A Model of Tobacco Smoke-Induced Airflow Obstruction in the Guinea Pig

A Model of Tobacco SmokeInduced Airflow Obstruction in the Guinea Pig* Joanne L. Wright, MD; and Andrew Churg, MD

Animal models have proven to be extremely worthwhile in the investigations of cigarette smokeinduced lung disease. We have found the guinea pig to be useful, with a large number of advantages and few disadvantages when used in our experimental circumstances. Other animal models exist, however, and the investigator should select the model that is most suited to the hypothesis to be tested. (CHEST 2002; 121:188S–191S) Key words: animal models; cigarette smoke; COPD; emphysema; guinea pig; pulmonary hypertension; species differences

there are many potential causes of airflow A lthough obstruction, this article will be restricted to discussions of airflow obstruction that is related to cigarette smoke, hereafter termed COPD. Animal experimentation has been crucial to our understanding of the mechanisms of COPD, and both large and small animals have been utilized. Although the guinea pig has long been recognized as a very useful model in investigations related to asthma, it is only in the last few years that it has been used extensively in research related to COPD. Investigations have centered on the lung parenchyma, most particularly examining for evidence of emphysematous destruction, although some studies have been directed specifically toward the effects of smoke on the airways. It is appropriate to note that the definition of emphysema in animal models is slightly different from that used in humans. In animals, emphysema is defined as follows: “. . . an abnormal state of the lungs in which there is enlargement of the airspaces distal to the terminal bronchiole. Airspace enlargement should be determined qualitatively in appropriate preparations and quantitatively by stereologic methods. Physiologic descriptions may or may not be used, depending on the purposes of the investigations, but in view of the great variability of the pathophysiology of human emphysema, it is not possible to establish specific physiologic requirements for animal models of emphysema.”1 Many investigators have used both morphologic and physiologic modalities and have thus provided additional information in the form of pathophysiologic correlations. *From the Department of Pathology, University of British Columbia, Vancouver, BC, Canada. This research was supported by grants from the Medical Research Council/Canadian Institutes of Health Research and the Heart and Stroke Foundation of British Columbia and Yukon. Correspondence to: Joanne L. Wright, MD, Department of Pathology, University of British Columbia, 2211 Westbrook Mall, Vancouver, BC, V6T 2B5 Canada; e-mail: jlwright@ interchange.ubc.ca 188S

Description of a Guinea Pig Model The value of using guinea pigs in investigations of the effects of cigarette smoke on the lung was promulgated by the description of a smoking apparatus designed specifically for guinea pigs,2 and its extensive use by the laboratory of Hogg and colleagues.3–5 These workers demonstrated that cigarette smoke induced increased vascular permeability, which appeared to be associated with neutrophil migration.6,7 More recently, there is evidence, again in the guinea pig, that increased microvascular permeability8 may be important in increased airways responsiveness,9 and that this may be mediated by peroxynitrite10,11 or superoxide.12 In addition, cigarette smoke appears to be able to inactivate the protective substance, airway neutral endopeptidase.13 In our laboratory, we selected the guinea pig as a model in which to investigate the effects of acute and chronic exposure to cigarette smoke on lung structure and function. After a short-term (10 min) exposure to smoke, there was airflow obstruction with air trapping, which was associated with neutrophilia in the peripheral blood and in the lung lavage fluid.14 Chronic exposure to cigarette smoke for periods of greater than 3 months was shown to produce emphysematous airspace enlargement.15 The increase appeared to be progressive with increasing lengths of exposure, with an increasing ratio to the measurements in the control animals. The mean chord length of alveoli and alveolar ducts were both increased, and this can be appreciated histologically in Fig 1. We found that the increased airspace size was associated with gas trapping and increased total lung volumes, and with a shift of both the pressure-volume and flow-volume curves.15 A cessation of smoke exposure appeared to halt, but not reverse, emphysematous lung enlargement.16 The protease-antiprotease paradigm suggests that inflammatory cells are responsible for an increase in the protease levels. Selman and colleagues17 found an increase in collagenolytic activity after 4 weeks of daily exposure to smoke from 20 cigarettes, and they identified collagenase messenger RNA expression in macrophages, epithelial cells, and interstitial cells. In our morphometric study of guinea pigs that had been exposed to the smoke of five cigarettes per day,18 we found decreased amounts of alveolar collagen after 3 months of exposure, but an increased amount after 6 months. Thus, although there is initial collagen destruction, there also appears to be a collagen-producing repair reaction. Our results therefore reiterate the paradox initially described in humans by Cardoso et al19 If the lung is destroyed, it may also be important to determine whether the destruction occurs randomly or occurs in a systematic fashion. Early workers suggested that the pores of Kohn, a normal anatomic feature responsible for collateral ventilation, were a site of destruction.20 To examine this hypothesis, we examined the lungs of guinea pigs that had been exposed to smoke for 12 months, and we found an increased number of alveolar holes, with a shift toward larger and more irregular hole configurations. These data imply that lung destruction COPD Symposium: Into the New Millennium

Figure 1. Left: photomicrograph taken of a control, sham-exposed animal after 12 months of smoke exposure. Right: photomicrograph taken of an animal exposed to cigarette smoke for 12 months. Note the enlarged alveolar ducts and alveoli compared to those seen in the left panel (hematoxylin-eosin, original ⫻16).

results from the formation of multiple holes rather than from a simple enlargement of existing pores of Kohn.21 In humans, there is goblet cell metaplasia of the airway epithelium of the membranous bronchioles of subjects who smoke cigarettes.22 In animal models, the correlate is secretory cell metaplasia, which is detected by histochemical staining against neutral and acid mucins. Guinea pigs exposed to smoke for periods of ⱖ 3 months develop secretory cell metaplasia.23 We presume that this finding is indicative of a response to chronic irritation. We also have used the guinea pig model to examine the effect of cigarette smoke on the pulmonary vasculature. In humans, a significant number of chronic smokers develop pulmonary hypertension,24 and this appears also to be true in the guinea pig.25 Interestingly, hypertension in the guinea pig does not appear to be due to capillary bed destruction.26,27 Long-term smoke exposure produces an increase in the percentage of muscularized small arteries and is associated with an increased level of cell proliferation in both muscular and partially muscularized vessels.28 However, there also appears to be a dynamic alteration of both airways and vessels, in that those animals with an increased pulmonary arterial pressure had greater airflow obstruction than those without.29 This would suggest that smoke alters the intrinsic tone of the muscle, in addition to altering the actual muscular structure.

Comparison of Guinea Pig to Rat or Mouse Models of Smoking-Induced Lung Disease Several other animal species have been exposed to smoke, usually in a short-term fashion, although some studies have lasted for months. Several studies have examined the effects of short-term or long-term smoke exposure on rats. Early studies by Huber et al30 and Heckman and Dalbey31 exposed animals to smoke for 6 and up to 30 months, respectively. Both groups, using an exposure level of 10 cigarettes per day, found a small, but significant, increase in airspace volume proportion. Saline solution pressure-volume curves demonstrated a loss of elastic recoil.30 Interestingly, Heckman and Dalbey31 noted that the volume proportion of airspace in the www.chestjournal.org

smoking group was not significantly increased when the exposure level was dropped to seven cigarettes per day, even if the length of exposure was increased. Using a smoke exposure of 10 cigarettes per day, Ofulue et al32 found an increase in airspace size after 2 months of exposure (123% of control values). After 6 months of exposure, the airspace size was further increased, although its ratio to the control value remained relatively stable (ie, 130% of control values). Rubio and colleagues33 exposed rats to two cigarettes a day for 10 weeks and morphometrically demonstrated an increased thickness of the walls of bronchioles that were ⬍ 100 ␮m in diameter, and they showed that this was associated with an abnormal nitrogen washout curve. In our own study,34 using a dose of five cigarettes per day, we found that smoke increased aging effects on pulmonary function. We could not demonstrate significant airspace enlargement or airway abnormalities to explain this phenomenon (data not reported) or find any alterations in the response of the smoke-exposed rats to methacholine challenge. The studies of Huber et al30 and Heckman and Dalbey31 demonstrated increased peribronchiolar chronic inflammatory cells, and Huber et al30 also found an increased number of metaplastic secretory cells in the tracheal epithelium. A morphometric study of the tracheal mucous glands35 after 30 consecutive days of smoke exposure for 30 min per day showed an increase in the volume proportion of glands, which was significant in female rats, but not in male rats, data that generally supported the conclusions of Jones et al.36 Ofalue et al32 used their rat model to investigate elastinolytic activity of the neutrophils and macrophages. Although lavage neutrophil numbers were increased after 1 month of smoke exposure, these numbers decreased to control levels thereafter. By contrast, the number of macrophages increased in both the BAL fluid and in the alveolar interstitium, with a concomitant increase in elastinolytic activity and an increase in levels of lavage elastin-derived peptides and desmosine. Mice are relatively recent models of cigarette smokeCHEST / 121 / 5 / MAY, 2002 SUPPLEMENT

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induced COPD, and their attractiveness as a model has been based on their ability to be transgenically modified. In addition, some isolated breeds normally develop emphysema as the animals age.37 Hautamaki and colleagues38 have exposed C57BL/6 and A/J mice to smoke for periods up to 6 months and have been able to document inflammation of the airways and within the airspaces, with associated airspace enlargement. The latter enlargement includes contributions from both the alveolar ducts and the alveolar spaces. Indeed, the mouse model has been instrumental in extending our knowledge of the mechanisms involved in emphysema to once again encompass the alveolar macrophage and metalloproteinases in addition to the serine proteinases produced by neutrophils.

Advantages of the Guinea Pig Model Animal The guinea pig is a gentle animal and does not bite. They accept materials willingly by mouth, and a gag has been described if intragastric lavage is necessary.39

Anatomy To calculate the surface area of the guinea pig,40 an equation has been established that is much like the one available for humans, although, unlike in humans, it depends on weight alone. Like humans, and compared to the mouse and even to the rat, a guinea pig has abundant lung tissue, allowing the research to partition the lungs of the same animal for investigations of, for example, molecular biology, histology, or lavage. The structure of the guinea pig lung is roughly analogous to the human lung, with three major lobes on the right and two major lobes on the left. Although the guinea pig, like the rat or mouse, does not have true respiratory bronchioles that would correspond to the three generations of respiratory bronchioles found in humans, they do have a well-defined terminal bronchiole with subtending alveolar ducts.

Responses to Smoke The guinea pig develops morphologic and physiologic alterations after exposure to cigarette smoke at roughly the same concentrations as humans (as assessed by carboxyhemoglobin levels). As noted above, rats require a significantly greater exposure before developing disease, but mice will develop emphysema after exposure to two cigarettes per day. A significant proportion of guinea pigs develop increases in pulmonary arterial pressure, although rat vessels clearly have a vascular proliferative response to smoke (at least after short-term exposures). We have no data that would indicate that they also have physiologic alterations. In our long-term exposure experiment, which was described above, we did not identify any alterations of vascular physiology, but it is also important to recognize that our exposures were at much lower levels than those used by others. 190S

Problems and Prospects of Model Anatomy Because of the anatomy of the pharynx, guinea pigs are extremely difficult to intubate, especially compared to the rat. It is not, however, impossible, and several articles41– 45 have described methodologies that can result in successful intubation for lavage or instillation into the lungs. It remains difficult to use intubation as a method for ventilation or for measurements of pulmonary function.

Molecular Biology The guinea pig has a very high level of endogenous ribonuclease. To obtain reliable results when measuring messenger RNA, which may be present in small quantities, it is necessary to take extensive precautions when handling the tissue and to make sure that the tissue is frozen or fixed appropriately as soon as possible after killing the animal. The number of gene sequences for the guinea pig is much lower than that available for the mouse or rat. However, the number of sequences listed in the Gene Bank is increasing rapidly, and this should not be a major deterrent in planning future experiments with this model. Protein sequences also are limited. However, there is extensive crossover of guinea pig to human, and Western blot analyses, enzyme-linked immunosorbent assays, or immunohistochemistry techniques are often successful when using the antihuman antibodies that are commercially available.

References 1 Snider GL, Kleinerman J, Thurlbeck WM, et al. The definition of emphysema: report of a National Heart, Lung, and Blood Institute; Division of Lung Diseases workshop. Am Rev Respir Dis 1985; 132:182–185 2 Simani AS, Inoue S, Hogg JC. Penetration of respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab Invest 1974; 31:75– 87 3 Hulbert W, Walker DC, Hogg JC. The site of leukocyte migration through the tracheal mucosa in the guinea pig. Am Rev Respir Dis 1981; 124:310 –316 4 Hulbert W, McLean T, Hogg JC. The effect of acute airway inflammation on bronchial reactivity in guinea pigs. Am Rev Respir Dis 1985; 132:7–11 5 Hulbert W, McLean T, Wiggs BR, et al. Histamine doseresponse curves in guinea pigs. J Appl Physiol 1985; 58:625– 634 6 Hulbert W, Walker DC, Jackson A, et al. Airway permeability to horseradish peroxidase in guinea pigs: the repair phase after injury by cigarette smoke. Am Rev Respir Dis 1981; 123:320 –326 7 Burns AR, Hosford SP, Dunn LA, et al. Respiratory epithelial permeability after cigarette smoke exposure in guinea pigs. J Appl Physiol 1989; 66:2109 –2116 8 James AL, Dirks P, Ohtaka H, et al. Airway responsiveness to intravenous and inhaled acetylcholine in the guinea pig after cigarette smoke exposure. Am Rev Respir Dis 1987; 136: 1158 –1162 9 Matsumoto K, Aizawa H, Inoue H, et al. Eosinophilic airway inflammation induced by repeated exposure to cigarette smoke. Eur Respir J 1998; 12:387–394 COPD Symposium: Into the New Millennium

10 Sugiura H, Ichinose M, Oyake T, et al. Role of peroxynitrite in airway microvascular hyperpermeability during late allergic phase in guinea pigs. Am J Respir Crit Care Med 1999; 160:663– 671 11 Sadeghi-Hashjin G, Folkerts G, Henricks PAJ, et al. Peroxynitrite induces airway hyperresponsiveness in guinea pigs in vitro and in vivo. Am J Respir Crit Care Med 1996; 153:1697– 1701 12 Nishikawa M, Kakemizu N, Ito T, et al. Superoxide mediates cigarette smoke-induced infiltration of neutrophils into the airways through nuclear factor-␬B activation and IL-8 mRNA expression in guinea pigs in vivo. Am J Respir Cell Mol Biol 1999; 20:189 –198 13 Dusser DJ, Djokic TD, Borson DB, et al. Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig. J Clin Invest 1989; 84:900 –906 14 Wright JL, Harrison N. Cardiopulmonary effects of a brief exposure to cigarette smoke in the guinea pig. Respiration 1990; 57:70 –76 15 Wright JL, Churg A. Cigarette smoke causes physiologic and morphologic changes of emphysema in the guinea pig. Am Rev Respir Dis 1990; 142:1422–1428 16 Wright JL, Sun J-P. The effect of smoking cessation on pulmonary and cardiovascular function and structure. J Appl Physiol 1994; 76:2163–2168 17 Selman M, Montano M, Ramos B, et al. Tobacco smokeinduced lung emphysema in guinea pigs is associated with increased interstitial collagenase. Am J Physiol 1996; 271: L734 –L743 18 Wright JL, Churg A. Smoke-induced emphysema in the guinea pig is associated with morphometric evidence of collagen breakdown and repair. Am J Physiol 1995; 268:L17– L20 19 Cardoso W, Sekhon H, Hyde DM, et al. Collagen and elastin in human pulmonary emphysema. Am Rev Respir Dis 1993; 147:975–981 20 Thurlbeck WM, Wright JL. Thurlbeck’s chronic airflow obstruction. 2nd ed. Hamilton, ON, Canada: BC Decker, 1999 21 Wright JL. The importance of ultramicroscopic emphysema in cigarette smoke induced lung disease. Lung 2002; 179: 71– 81 22 Wright JL, Cosio MG, Wiggs BR, et al. A morphologic grading scheme for membranous and respiratory bronchioles. Arch Pathol Lab Med 1985; 109:163–165 23 Wright JL, Ngai T, Churg A. Effect of long-term exposure to cigarette smoke on the small airways of the guinea pig. Exp Lung Res 1992; 18:105–114 24 Murphy ML, Bone RC. Cor pulmonale in chronic bronchitis and emphysema. New York, NY: Futura, 1984 25 Wright JL, Churg A. Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp Lung Res 1991; 17:997–1009 26 Yamato H, Sun J-P, Churg A, et al. Cigarette smoke-induced emphysema in guinea pigs is associated with diffusely decreased capillary density and capillary narrowing. Lab Invest 1996; 75:211–219

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27 Yamato H, Sun J-P, Churg A, et al. Guinea pig pulmonary hypertension caused by cigarette smoke cannot be explained by capillary bed destruction. J Appl Physiol 1997; 82:1644 – 1653 28 Wright JL, Sun J-P. Dissociation of chronic vascular cell proliferation and vascular contractility after chronic cigarette smoke exposure. Eur Respir J 1999; 14:832– 838 29 Wright JL. The relationship of increased pulmonary artery pressure and airflow obstruction to emphysema. J Appl Physiol 1993; 74:1320 –1324 30 Huber GL, Davies P, Zwilling GR, et al. A morphologic and physiologic bioassay for quantifying alterations in the lung following experimental chronic inhalation of tobacco smoke. Clin Respir Physiol 1981; 17:269 –327 31 Heckman CA, Dalbey WE. Pathogenesis of lesions induced in rat lung by chronic tobacco smoke inhalation. J Natl Cancer Inst 1982; 69:117–129 32 Ofulue AF, Ko M, Abboud RT. Time course of neutrophil and macrophage elastinolytic activities in cigarette smokeinduced emphysema. Am J Physiol 1998; 275:L1134 –L1144 33 Rubio ML, Sanchez-Cifuentes MV, Ortega M, et al. Nacetylcysteine prevents cigarette smoke induced small airways alterations in rats. Eur Respir J 2000; 15:505–511 34 Wright JL, Sun J-P, Vedal S. A longitudinal analysis of pulmonary function in rats during a 12 month cigarette smoke exposure. Eur Respir J 1997; 10:1115–1119 35 Hayashi M, Sornberger GC, Huber GL. Morphometric analysis of tracheal gland secretion and hypertrophy in male and female rats after experimental exposure to tobacco smoke. Am Rev Respir Dis 1979; 119:67–73 36 Jones R, Bolduc P, Reid LM. Goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with and without the antiinflammatory agent phenylmethyloxadiozole. Br J Exp Pathol 1973; 54:229 –239 37 Shapiro SD. Animal models for chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2000; 22:4 –7 38 Hautamaki RD, Kobayashi DK, Senior RM, et al. Macrophage elastase is required for cigarette smoke-induced emphysema in mice. Science 1997; 277:2002–2004 39 Smith BL, Flaoyen A, Embling PP. A simple gag for the intragastric dosing of guinea pigs (Cavia porcellus). Lab Anim 1993; 27:286 –288 40 Kibler HH, Brody S, Worstell D. Surface area and metabolism of growing guinea pigs. J Nutr 1947; 33:331–338 41 Kujime K, Natelson BH. A method for endotracheal intubation of guinea pigs. Lab Anim Sci 1981; 31:715–716 42 Blouin A, Cormier Y. Endotracheal intubation in guinea pigs by direct laryngoscopy. Lab Anim Sci 1987; 37:244 –245 43 Tremblay G, Gagnon L, Cormier Y. Sequential bronchoalveolar lavages by endotracheal intubation in guinea pigs. Lab Anim 1990; 24:63– 67 44 Turner MA, Thomas P, Sheridan DJ. An improved method for direct laryngeal intubation in the guinea pig. Lab Anim 1992; 26:25–28 45 Kramer K, Grimbergen JA, van Iperen DJ, et al. Oral endotracheal intubation of guinea pigs. Lab Anim 1998; 32:162–164

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