Rat models of asthma and chronic obstructive lung disease

ARTICLE IN PRESS

Pulmonary Pharmacology & Therapeutics 19 (2006) 377–385 www.elsevier.com/locate/ypupt

Review

Rat models of asthma and chronic obstructive lung disease$ James G. Martin, Meiyo Tamaoka Meakins Christie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Que., Canada H2X 2P2 Received 28 August 2005; accepted 25 October 2005

Abstract The rat has been extensively used to model asthma and somewhat less extensively to model chronic obstructive pulmonary disease (COPD). The features of asthma that have been successfully modeled include allergen-induced airway constriction, eosinophilic inflammation and allergen-induced airway hyperresponsiveness. T-cell involvement has been directly demonstrated using adoptive transfer techniques. Both CD4+ and CD8+ T cells are activated in response to allergen challenge in the sensitized rat and express Thelper2 cytokines (IL-4, IL-5 and IL-13). Repeated allergen exposure causes airway remodeling. Dry gas hyperpnea challenge also evokes increases in lung resistance, allowing exercise-induced asthma to be modeled. COPD is modeled using elastase-induced parenchymal injury to mimic emphysema. Cigarette smoke-induced airspace enlargement occurs but requires months of cigarette exposure. Inflammation and fibrosis of peripheral airways is an important aspect of COPD that is less well modeled. Novel approaches to the treatment of COPD have been reported including treatments aimed at parenchymal regeneration. r 2005 Elsevier Ltd. All rights reserved. Keywords: Early airway responses; Late airway responses; Cysteinyl leukotrienes; Proliferating cell nuclear antigen; Bromo-deoxyuridine; Adoptive transfer; Anti-oxidants; Emphysema

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Innate airway hyperresponsiveness . . . . . . . . . . . . . . . . Induced airway hyperresponsiveness . . . . . . . . . . . . . . . 3.1. Allergen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Cytokine-induced AHR . . . . . . . . . . . . . . . . . . . 3.3. Irritant-induced AHR . . . . . . . . . . . . . . . . . . . . . 3.4. Virally-induced AHR . . . . . . . . . . . . . . . . . . . . . 4. Allergen-induced early and late airway responses (LAR) . 5. Dry gas hyperpnea-induced airway narrowing . . . . . . . . 6. Airway remodeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Chronic bronchitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Emphysema. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Cigarette-induced emphysema . . . . . . . . . . . . . . . 8.2. Elastase-induced emphysema . . . . . . . . . . . . . . . . 9. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reference. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Supported by the Canadian Institutes of Health Research.

Corresponding author. Tel.: +514 398 3864x00137; fax: +514 398 7483.

E-mail address: [email protected] (J.G. Martin). 1094-5539/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2005.10.005

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1. Introduction Early studies of the regulation of airway function and its potential relationship to asthma and chronic obstructive pulmonary disease (COPD) employed large animals such as the dog, cat and monkey. The detailed study of respiratory system mechanics was easily performed in these animals whereas equipment to study smaller animals was not generally available. In the past 20 years the techniques available for small animals have improved substantially and have undergone extensive testing. Furthermore the focus has shifted from the investigation of the neural control of airway smooth muscle (ASM) tone and of neurogenic causes of airway hyperresponsiveness (AHR) to the study of airway inflammation. Small animals such as the rat have proven to be very useful for such studies. Although there have been a substantial number of studies employing murine models of asthma the measurement of pulmonary function in the mouse is somewhat more difficult to make than in the rat. Non-invasive techniques for the mouse have been developed to facilitate the assessment of pulmonary function but are associated with a substantial degree of uncertainty. The range of experimental techniques that can be practiced on the mouse will no doubt lead to further growth of the considerable enthusiasm for this animal for the modeling of respiratory disease. However, the rat is still an important species for drug testing so that it should continue to enjoy popularity for the study of experimental asthma and COPD. Asthma has three defining features, namely AHR, airway inflammation and airway obstruction that is at least partly reversible. All of these characteristics have been successfully reproduced in the rat. AHR can be induced by a variety of stimuli such as allergen challenge of actively or passively sensitized animals, through viral infections or irritant substances. However, AHR may also be innate, occurring without any particular trigger. The mechanisms of AHR are still unresolved. However, the phenomenon of AHR is a reflection of excessive airway narrowing for a given dose of contractile agonist administered. It is generally believed that changes in ASM properties may be important [1]. Post-receptor mechanisms must be important because responses to different agonists that employ distinct cell surface receptors reveal the hyperresponsiveness. Changes in contractile proteins (myosin isoforms, myosin light-chain kinase) or in signaling (inositol phosphate metabolism) causing increased smooth muscle contraction (force or velocity) could cause AHR. Alternatively, changes in mechanical impedances to airway narrowing such as the elastic properties of the lung parenchyma or local changes in the parenchyma adjacent to the airways causing mechanical uncoupling could also be a potential explanation [2]. Indeed it is likely that there are several mechanisms that account for AHR in different pathologies. Airway inflammation is usually studied after allergic challenge; it is pleiomorphic but it has a substantial

eosinophilic component when triggered by allergen. The eosinophilia is caused by cytokines and chemokines that are associated with the Th2 pattern of T-cell cytokines. Antigen presentation by dendritic cells to ovalbumin (OVA) specific T cells, predominantly CD4+ cells, is responsible for the generation of activated Th2 cells expressing interleukin (IL)-4, IL-5 and IL-13 [3]. Nonallergenic stimuli such as ozone are accompanied by neutrophilic inflammation. The role of T cells in these responses has not been well studied. Airway obstruction has generally been induced by allergic challenge. Allergen-induced bronchoconstriction involves an ‘‘immediate response’’ within minutes, also named the early response, and a delayed airway narrowing termed the late response. The latter is more strongly associated with airway inflammation than the isolated early airway response. Airway narrowing can also be evoked by dry gas hyperpnea, a model for exercise-induced asthma and various other non-allergic stimuli. 2. Innate airway hyperresponsiveness A comparison of agonist (serotonin or methacholine)induced airway responsiveness in the rat was made many years ago and demonstrated strain-related differences [4,5]. Fisher 344 rats have demonstrated consistent AHR compared to other strains, of which the Lewis was chosen as the most suitable for comparison. Several differences in the properties of ASM tissue have been identified that could contribute to or account for the differences. A greater amount of muscle has been shown by morphometry in airways of the Fisher rats compared to Lewis rats [6]. The volume sensitivity of induced bronchoconstriction is less marked in the Fisher rat than the Lewis rat. Tracheal responses to agonists are greater in the Fisher and also demonstrate increases in both sensitivity and maximal responses [7]. The increase in maximal response is consistent with the increase in muscle mass. However, the greater response seems to be in part related to nitric oxide production by epithelium; the Fisher rats have less cyclic guanosine monophosphate synthesis following treatment with a nitric oxide donor, nitroprusside, suggesting reduced guanylyl cyclase activity in the airway [8]. Inhibition of nitric oxide synthase by administration of L-NAME diminishes the differences in airway responsiveness between F344 and Lewis rats in vivo [9]. The Fisher rat retains the hyperresponsiveness of its ASM in vitro. Cultured explants from these rats have been used to show greater and faster contractions of the Fisher airways in situ to serotonin and methacholine [10,11]. The cells in culture show enhanced responses to serotonin and bradykinin in the form of calcium transients [12]. The greater calcium transients appear to be accounted for by reduced inositol trisphosphate phosphatase activity, resulting in larger IP3 transients in the Fisher rat [13]. Furthermore, the Fisher rat has greater expression of the 7 amino acid inserted myosin isoform associated with faster myosin contraction [14].

ARTICLE IN PRESS J.G. Martin, M. Tamaoka / Pulmonary Pharmacology & Therapeutics 19 (2006) 377–385 Table 1 Characteristics of hyperresponsive F344 compared to normoresponsive Lewis rats Pulmonary function Histology ASM properties

Hyperresponsive to methacholine and serotonin More smooth muscle in the airways Contracts with greater force and velocity Greater calcium responses to contractile agonists Resistance to nitric oxide donors (reduced guanylyl cyclase activity) Greater quantity of the fast isoform of myosin

This isoform is present in relatively greater amounts than the slow myosin isoform in phasic muscles (e.g. bladder compared to aortic smooth muscle). Phasic muscle is present in organs that require rapid adjustments to their dimensions such as the bladder. An increase in the ‘‘phasic’’ behavior of ASM might be expected to lead to more rapid and more complete airway narrowing following stimulation. The multiplicity of pathways that are altered in the ASM of the Fisher rat (summarized in Table 1) suggests that a common pathway for these differences must exist at the level of transcriptional regulation of the ASM phenotype, leading to a more phasic kind of ASM in the Fisher rat. 3. Induced airway hyperresponsiveness 3.1. Allergen The most suitable rat strain for the study of allergeninduced airway reactions is the Brown Norway (BN). This rat is naturally atopic and for many years has been know to have very high immunoglobulin E levels after active sensitization to experimental allergen [15]. It has many features in common with human allergic asthma (Table 2), including early and late allergic responses and T-cellmediated eosinophilic airway inflammation [16]. Allergen sensitization and subsequent challenge of the BN rat with allergen, usually chicken OVA, leads to the development of AHR by 24 h after challenge [17]. AHR is demonstrable using acetylcholine, serotonin and LTD4, suggesting postreceptor mechanisms of AHR. AHR can also be induced by allergen challenge of animals that are sensitized by the adoptive transfer of CD4+ T cells from sensitized donors [18,19]. CD8+ T cells seem to exert a protective effect against AHR; depletion of these cells using a specific antibody augments allergen-induced AHR [20]. The composition of the inflammatory cells in the BAL fluid after allergen inhalation demonstrates an initial neutrophilia within a few hours, a modest eosinophilia at 5–8 h, followed at 18–24 h by an increase in lymphocytes and a more marked eosinophilia. There is a significant correlation between airway responsiveness and eosinophil recovery at 5–8 and at 18–24 h after allergen exposure [21]. At

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Table 2 Characteristics of the Brown Norway rat High IgE producer Develops early and late allergic airway responses to allergen challenge Late responses can be adoptively transferred with CD4+ and CD8+ T cells Eosinophilia and Th2 cytokines are associated with allergen challenge Interferon-g deficiency contributes to atopic features Susceptible to respiratory viral bronchiolitis

18–24 h there is also a significant correlation between neutrophils and airway responsiveness. AHR is associated also with expression of Th2 cytokines. Indeed adoptive transfer of Th2 cells (CD4+ T cells cultured with IL-4 and anti-interferon (IFN)-g) and subsequent allergen challenge resulted in AHR [22]. Transfer of Th1 cells (CD4+ T cells cultured with IFN-g and anti-IL-4) in conjunction with Th2 cells prevented the development of AHR. Probing the roles of specific cytokines in AHR is not easy at present because there is a lack of reagents such as neutralizing antibodies or small molecule inhibitors and where commercial antibodies are available it is often impractical to attempt neutralization experiments because of cost. IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-5 are among several cytokines that have been shown to be increased in asthma. They mediate their effect via receptors that have a common beta subunit (bc) and blocking of this common bc using antisense (AS) phosphorothioate oligodeoxynucleotide (ODN) inhibited bc mRNA expression and immunoreactive cells within the lungs of BN rats when injected intratracheally [23]. Inhibition of bc significantly reduced eosinophilia in vivo in OVA-sensitized BN rats after antigen challenge and inhibited antigen-induced AHR to leukotriene D4. IL-5 alone causes AHR when given intratracheally but does not enhance AHR induced by OVA [24]. IL-2 alone in high doses is also a potent inducer of AHR (see below). However, even when administered to BN rats in low doses, insufficient to cause AHR, in conjunction with OVA sensitization and challenge it augments AHR to LTD4 [25]. IL-1b administered intratracheally can cause AHR to bradykinin and neutrophilic airway inflammation in the BN rat. However, it does not potentiate allergeninduced AHR [26]. IFN-g negatively regulates Th2 responses in the BN rat and inhibits AHR induced by allergen [27,28]. It appears to account for the inhibitory effects of Th1 cells and of CD8+ T cells on allergeninduced AHR [22]. Neutralization of endogenous IFN-g augments allergen-induced AHR. The biochemical mediators responsible for AHR are unclear. Although 5-HT and cysteinyl leukotrienes (cysLTs) are important mediators of the early and late responses to OVA, 5-HT, lipoxygenase and cyclooxygenase products and PAF do not appear to be involved in OVAinduced AHR in the BN rat [29]. Corticosteroids but not

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cyclosporine A inhibit OVA-induced AHR [30]. However, the multiple mechanisms of action of steroids prevent useful speculation as to mechanisms of AHR.

exposure to chlorine have not been reported. No mechanistic studies have been performed to date. 3.4. Virally-induced AHR

3.2. Cytokine-induced AHR A number of studies of IL-1b have shown that this cytokine can cause AHR. Intratracheal administration of IL-1b induces neutrophilic inflammation and increases AHR to bradykinin [26]. The effect is not potentiated by active sensitization. IL-2 potently induces AHR to methacholine by 45 min after its administration to Lewis strain rats [31]. Fisher rats do not show changes with the same dose of IL-2. There is an associated peribronchial edema and separation of the epithelium from the underlying connective tissue. When administered twice a day for 4.5 days, IL-2 causes a mixed cellular inflammation with a prominent eosinophilic component as well as AHR [32,25]. IL-5 administered intra-tracheally causes AHR in the BN rat [24]. IL-13 has not been tested in the rat although it has been shown to cause AHR in guinea pigs and mice. 3.3. Irritant-induced AHR Originally called reactive airways dysfunction syndrome (RADS), asthma induced by brief exposures to high concentrations of irritant substances has been recognized and relabeled irritant-induced asthma. Prolonged or repeated exposures to low doses of the same substances may also lead to asthma. Substances such as chlorine and ozone cause oxidant damage and may not be fundamentally different stimuli. Acute exposures cause airway damage with epithelial loss and a neutrophilic inflammation [33]. Bronchial responsiveness to methacholine is increased after exposures. A single exposure to low concentrations of ozone leads to AHR that is strain dependent, and it occurs in the absence of inflammation [34]. Repeated exposures to ozone lead to adaptation to some but not all of the effects of the exposure [35]. The extent of inflammation and neuropeptide release from sensory nerve fibers diminish but remodeling of the airways persists, with airway changes that extend to the acinus. The effects of ozone are no doubt in large part mediated by oxidant damage. Ozone-induced AHR to acetylcholine and bradykinin in the BN rat is prevented by the antioxidants allopurinol and deferoxamine, without any reduction in BAL neutrophil counts, however [36]. Chlorine has been less studied and is generally thought of in relation to occupational irritant-induced asthma whereas ozone exposures have a broader relevance. A single exposure to high-dose chlorine has been shown to cause histopathological changes that persisted for as long as 3 months after the exposure [33], consistent with the clinical observations of long-lasting effects on human subjects following single high-dose exposures. Dexamethasone reduces the inflammation, AHR and structural remodeling caused by chlorine [37]. The effects of repeated

The effects of a variety of experimental viral infections have been studied in the rat. Using Sendai and respiratory syncytial viral infection it has been possible to induce AHR [38,39]. Interestingly RSV infection causes upregulation of 5-lipoxygenase in the lungs and sensory nerve stimulation by capsaicin causes excessive increases in microvascular permeability in the infected animals by a mechanism involving cys-LTs. This cascade of events is not unlike the mechanisms operative in the changes in airway function induced by dry gas hyperpnea. The BN rat is also more susceptible to Sendai virus-induced bronchiolitis than the F344 rat and this susceptibility seems related to deficient IFN-g secretion by natural killer cells in the BN rat [39]. This rat also has deficient IFN-g secretion by CD8+ cells. The reduced IFN-g secretion may be secondary to reduced production of the IFN-g inducing cytokines IL-12 and IL18 by antigen presenting cells [40]. It is likely that these abnormalities are of importance in the atopic tendency of the BN which make it such a useful model of asthma. 4. Allergen-induced early and late airway responses (LAR) The study of allergic inflammation in the rat is best performed using the BN rat. This high IgE producer gives early and late allergic bronchoconstriction after sensitization to OVA and subsequent challenge by aerosol [16]. Unfortunately, neither the early responses nor LAR are large in magnitude because of the lack of responsiveness of the rat airways to the mediators of allergic airway narrowing. The early response is mediated by serotonin and cys-LTs and is virtually abolished by methysergide or a cys-LT receptor antagonist [41]. The LAR, which comprises a series of perturbations of the pulmonary resistance occurring from 3 to 8 h after the OVA challenge, is also markedly attenuated by a cys-LTR1 antagonist such as montelukast [42]. The analysis of cys-LTs excretion via the bile demonstrates that the principal metabolite N-acetylLTE4 is elevated after allergen challenge and shows a biphasic rise in animals in which a LAR is present [43]. An increase in bronchoalveolar lavage fluid levels of cys-LTs can also be measured after allergen challenge. There is an eosinophilic airway inflammation and Th2 cytokine expression by 8 h after challenge with OVA [28]. The source of the cys-LTs is not clear but mast cells or macrophages are likely candidates [44]. Rodent eosinophils do not synthesize cys-LTs. Inhaled corticosteroid inhibited the LAR and eosinophilia and reduced cys-LT levels in the bile when administered in two doses 18 and 1 h before the challenge with OVA [45]. Whether glucocorticosteroids act directly to inhibit the synthesis of cys-LTs or by inhibiting the influx or activation of cells is not clear. It has been possible to demonstrate early and LAR to OVA challenge

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in isolated airways in lung explant cultures, indicating that resident airway cells are sufficient for the development of these reactions [46]. Inhibition of LAR by corticosteroids or by anti-adhesion antibodies (anti-VLA-4) [47] is therefore likely to result from the inhibition of the activation of resident airway cells. It has been possible to reproduce the LAR in the BN rat by the technique of adoptive transfer of CD4+ T cells [48]. In the recipient animals there is no early response as expected but there is a LAR, Th2 cytokine expression and eosinophilic airway inflammation that is qualitatively indistinguishable from the response of an actively sensitized rat to OVA challenge. The T-cells-dependent LAR is also cys-LT mediated and abolished by the cys-LT1R antagonist pranlukast [49]. Adoptive transfer techniques have also been used to demonstrate that the CD8+ T cells are inhibitory of LAR and Th2 inflammation when administered to sensitized BN rats that undergo challenge 24 h after the receipt of the cells [50]. The inhibition is attributable to CD8+ gd T-cell receptor bearing cells whereas the CD8+ ab TCR bearing cells appear to behave like CD4+ cells, inducing LAR and typical Th2 cytokinedriven allergic inflammation [51,52]. This suggests that antigen is presented in association with major histocompatibility (MHC) class II molecules as expected and with MHC class 1 molecules also, which is less expected. The precise contribution of cross presentation of antigen to the allergic response is not known. CD8+ gd T cells are potent inhibitors of allergic airway responses [51] but the mechanism of their activation is not yet elucidated. A significant part of their inhibitory effect is mediated through IFN-g. 5. Dry gas hyperpnea-induced airway narrowing The modeling of exercise-induced asthma has be accomplished using dry gas hyperpnea as the challenge modality. Drying and/or cooling of the airways are likely to be the stimulus for exercise-induced asthma. From various animal models it has been possible to deduce that neurokinin release occurs and is accompanied by cys-LT synthesis. Cys-LTs may themselves release neurokinins from sensory C-fibers whereas there is also evidence that neurokinins may trigger cys-LT synthesis. There are few data on hyperpnea-induced airway narrowing in the rat. BN and ACI strains responded to challenge with an increase in pulmonary resistance whereas F344 and Lewis rats did not show these changes. NK1/NK2 receptor antagonists and the cys-LT1R antagonist, pranlukast inhibited the airway response to challenge [53]. 6. Airway remodeling The rat has proven to be useful for the study of airway remodeling in allergic asthma. The studies that have been performed have employed the BN rat. Most investigators have examined the remodeling involving ASM, since this

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form of remodeling is likely to be of the greatest significance for airway dysfunction [54]. Increase in ASM, assuming that the new muscle has a normal contractile phenotype is the best explanation for the progressive induction of asthma by exposure to sensitizing agents, as best exemplified by occupational asthma. Increase in ASM in the rat requires at least three challenges. Exposure of sensitized BN rats to three challenges at 3 to 5 day intervals results in an increase in ASM of approximately twofold [55]. Exposure has been done using chambers for awake animals and endotracheal intubation for anesthetized animals. The latter technique is associated with growth of ASM throughout the airway tree and the extent of growth of ASM is comparable in large and small airways. Animals exposed while in the awake state, however, appear to have preferential remodeling in the peripheral airways (R. Olivenstein, unpublished results), presumably because large particles of aerosolized allergen are effectively removed within the upper airways leaving particles that penetrate to the periphery of the lung. Hyperplasia of ASM seems to account for most if not all of the increase in ASM. Bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression have both been used successfully to demonstrate the presence of proliferating smooth muscle cells [56–58]. The technique of double immunostaining for PCNA and smooth muscle-specific alpha-actin has removed any uncertainty about whether the submucosal cells proliferating were ASM cells or not [58]. The process of ASM proliferation may be associated with loss of contractile proteins [59] that may result in a less than expected degree of AHR. The mechanisms of ASM growth induced by allergen challenge are still not entirely established. Cys-LTs, which are well-known bronchoconstrictive agents in asthma, have been shown to be implicated in ASM growth in the rat [60,61]. Cys-LTs are weak mitogens in vitro and it seems likely that they act through or in concert with classical growth factors. Epidermal growth factor expression increases in the lung tissues of the BN rats following three challenges with OVA. The level of EGF mRNA is reduced by the cys-LT1R antagonist montelukast, consistent with the idea that cys-LT-driven remodeling of ASM is at least in part growth factor dependent (M. Tamaoka unpublished results). There is convincing evidence also that endothelin is involved in ASM growth [62] but the precise mechanism of its involvement is also not clear. Not surprisingly corticosteroid pre-treatment prevents ASM growth [63]. The evidence that AHR is related to the increase in ASM mass is still circumstantial [55,64]. There is recent evidence that the airway epithelium may play a role in airway remodeling by novel mechanisms. Ressler et al. [65] demonstrated that airway epithelial cells in culture respond to compressive stresses to produce TGFb1 and endothelin-1. Perhaps the bronchoconstriction associated with allergen-induced airway responses may promote remodeling of the underlying ASM. Airway

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epithelium is stimulated to proliferate at any even faster rate than ASM [58]. It also is likely to have altered cellular phenotypes. Among the molecules it may express is inducible nitric oxide synthase (NOS2). Although nitric oxide is inhibitory of ASM growth in culture a NOS2 inhibitor had no effect on ASM proliferation in vivo [66] though it attenuated the allergen-induced increase in eosinophilia and CD4+ T-cell accumulation in the OVA challenged BN rat. The results may be complicated by the competing anti-inflammatory effects of cyclic-guanosine monophosphate and the pro-inflammatory effects of protein nitration mediated by oxidative species such as peroxynitrite. Other aspects of airway remodeling can also be addressed by the repeatedly OVA-challenged BN rat. Two weeks of alternate day challenges results in increased fibronectin deposition in the airways and measurable thickening. There is no significant collagen deposition at this time. After 12 weeks of exposure, various collagen subtypes are found in increased amounts. Fluticasone inhibits the increases in matrix proteins but at doses that exceed those required for the inhibition of eosinophilic inflammation [67]. Recent preliminary reports indicate altered proteoglycan deposition also [68]. The increase in ASM mass is not only a reflection of hyperplasia of cells but also apoptosis. This aspect of ASM remodeling has received little attention to date. In a recent study by Ramos-Barbon et al. [58] sensitized CD4+ T cells administered to naı¨ ve recipients adoptively transferred allergic sensitivity and animals had ASM and epithelial remodeling after three allergen challenges. The increase in ASM mass was attributable to both proliferation and a reduction in rates of apoptosis. Presumably survival signals derived from mediators released by allergen challenge were present at the same time as the growth factors responsible for hyperplasia. Although the mechanisms by which hyperplasia and reduction of apoptosis are coupled need elucidation, the direct contact between T cells and ASM leads to bidirectional effects including hyperplasia and reduction of apoptosis in vitro. 7. Chronic bronchitis The link between asthma and COPD has been debated for years. In fact there is not always a clear distinction between the histopathological changes in the airways of subjects with asthma and COPD, although COPD is certainly characterized by progressive and permanent loss of lung function whereas asthma has such a course infrequently. It is not surprising therefore that the modeling of chronic bronchitis has been performed using some of the same stimuli as used to model asthma. Chronic bronchitis has been modeled in the rat using ozone [34] which is a stimulus of considerable relevance to asthma. The airway changes induced by sulphur dioxide have also been used to model chronic bronchitis [69]. Both stimuli cause a transient neutrophilic inflammation in the rat.

After sulphur dioxide exposure airway remodeling occurs and comprises changes in ASM (hyperplasia) and epithelium. These changes are worsened by capsaicin pretreatment of animals, indicating a protective role for airway C-fibers and their complement of neurokinins [70]. Ozone has been shown to interact with endotoxin to promote mucus gene expression (Muc5AC) and mucus glycoprotein secretion from respiratory epithelium [71]. 8. Emphysema 8.1. Cigarette-induced emphysema The technique of exposing rats to cigarette smoke using a smoking apparatus and standardized research grade cigarettes is well described. Acute exposures to cigarette smoke induce airway narrowing. Acute inflammation is also induced which tends to diminish following more prolonged exposures. Oxidant damage is an important consequence of exposure to cigarette smoke, even for exposures as short as 3 days. A catalytic anti-oxidant has been shown to prevent epithelial metaplasia and to reduce inflammation in rats [72]. The hydrolysis of epoxyeicosatrienoic acids by soluble epoxide hydrolase contributes to the inflammation and damage by reducing the levels of endogenous EETs with anti-inflammatory properties [73]. However, short-term smoking has little effect on lung structure although it may be used in conjunction with elastase to accentuate elastase-induced damage. Emphysema can be induced by cigarette smoking in the rat but lesions take several months to develop. Reproducing centrilobular emphysema appears to be difficult in the rat and alveolar enlargement, more typical of centracinar emphysema, is more usual. The changes in lung function have been likened to lung aging [74]. Airway (peribronchial) inflammation is described but the extensive small airway disease seen in human subjects is not reproduced. Some alveolar septal fibrosis is also present in chronically smoked rats [75]. The mechanisms of the formation of the lesions of emphysema are still debated. Evidence is accumulating that favours the idea that macrophage derived products such as elastase are important. For example, the emphysema induced by cigarette smoke is not prevented by neutrophil depletion but is reduced by anti-CD11b and anti-CD18 antibody, indicating a more important role for the macrophage than the neutrophil in the genesis of emphysema in the rat [76]. Rat tracheal explants have been used to study airway remodeling caused by cigarette smoke. Procollagen gene expression and tissue hydroxyproline were increased in tissues exposed to smoke. Several scavengers of reactive oxygen species inhibited the observed changes in gene expression and collagen synthesis [77]. Epidermal growth factor was also implicated in the process [78]. Transforming growth factor (TGF)-b was also produced in situ in a tracheal explant by oxidant mechanisms, demonstrating the importance of oxidant

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effects on resident cells in the genesis of airway remodeling. These findings convincingly demonstrate the utility of the explant approach for the evaluation of direct effects of smoke and suggest that the effects of oxidant stress on structural cells in the airways may play an important role in the pathogenesis of the airway changes in COPD. The airway abnormalities are likely the consequence of a combination of direct damage to structural cells as well as the activation of inflammatory cells by smoke. Cigarette smoking induces AHR in rats after several months of exposure. There are potentially several mechanisms that could result in AHR. Changes in ASM mass or contractility may occur; there is evidence for ASM growth. Alternatively the impedances to ASM shortening in vivo such as lung elastic recoil could also alter responsiveness. It has been demonstrated that an increase in responsiveness after smoking may occur prior to observing changes in lung elastic recoil and is therefore unlikely to be mediated by effects of smoking on airway parenchymal interdependence [79]. How airway properties in the rat may be altered as a consequence of smoking is still an unresolved question. Currently mechanistic questions are being posed rather in the murine models.

8.2. Elastase-induced emphysema The administration of exogenous elastase has been frequently used to produce ‘‘emphysema’’. Porcine pancreatic elastase is often given intratracheally and within days significant airspace enlargement is produced. Rats develop larger lung volumes (total lung capacity) and show AHR that is associated with a failure of lung inflation to inhibit methacholine-induced bronchoconstriction [80]. In normal animals the application of a negative pressure to the chest so as to inflate the animals above the usual functional residual capacity by one tidal volume causes a substantial reduction of methacholine-induced airway narrowing, but not so after elastase administration. This finding has been interpreted to suggest that AHR is caused by loss of alveolar attachments to the airways, which normally prevent airway narrowing by transmitting lung recoil to the outer aspect of the airway wall. Several therapeutic strategies based on the re-growth of alveolar septa have been attempted in the rat. The report of all-trans-retinoic acid as a method of restoring alveolar architecture has raised the question of promoting alveolar re-growth as a more definitive treatment for emphysema [81]. This approach is far more attractive than the palliative measures currently available through existing drugs which have very modest effects on lung function and fail to alter significantly the natural history of the disease. However alltrans-retinoic acid may not be effective in reversing elastase-induced emphysema in other species [82]. Hepatocyte growth factor has been successfully used to reverse emphysema through viral transduction of pulmonary cells [83]. HGF increased alveolar septation and improved

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exercise tolerance and gas exchange in rats treated with porcine pancreatic elastase. 9. Conclusions The rat has long been used to model asthma and COPD. Virtually all of the defining features of asthma and COPD can be reproduced using various stimuli; allergen, irritant gas exposures, cigarette smoke or exogenous elastase. The modeling process is of necessity incomplete because the use of single stimuli does not mimic the rather varied and variably timed exposures of subjects in real life. However, the models show the utility of evaluating the biological responses of the rat as a guide to the pathological processes in human subjects affected by these diseases. Perhaps the modeling exercise has been more successful for asthma than COPD. Indeed much of our understanding of the immunology of asthma derived from these studies has stood the test of time for its relevance for human subjects. Reference [1] Solway J, Fredberg JJ. Perhaps airway smooth muscle dysfunction contributes to asthmatic bronchial hyperresponsiveness after all. Am J Respir Cell Mol Biol 1997;17(2):144–6. [2] Macklem PT. A theoretical analysis of the effect of airway smooth muscle load on airway narrowing. Am J Respir Crit Care Med 1996;153(1):83–9. [3] Lambrecht BN. Allergen uptake and presentation by dendritic cells. Curr Opin Allergy Clin Immunol 2001;1(1):51–9. [4] Pauwels R, van der Straeten M, Weyne J, Bazin H. Genetic factors in non-specific bronchial reactivity in rats. Eur J Respir Dis 1985;66: 98–104. [5] Martin JG, Opazo-Saez A, Du T, Tepper R, Eidelman DH. In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle. [Review]. Can J Physiol Pharmacol 1992;70:597–601. [6] Eidelman DH, DiMaria GU, Bellofiore S, Wang NS, Guttmann RD, Martin JG. Strain-related differences in airway smooth muscle and airway responsiveness in the rat. Am Rev Respir Dis 1991;144:792–6. [7] Florio C, Styhler A, Heisler S, Martin JG. Mechanical responses of tracheal tissue in vitro: dependence on the tissue preparation employed and relationship to smooth muscle content. Pulm Pharmacol 1996;9(3):157–66. [8] Jia Y, Xu L, Heisler S, Martin JG. Airways of a hyperresponsive rat strain show decreased relaxant responses to sodium nitroprusside. Am J Physiol 1995;269:L85–91. [9] Jia Y, Xu L, Turner DJ, Martin JG. Endogenous nitric oxide contributes to strain-related differences in airway responsiveness in rats. J Appl Physiol 1996;80(2):404–10. [10] Tao FC, Tolloczko B, Eidelman DH, Martin JG. Enhanced Ca(2+) mobilization in airway smooth muscle contributes to airway hyperresponsiveness in an inbred strain of rat. Am J Respir Crit Care Med 1999;160(2):446–53. [11] Wang CG, Almirall JJ, Dolman CS, Dandurand RJ, Eidelman DH. In vitro bronchial responsiveness in two highly inbred rat strains. J Appl Physiol 1997;82(5):1445–52. [12] Tao FC, Shah S, Pradhan AA, Tolloczko B, Martin JG. Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats. Am J Physiol—Lung Cell Mol Physiol 2003;284(1):L90–9. [13] Tao FC, Tolloczko B, Mitchell CA, Powell WS, Martin JG. Inositol (1,4,5)trisphosphate metabolism and enhanced calcium mobilization

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