MSU LIBRARIES 4-“. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. AIRWAY RESPONSES TO AEROSOLIZED HETHACHOLINE AND CITRIC ACID IN PONIES WITH RECURRENT AIRNAY DESTRUCTION (HEAVES) By Pamela Jane Armstrong A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Small Animal Clinical Sciences 1986 ABSTRACT AIRWAY RESPONSES TO AEROSOLIZED NETHACHOLINE AND CITRIC ACID IN PONIES WITH RECURRENT AIRWAY OBSTRUCTION (HEAVES) By Pamela Jane Armstrong We measured lung function and airway reactivity in response to aerosols of methacholine and citric acid in ponies with a history of heaves (principals) and in ponies with no history ofrespiratory disease . (controls). Principals were paired with controls, and measurements were made when principals were in clinicaI remission (Period A), following barn exposure when principals had acute airway obstruction (Period 8), and 1 and 2 weeks after they were returned to pasture (Periods C and 0). Differences between groups were primarily found at Period B. Barn housing (Period B) decreased dynamic compliance, increased pulmonary resistance, and caused airway hyperreactivity to methacholine and citric acid aerosols in principals but not in controls. InhaIation of 10% citric acid aerosol by principals at Period B induced changes only in pulmonary resistance. We conclude that ponies in clinical remission from heaves do not exhibit nonspecific airway hyperreactivity. Hyper- reactivity exists only during acute exacerbations of airway obstruction. ACKNOWLEDGEMENTS I wish most of all to thank the two members of my Graduate-Residency Committee who made this work possible, Drs. N. Edward Robinson and Frederik J. Derksen. ~To Dr. Robinson goes the credit for sparking and directing my interest in respiratory physiology early in my graduate program. My thanks go to Dr. Derksen for his enthusiasm and timeliness in giving assistance and constructive advice. I would also like to thank the rest of my committee, Drs. Mark Kittleson, George Eyster, and Robert G. Schirmer for providing assistance whenever it was requested. The technical expertise of Ms. Roberta Milar and Ms. Stephanie Berney was invaluable and is much appreciated. 1i II. III. IV. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION GENERAL LITERATURE REVIEW A. Bronchial Hyperreactivity in Asthma B. Animal Models of Airway Hyperreactivity 1. Specific antigen-induced bronchial hyperreactivity 2. Hyperreactivity asSociated with inflammation 3. Nonspecific airway hyperreactivity C. A Pony Model of Bronchial Hyperreactivity AIRWAY RESPONSES TO AEROSOLIZED METHACHOLINE AND CITRIC ACID IN PONIES WITH RECURRENT AIRWAY OBSTRUCTION (HEAVES) A. Methods 1. Pulmonary function measurements 2. Airway reactivity measurements 3. Statistical analysis B. Results 1. Lung function 2. Responses to aerosolized methacholine 3. Responses to citric acid aerosol C. Discussion SUMMARY AND CONCLUSIONS LIST OF REFERENCES iii iv 23 24 29 30 3O 35 38 43 45 LIST OF TABLES Table Page 1. Lung function of principal and control ponies at each 30 measurement period iv LIST OF FIGURES Figure 1. Schematic representation of instrumentation used for measurement of pulmonary function parameters. Diagrammatic representation of equipment used to generate and deliver aerosols of methacholine. Relationship between methacholine dose and dynamic compliance (Cdy n), pulmonary resistance (RL), and respiratory frequency (f). Doses of methacholine required to reduce dynamic compliance to 65% of the post- saline value (EDGSC W) and to double pulmonary resistance (EDzooRLg are shown. Also shown are RL and f at the ED,5Cdyn methacholine dose. Dose-response curves of Controi Pony 25 (left panel) and Principal Pony 5 (right panel). Pulmonary resistance (RL) and dynamic compliance (Cdy n) are shown as a func- tion of increasing methacholiney dose at 2 time periods. At Period A (closed circles), Principal Pony 5 was in clinical remission. At Period B (triangles), Principal Pony 5 had acute airway obstruction. Arrows indicate ED,5Cdy n. the dose of methacholine required to reduce n to 65% of the value observed after aerosol saline (cTrcled symbols). In Principal Pony 5, RL did not increase in response to methacholine at Period A; Engscdy n decreased at Period 8 in the principal, but not in they control pony. Logarithm of the concentration of methacholine required to reduce dynamic compliance to 65% of the value obtained after aerosol saline (log ED,5C W") in principal and control ponies. Methacholine concentration was in mg/ml; A, B, C, and 0 indicate different time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period 8 (asterick = significant dif- ference of principal group from Period A value; dagger = significant difference between principal and control groups within a time period). Page 24 26 28 32 33 Figure 10. Change in pulmonary resistance and dynamic compliance induced by 0.1 mg/ml methacholine aerosol (ARL0.1 and A%CWn 0.1, respectively); A, B, C, and D indicate dif- ferent time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (asterick = significant difference of principal group from Period A value; dagger = significant difference bet- ween principal and control groups within a time period). Pulmonary resistance at an ED,5Cd n dose of methacholine in principal and control ponies; , B, C, and D indicate different time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (asterick = significant difference of principal group from Period A value; dagger = significant difference bet- ween principal and control groups within a time period). Dynamic compliance (CW n) of principal and control ponies_ after aerosolized saline and 10% citric acid; A, B, C, and 0 indicate different time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B. Saline and citric acid failed to alter Cdyn in either group of ponies. Pulmonary resistance (RL) of principal and control ponies after aerosolized saline and 10% citric acid; A, B, C, and 0 indicate different time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B. Saline failed to alter RL but citric acid increased RL in principal ponies, particularly at Period B. Change in resistance (ARL) after aerosolized saline (upper panel) and 10% citric acid (lower panel) in prin- cipal and control ponies; A, B, C, and D indicate dif- ferent time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (dagger = significant difference between principal and control groups within a time period). vi Page 33 34 35 36 37 I . INTRODUCTION Asthma is a common respiratory disease that affects between three and seven percent of the American population,1:z and annually causes over four thousand deaths in this country.3 It is a leading cause of morbidity among infants and children and is responsible for more lost schooI days in the United States than any other singie chronic condition.4 __ Despite the impressive degree of morbidity and mortality‘ that exists with asthma, itv s StTII true that "in almost no other field is the gap between diagnostic and therapeutic knowledge and its general aPPIication so great."5 Eberle, a Philadelphia physician, defined asthma in 1830 as a "paroxysmal affection of the respiratory organs, characterized by a great difficulty of breathing, tightness across the breast, and a sense of impending suffocation, without fever or local inflammation."6 Despite tremendous strides forward in our understanding of the pathophy- siology of the respiratory tract since that time, bronchial asthma con- tinues to defy rigorous definition. In 1962, the .American Thoracic Society attempted to define asthma in terms of the clinical and func- tional abnormalities that occur.7 By this definition, asthma is "a disease characterized by increased responsiveness of the airways to various stimuli, manifested by prolongation of forced expiration, which changes in severity either spontaneously or as a result of therapy." This definition, by focusing attention on airway hyperreactivity as an essential feature of the disease, has paved the way for two and one half decades of intensive research on the physiological, immunological, biochemical, and pharmacological basis for this response. Widespread application of pulmonary function testing and bronchoprovocation tech- niques has made it possible to identify asthmatic patients who do not exhibit the classical signs of episodic wheezing and dyspnea. Unfortunately, the definition is sufficiently nonspecific that it also allows the criteria for asthma to be met when other pathological pro- cesses are clearly present.8 An additional problem with this definition arises from the observation that some asthmatic individuals, especially those who develop the condition late in life or suffer from it for many years, show little or no response to currently available treatments.9 As a result of such limitations, there is still no agreement on a uni- versal definition of asthma. Clearly, a more complete understanding of the physiological and pathological abnormalities that occur in bronchial asthma is needed to resolve this dilemma. Physiological studies on healthy volunteers and on asthmatic patients have greatly expanded our knowledge about this complex disease. There are, however, limits to which this type of investigation can be carried. Studies on the structural derangements in asthmatic lungs are largely based on information from the relatively few patients who die in status asthmaticus.10 It. is unlikely, therefore, that. we have an accurate picture of the changes that occur in the airways of patients who have mild to moderately severe asthma.10 As these limitations are reviewed, it is clear that there is a need for an animal model that parallels the clinical, physiological, and pathological abnormalities of human asthma. The dilemma posed by the difficulty in defining asthma becomes apparent when one considers that "a disease can be modelled only when the disease itself is adequately defined."11 Despite the problems involved in defining this particular disorder, it is widely accepted that the common denominator underlying the asthmatic diathesis is a nonspecific hyperirritability of the tracheobronchial tree.1 Clinical and animal research has revealed important clues as to the mechanisms of altered airway responsiveness in asthma. Evaluation of the currently available animal models of bronchial hyperreactivity has lead to the conclusion that recurrent air- way obstruction, or "heaves," in horses and ponies "may be the closest truly spontaneous disease yet described in animals that parallels the human disease of asthma."8 The purpose of the study described in this thesis was to further investigate the phenomenon of airway hyperreac- ,tivity in ponies with heaves by characterizing their response to aerosol delivery of a cholinergic agonist (methacholine) and a nonspecific irri- tant receptor stimulant (citric acid). II. GENERAL LITERATURE REVIEW A. Bronchial Hyperreactivity in Asthma From an etiological standpoint, asthma is a heterogeneous disease. It is useful for epidemiological and clinical purposes to classify the forms of this disorder by the principal stimuli that incite or are asso- ciated with acute episodes. The pathogenetic mechanisms responsible for producing the clinical signs of asthma appear to be multiple and complex. -It is important to emphasize, however, that nonspecific airway hyperresponsiveness is the central common~ feature yin. all forms of asthma. Thus, the distinction between various types of asthma may be artificial, and the response of patients within a given subclassifica- tion may be initiated by more than one type of stimulus.1 With this reservation in mind, asthma can be described in two broad groups: allergic and idiosyncratic.1 Allergic or extrinsic asthma usually affects children and young adults, and is frequently seasonal. This form of asthma is believed to occur as a result of inhalation, or less often ingestion, of an antigen that reacts with an immunoglobulin E (IgE) antibody.6 Allergic asthma is often associated *with a personal or family history of allergic diseases, a positive response to intradermal skin testing, increased serum concentrations of IgE, and/or a positive response to provocation tests involving the inhalation of specific antigen.12 Based on these defined immunological criteria, however, a significant segment of the asthmatic population cannot be readily classified. These patients are said to have idiosyncratic or intrinsic asthma. Bronchoconstriction in patients with idiosyncratic asthma can be triggered by diverse stimuli such as exercise, laughter, aspirin ingestion, and emotional stress.1 The observation that allergic patients exposed to allergens can develop both early and late reactions in the respiratory tract dates back more than a century.13 The early or immediate asthmatic response (EAR) occurs 10-30 minutes after allergen exposure and represents an IgE-mediated reaction to allergen.14 This aspect of the biphasic response has proven to be relatively easy to reproduce in experimental animals. Consequently a variety of in vivo model systems are available for study. Bronchospasm and edema characterize this phasé of the asth- matic response.15 A characteristic of the EAR is that beta-adrenergic agonists are effective in preventing or reversing bronchoconstriction.16 The late asthmatic response (LAR) occurs 2-12 hours after allergen expo- sure and appears to be largely due to the inflammatory component of allergic asthma.14:15 An important initial observation was made by Herxheimer17 in 1952, when he pointed out that patients with LARs had more severe asthma than patients without these reactions. It has sub- sequently been found that allergen-induced LARs are associated with a persistent increase in bronchial responsiveness to histamine and methacholine, while subjects with only EARs after inhalation challenge do not have this heightened responsiveness.18 As the foregoing discussion would imply, inhalation challenge testing has provided both the physician and researcher with a valuable tool. It is equally as useful for the identification and monitoring of asthmatic patients as it is for in vivo investigations into the pathogenesis of the disease. Bronchoprovocation may be defined as the administration of a stimulus to susceptible individuals, followed by measurements of the resulting bronchospasm.19 Inhalation challenge is a method of testing for bronchial reactivity following inhalation of either a specific antigen to which the patient may be sensitive or a "nonspecific" pharmacological agent. Early reports indicated that bronchospasm could be precipitated in asthmatic patients by injections of pilocarpinez0 and histamine.21 It has subsequently been shown that other drugs, including serotonin,22'24: bradykinin,25»26 prostaglandin an,27:23 and various cholinergic agents such as acetylcholine, methacholine, and carbachol29 have the same effect. Other stimuli such as exercise,30 rapid respiratory maneuvers,31:32 or inhalation of citric acid,33 dust,32:34 distilled water,35 or cold air35 can also provoke bronchoconstriction in susceptible individuals. Standardization of procedures for performing inhalation challenge testing using histamine and methacholine has greatly improved our understanding of asthma, and of bronchial hyperreactivity in particular.37'44 A variety of pulmonary function parameters can be serially evaluated in human subjects during inhalation challenge testing. A dose-response curve to the inhaled agonist is created by plotting the pulmonary function parameter of interest against the cumu- lative dose of agonist administered.19 The parameter used most widely to indicate a positive test is FEV1 (forced expiratory volume at 1 second). The provocative dose that causes a fall in FEV1 of 20% from the lowest post-saline value is determined from the dose-response curve by interpolation.19’38’39 By definition, asthmatics have hyperreactive airways, that is, they characteristically develop bronchoconstriction to a greater extent in response to smaller concentrations of various sti- muli than do healthy individuals.45 Over 99% of patients with current symptomatic asthma have a bronchoconstrictor response to an inhaled sti- mulus that is outside the normal range. Their sensitivity to an agonist such as methacholine varies from one hundred to several thousand times that of normal subjects.40 In establishing the diagnosis of asthma, bronchial provocation tests are more sensitive than standard pulmonary function tests when the latter are close to normal.46 The mean sen- sitivity of former asthmatics to inhalation challenge is approximately one tenth that of current asthmatics.41 In general, there is good agreement between bronchial hyperresponsiveness and the clinical severity of asthma.47 Concordance between airway reactivity and asth- matic state is not total, however. For example, not all patients with occupational asthma are hypersensitive to methacholine.48 Conversely, nonasthmatic patients with hay fever are nearly as sensitive to PGan as asthmatic patients, although the two groups are clearly separable with histamine or methacholine challenge.28 It has been recognized for some time that bronchial hyperreactivity is not unique to asthma.49:50 Nonspecific bronchial hyperreactivity is encountered in many patients with allergic rhinitis,37:49:50 chronic obstructive bronchitis,51 and in normal subjects following some viral upper respiratory tract infections.33 A very small proportion of healthy individuals may also have hyperreactive airways.37:41:52'54 Airway responsiveness can be temporarily heightened by events that cause inflammation. These include vaccination,55 natural exposure to allergens,56 specific occupational chemical sensitizers,48:57»58 and inhalation of ozone,59:60 or noxious fumes such as chlorine,38 nitrogen dioxide,61 and sulphur dioxide.62 The induction of increased bronchial reactivity by such means offers a way to reproduce this phenomenon, but these studies are probably only relevant to the study of asthma if the acquired bronchial hyperreactivity resembles the "endogenous" hyperreac- tivity associated with the disease.29 Acquired and endogenous hyperreactivity differ in both degree and duration, the bronchial sen- sitivity in asthma being more severe and persistent.29a40 In asymp- tomatic patients with a history of asthma, hyperreactivity is often present for over a year and may persist for up to 20 years.40:63964 It may be necessary for asthmatic subjects to be repeatedly exposed to antigen in order for bronchial hyperreactivity to persist.29 Cockcroft and co-workers65 surveyed the bronchial responsiveness to inhaled histamine in a large, randomly selected population.- They concluded that the distribution of histamine threshold values was unimo- dal, the asthmatic subjects representing a subgroup within the hyperresponsive distribution tail rather than a separate distribution peak. Such a unimodal distribution supports the concept of asthma as a heterogeneous polygenic and environmental disease. It also lends cre- dence to a continued search for common pathophysiological mechanisms to account for hyperreactivity of the tracheobronchial tree under a diver- sity of normal and abnormal conditions, and to the use of animal models of bronchial hyperreactivity to investigate the mechanisms involved. B. Animal Models of Airway Hyperreactivity It has been said that bronchial asthma is a disease that can be characterized clinically by reversible airway obstruction, physiologi- cally by increased bronchial reactivity to specific and/or nonspecific stimuli, and pathologically by airway inflammation.66 The relationship of' the clinical signs to the physiological changes has been partly addressed in the foregoing discussion. A number of animal models have been employed in an attempt to understand the link between the physiolo- gical and the pathological alterations seen in human asthma. 1. Specific antigen-induced bronchial hyperreactivity Bronchospasm induced by specific antigen challenge has beenhthe basis of most animal models of asthma. The traditional immunological model of antigen-induced airway constriction has been the guinea pig, which is easily sensitized to foreign proteins and in which the lung is the primary target organ in the anaphylactic reaction.67'7z Most such studies have used antigens such as bovine or egg albumin, which bear little resemblance to those incriminated in human extrinsic asthma. In addition to the guinea pig, other species including the sheep,73‘75 the monkey,75'79 and the d0980'89 have been used to assess airway reactivity and pulmonary mechanics in antigen-induced bronchoconstriction. In all allergic bronchoconstriction models, sensitized animals respond to anti- gen aerosol with increases in respiratory frequency and pulmonary resistance and decreases in dynamic lung compliance.90 These abnor- malities are associated with decreases in arterial oxygen tension.83 When functional parameters after antigen challenge have returned to nor- mal, bronchial responses to stimuli such as histamine, acetylcholine, and propranolol may be exaggerated.72 10 Dogs are often natively allergic to Ascaris .51le as a result of prior parasitic infestation with roundworms, which cross react with A, m. If this antigen is administered to a sensitized dog as\\‘an aerosol, airway constriction can occur.30'82 The systemic administra- tion of histamine produces physiological changes similar to those induced by specific antigen challenge.35 Histamine is released after systemic and local airway administration of specific antigen in allergic (1095.91'93 Similax- to the effects in human beings, canine plasma histamine levels correlate with circulatory and respiratory changes that occur during these reactions.85 Pretreatment with a combination of H1 and H2 antihistamines is not effective in preventing or altering speci- fic antigen-induced reactions.94 Studies by Bleeker and co-workers85 have shown that the products of arachidonic acid metabolism may be important in modulating immediate hypersensitivity, as the release of immunological mediators (PGDZ and leukotriene C4) into plasma and bronchoalveolar lavage fluids is temporally related to the development of hypotension and bronchospasm, as well as to an increase in the number of neutrophils in respiratory secretions. As the A_.~ suum sensitive canine model is only remotely related to clinical circumstances, considerable interest has been shown in the sub- set of the canine population that are ragweed-sensitive.81:85'89 A well-defined atopic disease entity occurs spontaneously in dogs.95:96 Ragweed-sensitized dogs and some spontaneously atopic dogs show IgE-mediated hypersensitivity reactions that bear many similarities to human allergic asthma.80:86:87:97 Common features include immediate and late—phase cutaneous responses, an immediate airway response after inha- 11 lation of specific antigen aerosol, and increased airway responsiveness to inhaled histamine and methacholine.37,95'98 Although the spontaneous occurrence of antigen-induced airway constriction mediated by an IgE-like antibody makes this an attractive model of asthma, it is necessary to screen large numbers of mongrel dogs to obtain a colony of such animals.99 Even when dogs from an established colony are used, approximately 35% of individuals do not demonstrate any increase in air- way reactivity after antigen exposure.86 Attempts to induce reaginic hypersensitivity in adult mongrel dogs have met with varying degrees of success.99 Success in inducing such hypersensitivity has been obtained using puppies from atopic parents and starting the immunization program at 4 weeks of age.100:101 In this situation, some correlation can be seen between skin and airway. responses to antigen.101 Ragweed- sensitized dogs exhibit nonspecific airway hyperresponsiveness for up to 4 months following antigen challenge,86 but a late-phase broncho- constriction has not been observed in such dogs.99 While immediate IgE-mediated reactions to allergen challenge are common in atopic asthmatics, the late phase responses are less common and poorly understood. It has been speculated that these late phase changes, which are poorly responsive to bronchodilators and often require corticosteroids for resolution, are important in the subacute and chronic symptoms of the more severely affected asthmatics.102 The late response can prevented by the administration of cromolyn sodium or corticosteroids prior to antigen challenge.13:16a103 The finding that inflammatory mediators are released during late-phase reactions in asth- matics after specific antigen exposure104 and after exercise105 or inha- 12 lation of cold dry air,106 suggests that the release of inflammatory substances may be central to the pathogenesis of disease in a much larger proportion of asthmatic patients than only those who experience symptoms immediately after exposure to a specific antigen.107 Late skin reactions that occur after allergy testing in man may be different from the LAR as late skin reactions do not occur in the absence of an early reaction.46 It is of interest nonetheless, to examine the results of studies that suggest late-phase cutaneous responses represent an IgE-dependent response.108»109 Late reactions would appear to be a more suitable model for the study of asthma than are acute reactions to antigen. To date, late- phase bronchoconstriction following speCific antigen challenge has only) been reproduced experimentally in conscious rabbits110 and sheep,111 although the response can also be simulated with nonantigenic agents such as toluene diisocyanate.8:48 In the rabbit model described by Shampain and colleagues,110 neonatal rabbits were immunized with Alternaria tenius extract and then boosted regularly. This immunization schedule takes advantage of the fact that neonatal rabbits exposed to antigen within 24 hours of birth will develop no detectable antibody response other than IgE.112 Such "IgE" rabbits exhibit changes in pulmonary function when challenged with aerosolized Alternaria extract at 3 months of age. An increase in pulmonary resistance and decrease in dynamic compliance begins within 15 minutes of challenge. These parame- ters again approach baseline or reach a plateau after 30 minutes and a second phase of bronchoconstriction begins after 105 minutes and lasts through the sixth hour. Rabbits immunized first at 7 days of age make 13 multiple antibody isotypes. The presence of IgG blunts, rather than enhances, pulmonary responsiveness. Further evidence in support of IgE mediation of the biphasic response was provided by transfusion of plasma containing anti-Alternaria IgE into control rabbits, producing, upon challenge, both early and late responses in pulmonary mechanics. These results are consistent with the report that asthmatic patients have increased levels of neutrophil chemotactic factor (a macromolecule asso- ciated with mast cell degranulation and IgE-mediated bronchoconstric- tion) during the late response.113 Abraham and co-workers111 documented that a dual response to inha- lation challenge with specific antigen occurs in conscious sheep with naturally-occurring Ascaris suum hypersensitivity. All animals selected for use in this study exhibited both a cutaneous reaction to intradermal injection of A, sggm extract and a bronchoconstrictor response to pre- vious inhalation challenge with A_._ M antigen. By 5 hours after challenge, mean specific pulmonary resistance and arterial oxygen ten- sion have returned to baseline values, but specific resistance increases again from 6.5 until 8 hours post challenge. Inhalation of cromolyn sodium prior to antigen challenge blocks both early and late responses, and parenteral steroid therapy 3 hours post challenge blocks the late response. The time course of both the immediate and late phase respon- ses approximates the time course observed in asthmatics, and phar- macological modification of both responses in allergic sheep is similar to that found in human patients. An ingenious in vivo model of the allergic reaction in man has recently been develOped.114 This model involves nasal challenge of an 14 allergic subject with specific allergens, followed by repetitive washing of the nose with a saline solution. Both early and late reactions are associated with the release of histamine, kinins and TAME (tosyl argi- nine methyl ester)-esterase activity into nasal washes.107 Prostaglandin 02 is liberated after the acute, but not the late phase response.115 Since P602 is a mast cell but not a basophil product, it seems likely that the basophil may play an important role in the late reactions.107 The observation that mast cells and basophils differ in their responsiveness to corticosteroids,115:117 is consistent with the fact that the acute response to antigen is not blocked by steroid pretreatment, while late reactions are sensitive to such intervention.103:107 This work indicates that IgE-mediated stimuli can initiate a series of events that can have a prolonged physiological response. Comparison of in vitr0113'120 and in vivo studies implicates the mast cell in acute reactions and the basophil in late reactions.107 2. Hyperreactivity associated with inflammation Bronchospasm induced by specific antigen challenge in a variety of animal species has been the basis of the models of asthma discussed to this point. Other models rely on changes induced by acute lung injury to provoke bronchial hyperreactivity. Pulmonary injury can be created by methods such as infusion of endotoxin or inhalation of air pollu- tants. Various species, particularly the dog,45’121 have been used in ozone-exposure studies. It has been found that airway responsiveness does not increase in all dogs after exposure to ozone, but only in those animals that have an increase in the number of neutrophils in the epithelium of the proximal122 and distal123 airways. As the development 15 of hyperresponsiveness and inflammation appear to be so closely related, this model continues to be used to further define this association. Unfortunately, when hyperresponsiveness develops after ozone exposure in this canine model, it is short-lived.124 This is in contrast to results obtained in healthy volunteers, 25% of whom have persistence of hyper- reactivity for more than one week post ozone exposure.125 The development of techniques for bronchoalveolar lavage has allowed for serial assessment of pulmonary inflammation in individual animals. Using this technique, Fabbri and coworkers]?3 determined that the level of bronchial responsiveness induced in dogs by ozone exposure was correlated with the number ofhneutrophils in the lavage fluid. Nadel Wand colleagues45. performed an elegant series of studies to investigate whether airway epithelial inflammation is the cause of ozone-induced hyperirritability. When circulating neutrophils were depleted in dogs prior to ozone exposure, the expected hyperreactivity was prevented.126 11> test the hypothesis that neutrophils release mediators that induce airway hyperresponsiveness, circulating neutrophils were harvested, activated in vitro and the supernatent removed.45 When this supernatent was aerosolized into the airways of healthy dogs, hyperresponsiveness of the airway smooth muscle resulted.121 .After finding that leukotriene B4 (LTB4), a leukotriene with potent neutrophil chemotactic properties, is released in signifi- cant quantities from inflamed canine tracheal epithelial cells,127 hyperirritability was reproduced in normal dogs by aerosol administra- tion of LTB4.45 Reproduction of airway hyperreactivity by this mediator suggests an important role for mediators released by the activated neutrophil in inducing hyperresponsiveness. 16 The guinea pig has been used extensively in studies examining the relationship between mucosal permeability and airway reactivity. Cigarette smoke is known to injure the airway' mucosa, resulting in migration of neutrophils into the airway epithelium within 4 to 6 hours.”8 Further investigations in the guinea pig resulted in the conclusion that the observed increase in airway reactivity occurs during the fluid exudative phase of the inflammatory response before the neutrophils migrate into the epithelium.129 'This conclusion is in agreement. with data that showed that smokers have increased airway permeability130:131 that is not associated with increased airway reactivity.132 This complex relationship between permeability and reactivity has been further studied in guinea pigs using intravenous and aerosol administrations of leukotrienes C4, D4, and E4, components of the slow-reacting substance of anaphylaxis.133 There are significant similarities between guinea pig134 and human135 studies. The leukotrienes produce potent, prolonged bronchoconstriction in both spe- cies. The time course of onset and resolution of bronchoconstriction is comparable to that elicited by inhaled antigen in each species, and is compatible with the leukotrienes having a causative role in allergic asthma.136 3. Nonspecific airway hyperreactivity It has been recognized for some time that the hyperreactivity that develops following specific antigen challenge, despite being charac- teristic and reproducible in individual animals, is variable from animal to animal.72 This latter characteristic is of interest in comparing animal models to human asthma. Studies by Douglas and colleagues70.137 17 suggest that there is a log normal distribution of nonspecific respon- siveness to inhaled chemical mediators in guinea pigs, a situation ana- lagous to that found in a random human population55 and in (1095.138 Attempts have been made to reproduce the human disease more closely by selecting animals at the hyperresponsive end of the distribution tail.139 Another interesting analogy with the situation in human asthma is the apparent genetic predisposition for guinea pigs to develop nonspecific bronchial hyperresponsiveness as suggested by the data of Takino and co-workers.139 Aflthough rats have not been used as exten- sively as guinea pigs in airway reactivity studies, an inbred line of hyperreactive rats has also been developed by selecting animals at the hyperrespOnsive end of the distribution curve.140 Further support for the hypothesis that acquisition of hyperreactivity requires both a gene- tic predisposition to the condition and an inciting event is provided by the work of Wardlaw,141 who reported that injection of pertussis vaccine induced hypersensitivity in some strains of mice and not in others. Considerable interest has been centered on selecting and breeding a population of dogs that possess both nonspecific airway hyperreactivity and allergic inhalant dermatitis (atopy). A line of basenji-greyhound crosses has been shown to possess these characteristics.99 Marked air— way hyperreactivity to methacholine, histamine, leukotriene D4, and citric acid has been demonstrated in these basenji-greyhound dogs com— pared to mongrel dogs of similar size.142:143 Moreover, the offspring of these basenji-greyhound dogs are also hyperreactive to methacholine and citric acid, whereas the offspring of allergic dogs lacking airway hyperreactivity are similarly unreactive to these stimuli.144 In the 18 basenji—greyhound dog, aerosol challenge with specific antigen results in changes in airflow resistance and dynamic compliance of far greater magnitude than has been reported in the mongrel dog.99 Additionally, increases in residual lung volume associated with antigen-induced bronchoconstriction occur in some of these basenji-greyhound dogs. Although the basenji-greyhound dog model has many similarities to human asthma, late bronchoconstriction following antigen challenge does not occur, and the bronchial hyperreactivity that these dogs exhibit is not associated with any clinical disease.99 In addition, inhalation challenge testing on these dogs requires general anesthesia, a factor that may depress airway smooth muscle responses.145.146 C. A Pony Model of Bronchial Hyperreactivity Spontaneous attacks of asthma-like signs are exceedingly rare in any nonhuman species except equids. The clinical signs associated with "heaves" or "broken wind" have been recognized since at least 333 B.C., when Aristotle described the characteristic active expiratory "heave" in affected horses.147 'The comparative importance of heaves to human asthma was first alluded to in 1726.148 AJthough Percivall warned in 1853 that many horses with broken wind did not have emphysema, St6mmer and others described the pathological changes in "heavey" horses in terms of their similarities to those 'hi. human patients with emphysema.148 It was almost a century before several investigators more accurately characterized the major pathological change in the lungs of affected horses as being a bronchiolitis.149:150 Over a combined obser- vation period of 23 months, Lowell151 recorded 16 attacks among 6 heavey horses while hay was being fed, whereas there were only 3 attacks in a 19 combined total period of 54 months when hay was not fed. Lowell also observed that severe manifestations of heaves could appear and disappear within a period of days upon manipulation of the diet. He concluded that the episodes of dyspnea were a result of transient bronchial obstruction caused by hypersensitivity to some component of hay, to which horses were exposed by inhalation. Evidence for an allergic etiology for the disease,151'154 combined with the facts that equine lungs are structurally similar to human lungs,155 and that airway inflammation is the characteristic pathological alteration in both asthma and heaves,156 provided justification for evaluating horses or ponies with heaves as a model for studying the pathophysiology of human asthma. The work of Alexander,157 Spbrri and Leeman,158 Gillespie and colleagues,15'39:160 McPherson and Lawson,161 Muylle and Oyaert,162 and Sasse163 characterized the pathophysiological changes that occur during spontaneous exacerbations of heaves. Increased work of breathing, increased change in intrapleural pressure (APpl) during tidal breathing, decreased dynamic compliance, increased pulmonary resistance, prolonged nitrogen washout, decreased maximal expiratory flows and hypoxemia have been consistently demonstrated. Such indicators of diffuse small and large airway obstruction lead Sasse to introduce the term "chronic obstructive pulmonary disease" (COPD) to the equine medical literature.163 Measurement of maximum APpl and arterial oxygen tension was suggested to establish diagnostic criteria for COPD.161 The observation that human patients with bronchial asthma are more sensitive to histamine than normal subjects was published by Weiss and 20 co-workers21 in 1929. Andberg, Boyd and Code164 demonstrated that the intravenous administration of histamine to normal horses would induce a complex of clinical signs closely resembling those seen in heaves. Obel and Schmitterliiw165 compared the histamine sensitivity of horses suf- fering from heaves with normal horses. In a landmark paper, they reported that affected horses exhibit bronchial hyperreactivity in response to intravenous histamine administration. Following work to determine the pulmonary effects of intravenous histamine administration in the conscious normal pony,166 Derksen and colleagues167 more fully characterized the changes in pulmonary mechanics that occur following intravenous histamine administration in ponies with a history of heaves. Dose-response curves were generated for affected ponies during periods of clinical remission, during acute dyspnea induced by barn exposure, and during recovery. It was found that bronchial reactivity differed between affected and control ponies only when ponies with a history of heaves had acute airway obstruction. Bronchoprovocation testing by specific inhalation challenge is known to produce bronchoconstriction in affected horses and ponies. As Lowell151 and others have shown, a crude natural inhalation challenge can be carried out by exposing horses to a barn environment, par- ticularly one containing moldy hay or straw.147 The onset of clinical signs following exposure to allergen can be sudden or gradual and shows considerable variability between individuals. The earliest response of an asymptomatic but affected horse to challenge occurs in about 2 hours. The more obvious "heaving" occurs 4 to 10 hours after exposure,”7 a time course similar to the LAR observed in asthmatic patients. Affected 21 horses are also sensitive to nebulization of culture extracts of speci- fic thermophilic actinomycetes (Micropolyspora faeni) and fungi (Asperqillus fumiqatus).147.154.168 It is interesting to consider the results of surveys for the pre- sence of serum precipitating antibodies and studies on intradermal testing for antigen sensitivity. Lawson et al.168 found that precipi- tating antibodies to M; faggi and A; fumigatus are present in the sera of both normal and COPD-affected horses, and many heavey horses without detectable precipitins respond clinically to inhalation challenge with those antigens. A positive reaction to an intradermal injection of antigen is evidence that a horse has been previously exposed to that antigen. Significantly more heavey horses show skin hypersensitivity to M; fagfli than do normal horses, and 90% of COPD cases with skin hyper- sensitivity to L flign_i also show bronchial hyperreactivity to this antigen.154 Using other antigens, positive skin reactions are as likely to occur in normal horses as in those with COPD. Halliwell and co-workers153 found a higher percentage of positive responses at 4 hours in affected horses compared to controls, but a positive response to spe- cific bronchial challenge was far more readily induced in animals with strong 30 minute reactions than those with strong 4-hour ones. Apparently, intradermal skin testing for antigen sensitivity is unre- liable as a means of identifying affected horses that are in clinical remission at the time of examination.147 Considering the many similarities between equine heaves and human asthma, it seemed logical to ask whether horses or ponies with a history of heaves exhibit that central common feature of asthma, nonspecific 22 airway hyperreactivity. To determine this, we measured lung function and airway reactivity to histamine administered by aerosol in two groups of ponies.169 .Affected and control ponies were studied at four time periods: when affected ponies were in clinical remission, following barn housing (when affected ponies had acute airway obstruction), and at one and two weeks after return to pasture. Ponies with a history of heaves were found to be hyperreactive to aerosolized histamine during acute exacerbations of their disease, but not during clinical remission. Histamine dose-response curves for dynamic compliance, pulmonary resistance and respiratory frequency were generated for each time period. The dose of histamine required to reduce dynamic compliance to 65% of the value obtained after saline challenge (EDSSCdyn) was calcu- lated by interpolation from these curves. During the period of acute airway obstruction, 50,,cdyn decreased by' 2.5-log' doses in affected ponies, but was unchanged in controls. In individual affected ponies, EDsstyn decreased by as much as 4.5 log-doses between studies performed during the period of clinical remission and following barn exposure. This study provided conclusive evidence that affected ponies have hyperreactive airways during periods of acute airway obstruction. The following study' was undertaken to determine whether the airways of affected ponies would similarly hyperreact to other nonspecific stimuli, namely methacholine and citric acid. III. AIRWAY RESPONSES TO AEROSOLIZED METHACHOLINE AND CITRIC ACID IN PONIES WITH RECURRENT AIRWAY OBSTRUCTION (HEAVES) A. Methods Five ponies (19.0 +/- 3.9 years of age and weighing 201.6 +/- 16.8 kg) with a history of heaves were designated as the principal group. They were matched for age, gender, and weight with 5 ponies (control group) (17.8 +/- 2.1 years of age and weighing 195.8 +/- 18.7 kg) historically free of respiratory disease. Chronic tracheostomies were created in the midcervical region, and a carotid artery was relocated to a subcutaneous site. All animals were kept at pasture and were not exposed to hay, straw, or a barn environment for at least 2 months prior to the first measurements of airway reactivity. All principal ponies were in clinical remission at the end of this period. For investigations of airway reactivity, principal and control ponies. were paired. To ensure that. both ponies received 'the same environmental exposure, each pair was always transported together, housed together, and studied on the same day. Baseline pulmonary func- tion data were collected, and airway reactivity to methacholine and citric acid was assessed in all ponies after' 2 months at pasture (Period A). Each pair was then housed in a barn, bedded on straw, and fed hay. An attempt was made to increase exposure to hay dust by shaking the hay in the stall 3 times daily. When the principal pony developed clinical signs of airway obstruction, pulmonary function and 23 24 airway reactivity were measured in both ponies (Period 8). Animals were then returned to pasture and studied 1 week (Period C) and 2 weeks (Period 0) after barn exposure. 1. Pulmonary function measurements Ponies were tranquilized with xylazine (Rompun; Haver Lockhart, Shawnee Mission, KS) (0.5 mg/kg of body' weight) and restrained in stocks. Each pony was intubated via a tracheostoma with a cuffed endotracheal tube (20 mm internal diameter, 45 cm long). A pneumo- tachygraph (Fleisch no. 4; Dynasciences, Blue Bell, PA) and an asso- ciated pressure transducer (Model PMS; Statham Instruments, Hato Rey, PR) were attached to the endotracheal tube. This system produced a signal proportional to air flow (V) that was electronically integrated to give tidal volume (VT) (Figure 1). VT f (Zuni IRL Pu 002 POCO: Figure 1. Schematic representation of instrumentation used for measure- ment of pulmonary function parameters. After each experiment, the system was calibrated by forcing known volumes and flows of air through the pneumotachygraph, using a 3-L 25 calibrated syringe (Three L Super Syringe; Hamilton Syringe Co., Reno, NV) and a rotameter flow meter (Model 10A3500; Fischer and Porter Co., Warminster, PA). An esophageal balloon (10 cm length, 3.5 cm perimeter, 0.06 cm wall thickness) was sealed over the distal end of a polypropylene catheter (3 mm internal diameter, 4.4 mm outside diameter, 140 cm length) that had several spirally arranged holes in the part covered by the balloon. The distance from the nares of the ponies to the midthoracic portion of the esophagus was visually approximated and marked on the esophageal balloon catheter with indelible ink. The esophageal balloon was passed via the nares into the midthoracic portion of the esophagus. Balloon volume was adjusted to 0.5 ml of air. The balloon was attached to a pressure transducer (Model PM 131; Statham) that was taped to the fore- lock. Transpulmonary pressure (PL) was defined as the difference bet- ween atmospheric and esophageal pressure. Transpulmonary pressure, VT, and flow signals were observed on an oscilloscope (Model VR12; Electronics for Medicine, White Plains, NY) and recorded on light- sensitive paper, and were entered into a pulmonary function computer (Model 6; Buxco Electronics, Sharon, CT) and data logger (Model DL12; Buxco) for the calculation of dynamic compliance (Cdyn), pulmonary resistance (RL), respiratory frequency (f), and minute ventilation (V5). The frequency responses of the pneumotachygraph transducer system and the esophageal pressure transducer system were matched to prevent phase differences between pressures and flows. This was accomplished by comparing pressure recorded with the esophageal catheter and transducer against pressure recorded with the pneumotachygraph transducer on the XY plotter while exposing all devices to the same oscillating pressure 25 source. Frequency responses were checked up to 5 Hz and were flat.170 In carotid artery samples, arterial oxygen tension (Paoz), carbon dioxide tension (P3C0,)s and pH were measured immediately prior to air- way reactivity measurements using a blood gas analyzer (Model ABL3; Radiometer, Copenhagen, Denmark). 2. Airway reactivity measurements Methacholine and citric acid challenges were performed on adjacent days. Three pairs of ponies always received methacholine challenge on the day prior to citric acid challenge; the order was reversed for the other 2 pairs. The apparatus used for generation and delivery of methacholine aerosols to the pony lung is shown in Figure 2. ~2 Transducer A0 liters j;— 0 0 5 Non rebreaihing valve fl ‘ =LZ‘< 0.05, means from each measurement period were compared using Tukey's w procedure.171 B. Results 30 TABLE 1 LUNG FUNCTION OF PRINCIPAL AND CONTROL PONIES AT EACH MEASUREMENT PERIOD Measurement Period Parameter A 8 C 0 Principal ponies P802. torr 79 1 3 65 1 4 76 1 4 85 1 3 Paco,.torr 4111 4212 3914 391i Cdyn. L-cmH,O“ 0.57 1 0.08 0.21 1 003'? 0.48 1 0.08 0.42 1 0.07 RL. cmH,O-L"-s 0.95 1 0.121 2.37 1 0.32'T 0.86 1 0.17 1.07 1 0.16 VT. L 2.32 1 0.36 1.70 1 0.10 2.21 1 0.31 2.08 1 0.39 WAmM” $5156 wo1um ws141 85131 rmw “1143 ae1s4 M3121 $2135 Control ponies Pao,.torr 8213 7615 8015 8619 Paco, ion 39 1 1 43 1 i 41 1 i 38 1 3 Cdyn, L'cmH,O“ 0.72 1 0.16 0.88 1 0.18 0.82 1 0.20 1.06 1 022' RL. cmH,O-L“-s 0.52 1 0.08 0.44 1 0.10 0.45 1 0.08 0.73 1 0.12 VT, L 2.34 1 0.23 2.41 1 0.32 2.22 1 0.32 2.11 1 0.19 WJmM” @0122 $9121 $0116 @6148 me 02109 "1107 m4124 $9128 Definition of abbreviations: Pao2 a arterial oxygen tensnon; Paco, a: arterial carbon dioxide tension; Cdyn - dynamic compli- ance, RL . pulmonary resustance; VT :2 tidal volume; V: = minute ventilation;l 1 respiratory frequency. ' Significant ditierence trom Period A I Signilicant ditterence lrorn control group 1. Luna function Table 1 shows pulmonary function data of principal and control ponies at all measurement periods. At Period A, principal ponies had a slightly higher mean RL than control ponies, despite the lack of clini- cal evidence of airway obstruction. After barn exposure (Period 8), there was no change in any values in the control ponies, but a signifi- cant decrease in Cdyn and an increase in RL occurred in ponies in the principal group. Both values were significantly different from those in the control group at Period B. Although Pao2 decreased in all principal ponies between Periods A and B, the magnitude of the decrease was variable. A trend towards decreased VT was seen in the principal ponies 31 at Period B. After removal from the barn environment, there was impro- vement in pulmonary function in the principal ponies, with values for Cdyn and RL at Periods C and 0 similar to those at Period A. Dynamic compliance in the control group increased at Period 0, resulting in a difference from the principal group at this measurement period. 2. Responses to aerosolized methacholine All ponies consistently responded to aerosolized methacholine by a decrease in Cdyn compared with the reference value obtained after aero- solized saline. In 38 of 40 observations (10 ponies at 4 time periods), an increase in RL accompanied the decrease in Cdyn- Examples of dose- response curves of principal and control ponies at Periods A and B are - provided in Figure4. In Principal Pony 5 at Period A, the decrease in Cdyn was unaccompanied by an increase in RL. At Period 8, the decrease in cdyn occurred at a lower dose of methacholine and was then accom- panied by a large increase in RL. In Cbntrol Pony 25, a decrease in Cdyn occurred at a similar dose of methacholine at Periods A and B, and in both cases was accompanied by modest increases in RL. The mean log £0,5an of methacholine is shown in Figure 5. At Periods A, C, and 0, log EDsstyn did not differ between principal and control groups. At Period B, log EDHCWn decreased significantly in principal ponies compared with that at Period A and was significantly lower than in control ponies at the same time period. The mean change in RL and the percent change in Cdyn induced by exposure to an aerosol of 0.1 mg/ml methacholine are shown in Figure 6. In control ponies, ARL0.1 and A%Cdyn0.1 did not differ between measure- ment periods. In the principal group at Period B, ARL0.1 and A%cdyn0-1 32 .510: pouueou ogu :— so: use pengu:pua on» c— m newcmm um commosuov czvuaucw u< $0,503 an asp—osuaguom on omconma&.=p ommouucp no: uvu 4: .m age; punpuc—Lg cm .Am—camhw uo—ospuV mew—um panacea sways vo>somno o:—a> 0;» me mm» on .cavu weaves cu 008.190; mew—osumnuom no anew as» .cavumunw mama—cc? whoss< .covuuasamao autswu maaum cu: m age: —mnpu:vsm .Amopmcmwsuv m 00—50; a< .covmmpmou pea—:w—u e. an: m >10; punpocpsm .Amopug—u vomopuV < v0.10; u< .muo—Loa on,» N as once may—ozoesuom ncpmuouuc. he :evuucaw m an craze use Acavuv macaw—ameu uvmuczv wee Aggy museum—mos anneal—:1 .Apocnn acmpsv m ace; —an—ucpsm can Apoeaa «Copy m~ ago; poLucou Ce mo>g=o omcoamesiomea .v «Lam—m :Eoe $3010.25: .59.. 31.619115: 0.0 0.“ 0. —O. —OO. '000. 05:0 0.0 0., p. '0. p00. —000. 05..” A a J a t . q . 1‘ a JI a q I N. (u m. p 0 O @ Oquox. 1 1 i. 0 cwxeu: @ r... o. moses. cuxo nusxcom 0.0 0.— '. '0. '00. p000. oczmm 0.n 0.— p '0. «00. .000. 06:00 .I d d A d d \M H W m ' Q l 1 0 10; Qm\4|| 0.— OIIOIIITIIIIIIII 0:: < e < O. 9... 0.... 0.1.160 C . 06:..1. musxcoa . 1.... 1o 0 04.1.3 o n msxcoa l o; .m Figure 5. Figure 6. 33 2__ Eflannchnus [3(2Nflnfls LOO I" EDBSCdyn l % o,_1fiFpZa - 'l" . i '2‘ A B c o Logarithm of the concentration of methacholine required to reduce dynamic compliance to 65% of the value obtained after aerosol saline (log EDsstyn) in principal and control ponies. Methacholine concentration was in mg/ml; A, B, C, and 0 indicate different time periods. At Period A, prin- cipal ponies were in clinical remission. At Period 8, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (asterick = significant difference of principal group from Period A value; dagger = significant difference between principal and control groups within a time period). ARI. o" cmHzO Ilsec -40 ~ 229mmmoe D Control: A. Bu (3 ID Change in pulmonary resistance and dynamic compliance induced by 0.1 mg/ml methacholine aerosol (ARL0.1 and 81C? n 0.1, respectively); A, B, C, and 0 indicate different t me periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (asterick.= significant difference of principal group from Period A value; dagger = significant difference between principal and control groups within a time period). 34 were significantly different from those in the control ponies. These parameters also differed within the principal group between Periods A and 8. Pulmonary resistance at the EDuCWn methacholine dose for both groups at all measurement periods is shown in Figure 7. Pulmonary resistance at 50,,cdyn followed the pattern of baseline RL, always being higher in principal ponies than in control ponies and increasing signi- ficantly in the principal group in response to barn exposure. We calcu- lated the change in RL that occurred between saline exposure and RL at ED,,cdyn (ARLEDss). ARLED,5 did not change significantly between time periods and did not differ in control and principal animals. Respira- "tory frequency at EDsscdyn did not differ between groups or at any measurement period. . 1 4 %.Princ1pols Rl atED Ody"3 % DCOI‘IIVOIS 65 % cmHzo 2 % Ilaec % / 2 a Figure 7. Pulmonary resistance at an ED,5C n dose of methacholine in principal and control ponies; Afha, C, and 0 indicate dif- ferent time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (asterick = significant difference of principal group from Period A value; dagger = significant difference between principal and control groups within a time period). 35 3. Responses to citric acid aerosol Mean CWn for principal and control ponies after sequential saline and citric acid inhalation is shown in Figure 8. Inhalation of saline or citric acid had no effect on Cdyn in either group. a Principals .Controls 13 .\ “W B c “'5‘ D Cdyn V“ . A UcmH O 2 c .. “_.Ilillbflgil=Ets.zi--;=,.— . g 3 B 1 :7fiéi4 6 eufi£é4.6 810 m 20 25. 30 Saline citric acid POST- POST-CITRIC AC|D(min) SALINE(min) Figure 8. Dynamic compliance (C n) of principal and control ponies after aerosolized saline and 10% citric acid; A, B, C, and 0 indicate different time periods. At Period .A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period 8. Saline and citric acid failed to alter CW" in either group of ponies. Mean values for RL are shown in Figure 9. Neither saline nor citric acid caused a change in RL of control ponies at any measurement period. In the principal group, the response to citric acid was highly variable in both magnitude and time of occurrence. The biggest changes in ih_ after citric acid inhalation were seen in principal ponies at Period 8. 36 RI. \jS/w o Prncupols A Controls cmHzolllsec 20- 7436” efé‘ééfz‘éééib .‘5 2‘0 25 36 Saline Citric acid POST- POST-CITRIC ACID(mln) SALINE(min) op) 0 no Figure 9. Pulmonary resistance (kg) of principal and control ponies after aerosolized saline and 10% citric acid; A, B, C, and 0 indicate different time periods. At Period A, principal ponies were in clinical remission. At Period B, they had acute heaves. Periods C and D were 1 and 2 wk after Period B. Saline failed to alter R but citric acid increased RL in principal ponies, particularly at Period B. We calculated the maximalchanges in RL that occurred after saline inhalation (ARL saline) and after citric acid (ARL citric acid) (Figure 10). Maximal ARL citric acid occurred 2 nfinutes after citric acid inhalation in 3 ponies, 15 minutes after citric acid in a fourth, and the fifth pony had a biphasic response with peaks at 2 and 30 minu- tes. Split-plot analysis of data showed a significant overall dif- ference between principal and control groups in response to citric acid but not in response to saline. 37 Principals UControls Figure 10. Change in resistance (ARL) after aerosolized saline (upper panel) and 10% citric acid (lower panel) in principal and control ponies; A, B, C, and D indicate different time periods. At Period A, principal ponies were in clinical remission. At Period 8, they had acute heaves. Periods C and D were 1 and 2 wk after Period B (dagger = significant difference between principal and control groups within a time period . It can be seen in Figure 10 that ARL saline did not differ between or within groups at any time period. At Period 8, ARL citric acid was significantly greater in principal than in control ponies. Within the principal group, ARL citric acid was highest at Period B and declined significantly after removal from a barn environment (Periods C and 0). Exposure to citric acid aerosol did not affect respiratory rate in either group of ponies at any measurement period. 38 C. Discussion The exposure of principal ponies to a barn environment resulted in changes in lung function similar to those previously reported by our group.1571169 A reduction in Cdyn and an increase in RL reflect the airway obstruction that is characteristic of horses with heaves.158'15311571159 The higher airway resistance of principal ponies at Period A compared with that in control animals may have been due to residual airway obstruction despite 2 months at pasture. The small but significant increase in Cdyn of control ponies at Period 0 remains unexplained. Both principal and control ponies responded to aerosols of metha- choline by decreasing Cdyn and increasing RL. In comparison to control animals, ponies with a history of heaves were hyperreactive to aeroso- lized methacholine at Period B, reacting at a lower dose of methacholine and undergoing a larger change in CW" and RL in response to an 0.1 mg/ml dose of methacholine. We used EDsstyn as a measure of bronchial reactivity because a decrease in Cdyn of 35% exceeds the normal variabi- lity in Cdyn observed in ponies.172 Furthermore, an EDGSCdyn dose of methacholine did not result in an increase in respiratory rate, which might have caused frequency-dependent decreases in Cdyn- We attempted to calculate the dose of methacholine needed to increase RL by a factor of 2 (EDzooRL). We were unable to do so 30% of the time, because as shown by the dose-response curve of Principal Pony 5 at Period A (Figure 4), some ponies did not double RL even when Cdyn decreased to almost 50% of the value recorded after saline administration. For this reason, we used ARL0.1 to compare the effects of treatment periods on resistance. In our previous study this with pony model,169 we were unable to express 39 reactivity by calculating the change in RL in response to a given aero- sol dose of histamine because of the extreme reactivity of our principal ponies at Period B. In the present study, a methacholine aerosol of 0.1 mg/ml consistently induced a change in resistance of less than 0.05 cm H20-1'1-sec‘1 'hi control animals. In principal ponies, the ARL0.1 was similar to that in control ponies and quite repeatable at Periods A and D. In contrast, at Period B, there was more than an eightfold increase in resistance in response to the same dose of methacholine. This hyperreactivity waned over 2 weeks after barn exposure. The hyperreactivity to aerosol methacholine only at time Period B in principal ponies is consistent with our previous observation of hyperreactivity to both intravenously administered and aerosolized histamine at this same time period.157:169 At this time period, bronchoalveolar lavage fluid contains increased numbers of neutrophils, indicative of airway inflammation.173 There was no consistent dif- ference in the RL response to aerosolized methacholine and histamine,169 i.e., the RL induced by an EDGSCdyn dose of either agonist was similar in control and principal ponies at all time periods. In heaves, the principal lesion is bronchiolitis with excess mucus production and leukocyte accumulation. While these lesions may be the cause of increased RL and decreased Cdyn at Period 8, constriction of central airways may also be occurring. Because hyperreactivity is pre- sent only in principal ponies at Period B, decreased baseline airway caliber must be considered as a possible cause of hyperreactivity.174 This explanation appears unlikely, as principal ponies were not hyperreactive at Period A, despite their resistance being significantly higher than that of control animals at this time period. Furthermore, 40 when we examined the change in reactivity of principal ponies between Period A and Subsequent measurement periods as a function of change in dynamic compliance, there was in) correlation. Several control ponies also had changes in lung function over the 4 measurement periods that were not associated with changes in reactivity. Even though statistical analysis demonstrated an overall difference between principal and control ponies in response to citric acid inhala- tion, the greatest change in RL occurred in principal ponies at Period 8. This is consistent with our previous observations of hyper- reactivity to aerosolized methacholine and intravenously administered167 and aerosolized169 histamine occurring only during periods of acute air— way obstruction. Curiously, unlike the response to methacholine, which involved an increase in RL and a decrease in CW", the response to citric acid was characterized by an increase in RL unaccompanied by a decrease in Cdyn: suggesting the response was occurring primarily in the central airways. The primarily central airway response can be explained in one of two ways. First, citric acid may have been deposited purely in the central airways as a result of the aerosol delivery system. Citric acid was inhaled because ponies would not tolerate ventilation for more than 2 minutes, whereas methacholine was delivered by positive pressure ven- tilation. In subjects with diffuse airway obstruction, as occurs in ponies with heaves, rapid inhalation results in central deposition of aerosol.175 SJower inhalation175 or forced ventilation176 SUCh as we used with methacholine favors deposition of aerosol in peripheral air- ways. Secondly, aerosol may have been deposited throughout the airways, but the mechanism of response to methacholine may have existed only in 41 central airways. There are two mechanisms that have been proposed to explain the response of airways to citric acid.3~21177'179 The response to short-term exposure (2 minutes or less) is transient and caused by a vagally-mediated reflex. This response is blocked by administration of anticholinergic drugs.32:178 Longer exposure to citric acid aerosol causes a more delayed and protracted response. Calcium chelation and release of leukotrienes have been suggested as mechanisms of this response.1781179 It is unlikely that the latter mechanism is restricted to central airways. In basenji-greyhound dogs in which these mechanisms have been investigated,178 both a decrease in Cdyn and an increase in RL occur in response to citric acid delivered by forced ventilation. If the response to citric acid is vagally mediated, vagal efferents would have to be predominantly distributed to the central airways to explain our data. The distribution of vagal efferents is unknown in the horse, but in the dog vagal efferents are extensively distributed throughout the tracheobronchial tree.180 It is, therefore, most likely that the different responses to citric acid compared with those to methacholine are due to varying sites of aerosol deposition. Small airways obstruction is the classic lesion of heaves.156 Lesions in central airways have not been described. The decrease in Cdyn and increase in RL in response to aerosolized methacholine could be explained either by diffuse peripheral airway obstruction or by genera- lized constriction of the tracheobronchial tree. Therefore, investiga- tions using methacholine do not allow differentiation of the site of airway reactivity. With histamine aerosol, we observed primarily a decrease in CW" with a lesser increase in RL, suggesting hyperreac- tivity of peripheral airways. In contrast, the selective response of RL 42 seen with citric acid clearly demonstrates that the central airways are also hyperreactive, despite the lack of description of lesions at this level. Heaves in horses has frequently been compared to human asthma,149»151:165 because, like atopic asthma, recurrent airway obstruction is characteristic of the disease and an allergy is suspected. With regard to airway reactivity, heaves clearly differs from asthma. Whereas asthmatics have hyperreactive airways during periods of clinical remission, ponies do not. Airway hyperreactivity in heaves occurs only during acute exacerbations of the disease. IV. SUMMARY AND CONCLUSIONS An ideal animal model of asthma would be a naturally-occurring syndrome that would duplicate the clinical, biochemical, physiological, immunological, and pathological characteristics of the human disease. Fundamental studies using such a model would facilitate the emergence of clinical and diagnostic correlates to the benefit of those involved in clinical investigation, diagnostics, and patient care. Unfortunately, despite investigations into a- plethora of in vivorasthma models, the ideal animal model does not appear to exist. Given this limitation, it becomes important to critically examine each available model system for its potential advantages and uses. The pony model of asthma has several advantages over other model systems. Heaves is a spontaneous disease, and the clinical signs resolve when affected indivuals are removed from a barn environment. We have shown that nonspecific airway hyperreactivity to aerosolized histamine,16g methacholine, and citric acid is present during acute exa- cerbations of airway obstruction. Thus, ponies with heaves fulfill the criteria of the 1962 American Thoracic Society definition of asthma.7 Because of their large size, it is practical in ponies to make multiple physiological measurements and to collect multiple samples of tissue and body fluids. It has proven to be possible to serially study individual animals over an extended time period. The investigation reported here was conducted on conscious ponies under xylazine tranquilization. These 43 44 animals, however, are generally cooperative and trainable. Studies on unmedicated ponies should, therefore, be possible. From this perspec- tive, ponies are more suitable than horses for such investigations. In addition, there is some epidemiological evidence that heaves may be more prevalent in ponies than in horses.181 A considerable body of clinical and experimental evidence supports the theory that inflammatory cells sequestered within the lungs mediate airway hyperreactivity, Available data suggest that the inflammatory reaction is the underlying process responsible for the histological appearance of the airways in asthma.10 This reaction can account for mucus hypersecretion and epithelial cell loss, as well as airway edema.10 Most of the pathological features of asthma can be accounted for by the release of mast cell and mast cell-associated inflammatory mediators.66 In contrast, the mechanism(s) of airway hyperreactivity have not yet been well defined, although they may be linked to airway inflammation.66 Identification of a broad spectrum of mediators derived from a variety of cell types has widened the scope of investigations into the role of mediators in airway hyperreactivity. Inhalation challenge and bronchoalveolar lavage procedures have evolved into con- venient methodologies for these studies. The pony model of asthma demonstrates both nonspecific bronchial hyperreactivity and airway inflammation173 during episodes of acute airway obstruction. Because of this, and its other similarities to human asthma, continued investiga— tions (using the pony model may help elucidate the complex interrela— tionship between cells, mediators, neural mechanisms, and airway responsiveness. LIST OF REFERENCES LIST OF REFERENCES 1. McFadden ER, Austen KF. Asthma. In Harrison's Principles of Internal Medicine, 10th edition. Petersdorf RG, Adams RD, Braunwald E, et al (eds). New York, McGraw Hill Book Company, 1983, pp 1512—1519. 2. Davies EJ. NIAID initiations in allergy research. J Allergy Clin Immunol 1972; 49:323-328. 3. Franklin W. Current concepts: Treatment of severe asthma. New Engl J Med 1974; 290:1469-1472. 4. 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