g MSU LIBRARIES “ RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. INVESTIGATION INTO PULMONARY FUNCTION DERANGEMENTS AND THE ROLE OF VAGAL MECHANISMS IN MODELS OF EQUINE LUNG DISEASE By Frederik Jan Derksen A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology l982 ABSTRACT INVESTIGATION INTO PULMONARY FUNCTION DERANGEMENTS AND THE ROLE OF VAGAL MECHANISMS IN MODELS OF EQUINE LUNG DISEASE By Frederik Jan Derksen After a standard method for the measurement of pleural pressure was determined, a technique for reversible vagal blockade was developed and reproducibility of pulmonary function tests was established, I studied pulmonary function derangements in 3-methylindole induced pulmonary toxicosis and ovalbumin aerosol challenge induced allergic lung disease before and after vagal blockade in standing ponies. Oral administration of 3-methylindole (3M1) increased reSpiratory rate (RR), minute ventilation (9min): functional residual capacity (FRC) and minimum volume (MV), decreased arterial C02 tension (PaCOz), dynamic compliance (Cdyn) and specific conductance (SGtot) and did not alter arterial oxygen tension (PaOz), total lung capacity (TLC) and quasista- tic compliance (Cstat)- Vagotomy following 3M1 treatment decreased RR, and increased SGtot but did not change the other variables. Histo- pathologic examination showed that 3M1 treatment resulted in necrotizing bronchiolitis and alveolar emphysema. I concluded that tachypnea was caused by stimulation of pulmonary receptors with afferents in the vagus nerve, and that 3M1 pulmonary toxicosis was characterized by small air- way obstruction, occurring independent of vagal mechanisms. Frederik Jan Derksen Bilateral ovalbumin aerosol challenge of locally and systemically sensitized ponies increased RR, Tim-n, total respiratory resistance (Rtot) and MV, decreased Cdyn and PaOz and was without effect on FRC and PaCOz. In the locally sensitized ponies, TLC and Cstat decreased following challenge but in systemically sensitized ponies these *variables did not change. Following aerosol ovalbumin challenge of the 'left lung, RR and left lung resistance (RtotL) increased while right lung resistance did not change. Vagal blockade following challenge ‘failed to decrease RtotL- HistOpathologically ovalbumin challenge resulted in bronchitis, bronchiolitis and pulmonary edema. I concluded that like the 3M1 model, tachypnea was mediated via pulmonary receptors with their afferents in the vagus nerve, and that local mechanisms were of primary importance in the mechanism of disease. Increased sen- sitivity to normal bronchomotor tone may have played a minor role in the pathogenesis of pulmonary disease in this model. DEDICATION This dissertation is dedicated to my wife Jano, because without her guidance and support this study would not have been conducted. ii ACKNOWLEDGMENTS In the first place, I wish to extend my special thanks to Dr. N. Edward Robinson who not only served as my major professor, but during the tenure of this investigation also supported me as an academic father and friend. . I also acknowledge the many people in the Department of Large Animal Clinical Sciences who with their personal and professional enthusiasm supported this project. Special thanks are extended to Dr. E.A. Scott whom I could always depend on for encouragement and personal and pro- fessional advice, Dr. J.A. Stick who with his surgical skills helped develop the vagal loop procedure, and Dr. R.F. Slocombe who helped to interpret pathologic data. I also thank Ms. Barbara Meining who with her unsurpassed typing skills made the preparation of the manuscript so much easier and Ms. Roberta Milar whose dependable technical assistance is greatly appreciated. Finally, I would like to thank the Guidance Committee members, Dr. J.B. Scott, Dr. C.M. Brown, Dr. R.A. Bernard, and Dr. w.R. Dukelow, for their academic guidance and support. This study was performed during the tenure of a National Institutes of Health post doctoral fellowship (#1-F32-HL-06073). TABLE OF CONTENTS LIST OF TABLES.................................................. LIST OF FIGURES................................................. INTRODUCTION.................................................... LITERATURE REVIEW............................................... Pulmonary innervation....................................... Allergic Lung Diseases: Derangements in Pulmonary Function and pathogeneSiSOOOOOOOO0.0.00.0.0...OOOOOOOOOOOOOOOOOOOO Naturally Occurring and Experimental Lung Diseases of the HorseOCOO0.0.0000...0.00.00...O...OOOOOOOOOOOOOOOOOOOOOOO Purpose Of this StUdy000000....0......OOOOOOOOOOOOOOOOOOO... CHAPTER 1: Esophageal and Intrapleural Pressures in the Healthy canSCIOus Pony...COO...00.0.0.0...000......OOOOOOOOOOOOOOOOO CHAPTER 2: Technique for Reversible Vagal Blockade in the Standing consc1ous PonyOOOOOOOOOOOOOOO0.0.000000000000000... CHAPTER 3: Pulmonary Function Tests in Standing Ponies: Reproducibility and Effect of Vagal Blockade................ CHAPTER 4: 3-methylindole Induced Pulmonary Toxicosis in the HorseOOOOOOOOOOOOOOOOOOO0....0......OOOOOOOOOOOOOOOOOOCOOOOO CHAPTER 5: Pulmonary Function in Ovalbumin Induced Allergic Lung Disease in the Awake Pony: Role of Vagal Mechanisms... CHAPTER 6: Response of the Locally Sensitized Equine Lung to Aerosol Ovalbumin Challenge: Role of Vagal Mechanisms...... CONCLUDING DISCUSSIONOOOCOOO0.00.00.00.0000000000000000000000000. SWY AND CONCLUSIONOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO LIST OF REFERENCESOOOOOOO0.0.000000000000000000000000000000...O. iv Page vi 11 19 22 27 53 68 "119 145 166 169 174 TABLE 1‘1. 1'2. 1'3. 3'1. 3'2. 3’3. 5'1. LIST OF TABLES Comparison of mean selected pressure changes (AP) as a funCtion 0f masuring SitelOOOOOOOOOOOO0.0.0.0.0000... Dynamic compliance (Cdyn) calculated from changes in pleural pressure measured at different sites............ Comparison of measurement technique used and dynamic compliance value obtained in normal standing horses..... Pulmonary function values (Y 1 SD). Derived from 6 studies repeated at hourly intervals.................... Pulmonary function values (Y + SD). Derived from at least 3 studies conducted at'Z month intervals.......... Pulmonary function values (§'+ SEM). Derived from 4 studies on 5 ponies, conducted at 2 month intervals..... Group 1 ponies: Bilateral aerosol antigen challenge. Arterial blood gas tensions and lung volumes (ix: SEM) during a prechallenge period, hourly after challenge for 5 hours and during 2 periods of vagal blockade...... Page 40 4O 47 85 86 132 LIST OF FIGURES FIGURE PAGE l-Ia. Pleural pressures and tidal volume during l breath. Ventral, middle and dorsal refer to the position of the pleural needles in the thoracic wall............... 35 1-lb. A comparison of pressures of the middle portion of the thoracic part of the esophagus and dorsal part of the thorax during 1 breath.......................... 35 1-2. Pleural pressure (i :_SEM) measured during the mid-- expiratory volume plateau in the ventral, middle, and dorsal thoraCIC "allOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 37 1-3. Esophageal pressure (2:1_SEM) measured during the mid- expiratory volume plateau in the cranial, middle, and caudal portions of the thoracic part of the eSOphagus.. 39 1-4. Expiratory limb of a quasistatic lung pressure-volume curve (from reference 8). Mean pressures measured during the mid-expiratory plateau at the ventral, middle, and dorsal thoracic positions are marked on the curve to show the possible regional variations in lung inflationOO00.00.000.000....OOOOOOOOOOOOOOOOOOOOOO 46 2-1. A left cervical vagal loop, l4 days after the surgical procedurEOOOOOOOOOOOOO0.0.0..OOOOOOOOOOOOOOOOOOOO0.0... 57 2-2. Right cervical vagal loop, 90 days after the surgical procedurEOOOOOO..0.000000000000IOOOOOOO0.00000000000000 59 2-3. Copper cooling coil used to refrigerate the vagal lOOpSOCOOOOOOOOOOOOOOOO0.0...0.0.0.0....OOOOOOOOOOOOOOO 61 2-4. Res iratory rate (RR), tidal volume (VT). heart rate (HR) and systemic blood pressure (Psyst) during a baseline period, after vagal cooling, during a second baseline period and after IV administration of 0.04 mg atropIHEIkg Of bOdy “EightOOOOOOOOOOOOOOOOOOOOOOOOOIOOO 65 3-1. Forced oscillation system used to measure total respiratory resistance................................. 74 vi LIST OF FIGURES--continued FIGURE 3‘2. 3'3. 3‘4. 4'1. 4'2. 4-3. 4‘4. 4‘5. 4'69 5‘10 5-20 Helium dilution system, used to measure functional res‘idua] capaCityIOO0.0.0.0.000000000000000000000000.00 Composite expiratory limbs of thoracic cage (T) and lung (L) pressure-volume curves. The dotted line is the best fit to a single rising exponential............ Total reSpiratory system resistance (Rtot) measured at increasing lung volumes, before and after vagal bIOCkadeOC0......O0.0.0.0000...OOOOOOOOCOOOOOO000...... Respiratory rate (RR).(§ + SEM), tidal volume (VT) and minute ventilation (Vm a), measured during a prechallenge period (PC), after 3-methylindole (3MI) treatment and after vagotomy‘(VC)...................... Arterial 02 tension (PaOz) (§:1_SEM), arterial C02 tension (PaCOz), and pH, measured during a prechallenge period (PC), after 3-methylindole (3MI) treatment and after vagotomy (VC).................................... Functional residual capacity (FRC) (i 1_SEM) and total lung capacity (TLC) measured during a prechallenge period (PC), after 3-methylindole (3MI) treatment and after vagotow (VC)OOOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOCO Total respiratory system resistance (Rtot)a specific conductance (SGtot) and dynamic compliance (Cd n) measured during a prechallenge period (PC), a er 3-methylindole (3MI) treatment and after vagotomy vc COICCCOOOCOOOOOIOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. Dynamic compliance (Cdyn) measured during a pre- challenge period (PC), after 3-methylindole (3MI) treatment and after vagotomy (VC)...................... Photomicrograph, showing bronchioles with epithelial degeneration and cellular debris in their lumen........ Respiratory rate (RR).(§ + SEM), tidal volume (VT) and minute ventilation (Vmifi) measured during a pre- challenge period, hourly after challenge for 5 hours and during 2 periods of vagal blockade................. Total respiratory resistance (Rtot) (E :_SEM) and dynamic compliance (Cdyn) measured during a prechal- lenge period, hourly after challenge for 5 hours and during 2 periods of vagal blockade..................... vii PAGE 77 81 83 101 103 105 107 109 111 127 129 LIST OF FIGURES--continued FIGURE 5‘3. 5’4. 6'1. 6'2. (5'3. Respiratory rate (RR) (x 1 SEM), left and right lung resistance (R otL and RtotR) measured during a pre- challenge period, hourly after challenge for 4 hours and during ipsilateral and bilateral vagal blockade.... Photomicrograph of a bronchiole, 5 hours after ovalbumin challenge, showing acute obstructive bronChiollitiSOOOOOOOOOIO0.0.0.0.00000000000000IOOOOOOO. Respiratory rate (RR) (R :_SEM), left and right lung resistance (R otL and RtotR) measured during a pre- challenge period, one hour after unilateral challenge and following unilateral and bilateral vagal blockade. (IVB and VB)OOOOOOOOOOOOOOOOO0.0...OOOOOOOOOOOOOOOOOOOO Respiratory rate (RR),(§ + SEM), tidal volume (VT), minute ventilation (VmifiT, total respiratory system resistance (Rto ), dynamic and quasistatic compliance (Cdyn and Csta I, total lung capacity (TLC), func- tional residua capacity (FRC) and PaOz, measured during a baseline period, one hour after bilateral challenge and following unilateral and bilateral vagal blockade (IVB and VB)............................ Photomicrograph of a bronchiole in the challenged lung, 5 hours after unilateral ovalbumin challenge..... viii PAGE 131 135 152 154 156' INTRODUCTION Pulmonary diseases in persons and domesticated animals are a major cause of morbidity and poor performance, resulting in enormous financial and social losses. Numerous etiologic agents including bacterial and viral agents, pollutants such as dust, ozone and allergens, and enzyme deficiencies have been identified as causes of some respiratory diseases while the etiology of other respiratory diseases remains unknown. Because of the large variety of etiologic agents and their preponderance in the daily environment, control and treatment measures directed at the agents themselves have enjoyed only partial success. Since it has become apparent that the lung uses only a limited number of mechanisms in response to insults, control and treatment measures aimed at mecha- nisms whereby lung diseases are expressed rather than at etiologic agents may have greater chance of success. Presently, this approach to the control of pulmonary disease in persons and animals is not widely used due to deficiencies in understanding of basic mechanisms. Because of this-lack of understanding and because vagal mechanisms are thought to be important in the pathogenesis of allergic as well as nonallergic pulmonary diseases in man and experimental animals, a major goal of this investigation was to elucidate the role of vagal mechanisms in the derangement of pulmonary function in an allergic and a nonallergic model of equine lung disease. The study of respiratory disease in the horse is appropriate because of the economic and social importance of respira- tory disease in this species. In addition the horse may be a good 1 model of some fOrms of respiratory disease in persons because anatomi- cally human and equine lungs are similar and because the horse is the only domestic species that commonly suffers from a naturally occurring asthma-like syndrome. Since at the outset of this investigation there was concern about the validity of pleural pressure measurement in the horse the first study was designed to compare esophageal pressures, measured at dif- ferent sites in the thoracic portion of the esophagus with pleural pressures measured at various sites in the pleural space. In order to facilitate the study of vagal mechanisms in control of pulmonary func- tion in health and disease I develOped a technique to reversibly block the cervical vagus nerves in the standing conscious hbrse. Subsequently I evaluated the reproducibility of pulmonary function tests used in this investigation, and determined the effect of vagal blockade on pulmonary function in normal ponies. Once these preliminary studies were completed I studied the role of vagal mechanisms in the pathogenesis of 3-methylindole induced pulmonary toxicosis and ovalbumin induced allergic lung diseaSe in the horse. LITERATURE REVIEW This literature review will first briefly describe the pulmonary innervation in order to provide a basis for subsequent discussion of the role of vagal mechanisms in asthma in persons and experimental lung diseases in animals. Experimental allergic lung diseases are most com- monly developed as models for asthma in humans and therefore I will describe the derangement‘in pulmonary function in both asthma and the available experimental lung diseases. This background information is necessary to develop an understanding of the range of functional abnor- malities that occur in various lung diseases in mammals. Subsequently I will review our current knowledge of “heaves”, a naturally occurring asthma-like disease in the horse, because its occurrence is one of the most persuasive reasons why the study of models of lung disease in the horse might add new basic knowledge. Finally, 2 new models of equine lung disease will be introduced and I will pose the questions investigated by this study. Pulmonary Innervation The lung has both an afferent and efferent nerve supply which has been studied with anatomical, histochemical and physiological tech- niques. This portion of the review will be restricted to the major phy- siologic studies designed to elucidate the functional importance of the PUlmonary innervation. As reviewed by Paintal (1973) three kinds of pulmonary afferent receptors have been identified. They are the bronchOpulmonary stretch receptor, the irritant receptor and the interstitial type J receptor. Pulmonary stretch receptors are those endings whose activity increases rhythmically in phase with each in5piration (Adrian, 1933). When a sudden and maintained inflation is applied, they respond with a discharge of impulses that adapts slowly, distinguishing them from the much more rapidly adapting irritant receptors which also respond to lung inflation with increased afferent activity (Mills et al, 1969). The histologic characteristics of pulmonary stretch receptors have not been established although it appears that these endings are associated with the smooth muscle of bronchi and bronchioles (Larsell et al, 1933, Elftman, 1943, Niddicombe, 19548). In all species studied so far, stretch receptor neurons are myeli- nated and run in the vagus nerve (Paintal, 1963). The natural stimulus for stretch receptor activation is the volume of air entering the lung. Hering and Breuer (1868) showed that inflation of the lungs of dogs led to a decrease in frequency and force of expiratory effort (Hering-Breuer reflex) and that deflation of the lung caused stronger and more frequent inspiratory efforts (inflation reflex). In 1933 Adrian showed that pulmonary stretch receptors were responsible for these reflexes. The importance of these reflexes in control of breathing varies between Spe- cies. Adrian (1933) showed that in cats the inflation reflex modifies the respiratory cycle substantially while Marshall et a1 (1958) showed that in man the reflex is weak and does not substantially modify the respiratory cycle. Niddicombe (1961) studied the inflation reflex in“ persons, monkeys, dogs, cats, rabbits, guinea pigs, rats, and mice and showed that the reflex was weakest in persons and strongest in rabbits. The Hering-Breuer reflex is so strong in rabbits that on occasion lung inflation induced apnea causes asphyxiation. The relative importance of the Hering-Breuer reflex in control of respiration in the horse is pre- sently unknown. In cats the effect of lung inflation on respiratory pattern is dependent upon bronchomotor tone and rate of inflation (Niddicombe, 1954A, Davis et al, 1956). When bronchomotor tone is increased (by administration of acetylcholine or histamine), the effect of pulmonary inflation on respiratory pattern is decreased; while following broncho- dilation (using adrenaline) the Hering-Breuer reflex is enhanced (Niddicombe, 1954A). Davis et a1 (1956) showed in cats that an increase in rate of inflation enhances the Hering-Breuer reflex and that changes in stretch receptor activity follow pleural pressure changes closely. Data from these studies support the hypothesis of Christie (1953) that the inflation and deflation reflexes adjust the rate and depth of breathing to be mechanically the most economical. Hhen studying the effects of pulmonary inflation and deflation on vagal afferent activity in cats, Knowlton et al (1946) described a group of afferent fibers whose activity was increased with lung infla- tion, but adapted rapidly to this stimulus. They called the receptors involved "rapidly adapting pulmonary stretch receptors“, now known as irritant receptors. Hiddicombe (1954A) found that in cats mechanical stimulation of the tracheal mucosa activated irritant receptors, and Idills et a1 (1969) in rabbits found similar receptors in the intra- IJulmonary airways, concentrated in the carina. Since the intrapulmonary 'irritant receptors are tonically active in the rabbit, while in the cat tracheal irritant receptors are inactive during normal breathing, these two types of receptors were thought to be different (Mills et al, 1969). It is now known that species differences, not differences bet- ween receptor types, are responsible for the differences in activity of irritant receptor (Paintal, 1973). Another difference between cat and rabbit irritant receptors is that the cat shows no increased activity after intravenous injection of phe- nyldiguanide, whereas the response in rabbits is marked (Mills et al, 1969, Paintal, 1953). Because species differences in irritant receptor activity exist, it is important to note that presently no reports on equine pulmonary irritant receptors are available. In addition to sensitivity to lung inflation and mechanical stimula- tion, irritant receptors increase firing frequency in response to chemi- cal stimulation of airway mucosa, anaphylaxis and microembolism (Nadel et a1, 1965, Mills et al, 1969, Sellick et al, 1969, 1971, Karczewski et al, 1969) resulting in tachypnea, bronchoconstriction and coughing. Mills et a1 (1969) and Paintal (1973) suggested that irritant receptor activity in response to various stimuli is enhanced by bronchoconstric- tion induced by histamine, or anaphylaxis, thereby producing a positive feedback system. However, it is not clear how the effect of broncho- constriction and the direct effects of histamine or anaphylaxis on irri- tant receptor activity can be separated. Since the direct effects of histamine or anaphylaxis on irritant receptors can explain experimental data, it is not necessary to postulate that bronchoconstriction enhances irritant receptor activity. In contrast with the previous two receptor types, the type J pulmo- nary receptor is served by nonmyelinated C fibers (Paintal, 1954). They were discovered by Paintal in 1954 when studying gastric receptors. The type J receptor is normally inactive and is located adjacent to pulmo- nary capillaries (Paintal, l969). Afferent activity is increased by a variety of chemical substances including halothane and phenyldiguanide, but the only physiologic stimulants capable of eliciting consistent responses are pulmonary vascular congestion and edema, produced by either chemical vasculitis or increased left atrial pressure (Paintal, 1973). It has been postulated that type J receptors function as interstitial stretch receptors, because they are located in series with the collagen elements of the pulmonary interstitium (Paintal, 1973). Chemical stimulation of J receptors, using phenyldiguanide results in apnea (Paintal, 1955). In addition, type J receptor activity inhi- bits somatic motor activity and produces the sensation of breathlessness (Kalia, l969). Efferent innervation of the lung is via the parasympathetic and sym- pathetic diversions of the autonomic nervous system. Parasympathetic neurons travel in the vagus nerve, while pulmonary sympathetic fibers originate in the cervical and first 5 thoracic sympathetic paravertebral ganglia (McKibben, 1975). Both autonomic divisions enter the lung via the hilum and exert a tonic influence on airway smooth muscle. Longet (1842) showed that the vagus nerve contained constrictor fibers to the bronchial muscle. He directly observed the bronchi, exposed by cutting through the lung of freshly killed horses and oxen and found that they contracted when the vagus was excited electrically. Since that time, many investigators have confirmed that vagal stimulation results in bronchoconstriction, while vagotomy is fellowed by bronchodilation (Dixon et al, 1903, 1912, Noolcock et a1, 1969, Hahn et a1, 1976). 8 As first demonstrated by Dixon et a1 (1912) sympathetic stimulation causes bronchodilation. This is attributed to the predominant presence of B-adrenergic receptors in the bronchial and bronchiolar smooth muscle (Castro de la Mata et al, 1962, Guirgis et al, 1969). Some authors have reported a paradoxical bronchoconstriction f01lowing sympathetic stimulation, but attributed this phenomenon to contamination of sym- pathetic nerves with parasympathetic fibers (Dixon et a1, 1912). However, Castro de la Mata et a1 (1962) showed in dogs that fOllowing B-adrenergic blockade, sympathetic stimulation resulted in broncho- constriction, and suggested that this could be explained by the presence) of a-adrenergic receptors in the lung. His conclusion was supported by .Fleisch et a1 (1970) who studied rats, guinea pigs, cats and rabbits but refuted by Foster (1966), Guirgis et a1 (1969), and Cabesas et a1 (1971) who studied guinea pigs, persons and dogs, reSpectively. Since these studies use pharmacologic techniques, differences in results may be due to the lack of specificity of various blocking agents at dif- ferent dosages. The distribution of autonomic fibers to various airway generations is not well established. Macklem et a1 (1967) introduced the retrograde catheter technique which was subsequently used to study distribution of autonomic innervation in the lung (Macklem et al, 1969). The technique uses a catheter inserted into a lung via the air- way opening and exited through the pleural surface. Pulmonary resistance, calculated as the difference between airway opening pressure and pleural pressure, divided by air flow, measured at the airway opening, is separated in central and peripheral components. Peripheral resistance is defined as the pressure difference between the retrograde catheter and pleural space divided by airflow, measured at the airway opening, while central resistance is the difference between pulmonary resistance and peripheral resistance. Macklem et a1 (1969) showed that in dogs, using the retrograde catheter technique, vagotomy resulted in preferential dilation of 3-8 mm bronchi, while Hoolcock et al (1969) using the same technique found that vagal stimulation increased central resistance in some dogs and peripheral resistance in others. In the latter study, catheters were wedged in bronchi between 2.7-0.8 mm diameter. Therefore if vagal innervation was predominantly in 3-8 mm bronchi, an increase in central resistance would be expected fOllowing vagal stimulation. Since the variable results of Noolcock et a1 cannot be explained based on retrograde catheter position, they are most likely caused by animal to animal variability in distribution of parasym- pathetic efferent innervation. Severinghaus et a1 (1955) reported that atropine increased dead space volume, suggesting that vagal tone has an important bronchoconstrictor effect on central airways. Using a radiographic technique, Cabesas et a1 (1971) confirmed Macklem's fin- dings and reported that vagal stimulation reduced airway diameter from the trachea to bronchioles 0.5 mm in diameter and had the greatest effect on airways 1-5 mm in diameter. Thus, although individual variation may be great, the majority of evidence suggests that in the dog vagal bronchomotor tone preferentially effects airways between l-8 nun diameter. The distribution of sympathetic pulmonary innervation is also in dispute, although the majority of evidence suggests that sympathetic fibers influence peripheral airways more than central airways. Hensly et al (1978) reported an increase in closing volume and decrease in 10 airway resistance without changes in dead space volume fOllowing treatment with a B-adrenergic agent, suggesting peripheral bronchodila- tion. This conclusion was suported by Ingram et al (1975) who showed that following isoproterenol treatment in persons maximum expiratory flow increased while elastic recoil did not change suggesting that sym- pathetic innervation predominantly influences peripheral airways. In contrast, Cabesas et a1 (1971) using a radiographic technique, showed that sympathetic stimulation dilated airways from 0.5-5 mm in diameter with the greatest effect on airways with diameters between 1-5 mm. These findings were recently confirmed by Russell (1980) who studied airways in vitro. The discrepancy between these studies is presently unexplained. Recently a third division of the autonomic nervous system (purinergic nervous system) was shown to be present in guinea pig trachealis muscle (Coburn et a1, 1973). Purinergic innervation of guinea pig airways was confirmed by others and the finding extended to human airways (Bando et al, 1973, Coleman, 1973, Richardson et al, 1976). The purinergic system, is also present in the gastrointestinal tract, uterus and guinea pig vas deferens (Burnstock, 1972). In the gastrointestinal tract and the lung following muscarinic and adrenergic blockade, vagal stimulation causes smooth muscle relaxation, suggesting that the purinergic system is inhibitory in nature. The chemical mediator of this inhibitory nervous system is not known, but there is extensive evidence to support adenosine triphosphate or another purine nucleotide as the mediator (Burnstock, 1972). The role of the puri- nergic nervous system in control of airway caliber in health and disease is presently unknown, although malfunction of this system may be 11 important in the pathogenesis of asthma-like syndromes in man and other animals. Allergic lung diseases: Derangements ingpulmonary function and pathogenesis In this section I will describe the derangement in pulmonary func- tion occurring in asthma in persons and the available experimental models of allergic lung disease. In addition, possible mechanisms involved in the pathogenesis of these diseases will also be discussed. In the last ten years there has been a great interest in models of allergic lung disease because of the lack of knowledge about and the prevalence of asthma in persons. Asthma has been defined as “widespread narrowing of the bronchial airways which changes in severity over short periods of time either spontaneously or under treatment and is not due to cardiovascular disease“ (CIBA symposium, 1959). As reviewed by Alexander et a1 (1921) an attack is triggered by a variety of stimuli, including allergens, chemical irritants, dust, smoke, cold, exercise, coughing, hyperinflation, laughter and excitement. Animal models have been developed to study asthma induced by allergens, i.e., asthma with a major allergic or immunologic component. Although approximately 75% of asthma cases have no major immunologic component, no animal model has been developed for these types of asthma (Stevenson, 1975). As reviewed by McFadden (1975) arterial oxygen tension (PaOz) decreases, while alveolar-arterial oxygen difference increases during an attack of asthma. In addition, specific conductance is increased, while dynamic compliance is decreased, suggesting bronchoconstriction involving both large and small airways. 'Small airway obstruction is also indicated by a depression of maximal expiratory flow rates 12 throughout the vital capacity (Despas et a1, 1972, McFadden et a1, 1973, 1975). Residual volume and functional residual capacity increase, probably because of airway closure (Hurtado et a1, 1934). After symp- tomatic improvement, specific conductance increases but maximum expira- tory flow rates, lung volumes and PaOz are still abnormal, suggesting central airway bronchodilation with persistence of peripheral airway narrowing (McFadden et al, 1969, 1973, 1975). It was postulated by McFadden (1975) that the persistent small airway narrowing may serve as a basis for recurrent attacks of airway obstruction. The mechanism of allergen induced bronchoconstriction may have several components. Combinations of antigen and antibody on the bronchial epithelial surface releases mast cell mediators which act on irritant receptors in the epithelium and elicit vagally mediated reflex bronchoconstriction (Weber, 1914). Alternatively, components of the complement cascade, lymphokines or polymorphoneuclear lysozymes could also stimulate these receptors (Cohen et al, 1979). The importance of vagal reflex bronchoconstriction in human asthma is presently in dispute. Arborelius et al (1962) administered specific antigen to only' one lung in each of two patients with allergic asthma. In both cases bronchoconstriction, indicated by delayed nitrogen washout, was observed in the challenged lung, while in the unchallenged lung, nitrogen washout characteristics remained normal. The authors concluded that vagal reflex mechanisms were not important in the pathogenesis of broncho- constriction in the two patients studied. A similar conclusion was reached by Rosenthal et al (1976) who showed that in a group of asth- matics, atropine pretreatment did not reduce the dose of antigen required to produce a 35% fall in specific conductance, although atropine did 13 increase baseline specific conductance. Yu et al (1972) arrived at an apposite conclusion. They reported that in 5 of 7 asthmatics, increased airway resistance due to antigen challenge was reversed or prevented by atropine treatment and pretreatment, respectively. They concluded that the parasympathetic nervous system was critically important in antigen induced bronchoconstriction is asthmatic patients. One explanation of the differences between various studies is that asthmatics are a heterogeneous papulation, with various mechanisms contributing to bronchoconstriction to various degrees. In support of this hypothesis, Orehek et a1 (1975) showed that in 10 asthmatic patients, pretreatment with scopolamine prevented increases in specific resistance in 5 patients and had no effect in 3 subjects and provided partial protection in two others. Other mechanisms that may be important in allergen-induced airway narrowing in asthmatics includes direct action of antigen-antibody complexes or chemical substances on smooth muscle, causing broncho- constriction, excess mucus production, edema, and inflammatory exudate (Huber et a1, 1922, Rebuck et a1, 1971, Bardana, 1976, Nadel, 1977). Alternatively, bronchoconstriction may be due to bronchial hyperreac- tivity, characteristic of the asthmatic patient. As reviewed by Boushey et a1 (1980), the hypothesized mechanisms of bronchial hyperreactivity include decreased baseline airway caliber, alterations in the amount or reactivity of smooth muscle, exaggerated parasympathetic response to stimulation of pulmonary mechanoreceptors, abnormalities of the sym- pathetic nervous system and changes in epithelial permeability which allow greater concentrations of antigen to contact subepithelial irri- tant receptors. Presently no animal model for bronchial hyperreactivity 14 is available although recent reports suggest that the Basenji-Greyhound, sensitized to Ascaris suum antigen has hyperreactive airways (Hirshman et al, 1980, 1981). A spontaneously occurring disease syndrome with clinical charac- teristics similar to those of human asthma is uncomnon in other mam- malian species except the equid (Cook, 1976). Since the horse is an unusual laboratory animal and since few baseline data on equine pulmo- nary function exist, most work with animal models has been done using experimentally induced allergic lung disease in other species. Experimental models of asthma have been developed in the dog, cat, rhe- sus monkey, guinea pig and rabbit (Dain et al, 1975, Drazen et al, 1975, Karcsewski et al, 1969, Mills et al, 1970). Sensitized animals are challenged by aerosol or intravenous administration of antigen, resulting in immediate bronchoconstriction. The mongrel dog, naturally sensitized to Ascaris spp. was first studied by Booth et a1 (1970) who reported an increased respiratory rate, decreased tidal volume and decreased peak expiratory flow rate, associated with abnormalities in gas exchange following challenge. Dain et a1 (1975) and Gold et a1 (1972A) characterized the pulmonary mecha- nical abnormalities in this model and showed that following aerosol challenge respiratory resistance increased, dynamic compliance and PaOz decreased without a change in functional residual capacity or C0 diffusion capacity. Tantalum bronchograms showed bronchoconstriction in all airways down to 1 mm diameter bronchi. Significantly, bronchodila- tors reversed the resistance and compliance changes, but hypoxia per- sisted suggesting that, like in asthma, gas exchange remains impaired after symptomatic improvement. A more detailed bronchographic study by 15 Kessler et a1 (1973) showed that following antigen exposure airway narrowing was slight in airways larger than 12 mm in diameter, moderate in airways 8-12 mm and maximal in airways 1-8 mm, but less in airways 0.5-1 mm. Since the distribution of airway constriction following anti- gen inhalation was identical to that observed during vagal stimulation and since atropine inhibited antigen-induced bronchoconstriction, it was concluded that antigen-induced airway constriction is mediated by the parasympathetic nervous system. The reflex nature of this mechanism was demonstrated by Gold et al (19728) who challenged one lung in sen- sitized mongrel dogs with homologous antigen. Challenge increased air- way resistance in both lungs, the increase was reversed by ipsilateral vagal blockade. They concluded that aerosol challenge activated pulmo- nary receptors resulting in a reflex bronchoconstriction mediated via the vagus nerve. Rubinfeld et a1 (1978) showed that ventilation- perfusion mismatch, characteristic of human asthma, also occurred in this dog model. The reflex nature of antigen-induced bronchoconstriction in the sen- sitized mongrel dogs does not go unchallenged, however, as Krell et a1 (1976) found that large intravenous doses of atropine did not result in significant reductions in the response to Ascaris antigen, although in some animals the increase in pulmonary resistance was attenuated. The variable result could reflect differences in antigen preparation, reac- tivity of individual dogs and experimental conditions. The Basenji- Greyhound, sensitized to Ascaris antigen, is distinguished from the mongrel dog by exaggerated bronchoconstriction in response to nonspeci- fic stimuli such as citric acid and methacholine (Hirshman et al, 1980). In support of the findings of Krell et a1 (1976), Hirshman et 16 a1 (1981) reported that in the Basenji-Greyhound the major component of antigen-induced bronchoconstriction is not cholinergically mediated as atropine pretreatment did not protect the dogs from antigen-induced bronchoconstriction. In summary, Ascaris aerosol challenge of the sensitized dog results in increased pulmonary resistance and decreased dynamic compliance and PaOz, in addition to ventilation-perfusion mismatch. These changes suggest generalized bronchoconstriction with impairment of gas exchange. The pathophysiology of the derangement in pulmonary function are pre- sently in dispute as some studies seem to clearly indicate the impor- tance of vagal reflex bronchoconstriction while others refute the major involvement of this reflex. The pulmonary response of the sensitized guinea pig to antigen challenge has been studied by a number of investigators (Ratner et al, 1927, Stein et a1, 1961, Mills et al, 1970, Richerson et al, 1972, Popa et al, 1974, Drazen et a1, 1975, Roska et al, 1977, Pare et al, 1979). Even though method of sensitization and challenge and therefore immunologic response varied between investigators (Richerson, 1972), in all studies pulmonary resistance increases and dynamic compliance decreases. Maximum changes occurred between 2 and 10 minutes after challenge with resolution occurring over 30 minutes. However, pulmonary resistance returns to normal before dynamic compliance recovers (Drazen et al, 1975). This suggests that, like in human asthma, central airway recovery preceeds return to normal caliber in small airways. The role of vagal mechanisms in antigen induced bronchoconstriction in the guinea pig was studied by Mills et a1 (1970) and Drazen et a1 (1975). Mills et al reported that vagotomy reduced by 75% the 17 increased resistance and halved the decreased compliance due to antigen challenge and concluded that vagal mechanisms played an important role in antigen induced bronchoconstriction in the guinea pig. Drazen et a1 (1975) found that atropine pretreatment prevented the decrease in pulmo- nary resistance but did not hinder the fall in dynamic compliance. This suggests that in the guinea pig, alterations in central airway tone resulting from antigen exposure are mediated predominantly by secondary cholinergic mechanisms while peripheral airway effects are mainly non- cholinergic. The pulmonary response to antigen challenge has also been studied in rabbits (Karcsewski et a1, 1968, Halonen et a1, 1976). Following challenge, dynamic compliance is reduced and pulmonary resistance is increased. Vagotomy decreased pulmonary resistance without changing dynamic compliance suggesting that, as in guinea pigs, cholinergic mechanisms play a role in central airway response, but is unimportant in small airway narrowing. In summary, the pulmonary response to antigen challenge in the small laboratory mammals is characterized by generalized bronchoconstriction occurring immediately following challenge. The majority of evidence suggests that central airways recover before peripheral airways dilate and that central airway narrowing but not peripheral airway constriction is vagally mediated. In the sensitized Rhesus monkey and sheep, antigen challenge also causes a decrease in dynamic compliance and increase in pulmonary resistance (Pare et al, 1976, Banner et a1, 1979). However, in these species, the role of vagal mechanisms in bronchoconstriction has not been studied to date. 18 As mentioned above, functional residual capacity and residual volume increase in acute attacks of asthma in persons (Hurtado et al, 1934). Following antigen challenge of the sensitized sheep and some Basenji-Greyhounds, functional residual capacity also increases, while in other Basenji-Greyhounds, the mongrel dog and monkey, challenge does not increase functional residual capacity (Dain et al, 1975, Pare et al, 1976, Wanner et al, 1979, Hirshman et al, 1981). The effect of challenge on lung volumes has not been studied in rabbits, but in guinea pigs, challenge results in an increased minimum volume in vitro, suggesting that residual volume and functional residual capacity may also be increased in vivo. Difference in lung volume changes following challenge between the mongrel dog and some Basenji-Greyhounds may be explained by the difference in magnitude of bronchoconstriction. ' Following antigen challenge of the Basenji-Greyhounds, pulmonary resistance increases 15-fold and dynamic compliance decreases by 73%, while in the mongrel dog pulmonary resistance increases only 3-fold and dynamic compliance decreases by 23.7% (Gold et al, 1972, Hirshman et al, 1981). These data suggest a more severe airway response in the Basenji-Greyhound model, which may result in gas trapping in the tidal volume range in some individuals. However, functional residual capacity does not increase in other challenged Basenji-Greyhounds with pulmonary mechanics changes as severe as seen in Basenji-Greyhounds that show significant increases in lung VOlumes (Hirshman et al, 1981). Thus within the Basenji-Greyhound model, the severity of bronchoconstriction does not correlate with changes in lung volumes. In monkey's, Pare et a1 (1976) reported similar findings. Following challenge, pulmonary resistance increased 7-fold while dynamic compliance decreased 82%. In 19 spite of this severe response, no changes in functional residual capacity were observed. It appears that the monkey is similar to the mongrel dog in its ability to ventilate peripheral lung units in spite of severe bronchoconstriction. In contrast, Wanner et a1 (1979) showed that in sheep, antigen induced decrease in pulmonary conductance of only 33% caused a significant increase in functional residual capacity, pre- sumably due to airway closure. Species difference in their ability to maintain ventilation distal to airway obstruction in the face of severe bronchoconstriction may be related to degree of collateral ventilation. The mongrel dog has low resistance collateral channels while collateral resistance is high in persons and in species with lobulated lungs such as sheep (Van Allen et a1, 1931). Since the pulmonary anatomy of the monkey lung is similar to dog lung, collateral ventilation is likely to offer a low resistance pathway in this species (McLaughlin et al, 1961). Therefore it appears that low resistance ventilation of obstructed lung units through collateral channels may prevent air trapping in the tidal volume range. If this is true, it is not apparent why, after antigen challenge, functional residual capacity increases in some Basenji-Greyhounds and not in others. Naturallyoccurringand experimental lung diseases of the horse Since the horse is an unusual laboratory animal, its use in these studies needs to be justified. The horse is the only domestic animal that commonly suffers from recurrent airway obstruction, clinically similar to asthma in man (Lowell, 1964, Thurlbeck et al, 1964) making it unique as an animal model fOr this disease. In addition, since the ‘ horse has a substantial financial and social value, study of the 20 condition itself is also of importance. The disease syndrome, characterized by recurrent airway obstruction, is commonly called heaves, chronic obsructive pulmonary disease or equine emphysema, but destructive emphysema is not a feature of the disease (Thurlbeck et a1, 1964). The cause of heaves is not known and the etiology may be multi- factoral (Gerber, 1973). Clinical signs may vary depending upon chroni- city but typically include eXpiratory and inSpiratory dyspnea, diffuse wheezing, increased sputum production, and reduced exercise tolerance (Gillespie et al, 1969, McPherson et al, 1978). Usually signs are intermittent but in advanced cases the animal may be continuously dysp- neic. Signs frequently begin after a viral reSpiratory infection (Platt, 1972). Subsequently animals exhibit periods of severe airway obstruction following exposure to organic dust in stables and clinical signs abate when animals are at pasture (Breeze, 1979). The onset of signs can occur acutely following exposure to dust but more typically signs occur 4 to 8 hours after exposure. ~Chronically affected animals typically have diffuse bronchiolitis with goblet cell metaplasia of the bronchioles, excessive mucus in the small airways and acinar overinfla- tion (Thurlbeck et al, 1964). Although centrilobular emphysema and alveolitis have been described, they are not a consistent finding (Gillespie et a1, 1966). Physiologic investigations in heaves have not been correlated with pathologic lesions so that multiple physiologic conditions may have been studied. Decreased dynamic compliance, increased pulmonary resistance, prolonged nitrogen washout, hypoxia and decreased maximal expiratory flows indicate diffuse small and large airway obstruction (Sporri, 1964, Gillespie et al,. 1966, Leith et al, 1971, Muylle et a1, 1973, Nilloughby 21 et al, 1979). Gas dilution functional residual capacity is not increased but thoracic gas volume measured plethysmographically increases suggesting extensive gas trapping (Leith et al, 1971). This is confirmed at necropsy as lungs at minimum volume are hyperinflated. Similarities of intermittent heaves to human asthma include natural occurrence of the disease, chronicity of the condition with intermittent exacerbations and multifactoral etiology. In addition, pathologic lesions and tests of lung function are also similar. Since the naturally occurring disease condition may have various etiologies and clinical manifestations and functional lesions may be diverse, I studied experimentally induced lung disease in normal ponies. Two models were studied. The first model of airway obstruction is an allergen induced bronchoconstriction caused by challenging sensitized horses with aerosol ovalbumin (Mansman, 1973). Following aerosol challenge, dyspnea develops gradually and peaks at about 4 hours. Animals appear clinically normal at 24 hours. Data indicate that this reaction is a type III Arthus hypersensitivity like farmer's lung syndrome and some forms of asthma in man (Dickie et al, 1958). This is contrast to other existing animal models in which reactions are imme- diate and of short duration. The mechanism of bronchoconstriction induced by this new model is not known and either one or a combination of the mechanisms offered above could play a role in the pathogenesis. In the second model, chronic small airway disease is created by the oral administration of 3-methylindole (3MI) (Breeze et al, 1978A). Clinical signs of dyspnea appear at about 24 hours, peak in 6 to 12 days and animals are clinically normal in about 30 days (Breeze et a1, 1978A). 3-methy1indole is a metabolite of L-tryptophan and is a cause 22 of atypical interstitial pneumonia in cattle, grazing on pasture rich in L-tryptophan (Carlson et al, 1975). Pulmonary disease has been eXperi- mentally reproduced in cattle, sheep, goats and horses by the oral admi- nistration of 3MI (Atkinson et al, 1977, Bradley et al, 1978, Breeze et a1, 1978B). A single dose of 3MI has a half-life of about 30 minutes, most being excreted in the urine as oxendole derivatives. 3-methylindole does not accumulate in the tissues and is not present in the urine (Breeze, 19788). The mixed function oxidase system, which is the main metabolic pathway of xenobiotics, appears to be involved in metabolism of 3MI and is an essential factor in the development of pneumotoxicosis (Hammond et a1, 1979). Goats, pretreated with pipero- nyl butoxide (an inhibitor of the MFO system) do not develop clinical signs or pulmonary lesions when given an intravenous infusion of 3MI, whereas animals pretreated with phenobarbital (an inducer of the MFO system) develop more severe clinical signs and pulmonary lesions (Bray et al, 1979). Lesions are those of an alveolitis and bronchiolitis, mainly involving the bronchiolar epithelium (Breeze, 1978A). This disease model is not allergic in nature, and was studied to provide a comparison between the role of vagal mechanisms in the pathogenesis of allergic and toxicologic lung diseases. Purpose of the studies At the outset of this investigation we were concerned about the validity of pleural pressure measurements in the horse. Since transpulmonary pressure (the pressure difference between airway opening pressure and pleural pressure) is an essential measurement in pulmonary function studies, this question needed to be resolved before any further ' 23 study could be undertaken. There was no standard technique for measur- ing pleural pressure in the horse but commonly used methods employed esophageal balloons or esophageal balloons made from condoms and direct puncture of the pleural space at various sites (Denac-Sikiric, 1970, Sasse, 1971, Sorenson et al, 1980). In persons and dogs, e50phageal pressure is commonly used as a measure of intrapleural pressure (Mead et al, 1955, Cherniack et al, 1955, Milic-Emili et a1, 1964). Although eSOphageal pressure may not always reflect absolute pleural pressure in these species, changes in eSOphageal pressure during breathing are simi- lar to changes in local pleural pressure (Daly et a1, 1963). In per- sons an eSOphageal pressure measuring technique has been standardized. Use is made of a 10 cm long esOphageal balloon containing 0.5 m1 of air, placed in the caudal portion of the thoracic part of the esophagus so as to minimize artifacts caused by heart beat, changes in posture, and pressure from mediastinal content (Milic-Emili et al, 1964). In all mammalian species studied, there is a gradient of pleural pressure from the dorsal to the ventral parts of the thorax (Krueger et al, 1961, Proctor et al, 1968, Fahri et al, 1969, Happin et a1, 1969, Hogg et al, 1969). In addition, regional changes in pleural pressure during breathing can be variable (Rousson et al, 1976, Engel ‘et al, 1977). If the latter is true in the horse, pleural pressure may vary with the site of measurement. Thus, the purpose of the first study, described in Chapter 1, was to compare intrapleural pressure uneasured at 3 sites in the thorax, with esophageal pressure at different ponnts in the thoracic part of the esophagus, using 2 commonly used balloons. In order to study the role of vagal mechanisms in our disease 24 models, I wanted to be able to reversibly block the vagus nerves in conscious chronic animals. As reviewed by Franz et a1 (1968) mammalian nerve conduction can be inhibited by cold block. When temperature of a nerve decreases, the maximum transmissible frequency of impulses decreases so that for example in a myelinated nerve with a conduction velocity of 40 meters second'], a dr0p in temperature from 20 to 10°C causes a decrease in maximum transmissible frequency from 240 to 40 impulses second-I. When the vagus nerve is cooled to 7°C, activity in myelinated fibers is almost completely blocked (Franz et al, 1968). However, low frequency activity in nonmyelinated fibers will continue to be conducted until a temperature of 4°C is reached (Paintal, 1971). Cooling of a portion of the vagus nerves is facilitated by the creation of cervical vagal leaps. Cooling of surgically prepared vagal 100ps is a commonly used technique for vagal blockade in dogs (Phillipson et al, 1975, Snapper et al, 1979). The purpose of the study presented in chapter 2 was to describe the adaptation and use of this technique in the standing conscious pony. Although pulmonary function tests have been used to evaluate horses with clinically normal lungs, few comprehensive studies of equine respiratory function have been made and the range of reported values is large (Mauderly, 1974, Orr et al 1975, Willoughby and McDonell, 1979). This may be due to differences in techniques used by the various investigators or because of real variation in values. Information about the repeatability of pulmonary function tests in normal horses was therefore necessary before models of lung disease could be studied. In addition, since I was interested in studying vagal mechanisms in disease, the role of the vagus nerve in control of pulmonary function in 25 healthy animals needed first be established. This information was not available for the equid. Therefore, the purpose of the study reported in chapter 3 was to assess the repeatability of pulmonary function measurements within a day, and over a 6-month period, to determine the effect of changes in lung volume on total respiratory resistance, to evaluate the effect of respiratory frequency on dynamic compliance and to study the effect of vagal blockade on pulmonary mechanics, lung volu- mes and gas exchange. 3-methylindole induced pulmonary toxicosis in the horse is unique because the horse is the only species studied so far in which oral or intravenous administration of 3MI produces a pure small airway obstruc- tion (Breeze, 1978). The study of this disease model was of interest because clinical signs of the disease are indistinguishable from the naturally occurring asthma-like syndrome in the horse and because it provided a comparison between the importance of vagal mechanisms in the pathogenesis of an allergic disease model (Chapter 5) and a pneumotoxi- cosis with no allergic etiology. Thus, in chapter 4 I report changes in pulmonary function in the early stages of 3MI induced pulmonary toxi- cosis, correlate functional changes with pathologic lesions and deter- mine the role of vagal mechanisms in the pathogenesis of disease. Chapter 5 represents an in-depth study of the role of vagal mecha- nisms in ovalbumin induced allergic lung disease in the sensitized horse. In awake sensitized ponies I studied the effect of aerosol ovalbumin challenge on ventilation, pulmonary mechanics, lung volumes and gas exchange over a five-hour period and before and after vagal blockade. I subsequently challenged one lung in a second group of sen- sitized ponies and measured respiratory rate and right and left lung 26 resistance (RtotR and RtotL) during the same time period and before and after both ipsilateral and bilateral vagal blockade. I reasoned that if vagal reflexes, originating in the challenged lungs or a challenge induced increase in efferent parasympathetic bronchomotor activity were responsible for airway narrowing in this disease model, unilateral aero- sol antigen challenge would result in airway narrowing in both lungs, abolished by either unilateral or bilateral vagal blockade. If aerosol antigen challenge increased the sensitivity of airway smooth muscle to normal vagal tone or if a decreased baseline airway caliber was impor- tant, left unilateral challenge would result in increase in RtotL only, abolished by either unilateral or bilateral vagal blockade, while if local mechanisms were important in airway caliber changes, unilateral challenge would only cause an increase in RtotLa unaffectd by vagotomy. Since pilot studies suggested that aerosol challenge fbllowing both systemic and local sensitization of the lung results in more severe dyspnea of rapid onset, using both unilateral and bilateral challenge protocols I investigated the pulmonary response to aerosol challenge in both systemically and locally sensitized ponies and studied the role of local and vagal mechanisms in this reSponse. In addition, I correlated functional changes with pathologic lesions as presently no information is available to document that changes in pulmonary function values have value in predicting location and relative severity of pathologic lesions in the equine lung. CHAPTER 1 Esophageal and Intrapleural Pressure in The Healthy Conscious Pony 28 Introduction Dynamic compliance and pulmonary resistance are measured as lung function tests in horses. To determine these values, measurements must be made of transpulmonary pressure, i.e., the pressure gradient between the airway opening and the pleural cavity. There is no standard tech- nique for measuring pleural pressure. Commonly used methods include using esophageal balloons or eSOphageal balloons made from condoms and direct puncture of the pleural space.1"10 Reported values fOr dynamic compliance vary, and we sought to determine whether the variation was partly due to different techniques for measuring pleural pressure. In persons and dogs, esophageal pressure is commonly used as a measure of intrapleural pressure.11'21 Although e50phageal pressure may not always reflect absolute pleural pressure, changes in esophageal pressure during breathing are similar to changes in local pleural pressure.11:12:14:15 In persons, an esophageal pressure measuring tech- nique has been standardized; use is made of a 10-cm long esophageal balloon containing 0.5 ml of air placed in the caudal portion of the thoracic part of the esophagus so as to minimize artifacts caused by heart beat, changes in posture, and pressure from mediastinal contents.15a20 In all mammalian species studied, there is a gradient of pleural Pressure from the dorsal to the ventral parts of the thorax.22'27 In addition, regional changes in pleural pressure during breathing can be variable.23-30.a.b If the latter is true in the horse, variability in ‘ a Kelly S, Roussos CS, Engel LA: Gravity independent sequential b emotying from topographical lung regions. Clin Res. 23:645A, 1975. Roussos CS, Genest J, Cosco MJ, et a1: Rib cage vs abdominal breathing and ventilation distribution. Clin Res. 23:648A, 1975. 29 reported values of dynamic compliance may be related to the site at which pleural pressure is measured. In the literature, there are no statistical comparisons of pleural and esophageal pressure in horses. The purpose of the present study was to compare intrapleural pressure (measured at 3 sites in the thorax) with esophageal pressure at different points in the thoracic part of the esophagus, using 2 commonly used balloons. Materials and Methods Six grade ponies, between 2 and 10 years old and weighing 160 to 180 kg each, were tranquilized with xylazinec to effect and were restrained in stocks. Using local anesthesia, a tracheostomy was performed and a 20-mm diameter endotracheal tube was introduced into the trachea. A Fleisch pneumotachograph (No. 4)d and associated pressure transducere were attached to the endotracheal tube. The pneumotachograph transducer system produced a signal pr0portional to flow which was electronically integrated to give tidal volume. This system was calibrated by fercing a known volume of air through the pneumotachograph after each experi- ment. Pleural pressure was recorded through three 6.5-cm blunt tipped 12 gauge needles, with 2 side holes near the tip. The needles were attached with 60-cm lengths of polyethylene tubing (ID = 1.67 mm, 00 = 2.42 mm) to 3 pressure transducers.f The transducers were taped to the thoracic wall, using elastic tape. The lst needle was introduced into C Rompum, Haver Hockhart, Shawnee Mission, Kan. d Dynasciences Bluebell, PA. e Model PMS, Statham Instruments, Hato Rey, Puerto Rico. f Model P2306 Statham Instruments, Hato Rey, Puerto Rico. 30 the pleural cavity at_a slight angle downwards through the right 10th intercostal space at the level of the point of the shoulder. A distinc- tive pop was felt when the needle penetrated the parietal pleura. The 2nd and the 3rd needles were introduced 10 cm and 20 cm, reSpectively, above the Ist needle. Two types of eSOphageal balloons were used. The first balloon as recommended by Milic-Emili et al15 for use in persons was made of rubber and had the following dimensions: length 10 cm, perimeter 3.5 cm, wall thickness 0.06 mm. The 2nd balloon, as described by GilleSpie et a12 and Willoughby and McDonell,3 was made from a condomg and was 15 cm long with a perimeter of 10 cm. Both balloons were sealed over the end of polyethylene catheters (ID =3 mm, 00 a 4.4 mm, length = 140 cm) which had a number of spirally arranged holes in the part covered by the balloons. Distances from the nares to the caudal, middle, and cranial por- tions of the thoracic part of the eSOphagus were visually approximated and marked on the eSOphageal balloon catheter with indelible ink. The same distances were used on all subjects due to the similarity in size. Esophageal balloon catheters were made rigid by introduction of a length of 18 gauge steel wire and passed via the nares into the cranial portion of the esophagus. The wire was removed and the balloon was attached to a pressure transducerh which was taped to the forelock. Balloon volume was adjusted to contain 0.5 m1 of air in the e50phageal balloon or 3.5 ml in the condom. Esophageal pressure, 3 pleural pressures, and tidal 9 Trojan-enz Youngs Rubber Co., Trenton, NJ. h Model PM131TC Statham Instruments, Hato Rey, Puerto Rico. 31 volume were amplified and recorded on a 6-channel recorder.1 Three pleural pressures and tidal volume were recorded continuously. Esophageal pressure was recorded for at least 5 breaths at each esopha- geal location. The sequence of introduction of the e50phageal balloon and condom was randomized. At each measuring site, dynamic compliance was calculated during at least 4 breaths from tidal volume and the change in the es0phageal or pleural pressure between the start and end of inspiration. Results were analyzed with a 2-way analysis of variance and Tukey's W procedure at the 0.05 level of significance.31 To avoid phase differences between various measuring devices, a check of frequency response was made. An alternating pressure was generated in a closed flask by means by a syringe. The interior of the flask was connected with a 1 cm long (ID = 0.5 cm) tube to a differen tial pressure transducer.'1 This recording system was assumed to measure the true pressure fluctuations within the container. The pleural pressure needle or the esophageal balloons were introduced into the flask through a side arm and connected to the opposite side of the pressure transducer with the same tubing used during the experiments. Using pressure variations up to 30 cm of water and a frequency of 5 Hz, a flat response was recorded. The frequency response of the pleural needles and catheters was matched to that of the esophageal balloons by attaching these devices to opposite sides of a differential pressure transducer and exposing them to a quasisinusoidal oscillating pressure. The frequency reSponse of both ports of the pneumotachograph were similarly matched. Finally, h Model PM131TC Statham Instruments, Hato Rey, Puerto Rico. 1 Model KA, Soltec Corp., Sun Valley, Calif. 32 the response of the pneumotachograph transducer system and the pleural pressure and esophageal pressure transducer systems were checked by comparing pressure recorded with esOphageal or pleural catheters and transducers against pressure recorded with the pneumotachograph trans- ducer on an XY plotterj while exposing all devices to the same oscillating pressure source. All frequency responses were checked up to 5 Hz and were flat. Results A biphasic expiratory pattern was observed in 5 of the 6 horses. A passive exhalation was followed by a pause and an abdominal excursion immediately preceding the next inhalation (Fig 1-1). The pleural pressure at the plateau which occurred just before the abdominal effort was relatively constant from breath to breath and seemed to correspond with the end of a passive exhalation, whereas pressures at the end of the abdominal effort were highly variable. The pressure at the eXpira- tory plateau was therefore recorded as pressure at the equilibrium position of the respiratory system. This pressure increased from the dorsal to the ventral thoracic positions (Fig 1-1 and 1-2). Pressures in the middle and caudal portions of the thoracic part of the eSOphagus were similar, and not significantly different from pressures measured at the middle and ventral thoracic positions (Fig 1-2 and 1-3). Pressures in the cranial portion of the thoracic part of the eSOphagus were significantly higher than pressures at the dorsal and middle thoracic positions and dorsal thoracic position pressures were significantly 3 Model VR6, E for M, White Plains, NY. 33 lower than esophageal pressures. Pressures at the expiratory plateau as measured by the 2 balloon types were not significantly different. Changes in pleural pressure during respiration were variable from breath to breath. To compare the eSOphageal pressure waves which were not recorded simultaneously, a pressure amplitude that occurred in all middle thoracic position tracings for each animal was selected as a standard with which the other pressure changes could be compared. Mean selected pressure changes are reported in Table 1-1. Variables are listed in order of magnitude with the lowest value lst and the highest value last. Pressure changes underscored by the same line do not differ significantly. These data can be interpreted to mean that pressure changes in the cranial portion of the thoracic part of the esophagus were the least, pressure changes in the middle and ventral thoracic positions the greatest, and the pressure changes in the dorsal thoracic position and middle and caudal portions of the thoracic part of the esophagus were intermediate. Cardiogenic pressure oscillations were obvious in the tracings from the cranial portion of the thoracic part of the esophagus and masked pressure changes of respiratory origin. These artifacts were not pre- sent in the middle and caudal portions of the thoracic part of the esophagus. Large positive deflections corresponding to swallowing were most frequent in the cranial portion of the thoracic part of the esophagus but were also present in the middle and caudal portions of the thoracic part of the esophagus. There was no significant dif- ference between the 2 balloon types with respect to pressures, ampli- tudes, or artifacts. Air (10 ml) was introduced through the needles into the pleural 34 .covuupmgxm maveau manna xgoueepnxo lu_a mg» ouoz .q: m_ :o_umc_qm=_ .m:_=ueea was—o> as» so .gueoga H mcvcsv xeeogu ogu so acne pomeou ecu mammgqomm as» yo “can upumgogu we» we wean o—uupe men we moczmmoea mo com_eoaeou < Anv .__oz upumcogp on» =_ mmpuoo: pmgsopn meg mo copuvmoq ogu op Loewe _mmeou use .opeupe .pmeu:o> .zuemgn H mcpeau man—o> _uv_u ecu mmeammmea ~mezm—e Amy alH «Lame; 35 . H-H eeem_a .2 .0 80¢. GEE. momm GEE. mwkhb mm euo I I I I I P I P I III emz. 0 2.2.5 . oE=_o> IO—l 628 .88 oexee 3028mm f 2252 . mi 238$ eeeafi gees. 35> eo um¢ 36 Figure 1-2 Pleural pressure (E : SEM) measured during the mid- expiratory volume plateau in the ventral, middle, and dorsal thoracic wall. Distance = height of middle and dorsal thoracic pleural cannulae above the ventral thoracic cannulae. 37 Pressure -4- cmHZO 1.— ? 1 Ventral ' Middle Dorsal o 10 20 Distance cm Figure 1-2 38 .moeec —mceouxo on» o» coo__ea Peommgqomm ago we a.» oz» Eon» ouceumwu "oucmumpa .msmmsaomo as» we peea upoeeogu on“ yo mcopueoa panama ecu .o—uupe .pevcaeu mg» :_ sumac—q mas—o> agoumewaxmluvs as» mcpeav vocameoe Axum +llp oesmmmgq peommgqomu mlH weaned 39 ? mm. .838 . m-H eeemma :8. 3.655 mm. 2222 NS .820. -m... UNI i... ONIEo To 838i 4o mango Pachmu Ho: .1. oz 89% 39% 3.9% ”85H 89% Scan 89% oz oz 5m H w ~¢m.o Num.o ~om.o moo.o m~¢.o Hum.c mum.o oz oz cm: mo 5mm; 0 mpvww: ~eeuco>, pumeoml. Eovcou cooppmm. soveoo cooppmm. sowzou coo-ham. m:o_owmom ——m: upuecosp mammszmo as» ea wmuegaomo oz» mo mzwozmomo ogu mo ween ovumeogu one “emu uFUogogu on» neon u_oaeo;u us» we copucoa puuaau we co—ugoq opuupz mo copueoa _a_cmgu mmu_m ucmcmem_o um noezmmoz weammmga .ueao—e cw omcogu sage oops—sopnudeaeuv occuppaeou upsecaa l NlH m4m

vmgoumgoeca .meaemvogq z mxuxuh anew: .mceoe mo mcommeeaeoo «paeupaemo ppamoe mzogm mcvcoumgoeca . am: we Eu mm.- “.ml mm.xl sm.sl ma.~ ismnml -.m o¢.m ma.H e m—uv_z pmeucm> pence: Acoo__uav cooppom soucou Aeoueouu soucou zoo—pan mamagqoma ago we mammgnomm we» we m:o_u_moa .me o.umeo;» neon u_ueeogu ogu msmognomo mo “Lon u.uaeogu as» mawecqomo as» we so easygoq o_vupz agoa ovuegogu as» we cowugoq o_vv_z wean u_omgo;u as» mo :oPuLoq .uuaou mo copueoa —o_:ucu ouemlmc_ezmeoz ea :o_uu::u a we Ne, —e:ewmee e—ewmmee we» seem e» e>e=e one go eexees ace meewuwmee eweeeege pemLee eee .epeewe .peeeees egg on eeeee—e ageueewexeiewe on» eeweee easemeoe meeemmeee eeoz .eeemmeee xeeeespeemeeee u meemmoee .Am eececewee Eeewv e>L=e eE=_e> eeemmoce mes. ewueumlwmeee e we eawp ageueewexm e-H ee=e_a e-H eeemwa ONIEe easemen— .253 22.5 e .83 e.m . em e._ n e 0.- ow- . a 46 --------------_----- -—---‘ em efix TOO— 47 memesmeme one we peeml eweeeezu use we eewueee owe lewz .eexee amaze me» ecu zen—e we» :eezuee mew— eeew—ee —eomeneeme eee m e ee eeeem peemeeeeefi 53 36 M wm.~ 23233... 7.26:. 39.3 o .83 M m3 e eoueeeee pee euwm mw.o + Nwfi aceseeemeee peeeowe eeeLwo N“ o.m¢ + emm Leeweegm one new eexeu Lone» eoozeee eew— l. l. e e ee eeeem .eemeeeeeee gees e~.e + m.~ .ee=e_e eeeeee em m.efl + mom mommoeeee emce> lmeeee geese— we wee—e peeee~wces on» :ewen cw m 3 ocean weemeecoefi 53 84 M N~.~ 22.8335 .233 32.5 S I owe a gene; we omen ea weeeeu no.9 + m~.e gee—pee peemegeemu ma m.m + one eeeem ~eumeuceuew saw as» ueeeecemeee weeee—e ueec l. N :e xecegu we eewgp wemeeo H.m H.H.m iwe wee meemmeceweememeemm mH m.m + woe eeee oewm we eeweewcemoa ow: we Sex; osewegeee ueosecemeez mange; Axum H.xv icewem exec we .ez meeeme_wx cw ugmwez seem. memeez weweeeum pescez cw eoeweueo ee—e> waxeov eecew—eeeu ewEeeza eee eon: osewegeow newcemeoz we eemwgeeeeu l ml“ mem weew>eee egmwm .lillimuillxhaallfl mlm meemwe N-N eeeeea .. 3...»? e a _. . 60 eee meeee N we meme one: wwweu .meee— puma) esp uww ee eeeegm .meeew weme> one eueeemwewee on new: wwee mewweee Leeeeu m-~ ee=e_a 61 m Nw eemwm 62 pressure transducerc were attached to measure flow. The flow signal was electronically integrated to give tidal volume.d After each experiment, the pneumotachograph was calibrated by forcing air at known flow rates through the instrument. The volume signal was calibrated using a three liter syringe.e A 20 gauge catheterf was inserted into one exteriorized carotid artery and connected to a pressure transducerg placed at the level of the shoulder. ReSpiratory rate, tidal volume, heart rate and mean systemic blood pressure were recorded on light sensitive paper. After control measurements were taken, cooling coils were wrapped around both vagal loops. Cooling coils were made from capper piping (I.D. 4 mm, 0.0. 5 mm) and consisted of two parts shaped to fit the vagal loop (Fig. 2). The coils were attached via tubing to a cir- culating coolerh containing methanol. The temperature of this fluid was maintained at -2°C Li.'2°C)- Measurements of reSpiratory rate, tidal volume, heart rate and mean systemic blood pressure were repeated while the vagi were cooled, five minutes after removal of the coils and after administration of 0.04 mg/kg Atropine I/V. In addition, a laryngoscopic examination was performed on three ponies before, during and after vagal cooling. Results were analyzed using two-way analysis of variance. Means were compared by the Student Newman Keul's test.4 c Model PM5. Statham Instruments, Hato-Rey, PR d Model VRG. Electronics for Medicine, White plains, NJ e Hamilton Syringe Co., Whittier, CA f Becton-Dickinson, Rutherford, NJ 9 ”099] 92303: Statham Instruments, Hato-Rey, PR h Model 90, Fisher Scientific Co., Livonia, MI 63 Results Cooling of the vagus nerve increased tidal volume, heart rate and mean systemic blood pressure and decreased respiratory rate (Fig 2-4). Control values were not significantly different before and after vagal cooling. Atropine administration did not alter the respiratory parame- ters but increased heart rate and mean systemic pressure to levels simi- lar to those measured during vagal cooling (Fig 2-4). Laryngoscopic examination revealed complete bilateral laryngeal paresis during vagal blockade and normal laryngeal mobility during the control periods. Discussion The technique we have described resulted in the incorporation of a functional vagus nerve in a skin 100p. The principal problem encoun- tered was a tendency for skin wounds to dehisce beneath the vagal loop because of excessive tension. The tension relieving incision on the ventral midline of the neck successfully alleviated this problem whe- never it was used. The functional integrity of the vagus nerve was indicated by the changes in respiratory rate, tidal volume, heart rate and blood pressure following vagal cooling and the return of these para- meters to control levels when the vagi were warmed. Great care was taken not to incorporate the recurrent laryngeal nerve into the skin 100p nor to excessively traumatize this nerve during separation from the vagus nerve. Although several ponies exhibited transient laryngeal hemiplegia, all returned to normal. Because of this transient hemiplegia, bilateral loops were never simultaneously created. The sympathetic trunk was also isolated from the vagus during surgery. Its functional integrity was indicated by the absence of 64 Figure 2-4 ReSpiratory rate (RR), tidal volume (VT), heart rate (HR), and systemic blood pressure (Psyst) during a base-line period, after vagal cooling, during a 2nd base-line period, and after IV administration of 0.04 mg of atrOpine/kg of body weight (x.1 SEM). *Indicates signifi- cant difference from control value. 141»1 12- R R , _l 10- breaihsmln 8" a: 6- 3.1 /‘*\‘/1 . VT 2. liters IB:[F?S§. '- 901 70 a: * HR 50.. beoismin" 30.. 160-1 * * Psysi '40. mmHg '20“ 100 Control Vagal Control Atropine Cooling Figure 2-4 66 Horner‘s syndrome following surgery or during blockade. Horner‘s syndrome is usually observed in horses following damage of the sym- pathetic trunk.5:6 Results of this experiment indicate that cooling of vagal 100ps caused reversible blockade of both afferent and efferent vagal nerve fibers in the standing conscious pony. The increased tidal volume and decreased respiratory rate observed after vagal cooling are also reported in dogs after vagotomy and are attributed to stretch receptor fiber blockade and subsequent interruption of the Hering-Breuer reflex.2.7»3 In addition, ponies exhibited increased heart rate and mean systemic blood pressure after vagal blockade. Similar changes in dogs are attri- buted to blockade of cardiac efferent preganglionic parasympathetic fibers.7»3a9a10 The increased heart rate is thought to increase cardiac output and therefore increase mean systemic pressure.9 Since heart rate and systemic pressure were not different during vagal cooling and following atropine, it appears that efferent cardiac preganglionic para- sympathetic fiber blockade was complete during vagal cooling. Laryngoscopy of three ponies during vagal cooling revealed complete bilateral laryngeal paresis. Since the recurrent laryngeal nerves were not included in the skin loops, the laryngeal paresis probably resulted from blockade of vagal fibers that subsequently form the recurrent laryngeal nerve.11 In two ponies, the endotracheal tube was removed during vagal blockade and the tracheostomy opening occluded. Animals breathed normally until a deep breath was taken. At that time, the larynx collapsed and animals became extremely dyspneic until the endotracheal tube was reinserted. A tracheostomy is therefore essential to insure a patent airway when blocking both vagi simultaneously. 67 References l. Phillipson EA, Murphy E, Kozar LF, et al: Role of vagal stimuli in exercise ventilation in dogs with eXperimental pneumonitis. J Appl Physiol 39:76-85, 1975. 2. Phillipson EA, Hickey RF, Bainton LR, et al: Effects of vagal blockade on regulation of breathing in conscious dogs. J Appl Physiol 29:475-479, 1970. * 3. Snapper JR, Drasen JM, Loring SH, et a1: Vagal effects on histamine, carbacol and prostaglandin an responsiveness in the dog. J Appl Physiol 47:13-16, 1979. 4. Steel GD, Torrie JH: Principles and Procedures of Statistics. McGraw-Hill Book Co, New York, 1960. 5. Firth EC: Horner's Syndrome in the horse: Experimental induc- tion and a case report. Equine Vet J 10(1):9-l3, 1978. 6. Smith JS, Mayhew IG: Horner's Syndrome in large animals. Cornell Vet 65:529-542, 1977. 7. MacCanon DM, Howath SM: Effect of bilateral cervical vagotomy in the dog. Am J Physiol 189:569-572, 1957. 8. Shepard RS, Whitty AJ: Bilateral cervical vagotomy: A long- term study on the unanesthetized dog. Am J Physiol 206:265-269, 1964. 9. Stone HL, Bishop VS: Ventricular output in conscious dogs following acute vagal blockade. J Appl Physiol 28:782-786, 1968. 10. Whitty AJ, Shepard RS: Role of the vagus in control of cardiac output in the unanesthetized dog. Am J Physiol 213:1520-1525, 1967. ll. Godinko HP, Getty R: The Anatomy of the Domestic Animal. ed 5, Philadelphia, Saunders Company, 1975, pp 660-663. CHAPTER 3 Pulmonary Function Tests in Standing Ponies: Reproducibility and Effect of Vagal Blockade 69 Introduction Although pulmonary function tests have been used to evaluate horses with clinically normal lungs and those with chronic lung disease, few comprehensive studies of equine respiratory function are presently available and the range of reported normal values is 1arge.l-6 This may be due to differences in techniques used by the various investigators or because of real variation in values. Information about the repeatabi- lity of pulmonary function tests in individual horses and groups of horses is therefore necessary to resolve this question. Clinical evidence suggests that the parasympathetic nervous system plays a role in the pathogenesis of chronic obstructive pulmonary disease, as many cases reSpond to atropine administration.7 Similarly vagal mechanisms play a role in pathogenesis of allergic lung disease in other species.3'10 In order to study vagal mechanisms in disease, the role of the vagus nerve in control of pulmonary function in healthy ani- mals must first be established. Presently this information is not 1 available for the equid. The purpose of this investigation was to assess the repeatability of pulmonary function measurements within a day and over a six-month period, to determine the effect of changes in lung volume on total respiratory resistance, to evaluate the effect of respiratory frequency on dynamic compliance, and to study the effect of vagal blockade on pulmonary mechanics, lung volumes and gas exchange. Materials and Methods Five ponies between two and ten years of age (2’= 6.6 years) weighing 199 i 27.0 kg (z 1.5EM) with bilateral cervical vagal loops and 7O ' exteriorized carotid arteries were used in the experiments.11 Prior to use, animals had been on pasture for at least two months and all were vaccinated for the common viral respiratory diseases. Animals were regularly examined to detect any signs of respiratory disease. Pulmonary Function Measurements Ponies were tranquilized with xylazinea (0.5 mg/kg) and restrained in stocks. A 20 mm diameter cuffed endotracheal tube was introduced into the trachea via a tracheostoma. A Fleisch pneumotachograph (n04)b and associated pressure transducerc were attached to the endotracheal tube. The pneumotachograph transducer system produced a signal proportional to flow which was electronically integrated to give tidal volume. After each experiment, this system was calibrated by forcing known volumes and flows of air through the pneumotachograph using a three liter calibrated syringed and a rotameter flow meter.e An esophageal balloon (length 10 cm, perimeter 3.5 cm, wall thickness 0.06 cm) was sealed over the end of a polyethylene catheter (ID = 3 mm, 0.0. = 4.4 mm, length 140 cm) which had a number of spirally arranged holes in the part covered by the balloon. The distance from the nares to the middle portion of the thoracic esophagus was visually approximated and marked on the esophageal balloon catheter with inde- lible ink. The esophageal balloon catheter was made rigid by intro- ducing a length of 18 gauge steel wire and passed via the nares into the a Rompun, Haver Lockhart, Shawnee, Mission, KS Dynasciences, Blue Bell, PA C Model PMS, Statham Instruments, Hato Rey, PR d 3 liter Super Syringe, Warren E. Collins Inc, Braintree, MA 9 Model 10A3500, Fisher & Porter Co, Warminster, PN 71 middle portion of the thoracic eSOphagus. The wire was removed and the balloon attached to a pressure transducerf which was taped to the fore- lock. The opposite side of the differential pressure transducer was attached to an identical balloon catheter system, with the balloon located just inside the distal end of the endotracheal tube to measure airway opening pressure (P30). Transpulmonary pressure (Ptp) was defined as the pressure difference between the airway opening pressure (Pao) and esophageal pressure (Pes)- Balloon volumes were adjusted to contain 0.5 ml of air. Transpulmonary pressure, tidal volume (VT) and flow were recorded on light sensitive paper.9 From these traces, dyna- mic compliance (Cdyn). respiratory rate (RR) and minute ventilation (Vmin) were calculated.12 A pressure cycled ventilatorh was attached to the endotracheal tube via the pneumotachograph. Animals were force ventilated to 20 cm H20 Ptp for two breaths to insure constant lung volume history prior to recording quasistatic pressure volume curves. Quasistatic pressure volume curves of lung and chest wall were generated by inflating the respiratory system to Ptp = 20 cm H20 and allowing it to deflate slowly to functional residual capacity (FRC). To minimize flow resistive far- ces, rate of deflation was slowed by a retard valve on the expiratory line of the ventilator. Lung and thoracic cage pressure volume curves were recorded by plotting Ptp and Pes respectively against lung volume on an x-y plotteri during at least two quasistatic pressure volume f Model PM 131 TC, Statham Instruments, Hato Rey, PR 9 Model VR6, Electronics for Medicine, White Plains, NY 0 Mark 9, Bird Co, Palm Springs, CA 1 Model XY575, Esterline Angus Co, Indianapolis, IN 72 maneuvers. Using a digital computeri the deflation limb of the lung pressure volume curves was empirically described as a single rising exponential.13 V = vmax (l-e’“ PtP) (I) where V = lung volume at a given transpulmonary pressure (Ptp), Vmax is the lung volume at which the slope of the curve is zero (i.e., at infi- nite Ptp) and 0 defines the rate of rise of the curve from FRC to the Vmax- Quasistatic compliance (Cstat) was calculated from the first derivative of equation #1 at Ptp = 3 cm H20. Subsequently, animals were force ventilated four times up to a transpulmonary pressure of 20 cm H20 to create a period of apnea, lasting between 10 and 30 sec. During this period of apnea, an oscilla- tion system consisting of a sine wave generator,k an amplifier and a 12" speaker in box (Fig 3-1), was attached to the endotracheal tube via the pneumotachograph. Pressure and flow were recorded on an oscilloscope as sinusoidal flow oscillations were applied to the lung via the l endotracheal tube. Oscillation frequency was modulated until the pressure flow loop closed, usually between 5 and 10 Hz. The closed pressure flow loop was recorded on light sensitive paper and total respiratory resistance was calculated as the slope of the line. At least two recordings were made for each measurement. In order to prevent phase differences between pressure and flows, frequency responses of catheter systems were carefully evaluated as pre- viously described.14 In addition, the airway Opening pressure signal and flow signal used to measure oscillatory resistance were evaluated up to 3 Model PDPll, Digital Equipment Co, Maynard, MA k Model 200, Continental Specialties Co, New Haven, CT 73 .eeceumwmec xceeeewemee Pepe» ecumees eu new: Eeemxm :eweewwweme eeeeea H-m oeem_a 74 H-m ocemwe mozmwzmo m><3 wz.m .3332 w... .33 > .U .U 3:3» 0.5mmown. WWW I mucjaze .HHHUUHD >._._..mo_m 10.: mwoaowzgao we:_o>-og=mmacq AAV m==_ new Ahv ammo uFumcogu mo mnsw— agoumcwqu mu_moqsou m-m ma=m_u 81 m-m ma:m.u ouxeo Ba .5 3n. ommNmNVNNNONm. m. S QC. 0 m .V N o N: _ \ ‘ “I“ 3.: T om... om .2. 0.: o\o Fom .om 82 .xuvuuauu mcap Page» u ugh moumxoopn pumm> n m> .mumxuo_a ~umm> gmuea new mcoeon .mmE=_o> wasp m:_momsu=_ an vogammms Auoumv mucmum_mog soumxm xgoumcpqmmg page» ¢-m ma=m_a 83 ¢-m mesmwm o.:.o\o oo. om om. on om ILIIIIIPIIILIILIL Om LII m. .2: ooméNIEo 8;... =o_umpgm> mo u=m_u_mwoou u >0 «mu—uaamo m::_ .auou u ugh »a_uuaau —a=v_mwg po=o_uu:=$ u emu ”mocuwpasou u_uoum_ma=c u uuumu mocaw_qsou u_sm=av a cave «mucmumvmmg gunman agouucpammc peace a uoua copuupwa=o> ou==_e u =_e> mosspo> —mv_u u h> ”one; xgouogpammg u «a ~.m a.m w.m m.- c.0H m.m H.N~ N.- m.NH c.m m.~ >u mc.o m¢.o No.o m¢.c mo.c m¢.o oo.o ae.o Ush\uma m.H ¢.oH m.H m.o~ an.“ ~.a~ ¢.~ a.¢~ Amman 4v use ~¢.c a.» ..o o.- ~m.e a.» ~e.o m.~ Amaua 4v uma o¢~.o can.” mH~.c oH~.H mm~.o mmm.~ Nom.o -m.c A~-c~= so by young mmfi.o Hca.c ooH.o .mm.o -H.¢ ¢m~.c Nao.o mom.o AH-o~= so by chug amc.o m~¢.o m.o.o mmm.c m~o.o om¢.o mmo.o Nam.o AH-4 com om: gov doom m.“ o.m~ ~.m o.~m m.m m.Hm ~.¢ o.- A~-=_s 4V =_so m~.o m.H -.o m.~ NH.° m.~ m~.c om.“ Angus 4V h> o.~ m.e~ m.“ ~.mH c." N.N. o.~ e.¢H AH-=_EV «a m.¢ H.~¢ m.H o.H¢ m.~ o.mm ~o.c N... Aaaopv Nouns H.m m.~m ~.e ”.mm a.” H.nw mo.H m.~m Aacopv Nona o.om~ m.¢o~ m.dmfi o.m¢~ Amxv a=a_a= scam xm mu .8 m. xm m. xm m. v‘ Agog m* Agog ufi xgom Ha xcom ucosagamamz .mpu>gou:_ a—gaog an umumoaac mm_v=um o sage um>pcmu Ace—uu_>ov vgoucnum ago gumsv mm=_u> :o_uu==m agacospam Hum m—nm» 85 covuopcm> mo u:m_u_$wmou u >9 mxuyuoaou mes. ~uuo» a gap Aupuaquo pmzupmmg _m:o_uo:=m u um; moo:u__aeou o_paum_ma:c a young mucnvpasou u_su=xu u exec «oucuumpmac soumxm agoucgwammg ~oaou u Hoax :o_uu_wu:m> muacps u :_s> mosapo> pun.» u »> «one; xyoumcpqmmg n ma o... mc.o ~m.o so.o om.o oo.o ¢¢.o mo.c om.o mc.o mm.o ush\u¢a ~.m~ m.m c.H~ ~.m o.o~ o.m m.m~ e.” m.m~ m.H ".mH Amaun by use o.H~ m.~ o.HH ~.N o.oH N.N m.HH m.~ ~.cH m.~ H.¢ Aways by age o.m~ ~m~.a omn.~ emm.o new." ¢~¢.o m~¢.H omm.° awe.“ Ho~.o woo.“ A~-o~= so by “mama o.mm mm¢.o «ow.c a-.o em~.c ~a~.o mm~.o omm.c mko.o Hm~.c mmk.c A~-o~= 50 4V csvu m.m~ omo.o eke.c a~H.o mom.o emo.o mNm.o “mo.c om¢.o mm~.o om¢.c A.-4 um. om: gov Hoax H.m~ m.~H o.e~ m.m k.m~ m.o c.H~ m.o H.H~ ~.~ ~.m~ “H-=_a 4V =.e» o.¢~ mm.c ~.~ Hm.o H.N km.c o.~ -.o . a.” mH.o m.H Away; by h> ”.mH m.” ~.oH ~.~ k.~H m.~ m.oH m." ~.~H N.” o.m~ AH-=.av «a ¢.m o.~ ~.o¢ H.~ H.~¢ a.“ a... o.~ m.km A.“ c.H¢ Aesopv Noun. ~.m m.~ H.Nm M.“ a.mm m.~ H.mm e.m_ ~.¢w m.e m.mm Attacv Nona ~.¢ c.m¢ o.-~ m.¢H o.~¢~ o.c c.¢o~ ~.HH H.mm~ o.- o.mm~ Amxv pgm.m= swam Id... .3 w xm w xm w .3 w .3 m. m‘ xcom ¢* xcog m; ace; ~§ ago; am xcom acasmgsmuoz .m—m>gaa=_ gucosaozu um vouuaucou mm_v=um m umump an 56;; vo>_gav AgopuuF>oe ugavcoum ucm cumsv mozpu> :o_uu==p xgocoepzm mum «pan» 86 auvuoaau .uaupmmg paccpuucam a gag “moan. xuvuuauu m==_ papa» pqeoo u_umumpmm=a u u ugh «mung mu:a_—asoo upsucxu u :xvo "mucoumwmmc Eoumxm xgoumcpnmuc puuou a yoga copuu—'u=m> «wasps u :_s> mosa—o> pmu_u u h> ”mung xgoungwammc u «a Amo.ov Nm.o QHH12 mfifiufim 36 H 33 .5... .+. m8... m8... H 8.5 fisumém ofioqu Qflufiz Naumae ~.¢_H.o.mm ”.8 H SN in H m. v me_h 30.3 35 mm; H as ROHQ: :3 H 33 3c... M :3. .85 H 8.5 meme: 86 H 8; 9~n92 3 H 3: 3 u. 1.3 qausu in HR m «EFF Amc.ov m¢.o 3 .+. «.8 :5 .+. 3.. 9%... H 33 83 H $3 ~85 H 33 2 H ~.- 2... .+. 8; RHHfiS 3. u. 3:. o.» H «.8 9% H m8 2% Hm. a: oz 3... .+. 2: oz 82. H OB... >86 .+. 8:. 3 M 3m aduom 3 H 9: o; H 98 5 H13 SN H 8. 2mm Hm. N mark a mark uuh\u¢u Amaua 4v use Away; by age .H-o~= so by uubmo AH-o~= so by exec Agug uwm om: Euv uoua Amncps 4v awe» Ana»; 4v h> Aanzwsv mm ALLOHV Noun. Aceohv Noam Amxv u=m_m= seem acosmcamumz nouuaucou .mowcoa a>pw umuuaucoo mo_v:um ¢ soc» vm>Pgou Axum.fi .m_u>gmu:_ gucosuozu an m» moapu> copuucsm agocos—am mum «pan» 87 defined by equation l. Alpha averaged 0.1594 1 0.0135 cm H20'1. The volume at which thoracic cage and lung elastic recoil were equal and opposite was not significantly different from FRC measured by He equilibration. Vagal blockade increased VT and decreased RR. In addition respira- tory resistance at functional residual capacity was decreased by vagal blockade from 0.496 :.0.054 to 0.36l 1_ 0.03 cm H20 sec liter-1 but arterial blood gas tensions, 9min» cdyna Cstat FRC, TLC and lung and. thoracic cage pressure-volume curves were unaffected. Baseline values of all variables were the same before and after vagal blockade. Figure 3-4 shows the effect of vagal blockade and lung volume on Rtot- Resistance decreased significantly with increasing lung volume. Vagal blockade significantly decreased Rtot at FRC but was without effect at higher lung volumes. Dynamic compliance did not change as respiratory frequency increased from lS-GO breaths/minute. Discussion This study has documented the daily and monthly variability of pulmonary function measurements in standing conscious ponies. Variability of TLC and arterial blood gas tensions was small over both the short and long-term measurement periods. This finding is not surprising. Total lung capacity is a fixed volume probably defined by the elastic limits of the lung. In the case of arterial blood gases, respiratory control mechanisms maintain these values within fairly tight limits to ensure adequate gas transport to and from the tissues. The variability in cstat was surprising since the elastic properties 88 of the lung would appear to be determined by lung structure. However, airway closure also affects the shape of the pressure-volume curve and may have been responsible for some of the variability in Cstat even though we attempted to eliminate this possibility by inflating the lung to Ptp-ZO cm H20 prior to recording the lung pressure-volume curve. Variability of FRC, Rtot» Cdyn: lmina VT, and RR was considerable over the long term. The variability in FRC may be the result of variations in posture, respiratory muscle tone and changes in abdominal filling caused by alterations in diet and fat deposition. As shown in Fig. 3-4, changes in lung volume result in changes in Rtot and it is possible that the variability in FRC was responsible in part fOr the variability in resistance. Since Cdyn is determined by both lung elastic recoil (indicated by Cstat) and the resistance of airways (indicated by Rtot) and since both Cstat and Rtot were quite variable, the variability in Cdyn is not surprising.17 Furthermore, calculation of Cdyn assumes inertial fOrces are negligible during tidal breathing.18 This assumption may not be valid in the horse as there are rapid rates of change of flow par- ticularly between inhalation and exhalation. The variability in 9min: VT, and RR was not surprising since 9min is determined in part by metabolism and the possible combinations of RR and VT for a given 9min are limitless. Hith the exception of the variability in Cdyna variability of our measurements was similar to that reported in conscious calves and dogs studied at daily and monthly intervals, respectively.19:20 When data from five horses was grouped there was no significant change in any variable over the six-month study period. These data suggest that with 89 the exception of arterial blood gas tensions, the results of pulmonary function tests described in this paper are too variable to be useful in detecting individual horses with mild or moderate lung disease but may be useful in assessing the effects of treatments on lung function in a group of horses studied over a period of days or months. He have previously reported a midexpiratory cessation of air flow in tracheostomized ponies.14 This midexpiratory pause appears to occur at a relatively constant lung volume whereas lung volume at end expiration varies with the amount of abdominal eXpiratory effort. Leithn has suggested that the midexpiratory pause represents the equilibrium point of the respiratory system where lung and thoracic cage recoil are equal and opposite. Examination of lung and thoracic cage pressure-volume curves (Fig 3-3) shows that in our ponies, equilibrium volume was not significantly different from lung volume at the midexpiratory pause suggesting this volume is determined by passive relaxation of the respiratory system. However this conclusion must be tempered with caution because we did not ascertain that the respiratory muscles were relaxed although we did provide two deep breaths to induce apnea prior to recording pressure-volume curves. Salazar et al showed empirically that the expiratory limb of the dog quasistatic pressure-volume curve can be described by a single rising exponential.13 Our data suggest that this is also true in ponies. Alpha (the parameter describing the rate of rise of the expira- tory limb of the pressure-volume curve) was similar to the value calcu- lated from data obtained in anesthetized horses suspended upright.21.22 " Leith DE, Personal communications, 198l 90 Static compliance calculated from the first derivative of the single rising exponential at Ptp=3 cm H20 was also similar on a body weight basis to values reported in anesthetized upright horses but greater than values in anesthetized ponies in which there may have been considerable airway closure.°:3:4 The effects of vagal blockade in ponies are similar to effects in dogs.23:24 Tidal volume increased and respiratory rate decreased pro- bably as a result of blockade of vagal afferents from pulmonary recep- tors. Vagal blockade had no effect on lung and thoracic cage pressure- volume behavior. The primary effect of vagal blockade was a decrease in respiratory resistance at FRC but not at higher lung volumes. This interaction of parasympathetic tone and lung volume in determining resistance was also reported in dogs by Macklem et al who proposed the following explanation based on pressure diameter behavior of airways with and without bronchomotor tone.25 Isolated bronchi lacking broncho- motor tone increase maximally in diameter with only small changes in transmural pressure (+ 3 cm H20) whereas intact bronchi with parasym- pathetic tone increase in diameter progressively as transmural pressure increases to 30 cm H20. In the ponies with vagal tone, resistance therefore decreases progressively with increasing lung volume. In contrast fOllowing vagal blockade airways are probably almost maximally dilated at FRC and increasing lung volume causes only a slight decrease in resistance. Frequency dependence of lung compliance results when there is ine- quality of time constants in peripheral parallel units in the lung and ° Leith DE, Gillespie JR: Respiratory mechanics of normal horses and one with chronic obstructive lung disease. .Egd Proc 30:556, l97l. 91 for this reason is suggested as an indicator of peripheral airway obstruction.26 The lack of frequency dependence in our normal ponies suggests equality of time constants and a lack of peripheral airway obstruction. However, Macklem et al calculated that a five-fold variation in time constant would cause only a 25% reduction in Cd," at a respiratory frequency of 60 breaths/minute and a two-fold difference in time constants would not be detectable.25 Thus considerable variability in peripheral time constants may exist in our normal ponies despite the lack of frequency dependent compliance. Even with these limitations frequency dependence of compliance is still one of the most sensitive tests of small airway obstruction in persons and may prove to be a valuable test in the detection of small airway disease in the horse.26 92 References l. Mauderly JL: Evaluation of the grade pony as a pulmonary func- tion model. Am J Vet Res 35:1025-1029, 1974. 2. Purchase IFH: The measurement of compliance and other reSpira- tory parameters in horses. Vet Record 78:613-616, 1966. 3. Rawlings CA, Birnbaum ML, Bisgard GE: Static pulmonary compliance in ponies. J Appl Physiol 38:657-660, 1975. 4. Muylle E, Dyaert H: Lung function tests in obstructive pul- monary disease in horses. Equine Vet J 5:37-43, 1973. 5. Willoughby RA, McDonell NN: Pulmonary function testing in hor- ses. Vet Clinics of N Am l:l7l-l96, 1979. 6. Drr JA, Bisgard GE, Forster HV, et al: Cardiopulmonary measure- ments in nonanesthetized, resting normal ponies. Am J Vet Res 36:1667-1670, 1975. 7. Murphy JR, McPherson EA, Dixon PM: Chronic obstructive pulmo- nary disease (CDPD): Effects of bronchodilator drugs on normal and affected horses.. Equine Vet J 12(1):10-l4, 1980. 8. Gold HM, Kessler GF, Yu DYC: Role of vagus nerves in experimen- tal asthma in allergic dogs. J Appl Physiol 33:719-725, 1972. 9. Kessler GF, Austin JHM, Graf PD, et al: Airway constriction in experimental asthma in allergic dogs: Tantalum bronchographic studies. J Appl Physiol 35:703-708, 1973. 10. Yu DYC, Galant SP, Gold HM: Inhibition of antigen-induced bronchoconstriction by atropine in asthmatic patients. J Appl Physiol 32:823-828, 1972. ll. Derksen FJ, Robinson NE, Stick JA: Technique for reversible vagal blockade in the standing conscious pony. Am J Vet Res In press. 12. Mead J, Hhittenburger JL: Physical properties of human lungs measured during spontaneous respiration. J Appl Physiol 5:779-796, 1953. 13. Salazar E, Knowles JH: An analysis of pressure-volume charac- teristics of the lungs. J Appl Physiol 19:97-104, 1964. 14. Derksen FJ, Robinson NE: E50phageal and intrapleural pressures in the healthy conscious pony. Am J Vet Res 41:1756-1761, 1980. 15. Denac-Sikiric M: Die functionelle Residualkapazitat und Helium- Einmischzeit gesunder and lungenkranker Pferde. Zbl Vet Med 23:195-205, 1976. 93 16. Steel RGD, Torrie JH: Principles and Procedures of Statistics. McGraw-Hill, New York, 1960. 17. Otis AB, McKerrow CB, Bartlett RA, et al: Mechanical factors in distribution of pulmonary ventilation. J Appl Physiol 8:427-443, 1956. 18. Mead J: Measurement of inertia of the lungs at increased ambient pressure. J Appl Physiol 9:208-212, 1956. 19. Dain D, Gold NM: Mechanical properties of the lung and experi- mental asthma in conscious allergic dogs. J Appl Physiol 38:96-100, 1975. 20. Kiorpes AL, Bisgard GE, Manchar M: Pulmonary function values in healthy Holstein-Friesian calves. Am J Vet Res 39:773-778, 1978. 21. Schroter RG: Quantitative comparisons of mammalian lung pressure-volume curves. Respiratory Physiol 42:101-107, 1980. 22. Leith DE: Comparative mammalian respiratory mechanics. Physiologist 19:405-510, 1976. 23. McCanon DM, Howath SM: Effect of bilateral cervical vagotomy in the dog. Am J Physiol 189:569-572, 1957. 24. Hahn HL, Graf PD, Nadel JA: Effect of vagal tone on airway diameters and on lung volume in anesthetized dogs. J Appl Physiol 41:581-589, 1976. 25. Macklem PT, Hoolcock AJ, Hogg JC, et al: Partitioning of pulmo- nary resistance in the dog. J Appl Physiol 26:798-805, l969. 26. Hoolcock AJ, Vincent NJ, Macklem PT: Frequency dependence of compliance as a test for obstruction in the small airways. J Clin Invest 48:1097-1106, 1969. CHAPTER 4 3-methylindole Induced Pulmonary Toxicosis in the Horse 95 Introduction Horses commonly suffer from chronic obstructive pulmonary disease, the etiology of which is unknown but hypersensitivity to molds, viral and bacterial infections, and dietary factors have been incriminated.1-5 Recently, 3-methylindole (3MI) has been suggested as a possible etiolo- gic agent of the disease syndrome in horses.6 This compound is a meta- bolite of the amino acid L-tryptophan and is found in the feces of mam- mals as well as in tobacco smoke.7 Dral administration of 3MI to horses results in dyspnea, tachypnea and impaired gas exchange, most evident 7 days post treatment but to date there are only preliminary reports on the pathologic and physiologic changes induced by 3MI.6 There is clinical evidence to suggest that the parasympathetic ner- vous system plays a role in the pathogenesis of chronic obstructive pulmonary disease in the horse, because many cases respond to atropine administration.8 The parasympathetic nervous system also plays a role in the pathogenesis of experimentally induced airway diseases in dogs, guinea pigs, and rabbits and some forms of asthma in man.9'13 I there- fore wondered if vagal reflexes were also involved in the pathogenesis of 3MI induced pulmonary toxicosis. The purpose of this paper is to report changes in pulmonary function occurring in the early stages of 3MI induced pulmonary toxicosis, to correlate functional changes with pathologic lesions and to determine the role of vagal reflexes in the mechanism of disease. 96 Materials and Methods Ten ponies between 2 years and 15 years of age (x'a 8.5 years) weighing 167.9 :_8.7 kg (x :_SEM) were used in the experiments. Animals had been on pasture for the previous 2 months and all were vaccinated for the common viral respiratory diseases. Animals were regularly observed during this period, to detect any signs of respiratory disease. Surgical Preparation Horses were prepared for experiments by surgically exposing the vagus nerves, cannulating a carotid artery, and performing a tracheostomy. Anesthesia was induced with intravenous sodium thiamylal (10 mg/kg BH) and maintained with inhalation anesthesia using halothane. With the pony in left lateral recumbency the right cervical region was prepared for aseptic surgery. A 10 cm linear skin incision was made just dorsal to the jugular vein in the mid cervical region. The vagus nerve was exposed and a 1 cm section dissected free. A silk suture was looped around the nerve and tied loosely. The wound was closed in a routine manner, allowing the ends of the silk suture to exit through the skin. The same procedure was repeated on the right side of the neck and in addition a 1.19 ID, 1.70 DD (PE190) polyethylene catheter was placed in the right carotid artery and allowed to exit through the skin at a site distant from the incision. Lastly a ventral midline tracheostomy was performed in the mid cervical region. Animals were allowed to recover for 24 hours. The carotid catheter was flushed every 4 hours with 2 ml of heparinized saline. 97 Methods of Pulmonary Function Testing Twenty-four hours after surgical preparation, animals were tranquilized with intravenous xylazinea (0.5 mg/kg of body weight) and restrained in stocks. The methods of pulmonary function measurement have been previously described.14 Briefly, air flow (V) and tidal volume (VT), measured using a pneumotachographb transducer systemc attached to a cuffed endotracheal tube and inserted into the trachea via a tracheostoma, were recorded on light sensitive paper.d Transpulmonary pressure (Ptp) was measured as the pressure dif- ference between the mid’portion of the thoracic esophagus and the airway opening, using identical catheter systems. From the recording of Ptp, V and VT, dynamic compliance (Cdyn). respiratory rate (RR) and minute ven- tilation (6min) were calculated. Quasistatic pressure-volume curves were generated on an x-y plotter,e using an air driver pressure cycled ventilator.f The defla- tion limb of the quasistatic pressure-volume curve was empirically described as a single rising exponential, using a digital computer.9.15 v = Vmax (l-e-a Ptp) (1) Where V = lung volume at a given Ptp, Vmax is the volume at which the slope of the curve is 0 (i.e., Ptp is infinite) and 0 describes the rate of rise of the curve from functional residual capacity (FRC) to Vmax- Quasistatic compliance (Cstat) was calculated from the first derivative a Rompun, Haver Lockhart, Shawnee Mission, Kansas Dynasciences, Bluebell, Pennsylvania C Model PMS, Statham Instruments, Hato Rey, Puerto Rico d Model VR6, Electronics for Medicine, White Plains, New York e Model XY575, Esterline Angus Co., Indianapolis, Indiana f Mark 9, Bird Co., Palm Springs, California 9 Model PDPll Digital Equipment Co., Maynard, Massachusetts 98 of equation #1 at Ptp = 3 cm H20. Functional residual capacity was measured by helium equilibration and total lung capacity (TLC) was defined as the total lung volume at Ptp = 30 cm H20. Total respiratory system resistance (Rtot) was measured using a forced oscillation technique. During hyperventilation induced apnea, the respiratory system was oscillated at its resonant frequency and air- way opening pressure (Pao) and flow were plotted on an x-y plotter.d Total respiratory system resistance was calculated as the slope of the resulting line. Specific conductance (SGtot) was calculated as the ratio of conductance (Rtot'I) and FRC. Experimental Protocol Ten ponies were randomly divided into two groups. Arterial blood gas tensions, pulmonary mechanics and lung volumes were determined in all ponies 24 hours post surgery to establish baseline values. Immediately after these measurements were taken, the four ponies in group 1 received 0.5 liters of corn oil while the six ponies in group 2 received 100 mg/kg of 3MI in 0.5 liters of corn oil, both via naso- gastric tubes. Measurements were repeated 24 hours after treatment and if Rtot had not increased and Cdyn had not decreased from baseline values, again at 48 hours post treatment. Subsequently, the vagus ner- ves of ponies in group 2 were exposed through the surgical wounds using the silk sutures and were transected. Ten minutes after bilateral vago- tomy, arterial blood gases, pulmonary mechanics and lung volumes were measured again. 99 Data Reduction and Statistical Treatment Curve fitting routines were performed by a digital computer, using the nonlinear least squares method of Bevington.16 Data were analyzed using the students t test for paired data.‘7 Significance was deter- mined at.P < 0.05. Postmortem Examination After the last measurement was taken, the six animals in group 2 were euthanized with an overdose of pentobarbital and exsanguinated. After the gross appearance of the lung was noted, the lungs were removed and minimum lung volume determined in 5 ponies by water diSplacement. The minimum lung volume was compared to that of 5 ponies free of clini- cally apparent lung disease, euthanatized and exsanguinated in the same manner. Random sections of tissue were taken from the lungs, fixed in buf- fered formalin, paraffin embedded, sectioned at 5 microns, and stained with H and E. Additional fixed tissues were post-fixed in osmium tetroxide, dehydrated in graded alcohols, critical point dried, coated with 20 nm of gold and viewed under a JEDL JSM-BSC scanning electron microscope. Results Gas exchange, pulmonary mechanics and lung volumes remained unchanged in ponies in group one up to 48 hours after corn oil treat- ment. Twenty-four hours after 3MI treatment, Cdyn and Rtot had not changed from baseline values in two ponies in group 2. In these ponies, measurements were repeated 48 hours post treatment. Figures 4-l and 4-2 100 .mz—o> m:_uaomeq sage mucmemue_u acme—wvcmpm mumuwuc. macaw .%u>v xsouomm> emcee use newswomeu “Hzmv mpou:_pxgumslm gmumm .Aumv nopeoa mmcflhhonomc a m:_e=u vacamams .A=_s>v cowum_wu=m> mu==_s van Ah>v we:_o> paw.» .Azum + My Aggy mung xgouoepammm gic mesa—u 101 Hi¢ wezmwm o> in on. .0. 0.0.0.0 0.0. 00000 0.0. °0°0 0.0.0.0... . 0'0'0'0'0 0 0.0.0. . 0.0.0.0.0.0 .0 '0'0'0'0' ° °0°0°0°0 0 0 0.0.0 0.0. 0 0.0.0.00000000000 0. .0 .. .0 0.. 0:0 0 0 0. s 6 O O O 0.00.00.00.0000000 ’0 ’0 ’0 0 O of 0 ’0 ’0‘0’0‘0 0.0.0.0 0000 0A.... .. 0: 0. ... 0.0 f0? 0 6 0’0 ‘0 ’0 .0 0.0 O '0 ’0 0? .0 10¢ 102 .m=_m> m:_uwumgq Eocm mucmgmm$_u acmu_w_=mvm oumupucp mcmum .Ao>v xsou lame> gages vac “coaummeu AHzmv mpovcwp»;umsim emcee .Aunv uopgoq omcoppmgomgq m mcpezu umgzmmms .zq new .Amoumav copmcou moo —opemuea .Azum +_mV Auomav copmcou No popeoue< «-4 eeem.e 103 OOVOOQOQQQOQQOOOQCCQQQOOQO'OOOO 0.0...0.0.0000000000QOOOOOOOOOOOOG 0.00 00.00.000.90... 0.. 9 .000 O...:.0.0.0.0.:.:.O O...’.0.0.0.0.0. 000 ’0?0°.0?0’0’0°0?090_0. . .0.0.0. . .0?0.0.0.0. . - .0.0.0.0.0.0. \ICI vvflvvvvvvvvvvvvvvvv vv vv v v v v v. 0’0’0’0’0’0’0’0’0’0’0’0’0’0’0‘0’0’0°0’0’0’0’0’0’0’0’0.0.0.0. ’ 0°0°0°0’0°0’0’0°0’0’0°0°0’0’0’0’0’0’0’0’0’0°0°0°0°0°0°0’0’0 ’ . . 00.0.0.” ° ’0’0’0’0’0’0’090...‘ . 909030?0?0?090?0?0‘ b’0'0’0’0’0'0’0’0’0’0’0’0.0.0’0’0’0’0’0’0’0’0'0'0'0'0'0'0'0'0'0'0'0‘ 000000000000000000000000000000000i ’0’0’0’0’0’0’0’0’0’0’0’0’0’0.0°0°0°0’0’0°0°0°0°0’0°0’0’0’0’0’0’0’0‘ 50.0.0.0. .0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0. 13(3 ESIMI '715- V""""'."' 0°0°0’0’0’0’0‘0’0'0’0z0’0’0 0 0.03.0.0.0.0.0.0.0~.0.:.0.0.0.0 ’0’09090909090909090 090 090.0% ...... ’ 0'0‘ 0 0 0 0 0 0 0.:.0.0.0.:.0. .0; 0:0:0’0°0'0’0'0 0°0’0'0'0’0’0'0 O VH3 ’0'0'0'0'0'0'0'0 0 0 0 0'0 0 '0’0'0’0’0’0’0 0 {0:0‘0°0°0’0°0:0 0 0 0 0 0 0 0 0 0:0:0z0z0:0:0:0’ 0.0.0.0.0.0.0.0.0. 0 . . . . . :33.33.03.033333333333. 33.033.033.033.0.0.03.03.03.03.03.0.0.0.0-0.0.0.0.0.0.0. 0.0 . 0 O ,_. ------- O-9'".""""'v""vv'v'v 0 0 0 0 0 0 0.0.0.0.0.0 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0. . 00000000000000000000000000 . . ° ° °0°0°0.0°0°0°0°0°0°0°0’0.0.0’0’0’0’0’0’0‘0’0’0’0’0’0’0’0’0’0 0 0’ 0°0°0°0°0°0’0°0°0 0’0 0 . .233. 33333333333.0.0.0.0.0.00.0.0.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 09 .v -.- -. vvvvvvvv VCC'V'VVV """"""vvvvvvvv'""' V 0 ’ ’0’0’0’0’0’0’0’0‘0’0’0’0’0’)0’0‘0‘0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0’0 0 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0. r90’0?0,0?0,0?O’090’0.0’0.0’0 , 0.0.0’.’A’WA’A’A’A’A’0’0.0...g.0.......¢.A.0.A‘A’;’A’.’.’A’A’A’A.A.A.A.A.A.A.n O O O O O 0 O O O D O O O I 6 . I 8 a O :5 095 Figure 4-2 104 .o=_o> mcpuooaea ace» oucoeamewu acuuvypgmpm munopucw mgoum .Ao> asap iomm> gmuuu can «enzymes» A~zmv upou=¢_»;aoeim tmweu .Aum uovgoa omcmppoguuca a event vocamuos Aughv Appuoquu was. page» wen Axum +_mv Aug; auvuaaau ~u=upmog —aco.au==u mic us:m.m 105 0000 0 0 0000 0 0 000000000000000000000000000000000000000000000c %flhhflfiQQQflfiflQflfifiQfifififififififififififififififififififififififififi’” AA... 0000000000vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv E 0 0 0 0.0.0 0.0.0.0 0.0.0.0.0.0.0.0.0.0.0 0 0 0 0.0.0.0 0 0 0 0.0.0 0.0.0.0.0.0.0.0.0.0.0.i #9999990UNVUUVVVV005 t:’:°:’:°:’0’:’:’:’:’:’:’:’:’:’:’ ’0 0 0 0 0 0’0’0’0°0°:'0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O .0. 0 . 0. 0. 0. .............g.g..~....A...A.A.A...A.A.A.A.A.A.A.A.A.L’A.A.A.A.‘.A.A.A.A .‘.’.‘.V.v.v.v.u.v.u.-.-.-.v.-.v.v.u.o.w.~.~. 0 00.000000000000000... 0 0 0 0 0 0 0 0.0.0.0.0.0.0.0.0.0.0.0.0.0.0 vvvvvvvvvv 'V V'vvv v V'VVVVVVvvvvvvvvv 00000 EVVVVVVUVVV5€005éVVVVVVVUVVVVVVVVVVVVVUVfi 00000000000000000 00000000 000000000000000000000000000000000000 fifififififififififififififififi!fifififififif?$§£3fifl£fiéfl颒 TLC Liters Qhhflfifififlflfiflfiflflfiflflflflfiflfififlflfl VV?V”%%%%WV~VVVW~W~WVV~W§% g’000000000000§§§hfihfifififififi 000$?Waafifii flfiflflflflhflVVQ .0000000000000000000000000 k.A’A’A’A.5,A’A.A’A.A.AOA.A.A.A.A’A.A.A.A.A.A.A.A.A. PC 3M1 .1 FRC Liters Figure 4-3 106 .mapc> m:_uuumea soc» oucmgmmepu ucuu_mv:m.m uuuupucp meuum .Au>v xEou iomm> scuba use acoEHmmeu AHzmv o_ou:__z=aosim emuwo .Aoav .uopemq mace—posuaea a mcpgsv umezmmms Auouomv mocuuuauccu u_$Pumqm new Auoumv mocuum_mog soumam agouue_ammc .muoh «-0 ae=a_e 107 0 .3: .0 0 .0 .O ‘% O 0 .. 0 0 ‘3 00 ‘% 0 O we: 0 0 0 9090909 0 T om“. . 7.21.. .2 om .Nd ..¢.O 3.0 e-e aesm.a Frau ”.9 om ' 0 .0 0 #53 0 0. ’0 0'0'0 0 0 .0 0 %. yy 00 000 0000 fififi’ .md 5.0 8:. 08.0 :50 BE 1 108 Figure 4-5 Dynamic compliance (Cd n) measured during a prechallenge period (PC) after 3-me hylindole (3MI) treatment and after vagotomy (VC). Stars indicate significant difference from preceding value. 109 1.0 30000000000000.000000 w0.0m0w00 w 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 e. 000000000 0 0 00 00 ””.”’b’,”””” var r fi. . » > > v » >0w0n0n0.0w0w. 000 0000 0000000000 v.0.0.0.0.0..04040.0l0l0¢0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0.0. * .000000u0u0u000000000u0u0u0u0u0n0u0u0u0u0n0u u 0 0 u u 0 & caaxvaaavRaavahcwnaanhanhxvnhannan. ‘ vnwuwuwuwduwumvv w .0... w w0.0w w0w «0w0w0w0w w0w wuwnw0w0w0w0. w0w0w0w0w w0w0w0w M0» 0 0 0 0 00000000000000000 00000000000000... 00000000000000000000000000000000000000000 r.$5955....050.0%...5gvr.areasvgrvvvvvvwe 0.8 VC Figure 4-5 PC 3MI 110 .coES— spas» c. upcnmu ee—appmu use copumgacammv pay—mgu_qa sue: mm_o.:u=oeg mcvzogm .zaacmogupeouagm oi¢ mgamvm 112 show the effects of treatments on respiratory rate, tidal volume, minute ventilation, Pa02, PaCDz and pH in group 2. Respiratory rate and minute ventilation were increased significantly by 3MI treatment and were decreased after vagotomy. Tidal volume was unaffected by 3MI but increased significantly after vagotomy. PaCDg decreased significantly after 3MI treatment and remained the same after vagal section. PaDz was unaffected by either treatment. Figure 4-3 shows the effect of treatments on FRC and TLC. FRC was increased significantly by 3MI and remained increased after vagotomy. TLC was unaltered by either treatment. Total respiratory resistance was increased by 3MI and decreased again following vagotomy (Fig 4-4). Since changes in lung volume alter Rtot and since 3MI increased FRC, Rtot was corrected f0r changes in lung volume by calculation of SGtot- Specific respiratory conductance decreased 46% following 3MI and was significantly increased by vagotomy (Fig 4-4). Dynamic compliance was decreased by 3MI but unaffected by vagotomy (Fig 4-5). Quasistatic compliance was 0.887 :_0.04 L cm H20'1 and was unaffected by treatments. Minimum lung volume per kg of body weight of 5 3MI treated animals determined at necropsy was 43.3 1 2.4 ml/kg (z 1 SEM) as compared to 20.8‘: 1.4 m1/kg for 5 untreated ponies. 0n gross pathologic examination lungs from 3MI treated horses appeared distended and palpation revealed crepitus. Histologically, lesions were restricted principally to the bronchioles, where there was widespread epithelial degeneration and a mild to moderate mixed inflam- matory response in peribronchiolar areas (Fig 4-6). In addition, in two of the ponies there was diffuse alveolar and peribronchiolar edema. The 113 edema fluid was cell free but contained fibrillar material suggestive of fibrin. Scanning electron micrographs indicated that bronchiolar epithelial surfaces were damaged and were covered by scattered accumu- lations of cellular debris. Surfaces of the larger airways were normal. Discussion Derangement of pulmonary function after oral administration of 3MI was characterized by decreased Cdyn and SGtot and'an increased FRC and MV. A decrease in Cdyn may be produced by decreased static compliance (Cstat) or by the production of time constant inequalities between parallel lung units.18 In addition, if significant time constant ine- qualities pre-exist, an increase in RR will decrease Cdyn-lg In normal ponies, Cdyn is not frequency dependent over a range of 15 to 60 breaths per min.14 Therefore it is unlikely that the decreased Cdyn after 3MI was due to increased RR acting on preexisting time constant inequali- ties. Since Cstat was unchanged by 3MI, the observed decrease in Cdyn was most probably due to the production of time constant inequali- ties, characteristic of small airway obstruction. In dogs, resistance to flow resides mainly in large airways with peripheral airways contributing approximately 20% of pulmonary resistance of FRC.20 If the distribution of resistance is similar in ponies, the decreased conductance caused by 3MI may be attributed to a decrease in caliber of central airways, a massive small airway obstruc- tion or a combination of large and small airway obstruction.21 Factors that may increase FRC include small airway closure, increased expiratory time constants of peripheral lung units and tonic activation of inspiratory muscle groups. Muller et al reported 114 recently that the increase in FRC seen in persons after histamine admi- nistration may be due in part to persistence of inSpiratory muscle acti- vity during exhalation, while Slocombe et al report that in calves the increased FRC caused by histamine is abolished by vagotomy, suggesting stimulation of pulmonary receptors with afferents in the vagus is the cause of the increased FRC.ha21 In the present study, vagotomy did not reverse the increase in FRC. It is therefore unlikely that pulmonary .vagal reflexes were involved and the increase in FRC was probably caused by airway obstruction and prolongation of expiratory time constants. Increased minimal volume results from premature small airway closure which results from decreased elastic recoil of the lung, increased smooth muscle tone in airways or plugging of airways by secretions or debris. He did not evaluate the elastic properties of the lung at MV but the pressure volume behavior of the lung above FRC was not changed by 3MI. Thus the increase in minimum volume observed may be attributed to small airway obstruction resulting from either increased airway smooth muscle tone or accumulation of secretions or debris. The decrease in Cdyn. increase in Rtota FRC and MV after 3MI treatment suggest small airway obstruction. It is surprising that in the face of small airway disease horses were able to maintain normal arterial oxygen tensions. Data suggest however that despite normal PaOz, gas exchange was impaired. After 3MI treatment, tidal volume remained at baseline values but RR increased significantly. If dead space volume did not change, alveolar ventilation must have increased, resulting in the —_ " Slocombe RF, Robinson HE: Vagotomy abolishes the histamine induced increase in functional residual capacity in neonatal calves. Fed Proc 40:387, 1981. 115 significant decrease in PaCDz. Since the alveolar gas equation states that PAOz = K1-K2 PaCOz (2) where PADz = alveolar oxygen tension and K1 and K2 are constants, a decrease in PaCDz must have resulted in an increased PA02.22 Thus, if PADz increased and Pa02 was unchanged by 3MI, the alveolar arterial oxy- gen tension difference increased indicating impaired gas exchange. Considering that minute ventilation more than doubled after 3MI, the decrease in PaC02 is small. This suggests either that C02 production or the dead space tidal volume ratio increased. While our data do not dif- ferentiate these possibilities, the tachypnea induced by 3MI may have increased both parameters. Following vagotomy, PaCDz remained the same as following 3MI despite a decrease in minute ventilation. The increased tidal volume resulting in a smaller deadspace tidal volume ratio would adequately explain these latter observations. The increase in V7 and decrease in RR and Rtot after vagotomy are similar to changes reported in normal conscious ponies.23 The marked decrease in RR after vagotomy suggests that the tachypnea induced by 3MI was a vagally mediated response. Vagal afferents known to affect RR include aortic chemoreceptors, pulmonary J receptors located in the interstitium, and irritant receptors located in the submucosa of con- ducting airways.24 Since Pa02 and arterial PH were unchanged by treat- ments and PaC02 decreased after 3MI treatment, stimulation of aortic body chemoreceptors did not occur. Therefore the tachypnea was due to stimulation of pulmonary afferent receptor systems. The decrease in Rtot and increased SGtot after vagotomy suggests dilation of large or small airways. Since Cdyn remained unaltered after 116 vagotomy, it appears that dilation of small airways did not occur. Interruption of normal parasympathetic bronchomotor tone to large air- ways would adequately explain the observed decrease in Rtot and increase in SGtot after vagotomy.14 Histopathologically, 3MI pneumotoxicosis was characterized by necro- tizing bronchiolitis and alveolar emphysema without involvement of bronchi. In addition to bronchiolitis, alveolar edema was also present in two of six horses. Alveolar edema has not been previously reported in horses after 3MI treatment. Bronchiolitis with acinar over-inflation is also characteristic of naturally occurring chronic obstructive lung disease in the horse,1‘3 but the role of 3MI pneumotoxicosis in the disease is presently unknown. Histologic findings support the physiologic data and both suggest that 3MI toxicosis is characterized by small airway obstruction and aci- nar over-inflation without involvement of bronchi. Tachypnea produced by 3MI appears to be due to stimulation of pulmonary receptors with afferents in the vagus. 3-methylindole induced pulmonary toxicosis may have broad biolo- gic implications because the fecal flora of man, horses and other domestic species is capable of producing 3MI from tryptophan and some of its metabolites.25 In addition, potentially significant amounts of 3MI are found in tobacco smoke.7 117 References 1. Cook NR: Chronic bronchitis and alveolar emphysema in the horse. Vet Rec 99:448-451, 1976. 2. Muylle E, Dyaert H: Lung function tests in obstructive pulmo- nary disease in horses. Equine Vet J 5(1):37-43, 1973. 3. Gerber H: Chronic pulmonary disease in the horse. Equine Vet J 5(l):26-32, 1973. 4. McPherson EA, Lawson GHK, Murphy JR et al: Chronic obstructive pulmonary disease (CDPD): Factors influencing the occurrence. Equine Vet J 11(3):l67-l7l, 1979. 5. Beech J: Diseases of the lung. Veterinary Clinics of North America: Large Animal Prctice. 1:149-169, 1979. 6. Breeze RG, Lee HA, Grant BD: Toxic lung disease. Mod Vet Pract 59:302, 1978. 7. Hoffman D, Rathkamp G: Quantitative determination of l- alkylindoles in cigarette smoke. Anal Chem 42:366-370. 1970. 8. Murphy JR, McPherson EA, Dixon PM: Chronic obstructive pulmo- nary disease (CDPD) Effects of bronchodilator drugs on normal and affected horses. Equine Vet J 12(11):10-14, 1980. 9. Gold HM, Kessler GF, Yu DYC: Role of vagus nerves in experimen- tal asthma in allergic dogs. J Appl Physiol 33:719-725, 1972. 10. Mills JE, Hiddicombe JG: Role of the vagus nerves in anaphy- laxis and histamine induced bronchoconstrictions in guinea pigs. Br J Pharmacol 39:724-731, 1970. ll. Drazen JM, Austen KF: Pulmonary response to antigen infusing in the sensitized guinea pig. Modification by atropine. J Appl Physiol 39:916-919, 1975. 12. Karczewski H, Hiddicombe JG: The role of the vagus nerves in the respiratory and circulatory reactions to anaphylaxis in rabbits. J Appl Physiol 201:293-304, 1969. 13. Yu DYC, Galant SP, Gold HM: Inhibition of antigen-induced bronchoconstriction by atropine in asthmatic patients. J Appl Physiol 32:823-828, 1972. 14. Derksen FJ, Robinson NE, Slocombe RF et al: Pulmonary function in standing ponies: Reproducibility and effect of vagal blockade. Am J Vet Res (in press). 15. Salazar E, Knowles JH: An analysis of pressure-volume charac- teristics of the lung. J Appl Physiol 19:97-104, 1964 118 16. Bevington P: Data Reduction and Error Analysis for the Physical Sciences. New York, McGraw-Hill, 1969. 17. Steel RGD, Torrie JA: Principles and Procedures of Statistics. New York, McGraw-Hill, 1960. 18. Otis AB, McKerrow CB, Baitlett RA et a1: Mechanical factors in distribution of pulmonary ventilation. J Appl Physiol 8:427-443, 1956. 19. Ingram RH, Schilder DP: Association of a decrease in dynamic compliance with a change in gas distribution. J Appl Physiol 23:911-916, 1967. 20. Macklem PT, Hoolcock AT, Hogg JC, et al: Partitioning of pulmo- nary resistance in the dog. J Appl Physiol 26:798-805, 1969. 21. Muller N, Bryan AC, Zanrel N: Tonic inspiratory muscle activity as a cause of hyperinflation in histamine induced asthma. J Appl Physiol 49: 869- 874, 1980. 22. Murray JP: The Normal Lung. Philadelphia, H.B. Saunders, 1976, p 172-176. 23. Derksen FJ, Robinson NE, Slocombe RF et al: Technique for reversible vagal blockade in the standing conscious pony. Am J Vet Res 42:523-525, 1981. 24. Paintal AS: Vagal sensory receptors and their reflex effects. Physiolocal reviews 53:159-226, 1973. 25. Yokoyama MT, Carlson JR: Microbial metabolites of tryptophan in the intestinal tract with special reference to skatole. Am J Clin Nutr 32:173-178, 1979. CHAPTER 5 Pulmonary Function in Ovalbumin Induced Allergic Lung Disease in the Awake Pony: Role of Vagal Mechanisms 120 Introduction In some experimental and Spontaneous lung diseases, vagal mechanisms are believed to be involved in the pathogenesis of airway obstructionlaza3 while in others the vagus nerve plays no significant role.4:5o5 Alleviation of bronchoconstriction by vagal blockade may be attributed to several different mechanisms. Bronchoconstriction may be induced by a vagal reflex originating in pulmonary receptors, by a central nervous system induced increase in efferent vagal activity, or by an increase in sensitivity of airway smooth muscle to vagal influences.7 In only one disease model has a true vagal reflex broncho- constriction been described. Gold et al1 showed that in the sensitized mongrel dog, unilateral Ascaris suum aerosol challenge induces bilateral bronchoconstriction reversible by unilateral vagal blockade. In order to study vagal mechanisms in another immunologically mediated lung disease, I assessed the derangement in pulmonary function and the effect of vagal blockade during ovalbumin induced allergic lung disease in the sensitized conscious pony. The equid was chosen fer study because it is the only domestic animal that commonly suffers from chronic recurrent airway disease8 and because of its size, intubation of mainstem bronchi could be relatively easily accomplished in the awake animal allowing investigation of vagally mediated reflexes following antigen challenge of only one lung. Materials and Methods Eight grade ponies between 2 and 10 years of age (Y's 3.9 years) weighing 149.5 3 17.8 kg were used in the experiments. Four animals had bilateral cervical vagal loops and exteriorized carotid arteries. Prior 121 to use, animals had been on pasture fer at least two months and all were vaccinated against the common viral respiratory diseases. Animals were regularly examined to detect any signs of respiratory disease. Sensitization of Ponies Animals were sensitized with 10 mg ovalbumin dissolved in 2 ml of phosphate buffered saline solution and emulsified in 2 ml of complete Freund‘s adjuvant. The emulsified ovalbumin was divided and injected deep into the right and left triceps and semimembranosus muscles. This protocol was repeated two months later. Aerosol challenges were con- ducted at least 2 weeks after the last sensitization. Aerosol Challenge In bilateral challenge experiments an endotracheal tube was inserted into a tracheostoma. An ultrasonic nebulizer (Devilbis model 65) was attached to the endotracheal tube via a one-way valve assembly so that animals inhaled through the nebulizer. Forty ml of solution were aero- solized in 20 minutes. In unilateral challenge experiments the right and left mainstem bronchi were intubated with specially prepared cuffed tubes via a tracheostoma created in the lower 1/3 of the cervical trachea. The distal ends of the endobronchial tubes were coated with a thin layer of anesthetic cream to prevent excessive coughing during intubation. A side hole catheter, incorporated into the diStal end of the tubes, was used to measure bronchial pressure. Isolation of the lungs was verified by 1) visual inspection of the cuffs of the tubes using a fiberoptic bronchoscope, and 2) ventilation of the left lung with 80% He, 20% 02 mixture for 10 minutes and failure to measure 122 helium in the gas expired from the right lung. In unilateral challenge experiments, the ultrasonic nebulizer and one-way respiratory valve assembly were used to deliver 20 ml of solution in 20 minutes. Pulmonary Function Measurements Experiments were performed with animals restrained in stocks and tranquilized with intravenous xylazine (0.5 mg/kg of body weight). The methods and reproducibility of pulmonary function measurements in ponies have been previously described.9a10 Briefly, air flow (V) and tidal volume (VT), measured using a pneumotachograph (Fleisch #4, Dynasciences, Blue Bell, PA) and transducer (Model PM5, Statham Inst., Hato Rey, PR) attached to a cuffed endotracheal tube and inserted into the trachea via a tracheostoma, were recorded on light sensitive paper (Model VR6, Electronics for Medicine, White Plains, New York). Transpulmonary pressure (Ptp) was measured as the pressure difference between the mid portion of the thoracic esophagus and the airway opening, using identical balloon catheter systems attached to a dif- ferential pressure transducer (Model P131, Statham Inst., Hato Rey, PR). From the recording of Ptp, V and VT, dynamic compliance (Cdyn), respira- tory rate (RR) and minute ventilation (9min) were calculated. Quasistatic pressure-volume curves between functional residual capa- city (FRC) and total lung capacity (TLC) were generated on an xey plotter (Model XY575, Esterline Angus, Indianapolis, IN) using an air driven pressure cycled ventilator (Mark 9, Bird Corp., Palm Springs, CA). The deflation limb of the quasistatic pressure-volume curve was empirically described as a single rising exponential. The curve was fit by computer to the equation 123 V = Vmax (1-e‘a'PtP) (1) Where V = lung volume at a given Ptp, Vmax is the volume at which the slope of the curve is O (i.e., Ptp is infinite) and describes the rate of rise of the curve from FRC to Vmax-ll Quasistatic compliance (Cstat) was calculated from the first derivative of equation #1 at Ptp a 3 cm H20. Functional residual capacity was measured by helium equilibration and TLC was defined as the total lung volume at Ptp = 30 cm H20. Prior to measurement of total respiratory system resistance (Rtotla animals were force ventilated 4 times up to an airway opening pressure of 30 cm H20 to ensure a constant volume history and to create a period of apnea lasting between 2 and 30 sec. During this period of apnea, the respiratory system was oscillated at its resonant frequency and airway opening pressure (Pao) and flow were plotted on photorecording xey plotter (Model VR6, Electronics for Medicine, White Plains, NY). Total respiratory resistance was calculated as the slope of the resulting line. In the experiments in which left and right lungs were intubated separately, the two endobronchial tubes were connected with a y-tube so that a volume history was provided simultaneously to both lungs. The right and left lungs were then oscillated separately at their resonant frequencies and bronchial opening pressure and flow were plotted on the x-y plotter.. Left and right lung resistances (RtotL and RtotR) were calculated as the slope of the resulting lines. ‘Vggal Blockade In 4 ponies with bilateral cervical vagal loops, the vagus was reversibly blocked by circulating coolant at a temperature of -2°C through copper coils, wrapped around both loops. In an earlier study on 124 the same ponies,12 we established criteria of bilateral cervical vagal blockade: tachycardia, slow deep breathing and paresis of the cricoary- tenoideus dorsalis muscle. The latter was determined by lack of move- ment of the arytenoid cartilages during tidal breathing, as observed through an endoscope (Model BF type B2, Olympus Co., New Hyde Park, NY). In the remaining ponies, vagal blockade was achieved by vagal sec- tioning, performed under local anesthesia. Experimental Protocol Ponies were divided in two groups of 4 animals each. Group 1 ponies had bilateral vagal loops and both lungs were challenged with aerosol antigen via the endotracheal tube, while in group 2 ponies only the left lung was challenged through the left endobronchial tube. Group 1 ponies: Bilateral aerosol antigen challegg_. The fOur ponies with bilateral cervical vagal loops and exteriorized carotid arteries were challenged with 40 ml of saline, 2 g of bovine v globulin in 40 ml saline and 2 g of ovalbumin in 40 ml of saline on separate days. Pulmonary function measurements were made during a baseline period, and hourly after the beginning of challenge f0r 5 hours. In the ovalbumin group, measurements were also made during two periods of vagal blockade, at 1L2 and 4L2 hours after the beginning of challenge. Group 2.ponies: Unilateral aerosol antigen challenge. The left lungs of group 2 animals were challenged with l g of ovalbumin in 20 ml of saline. Respiratory rate, RtotL and RtotR were measured during a baseline period, hourly after the beginning of challenge f0r 4 hours, and after both ipsilateral and bilateral vagal sectioning which was 125 performed after the 4 hour measurement. Subsequently, animals were eutha- nized and subjected to postmortem examination. Postmortem Examination Animals were euthanized with an overdose of pentobarbital and exsanguinated. After the gross appearance of the lung was noted, mini- mal volume (MV) of both challenged and unchallenged lungs was deter- mined by water displacement. Random sections of tissue were taken from the dorsal, middle and ventral regions of the lung and from a main stem bronchus. Sections were fixed in phosphate buffered f0rmalin, sectioned at 5 microns and stained with H & E. Qualitative comparisons were made between challenged and unchallenged lungs and between regions of lungs. Statistical Treatment The effect of aerosol challenge and vagal blockade on pulmonary function variables was analyzed using two-way analysis of variance in a randomized complete block design.13 Differences between means were determined using the Student-Newman Keul's test. The effects of aerosol challenge were assessed by comparing the 1, 2, 3, 4 and 5 hour measure- ment periods with baseline values while the effects of vagal blockade were assessed by comparison of the first period of vagal blockade with the 1 and 2 hour measurement periods and the second period of vagal blockade with the 4 and 5 hour measurement periods. In the unilateral challenge experiments, the effect of ipsilateral and bilateral vagal section were compared with the 4 hour measurement period. At necropsy minimum volume data were analyzed using the students' t test f0r paired data. Significant was set at a < 0.05. Figure 5-1 126 Respiratory rate (RR).(§ 1 SEM), tidal volume (VT) and minute ventilation (V min) measured during a prechallenge period (PC), hourly after challenge fer 5 hours and during two periods of vagal blockade (VB). Round stars indicate significant differences from prechallenge value, while pointed stars indicate significant effect of VB, compared to adjacent measurement periods. ’00 127 000 00M0M00 0 . 000n00 0 0 0 040400040. 00 0 0 0 000000 00 r 0 00 . Q O TI M E hours after beginning of challenge Figure 5-1 128 .muopcwq ucmsmesmome acmumaue o» umgmasou. m> eo uumewm ucauwwvcmwm mueu_ucp macaw emucpoa apes: m=_~> omcmmegomea sage mmucmemem_v pcoowewem_m mamu_ucp meaum ucsoa wmm>v muoxuopn Fumes we muopemn N m:_g=u ewe meaog m so» mmcm——a;u eouem xpeso; Auaw uo_e mace—pmgumea m mcvgzv umeammms A: vuv mu=a_.aeou upsacxv can Axum + iv Ago my mocmumpmme awumxm agopmeeqmoe pouch mum mgamvm Nim manure 85:20 yo 05563 .23 «.39. 92:. m 91.. m m m>_ ooa 129 0 0 . 0’0 0 00 9080 00 0 00 .00 0 .0 0 0 .0 0 0.0 0 0 0 0 0 0 0 ’0 0.0.0 0 0 0.0.0 0 0 O 0 0’0’ 0 0 ‘0 . 0 0 ’0 0 ‘0 0 0 0 0 0 A 0 0 0 0 0 A.A ... .0 0 0 :.0 »2 fl '1’. O c 5‘4 0 .0.. 0? A 0 .2 0 0 .0 0 0 0 0 0 0 A .0 .0 0 0 0 0 A 0 .0 0 0 . 0.0. 0? 0? 0. 0 0 .A 0 0 .A 0 0 0 0.0 0’0’ 0 0 0 .0. 0 ’0 .._.. . - . .0 .0 0 0.. 20? 0 0 .‘.A . 0° 0 . .0 A 0 0 0 0 0 A.A. A 0 '0 .0 0 .0 . 0°. 0 0 .0 0 0 '0 0 0 .0 0 '0 0 0 .0. .0 0 0 ’0 0 0 '0 0 0 .0 0 A 0 0 0. 0 .0. '0 0 0 0 ’0 .0 ’ 0 0 0 9. 0 A .8 ’0 0 000 00. :0 0 0 .0 .0.0. .0 0 0 '0’ .0 .0 ' '0 0 0 .0 .0 0 0 0 .090 .mco_gmq ucmamgsmums gmguo ppm sag; mucogommwu u:~u_w_=m_m oumuwuc_ mgoum «peace .uo.goa acmsmgsmame gao; e ogu cg MW umguqsou .z> mo uumwwm ucmu_$wcm_m mmamu_c:. ¥m_gmum< .ospa> mace—pocuaga sag» mucmgomw_u 1. acmu_mpcm_m mmumu_ucp Loam upmcwm .Am> can m>~v mumxuopn ~mmu> PagmuuFPn new —mgmum_wma_ mcpgzu vac mgao; w go» mm:m_Pu;u Lmumo »_g=og Romy vo_gma mmcmhhozumga a mcwgav umgsmmms Amuoum van 49o av mucuumvmmg wasp a;m_g use pump wen .Azum +.mv Aggy one; xgoumgpqmoa m-m mgamwm 131 IVB VB 4 hours Time After Beginning Of Challenge Figure 5-3 132 m=_u> mc—pomoa sage mmmmguou acme—mpcmwm mmumu_u=w « aucowpqeou ovuaumwmmno u an mu xupuaqou m=:_ peach u QAH aupumamu pmauwmmg —~:o_uu==m a cam m_.quwwa.o No.qufloa.o ~_.QHwo._ c_.quu_._ __.qudo._ m_.quu_._ c_.qu_a.o m_~.quw~._ H om:%wumu o.quw.m_ o.qu.m_ _.euv.m_ ..mw..mp m.HH#.m_ m.F“w.m_ N.nfifl.mp _.Nuw.o~ mgoup_ ugh ~.ouw._. o.QH¢.P_ ¢.QHG.__ ~.eu..pp ~.quw.__ m.quw.o_ m.quw.__ _N.q“w.~_ new... um. m.~Hw.em m.~H~.mm _.~Hw.mm ¢.Nuo.mm m.nuw.mm m.nfla._¢ m.~u~.~¢ ~.nH¢.Pe ego» Noun; .¢.nua.m~ .m.¢u~.¢~ .m.nfi~.- .m.¢uw.- .¢.¢Hw.- o.m.nuw.¢~ .m.¢uw.m~ ~.~H~.mm Leo» Noam ~o.qu.¢.~ ~o.q“a¢.~ Nc.qu~¢.~ No.QH~¢.~ ~o.quu¢.~ No.QHa¢.~ NQ.QH¢¢.~ .o.quu¢.~ zg sac: m HH m> Lao: e Lao: m use: N _ H m> Lao: — ocppamum m m a . ea .mm>v aumxumpn wmmu> we muphmaq .3... an ammonfifim wan Hug“. mathvfim ewuwfimuzm ”muncfifiom z 2.: 133 Results Group 1 ponies: Bilateral aerosol antigen challenge. Aerosol challenge using saline or bovine v globulin did not alter arterial blood gas tensions, pulmonary mechanics, lung volumes or rectal temperature. Fig S-l shows the effect of ovalbumin aerosol challenge on RR, VT and Qmin- Respiratory rate and 6min were increased significantly at l, 2 and 4 hours post challenge and at l and 2 hours post challenge reSpec- tively, but tidal volume did not change with time. During both periods of vagal blockade, RR and 9min decreased while VT increased. The effect of ovalbumin aerosol challenge on Rtot and Cdyn is shown in Fig 5-2. Dynamic compliance decreased significantly after challenge at all measurement periods and was not significantly altered by vagal blockade. Total respiratory resistance increased significantly at 2, 4 and 5 hours post challenge. Vagal blockade decreased Rtot during the second period of vagal blockade. PaOz decreased significantly 1 hour after ovalbumin aerosol challenge and remained depressed at all subsequent measurement periods. PaCOz, arterial blood pH, lung volumes, pressure-volume characteristics of the lungs and rectal temperature were not changed by ovalbumin challenge or vagal blockade (Table S-l). Group ngonies: Unilateral aerosol antigen challenge. Challenge of the left lung increased RR significantly at l and 2 hours after challenge. At 3 and 4 hours, RR decreased but was still significantly higher than the prechallenge value (Fig 5-3). Ipsilateral vagal sec- tioning did not affect RR but bilateral section reduced RR below prechallenge levels. Left lung resistance was increased after challenge and remained elevated at all measurement periods, while RtotL was not 134 uzc . .mwuwpowgucocn o>wu . “mac mango mcwzosm mace—.ezu cvs=n_m>o Lmuma meson m upowguocn a mo gqmgmogupeouogm ¢-m a.=m_d 135 ¢-m mesmwu 136 affected by challenge. Vagal section did not change RtotL- Right lung resistance was decreased after bilateral but not unilateral vagal sec- tion. Gross Pathology. Following unilateral aerosol antigen challenge, the difference in color, size and texture of the two lungs was striking. The left lung (exposed to ovalbumin) was discolored, blotchy red and of rubbery consistency. Minimal volume, of left and right lungs were 3.%: 0.2 (§::_SEM) and 1.5: 0.3 liters respectively. On cut surface the left lung parenchyma was wet and many airways had excessive frothy fluid or mucus. Histopatholggy. Focal pulmonary lesions of a chronic nature were found in varying degrees of severity in most sections examined and were assumed not to be associated with the disease processes induced by aero- sol ovalbumin exposure. The left lung differed from the right in that the former had acute diffuse lung edema and widespread severe acute obstructive bronchiolitis (Fig. 5-4). Airway lesions in the left lung were most severe in the bronchioles but exudate consisting of fibrino- purulent material was also noted in more central airways. The bronchiolar epithelium was vacuolated, degenerating and in fbcal areas had undergone necrosis. Fibrinous thrombi were noted occasionally in the bronchiolar lumens and to a lesser extent in peribronchial lympha- tics and within alveoli. Alveolar walls were thickened by edema fluid containing numerous neutrophils. A similar material was found free in many alveolar lumens. Pulmonary congestion and hemorrhage were observed around affected bronchioles. Unaffected bronchioles and alveoli were markedly distended in comparison to those of the right lung. Lymphatics were distended with proteinaceous fluid and margination of leucocytes, 137 predominantly neutrophils, was noted occasionally in peribronchiolar vessels. Mild to moderate numbers of eosinophils were found associated with the airways and the lymphatics but were not observed in excessive numbers in the alveolar edema fluid. The lesions appeared most severe in the ventral lung. Discussion Bilateral aerosol challenge with saline or bovine Y globulin did not significantly alter pulmonary function. Therefore the response to inhaled ovalbumin antigen was immunologic in nature and not due to nonspecific airway irritation. The pulmonary response to bilateral aerosol antigen challenge of sensitized ponies with ovalbumin was biphasic. The early response, evi- dent at l and 2 hours after the onset of challenge was characterized primarily by tachypnea, while the late response was characterized pri- marily by an increased Rtot- Response to antigen challenge depends upon the manner of sensitization. Large quantities of antigen in complete Freund's adjuvant encourages delayed responses while sensitization with ‘ antigen saline solutions or antigen in incomplete Freund's adjuvant encourages an immediate response.14 Since we sensitized with antigen in complete Freund's adjuvant, the early tachypneic reSponse was unexpected. Tachypnea is observed in experimentally induced allergic lung disease in animals and acute attacks of asthma in man.15:16 Mechanisms proposed for the increased RR include an increase in PaCOz and pH or decrease in PaOz, a rise of core temperature, anxiety, antigen induced changes in mechanical properties the lungs, and stimulation of pulmonary receptors with vagal afferents.15 In these experiments, rectal 138 temperature, PaCOz and pH did not change and anxiety was not likely to be involved because challenge with saline and bovine Y globulin did not alter RR. Although the Paoz decreased, from 85.2 torr to 75.3 torr, the tachypneic response was too severe to be explained solely on this basis.17 In addition the tachypneic response followed a clearly dif- ferent time course than both the PaOz and the pulmonary mechanics changes. Therefore it is most likely that in the pony, as in the dog15 afferent vagal pathways mediate the ventilatory response to inhaled antigen. The time course of tachypnea in unilateral and bila- teral challenge experiments was similar. In addition, in unilateral challenge experiments, tachypnea was only abolished after bilateral vagal blockade, suggesting that in the horse pulmonary afferent vagal fibers cross over to the contralateral vagus nerve within the thorax. Pulmonary receptors which may have been involved in the stimulation of respiration include irritant receptors present in the submucosa of con- ducting airways and J receptors, located in the pulmonary interstitium.18 Our data do not allow identification of responsible receptor systems. After bilateral aerosol antigen challenge, Rtot increased gradually and was greatest 4 hours after challenge. If the central airways account fbr the majority of resistance to airflow in horses as they do in dogs19 the 3-fold increase in Rtot at 4 hours after challenge com- bined with a small decrease in Cdyn suggests that large airways were involved in the antigen induced airway narrowing.20 Vagal blockade decreased Rtot at 4%? hours after challenge. Although in normal ponies vagal blockade also decreases Rtotg the decrease in Rtot in this experiment was too large to be explained by interruption of normal bronchomotor tone alone. This suggests the involvement of a 139 vagal mechanism such as a vagal reflex originating in pulmonary recep- tors, a central increase in efferent vagal activity or an increase in sensitivity of airway smooth muscle to normal vagal tone. Vagal blockade did not reduce Rtot below baseline value as it should have done if vagal mechanisms alone were responsible for the increased Rtot-9 Therefore, local mechanisms also play a role in aerosol antigen induced airway caliber changes. This latter conclusion is supported by the failure of vagal blockade to reduce Rtot below baseline value at 1L2 hours after challenge. In order to determine the relative importance of vagal and local mechanisms in aerosol antigen induced airway narrowing in the horse, we challenged the left lungs of ponies through an endobronchial tube and determined RtotL and RtotR hourly for 4 hours after the beginning of challenge and following unilateral and bilateral vagal sectioning. He reasoned that if vagal reflexes, originating in the challenged lungs or a challenge induced increase in efferent parasympathetic bronchomotor activity were responsible for airway narrowing in this disease model, unilateral aerosol antigen challenge would result in airway narrowing in both lungs, abolished by either unilateral or bilateral vagal blockade. If aerosol antigen challenge increased the sensitivity of airway smooth muscle to normal vagal tone or if the baseline airway caliber was impor- tant, left unilateral challenge would result in increase in RtotL only, abolished by either unilateral or bilateral vagal blockade, while if local mechanisms were important in airway caliber changes, unilateral challenge would only cause an increase in RtotL: unaffected by vagotomy. Since unilateral aerosol antigen challenge of the left lung resulted in a marked increase in RtotL: without altering RtotR. a vagal reflex 140 bronchoconstriction originating in the left lung or a central increase in parasympathetic bronchomotor tone were not responsible for the airway narrowing. The increase in RtotL was of greater magnitude than the increase in Rtot following bilateral challenge. Because the endobron- chial tubes were more peripherally located and had less deadspace, dif- ferences in amount and location of aerosol deposition may have been responsible for this discrepancy. Although a trend was apparent, unilateral and bilateral vagal blockade did not significantly decrease Rtoth suggesting that increased responsiveness of airway smooth muscle to normal vagal tone or decreased baseline airway caliber was not the most important mechanism in the pathogenesis of antigen induced airway narrowing. Thus these data clearly indicate that local mechanisms such as direct effects of mediators of inflammation on airway smooth muscle or mechanical obstruc- tion of airway lumens with debris or edema fluid are of critical impor- tance in the antigen aerosol induced airway obstruction in ponies. Although local mechanisms appear to be the most important in the increase in RtotL following unilateral challenge, data suggest a minor role for increased responsiveness of airway smooth muscle to normal vagal tone. Following vagal blockade there was a trend towards a decrease in RtotL- Although this decrease was not statistically signi- ficant because of variability in response to both challenge and vagal blockade, the magnitude of decrease in RtotL was larger than the decrease in RtotR and similar to the magnitude of decrease in Rtot during the second period of vagal blockade in the bilateral challenge experiment. If a decrease in baseline airway caliber was important, the decrease in RtotL following vagal blockade would have 141 been much larger than the decrease in RtotR- Therefore these data suggest that in challenged lungs, the effect of vagal blockade on airway smooth muscle is enhanced, i.e., that following antigen aerosol challenge airway smooth muscle responds more vigorously to normal vagal tone. This mechanism may have been responsible for the decrease in Rtot during the second period of vagal blockade in the bilateral challenge experiment, but because of an enhanced local effect was of minor importance in the response to antigen aerosol in the unilateral challenge experiment. A change in Cdyn can be produced by changes in FRC, by an alteration in the elastic prOperties of the lung, or by the production of time constant inequalities between parallel lung units.20 In addition, if significant time constant inequalities preexist, an increase in RR will cause a fall in cdyn.21 Since neither the quasistatic pressure-volume curve nor FRC changed with bilateral aerosol antigen challenge, the fall in Cd," observed must have been caused by the production of time constant inequalities. Significant time constant inequalities did not preexist in these ponies, as in a previous study using the same animals we demonstrated that within a range of frequencies between 15 and 60 breaths per minute, Cd," did not change.9 The increase in Rtot and decrease in Cdyn following challenge suggests that both central and peripheral airways narrowed in response to challenge. Similar findings were reported by Drazen et al and Mills at 3122.3 in guinea pigs and Karczewskiz in rabbits who concluded that antigen challenge in these species results in generalized bronchoconstriction. Minimal volume increased but FRC was not changed fbllowing ovalbumin challenge, suggesting that ovalbumin challenge resulted in airway clo- sure at lung-volumes greater than MV, but not at FRC. Since the helium 142 equilibration method f0; FRC measurement only detects gas volumes in communication with the airways, gas trapping may have gone undetected. However in 3-methylindole (3MI) induced diffuse small airway disease in ponies, we measured a similar increase in MV, and a significant increase in FRC suggesting gas trapping at both these lung volumes in the 3MI disease model23 and showing that helium equilibration could measure an increase in FRC. Our data therefore suggest that FRC did not increase following ovalbumin aerosol challenge. In persons and sheep, allergic lung disease increases FRC,24.25 while in dogs and monkeys, no increase .in FRC has been observed.25:27 The reasons fbr these species differen- ces are not clear, as the severity of the airway response to challenge does not correlate well with increases in FRC. The results of this study show that ovalbumin aerosol challenge of sensitized ponies causes both large and small airway obstruction, characterized physiologically by an increase in Rtot and NV, and decrease in Cd," and Paoz and pathologically by acute fibrinopurulent obstructive bronchiolitis, bronchitis, pulmonary edema and alveolar distension. Results further show that local mechanisms such as direct effects of mediators of inflammation on airway smooth muscle or mechani- cal obstruction of airway lumens with debris or edema fluid are of cri- tical importance in the pathogenesis of airway obstruction in this disease model. In addition, increased sensitivity of airway smooth muscle to normal vagal tone may also play a role in the pathogenesis of ovalbumin challenge induced airway obstruction. Tachypnea fbllowing ovalbumin challenge is caused by increased activity of pulmonary recep- tors. 143 References 1. Gold, NM, Kessler GF, Yu DYC: Role of Vagus Nerves in Experimental Asthma in Allergic Dogs. J Appl Physiol 33: 719-725, 1972. 2. Karczewski H, Niddicombe JG: The Role of the Vagus Nerve in the Respiratory and Circulatory Reactions to Anaphylaxis in Rabbits. J Physiol 201: 293-304, 1969. 3. Mills JE, Hiddicombe JG: Role of the Vagus Nerves in Anaphylaxis and Histanine-Induced Bronchoconstrictions in Guinea-Pigs. Br J Pharmac 39: 724-731, 1970. 4. Arborelius M, Ekwall B, Jernerus B, Lundin G, Svanberg L: Unilateral Provoked Bronchial Asthma in Man. J Clin Invest 41: 1236-1241, 1962. 5. Hirshman CH, Downes H: Basenji-Greyhound Dog Model of Asthma: Influence of Atropine on Antigen-Induced Bronchoconstriction. J Appl Physiol: Respirat Environ. Exercise Physiol 50: 761-765, 1981. 6. Krell RD, Chakrin LN, Nardell JR: The Effect of Cholinergic Agents on a Canine Model of Allergic Asthma. J Allergy Clin Immunol 58: 19-30, 1976. 7. Boushey HA, Holtzman MJ, Sheller JR, Nadel JA: Bronchial Hyperreactivity. Am Rev Resp Dis 121: 389-413, 1980. 8. Thurlbeck NM, Lowell FC: Heaves in Horses. Am Rev ReSp Dis 89: 82-88, 19640 9. Derksen FJ, Robinson NE, Slocombe RF, Riebold TH, Brunson DB: Pulmonary Function Tests in Standing Ponies: Reproducibility and Effect of Vagal Blockade. Am J Vet Res: in press. 10. Derksen FJ, Robinson NE: Esophageal and Intrapleural Pressures in the Healthy Conscious Pony. Am J Vet Res 41: 1756-1761, 1980. 11. Salazar E, Knowles JH: An Analysis of Pressure-Volume Characteristics of the Lung. J Appl Physiol 19: 97-104, 1964. 12. Derksen FJ, Robinson NE, Stick JA: Technique for Reversible Vagal Blockade in the Standing Conscious Pony. Am J Vet Res 42: 523-525,1981. 13. Steel GD, Torrie JH: Principles and Procedures of Statistics. New York, McGraw Hill Book Co., 1960. 14. Richerson HB: Varieties of Acute Immunologic Damage to the Rabbit lung. Ann NY Acad Sci 221: 340-360, 1974. 144 15. Cotton DY, Bleecker ER, Fischer SP, Graf PD, Gold NM, Nadel JA: Rapid Shallow Breathing After Ascaris Suum Antigen Inhalation: Role of Vagus Nerves. J Appl Physiol Respirat Evniron. Exercise Physiol 42: 101-106, 1977. 16. McFadden, ER: Exertional dyspnea and cough as preludes to acute attacks of bronchial asthma. New Eng J Med 292: 555-569, 1975. 17. Muir NH, Moore CA, Hamlin RL: Ventilatory alterations in normal horses in response to changes in inspired oxygen and carbon dioxide. Am J Vet Res. 36: 155-166, 1975. 18. Paintal AS: Vagal Sensory Receptors and Their Reflex Effects. Physiological Reviews 53: 159-227, 1973. 19. Machlem PT, Hoolcock AT, Hogg JC, Nadel JA, Wilson, NJ: Partitioning of Pulmonary Resistance in the Dog. J Appl Physiol 26: 798-805, 1969. 20. Otis AB, McKerrow CB, Bartlett RA, Mead Y, McIlroy MB, Selverstone NJ, Radford EP: Mechanial Factors in Distribution of Pulmonary Ventilation. J Appl Physiol 8: 427-443, 1956. 21. Brown R, Hoolcock AJ, Vincent NJ, Macklem PT: Physiological Effects of Experimental Airway Obstruction Nith Beads. J Appl Physiol 27: 328-335, 1969. 22. Drazen JM, Austen KF: Pulmonary Response to Antigen Infusion in the Sensitized Guinea-Pig: Modification by Atropine. J Appl Physiol 39: 916-919, 1975. 23. Derksen FJ, Robinson NE, Slocombe RF, Hill RE: 3-Methylindole Induced Pulmonary Toxicosis in the Horse. Am J Vet Res: in press. 24. McFadden ER: The Chronicity of Acute Attacks of Asthma: Mechanical and Therapeutic Implications. J Allery Clin Immunol 56: 18-26, 1975. 25. Hanner A, Mezey RJ, Reinhart ME, Eyre P: Antigen Induced Bronchospasm in Conscious Sheep. J Appl Physiol: Respirat Environ Exercise Physiol 47: 917-922, 1979. 26. Gold NM, Kessler GF, Yu DYC, Frick 0L: Pulmonary Physiologic Abnormalties in Experimental Asthma in Dogs. J Appl Physiol 33:496-501, 1972. 27. Pare P0, Michoud MC, Hogg JC: Lung Mechanics Following Antigen Challenge of Ascaris-Suum Sensitive Rhesus Monkeys. J Appl Physiol 41: 668-676, 1976. CHAPTER 6 Response of the Locally Sensitized Equine Lung to Aerosol Ovalbumin Challenge: Role of Vagal Mechanisms 146 Introduction In the previous chapter I investigated the role of vagal mechanisms in the response of the equine lung to aerosol antigen challenge.1 Ponies were systemically sensitized by intramuscular injection of ovalbumin in complete Freund's adjuvant. During an initial aerosol challenge, resistance increased gradually and was 300% above baseline at 4 hours after challenge. Vagal mechanisms were involved in the genesis of tachypnea and both local and vagal mechanisms were responsible for the increase in total respiratory system resistance. Since pilot studies suggested that aerosol challenge following both systemic and local sensitization of the lung results in more severe dyspnea of rapid onset, in the present study I investigated the pulmo- nary response to aerosol challenge in ponies sensitized both systemi-l cally and locally. In addition, I studied the role of local and vagal mechanisms in the response to challenge and correlated the physiologic findings with histologic lesions in the lung. Materials and Methods Four grade ponies between 2 and l0 years of age (E's 5.7 years) weighing l99.4 :_28.3 kg were used in the experiments. The animals had bilateral cervical vagal loops and exteriorized carotid arteries.2 Prior to use, ponies had been on pasture for at least two months and all were vaccinated against the commdn viral respiratory diseases. Animals were regularly examined to detect any signs of respiratory disease. 147 Sensitization of Ponies Ponies were sensitized systemically by intramuscular injection and locally via aerosol. Ten mg ovalbumin dissolved in 2 ml of phosphate buffered saline solution and emulsified in 2 ml of complete Freund's adjuvant was divided and injected deep into the right and left triceps and semimembranosus muscles. This protocol was repeated two months later. Three weeks following the last intramuscular injection, an endotracheal tube was inserted into a tracheostoma. An ultrasonic nebu- lizer Devilbis model 65 was attached to the endotracheal tube via a one- way respiratory valve assembly. Two grams of ovalbumin in 40 ml of saline were aerosolized in 20 minutes. Aerosol Challenge In bilateral challenge experiments aerosol challenge was accom- plished by delivery of 2 g of ovalbumin in 40 ml saline via a tracheostomy tube and one-way valve assembly using a Devilbis model 65 ultrasonic nebulizer. In unilateral challenge experiments the right and left mainstem bronchi were intubated with specially prepared cuffed tubes via a tracheostoma created in the lower l/3 of the cervical trachea. The distal ends of the endobronchial tubes were coated with a thin layer of anesthetic cream to prevent excessive coughing during intubation. A side hole catheter, incorporated into the distal end of the tubes, was used to measure bronchial airway opening pressures. Seal of the cuffs was ascertained by l) visual inspection using a fiberoptic bronchoscope, and 2) ventilation of the left lung with 80% He, 20% 02 mixture fbr l0 minutes and failure to measure helium in the gas expired from the right lung. 148 In unilateral challenge eXperiments, the ultrasonic nebulizer and one-way respiratory valve assembly were used to deliver 1 gm ovalbumin in 20 ml of saline in 20 minutes. Pulmonary Function Measurements Experiments were performed with animals restrained in stocks and tranquilized with intravenous xylazine (0.5 mg/kg of body weight). The methods and reproducibility of pulmonary function measurements have been previously described.3a4 Briefly, air flow (V) and tidal volume (VT), measured using a pneumotachographb (Fleisch #4, Dynasciences, Blue Bell, PA) transducer (Model PM5, Statham Inst., Hato Rey, PR), attached to a cuffed endotracheal tube and inserted into the trachea via a tracheostoma, were recorded on light sensitive paper (Model VR6, Electronics fbr Medicine, White Plains, NY). Transpulmonary pressure (Ptp) was measured as the pressure difference between the mid portion of the thoracic es0phagus and the airway Opening, using identical catheter systems attached to a differential pressure transducer (Model P131, - Statham Inst., Hato Rey, PR). From the recording of Ptp, V and VT, dynamic compliance (Cdyn): respiratory rate (RR) and minute ventilation (6min) were calculated. Quasistatic pressure-volume curves between functional residual capa- city (FRC) and total lung capacity (TLC) were generated on an x-y plotter (Model XV 575, Esterline Angus, Indianapolis IN), using an air driver pressure cycled ventilator (Mark 9, Bird Corp., Palm Springs, CA). The deflation limb of the quasistatic pressure-volume curve was empirically described as a single rising exponential. The curve was fit by computer to the equation 149 v = Vmax (I-e-a Ptp) (I) where V = lung volume at a given Ptp, Vmax is the volume at which the slope of the curve is 0 (i.e., Ptp is infinite) and a describes the rate of rise of the curve from functional residual capacity (FRC) to Vmax-S Quasistatic compliance (Cstat) was calculated from the first derivative of equation #1 at Ptp s 3 cm H20. Functional residual capacity was measured by helium equilibration and total lung capacity (TLC) was defined as the lung volume at Ptp = 30 cm H20. Prior to measurement of total reSpiratory system resistance (Rtot) animals were force ventilated 4 times up to an airway opening pressure of 30 cm H20 to ensure a constant volume history and to create a period of apnea lasting between 2 and 30 sec. During this period of apnea, the respiratory system was oscillated at its resonant frequency (3-5 Hz) and airway opening pressure (Pan) and flow were plotted on a photorecording x-y plotter (Model VR6, Electronics for Medicine, White Plains, NY). Total respiratory resistance was calculated as the slope of the resulting line. In the experiments in which left and right lungs were intubated separately, the two endobronchial tubes were connected with a y tube so that a volume history was provided simultaneously to both lungs. The right and left lungs were then oscillated separately at their resonant frequencies and bronchial opening pressure and flow were plotted on an x-y plotter. Left and right lung resistances (RtotL and RtotR) were calculated as the slope of the resulting lines. ‘Vggal Blockade The vagus was reversibly blocked by circulating coolant at a tem- perature of -2°C through copper coils, wrapped around both loops. In an 150 earlier study on the same ponies,2 we established criteria of bilateral cervical vagal blockade: tachycardia, slow deep breathing, and paresis of the crycoarytenoides dorsalis muscle, determined by failure of the arytenoid cartilages to abduct during inhalation as observed through an endoscope (Model BF type 82, Olympus Co., New Hyde Park, NY). Experimental Protocol The left lungs of ponies were challenged with l g ovalbumin in 20 ml of saline. Arterial blood gas tensions, RR, RtotL and RtotR were measured during a baseline period, one hour after challenge and during both ipsilateral and bilateral vagal blockade. At least 3 months later, ponies were challenged through the endotracheal tube with 2 g ovalbumin in 40 ml saline. Pulmonary function measurements were made during a baseline period, one hour after challenge and fbllowing bilateral vagal blockade. Following this protocol animals were euthanized and subjected to postmortem examination. Postmortem Examination Animals were euthanized with an overdose of pentobarbital and exsanguinated. After the gross appearance of the lung was noted, mini- mum volume of the lungs was determined by water displacement. The mini- mum volume was compared to that of 5 ponies, free of clinical apparent lung disease, euthanized and exsanguinated in the same manner. Tissue sections were fixed in phosphate buffered fbrmalin, sectioned at 5 microns, and stained with H & E. 151 Figure 6-1 Respiratory rate (RR) (i :_SEM), left and right lung resistance (RtotL and RtotR) measured during a prechallenge period, one hour after unilateral challenge and following unilateral and bilateral vagal blockade. (IVB and VB). Stars indicate significant differences from prechallenge value. Asterisk indicates significant effect of V8 compared to the challenge measurement period- 152 .0: l Dolor-I . . IIItCIItuAI-hll . . . . . . . J q q R 101 L cml-l O-sec-L ' Riot n cml-i O-sec-L" PC C IVBVB Figure 6-1 153 .ez—a> mcwvoumgq 555$ moucmcaumpv p:.u...=m.m mo.o_e=_ steam .Am> we. m>Hv meaxuo_n .em.> .aaao.__n we. Pegauup c: mcvzop—ow can omcm——ogu pecmuu—pa Loueu use: one .uo_goa m:_pomon a u=.g:u enhances . we; use Aoumv aHFouauo poauwmac —o=o_uo==w . ugpv »u_ueqoo asap page» .Aueumu van : uo au=o__qsou uFueumvmeza ego uvsocxu .Auo av uuemmmpmmg Seaman agopmcpammg _oaou .A:_ >V copum—_u:o> ooze—E .Ap>v oe:_o> pee.» .Axum + my Aazv one; xuoaugwqmom ~-e mesa.“ N-o maamwa w. 0.. 92.5: a. .08 ...:_E a. .4 1...... a .. . 154 .0. .0.. 2:... .. A... .-o~:§ ... :2... .8 0.: .. .0» AI 5.. 5.0 _._ _ . m 6.0 .0.... . 22... . ,. a a. . 5... A. 6. om... .3 8:... 6.. mm A. 0. .3 0 fan 155 .om:o_~u;u cps nan—«>0 pucmum—_== cmuee mono; m .m==_ amaze—Pogo as» c. opopgucoca a mo gaacmogu.souoga mum assay; 4 «4. Figure 6-3 ' IT“;- ’ r51 ~' . )' .L‘I-‘NVA'LR J.‘ ‘\ '- “K . J‘&-¢.~,w. ( . ,1 )_4§f;95... (‘ kit”, . ”.3gf.) . 157 Statistical Treatment Data were analyzed using two-way analysis of variance. Differences between means was determined by using the Student Neuman-Keul's test. Significance was determined at P < 0.05.6 Results Fig 6-1 shows the effect of left unilateral challenge and ipsila- teral and bilateral vagal blockade on RR, RtotL and RtotR° Respiratory rate increased after challenge. Bilateral vagal blockade decreased RR significantly while ipsilateral blockade had no effect. Ovalbumin challenge increased RtotL but did not change RtotR- Vagal blockade had no effect on either parameter. After challenge Pa02 decreased from 83.3 1 LS torr (3?; SEM) to 66.7 1 6.7 torr. PaCOz and pH (39.8 I. 2.7 torr and 7.4l‘:_0.02, respectively) did not change with challenge. Blood gas tensions and pH were unaltered by vagal blockade. Results of bilateral aerosol challenge are shown in Fig 6-2. Bilateral aerosol challenge increased RR and 9min and decreased VT. Vagal blockade reversed these changes. Total respiratory system resistance increased and cdyn and Cstat decreased following ovalbumin challenge. There was a small but significant increase in Cdyn following vagal blockade but Rtot~and Cstat were not changed. Ovalbumin challenge also resulted in a significant decrease in Pa02 but no change in PaCOz or arterial blood pH which were 40.0 1 2.5 torr and 7.4l :_0.02, respectively. Pa02 remained depressed after vagal blockade and Pacoz and pH were also unaffected by this treatment. Tbtal lung capa- city decreased after challenge but FRC was not changed. Vagal blockade did not reverse the decrease in TLC. 158 Gross Pathology Lungs failed to collapse after removal from the thorax, with clearly delineated rib impressions as a result of lung hyperinflation. The lungs were blotchy dark red and firm. Numerous petechia were present. Minimum volume per kg of body weight of the challenged lungs was 35.7;: 1.3 ml/kg (x :.SEM) as compared to 20.8 1 1.4 ml/kg for the lungs of 5 control ponies. Histopathology The most striking lesions were present in the smaller airways but pathologic changes were not restricted to these areas. Peribronchiolar areas and bronchiolar lumens had large accumulations of a cellular exu- date consisting principally of neutrophils, but wdth lesser numbers of eosinophils. Neutrophils were frequently present within the bronchiolar wall (Fig 6-3). The bronchiolar mucosa was extensively fblded and the smooth muscle in the wall was especially prominent, suggesting the pre- sence of considerable airway constriction. The gas exchange areas of the lung were not uniformly affected. Multifocal areas of alveolar edema and hemorrhage were scattered throughout the lung. There were mild focal aggregates of neutrophils in alveolar walls and some of these areas also had accompanying congestion, hemorrhage and edema. Except fbr the fbci of hemorrhage and edema the lung parenchyma was well inflated. Patchy hyperinflation was observed, particularly in subpleural areas. The lumens of the bronchi and trachea had small accumulations of proteinateous fluid containing variable numbers of neutrophils. The mucosa was also infiltrated with neutrophils and the submucosa was 159 congested and had prominent focal aggregates of neutrophils. In contrast to the smaller airways, where the lumens were plugged with cellular exudate, the exudate in these larger airways was less cellular and did not obstruct the lumens. In addition to these acute pathologic changes, focal lesions of a more chronic nature were occasionally observed, and were assumed to be the result of pre-existing lung disease. Discussion The results of aerosol antigen challenge in this group of ponies which were sensitized by intramuscular and aerosol exposure are clearly different from the results of a previous study where ponies were sen- sitized only by the intramuscular route.1 In the present study, following bilateral challenge, Rtot increased by 300% and Cdyn decreased to 18% of control within one hour. In the previous study, there was n0‘ significant increase in Rtot at l hour but 4 hours after challenge resistance had increased 300%, while Cdyn decreased to 75% of control. In addition, aerosol challenge following intramuscular sensitization alone produced a smaller decrease in Pa02 and no change in Cstat and TLC. Therefore it appears that aerosol challenge fbllowing both syste- mic and local sensitization of the lung results in a more severe and more rapid response than occurs following challenge of ponies, sen- sitized by the systemic route alone. Differences in response to antigen challenge, dependent upon route and method of sensitization, have been previously reported in other species.7 In contrast to the differences in the mechanical response of the lung to challenge in the two studies, changes in RR were independent of 160 the route of sensitization. Both systemic and systemic and local sensitization resulted in tachypnea within l hour after challenge. The tachypnea may have been caused by increase in core temperature, anxiety, pulmonary mechanical changes, changes in arterial blood gas tensions and pH, or increased activity of pulmonary receptors, with their afferents in the vagus nerve.8 Rectal temperature did not change during the experiments and anxiety is not likely to have played a role in the tachypnea, as challenge with saline or bovine Y globulin does not change RR in ponies.1 Respiratory rate changes were independent of pulmonary mechanics changes, because pulmonary mechanics were unaltered by vagal blockade, while RR decreased. Although Pa02 decreased following challenge, the magnitude of decrease is insufficient to solely account for the tachypnea.9 Since vagal blockade reversed the increase in RR, these data suggest that in ponies as in dogs tachypnea fbllowing aerosol antigen challenge is mainly caused by increased activity of pulmonary receptors with their afferents in the vagus nerve. The conclusion is supported by results from the unilateral challenge experiment in which bilateral vagal blockade eliminated tachypnea. Since RR did not decrease following left unilateral vagal blockade alone, these results further suggest that vagal afferent fibers crossover to the contrala- teral vagus nerve in the thorax. In both the unilateral and bilateral challenge experiments, fbllowing vagal blockade RR did not decrease below baseline value as it should have done if vagal mechanisms alone were responsible fbr the tachypnea observed. Muir et al9 reported in horses that a decrease in Pa02 from 89.2 to 55.9 mmHg as occurred following bilateral ovalbumin challenge results in an increase in RR of approximately l2 breaths per minute. This increase is similar to the 161 difference between observed and expected RR following vagal blockade, suggesting that the decreased Pa02 was responsible for the failure of RR to decrease below baseline value. Following bilateral ovalbumin challenge, Rtot increased 300%. In the dog, the majority of resistance to flow resides in the central air- ways, while the peripheral airways contribute little to Rtot-lo If this is also true in the horse, the increase in Rtot» combined with a large decrease in Ody" in the bilateral challenge experiments, may be atti- buted either to a modest large airway narrowing or to the massive small airway obstruction which was observed histologically.11 Failure of vagal blockade to alter Rtot suggests that vagal mechanisms were not involved in the increased Rtot following challenge. This conclusion is supported by data from the unilateral challenge experiment because challenge of the left lung alone increased RtotL but did not change RtotR and because vagal blockade had no effect on RtotL- Since vagal mechanisms were not involved, the increase in Rtot following challenge must be due to local mechanisms, such as direct effect of mediators of inflammation on airway smooth muscle or mechanical obstruction of airways by debris or edema. This conclusion is slightly different from that reached in systemi- cally sensitized ponies.1 In this latter group of ponies, vagal blockade partially reversed the increase in Rtot following bilateral challenge. He concluded that in addition to local mechanisms increased sensitivity of airway smooth muscle to normal vagal tone also played a role in the response of the lung to antigen challenge.1 ’ The increase in RtotL following left unilateral ovalbumin challenge was nearly an order of magnitude greater than the increase in, Rtot following bilateral challenge. Because the endobronchial tubes 162 were shorter and narrower than the endotracheal tube and located in a mainstem bronchus rather than the trachea, differences in amount and deposition of aerosol may account fOr this discrepancy. Using the same challenge technique we previously reported a similar difference in response to unilateral and bilateral antigen challenges in systemically sensitized ponies.1 Dynamic compliance can be decreased by a change in lung volume at which tidal breathing is accomplished by an alteration of the elastic properties of the lung or by a prolongation of peripheral time constants. The decrease in Cd," following challenge was not caused by an increase in lung volumes as FRC did not change. Although - Cstat decreased, the magnitude of this change was too small to solely account fbr the decrease in Cdyn- Therefore the marked decrease in Cdyn suggests that ovalbumin aerosol challenge resulted in prolongation of peripheral time constants, probably caused by the small airway obstruction which was observed histologically. Small airway obstruction probably also resulted in the increase in MV and decreased Pa02. Since following small airway obstruction Cdyn may become frequency dependent,12 the small but significant rise in Cdyn following vagal blockade may have been due to the concurrent decrease in RR. Functional residual capacity was unaltered by challenge or vagal blockade. Following antigen challenge in some species FRC increases,13 while in others FRC does not change.14:15 The reason fbr this discrepancy is not clear as the change in FRC does not correlate with the severity of airway response as judged by changes in resistance and Cdyn- The decrease in both TLC and Cstat following challenge may also have 163 resulted from diffuse peripheral airway obstruction and failure to recruit obstructed air spaces during inflation of the lung to 30 cm H20 Ptp. In addition, because Cstat decreased and FRC was unchanged, specific com- pliance of the lung also decreased. A decrease in TLC fallowing antigen challenge has also been reported in the guinea pig16 but not in the monkey or dog.14s15 In the guinea pig, this decrease in TLC was rever- sible by vagal blockade, suggesting that alveolar duct constriction was an important factor. This does not appear to be the case in the pony. Distribution and severity of histopathologic lesions correlated with pulmonary function data. The most dramatic changes in pulmonary func- tion were the large decrease in Cdyn and increase in MV. These changes were probably associated with the principal histologic lesions of severe necrotizing bronchiolitis and bronchiolar obstruction. The multifocal alveolitis and edema probably resulted in the change in the elastic pro- perties of the lung and the decrease in TLC. Histologically, large air- ways were less affected by ovalbumin challenge than small airways. However, bronchoconstriction may have occurred in response to challenge and therefore we cannot determine whether the 3-fold increase in Rtot following challenge was due to massive small airway obstruction or large airway narrowing. In this study we have shown that the tachypnea following ovalbumin aerosol challenge of conscious ponies is mediated via vagal afferents but that airway obstruction fbllowing challenge is caused by local mechanisms such as the obstruction of airway with exudate, debris, mucus and edema fluid. He also conclude that in this model of lung disease, changes in pulmonary function following challenge are predictive of the major histologic lesions. 164 1. Derksen FJ, Robinson NE, Slocombe RF: Pulmonary function in ovalbumin induced allergic lung disease in the awake pony: role of vagal mechanisms. J Appl Physiol: Respirat Environ Exercise Physiol, in press. 2. Derksen FJ, Robinson NE, Stick JA: Technique fbr refersible vagal blockade in the standing conscious pony. Am J Vet Res 42:523-525, 1981. 3. Derksen FJ, Robinson NE: Esophageal and intrapleural pressures in the healthy conscious pony. Am J Vet Res 41:1756-1761, 1980. 4. Derksen FJ, Robinson NE, Slocombe RF, Riebold TH, Brunson DB: Pulmonary function tests in standing ponies: reproducibility and effect of vagal blockade. Am J Vet Res, in press. 5. Salazar E, Knowles JH: An analysis of pressure-volume charac- teristics of the lung. J Appl Physiol 19:97-104, 1964. 6. Steel GD, Torrie JH: Principles and Procedures of Statistics. New York, McGraw Hill Book Co., 1960. 7. Richerson HB: Varieties of acute immunologic damage to the rab- bit lung. Ann NY Acad Sci 221:340-360, 1974. 8. Cotton DJ, Bleecker ER, Fischer SP, Graf PD, Gold HM, Nadel JA: Rapid shallow breathing after ascaris serum antigen inhalation: role of vagus nerves. J Appl Physiol: Respirat Environ Exercise Physiol 42:101-106, 1977. 9. Muir NH, Moore CA, Hamlin RL: Ventilatory alterations in normal horses in response to changes in inspired oxygen and carbon dioxide. Am J Vet Res 36:155-166, 1975. 10. Machlem PT, Hoolcock AT, Hogg JC: Partitioning of pulmonary resistance in the dog. J Appl Physiol 26:798-805, 1969. 11. Otis AB, McKerrow CB, Bartlett RA, Mead J, McIlrdy MB, Selverstone NJ, Radford EP: Mechanical factors in distribution of pulmonary ventilation. J Appl Physiol 8:427-443, 1956. 12. Brown R, Hoolcock AJ, Vincent NJ, Macklem PT: Physiological effects of experimental airway obstruction with beads. J Appl Physiol 27:328-335, 1969. 13. Harner A, Mezey RJ, Reinhart ME, Eyre P: Antigen induced bronchospasm in conscious sheep. J Appl Physiol Respirat Environ Exercise Physiol 47:917-922, 1979. 14. Gold HM, Kessler GF, Yu DYC, Frick 0L: Pulmonary physiologic abnormalities in experimental asthma in dogs. J Appl Physiol 33:496-501, 1972. 165 15. Pare P0, Michoud MC, Hogg JC: Lung mechanics fellowing antigen challenge of Ascaris-Suums-sensitive Rhesus monkeys. J Appl Physiol 41:668-676, 1976. 16. Drazen JM, Louing SH, Venugopalan C: Lung volumes after antigen infusion in the guinea pig in vivo: effects of vagal section. J Appl Physiol: Respirat Environ Exercise Physiol 45:957-961. 1978. CONCLUDING DISCUSSION When comparing the derangement in pulmonary function induced by the oral administration of 3MI and ovalbumin challenge of locally and syste- mically sensitized ponies, certain similarities are striking. Tachypnea characterized both the 3MI induced pulmonary toxiosis and the 2 allergic disease models and was mediated by pulmonary receptors with their afferent neurons in the vagus nerve. Tachypnea is characteristic of a variety of lung diseases in mammals and this study suggests that activa- tion of pulmonary receptors with afferent neurons in the vagus nerve may be a stereo typical response to lung injury in the equid. A decrease in Cdyn, an increase in MV and impairment of gas exchange occurred in both the 3MI model as well as the ovalbumin induced allergic disease models. These changes in pulmonary function are suggestive of small airway obstruction, which was confirmed at necropsy, as the major histopathologic lesion in all 3 pulmonary disease models was an obstruc- tive bronchiolitis. Vagal blockade did not change Cdyn or gas exchange, suggesting that vagal mechanisms were not involved in the small airway obstruction in these pulmonary disease models. Interestingly, naturally occurring obstructive pulmonary disease in the horse and in persons is also characterized by obstructive bronchiolitis and, therefore, it appears that the small airways are a weak link in the mammalian pulmo- nary defence system. Following 3MI administration the increase in Rtot was 30% while following ovalbumin challenge Rtot increased 300%. This 166 167 suggests that the pulmonary lesions induced by 3MI were primarily in the small airways, while ovalbumin challenge also resulted in large airway obstruction. This was supported by histopathologic findings as no large airway lesions were present in 3MI treated horses while bronchitis was described following ovalbumin challenge. The studies presented in this dissertation suggest that the role of vagal mechanisms in airway obstruction in the 3 pulmonary disease models is minor in importance. Although Rtot decreased following vagal blockade in 3MI treated ponies, this change in Rtot could be attributed to the interruption of normal parasympathetic bronchomotor tone. In the allergic pulmonary disease models it was clearly demonstrated that local mechanisms were of major significance in the pathogenesis of airway obstruction. However, an increased sensitivity to normal vagal tone may have contributed to the airway obstruction in systemically sensitized ponies fbllowing ovalbumin challenge. In some species, small airway obstruction is accompanied by an increase in FRC, while in others it is not. The reason fbr this discre- pancy is not clear as the severity of the obstruction does not correlate with changes in FRC. In this study fbllowing 3MI treatment FRC increased while following ovalbumin challenge FRC did not change. The reason fbr this discrepancy remains uneXplained. Total lung capacity and quasistatic compliance decreased only following ovalbumin challenge in ponies sensitized both via the intra- muscular and aerosol routes. Since the decrease in Cdyn in this model of lung disease was an order of magnitude larger than the decrease in Cdyn following 3MI treatment of ovalbumin challenge of systemically sen- sitized ponies, a more severe small airway obstruction may have resulted 168 in failure to recrute obstructed lung units at a Ptp of 30 mm H20. Morphometric studies were not performed on the histopathologic specimens and, therefore, this hypothesis could not be tested. SUMMARY AND CONCLUSION In the study reported in Chapter 1, pleural and esophageal pressures were compared in 6 standing sedated ponies. Pleural pressure was measured with blunt needles attached to transducers and inserted in the l0th intercostal space level with and l0 and 20 cm above the point of the shoulder. Two balloons (a condom and an esophageal balloon) attached to transducers measured esophageal pressure in the cranial, middle, and caudal portions of the thoracic part of the esophagus. Tidal volume was measured by integrating a flow signal derived from a pneumotachograph attached to an endotracheal tube inserted through a tracheostomy. Frequency responses of all measuring systems were matched. The change in pleural pressure during respiration was greatest in the middle and ventral portions of the thorax, less in the dorsal portion of the thorax and in the middle and caudal portions of the thoracic part of the eSOphagus, and least in the cranial portion of the thoracic part of the esophagus. The type of esophageal balloon had no effect on the measured pressure change and using either balloon, changes in e50phageal pressure reflected local changes in pleural pressure. Regional variations in eSOphageal or pleural pressure during breathing caused variations in the calculated dynamic compliance. Pleural pressure gradients of 0.33 cm of water/cm of descent and 0.484 cm of water/cm of descent were recorded in the dorsal and ventral halves of the thorax, respectively, and may result in regional variations in lung 169 17D inflation similar to those observed in persons. In Chapter 2, a surgical technique is described fer preparation of chronic cervical vagal loops in ponies. Vagal blockade was induced by circulating methanol (-2°C) through coils which enclosed the loops. Vagal blockade increased tidal volume, heart rate, and systemic blood pressure and decreased respiratory rate. Atropine 0.04 mg/kg intrave- nously increased heart rate and systemic pressure but did not alter respiratory parameters indicating vagal cooling caused both afferent and efferent blockade. The effects of vagal blockade were rapidly reversed when refrigerated coils were removed. In order to determine the short and long-term reproducibility of pulmonary function tests in ponies. Arterial blood gas tensions, pulmo- nary mechanics and lung volumes were measured in 4 sedated animals every hour for 6 hours and in 5 animals 4 times at 2 monthly intervals. (Chapter 3) Variability in blood gas tensions was small over both the short and long-term measurement periods, while the variability in total respiratory resistance (Rtot) and functional residual capacity (FRC) was small over the short term but larger over the long term. The variabi- lity in tidal volume (VT), minute ventilation (7min). respiratory rate (RR) and dynamic and quasistatic compliance (Cdyn and Cstat) was relati- vely large over both the short and long term. When data from five ponies was pooled no significant change occurred in any of the variables over a period of six months. Vagal blockade increased VT and decreased RR and Rtot: but arterial blood gas tensions, 9min. Cdyn: Cstats FRC and lung and thoracic cage pressure-volume curves were unaffected. 171 Total respiratory resistance decreased with increasing lung volume with the vagus intact. Following vagal blockade the decrease in Rtot with lung volume was minimal. Dynamic compliance was frequency independent over a range of 15-60 breaths min-1, suggesting that significant inhomogeneity of peripheral time constants did not exist in our normal ponies. Chapter 4 reports changes in arterial blood gas tensions, pulmonary mechanics and lung volumes, 24 to 48 hours after oral administration of either 500 ml of corn oil or l00 mg/kg body weight of 3-methylindole (3MI) in 500 ml of corn oil. In the latter group, variables were also measured after bilateral cervical vagotomy. Respiratory rate (RR) and minute ventilation (7min) were increased by 3MI treatment and decreased after vagotomy, suggesting that the tachypnea induced by 3MI was vagally mediated. Pao2 was unaffected but PaCOz decreased below baseline following 3MI and vagotomy. Both specific respiratory conductance (SGtot) and dynamic compliance were decreased by 3MI. Following vago- tomy SGtot was increased but remained below baseline level, suggesting that local mechanisms were involved in the pathogenesis of airway narrowing. The increase in SGtot following vagotomy may have been due to interruption of normal parasympathetic bronchomotor tone. Functional residual capacity, which increased following 3MI, was unaffected by vagotomy. Total lung capacity and quasistatic compliance were unaf- fected by either treatment. Minimal volume was larger in 3MI treated ponies than in a group of untreated ponies. Decreased dynamic compliance and specific respiratory conductance and increased functional residual capacity and minimal volume are all compatible with small 172 airway obstruction produced by the necrotizing bronchiolitis and bronchiolar obstruction observed histologically in 3MI treated ponies. In Chapter 5, in awake sensitized ponies, we studied the effect of aerosol ovalbumin challenge on ventilation, pulmonary mechanics, lung volumes and gas exhange before and after vagal blockade. He also challenged the left lung and measured respiratory rate (RR), and right and left lung resistance (RtotRa RtotL) before and after both left and bilateral vagal section. Bilateral ovalbumin aerosol challenge increased RR, minute ventilation (7min). respiratory resistance (Rtot) and minimal volume, decreased dynamic compliance and arterial oxygen tension, and was without effect on functional residual capacity, total lung capacity, quasistatic lung compliance, and arterial carbon dioxide tension. Vagal blockade reversed the increase in RR, 7min and Rtot and increased VT. Challenge of the left lung increased RR and RtotL but did not alter RtotR- Bilateral vagal section reversed the tachypnea but unilateral section did not. Histopathologic lesions included acute fibrino-purulent obstructive bronchiolitis, bronchitis, edema and alveolar distension. He conclude that local mechanisms are of critical importance in the pathogenesis of ovalbumin induced airway obstruction in ponies, that increased sensitivity of airway smooth muscle to normal vagal tone may also play a role and that tachypnea fellowing challenge is caused by activity of pulmonary receptors with vagal afferent fibers. Since pilot studies suggested that aerosol challenge fbllowing both systemic and local sensitization of the lung results in more severe dyspnea of rapid onset, in Chapter 6, in awake ponies, sensitized syste- mically by intramuscular injection and locally via aerosol, we studied ventilation, pulmonary mechanics, lung volumes and gas exchange before 173 and one hour after bilateral aerosol ovalbumin challenge and after left unilateral and bilateral vagal blockade. He also challenged the left lung and measured reSpiratory rate (RR) and right and left lung resistance (RtotR and RtotL) during the same measurement periods. Bilateral ovalbumin aerosol challenge inereased RR, minute ventilation (7min): respiratory system resistance (Rtot) and minimum volume, decreased dynamic compliance, quasistatic compliance, Pa02, tidal volume, total lung capacity and was without effect on functional resi- dual capacity and PaC02. Bilateral and not unilateral vagal blockade decreased RR, 9min» and increased VT and Cdyn- Challenge of the left lung increased RR and RtotL but did not alter RtotR- Bilateral vagal blockade reversed the tachypnea but unilateral blockade did not. Pulmonary function changes following challenge in this group of ponies was more severe than in ponies sensitized only by intramuscular injec- tion. Histopathologic lesions included acute fibrinopurulent obstruc- tive bronchiolitis, bronchitis, and alveolar distension. We conclude that in this disease model local mechanisms are of critical importance in the pathogenesis of airway obstruction, that tachypnea fbllowing challenge is caused by increased activity of pulmonary receptors with vagal afferent fibers and that changes in pulmonary function following challenge are predictive of the major histologic lesions. LIST OF REFERENCES LIST OF REFERENCES 1. Adrian ED: Afferent impulses in the vagus and their effect on respiration. J Physiol (London) 79:332-358, l933. 2. Alexander HL, Paddock R: Bronchial asthma: response to pilo- carpine and epinephrine. Arch Int Med 27:l84-l9l, l92l. 3. Arborelius M, Ekwall B, Jernerus R, Lundin G, Svanberg L: Unilateral provoked broncheal asthma in man. J Clin Invest 4l:l236- 1241, l962. 4. Atkinson G, Bogan JA, Breeze RG, Schman IE: Effects of 3-methylindole in cattle. Brit J Pharmac 6l:285-290, l977. 5. Bando T, Skindo N, Skimo Y: Non-adrenergic inhibitory nerves in tracheal smooth muscle of guinea pig. J Physiol Soc Jrn 35:508-509, l973. 6. Bardana EJ: Modern aspects of diagnosis and treatment of the asthmatic patient. Clinical notes on R0. l5:3-l3, I976. 7. Booth BH, Patterson R, Talbot CH: Immediate type hypersen- sitivity in dogs: cutaneous anaphylactic and respiratory responses to Ascaris. J Lab Clin Med 76:l8l-l89, 1970. 8. Boushey HA, Holtzman MJ, Sheller JR, Nadel JA: State of the art. Bronchial hyperreactivity. Am Rev Resp Dis lZl:389-4l3, l980. 9. Bray TM, Carlson JR: Role of mixed-function oxidase in 3-methylindole induced acute pulmonary edema in goats. Am J Vet REs 40:1268-1272, l979. 10. Bradley BJ, Carlson JR, Dickson E0: 3-Methylindole-induced pulmonary edema and emphysema in sheep. Am J Vet Res 39:l355-l358, I978. 11. Breeze RG: Heaves. Vet Clin N Amer Large Animal Ed, l:2l9-230, l979. 12. Breeze RG, Lee HA, Grant BD: Toxic lung disease. Mod Vet Pract 59:301-302, l978. 13. Breeze RG: Fog fever and heaves: study on the respiratory diseases of adult cattle and horses. Proceedings ACVIM 87-ll9, l978. 175 176 14. Briscoe NA, McLemore GA: Ventilatory function in bronchial asthma. Thorax 7:66-77, l952. 15. Burnstock G: Purinergic nerves. Pharmacol Rev 24:509-58l, l972. 16. Cabezas GA, Graf PD, Nadel JA: Sympathetic versus parasym- pathetic nervous regulation of airways in dogs. J Appl Physiol 3l:65l-655, l97l. 17. Carlson JR, Dickinson E0, Yokoyama MT, Bradley BJ: Pulmonary edema and emphysema in cattle, after intraruminal and intravenous admi- nistration of 3-methylindole. Am J Vet Res 36:l34l-l347, l975. 18. Castro de la Mata R, Penna M, Aviado 0M: Reversal of sym- pathomimetic bronchodilation by dichloroiSOproterenol. J Pharmacol Exp Ther l35:l97-20l, l962. 19. Cherniack RM, Farhi LE, Armstrong BH, Proctor OF: A comparison of esophageal and intrapleural pressure in man. J Appl Physiol 8:203-2ll, l955. 20. Christie RV: DySpnea in relationship to the viscoelastic pro- perties of the lungs. Proc Roy Soc Mod 46:38l-386, l953. 21. CIBA Symposium: Terminology, definitions, and classification of chronic pulmonary emphysema and related conditions. Thorax 14:286-287, l959. 22. Coburn RF, Tbmita T: Evidence for nonadrenergic inhibitory nerves in the guinea pig trachealis muscle. Am J Physiol 224:l072-l080, T973. 23. Cohen S, Yoshida T: Lymphokine-mediated reactions. In Mechanisms of Immunopathology, 49-68, Cohen S ed. John Hiley & Sons, New York, l979. 24. Coleman RA: Evidence for a non-adrenergic inhibitory nervous pathway in guinea pig trachea. Br J Pharmacol 48:360P-36lP, l973. 25. Cook HR: Chronic bronchitis and alveolar emphysema in the horse. Vet Rec 99:448-451, 1976. 26. Dain 0, Gold HM: Mechanical properties of the lungs and experimental asthma in conscious allergic dogs. J Appl Physiol 38:96-100, l975. 27. Daly HJ, Bondurant 5: Direct measurement of respiratory pleural pressure changes in normal man. J Appl Physiol 18:5l3-5l8, 1963. 177 28. Davis HL, Fowler HS, Lambert EH: Effect of volume and rate of inflation and deflation on transpulmonary pressure and response of pulmonary stretch receptors. Am J Physiol 187:558-566, l956. 29. Denac-Sikiric M: Untersuchungen der Dehnbarkeit des Lungengewebes bei gesunden und emphysemkranken Pferden. Schweiz Arch Tierheilkd ll2:606-6l5, 1970. 30. Despas PJ, Leroux M, Macklem PT: Site of airway obstruction in asthma as determined by measuring maximal expiratory flow breathing air and a helium-oxygen mixture. J Clin Invest 51:3235-3243, l972. 31. Dickie HA, Rankin J: Farmer's lung: an acute granulomatous interstitial pneumonitis occurring in agricultural workers. JAMA 167:l069-l076, l958. 32. Dixon HE, Brody TG: Contributions to the physiology of the lungs. Part I. The bronchial muscles, their innervation, and the action of drugs upon them. J Physiol (London) 29:97-l73, l903. 33. Dixon HE, Ransom F: Bronchodilator nerves. J Physiol (London) 45:4l3-428, l9l2. 34. Drazen JM, Austen KF: Pulmonary response to antigen infusion in the sensitized guinea pig: modification by atropine. J Appl Physiol 39:9l6-9l9, l975. 35. Drazen JM, Loring SH, Venugopalan C: Lung volumes after anti- gen infusion in the guinea pig-lg vivo: effects of vagal section. J Appl Physiol 45:957-96l, l978. 36. Elftman AG: The afferent and parasympathetic innervation of the lungs and trachea of the dog. Am J Anat 72:l-28, l943. 37. Engel LA, Macklem PT: Gas mixing and distribution in the lung. Respir Physiol l4:37-83, l977. 38. Farhi L, Otis AB, Proctor 0F: Measurement of intrapleural pressure at different points on the chest of the dog. J Appl Physiol 10:15-18, 1957. 39. Fleish JH, Maling HM, Brodie BB: Evidence of alpha-adrenergic receptors in the mammalian trachea. Am J Physiol 218:596-599, 1970. 40. Foster RH: The nature of adrenergic receptors in the trachea of the guinea pig. J Pharm Pharmac l8:l-l2, l966. 41. Franz DN, 1990 A: Conduction failure in myelinated and non- ?yeginated axons at low temperatures. J Physiol (London) l99:3l9-345, 96 . 42. Gerber H: Chronic pulmonary disease in the horse. Equine Vet J 5:26-33, l973. 178 43. Gillespie JR, Tyler HS: Chronic alveolar emphysema in the horse. Adv Vet Sci l3:59-93, l969. 44. Gillespie JR, Tyler HS, Eberly VE: Pulmonary ventilation and resistance in emphysematous and control horses. J Appl Physiol 2l:416-422, l966. 45. Gold HM, Kessler GF, Yu DYC, Frick 0L: Pulmonary physiologic abnormalities in experimental asthma in dogs. J Appl Physiol 33:496-501, l972. 46. Gold HM, Kessler, GF, Yu DYC: Role of vagus nerves in experi- mental asthma in allergic dogs. J Appl Physiol 33:719-725, 1972. 47. Guirgis HM, McNeill RS: The nature of adrenergic receptors in isolated human bronchi. Thorax 24:613-6l5, l969. 48. Hahn HL, Graf PD, Nadel JA: Effect of vagal tone on airway diameters and on lung volume in anesthetized dogs. J Appl Physiol 4l:58l-589, l976. 49. Halonen M, Fisher HK, Blair L, Butler C, Pinckard RN: IgE-induced respiratory and circulatory changes during systemic anaphy- laxis in the rabbit. Am Rev Res Dis ll4:96l-970, 1976. 50. Hammond AC, Carlson JR, Hillett JD: The metabolism and dispo- sition of 3-methylindole in goats. Life Sciences 25:130l-l306, l979. 51. Hensley MJ, D'Cain CF, McFadden ER, Ingram RH: Distribution of bronchodilation in normal subjects: beta agonist versus atr0pine. J Appl Physiol 45:778-782, l978. 52. Hering E, Breuer J: Die selbststerdrung der atemung durch den nervus vagus. Sitzber Akad Hiss Hien 57:672-677. l868. 53. Hirshman CA, Downes M: Basenji-Greyhound dog model of asthma: Influence of atropine on antigen-induced bronchoconstriction. J Appl Physiol 50:76l-765, l98l. 54. Hirshman CA, Leon DA, Bergman NA: The Basenji-Greyhound dog: Antigen-induced changes in lung volumes. Respiration Physiol 43: 377- 388, l98l. 55. Hirshman CA, Malley A, Downes H: Basenji-Greyhound dog model of asthma: reactivity to Ascaris suum citric acid and methacholine. J Appl Physiol 49:953-957, l 56. Hoppin FG, Green ID, Mead J: Distribution of pleural surface pressure in dogs. J Appl Physiol 27: 863- 873, l969. 57. Hogg JC, Nepszy S: Regional lung volume and pleural pressure gradient estimated from lung density in dogs. J Appl Physiol 27:l98-203, l969. 179 58. Huber HL, Koessler KK: The pathology of bronchial asthma. Arch Int Med 30:689-760, l922. 59. Hurtado A, Kaltreider NL: Studies of total pulmonary capacity and its subdivisions. Observations during the acute respiratory distress of bronchial asthma and following the administration of epi- nephrine. J Clin Invest l3:l053-l062, 1934. 60. Ingram RH, Hellman JJ, McFadden ER, Mead J: Relative contribu- tion of large and small airways to flow limitation in normal subjects before and after atropine and isoproterenol. J Clin Invest 59:696-703, 1977. 61. Kalia M: Cerebral pathways in reflex muscular inhibition from type J pulmonary receptors. J Physiol (London) 204:92P-93P, l969. 62. Karczewski H, Hiddicombe JG: The role of the vagus nerves in the respiratory and circulatory reactions to anaphylaxis in rabbits. J Physiol 201:293-304, 1969. 63. Kessler GF, Austin JH, Graf PD, Gamsu G, Gold HM: Airway constriction in experimental asthma in dogs: Tantalum bronchographic studies. J Appl Physiol 35:703-708, 1973. 64. Knowlton GC, Larrabee MG: A unitary analysis of pulmonary volume receptors. Am J Physiol l47:l00-ll4, l946. 65. Krell RD, Chakrin LH, Hardell JR: The effect of cholinergic agents on a canine model of allergic asthma. J Allergy Clin Immunol 58:19-30, 1976. 66. Krueger JJ, Bain T, Patternson JL: Elevation gradient of intrathoracic pressure. J Appl Physiol 16:465-468, l961. 67. Larsell 0, Dow RS: The innervation of the human lung. Am J Anat 52:l25-l46, l933. 68. Leith DE, Gillespie JR: Respiratory mechanics of normal horses and one with chronic obstructive lung disease. Fed Proc 30:556, l97l. 69. Longet M: Recherches experimentales sur la nature des mouve- ments intrinsique du poumon et sur une nouvelle cause d'emphyseme pulmo- naire. Comptes Rendus l5:500-503, l842. 70. Lowell FC: Observations on heaves, an asthma-like syndrome in the horse. J Allergy 35:322-330, l964. 71. Macklem PT, Mead J: Resistance of central and peripheral air- ways measured by a retrograde catheter. J Appl Physiol 22:395-40l, 1967. 72. Macklem PT, Hoolcock AJ, Hogg JC, Nadel JA, Hilson NJ: Partitioning of pulmonary resistance in the dog. J Appl Physiol 26:798-805, l969. 180 73. Mauderly JL: Evaluation of the grade pony as a pulmonary func- tion model. Am J Vet REs 35:l025-l029, l974. 74. Mansman RA: The pathogenesis of chronic bronchiolitis in the horse. PhD Thesis, U of California, l973. 75. Marshall R, Hiddicombe JG: The weakness of the Hering-Breuer reflex in man. J Physiol l40:36P, l958. 76. McFadden ER: The chronicity of acute attacks of asthma: mechanical and therapeutic implications. J Allergy Clin Immunol 56:l8-26, l975. 77. McFadden ER, Kiser R, DeGroot HJ: Acute bronchial asthma. Relations between clinical and physiologic manifestations. N Eng J Med 288:22l-225, l973. 78. McFadden ER, Lyons HA: Airway resistance and uneven ven- tilation in bronchial asthma. J Appl Physiol 25:365-370, 1968. 79. McKibben JS: Cervical and thoracic autonomic innervation. In The Anatomy of Domestic Animals, Getty, 5th ed. 688-696, HB Saunders, Philadelphia, 1975. 80. McLaughlin RF, Tyler HS, Canada R0: A study of the subgross pulmonary anatomy in various mammals. Am J Anat l08:l49-l65, l96l. 81. McPherson EA, Lawson GHK, Murphy JR, Nicholson JM, Fraser JA, Breeze RG, Pirie HM: Chronic obstructive pulmonary disease: iden- tification of affected horses. Equine Vet J l0(l):47-53, l978. 82. Mead J, McIlroy MB, Selverstone NJ, Kriete BC: Measurement of intraesophageal pressure. J Appl Physiol 7:49l-495, l955. 83. Milic-Emili J, Mead J, Turner JM, Glauser EM: Improved tech- nique for estimating pleural pressure from esophageal balloons. J Appl Physiol l9:207-le, l964. 84. Mills JE, Sellick H, Hiddicombe JG: Activity of lung irritant receptors in pulmonary micro-embolism, anaphylaxis and drug induced bronchoconstrictions. J Physiol (London) 203:337-357, l969. 85. Mills JE, Hiddicombe JG: Role of the vagus nerves in anaphy- laxis and histamine induced bronchoconstriction in guinea pigs. Br J Pharmac 39:724-73l, l970. 86. Muylle E, Dyaert H: Lung function tests in obstructive pulmo- nary disease in horses. Equine Vet J 5(l):37-43, l973. 87. Nadel JA: Autonomic control of airway smooth muscle and airway secretions. Am Rev Resp Dis ll5:ll7-l26, l977. 181 88. Nadel JA, Salem H, Tamplin B, Tokiwa Y: Mechanism of broncho- constriction during inhalation of sulfur dioxide. J Appl Physiol 20:l64-l67, l965. 89. Orehek J, Gayrard P, Grimand C, Charpin J: Effect of beta adrenergic blockade on bronchial sensitivity to inhaled acetylcholine in normal subjects. J Allergy Clin Immunol 55:164-169, 1975. 90. Orr JA, Bisgard GE, Foster HV, Rawlings CA, Buss 00, Hill JA: Cardiopulmonary measurements in nonanesthetized resting normal ponies. Am J Vet Res 36:1667-l670, l975. 91. Paintal AS: Vagal sensory receptors and their reflex effects. Physiological Reviews 53:l59-227, 1973. 92. Paintal AS: A comparison of the nerve impulses of mammalian nonmodulated nerve fibers with those of the smallest diameter medulated fibers. J Physiol (London) 193:523-533, 1967. 93. Paintal AS: The J reflex. Proc Inter Union Physiol Sci 7:79-80, l97l. 94. Paintal AS: Mechanism of stimulation of type J pulmonary receptors. J Physiol (London) 203:5ll-532, I969. 95. Paintal AS: Vagal afferent fibers. Ergeb Physiol 52:74-l56, l963. 96. Paintal AS: Impulses in vagal afferent fibers from specific pulmonary deflation receptors. The response of these receptors to phe- nyldiguanide, potato starch, 5 hydroxytryptamine and nicotine and their role i? {esgiratory and cardiovascular reflexes. Quart J Exptl Physiol 40:89- l , 955. 97. Paintal AS: The response of gastric stretch receptors and cer- tain other abdominal and thoracic vagal receptors to some drugs. J Physiol (London) l26:27l-285, l954. 98. Paintal AS: The response of pulmonary and cardiovascular vagal receptors to certain drugs. J Physiol (London) l2l:l82-l90, l953. 99. Pare PD, Michoud MC, Hogg JC: Lung mechanics fbllowing antigen challenge of Ascaris suum sensitive Rhesus monkeys. J Appl Physiol 41:668-676, 1976. 100. Pare PD, Michoud MC, Boucher RC, Hogg JC: Pulmonary effects of acute and chronic antigen exposure of immunized guinea pigs. J Appl Physiol 46:346-353, l979. 101. Patterson R, Mellies CJ, Kelly JF, Harris KE: Airway responses of dogs with ragweed and ascaris hypersensitivity. Chest 65:488-492. 182 102. Phillipson EA, Murphy E, Kozar LF, Schultze RK: Role of vagal stimuli in exercise ven tilation in dogs with experimental pneumonitis. J Appl Physiol 39:76-85, 1975. 103. Platt H: The role of respiratory viruses in equine disease. Vet Rec 9l:33-36, l972. 104. Popa V, Douglas JS, Bouhuys A: Airway responses to histamine, acetylcholine and antigen in sensitized guinea pigs. J Lab Clin Med 84:225-234, l974. 105. Proctor 0F, Caldini P, Permutt S: The pressure surrounding the lungs. Respir Physiol 5:l30-l44, l968. 106. Ratner B, Jackson HC, Gruehl HL: Respiratory anaphylaxis; sen- sitization, shock, broncheal asthma and death induced in the guinea pig by the nasal inhalation of dry horse dander. Am J Dis Child 34:23-52, 1927. 107. Rebuck AS, Read J: Assessment and management of severe asthma. Am J Med 5l:788-798, l97l. 108. Richardson JB, Beland J: Non-adrenergic inhibitory nervous system in human airways. J Appl Physiol 41:764-771, l976. 109. Richerson HB: Acute experimental hypersensitivity pneumonitis in the guinea pig. J Lab Clin Med 79:745-757, l972. 110. Rosenthal RR, Norman PS, Summer HR, Permutt S: Role of the parasympathetic system in antigen induced bronchospasm. J Appl Physiol 42:600-606, 1977. 111. Roska AKB, Garancis JC, Moore VL, Abramoff P: Immune-complex disease in guinea pig lungs. Clin Immunol Immunopathol 8:2l3-224, T977. 112. Roussos CS, Fukuchi Y, Macklem PT, Engel LA: Influence of diaphragmatic contraction on ventilation distribution in horizontal man. J Appl Physiol 40:4l7-424, l976. 113. Rubinfeld AR, Hagner PD, Hest JB: Gas exchange during acute experimental canine asthma. Am Rev of Resp Dis ll8:525-536, l978. 114. Russell JA: Noradrenergic inhibitory innervation of canine airways. J Appl Physiol 48:l6-22, l980. 115. Sasse HHL: Some pulmonary function tests in horses. Rotterdam, Bronder offset, l97l. 116. Sellick H, Hiddicombe JG: The activity of lung irritant recep- tors during pneumothorax, hyperpnea, and pulmonary vascular congestion. J Physiol (London) 203:359-381, l969. 183 117. Sellic M, Hiddicombe JG: Stimulation of lung irritant recep- tors by cigarette smoke, carbon dust and histamine aerosol. J Appl Physiol 3l:l5-l9, l97l. 118. Severinghaus JH, Stupfel M: Respiratory dead space increase following atropine in man and atropine, vagal or ganglionic blockade and hypothermia in dogs. J Appl Physiol 8:8l-87, 1955. 119. Snapper JR, Drazen JM, Loring SH, Braasch PS, Ingram RH: Vagal effects on histamine, carbachol and prostaglandin F2 responsiveness in the dog. J Appl Physiol 47:l3-16, l979. 120. Sorenson PR, Robinson NE: Postural effects on lung volumes and asynchronous ventilation in anesthetized horses. J Appl Physiol 48:97-103, l980. 121. Sporri VH, Leemann H, Zur Untersuchung der lungenmechanik bei grobtieren. Schweiz Arch Tierkeilkd 106:699-7l4, l964. 122. Stanescu DC, Teculescu DB: Exercise and cough induced asthma. Respiration 27:377-383, l970. 123. Stein M, Schiavi RC, Ottenberg P, Hamilton C: The mechanical properties of the lungs in experimental asthma in the guinea pig. J Allergy 32:8-l6, l96]. 124. Stevenson DD, Mathison DA, Tan EM, Vaughan JH: Provoking fac- tors in broncheal asthma. Arch Intern Med l35:777-783, l975. 125. Thurlbeck HM, Lowell FC: Heaves in horses. Am Rev Resp Dis 89:82-88, l964. ~ 126. Van Allen CM, Lindskog GE, Richter HG: Collateral respiration. Transfer of air collaterally between pulmonary lobules. J Clin Invest l0:559-590, l93l. 127. Harner A, Mezey RJ, Reinhard ME, Eyre P: Antigen-induced bronchospasm in conscious sheep. J Appl Physiol 47:917-922, 1979. 128. Heber E: Uber experimentelles asthma und die innervation der bronchialmuskeln. Arch J Physiol 63-l54, l9l4. 129. Hiddicombe JG: Some experimental models of acute asthma. J Roy Coll Phycns ll:l4l-l55, l977. 130. Hiddicombe JG: Respiratory reflexes in man and other mammalian species. Clin Sci 2l:l63-l70, l96l. 131. Hiddicombe JG: Receptors in the trachea and bronchi of the cat. J Physiol (London) l23:7l-104, l954. 132. Hiddicombe JG: The site of pulmonary stretch receptors in cats. J Physiol 125:336-351, l954. 184 133. Hilloughby RA, McDonell HN: Pulmonary function testing in hor- ses. Vet Clin N Amer Large Animal Ed 1(1):l7l-l96, l979. 134. Hoolcock AJ, Macklem PT, Hogg JC, Hilson NJ, Nadel JA, Frank NR, Brain J: Effect of vagal stimulation on central and peripheral air- ways in dogs. J Appl Physiol 26:806-8l3, l969. 135. .Yu ovc, Galant 59, Gold HM: Inhibition of antigen-induced bronchoconstriction by atropine in asthmatic patients. J Appl Physiol 32:823-828, I972. 136. Zeballos RJ, Shturman-Ellstein R, McNally JF, Hirsch JE, Souhrada JP: The role of hyperventilation in exercise induced broncho- constriction. Am Rev Resp Dis llB:877-884, l978.