tachykinin receptor antago-nists: potential in airways diseases. Curr Opin Pharmacol1:235–241

Kodavanti UP, Jackson MC, Ledbetter AD, Starcher BC, Evan-sky PA, Harewood A, Winsett DW, Costa DL (2000) Thecombination of elastase and sulfur dioxide exposure causesCOPD-like lesions in the rat. Chest 117:299–302

Kumar RK, Foster PS (2002) Modeling allergic asthma in mice.Pitfalls and opportunities. Am J Respir Coll Mol Biol27:267–272

Kumar RK, Herbert C, Thomas PS, Wollin L, Beume R,Yang M, Webb DC, Foster PS (2003) Inhibition of in-flammation and remodeling by Roflumilast and dexam-ethasone in murine chronic asthma. J Pharmacol Exp Ther307:349–355

Lappalainen U, Whitsett JA, Wert SE, Tichelaar JW, Bry K(2005) Interleukin-1β causes pulmonary inflammation,emphysema, and airway remodeling in the adult murinelung. Am J Respir Cell Mol Biol 32:311–318

Lee JH, Lee DS, Kim EK, Choe KH, Oh YM, Shim TS, Kim SE,Lee YS, Lee SD (2005) Simvastatin inhibits cigarettesmoking-induced emphysema and pulmonary hypertensionin rat lungs. Am J Respir Crit Care Med 172:987–993

Martorana PA, Beume R, Lucattelli M, Wollin L, Lungarella G(2005) Roflumilast fully prevents emphysema in micechronically exposed to cigarette smoke. Am J Respir CritCare Med 172:848–853

Meshi B, Vitalis TZ, Ionescu D, Elliott WM, Liu C, Wang XD,Hayashi S, Hogg JC (2002) Emphysematous lung destruc-tion by cigarette smoke. The effects of latent adenoviral in-fection on the lung inflammatory response. Am J RespirCell Mol Biol 26:52–57

Romano SJ (2005) Selectin antagonists: therapeutic potential onasthma and COPD. Treatments Respir Med 4:85–94

Sturton G, Fitzgerald M (2002) Phosphodiesterase 4 inhibitorsfor the treatment of COPD. Chest 121.� 5 Suppl):192S

Wright JL (2001) The importance of ultramicroscopic em-physema in cigarette smoke-induced lung disease. Lung179:71–81

Wright JL, Churg A (2002) Animal models of cigarette smoke-induced COPD. Chest 122 [6 Suppl]:301S

D.3Antitussive Activity

D.3.0.1Antitussive Activity After Irritant Inhalationin Guinea Pigs

PURPOSE AND RATIONALECough is thought to be caused by a reflex. The sensi-tive receptors are located in the bronchial tree, particu-larly in the bifurcation of the trachea. These receptorscan be stimulated mechanically or chemically, e. g., byinhalation of various irritants. Nerve impulses then ac-tivate the cough center in the brain. Several animalsspecies and several irritants have been used, most fre-quently the citric acid induced cough in guinea pigs

(Charlier et al. 1961; Karlsson et al. 1989 Braga et al.1993). The pharmacology of cough was reviewed byReynolds et al. (2004).

PROCEDUREGuinea pigs of either sex weighing 300–400 g areused. The animal is placed in a cylindrical glass vessel,with 2 tubes at either ends. One serves as the entranceof the aerosol, the other for its efflux. The latter tubehas a side-arm connecting to a tambour, from whichchanges in pressure can be registered. A pinch-clampwith a variable screw is placed on the efflux tube be-yond the side arm, permitting the regulation of the sen-sitivity of the system, so that the normal respiration isnot registered, while the displacement of air in the en-closure caused by coughing of the animal is registered.The guinea pig is exposed to the aerosol of 7.5% cit-ric acid in water for 10 min. Each animal is tested firstto obtain the control response. The number of tussiveresponses is registered. One hour later, the test sub-stance is applied either s.c. or orally, and 30 min laterthe guinea pig is subjected to the aerosol again. Thenumber of coughs during 10 min is recorded.

EVALUATIONThe number of coughs after treatment is expressed aspercentage of the control period. Using various doses,ED50 values can be calculated.

CRITICAL ASSESSMENT OF THE METHODThe citric acid induced coughing in guinea pigs hasbeen proven to be an effective method to test antitus-sive agents.

MODIFICATIONS OF THE METHODThe citric cough model in guinea pigs was used byAdcock et al. (1988) to study the effects of codeine,morhine and an opioid pentapeptide, by Hay et al.(2000) to study a potent and selective neurokinin-3 re-ceptor antagonist, and by Brown et al. (2004) to studyantitussive activity of sigma-1 receptor agonists.

Cough elicited by capsaicin inhalation in guineapigs was used by Bolser et al. (1993, 1997) to studyantitussive effects of GABAB agonists or NK1 andNK2 tachykinin receptor antagonists, by McLeod et al.(1998) to study the antitussive action of antihis-tamines, and by Trevisani et al. (2004) to investigatethe activity of iodo-resiniferatoxin, an ultra potent an-tagonist of the transient receptor potential vanilloid-1.

Forsberg et al. (1988) studied cough and bron-choconstriction mediated by aerosols of capsaicin orcitric acid or nicotine or histamine in guinea pigs.


Püschmann and Engelhorn (1978) studied the inhi-bition of the coughing reflex induced by inhalation ofa citric acid spray in rats.

Other irritants have been used to induce cough,e. g. ammonia in dogs, guinea pigs, and cats (Rosiereet al. 1956; Källqvist and Melander 1957; Chen et al.1960; Sallé and Brunaud 1960; Ellis et al. 1963), ornebulized sulfuric acid or sulfur dioxide in guinea pigs,rats, cats or dogs (Eichler and Smiatek 1940; May andWiddicombe 1954; Winter and Flakater 1952, 1954;Friebel et al. 1955; Reichle and Friebel 1955; Wiede-meijer et al. 1960; Chermat et al. 1966; Karttunen et al.1982). Capsaicin aerosol was used by Forsberg andKarlsson (1986), Gallico et al. (1997).

Winter and Flakater (1955) exposed sensitizedguinea pigs to aerosol of a specific antigen.

Kamei et al. (1989) induced cough in rats by a nebu-lized solution of capsaicin. The cough reflex was mea-sured as airflow into or out of the chamber of a bodyplethysmograph by a pneumotachometer head.

Carotis sinus excitation in dogs induced by injectionof lobeline resulting in coughing could be suppressedby codeine (Gross 1957).

Sanzari et al. (1968) induced cough in cats anes-thetized with α-chloralose by intravenous injection of1,1-dimethyl-4-phenylpiperazinium iodide (DMPP),a ganglionic stimulant which is more potent than lo-beline and possesses only marginal ganglionic block-ing properties. The number of coughs was found to bea linear function of the dose of the irritant. Coughswere recorded as spikes superimposed on the respi-ratory pattern. The method is suitable for quantitativeevaluation of antitussive activity.

REFERENCES AND FURTHER READINGAdcock JJ, Schneider C, Smith TW (1988) Effects of codeine,

morphine and a novel opioid pentapeptide BW443C, oncough, nociception, and ventilation in the unanesthetizedguinea pig. Br J Pharmacol 93:93–100

Bolser DC, Aziz SM, DeGennaro FC, Kreutner W, Egan RW,Siegel MI, Chapman RW (1993) Antitussive effects ofGABAB agonists in the cat and guinea pig. Br J Pharma-col 110:491–495

Bolser DC, DeGennaro FC, O’Reilly S, McLeod RL, Hey JA(1997) Central antitussive activity of the NK1 and NK2tachykinin receptor antagonists, CP-99,994 and SR 48968,in the guinea pig and cat. Br J Pharmacol 121:165–170

Braga PC, Bossi R, Piatti G, Dal Sasso M (1993) Antitussive ef-fect of oxatomide on citric acid-induced cough in consciousguinea pig. Arzneim Forsch/Drug Res 43:550–553

Brown C, Fezoui M, Selig WM, Schwartz CE, Ellis JL (2004)Antitussive activity of sigma-1 receptor agonists in theguinea pig. Br J Pharmacol 141:233–240

Charlier R, Prost M, Binon F, Deltour G (1961) Étude pharma-cologique d’un antitussif, le fumarate acide de phénéthyl-1 (propyne-2-yl)-4-propionoxy-4 pipéridine. Arch internPharmacodyn 134:306–327

Charmat R, Kornowski H, Jondet A (1966) Technique desélection rapide des substances antitussives. Application àl’évaluation de l’activité d’un dérivé de la prométhazine.Ann pharmaceut franç 24:181–184

Chen JYP, Biller HF, Montgomery EG (1960) Pharmacologicstudies of a new antitussive, alpha-(dimetylaminomethyl)-ortho-chlorobenzhydrol hydrochloride (SL-501, Bayer B-186) J Pharmacol Exp Ther 128:384–391

Eichler O, Smiatek A (1940) Versuche zur Auswertung vonMitteln zur Bekämpfung des Reizhustens. Arch Exp PathPharm 194:621–627

Ellis GP, Goldberg L, King J, Sheard P (1963) The synthesisand antitussive properties of some cyclopentane derivates.J Med Chem 6:111–117

Forsberg K, Karlsson JA (1986) Cough induced by stimulationof capsaicin-sensitive sensory neurons in conscious guineapigs. Acta Physiol Scand 128:319–320

Forsberg K, Karlsson JA, Theodorsson E, Lundsberg JM, Pers-son CG (1988) Cough and bronchoconstriction mediatedby capsaicin-sensitive sensory neurons in the guinea pig.Pulmon Pharmacol 1:33–39

Friebel H, Reichle C, v. Graevenitz A (1955) Zur Hemmungdes Hustenreflexes durch zentral angreifende Arzneimittel.Arch exp Path Pharm 224:384–400

Gallico L, Borghi A, Dalla Rosa C, Ceserani R, Tognella S(1994) Mogusteine: a novel peripheral non-narcotic anti-tussive drug. Br. J Pharmacol 112:795–800

Gross A (1957) Etude expérimentale, chez le chien choralosé,de l’action antitussive de la codéine, au moyen du réflexepleurotussigène et de la toux lobélinique. C R Soc Biol,Paris 151:704–707

Hay DWP, Giardina GAM, Griswold DE, Underwood DC, Kot-zer CJ, Bush B, Potts W, Sandhu P, Lundberg D, Fo-ley JJ, Schmidt DB, Martin LD, Kilian D, Legos JJ,Barone FC, Luttmann MA, Grugni M, Raveglia LF, Sa-rau HM (2002) Nonpeptide tachykinin receptor antag-onists. III. SB235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor an-tagonist, inhibits citric acid-induced cough and airwayshyper-reactivity in guinea pigs. J Pharmacol Exp Ther300:314–323

Källqvist I, Melander B (1957) Experimental and clinical eval-uation of chlorcyclizine as an antitussive. Arzneim Forsch7:301–304

Kamei J, Tanihara H, Igarashi H, Kasuya Y (1989) Effects ofN-methyl-D-aspartate antagonists on the cough reflex. EurJ Pharmacol 168:153–158

Karlsson JA, Lanner AS, Persson CGA (1989) Airway opi-oid receptors mediate inhibition of cough and reflex bron-choconstriction in guinea pigs. J Pharmacol Exp Ther252:863–868

Karttunen P, Koskiniemi J, Airaksinen MM (1982) An improve-ment to the use of sulfur dioxide to induce cough in exper-imental animals. J Pharmacol Meth 7:181–184

May AJ, Widdicombe JG (1954) Depression of the cough reflexby pentobarbitone and some opium derivatives. Br J Phar-macol 9:335–340

McLeod RL, Mingo G, O’Reilly S, Ruck LA, Bolser DC,Hey JA (1998) Antitussive action of antihistamines is in-dependent of sedative and ventilation activity in the guineapig. Pharmacology 52:57–64

Püschmann S, Engelhorn R (1978) Pharmakologische Un-tersuchungen des Bromhexin-Metaboliten Ambroxol.Arzneim Forsch/Drug Res 28:889–898

Reichle C, Friebel H (1955) Zur Hemmung des Hustenre-flexes durch zentral angreifende Arzneimittel. II. Mit-teilung. Arch exp Path Pharm 226:558–562

D.3 · Antitussive Activity 553

Reynolds SM, Mackenzie AJ, Spina D, Page CP (2004) Thepharmacology of cough. Trends Pharmacol Sci 25:569–576

Rosiere CE, Winder CV, Wax J (1956) Ammonia cough elicitedthrough a tracheal side tube inn unanesthetized dogs. Com-parative antitussive bioassay of four morphine derivativesand methadone in terms of ammonia thresholds. J Pharma-col Exp Ther 116:296–316

Sallé J, Brunaud M (1960), Nouvelle technique d’enregistrementdes mouvements de toux provoqués par l’inhalation devapeurs irritantes chez le cobaye. Arch Int Pharmacodyn126:120–125

Sanzari NP, Fainman FB, Emele JF (1968) Cough induced by1,1-dimethyl-4-phenylpiperazinium iodide: a new antitus-sive method. J Pharmacol Exp Ther 162:190–195

Shemano I (1964) Techniques for evaluating antitussive drugsin animals. In: Nodine JH, Siegler PE (eds) Animal andclinical pharmacologic techniques in drug evaluation. YearBook Medical Publishers, Inc. Chicago, pp 456–460

Trevisani M, Milan A, Gatti R, Zanasi A, Harrison S,Fonatana G, Morice AH, Geppetti G (2004) Antitussiveactivity of iodo-resiniferatoxin in guinea pigs. Thorax59:769–772

Wiedemeijer JC, Kramer HW, deJongh DK (1960) A screeningmethod for antitussive compounds. Acta Physiol Pharma-col Neerl 9:501–508

Winter CA, Flakater L (1952) Antitussive action of d-iso-methadone and d-methadone in dogs. Proc Soc Exp BiolMed 81:463–465

Winter CA, Flakater L (1954) Antitussive compounds: Testingmethods and results. J Pharmacol Exp Ther 112:99–108

Winter CA, Flakater L (1955) The effects of drugs upon a gradedcough response obtained in sensitized guinea pigs exposedto aerosol of specific antigen. J Exp Med 101:17–24

D.3.0.2Cough Induced by Mechanical Stimulation

PURPOSE AND RATIONALECough can be induced by mechanical stimulation ofthe trachea in anesthetized guinea pigs (Takagi et al.1960; Gallico et al. 1994).

PROCEDUREMale guinea pigs weighing 350–400 g are maintainedin conditioned quarters (temperature 21 ± 2°C, relativehumidity 55 ±10%, 12 h on-12 h off light cycle) withfood and water ad libitum for at least 1 week beforeuse.

After overnight fasting with water ad libitum, theguinea pigs are lightly anesthetized with 25% urethane(4 ml/kg i.p.) which induces surgical levels of analge-sia without depressant effects on respiratory function.Analgesia is monitored throughout the experiment asthe disappearance of head shaking in response to earpinch. The animals are maintained at a constant bodytemperature of 37°C by means of a heated plate. A thinsteel wire is gently inserted into the trachea througha small incision near the cricoid cartilage. Coughs areevoked by pushing the steel wire to reach the bifurca-

tion of the trachea 35 and 5 min before oral drug ad-ministration and 30, 60 and 120 min after treatment.One violent cough occurs upon each stimulation. Onlythose animals that respond to both mechanical stimu-lations before dosing are selected and then randomlyassigned to receive the test drug at various doses or thestandard (codeine 15, 30 and 60 mg/kg). Ten animalsper dose are used.

EVALUATIONEvaluation of the statistical significance of the re-sults is performed with Student’s t-test for paired data.ED50 values are determined by logit transformation.

MODIFICATIONS OF THE METHODSeveral other ways of mechanical stimulation havebeen used, e. g., by a nylon-bristled stimulator thrustinto the trachea in dogs (Kasé 1952, 1954), or by a sil-ver thread in decerebrated guinea pigs (Lemeignanet al. 1966), or by vibration of an iron slung in the tra-chea of a dog induced by an electromagnet (Tedeschiet al. 1959) or electrical stimulation of the trachea viaa bronchoscope (Gross et al. (1958) or through im-planted copper electrodes (Stefko and Benson 1953;Benson et al. 1953; Granier-Doyeux et al. 1959; Ste-fko et al. 1961).

Hara and Yanaura (1959), Yanaura et al. (1974,1982) induced cough in unrestrained animals after im-plantation of electrodes in the trachea.

Combined mechanical and chemical stimulationhas been applied by Kroepfli (1950).

Bolser et al. (1999, 2001) and McLeod et al. (2002)studied the influence of antitussive drugs on the coughmotor pattern in anesthetized cats. Coughing was pro-duced by mechanical stimulation of the intrathoracictrachea with a thin flexible polyethylene cannula for10 s per stimulus trial. During each trial, the can-nula was repetitively moved in the trachea at a fre-quency of ∼ 2 Hz. EMGs from the diaphragm and rec-tus abdominis muscles were recorded with the use ofbipolar tungsten-wire electrodes. The diaphragm elec-trodes were placed through a small midline abdomi-nal incision, which was subsequently closed. Cough ischaracterized by coordinated bursts of activity in in-spiratory and expiratory muscles. Cough was definedas a large burst of EMG activity in the diaphragm thatis immediately followed by a burst of EMG activity inthe rectus abdominis muscle. This definition differen-tiates augmented breaths, the aspiration reflex, or theexpiration reflex from cough. The antitussive activityof selected drugs was evaluated from cumulative doseresponses after intravertebral artery administration of


each compound. The protocol consisted of the appli-cation of five consecutive mechanical stimulus trialsafter vehicle administration. Stimulus trials were ap-plied at 1-min intervals after each dose of compound,for a total of five stimulus trials between doses. Ap-proximately 7 min elapsed between each dose of com-pound.

Kasé et al. (1976) studied the antitussive activityof d-3-methyl-N-methylmorphinan in conscious mon-grel dogs. Coughing was induced by mechanical stim-ulation with a stimulator consisting of 5 hog bris-tles on the mucosa of tracheal bifurcation througha chronically-built tracheal fistula and in lightly anes-thetized cats with a stimulator consisting of 5 whiskersof a rabbit.

REFERENCES AND FURTHER READINGBenson WM, Stefko PL, Randall LO (1953) Comparative

pharmacology of levorphan, racemorphan and dextromor-phan and related methyl esters. J Pharmacol Exp Ther109:189–200

Bolser DC, Hey JA, Chapman RW (1999) Influence of centralantitussive drugs on the cough motor pattern. J Appl Phys-iol 86:1017–1024

Bolser DC, McLeod RL, Tulshian DB, Hey JA (2001) Antinoci-ceptive action of nociceptin in the cat. Eur J Pharmacol430:107–111

Gallico L, Borghi A, Dalla Rosa C, Ceserani R, Tognella S(1994) Mogusteine: a novel peripheral non-narcotic anti-tussive drug. Br. J Pharmacol 112:795–800

Granier-Doyeux M, Horande M, Kucharski W (1959) Méthoded’évaluation quantitative des agents antitussigènes. ArchInt Pharmacodyn 121:287–296

Gross A, Lebon P, Rambert R (1958) Technique de toux ex-périmentale chez le Chien., par excitation faradique, sousbronchoscopie, de l’éperon trachéal. C R Soc Biol Paris152:495–497

Hara S, Yanaura S (1959) A method of inducing and recordingcough and examination of the action of some drugs withthis method. Jap J Pharmacol 9:46–54

Kasé Y (1952) New methods of estimating cough depressingaction. Jap J Pharmacol 2:7–13

Kasé Y (1954) The “coughing dog” – an improved methodfor the evaluation of an antitussive. Pharm Bull (Jpn)2:298–299

Kasé Y, Kito G, Miyata T, Uno T, Takahama K, Ida H (1976) An-titussive activity and other related pharmacological proper-ties of d-3-methyl-N-methylmorphinan (AT-17). ArzneimForsch/Drug Res 26:353–360

Kroepfli P (1950) Über das Verhalten einiger Atmungsgrößenbeim Husten. I. Mitteilung über den Hustenmechanismus.Helv Physiol Acta 8:33–43

Lemeignan M, Streichenberger G, Lechat P (1966) Del’utilisation du Cobaye décérébré pour l’étude des antituss-sifs. Thérapie 21:361–366

McLeod RL, Bolser CD, Y J, Parra LE, Mutter JC, Wang X,Tulshian DB, Egan RW, Hey JA (2002) Antitussive effectof nociceptin/orphanin FG in experimental cough models.Pulmon Pharmacol Ther 15:213–216

Stefko PL, Benson WM (1953) A method for the evaluation ofantitussive agents in the unanesthetized dog. J PharmacolExp Ther 108:217–223

Stefko PL, Denzel J, Hickey I (1961) Experimental investigationof nine antitussive drugs. J Pharm Sci 50:216–221

Takagi F, Fukuda H, Yano K (1960) Studies on antitussives. I.Bioassay of antitussives. Yakugaku Zasshi 80:1497–1501

Tedeschi RE, Tedeschi DH, Hitchens JD, Cook L, Mattis PA,Fellows EJ (1959) A new antitussive method involving me-chanical stimulation in unanesthetized dogs. J PharmacolExp Ther 126:338–344

Yanaura S, Iwase H, Sato S, Nishimura T (1974) A newmethod for induction of the cough reflex. Jap J Pharmacol24:453–460

Yanaura S, Kitagawa H, Hosakawa T, Misawa M (1982) A newscreening method for evaluating antitussives in consciousguinea pigs. J Pharm Dyn 5:965–971

D.3.0.3Cough Induced by Stimulationof the Nervus Laryngicus Superior

PURPOSE AND RATIONALEThe probable pathways in the cough reflex arc are re-ceptors in the area of the trachea and the large bronchi,afferent nerves mainly in the branches of the vagusnerve, a “cough center” located in the medulla ob-longata, and efferent nerves closing the glottis andreinforcing the expiratory thrust. Stimulation of theNervus laryngicus superior induces coughing. Antitus-sive agents with predominantly central action suppressthe coughing reflex.

PROCEDURECats of either sex weighing 2–3 kg are anesthetizedwith 40 mg/kg i.p. pentobarbital, placed on a heatedoperating table and their extremities secured. Sincedeep anesthesia suppresses coughing the dose of pen-tobarbital has to be adjusted. The fur is shaved ven-trally at the neck. Small incisions are made at bothsides of the larynx. The superior laryngeal nerves(forming a loop) are prepared carefully. After a me-dian skin incision, the trachea is exposed and cannu-lated. The cannula is connected with a Fleisch-tube(size 00). One femoral artery is cannulated for regis-tration of blood pressure via a Statham pressure trans-ducer. One femoral vein is cannulated for intravenousapplication of test substances. Small hook electrodesare attached to each laryngeal nerve. At the end of aninspiration square wave impulses with a frequency of50 Hz, an impulse width of 0.5 ms, an amplitude of0.2–1.0 Volt, and a duration of 1–10 s are applied every5 min. The intensity of the forced expiration is mea-sured by the Fleisch pneumotachograph and recordedsimultaneously with blood pressure on a polygraph.Prior to the intravenous application of the test com-pound, the response to three stimuli is recorded servingas control. After injection of the test compound or the

D.4 · Effects on Tracheal Cells and Bronchial Mucus Secretionand Transport 555

standard the stimuli are repeated every 5 min. Suppres-sion or diminution of the forced expiration is recordedover 1 h. Then, the next dose or the standard (codeinephosphate 1–2 mg/kg i.v.) is applied.

EVALUATIONTotal or partial suppression of the forced expiration arerecorded over time and expressed as percentage of con-trol. Intensity and duration of the effect are comparedwith the standard.

CRITICAL ASSESSMENT OF THE METHODThe method described by Domenjoz (1952) is veryuseful to detect centrally active antitussive agents likecodeine, but by definition can not determine com-pounds which act on cough receptors in the bronchialarea. Moreover, even light anesthesia influences thecough reflex.

MODIFICATIONS OF THE METHODSeveral other assays have been described which elicitthe cough reflex by central or nerve stimulation. Tonerand Macko (1952) also stimulated the superior laryn-geal nerve in anesthetized cats to induce a definitecough as indicated by rapid contractions of the abdom-inal musculature.

Mattalana and Borison (1955), Chakravarty et al.(1956) used decerebrated cats to study the centraleffects of antitussive drugs on cough and respira-tion. Cough responses were obtained by electricalstimulation of the dorsolateral region of the medullawith bipolar needle electrodes oriented by means ofa stereotactic instrument.

Lindner and Stein (1959) evaluated derivatives ofdiphenyl-piperidono-propan, a series of antitussivedrugs using a modification of the method originally de-scribed by Domenjoz (1952).

Schröder (1951) and Bobb and Ellis (1951) elicitedcough in conscious dogs by stimulation of the va-gus nerve in a surgically prepared skin loop. In anes-thetized cats, coughs were elicited by electrical stimu-lation of the dorsolateral region in the upper medulla(Kasé et al. 1970).

REFERENCES AND FURTHER READINGBobb JRR, Ellis S (1951) Production of cough and its suppres-

sion in the unanesthetized dog. Am J Physiol 167:768–769Braga PC (1989) Experimental models for the study of cough.

In: Braga PC, Allegra L (eds) Cough. Raven Press, Ltd.New York, pp 55–70

Chakravarty NK, Mattalana A, Jensen R, Borison HL (1956)Central effects of antitussive drugs on cough and respira-tion. J Pharm Exp Ther 117:127–135

Domenjoz R (1952) Zur Auswertung hustenstillender Arznei-mittel. Arch exper Path Pharmakol 215:19–24

Kasé Y, Wakita Y, Kito T, Miyata T, Yuizono T, Kataoka M(1970) Centrally-induced coughs in the cat. Life Sci9:49–59

Lindner E, Stein L (1959) Abkömmlinge des Diphenyl-piper-idono-propans – eine neue Reihe hustenstillender Mittel.Arzneim Forsch/Drug Res 9:94–99

Mattalana A, Borison HL (1955) Antitussive agents and cen-trally-induced cough. Fed Proc 14:367–368

Schröder W (1951) Die Verwendung des Vagusschlingenhundesfür die Wertbestimmung hustenstillender Substanzen. ArchExp Path Pharmakol 212:433–439

Toner JJ, Macko E (1952) Pharmacological studies on bis-(1-carbo-β-diethyl-aminoethoxy)-1-phenylcyclopentane)-ethane disulfonate. J Pharm Exp Ther 106:246–251

van Dongen K (1956) The effect of Narcotine, Ticarda andRomilar on coughs and on the movements of the cilia inthe air passages. Acta Physiol Pharmacol Neerl 4:500–507

D.4Effects on Tracheal Cells and BronchialMucus Secretion and Transport

D.4.0.1In Vitro Studies of Mucus Secretion

PURPOSE AND RATIONALEMucus secretion has been studied in isolated tracheasfrom ferrets and dogs (Borson et al. 1980; Kyle et al.1987).

PROCEDUREFerrets of either sex weighing 0.6 to 1.5 kg are anes-thetized with sodium barbital intraperitoneally. Thetrachea is exposed and cannulated with a special Per-spex cannula about 5 mm below the larynx. The animalis then sacrificed with an overdose of the anestheticand the chest is opened along the midline. The tracheais exposed to the carina, cleared of adjacent tissue,removed from the animal and cannulated just abovethe carina. The trachea, with its laryngeal end down,is then mounted in a water-jacketed organ bath andbathed on its submucosal site with Krebs-Henseleit so-lution plus 0.1% glucose at 37°C and bubbled with95% O2 and 5% CO2. The lumen of the trachea re-mains air-filled. A plastic catheter is inserted into thelower cannula to form an airtight seal into which se-cretions can periodically be withdrawn and collected.Volumes of secretions are estimated by the weight dif-ference of catheter lengths with and without secretions.

Simultaneous measurements of both mucus secre-tion and changes in tissue volume in vitro are achievedby mounting portions of ferret trachea cut longitudi-nally along the posterior wall, flattened out and pinnedto a Perspex chamber. Krebs-Henseleit solution at


37°C and gassed with 95% O2 and 5% CO2, is cir-culated on the submucosal side of the tissue, while theluminal side is exposed to the atmosphere. The surfacearea of the exposed tissue is about 50 mm2. Mucus se-cretion is promoted by electrical field stimulation at50–100 V, 20 Hz, 1–2 ms duration, applied through thepins holding the tissue. Before the start of each exper-iment, surface fluid is gently wiped off from the lu-minal surface with a tissue pledglet. The epithelium iscoated with a layer of powdered tantalum dust; as mu-cus secretion from submucosal glands occurs throughgland ducts, the layer of tantalum effectively traps thesecreted mucus above the duct and under the tantalumlayer. Nearly hemispherical hillocks are formed. Thesurface is photographed at intervals through a dissect-ing microscope and hillock diameters are measured.Assuming the hillocks to be hemispheres, the secretionvolume per unit area is calculated. Drugs are added tothe submucosal bath.

EVALUATIONSecretory response after electrical stimulation in thepresence or absence of drugs is recorded after 45, 90,and 135 min.

MODIFICATIONS OF THE METHODQuinton (1979) used isolated tracheae from cats.A segment of the trachea was mounted in a cham-ber such that the serosal side was constantly bathedin Ringer solution, whereas the epithelial surface wascoated with water-saturated paraffin oil. Secretion wasstimulated by adding appropriate drug concentrationsto the bath. Under a dissecting microscope, smalldroplets of secretory fluid were observed to form onthe tracheal epithelial surface shortly after stimula-tion. Timed collection of droplets secreted from threeto four glands were taken up between oil blocks inconstant-bore capillaries (78 µm inner diameter), anddroplet volumes were measured for rate determina-tions usually over a period of 5 min.

CRITICAL ASSESSMENT OF THE METHODSBoth modifications of the in vitro methods need at leastas many animals and are as time consuming as the invivo methods.

REFERENCES AND FURTHER READINGBorson DB, Chinn RA, Davis B, Nadel JA (1980) Adrenergic

and cholinergic nerves mediate fluid secretion from tra-cheal glands of ferrets. J Appl Physiol Respir Environ Ex-ercise Physiol 49:1027–1031

Kyle H, Robinson NP, Widdicombe JG (1987) Mucus secretionby tracheas of ferret and dog. Eur J Resp Dis 70:14–22

Quinton PM (1979) Composition and control of secretions fromtracheal bronchial submucosal glands. Nature 279:551–552

Robinson N, Widdicombe JG, Xie CC (1983a) In vitro collectionof mucus from the ferret trachea. J Phys 340:7P–8P

Robinson N, Widdicombe JG, Xie CC (1983b) In vitro measure-ment of submucosal gland secretion in the ferret tracheaby observation of tantalum dust-coated “hillocks”. J Phys340:8P

Widdicombe JG (1988) Methods for collecting and measur-ing mucus from specific sources. In: Braga PC, Allegra L(eds) Methods in Bronchial Mucology. Raven Press, Ltd.,pp 21–29

D.4.0.2Acute Studies of Mucus Secretion

PURPOSE AND RATIONALEMany diseases of the respiratory tract cause both qual-itative and quantitative changes in the mucus that cov-ers and protects the airway epithelium. To study theinfluence of drugs, methods of collecting bronchialmucus are necessary (Braga 1988). Perry and Boyd(1941) described a method for collecting bronchialmucus from the rabbit.

PROCEDURERabbits weighing 2.5 to 3.5 kg are anesthetized by in-traperitoneal injection of 1.1 to 1.4 g/kg urethane. Thetrachea is exposed by blunt dissection and half opened,2 cm below the cricoid cartilage. One arm of a T can-nula with a large enough diameter to slightly distendthe trachea is inserted into the trachea. The perpendic-ular arm is connected to an air outlet of a humidifier(temperature 35–38°C, relative humidity 80%). Theother arm is connected to a collection tube. The rab-bit is restrained in the supine position on a 60-degreeinclined board with his head downward. Respiratorytract fluids are collected in centrifuge tubes at one hourintervals. Mucus secretion can be stimulated by vagalstimulation or by ammonium chloride given by stom-ach tube or by pilocarpine given i.p.

EVALUATIONTime response curves after stimulants of mucus secre-tion are compared with data from untreated animals.

MODIFICATIONS OF THE METHODA method for collecting mucus from cats, using a seg-ment of cervical trachea about 5 cm long isolated insitu, with nerve and blood supplies intact and a glasscannula inserted to each end, has been described byGallagher et al. (1975).

A method to collect mucus from the upper tract tra-chea and the nasopharynx in dogs in acute experimentshas been proposed by Proctor et al. (1973).


Engler and Szelenyi (1984) described a new methodfor screening mucosecretolytic compounds using tra-cheal phenol red secretion in mice. Phenol red ata dose of 500 mg/kg was injected intraperitoneally tomale mice. Thirty min later, the animals were sacri-ficed by carbon dioxide. The whole trachea was dis-sected free from surrounding tissue and excised. Eachtrachea was washed for 30 min in 1 ml physiologicalsaline. Afterwards, 0.1 ml 1 M NaOH was added to thewashing to stabilize the pH of the lavage fluid. Theconcentration of phenol red was measured photomet-rically. Agonists were administered subcutaneously15 min or intragastrally 30 min before phenol red wasinjected. Antagonists were given 5 min prior to the ad-ministration of agonists.

Other dyes, such as Evans blue or sodium fluores-cein also are reported to be eliminated in the respira-tory tract fluid of mice (Graziani and Cazzulani 1981).

Dye methods reported for mice can also be used forrats (Quevauviller and Vu-Ngoc-Huyen 1966). Alcianblue was used to stain the normal bronchial tree. Af-ter chronic treatment with sulfur dioxide, there werechanges in bronchial coloration. Administration ofdrugs protected against the effects of sulfur dioxide.

Secretion from tracheal submucosal glands can bestudied in dogs (Davis et al. 1982; Johnson and McNee1983, 1985). In anesthetized dogs the epithelial surfaceof the upper trachea is exposed and coated with pow-dered tantalum. Secretions from the submucosal glandducts form elevations (hillocks) in the tantalum layer.The number of hillocks that appear in a 1.2 cm2 fieldis counted.

A micropipette method for obtaining secretionsfrom single submucosal gland ducts in vivo in cattracheas has been described (Ueki et al. 1979, 1980;Leikauf et al. 1984). In anesthetized cats an endotra-cheal tube was inserted into the lower trachea and con-nected to a constant volume respirator. The remainderof the trachea above the endotracheal tube was thendissected open by a midline incision. Paraffin oil equi-librated with HEPES buffer was then placed on the ex-posed mucosa to prevent drying and to aid visualiza-tion of the gland duct openings. The secretions fromthe gland duct openings were collected with constant-bore (99 µm ID) glass micropipettes. The volume andthe viscosity of the secreted mucus were determined.

CRITICAL ASSESSMENT OF THE METHODSFor the methods of mucus collection in rabbits, catsor dogs a rather high number of animals is neces-sary to achieve data suitable for statistical analysis. Forscreening procedures the methods using dye elimina-

tion into the trachea of mice or rats seem to be prefer-able.

REFERENCES AND FURTHER READINGBraga PC (1988) Methods for collecting and measuring airway

mucus in animals. In: Braga PC, Allegra L (eds) Methodsin Bronchial Mucology. Raven Press, Ltd., pp 3–11

Davis B, Chinn R, Gold J, Popovac D, Widdicombe JG, Na-del JA (1982) Hypoxemia reflexly increases secretion fromtracheal submucosal glands in dogs. J Appl Physiol RespEnviron Exercise Physiol 52:1416–1419

Engler H, Szelenyi I (1984) Tracheal phenol red secretion, a newmethod for screening mucosecretolytic compounds. J Phar-macol Meth 11:151–157

Gallagher JT, Kent PW, Passatore M, Phipps RJ, Richardson PS(1975) The composition of tracheal mucus and the nervouscontrol of its secretion in the cat. Proc Roy Soc London192:49–76

Graziani G, Cazzulani P (1981) Su un metodo particolarmenteindicato per lo studio dell’attivita espettorante nei piccolianimali. Farmaco/Ed Pr 36:167–172

Johnson HG, McNee ML (1983) Secretagogue responses ofleukotriene C4,D4: comparison of potency in canine tra-chea in vivo. Prostaglandins 25:237–243

Johnson HG, McNee ML (1985) Adenosine-induced secretionin the canine trachea: Modification by methylxanthines andadenosine derivatives. Br J Pharmacol 86:63–67

Leikauf GD, Ueki IF, Nadel JA (1984) Autonomic regulationof viscoelasticity of cat tracheal gland secretions. J ApplPhysiol Respir Environ Exercise Physiol 56:426–430

Perry WF, Boyd EM (1941) A method for studying expectorantaction in animals by direct measurement of the output ofrespiratory tract fluids. J Pharmacol Exp Ther 73:65–77

Proctor DF, Aharonson EF, Reasor MJ, Bucklen KR (1973)A method for collecting normal respiratory mucus. BullPhysiopath Respir 9:351–358

Quevauviller A, Vu-Ngoc-Huyen (1966) Hypersecretion ex-périmentale du mucus bronchique chez le rat. I. Meth-ode de appreciation anatomopathologique. C R Soc Biol160:1845–1849

Ueki I, German V, Nadel J (1980a) Direct measurement of tra-cheal mucus gland secretion with micropipettes in cats. Ef-fects of cholinergic and α-adrenergic stimulation. Clin Res27:59A

Ueki I, German VF, Nadel JA (1980b) Micropipette measure-ment of airway submucosal gland secretion. Autonomic ef-fects. Am Rev Resp Dis 121:351–357

D.4.0.3Studies of Mucus Secretion With Chronic Cannulation

PURPOSE AND RATIONALESeveral techniques have been developed for chroniccollection of mucus (Wardell et al. 1970; Yankell et al.1970; Scuri et al. 1980).

PROCEDUREBeagle dogs weighing 9–11 kg are anesthetized byintravenous injection of 35–40 mg/kg pentobarbitalsodium. The cervical trachea is exposed by a mid-line skin incision and blunt dissection of the mus-cles. A segment approximately 10 rings in length,


with an intact blood and nerve supply, is transected.The cephalic and caudal parts of the trachea are anas-tomosed end-to-end with interrupted gut sutures toreestablish a patent airway. The isolated segment isloosened slightly from the surrounding tissue andturned 180° to reverse cilia movement. A funnel-shaped silicone cannula is attached to the outer sur-face of the proximal end of the tracheal segment withsurgical mesh and sutured in place. With cannulationcompleted, the tracheal segment is placed in a pocketbelow the sternohyoid muscle and the cannula broughtto the surface and exteriorized through a stab wound.Alternatively, the isolated segment is closed at its cau-dal end with interrupted gut sutures. The mucosal sur-face of the cervical end of the isolated tracheal segmentis sutured with interrupted silk sutures to the overly-ing subcutaneous tissue through a small incision in thecervical skin. Muscles and skin are sutured normally.In two or three weeks the skin heals over the smallstoma resulting in a subcutaneous pouch of function-ing tracheal tissue. Mucus samples can be collected formonths. In this modification, a balloon can be placedinto the pouch. Pressure changes in the balloon dueto contraction of the smooth tracheal muscles afterphysostigmine injection or vagal stimulation or relax-ation after atropine injection are recorded demonstrat-ing parasympathetic innervation.

EVALUATIONParasympathomimetic stimulation (0.5 mg/kg pilo-carpine s.c.) increases the flow rate of tracheal flu-ids. Pressure changes in the balloon after injection ofparasympathomimetic or sympathomimetic drugs arecompared with baseline values.

MODIFICATIONS OF THE METHODScuri et al. (1980) inserted a T-shaped cannula intothe trachea of anesthetized rabbits. The wound wassutured, the third arm was connected with a collect-ing tube, and after 3 days of antibiotic administra-tion, the mucus was collected at different times to es-tablish basal production. For the experiments, mucuswas collected during a 4 h control period, then drugswere given intravenously, orally or as aerosol inhala-tion. Mucus was further collected during the periodsof 0 to 4 and 4 to 24 h and analyzed for sialic acid,fucose, and protein content.

A tracheal pouch method in ferrets has been de-scribed by Barber and Small (1974).

Several authors published methods to determineviscoelastic properties and rheological behaviorof tracheal and bronchial mucus: Philippoff et al.

(1970), Lopez-Vidriero and Das (1977), Martin et al.(1980) Kim et al. (1982), Braga (1988), King (1988),Majima et al. (1990).

CRITICAL ASSESSMENT OF THE METHODSThe methods using pouches in dogs may be usefulfor physiological studies, but for pharmacological pur-poses the rabbit method of Scuri et al. (1980) seemspreferable.

REFERENCES AND FURTHER READINGBarber WH, Smal Jr PAl (1974) Construction of an im-

proved tracheal pouch in the ferret. Am Rev Respir Dis115:165–169

Braga PC (1988) Dynamic methods in viscoelasticity assess-ment. Sinusoidal oscillation method. In: Braga PC, Alle-gra L (eds) Methods in Bronchial Mucology. Raven Press,Ltd. pp 63–71

Kim CS, Berkley BB, Abraham WM, Wanner A (1982) A mi-cro double capillary method for rheological measurementsof lower airway secretions. Bull Eur Physiopath Resp18:915–927

King M (1988) Magnetic microrheometer. In: Braga PC, Alle-gra L (eds) Methods in Bronchial Mucology. Raven Press,Ltd, pp 73–83

Lopez-Vidriero MT, Das I, Reid LM (1977) Airway se-cretion: Source, biochemical and rheological properties.In: Brain JD, Proctor DF, Reid LM (eds) Respiratory De-fense Mechanisms. Part I, Marcel Dekker, Inc., pp 289–356

Majima Y, Hirata K, Takeuchi K, Hattori K, Sakakura Y(1990) Effects of orally administered drugs on dynamicviscoelasticity of human nasal mucus. Am Rev Respir Dis141:79–83

Martin M, Litt M, Marriott (1980) The effect of mucolyticagents on the rheological and transport properties of caninetracheal mucus. Am Rev Resp Dis 121:495–500

Philippoff W, Han CD, Barnett B, Dulfano MJ (1970) A methodfor determining the viscoelastic properties of biological flu-ids. Biorheology 7:55–67

Scuri R, Frova C, Fantini PL, Mondani G, Riboni R, Alfieri C(1980) Un nuovo metodo per lo studio della mucopro-duzione nel coniglio. Boll Chim Farm 119:181–187

Wardell Jr, Chakrin LW, Payne BJ (1970) The canine trachealpouch. A model for use in respiratory mucus research. AmRev Resp Dis 101:741–754

Widdicombe JG (1988) Methods for collecting and measur-ing mucus from specific sources. In: Braga PC, Allegra L(eds) Methods in Bronchial Mucology. Raven Press, Ltd.,pp 21–29

Yankell SL, Marshall R, Kavanagh B, DePalma PD, Resnick B(1970) Tracheal fistula in dogs. J Appl Physiol 28:853–854

D.4.0.4Bronchoalveolar Lavage

PURPOSE AND RATIONALEIsolation of bronchial cells from bronchoalveolarlavage was described by Myrvik et al. (1961), Bas-sett et al. (1988), Fryer et al. (1994, 1997), Wang et al.(1997).


PROCEDUREAfter determination of mechanical respiratory param-eters in anaesthetized guinea pigs, bronchoalveolarlavage is performed via the tracheal cannula. Thelungs are lavaged with 5 aliquots of 10 ml phosphate-buffered saline containing 3 mM EDTA and 100 µMisoproterenol (pH 7.2–7.4). The recovered lavage fluid(40–45 ml) is centrifuged, the cells are resuspended in20 ml of phosphate-buffered saline, and total cells arecounted using a hemacytometer. The remaining aliquotis centrifuged again and cells are stained to determinecell differentials.

EVALUATIONThe differences in cells recovered from bronchoalveo-lar lavage between treatment groups are tested by useof a one-factor analysis of variance. P < 0.05 is consid-ered significant.

MODIFICATIONS OF THE METHODGossart et al. (1996) determined TNF-α activity in thesupernatant of bronchoalveolar lavage by the cytotoxi-city against TNF-α-sensitive L929 murine fibroblasts.

REFERENCES AND FURTHER READINGBassett DJP, Bowen Kelly E, Brewster EL, Elbon CL, Re-

ichenbaugh SS, Bunton T, Kerr JS (1988) A reversiblemodel of acute lung injury based on ozone exposure. Lung166:355–369

Fryer AD, Yarkony KA, Jacoby DB (1994) The effect of leuko-cyte depletion on pulmonary M2 muscarinic receptor func-tion in parainfluenza virus-infected guinea pigs. Br J Phar-macol 112:588–594

Fryer AD, Costello RW, Yost BL, Lobb RR, Tedder TF, Stee-ber DA (1997) Antibody to VLA-4, but not to L-selectin,protects neuronal M2 muscarinic receptors in antigen-chal-lenged guinea pig airways. J Clin Invest 99:2036–2044

Gossart S, Cambon C, Orfila C, Séguélas MH, Lepert JC,Rami J, Carré P, Pipy B (1996) Reactive oxygen intermedi-ates as regulators of TNF-α production in rat lung inducedby silica. J Immunol 156:1540–1548

Myrvik QN, Leake ES, Fariss B (1961) Studies on pulmonaryalveolar macrophages from the normal rabbit: A techniqueto produce them in a high state of purity. J Immunol86:128–132

Wang S, Lantz RC, Rider RD, Chen GJ, Breceda V, Hays AM,Robledo RF, Tollinger BJ, Dinesh SVR, Witten ML (1996)A free radical scavenger (Lazaroid U75412E) attenuatestumor necrosis factor-alpha generation in a rabbit smoke-induced lung injury. Respiration 64:358–363

D.4.0.5Ciliary Activity

PURPOSE AND RATIONALECiliary activity is a natural defense mechanism of themucosa in the respiratory tract against harmful extra-neous agents resulting in a continuous transportation

of secreted mucus. Many attempts have been made tovisualize and to quantify this phenomenon. Several au-thors used the light beam reflex method which can beused for in vivo as well as for in vitro experiments(Dalhamn 1956, 1964; Dalhamn and Rylander 1962;Hakansson and Toremalm 1963; Mercke et al. 1974;Baldetorp et al. 1976; Lopez-Vidriero et al. 1985).

PROCEDURERats are anesthetized by intraperitoneal injection of tri-bromo-ethanol (Avertin). The trachea is exposed andits soft parts are incised by electrocoagulation so thatbleeding is avoided. The cartilaginous rings are openedsufficiently to permit microscopy. The rat is immedi-ately placed in a moist chamber. The trachea openingis linked to a microscope (Leitz Ultropak) by meansof a rubber bellows which is fitted around the lens ofthe microscope and is made to embrace the trachea bymeans of a piece of rubber tubing that is slit along itslength and secured to the bellows. The beam of an il-luminating lamp is concentrated to a surface of aboutone mm2. By placing a heat-reflecting filter in the pathof the beam the rise in temperature can be reduced.For registration of the reflected light high speed cam-eras with a speed of 220 exposures are used. Alter-natively, the reflected light from the microscope is di-rected to a TV camera and amplified to be displayed ona TV screen. The frequency of ciliar beats is recordedover one hour.

EVALUATIONThe beat frequency of treated animals is comparedwith that of controls.

MODIFICATIONS OF THE METHODMercke et al. (1974) described a stroboscopic methodfor standardized studies of mucociliary activity in rab-bit tracheal mucosa.

Lierle and Moore (1935) inserted windows intoanesthetized rabbits for observation of the ciliary ac-tivity in the maxillary sinus of living animals.

With modern equipment, a similar technique hasbeen used by Hybbinette and Mercke (1982a–c), Lind-berg and Mercke (1986), Lindberg et al. (1986), Mer-cke et al. (1987) to study the role of several pharma-cologic agents in the mucociliary defense of the rabbitmaxillary sinus.

Corssen and Allen (1958) compared the toxic ef-fects of various local anesthetic drugs on human cil-iated epithelium in vitro by observation of rotatingglobes of human tracheal epithelium in tissue culture.


Cheung (1976) performed high speed cinemicro-graphic studies on rabbit tracheal (ciliated) epithelia.

Iravani (1967, 1971, 1975) studied the ciliary activ-ity in the intrapulmonary airways of rats by incidentlight microscopy.

Lee and Verdugo (1976), Verdugo et al. (1980) rec-ommended laser light-scattering spectroscopy for thestudy of ciliary activity.

Manawadu et al. (1978) studied the effects of lo-cal anesthetics on ciliary activity using ferret trachealrings in vitro. The ferret possesses a long neck, and100 or more tracheal rings can be obtained from a sin-gle ferret. The tracheal rings were maintained in ster-ile tubes. The ciliary activity was graded by determin-ing the percentage of cilia beating on each ring, usingtransmitted light and the 10 × objective of an invertedmicroscope.

Rutland and Cole (1980) and Hesse et al. (1981)used a non-invasive method for obtaining nasal cili-ated epithelium which is suitable for measurement ofciliary beat frequency.

Van de Donk et al. (1980) used isolated chicken em-bryo tracheas to measure the effects of preservativeson ciliary beat frequency. Maurer et al. (1982) stud-ied the role of ciliary motility in acute allergic mu-cociliary dysfunction in cultivated ciliated cells fromsheep.

Lopez-Vidriero et al. (1985) studied the effect ofisoprenaline on the ciliary activity of an in vitro prepa-ration of rat trachea.

Braga et al. (1986) described a simple and precisemethod for counting ciliary beats directly from the TVmonitor screen using specimens of human ciliated ep-ithelium obtained by brushing the nasal mucosa.

Curtis and Carson (1992) used pieces of humannasal epithelium for computer-assisted video measure-ment of ciliary beat frequency in vitro. Ciliary beat fre-quency was viewed with a microscope equipped witha phase contrast objective. The microscopic image wasrecorded by a camera and data stored by a video-recorder. For measuring ciliary beating, tapes were dis-played with amplification on a monitor. A photoelec-tric transducer was positioned over the video image ofthe cilia. Movement of the cilia interrupting the lightpath caused changes in light intensity recorded by thephotocell transducer.

REFERENCES AND FURTHER READINGBaldetorp L, Huberman D, Håkanssson CH, Toremalm NG

(1976) Effects of ionizing radiation on the activity of theciliated epithelium of the trachea. Acta Radiol Ther PhysBiol 13:225–232

Braga PC, Dall’Oglio G, Bossi R, Allegra L (1986) Simple andprecise method for counting ciliary beats directly from theTV monitor screen. J Pharmacol Meth 16:161–169

Cheung ATW (1976) High speed cinemicrographic studies onrabbit tracheal (ciliated) epithelia: Determination of thebeat pattern of tracheal cilia. Pediat Res 10:140–144

Corssen G, Allen CR (1958) A comparison of the toxic effects ofvarious local anesthetic drugs on human ciliated epitheliumin vitro. Texas Rep Biol Med 16:194–202

Curtis LN, Carson JL (1992) Computer-assisted video measure-ment of inhibition of ciliary beat frequency of human nasalepithelium in vitro by xylometazoline. J Pharm ToxicolMeth 28:1–7

Dalhamn T (1956) Mucous flow and ciliary activity in the tra-chea of healthy rats and rats exposed to respiratory irri-tant gases (SO2, H3N, HCHO). A functional and morpho-logic (light microscopic and electron microscopic) study,with special reference to technique. Acta Physiol Scand 36,Suppl 123:1–161

Dalhamn T (1964) Studies on tracheal ciliary activity. Am RevRespir Dis 89:870–877

Dalhamn T, Rylander R (1962) Frequency of ciliary beat mea-sured with a photo-sensitive cell. Nature 196:592–593

Hakansson CH, Toremalm NG (1963) Studies on the physiol-ogy of the trachea. I. Ciliary activity indirectly recorded bya new “light beam reflex” method. Ann Otol 74:954–969

Hesse H, Kasparek R, Mizera W, Unterholzner Ch, Konietzko N(1981) Influence of reproterol on ciliary beat frequency ofhuman bronchial epithelium in vitro. Arzneim Forsch/DrugRes 31:716–718

Hybbinette JC, Mercke U (1982a) A method for evaluating theeffect of pharmacological substances on mucociliary activ-ity in vivo. Acta Otolaryngol 93:151–159

Hybbinette JC, Mercke U (1982b) Effects of the parasympath-omimetic drug methacholine and its antagonist atropine onmucociliary activity. Acta Otolaryngol 93:465–473

Hybbinette JC, Mercke U (1982c) Effects of sympathomimeticagonists and antagonists on mucociliary activity. Acta Oto-laryngol 94:121–130

Iravani J (1967) Flimmerbewegung in den intrapulmonalenLuftwegen der Ratte. Pflügers Arch 207:221–237

Iravani J (1971) Physiologie und Pathophysiologie der Cilien-tätigkeit und des Schleimtransports im Tracheobron-chialbaum. (Untersuchungen an Ratten). Pneumonologie144:93–112

Iravani J, Melville GN (1975) Mucociliary activity in the respi-ratory tract as influenced by prostaglandin E1. Respiration32:305–315

Lee WI, Verdugo P (1976) Laser light-scattering spectroscopy.A new application in the study of ciliary activity. Biophys J16:1115–1119

Lierle DM, Moore PM (1935) Further study of the effects ofdrugs on ciliary activity: a new method of observation inthe living animal. Ann Otol 44:671–684

Lindberg S, Mercke U (1986) Bradykinin accelerates mucocil-iary activity in rabbit maxillary sinus. Acta Otolaryngol(Stockh) 101:114–121

Lindberg S, Hybbinette JC, Mercke U (1986) Effects of neu-ropeptides on mucociliary activity. Ann Otol Rhinol Laryn-gol 95:94–100

Lopez-Vidriero MT, Jacobs M, Clarke SW (1985) The effect ofisoprenaline on the ciliary activity of an in vitro preparationof rat trachea. Eur J Pharmacol 112:429–432

Manawadu BR, Mostow SR, LaForce FM (1978) Local anes-thetics and tracheal ring ciliary activity. Anesth Analg57:448–452


Maurer DR, Sielczak M, Oliver Jr W, Abraham WM, Wanner A(1982) Role of ciliary motility in acute allergic mucociliarydysfunction. J Appl Physiol 52:1018–1023

Mercke U, Håkanson CH, Toremalm NG (1974) A method forstandardized studies of mucociliary activity. Acta otolaryng78:118–123

Mercke U, Lindbergh S, Dolata J (1987) The role of neu-rokinin A and calcitonin-related peptide in the mucociliarydefense of the rabbit maxillary sinus. Rhinology 25:89–93

Rutland J, Cole PJ (1980) Non-invasive sampling of nasal ciliafor measurement of beat frequency and study of ultrastruc-ture. Lancet ii, 564–565

Suzuki N (1966) Motor control of the ciliary activity in the frog’spalate. J Faculty Sci, Hokkaido Univ Ser VI, 16:67–71

Van de Donk HJM, Muller-Platema IP, Zuidema J, MerkusFWHM (1980) The effects of preservatives on the cil-iary beat frequency of chicken embryo tracheas. Rhinology18:119–133

Verdugo P, Johnson NT, Tam PY (1980) β-adrenergic stim-ulation of respiratory ciliary activity. J Appl Physiol48:868–871

D.4.0.6Studies of Mucociliary Transport

PURPOSE AND RATIONALEMucus flow has been studied in in vitro and in vivoexperiments (Iravani 1971; Ahmed et al. 1979). Therate of mucus flow can be estimated by measuring thetime needed for certain particles to travel a knowndistance in the trachea. Numerous substances havebeen used such as charcoal particles (Dalhamn 1956),Teflon discs (Ahmed et al. 1979), and other pulverizedmaterials (Deitmer 1989). The effect of local radioac-tivity on tracheal mucous velocity of sheep has beenstudied in vitro and in vivo by Ahmed et al. (1979).

PROCEDUREFor in vitro experiments, sheep are sacrificed duringanesthesia, the trachea is exposed, clamped, and re-sected just below the cricoid cartilage. The chest isopened and the trachea is resected at the level of thecarina. Then the trachea is slit open along the poste-rior membranous wall, pinned with gentle stretchingon a board, and slanted upward at an angle of 25°.A metric ruler is placed along the board as a mea-suring reference. The board is then placed in a Plexi-glas chamber with a constant temperature of 37°C and100% humidity. Teflon discs are spread on the mucouslayer. Tracheal mucus velocity is estimated by filmingthe movements of the radioopaque Teflon discs visu-alized on a television monitor connected to a camera.Disc motion is recorded for 1–2 min on a videotape,and the distance measured during the elapsed time isobtained from the videomonitor.

For in vivo measurements of tracheal mucus veloc-ity, the roentgenographic method of Friedman et al.

(1977), Sackner et al. (1977) is used. The sheep are re-strained, and their heads are immobilized with a sling.The nasal mucosa is sprayed with a 2% lidocaine solu-tion for topical anesthesia. A bronchofiberscope is in-serted transnasally, and its tip is placed just below thevocal cords. Radioopaque Teflon discs 1.0 mm in di-ameter, 0.8 mm thick, and weighing 1,76 mg are blownthrough the inner channel of the bronchofiberscopeonto the tracheal mucosa in a circumferential distribu-tion. The cervical trachea containing the discs is visu-alized in the lateral projection with a television moni-tor. Disc motion is recorded on a videotape while thetime is displayed on a digital clock. The disc image ismarked to obtain the distance traveled. This distanceis measured with a ruler, and the linear velocity ofthe disc is computed by dividing the distance by theelapsed time. This procedure is repeated for all discs inthe field of view. To compute a mean tracheal mucusvelocity, data from 10–15 discs are obtained in eachfilmed run.

EVALUATIONDisc velocities measured in vitro or in vitro are com-pared before and after treatment.

MODIFICATIONS OF THE METHODMucociliary transport has been studied on the hardpalate of decapitated frogs measuring the transport ve-locity of small particles, e. g., pieces of cork or char-coal (Kochmann 1930; Sadé et al. 1970).

Suzuki (1966) measured the movement of a stan-dard object (1 mm2 aluminum foil) on the ciliated sur-face of the palate of frogs.

Mucociliary clearance by the in vitro frog methodwas used by Leitch et al. (1985) to study the effects ofethanol.

Movement of poppy seeds in rabbit tracheal prepa-rations was studied by Kensler and Battista (1966).

In chicken nasal mucosa the interaction betweenmucociliary transport and the ciliary beat was studiedby Ukai et al. (1985).

Mucus transport in the respiratory tract of anes-thetized cats was measured with uniform particles oflycopodium spores triturated with lamp black (Carsonet al. 1966).

Mucociliary clearance velocities were determinedby a radioisotopic method in dogs (Giordano et al.1977, 1978).

REFERENCES AND FURTHER READINGAhmed T, Januskiewicz AJ, Landa JF, Brown A, Chapman GA,

Kenny PJ, Finn RD, Bondick J, Sackner MA (1979) Effect


of local radioactivity on tracheal mucous velocity of sheep.Am Rev Resp Dis 120:567–575

Battista SP (1971) Agents affecting mucociliary activity.In: Turner RA, Hebborn P (eds) Screening Methods inPharmacology, Vol II. Academic Press, New York and Lon-don. pp 167–202

Carson S, Goldhamer R, Carpenter R (1966) Mucus transport inthe respiratory tract. Am Rev Resp Dis 93:86–92

Dalhamn T (1956) Mucous flow and ciliary activity in the tra-chea of healthy rats and rats exposed to respiratory irri-tant gases (SO2, H3N, HCHO). A functional and morpho-logic (light microscopic and electron microscopic) study,with special reference to technique. Acta Physiol Scand 36,Suppl 123:1–161

Deitmer Th (1989) Physiology and pathology of the mucocil-iary system. Special regards to mucociliary transport in ma-lignant lesions of the human larynx. Karger Basel, Chap-ter 5: Methods of investigation of mucociliary transport,pp 26–34, Chapter 9: Pathophysiology and pharmacologyof the mucociliary system, pp 47–54

Friedman M, Stott FD, Poole DO, Dougherty R, Chapman GA,Watson H, Sackner MA (1977) A new roentgenographicmethod for estimating mucus velocity in airways. Am RevRespir Dis 115:67–72

Giordano AM, Shih CK, Holsclaw DS, Khan MA, Litt M (1977)Mucus clearance: in vivo canine tracheal vs. in vitro bull-frog palate studies. J Appl Physiol 42:761–766

Giordano AM, Holsclaw D, Litt M (1978) Mucus rheology andmucociliary clearance: normal physiologic state. Am RevResp Dis 118:245–250

Iravani J (1971) Physiologie und Pathophysiologie der Cilien-tätigkeit und des Schleimtransports im Tracheobronchial-baum. (Untersuchungen an Ratten). Pneumonologie144:93–112

Kensler CJ, Battista SP (1966) Chemical and physical factorsaffecting mammalian ciliary activity. Am Rev Resp Dis93:93–102

Kochmann M (1930) Zur Pharmakologie der Expektorantien.Wirkung auf die Flimmerbewegung. Naunyn-Schmiede-berg’s Arch Exp Path Pharmakol 150:23–38

Leitch GJ, Frid LH, Phoenix D (1985) Effects of ethanol on mu-cociliary clearance. Alcoholism Clin Exp Res 9:277–280

Sackner MA, Reinhart M, Arkin B (1977) Effects of be-clomethasone diproprionate on tracheal mucus velocity.Am Rev Resp Dis 115:1069–1070

Sadé J, Eliezer N, Silberberg A, Nevo AC (1970) The role ofmucus in transport by cilia. Am Rev Respir Dis 102:48–52

Ukai K, Sakakura Y, Saida S (1985) Interaction between mu-cociliary transport and the ciliary beat of chicken nasal mu-cosa. Arch otorhinolaryngol 242:225–231

D.4.0.7Culture of Tracheal Epithelial Cells

PURPOSE AND RATIONALEMarked morphological changes of the airway epithe-lium, up to severe damage, are frequently observed ininflammatory airway diseases and appear to play animportant role in the pathogenesis of the broncho-ob-structive symptoms (Webber and Corfield 1993; Hayet al. 1994). Freitag et al. (1996) studied the effects oflipopolysaccharides (LPS) and TNF-α on cultured rattracheal epithelial cells and determined NO synthaseactivity.

PROCEDURETrachea of newborn rats, cut into small pieces, areexplanted with the epithelial surface downwards onto60 mm culture dishes (Lechner et al. 1985) andcultured in low-calcium (60–80 µmol/l) RPMI-1640medium at 37°C and 5% CO2. The medium con-taining 16% fetal calf serum is supplemented withepidermal growth factor and other growth promot-ing factors, such as 80 ng/ml cholera toxin, 2.6 ng/mlestradiol, 180 ng/ml hydrocortisone, 2.5 µg/ml in-sulin, 12.5 µg/ml transferrin, and 100 U/ml penicillin,100 µg/ml streptomycin and 3.5 µg/ml amphotericin Bto allow a selective outgrow of epithelial cells (Emuraet al. 1990). Confluent epithelial layers are obtainedafter about 6–10 weeks at which time all cells showa positive staining with a pancytokeratin-antibody.Confluent cells of following passages are treated forfour subsequent days with LPS (10 µg/ml) or TNF-α(500 U/ml).

The morphology of the cells is evaluated is evalu-ated by daily inspection using a phase contrast micro-scope with photographical documentation. Cell den-sity in the culture is determined in each culture dish bycounting daily the number of cells in four marked ar-eas (each 2000 µm2). For immunocytochemical stain-ing, the cells are fixed by incubation in 100% methanolat –20°C for 20 min. The cells are washed with Ca/Mg-free phosphate buffered saline and several areas aremarked of by nail-varnish. A solution of the anti-cytokeratin(pan)-antibody (monoclonal mouse IgG,Boehringer Mannheim) is added, incubated for 2 hat room temperature and after washing incubated for30 min with a secondary antibody (FITC coupled,polyclonal rabbit anti-mouse IgG, Sigma). After wash-ing, fluorescence microscopy is performed using a mi-croscope with a FITC-specific filter combination. Un-specific fluorescence is excluded by performing thestaining procedure in a different area of the same cul-ture, but without the addition of the primary antibody.

For determination of NO synthase activity, con-fluent cultures are washed with oxygenated and pre-warmed (37°C) Krebs-HEPES medium. Then the cellsare incubated for 1 h in the same medium contain-ing 37 kBq 3H-L-arginine (100 nmol/l). After collec-tion of the supernatants the cells are extracted in 1 mlof 0.4 mol/l HClO4 for 2 h at 0–4°C.

By HPLC analysis 3H-compounds (3H-L-ci-trulline, 3H-L-ornithine and 3H-L-arginine) in incuba-tion media and cell extracts are separated on a reversephase column (length 250 mm, inner diameter 4.6 mm,pre-packed with Shadon ODS-Hypersil, 5 mm) usingas mobile phase 0.1 mmol/l sodium phosphate buffer


(adjusted to pH 1.8) which contains octane sulphonicacid sodium salt (400 mg/l), Na2EDTA (0.3 mmol/l)and methanol (6.25% v/v) with a flow rate of 1 ml/min(Hey et al. 1995). The eluate is collected in 1 minfractions into counting vials. After addition of a com-mercial scintillation cocktail the radioactivity is deter-mined by liquid scintillation spectrophotometry. Ex-ternal standardization is used to correct for countingefficiency. The retention time is determined by theuse of 14C-labelled (L-citrulline or L-ornithine) or 3H-labelled (L-arginine) standards. Protein content in cellsis determined by a commercially available assay.

EVALUATIONThe amounts of 3H-L-citrulline in supernatants or cellextracts are expressed as DPM/µg protein. Changes incell density are expressed as % of the density observedin each culture dish at the start of the experiment. Meanvalues are given ± SEM. The significance of differ-ence is evaluated by Student’s t-test.

REFERENCES AND FURTHER READINGEmura M, Riebe M, Ochiai M, Aufderheide M, Germann P,

Mohr U (1990) New functional cell-culture approach topulmonary carcinogenesis and toxicology. Cancer Res ClinOncol 116:557–562

Freitag A, Reimann A, Wessler I, Racké K (1996) Effect of bac-terial lipopolysaccharides (LPS) and tumor necrosis factor-α (TNF-α) on rat tracheal epithelial cells in culture: mor-phology, proliferation and induction of nitric oxide (NO)synthase. Pulmon Pharmacol 9:149–156

Hay DWP, Farmer SG, Goldie GR (1994) Inflammatory medi-ators and modulation of epithelial/smooth muscle interac-tions. In: Goldie RG (ed) Handbook of Immunopharma-cology: Immunopharmacology of Epithelial Barriers. Aca-demic Press, London, pp 119–146

Hey C, Wessler I, Racké K (1995) Nitric oxide (NO) synthaseis inducible in rat, but not in rabbit alveolar macrophages,with a concomitant reduction in arginase activity. NaunynSchmiedeberg’s Arch Pharmacol 351:651–659

Lechner JF, LaVeck MAA (1985) A serum-free method for cul-turing normal bronchial cells. J Tissue Cult Meth 9:43–48

Webber SE, Corfield DR (1993) The pathophysiology of air-way inflammation and mucosal damage in asthma. In: An-drews P, Widdicombe J (eds) Pathophysiology of the Gutand Airways. Portland Press, London, pp 67–77

D.4.0.8Alveolar Macrophages

PURPOSE AND RATIONALEAlveolar macrophages have been used for variouspurposes. A rat pulmonary alveolar macrophage cellline (NR8383) was initiated in culture by Helmkeet al. (1987) in the presence of a gerbil lung con-ditioned medium and has been propagated continu-ously in culture. Sun et al. (1999) tested the inhibition

of Ca2+ influx by pentoxifylline in NR8383 alveolarmacrophages.

PROCEDURECell CultureThe NR8383 alveolar macrophages cell line is grownin plastic tissue culture flasks in Ham’s F12 mediumcontaining 15% fetal bovine serum, 100 g/ml peni-cillin and 100 U/ml streptomycin sulfate. The alveolarmacrophages are cultured at 37°C in an atmosphere of5% CO2 in air. The medium is routinely changed twiceweekly. The viability of alveolar macrophages is rou-tinely measured by trypan blue exclusion before and at1.5 h after fura-2 loading, when all [Ca2+]i determina-tions are completed.

Determination of [Ca2+]i[Ca2+]i is determined using the Ca2+-sensitive fluores-cent indicator fura-2 (Zhang et al. 1997; Mörk et al.1998). NR8383 alveolar macrophages are loaded withfura-2 by incubation in PSS containing 2 mM (fi-nal concentration) fura-2/AM and 0.01% BSA for 20min at 37°C. The cells are then rinsed twice withPSS containing 0.01% BSA and resuspended in thesame medium (1.75 × 106 cells/ml). For [Ca2+]i deter-mination, a 2-ml aliquot of fura-2-loaded cells is cen-trifuged at 50 g for 2 min, resuspended in PSS contain-ing 0.01% BSA and placed in a 4-ml cuvette. [Ca2+]iis measured at 37°C using a PTI Deltascan fluorome-ter (PTI, South Brunswick, N.J., USA). The excitationwavelengths used are 340 and 380 nm and the emis-sion wavelength is 505 nm. Calibration of [Ca2+]i isperformed for each measurement trace. Then, 1 mMCaCl2 (for Ca2+-free medium) and 50 mM ionomycinare added to obtain the limiting ratio for the Ca2+-saturated form (Rmax) of fura-2. Then, 0.0005% digi-tonin and 10 mM EGTA are added to obtain the lim-iting ratio for the free form of fura-2 (Rmin. Fluores-cence ratios of the 340- and 380-nm excitation and505-nm emission are converted to [Ca2+]i accordingto Grynkiewicz et al. (1985) using 224 nM as the Kdof fura-2 for Ca2+.

Measurement of Store-Depletion-Activated Ca2+ InfluxThe Ca2+ influx activated by depletion of the inositol1,4,5-trisphosphate- (IP3-) sensitive intracellular storeis measured according to Zhang et al. (1997) and Zhuet al. (1998). Cells are exposed to either ATP or thap-sigargin (TG) in Ca2+-free medium for 5 min, and 1mM Ca2+ is then added to initiate Ca2+ influx. The ini-tial linear portion of [Ca2+]i changes after addition ofCa2+ is used to calculate Ca2+ influx rate (nM/min).


EVALUATIONResults are presented as the mean± SEM of separatedeterminations using different cell preparations. Com-parisons are made using the unpaired Student’s t-testor the analysis of variance. P values <0.05 are consid-ered significant.

MODIFICATIONS OF THE METHODSirois et al. (2000) studied the influence of histamine inthe cytokine network in the lung through H2 and H3 re-ceptors. Alveolar macrophages from humans, SpragueDawley rats and the alveolar macrophage cell lineNR8383 were treated with different concentrations ofhistamine prior to their stimulation with suboptimalconcentrations of lipopolysaccharide (LPS). Releaseof tumor necrosis factor (TNF) and interleukin-10 (IL-10) was measured.

Using the rat alveolar macrophage cell lineNR8383, Gazin et al. (2004) found that uranium in-duces TNF-α secretion and activates the p38 mitogen-activated protein kinase (p38 MAPK).

Yang et al. (2004) studied in mice the synergy be-tween a signal transducer and activator of transcription3 and retinoic acid receptor-α in the regulation of thesurfactant protein B gene in the lung.

REFERENCES AND FURTHER READINGGazin V, Kerdine S, Grillon G, Pallardy M, Raoul H (2004)

Uranium induces TNFα secretion and MAPK activation inrat alveolar macrophage cell line. Toxicol Appl Pharmacol194:49–59

Grynkiewicz G, Poenie M, Tsien RY (1985) A new generationof Ca2+ indicators with improved fluorescence properties.J Biol Chem 260:3440–3450

Helmke RJ, Boyd RL, German VF, Mangos JA (1987) Fromgrowth factor dependence to growth factor responsiveness:the genesis of an alveolar macrophage cell line. In VitroCell Dev Biol 23:567–574

Mörk AC, Helmke RJ, Martinez JR, Michalek MT, Patchen MI,Zhang GH (1998) Effects of particulate and soluble(1–3)-α-glycans on Ca2+ influx in NR8383 alveolarmacrophages. Immunopharmacology 40:77–89

Sirois J, Ménard G, Moses AS, Bissonette EY (2000) Impor-tance of histamine in the cytokine network in the lungthrough H2 and H3 receptors: stimulation of IL-10 produc-tion. J Immunol 164:2964–2970

Sun X, Martinez JR, Zhang GH (1999) Inhibition of Ca2+ in-flux by pentoxifylline in NR8383 alveolar macrophages.Immunopharmacology 43:47–58

Yang L, Lian X, Cowen A, Xu H, Du H, Yan C (2004) Syn-ergy between signal transducer and activator of transcrip-tion 3 and retinoic acid receptor-α in the regulation ofthe surfactant protein B gene in the lung. Mol Endocrinol18:1520–1532

Zhang GH, Helmke RJ, Mörk AC, Martinez RJ (1997) Regu-lation of cytosolic free Ca2+ in cultured rat macrophages(NR8383). J Leukoc Biol 62:341–348

Zhu X, Birnbaumer L (1998) Calcium channels formed by mam-malian Trp homologues. News Physiol Sci 13:211–217

D.5Safety Pharmacologyof the Respiratory System

See Murphy (2006).

REFERENCES AND FURTHER READINGMurphy DJ (2006) Respiratory function assays in safety phar-

macology In: Drug Discovery and Evaluation Safety andPharmacokinetic Assays, Chapter I.F, pp 141–149 H.G. Vogel (ed), F.J. Hock, J. Maas, D. Mayer (Co-eds)Springer-Verlag Berlin Heidelberg New York

drug discovery and evaluation || respiratory activity

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