THE PULMONARY VASCULAR RESPONSES TO UNILATERAL VAGAL 0R SYMPATHEHC STIMULATIONS lN lNTACT‘ DOGS. CATS. AND RABBITS Thesis for the Degree of Ph, D. MICHIGAN [STATE UNEVERSW DAVIUOTTO DeFOUW .1 9.7 2 A.“ LIBRARY Michigan 5:336 UnivcfSitY ‘ BINDING BY ‘E ; “HEAD & SDNS' ABSTRACT THE PULMONARY VASCULAR RESPONSES TO UNILATERAL VAGAL OR SYMPATHETIC STIMULATIONS IN INTACT DOGS, CATS, AND RABBITS By David Otto DeFouw Regulation of the pulmonary circulation by neuro- humoral mechanisms remains questionable. Assessment of remote pulmonary vasomotor control requires examination of pulmonary vascular architecture and innervation. Hyman (1966) reported a well-developed smooth muscle tunica media in pulmonary arteries (30-100u diameter) while com- parably sized veins contained relatively little smooth muscle. Hirsch and Kaiser (1969) described vagal and sympathetic motor nerve fibers forming a terminal reticulum within the smooth muscle vascular media. Daly and Hebb (1966), employing isolated perfused lung preparations, recorded evidence of direct nervous control of pulmonary vascular perfusion. Pulmonary vascu- lar resistance increased after sympathetic stimulation and decreased after vagal activation. In contrast, Krahl (1968) described vagally-induced arteriolar precapillary sphincter constriction. The i§_vivo lung observations also David Otto DeFouw demonstrated pulmonary arteriolar relaxation after sympa- thetic excitation. The present investigation attempted to clarify the nervous component of pulmonary microvascular control. Uni- lateral stimulations of the left sympathetic or parasympa- thetic nerves in closed-chest dogs, cats, and rabbits were performed. Fluctuations in pulmonary vascular pressures were evaluated in conjunction with changes in cardiomotor and/or bronchomotor activity. After general anesthesia, catheters, under fluor- oscopic control, were positioned in the left ventricle and left pulmonary artery. Adaptation of a respirometer and volumetric-pressure transducer to the tracheal cannula enabled measurement of airway volume and pressure changes. After recording responses to unilateral stimulations, a final stimulation was performed with a concomitant intra- venous Pelikan ink injection (volume approximated pulmonary blood volume). To prevent the heart from pumping ink past the pulmonary circuit, carbachol, a parasympathomimetic agent produced immediate cardiac arrest via left intra- ventricular injection. After pressure controlled formalin instillation into the airways, the lungs were removed and the extent of ink perfusion in stimulated (ipsilateral) and control (contralateral) lungs was recorded. In addition, a second group of non-stimulated animals was similarly infused with Pelikan ink. This enabled an additional David Otto DeFouw comparison between ink perfusion patterns in the stimulated and non-stimulated preparations. The lungs from both groups were then fixed under constant inflation pressure (25cm H20) for 24 hours. Serial, histologic sections (lOu) were taken from hilus to periphery of the middle lobes and mounted on 35 mm leader film. Stereologic evaluations (Weibel, 1963) determined the absolute volumes of the alveolar capillaries, larger pulmonary vessels and the conductive airways. Correlations of pulmonary airway and vascular pres- sure changes with the ink perfusion patterns and stereo- logic determinations defined the nervous contribution toward pulmonary vasomotor control. Unilateral vagal stimulation produced bilateral decreases in pulmonary vascular perfusion in response to the vagally-induced de- cline in cardiac activity. This indirect vagal control of pulmonary vascular perfusion was most evident in the cat. Unilateral sympathetic stimulations produced ipsilateral decreases in pulmonary vascular compliance. Sympatheti- cally-induced elevations in cardiac output failed to distend the pulmonary vascular tree while alveolar capillary perfusion tended to increase. Thus, the increased pulmonary inflow appeared to utilize the vascular reserve capacity rather than to passively dilate normally perfused channels. Vascular stiffening was more prominent in the rabbit than either the dog or cat. THE PULMONARY VASCULAR RESPONSES TO UNILATERAL VAGAL OR SYMPATHETIC STIMULATIONS IN INTACT DOGS, CATS, AND RABBITS By David Otto DeFouw A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1972 DEDICATED TO: Linda and Jonathan ii ACKNOWLEDGMENTS The author wishes to express his deep and sincere appreciation to Dr. Robert Echt for his guidance, considera- tion, and friendship throughout this study. Appreciation is also extended to Drs. T. W. Jenkins, C. W. Welsch, and J. M. Dabney for serving on the guidance committee and taking time to read this manuscript. The author wishes to thank Mr. J. Irvin Nichols, Executive Director, and the Board of Trustees of the Michigan Tuberculosis and Respiratory Disease Association for their kind consideration and fellowship support. Special thanks to Mr. Robert C. Paulson whose photographic professionalism aided the author throughout this study. Graditude is also expressed to Drs. G. E. Eyster and U. V. Mostosky for use of laboratory facilities and their expertise assistance. Appreciation is also extended to Dr. F. J. Haddy, Chairman of the Department of Physiology, for use of depart- ment equipment. Thanks are due to Harold Roth for his friendship and assistance. iii A note of special graditude is extended to my wife, Linda, for the long hours spent typing the original manu- script and her continued encouragement and patient under- standing. iv TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . 4 Subgross Pulmonary Anatomy . . . . . . . . . . 4 Architecture of Pulmonary Circulation . . - 6 Innervation of Pulmonary Circulation 9 Parasympathetic Mechanisms in Pulmonary Circulation : 12 Sympathetic Mechanisms in Pulmonary Circulation . . . 21 summary 0 O O O O O O O O O O O O O O O O O O O O O O 31 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 32 Dogs . . . . . . . . . . . . . . 32 Parasympathetic Stimulations . . . . . . . . . . . 35 Sympathetic Stimulations . . . . . . . . . . . 38 Lung Photography and Gross Analysis . . . . . . . 38 Microscopic Analysis . . . . . . . . . . . 39 Stereologic Evaluation . . . . . . . . . . .‘. . . 44 Cats . . . . . . . . . . . . . . 54 Parasympathetic Stimulations . . . . . . . . . . . 55 Sympathetic Stimulations . . . . . . . . . . . . 56 Gross and Microscopic Evaluations . . . . . . . . S7 Rabbits . . . . . . . . . . . . . . . 58 Parasympathetic. Stimulations . . . . . . . . . . . 58 Sympathetic Stimulations . . . . . . . . . . . 59 Gross and Microscopic Evaluations . . . . . . . . 59 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . 60 Dog . . . . . . .~. . . . . . . . . . . . . . . . . . 60 Parasympathetic Studies . . . . . . . . . . . . . 68 Sympathetic Studies . . . . . . . . . . . . . . . 73 Cat . . . . . . . . . . . . . . . .». . . . . . . . . 88 Parasympathetic Studies . . . . . . . . . . . . . 91 Sympathetic Studies . . . . . . . . . . . . . . . 97 Rabbit . . . . . . . . . . . . . . . . 105 Parasympathetic Studies . . -.- . . . . . . . . . 105 Sympathetic Studies . . . . . . . . . . . . . . . 114 GENERAL DISCUSSION OF TECHNIQUE . . . . . . . . . . . . 123 Page SUMMARY AND CONCLUSIONS 126 LITERATURE CITED . 128 APPENDICES A. DOG Individual Volumes and Pressures (Dog) . . 137 Lobar Perfusion Percentages (Dog) . . . . 139 Stereologic Count Totals (Dog) . . . . 140 Airway Volume Fractions in Dog Lungs . . . 141 Large Vessel Volume Fractions in Dog Lungs . 142 Large Vessel Relative Lengths in Dog Lungs . . 143 Ink- Filled Capillary Volume Fractions in Dog Lungs . . . . . . . . . . . . 144 B. CAT Individual Volumes and Pressures (Cat) 145 Lobar Perfusion Percentages (Cat) . . 147 Stereologic Count Totals (Cat) . . . 148 Airway Volume Fractions in Cat Lungs . 149 Large Vessel Volume Fractions in Cat Lungs 150 Large Vessel Relative Lengths in Cat Lungs 151 Ink- Filled Capillary Volume Fractions in Cat Lungs . . . . . . . . 152 C. RABBIT Individual Volumes and Pressures (Rabbit) 153 Lobar Perfusion Percentages (Rabbit) . . . 155 Stereologic Count Totals (Rabbit) . . . . . 156 Airway Volume Fractions in Rabbit Lungs . . . 157 Large Vessel Volume Fractions in Rabbit Lungs . 158 Large Vessel Relative Lengths in Rabbit Lungs . 159 Ink- Filled Capillary Volume Fractions in Rabbits Lungs 160 D. ANALYSIS OF COVARIANCE TABLES FOR DOG, CAT, AND RABBIT Dog Parasympathetic Trials 161 Cat Parasympathetic Trials 162 Rabbit Parasympathetic Trials 164 Dog Sympathetic Trials 166 Cat Sympathetic Trials 167 Rabbit Sympathetic Trials 168 vi Table LIST OF TABLES Effects of autonomic nerve stimulations on pressure and volume parameters in the dog Stereologic evaluations in the dog lung . . . Effects of autonomic nerve stimulations on pressure and volume parameters in the cat Stereologic evaluations in the cat lung . . . Effects of autonomic nerve stimulations on pressure and volume parameters in the rabbit Stereologic evaluations in the rabbit lung . . vii Page . . 89 90 106 . 107 LIST OF FIGURES Figure 1. Lung inflation-fixation apparatus 2. Serial sections of the ink- -perfused middle lobar segments . . . . . . . . . . . 3. The plexiglass film transport device used to mount t e serial histologic sections on plastic leader film . . . . . . . . . . 4. The mounting device, containing the plastic leader film, is shown lowered into the water bath 0 O O O O O O O O O O O O C 5. Plastic dishes used in staining the mounted histologic sections . . . . . . . . . 6. The plexiglass film holding device helped to insure rapid and efficient microscopic evaluation of the serial sections . . . 7. Stereologic test system consisted of a square test area and 42 test points . 8. Complete microscopic test system employed in the stereologic analysis of the serial histologic sections . . . . . . . . . 9. Close-up view of the projection head and stereologic test system. Note the test point "hits" within the lumen of the centrally placed airway . . . . . . . . . . 10. Arbitrary division was made of each section into nine unit areas . . . . . . . . . . 11. Representative dog lungs after unilateral vagal stimulation 12. Average lobar perfusion percentages after left vagal stimulation in the dog . viii Page 43 43 43 47 47 47 47 SO 50 50 65 67 Figure Page 13. Average lobar perfusion percentages after left middle cervical ganglion stimulation in the dog . . . . . . . . . . . . . . . . . . . . . . 67 14. The dog lungs after left middle cervical ganglion stimulation . . . . . . . . . . . . . . 75 15. Histologic section from dog lung after vagal stimulation . . . . . . . . . . . . . . . . . . 77 16. Section from hilar region of left middle lobe after left sympathetic stimulation in the dog . 77 17. Physiologic pressure measurements during control and stimulation periods in the dog . . . 81 18. The cat lungs after left vagal stimulation . . . . 93 19. Average lobar perfusion percentages after left vagal stimulation in the cat . . . . . . . . . . 95 20. Average lobar perfusion percentages after_left stellate ganglion stimulation in the cat . . . . 95 21. Thorough ink perfusion appears in the left and right lungs after left stellate ganglion stimulation in the cat . . . . . . . 99 22. Histologic section from cat lung after vagal stimulation . . . . . . . . . . . . . . . . . . 101 23. This figure depicts a higher magnification of the ink-filled feline pulmonary microcircula- tion . . . . . . . . . . . . . . . . . . . . . . 101 24. Physiologic pressure measurements during control and stimulation periods in the cat . . . 104 25. The rabbit lungs after left vagal stimulation . . 110 26. Average lobar perfusion percentages after left vagal stimulation in the rabbit . . . . . . . . 112 27. Average lobar perfusion percentages after left inferior cervical ganglion stimulation in the rabbit O O O O O O O O O O O O O O O O O O O O O 112 ix Figure Page 28. Even bilateral ink distribution appears after left sympathetic stimulation in the rabbit . . . 116 29. Histologic section from rabbit lung after vagal stimulation . . . . . . . . . . . . . . . 118 30. A muscular pulmonary artery, located in the middle segment of the rabbit's middle lobe, is shown . . . . . . . . . . . . . . .g . . . 118 31. Physiologic pressure measurements during control and stimulation periods in the rabbit . . . . . 121 INTRODUCTION Whether neurohumoral regulation of pulmonary blood vessels exists has long been a controversy.‘ The mechanical hypothesis, an alternative to neurohumoral regulation, rests on two propositions. First, the pulmonary circuit, con— structed as a short pathway between the right and left ventricles, adjusts automatically to changes in cardiac behavior without intervention of a special control system. Secondly, nervous and/or humoral regulation of the pulmon- ary vasculature would serve no useful purpose. Since all parts of the lung function similarly, selective changes in resistance producing regional differences in blood flow would be insignificant. Daly and Hebb (1966a) appreciated the major role of passive or mechanical forces in controlling pulmonary perfusion. While such forces might be sufficient to obScure or overwhelm the active component of control, its physio- logic significance should not be negated. The authors regarded nervous pulmonary vascular control as a modulating influence operating on a changing background of flow and pressure. Results from numerous isolated perfused lung preparations, provided these investigators with evidence of an active pulmonary vasomotor control system. Aviado (1965a) reviewed evidence of autonomic nervous input to the pulmonary circulation. Electrical stimulation of parasympathetic or sympathetic pulmonary nerves and intra- vascular injections of neurohormones demonstrated direct pulmonary vasomotor activity. Failure to detect pulmonary response was also reported. The author hypothesized that nervous actions of bronchial smooth muscle and cardiac out- put complicated a simple pulmonary vasoconstriction or dilation. Most investigations, depicting direct vasomotion as an important determinant of pulmonary blood flow, were obtained by measurements of pressure differences and flow rates across the lung. In addition, Krahl (1968), employing in XEXE observations of rabbit lung, described local control of alveolar capillary perfusion on a lobular basis. Sub- sequent histologic examinations of ink perfused lungs demon- strated parasympathetically induced constriction of arteriolar precapillary sphincters. With these studies in mind, the present investiga- tions attempted to define pulmonary vascular reactivity to the autonomic nervous system. Using closed-chest dogs, cats, and rabbits, evaluations were based upon physiologic pres- sure responses to unilateral sympathetic or parasympathetic nerve stimulations. The lungs, also ink-perfused during the final stimu- lations, were serially sectioned from hilus to periphery. Stereologic principles, defined by Weiber (1963), enabled histologic evaluations of the 10p serial sections. Uni- lateral nerve stimulations enabled the contralateral lung to serve as control during histologic examinations. In conjunction with the preceding results, the following questions were asked: 1. Does the autonomic nervous system evoke active pulmonary vasomotion? 2. Do passive pulmonary responses, induced by broncho- motor and/or cardiomotor actions, dominate an active response? 3. If an active reaction predominates, what specific site(s) is responsible for the activity? 4. Are both passive and active pulmonary vascular responses nonexistent after autonomic nervous stimulation? REVIEW OF LITERATURE Subgross Pulmonary Anatomy Variations in pulmonary structure among the mamma- lian species suggest comparable differences in physiologic behavior. Therefore, specific morphologic characteristics and relationships must be recogniZed in considering control of pulmonary vascular and airway mechanics. Miller (1947) described connective tissue septae extending from pleural tissue into the lung parenchyma and marking the boundaries between adjacent secondary lobules. The primary lobule, consisting of a single alveolar duct and its accompanying alveolar sacs and alveoli, defined the basic unit of lung structure. Fifty or more primary lobules comprised a secondary lobule. Tyler, McLaughlin, and Canada (1961) studied 3mm serial sections of latex-injected lungs from several mam- malian species. Three principle subgross patterns were observed. Type I was found in cow, sheep, and pig. This group displayed well-developed secondary lobules with marked interlobular septae and a thick visceral pleura. The distal airways consisted of terminal bronchioles leading directly into alveolar ducts without intervention of respiratory bronchioles. The pulmonary vein closely followed the course of the bronchus and pulmonary artery from hilum to periphery. The bronchial artery, also paralleling the airways, termi- nated in a common capillary network with the pulmonary artery at the terminal bronchioles' distal end. A number of bronchial-pulmonary arterial anastomoses were found. Type II was found in dog, cat, rabbit, and monkey. Contrary to type I, there was an absence of secondary lobules with haphazard intraparenchymal septae and an extremely thin visceral pleura. The canine visceral pleura, however, was intermediate in thickness relative to types I and II. The type II distal airways included well-developed respiratory bronchioles leading into large alveolar ducts. The bronchovascular tree resembled type I, however, the pulmonary vein maintained an independent course along con— nective tissue septae to the hilum and no bronchial- pulmonary arterial anastomoses were found. Type III was identified in horse and man. Incom- pletely defined secondary lobules and a thick, highly vascular pleura characterized this group. Distal airway design paralleled that of type I. However, occasional, poorly developed respiratory bronchioles intervened between the terminal bronchioles and alveolar ducts. The broncho- vascular tree was similar to type II with the exception of numerous proximally located (hilar region) bronchial- pulmonary arterial shunts. Architecture of Pulmonary Circulation According to Daly and Hebb (1966b), intrapulmonary arterial bronches accompanied the airways distally to the alveoli in mammalian lung. However, Reid (1968), working with formalin-fixed human lungs, reported a striking dif- ference between branching patterns of the bronchial and arterial trees. While each bronchial branch was accompanied by a pulmonary arterial division, termed a conventional arterial branch, the pulmonary artery also generated addi- tional side branches. These supernumerary arterial branches occurred throughout the length of an artery from hilum to the capillaries. Being most numerous at the periphery, the supernumerary also outnumbered the conventional at all pulmonary levels. Ferenz (1969) reported a basic histologic structure of pulmonary vessels similar in all mammals including man. Four vessel types were defined: diameters l. elastic arteries greater than 500p 2. transitional arteries lOO-SOOu 3. muscular arteries 30'1ISU‘ 4. endothelial vessels 15—40u (arterioles) A brief description of histologic design clarified this arterial classification. The large elastic arteries possessed numerous circumferentially arranged layers of elastic tissue, interspersed with collagen and sparse smooth muscle. The transitional vessels possessed a muscular wall containing scattered elastic fibers not arranged in parallel laminae. The muscular arteries consited of a circular layer of smooth muscle bounded by two elastic laminae. The endo- thelial arterioles were thin-walled precapillary vessels of a single elastic lamina supporting an endothelium. Hyman (1966) described muscular pulmonary arteries (lOO-lOOOu diameter) accompanying respiratory bronchioles and alveolar ducts in mammalian lung. Nonmuscular arterioles arose at right angles from these arteries and fed the alveo- lar capillary networks. VonHayek (1960a) reported similar small pulmonary arteries in human lungs. These vessels had an incomplete muscular tunica media with distinct muscular rings surrounding right-angled origins of nonmuscular pre- capillary branches which abruptly divided into alveolar capillaries. Sobin (1966) failed to identify smooth muscular precapillary sphincters. However, the distribut- ing artery (60-100u diameter) was defined by the author as partially muscular in dog, cat, and rabbit. Knisely (1960) and Reeves, Leathers, and Quigley (1965) defined the morphology of the rabbit pulmonary micro- circulation. Small (30-150u) rapidly tapering arteries (termed arterioles by these authors) branched at right angles in contrast to simple dichotomous systemic arteriolar ‘branching. In addition, two types of right-angled branches arose from the arterioles: l) precapillaries (15-20u dia- lneter) supplied alveoli contiguous to the arteriole; and 2) precapillary arterioles (up to lOOu diameter) supplied more distant alveoli. The alveolar capillaries (10-12u diameter) were drained by short right-angled venules which fed into larger pulmonary veins. Krahl (1964) likened this right-angled branching pattern to the teeth of an elastic comb. Stretching the parent artery (comb's base) simply moved the right-angled branches (comb's teeth) further apart. Upon relaxation, the vessels returned to their original position. Similarly, during the respiratory cycle undue traction was not placed on the arteriolar branches during shortening (expiration) or lengthening (inspiration) of the parent vessel. VonHayek (1960b) described the histologic design of postcapillary vessels found in human lung. Union of alveolar capillaries formed amuscular SOu diameter post- capillaries which led into sparsely muscularized pulmonary venules. Postcapillaries approached the venules at right angles and their entrance sites were marked by smooth muscular rings. Draining the venules were larger pulmonary veins which possessed generally thinner and less muscular walls than comparably sized arteries. The presence of smooth muscle fibers within pul- monary veins of mammalian lung was reported by Hyman (1966). In contrast to the pulmonary arterial vessels, veins and venules less than 1000p diameter contained relatively little muscle. The adventitia of large pulmonary veins within one or two cm of the left atrium contained a tube- like extension of cardiac muscle. Hyman also described a second architectural contrast between the pulmonary arterial and venous systems. Whereas arterial branches were inti- mately associated with respiratory tissue and thus subjected to mechanical effects of breathing, the veins in most species coursed through connective tissue interlobular septa distant from the airways. Thus, respiratory movements would have considerably less mechanical effect on pulmonary venules and veins than on arterial vessels. Innervention of Pulmonary Circulation Histologic evidence reviewed by Daly and Hebb (1966c) indicated that efferent fibers belonging to both the parasympathetic and sympathetic divisions may innervate pulmonary blood vessels 30u diameter or larger in mammalian lung. The authors also reported a predominately ipsilateral distribution of the autonomic nerve fibers. Unilateral pneumonectomy produced chromatolysis in cells of the ipsi- lateral middle cervical, stellate, and nodose ganglia in the dog. Additional chromatolysis was observed in the con- tralateral ganglia, however, the relative number of cells affected was minor. In cats, unilateral lung removal had no effect on the contralateral ganglia. 10 Hebb (1969) studied the distribution of choe linesterases and catecholamines in rabbit, dog, and cat lungs as means of identifying intrapulmonary nerve fibers. The following variations among species were reported: Parasym athetics: Rab it: Cholinergic fibers appeared most evident in large and medium sized arteries. Fibers were distributed perimedially in the largest intra- pulmonary arteries, while arteries SOOu or less showed intramedial fibers. The large veins also received perimedially distributed Cholinergic fibers. No vessels less than lOOu appeared innervated. Cat: Cholenergic fiber distribution was similar to the rabbit; however, arterial vessels down to 40u diameter were innervated. Dog: Both large pulmonary arteries and veins demon- strated Cholinergic fibers running perimedially. Arteries down to 30p diameter demonstrated Cholinergic innervation, however, similarly sized veins lacked a nervous input. Sympathetics: Rabbit: Pulmonary arterial branches down to 70p dia- meter were innervated. The highest density of fiber distribution appeared in the smaller arteries. Adrenergic fibers appeared in large and medium sized (greater than 500p) veins. Cat: The arterial innervation pattern was similar to the rabbits' adrenergic design except that vessels 30-40u were more intensely innervated. A profuse pulmonary venous innervation was an outstanding feature of the feline lung. Dog: The adrenergic innervation appeared identical to the Cholinergic fiber distribution. Spencer and Leof (1964) presented histologic evidence (of pulmonary vascular innervation in humans. Intrapulmonary :nerwes accompanying the vascular tree, contained few thick and many thin fibers. In large arteries, fibers terminated 11 within the adventitia or at the media-adventitial interface. The remainder of the arterial tree, extending distally to the arterioles, was-innervated by thin fibers positioned within the tunica media. Perivenous fibers, after supplying the large pulmonary veins, continued distally with a predomie nately thin fiber distribution. The pulmonary tissue was vitally stained with methylene blue. However, the authors did not differentiate between Cholinergic and adrenergic fibers. The most extensive investigation of mammalian lung innervation was performed by Hirsch and Kaiser (1969). Serial, paraffin-embedded sections routinely stained with Bielschowski, followed by Gomori trichrome were observed from normal lung tissue. Intrapulmonic vagal and sympa- thetic distributions were also traced by cervical vagal and thoracic sympathetic neurectomines. Histologic changes of the subsequent Wallerian degeneration demonstrated intrinsic nervous tissue designs. Parasympathetic and sympathetic nerves entered at the ipsilateral hilus and extended in the arterial and air- way adventitial tissue to the alveolar ducts. At this point, terminal branches arose and passed into the inter- alveolar septae. The structural complex of these nerves identified a parasympathetic predominance and their abund- ance implied a large pulmonary vagal supply. Both motor and sensory fibers were identified. The great number of 12 myelinated axons associated with clusters of ganglion cells suggested a predominance of vagal (mostly afferent) fibers. Less numerous sympathetic nerves consisted mainly of un- myelinated (motor) axons. Fascicles of nerve fibers extended from the adventi- tal vagal and sympathetic plexuses to supply both the bronchial and arterial trees. Thick, sensory fibers arose from the periarterial plexuses and terminated as small tight-meshed arborizations or glomerular spindles within the adventitia. Thin, motor (vagal and sympathetic) fibers, which also originated from the main plexuses, formed a terminal reticulum of wide-meshed arborizations within the smooth muscular media. The terminal branches extending into the interalveolar septae, consisted of curved argyro- philic segments composed of a thick, sinuous fiber and one or more thin fibers. Associated with these dense argyro- philic receptors were rich plexuses of tortuous fiber and fibril branches which curved around the alveolar capillaries. Parasympathetic Mechanisms ii Pulmonary Circulation Original investigation of pulmonary vasomotor response to acetylcholine appeared in the 1930's. Sub- sequently, a number of investigators, utilizing various methods, have observed parasympathetic activity within the pulmonary vascular tree. 13 In 1932, VonEuler recorded pulmonary arterial pres- sure during vagal stimulation in the rabbit. The animal, with opened thorax, was sealed under negative pressure within a wooden box. A pump delivered constant inflow perfusion into the cannulated pulmonary artery. With vagal stimulation or direct injection of acetylcholine, pulmonary arterial pressure was elevated. This vasoconstrictor effect was abolished by atropine. Gaddum and Holtz (1933) perfused isolated dog and cat lungs in a nearly deflated condition with heparinized autologous blood under constant inflow. In both species small doses of acetylcholine (l-3ug/kg) produced vasodila- tion followed by constriction with increased dosage (10-20 ug/kg). Initially, at constant inflow, arterial pressure dr0pped (vasodilation). A secondary rise in pres- sure (vasoconstriction) followed increased acetylcholine injection. Both responses were prevented by atropine. Improvements of these isolated perfused lung pre- parations led to the technique described by Daly, Hebb, and Petrovskaia (1941). To insure viability of the pulmonary nervous input, the bronchial circulation was perfused in conjunction with the pulmonary vasculature. Heparinized blood was perfused at constant inflow into the cannulated pulmonary artery. Blood from the pulmonary veins was returned via a cannula from the left atrium to a venous reservoir. A second pump perfused the bronchial circulation. 14 The arterial cannula was inserted into the thoracic aorta from which all vessels had been ligated with exception of those supplying the bronchial tree. Blood from the bron- chial veins was returned to the reservoir from a cannula inserted into the azygos vein. The entire preparation was placed in an air-tight chamber where the lungs were respired by application of rhythmic negative pressure or the lungs were artifically ventilated with positive pressure. Gener- ally, the pulmonary and bronchial circulations were perfused at constant-volume inflow and changes in arterial inflow pressures were recorded. Employing the perfused lung preparation, Daly and Hebb (1952) described atropine-sensitive vasodilator fibers within the cervical vagosympathetic trunk in dogs. Stimula- tion of these fibers decreased pulmonary vascular resistance without affecting ventilation volumes. However, the associa- tion of these vasodepressor fibers with atropine-resistant adrenergic pulmonary vasoconstrictor fibers prevented large vasodepressor responses to vagosympathetic trunk stimulation. Dirken and Heemstra (1948) perfused isolated rabbit lungs with constant inflow pressures before and after unilateral vagotomy. Inflow volume through the ipsilateral lung was unchanged after section of the vagus nerve. In addition, unilateral, hypoxia-induced pulmonary vasocon- striction was unaffected by vagotomy. 15 Suggestion of acetylcholine's pulmonary vasocon- strictor potential was presented by Bohr, Goulet, and Taquini (1961). Helical strips were cut from pulmonary arteries (rabbit and dog) ranging from 200-300u in diameter. The strips, bathed in Kreb's solution, were mounted on a displacement transducer which recorded phasic changes in tension. Acetylcholine (30-100ug) produced arterial smooth muscular contraction. According to Aviado (1965b) and Fishman (1961), it was most difficult to demonstrate effects of vagal stimula- tion or acetylcholine injection on pulmonary blood vessels. Most results demonstrated decreased pulmonary vascular resistance. However, an increased resistance or simply no response had also been revealed. For example, Borst, Berglund, and McGregor-(1957) injected acetylcholine into isolated perfused dog lungs. The vasomotor changes were always of small magnitude and varied as dilation or con- striction. The constrictor response appeared minimal in absence of increased airway pressure or bronchoconstriction. A passive arterial response was suggested. Failure to record consistant vasodilation was attributed to low basal or "control" tone in the pulmonary arteries. Similarly, Harris (1957) and Fritts, Harris, Clauss, and O'Dell (1958) failed to detect a fall in pulmonary arterial pressure following acetylcholine injection (0.5ug/kg) in normal humans. However, patients with mitral stenosis or other 16 hypoxic conditions (well-known pulmonary constrictors) responded with convincing pulmonary vasodilation. Pulmon- ary arterial pressure fell while cardiac output and left atrial pressure (measured as pulmonary wedge pressure) remained unchanged. The authors related pulmonary vascular responsiveness to acetylcholine with initial pulmonary arterial pressure or tone. Alternate pulmonary vascular responses to acetyl- choline have been recorded in isolated, perfused dog lungs. Bell (1961) and Shimomura, Pierson, Krstulovic, and Bell (1962) demonstrated vasoconstriction in wedged pulmonary segments. Perfusion of acetylcholine (0.5-50ug/kg) into a catheter wedged in a small pulmonary artery produced an immediate rise in perfusion pressure prior to change in left atrial pressure or heart rate. Niden, Burrow, and Barclay (1960) reported variable pulmonary arterial pres- sure responses to acetylcholine. Generally, arterial pressure rose slightly but occasionally a fall or diphasic response appeared. Coupled with these pressure variations was a decrease in systemic arterial oxygen saturation. Respiratory depression with occasional apnea closely par- alleled the oxygen content changes. The major effect of acetylcholine appeared to be dimunition of overall ventila- tion. However, persistance of the drug response with «constant ventilation and perfusion indicated either physio- logic shunting (local changes in ventilation-perfusion 17 ratiO) or anatomic shunting via arterio-venous anatomoses. Bean, Mayo, O'Donnell, and Gray (1951) observed decreased pulmonary outflow with constant perfusion pressure (25mmHg) following acetylcholine injection. Increased air eXpulsion from the lungs (bronchoconstriction) with an attendant in- creased total lung blood volume implied retention of blood within the lungs. Therefore, the reduced outflow was attributed to pulmonary arterial dilation coupled with venous constriction. Pulmonary vasomotor responses to acetylcholine in unanesthetized dogs and rabbits were also reported. Rudolph and Scarpelli (1964), following initial anesthesia, placed electromagnetic flowmeters around the main pulmonary trunk and left pulmonary artery in dogs. After the cannula- tions, leads from the catheters were exteriorized in pre- paration of the animals' recovery from anesthesia. Acetylcholine, infused into the main pulmonary trunk did not change total or left pulmonary arterial flow. However, its injection into the left pulmonary artery produced marked decrease in ipsilateral flow without altering total pulmonary flow. Site of this local vasoconstriction was not predicted because pre and postcapillary changes were not differentiated. Lehr, Tuller, Fishman, and Fisher (1963) recorded color patterns produced by India ink injections into the Infihnonary vasculature of unanesthetized rabbits. Under 18 preliminary anesthesia, a catheter was introduced into the right ventricle via the external jugular vein. Following recovery from anesthesia, five ml of India ink immediately followed byzifive to kn ml solution of sodium pentobarbital and potassium chloride, which produced cardiac arrest, were injected into the cannula. The lungs demonstrated a "patchy" appearance, that is, hilar portions were black while the periphery appeared pink or grayish-pink. Urokon, an angiographic tracer, caused hemorrhage or edema depending on its contact duration with capillary endothelium. The ink-Urokon-blood mixture had traversed more rapidly through pink areas (edematous) indicating an increased resistance to flow through the black (hemorrhagic) regions. This "patchy" lung was not prevented by atropine, vagotomy, or vagal stimulation. The authors hypothesized that right ventricular catheterization initiated a reflex culminating in pulmonary vasoconstriction which affected hilar portions more than the periphery. Utilizing India ink injections, Krahl (1968) de- tected a regulatory mechanism which altered pulmonary capillary perfusion on a secondary lobular basis. Under anesthesia, an ink-saline-potassium chloride mixture was injected directly into the right ventricle of the closed- chest rabbit. Lung surface color patterns presented a mosaic of black, grayish-pink, and pink. Subsequent ink injections with simultaneous unilateral vagal stimulation 19 produced a predominately pink lung ipsilaterally while the contralateral lung displayed the mottled pattern of lobular ink distributions. Histologic sections from adjacent black and pink areas of the normal and vagally stimulated lungs were observed. Muscular arteries, which accompanied the respiratory bronchioles, contained similar quantities of ink in both black and pink regions. Ink was prevented from entering pulmonary capillary beds of pink areas by marked contraction of smooth muscular sphincter mechanisms located at origins of short, right-angled supply vessels of alveolar capillaries. In blackened areas this muscular apparatus was relaxed, permitting unobstructed entry of ink into alveolar capillary networks. Thus, vagally- mediated precapillary arteriolar sphincter contriction was demonstrated in rabbits. Demonstration of active pulmonary vascular re- sponses to parasympathetic stimuli demands exclusion of passive effects which may simulate or modify direct actions on the vessels. Fishman (1961) linked discordant results of actelycholine injections with difficulty of distinguish- ing between active and passive vascular responses. Passive responses were attributed to vagally induced cardiomotor and bronchomotor effects. Daly and Hebb (1966d) reported bronchoconstrictor activity of acetylcholine in most mammalian species. Rodbard.(1966) believed pulmonary blood flow was dependent 20 upon mechanisms extrinsic to the vasculature. Thus, flow was intimately asSociated with respiratory function via bronchiolar tone. Administration of acetylcholine caused decreased pulmonary blood flow with concomitant bronchocon- striction. The airway contriction increased alveolar pressure thus compressing alveolar capillaries with con- sequent decrease in vascular conductance. As previously mentioned, Borst, et a1. (1957) suggested similar passive vasoconstriction in response to active bronchoconstriction. Campbell and Haddy (1949) studied effects of vagally mediated bradycardia upon pulmonary vascular pressure in anesthetized dogs. Prompt increase in mean venous pressure accompanied by smaller decreases in mean arterial pressure were observed. With continuous vagal stimulation, pulmonary arterial pressure eventually rose above control levels, while venous pressure remained elevated. Similarly, Johnson, Hamilton, and Katz (1937) recorded lowered systemic and pulmonary arterial pressures following acetylcholine infu- sion into the cannulated pulmonary trunk. Cardiac slowing and subsequent decreased cardiac output passively induced a decreased pulmonary arterial pressure. Therefore, local pulmonary vasomotor activity was discounted. Eliakim, Rosenberg, and Braun (1957) accorded a rise in pulmonary arterial pressure after acetylcholine injection (l-SOOug/kg) to both bradycardia and bronchocon- striction. Working with anesthetized dogs, pulmonary 21 arterial pressure failed to rise until systemic arterial pressure had fallen. In addition, aminophylline (potent bronchodilator) appeared to diminish slightly the increased pulmonary arterial pressure. Sympathetic Mechanisms in Pulmonary Circfilation Numerous isolated perfused lung preparations have established pulmonary vasomotor activity of epinephrine and norepinephrine. Additional evidence has evolved from in viva observations of the pulmonary microcirculation and from interpretations of pulmonary pressure changes observed in intact animal preparations. Daly, Duke, Hebb, and Weatherall (1948) demonstrated pulmonary vasopressor responses in perfused dog lungs to electrical stimulation of the upper thoraic sympathetic outflow from Tl-TS, the stellate and middle cervical ganglia and their branches, and the main thoracic sympathetic chain. Nervous stimulation during constant inflow perfusion pro- duced 10-15 percent elevations in pulmonary arterial pressure. More recently, Daly, Ramsay, and Waaler (1970) attributed the adrenergic vasopressor response to pulmonary venoconstriction. A greater response during reverse perfu- sion of isolated dog lungs suggested more extensive nervous «control of the venous bed. However, abnormal direction of l1lood flow during reverse perfusion, may have initiated 22 turbulence within the blood stream and thus created greater resistance to flow. Eliakim and Aviado (1961) reported comparable increases in total pulmonary and extrapulmonary venous resistances following stimulation of postganglionic fibers from the stellate ganglion in isolated dog lungs. Since constant pulmonary perfusion was maintained, resist- ance changes were directly proportional to pressure gradient changes across the isolated vascular segments. Therefore, the pressure gradient between the pulmonary artery and left atrium and that between the pulmonary vein and left atrium were both elevated. Duke and Stedeford (1960) stimulated efferent fibers from the stellate ganglion in isolated cat lungs and recorded an increased pulmonary vascular resistance. Conversely, stellectomy produced decreased pulmonary resistance. These resistance changes were calculated by subtracting left atrial pressure from pulmonary arterial pressure and divid- ing by the constant inflow volume. Ingram, Szidon, Skalak, and Fishman (1968) employed an isolated lobe preparation to investigate effects of stellate ganglion stimulation in dogs. A pulsatile pump perfused the left caudal lobe at constant inflow while the remainder of the pulmonary circulation was perfused by the right ventricle. Venous effluent from the isolated lobe was collected and measured in a blood reservoir maintained at 37°C. A constant volume pump ventilated the lungs. The 23 ventilatory pump's outflow was submerged under three cm of water to maintain a constant expiratory pressure. To avoid complicating effects of positive pressure inflation or lung volume changes on pulmonary hemodynamics, all measurements were taken while the respiratory pump was st0pped in expira- tion. Mean pulmonary arterial pressure in the isolated lobe remained virtually unchanged during the stimulation. Because stroke volume and rate were held constant by the pump, calculated resistance was unchanged. However, both amplitude and contour of the arterial pressure pulse were altered during the stimulation. This suggested a sympa- thetically induced change in large pulmonary arterial physical properties. Therefore, to evaluate compliance changes within the isolated lobar precapillary bed, pressure- volume relationships were examined by lobar flow loading. Doubling pump rate produced the flow load with associated increases in lobar blood volume and intravascular pressure. Blood volume increases during flow loading coupled with nerve stimulation were consistently less than during the control state. Therefore, a less distensible vascular bed, rather than calculated pulmonary vascular resistance, pre- sented a more meaningful measure of heightened sympathetic activity. In addition, Szidon and Fishman (1971) reported that effects of hypothalamic (preoptic area) stimulation on the isolated lobar circulation were similar to the stellate ganglion stimulations. 24 Hauge,-Lunde, and Waaler (1967) also recorded stiff- ening of capacitance vessels after norepinephrine or epinephrine injection into isolated rabbit lungs. Marked reductions in lung weight regardless of changes in pulmonary vascular resistance rapidly followed infusion of both sub- stances. Localization of capacitance vessels within the pulmonary vascular bed reacting to the catecholamines was not determined. However, the lungs' vascular capacity was markedly reduced with a slight elevation in resistance to blood flow. Therefore, the authors suggested the venous compartment was primarily responsible for decreased pul- monary blood volumes. Similarly, Aarseth, Nicolaysen, and Waaler (1971) recorded lung weight reductions following vagosympathetic nerve stimulations in isolated dog lungs. The dependence of weight reduction on pulmonary outflow pressures (regulated by varying left atrial pressure) indi- cated a predominately venous response. That is, marked weight reduction after nerve stimulation occurred only when left atrial pressure remained abOve one cm of water. In addition, mean pulmonary arterial pressure remained unchanged after stimulation. Thus, reduced vascular com- pliance rather than increased resistance typified the response. McGaff and Leight (1963), using fluorosc0py, cath- eterized the main pulmonary arterial trunk and the left atrium in dogs. Cardiac output and pulmonary blood volume 25 were measured via dye dilution by sampling from the femoral artery and left atrium respectively. Following sympathetic ganglionic blockage, cardiac output and pulmonary arterial and left atrial pressures were markedly decreased while pulmonary blood volume was elevated. Autonomic regulation of pulmonary vascular tone was suggested; however, the site of vascular relaxation after ganglionic blockage was not determined. Bauman and Fletcher (1967) induced pulmonary vaso- constriction with hypoxia as evidenced by increased pulmonary arterial pressure in dogs. Femoral arterial blood was monitored for oxygen saturation and systemic pressure. After sympathetic blockage via segmental epidural anesthesia from T1 to T7, systemic arterial oxygen saturation in hypoxic dogs was increased and pulmon- ary arterial pressure was decreased. Improvement of arterial oxygen saturation was attributed to decreased pulmonary vascular resistance. In_vivg observations of the pulmonary microcircula- tion with the fused quartz rod illuminator (Knisely, 1936) also depicted adrenergic vasomotor activity. After in- cising an intercostal space and parietal pleura (visceral pleura remained intact), the ribs were retracted and a :marginal portion of the lung, permanently distended by positive pressure with 100 percent oxygen, was transillumi- 'nated with light conducted down the quartz rod. The 26 peripheral pulmonary microcirculation was then observed microscopically. Wearn (1934), using the quartz rod illumi- nator, also observed the cats' pulmonary vasculature. Spontaneous opening and closing of alveolar capillary beds was noted. The intermittent flow pattern was completely independent of changes in systemic blood pressure. There- fore, arteriolar contractility was hypothesized as the governing factor. Furthermore, arteriolar tone was altered after epinephrine injection. An arteriOle, that previous to injection was allowing only single erthyrocyte passage, dilated sufficiently to allow passage of eight or more red blood cells abreast. Irwin and Burrage (1958), also employ- ing the quartz rod transilluminator, observed similar intermittent alveolar capillary flow in rabbits. After epinephrine injection, initial pulmonary arteriolar and venular constriction was rapidly replaced by dilation. Wagner and Filley (1965) combined the quartz rod with inser- tion of a thoracic window, consisting of teflon stretched across a lucite frame, at the second intercostal space. After epinephrine injection, erthyrocyte velocity through the dogs' pulmonary microcirculation was visually increased. There was, however, no evident change in vessel lumen dia- meter. The increased flow velocity was attributed to epinephrine's positive inotropic effect in contrast to direct pulmonary vasomotor activity. 27 Hirschman and Boucek (1963) employed pulmonary angiography in dogs to examine pulmonary vasomotion. A pulmonary catheter, under fluorosc0pic control, was advanced into an arterial segment of the caudal lobe. Injection of opacifying dye during early systole enabled maximum visuali- zation of small muscular pulmonary arteries. Angiograms obtained after epinephrine injection (.Olmg) depicted obvious alterations in the precapillary bed. Bands or collars of constriction appeared at bifurcation sites of l-me arteries. Beading occurred along arteries of 0.5-l.5mm size and spiralling developed along visible arteries down to 0.1mm. Patel and Burton (1957) reported gnarling and bands of constriction within pulmonary arte- rial vessels (25-100u diameter) after norepinephrine (2-8mg) injection in rabbits. After thoracotomy and initiation of positive pressure respiration, a needle was inserted into the main pulmonary trunk for vinyl acetate injection at lOOmmHg perfusion pressure. After hardening, plastic casts of the pulmonary arterial tree depicted the pressor response to norepinephrine. Further confirmation of cathecholamines' pulmonary ‘vasoconstrictor potential was presented by Somlyo and Sonflyo (1964). Main pulmonary arteries, removed from anesthetized dogs, were mounted on displacement transducers 'resting in chamber bathes containing 37°C Kreb's solution. lhyth epinephrine (2.5ug) and norepinephrine (5ug), when 28 added to the baths, induced arterial smooth muscular con- traction. Bevan and Nedergaard (1968) observed contractile responses of isolated rabbit pulmonary arterial segments with attached sympathetic nerve supply. Stimulations were applied to the nerve and contractile responses recorded with an isometric strain gauge and pen recorder. Consistent vasoconstriction was displayed. Conversely, Bohr, et al. (1961) observing isolated pulmonary arterial vessels with 200-300u diameter from dogs and rabbits, found no response to epinephrine or norepinephrine. Pulmonary vasomotor effects of intravascularly injected norepinephrine and epinephrine have also been examined in isolated, blood-perfused lung preparations. Daly, Foggie, and Hebb (1940) injected epinephrine (10- ZSug) into the bronchial arterial system of isolated dog lungs. It was assumed the drug would reach the pulmonary vessels only through confluence of the bronchial and pul- monary capillary beds. Therefore, during normal perfusion epinephrine, injected into the bronchial artery, would reach only the pulmonary capillaries, venules, and veins. During reverse perfusion, only the capillaries, arterioles, and arteries would be effected. An apparent constriction of post and precapillary vascular beds was recorded after epinephrine infusion during normal and reverse perfusion respectively. 29 Gilbert, Hinshaw, Kuida, and Visscher (1958) reported increased pulmonary venous pressure (venoconstriction) after epinephrine infusion in isolated dog lungs. Rose, Kot, Cohn, Fries, and Eckert (1961) demonstrated direct pulmonary vasoconstriction with a rise in pulmonary arterial pressure and decrease in pulmonary venous outflow after norepineph- rine infusion (lOug/kg) into isolated dog lungs. Takaski and Ahlquist (1963) recorded identical results with epin- ephrine injection (lOug/kg) into isolated, perfused lobes of dog lungs. Patel, Mallos, and deFreitas (1961) re- ported a 27 percent increase in pulmonary vascular resist- ance after norpinephrine injection (0.5-4.5ug/kg) into isolated dog lungs. Passive bronchomotor effects were eliminated by momentarily halting artifical ventilation at three cmHZO pressure during data collection. Fenyvesi and Kallay (1966) intravenously adminis- tered physiologic doses of norpinephrine (0.20-0.47ug/kg) into the pulmonary circulation of intact dogs. The authors concluded that the sympathetic nervous system played a minor role in direct regulation of the pulmonary vascula- ture. Feely, Lee, and Milor (1963) also observed the actions of norepinephrine and epinephrine in intact dog lungs. Two effects were recorded: 1) increased contrac- tion of pulmonary vessels thus increasing vasomotor tone Oneasured as an increased pulmonary vascular resistance) and 2) increased transmural pressure, resulting from 30 actions on the heart, which tended to distend the pulmonary vessels. Therefore, the end result depended on relative magnitude of each effect. Generally, vascular distention predominated and thus, pulmonary volume rose and resistance fell. Witham and Fleming (1951) catheterized the pulmonary artery of lightly anesthetized human subjects for recording pressure responses to epinephrine injections. Pulmonary arterial pressure increased within eight to ten seconds after the injections, while pulse rate and cardiac output increased immediately. Therefore, passive pulmonary arte- rial response was defined. Fowler (1951) reported increased pulmonary arterial pressure in humans after norepinephrine injection. However, it was attributed to increased cardiac output rather than pulmonary arterial constriction. Bousvaros (1962) observed a rise in pulmonary vascular resistance, calculated from measurements of cardiac output and pressure gradient between the-main artery and tip of a wedged catheter, after norepinephrine (20-40ug) injections in humans. The author noted, however, increased resistance 'was soon obscured by passive pulmonary vascular dilation arising from increased left atrial pressure. Dollery and Glazier (1966) recorded similar increases in pulmonary vascular resistance after norepinephrine (20-40ug) infusion, however, secondary passive responses were not observed. 31 Summary Neurohumoral activity within the pulmonary circula- tion has been viewed with a variety of experimental methods. Direct autonomic nerve stimulations and intravascular neurohormone injections in isolated perfused lungs and intact animal preparations have been reported. After re- viewing the results from these investigations, an obvious controversy concerning nervous control of pulmonary per- fusion was established. Evidence of pulmonary vasodilation after acetylcholine or norepinephrine infusions has been presented. However, equal support has evolved suggesting that these humoral agents elicit a primary pulmonary vaso- constrictor response. Thus, after viewing pulmonary vascular function and its regulation by a nervous input, further assessment of structure-function relationships was required. The present study, coupling physiologic measurements and morphometric evaluations, provides an essential step in further investigation of this problem. MATERIALS AND METHODS Pulmonary vascular responses to unilateral stimula- tion of left vagal or sympathetic nerves were observed in closed-chest dogs, cats, and rabbits. The contralateral lung served as control and afforded immediate comparison to the experimental side. Dog§_ Ten mongrel dogs weighing approximately 6.75-9.0kg, were studied. Five sympathetic and five parasympathetic evaluations were made. Specific technical differences are presented at appropriate places below. The animals were anesthetized with sodium pentoF barbital (32mg/kg) injected intravenously (cephalic vein) and secured in the supine portion. The skin and superficial fascia were incised and the right external jugular vein was exposed. After removing the surrounding fascia, a 100p of umbilical tape was loosely placed around the vessel. A second loop of surgical suture, positioned three to four cm cephalad to the umbilical tape, was tightly secured to occlude venous blood flow. A blunt probe was placed immedi- ately behind the umbilical tape and elevated slightly to position the vein for cannulation. An incision, extending 32 33 through three-quarters of the vessel's circumference, was made. Fine forceps were applied on the incision's edge to facilitate entrance of the catheter. A 50cm, 6F radio- opaque Lehman-Ventriculographic cardiac catheter (U.S. Catheter Co., New York) was inserted into the vessel and secured by gently tightening the umbilical tape. The catheter remained undisturbed until later positioning in the left pulmonary artery. The catheter's free end was connected to a P23AC Statham strain gauge which recorded blood pressures on a Grass ink writing oscillograph (5D polygraph). To insure catheter patency, periodic flushing with heparized saline (64mg/l) was maintained at approxi- mate two minute intervals throughout the procedure. Zero reference level for all pressures was five cm above the surgical table. This approximated the midpoint of the right atrium. The sternohyoid and sternothyroid muscles were separated at the midline to expose the trachea. An umbili- cal tape loop served to isolate and to prepare the trachea for later cannulation. Lateral retraction of the right sternohyoid and sternothyoid muscles exposed the right common carotid artery. Following identical cannulation procedures employed for the vien, the artery was cath- eterized with a 50cm, 5F, radio-opaque Lehman-Ventriculo- graphic cardiac catheter and left undisturbed until later positioning in the left ventricle. 34 After preliminary cannulation procedures, the animals were placed in the left lateral recumbent posi- tion. Fluoroscopy was essential in controlling advancement and location of the catheters. The intravenous catheter was carefully guided into the right ventricle. Its flexi- ble design permitted a 180° bend necessary to reach the pulmonic valve. Slight force advanced the catheter through the valve into the left pulmonary artery. The intra- arterial catheter bent slightly at the aortic valve and then snapped into the left ventricle. Following catheter placement and animal replacement in the supine position, trachetomy enabled intratracheal insertion of a five-sixteenth inch diameter glass cannula. It was secured within the tracheal lumen by tightening the previously placed umbilical tape. The cannula's open end was adapted to a glass T-tube. One outlet was connected to a Grass PTS volumetric-pressure transducer which recorded intratracheal pressures during respiratory movements. The other outlet was connected to a Wright Respirometer (Harris Calorific Co., Ohio) which directly measured tidal volumes during expiration. A one-way valve enabled continuous recording during expiration without measuring accompanying inspiratory volumes. After securing the tracheal cannula and its attachments, subcutaneous electrodes (20 gauge needles) were applied to each forelimb and hindlimb. From these limb leads, standard EKG'S were recorded on the Grass 3S oscillograph. Generally, lead 11 presented the best measuring source of cardiac activity. Prior to isolation of the vagosympathetic trunk and subsequent nerve stimulation, three, one minute control periods were recorded for the following measurements:‘ left pulmonary arterial pressure left ventricular pressure intratracheal pressure minute volume and frequency of respiration After EKG control data had been collected, the left vagosympathetic trunk was isolated and severed mid- cervically. Electrical stimulations of either the sympa- thetic or parasympathetic component during one minute experimental periods were subsequently performed. Parasympathetic Stimulations Specific parasympathetic stimulations required pharmacologic sympathetic blockage. Phenoxybenzamine (Smith, Kline, and French), an alpha-receptor blocker, and propranolol (Ayerst), a beta-receptor blocker, inhi- bited sympathetic response to vagosympathetic stimulation. Doses of 2mg/kg and lmg/kg were given respectively. Each blocking agent, in solution with 40cc saline, was adminis- tered via intravenous drip through the cephalic vein. The I.V. perfusion unit, inserted immediately following anes- thesia, delivered each agent over a 45-50 minute duration. 36 The severed left vagosympathetic trunk's distal stump was positioned around a silver wire stimulating electrode. The electrode was connected to a Grass S-8 stimulator which generated monophasic square wave stimuli with parameters of 2-3 volts/2.5 msec/lO cps. Pressure and volume measurements during three, one minute stimula- tions were recorded. Bradycardia, demonstrated on the EKG tracings, verified the parasympathetic responses. After three stimulations, the glass T-tube was detached from the tracheal cannula and thus eliminated the respirometer and volumetric-pressure transducer. The pul- monary arterial catheter was removed from the external jugular vein and replaced with a short segment of PE 190 polyethylene tubing. The tubing's free end was attached to a syringe containing biologic Pelikan ink (No. Cll-1431A J. Herschel Co., New York). To insure physiologic pH, the ink was buffered with 1.0 N HCl to pH of 7.4. The ink's volume approximated the animal's pulmonary blood volume which represented one-tenth of the total blood volume. The animal's total blood volume (cc) was calculated as eight percent of body weight measured in grams (Schmer, 1967). The stimulating electrode was repositioned and activated. After 15 seconds, the ink syringe was rapidly emptied (approximately 10 seconds) during continuous stimu- lation. After three-quarters of the ink infusion, one cc of 50 percent carbachol solution (Mereck, Sharpe, and Dohme) 37 was injected directly into the left ventricle via the intra- ventricular catheter. Carbachol, a parasympathomimetic drug, caused immediate cardiac arrest upon entering the coronary circulation. This arrest was presumably due to extensive hyperpolarization of cardiac muscle fibers associ- ated with selective permeability increase to potassium (Koelle, 1970a). The prompt cessation of cardiac output prevented the heart from pumping ink past the pulmonary circuit. Thus, ink perfusion patterns within the pulmonary vasculature, coupled with changes in recorded intrapulmon- ary pressures demonstrated parasympathetic effects on the pulmonary vasculature. Immediately following ink injection, a 25cm segment of rubber tubing (five-sixteenth inch external diameter) was connected to the tracheal cannula's free end. The tubing led to the sidearm of a 500ml pyrex aspirator bottle containing approximately 250cc of 20 percent buf- fered formalin. The bottle, elevated 25cm above the trachea, established lung fixation at 25cm H20 pressure which approximated the physiologically inflated state (Heard, 1962). After a 30-60 second period of formalin instilla- tion into the airways, the lungs were carefully removed from the thorax. First, the abdominal cavity was incised. The sternum's xiphoid process was clamped with a hemostat and slightly elevated to allow for an incision through the 38 ventral thoracic wall. The costochondral junction of each rib was severed and the lungs exposed by retraction of the cut edges. The visceral contents of the thoracic cavity were then removed by careful dissection. Sympathetic Stimulations Evoking sympathetic activity from vagosympathetic nerve stimulations necessitated parasympathetic blockage. Therefore, atropine (National Biochemical Corp., Cleveland, Ohio) was delivered intravenously (2mg/kg) directly into the cannulated external jugular vein. To activate pulmonary sympathetic nerves, the left middle cervical ganglion was electrically stimulated during one minute periods and fluctuations in pulmonary pressures and volumes were assessed relative to control valves. The stimulation parameters, intravascular catheterizations, Pelikan ink infusions, and lung fixation were identical to those in the parasympathetic studies. Lung Photography and Gross Analysis After lung removal, the ink's distribution as dis- played by lung surface color patterns, was evaluated. Each lobe was assessed according to percent black (ink perfused) versus percent pink (non-ink perfused). Duplicate blind examfinations were conducted on each specimen to test 39 consistency of this subjective judgement. The lungs were photographed after color evaluations for permanent records of surface perfusion patterns. A Nikon F. camera with a 55mm automicro Nikkor lens was used for all photography. High speed Ektachrome B film (Kodak) was used for color photographs and Panatomic-X film for black and white photo- graphy. Microsc0pic Analysis Gross lung evaluations depicted only ink distribu- tion within the peripheral pulmonary circulation. To augment evaluations of the lungs' surface color patterns, histologic serial sections were prepared from hilus to periphery of experimental and control lungs. After photography, the lungs underwent 24 hour fixation as described by Weiss and Tweeddale (1966). The lungs were placed in a plastic pan containing approximately six liters of 20 percent buffered formalin (Fig. l). The tracheal connula's free end was attached to polyethylene outflow tubing (five-sixteenth inch diameter) extending from a 2000ml pyrex aspirator bottle. The bottle was supported by a ringstand at 30cm above the lungs. A small aquarium pump (Little Giant Pump Co., Oklahoma City, Okla.), placed within the formalin-filled pan, continuously filled the aspirator bottle. A double hose system was employed by threading a 40cm segment of polyetheylene tubing 40 (five-sixteenth inch diameter) through a penrose tubing drain. The plastic tubing, attached to the outflow port of the pump, rose to and entered the top of the bottle. The surrounding penrose drain, used as an overflow system, automatically regulated pressure at 30cm H20. It was securely attached to the neck of the aspirator bottle and was allowed to fall unobstructively back into the formalin- filled pan. For safety purposes, the inflation-fixation procedure was performed within a well-ventilated hood system. After 24 hour inflation-fixation, the lungs were removed from the pump and stored in formalin for at least three days. Following prOper fixation, the right and left middle lobes were excised at the hilus. The tertiary bronchus and artery were located and centered within the hilar surface of the lobar boundaries. With these struc- tures centered, the lobes were trimmed along their borders into approximate two cm squares. In preparation for paraffin impregnation, the lobar tissue was dehydrated with ethyl alcohol and cleared with toluene: l. 70% alcohol --------------- overnight 2. 80% alcohol --------------- one hour under 375mmHg vacuum 3. 95% alcohol --------------- two, one hour changes under vacuum 4. 100% alcohol -------------- three, one hour changes under vacuum 5. toluene ------------------- two, one hour changes under vacuum 41 6. 50% toluene, 50% paraffin--one hour under vacuum 7. paraffin ------------------- three, one hour changes under vacuum 8. embedded with paraffin and kept overnight at 0°C. The paraffin-embedded lobar segments were positioned on a Spencer rotary microtome for cutting. Serial section- ing at lOu thickness produced continuous paraffin ribbons. A paper trough extending from the microtome blade, supported the paraffin strips (Fig. 2). After collecting 25—30 sec- tions, the ribbon was severed several sections from the blade and removed to an adjacent table for subsequent mounting procedures. The serial sectioning was repeated across the lobes from hilus to periphery. To mount the tissue sections, a film transport device, modified after Wilson and Pickett (1970), was employed (Fig. 3). The plexiglass unit supported a kinder- man guide and developing reel (Ehrenreich Photo-optical Industries Inc., Garden City, New York). The transport system was lowered into a seven and one-half inch water bath maintained at 45-50°C. Five feet segments of 35mm P40B Cronar motion picture leader film (E.T. duPont de Nemours and Co., Wilmington, Delaware) were guided under the roller of the plexiglass transporter,through the Kinderman film guide, and secured in the developing reel. The film was continually coated with albumin-glycerol 'before entering the water bath to insure tissue adherence. 42 Figure 1 Figure 2 Lung inflation-fixation Serial sections of the apparatus. The aspirator ink-perfused middle bottle was continuously pump- lobar segments. The filled with formalin. Thus, continuous paraffin the lung airways were steadily ribbons are displayed. perfused with the fixative. Figure 3 The plexiglass film transport device used to mount the serial histologic sections on plastic leader film. 43 Figure l Figure 3 44 The 25-30 section paraffin ribbons were divided into smaller segments and consecutively placed in the water bath (Fig. 4). Each segment was positioned to join the center of the film as it emerged from the water onto the Kinderman guide. The pick-up reel was slowly turned as the paraffin strips were guided onto the film. Consecutive reels were employed until the entire lobe had been mounted. After drying at room temperature for 24 hours, each reel was routinely stained with hematoxylin and eosin. To enable efficient staining of the reels, ordinary round plastic storage containers were used (Fig. 5). Next, the reels were dipped in liquid plastic (Lab-line Instruments, Inc., Melrose Park, Illinois) for two minutes. After allowing excess plastic to drain, the reels were placed for 30-60 seconds in staining dishes containing 20cc xylene which removed plastic from the films' edge and facilitated subsequent stereologic evaluations. Prior to evaluation, the film strips were hung freely from the laboratory ceiling and dried for 24 hours. The film segments were then sequentially spliced together with liquid film cement (Craig Inc., Plainville, Conn.). Stereologic Evaluation The film-mounted histologic sections represented two dimensional presentations of three-dimensional lung compon- ents. Physiologic evaluations of these preparations 45 necessitated application of sterologic principles (Elias, Henning, and Schwartz, 1971). First, to insure film strip stabilization upon the microscope stage, a film holding device (Fig. 6) was modi- fied after Wilson and Pickett (1970). Two strips of etched one-eighth inch plexiglass, glued upon a one-eighth inch glass base, served to guide the film beneath the microscope objectives. The device was taped to the micro- scope stage and thereby followed all horizontal and vertical movements of the stage. Next, a stereologic test system based upon an eye- piece grid, which presented test points within a test area (Weibel, Kistler, and Scherle, 1966) was constructed. The guide lines and points were etched upon a rounded one- sixteenth inch plexiglass plate with a three and one-half inch diameter. The etched grooves were filled with black wax and the excess wiped off (Fig. 7). The square test area contained 21 lines of constant length, 2 (10mm), arranged on seven equidistant and parallel rows, whereby distance between the 42 end-points was Z in every direc- tion. The circular viewing plate was taped on the screen of a three and one-half inch Nikon projection head (cat. no. 76865). A Nikon universal eyepiece adapter (cat. no. 77111) joined the projection head with a vertical monocular photo tube (cat. no. 77745) which contained a high eyepoint 46 .mpcwom owe» me dam mops umou ohmscm a mo woumfim -coo Eopmxm umou owmoaoohopm one u oHSMHm .mucoEwom honoa opwuno may :wmum ou com: one: mHooH o>fipsoom -cou .maofipoom ofimofloumfln wouanoe on“ mcwnwmum :fi com: moAmfiw owpmmfim m oyzmfim .mewpoom Hmfiuom may mo :owumsam>o owmoomohofie uaoflofimmo use pagan exams“ ow womao: oofi>ow mcfivaoa Eaflw mmmHMonHm 039 o oHDMfim .nwhum anm mcwmuoEo on“ cfiom op commuzm hops: map so wocofipflmom connfih :«mmmhmm mo unoEmom whoam opp opoz .npmn pope: may oucfi wopoon czoam ma .Eafim novmoa oflpmmam ogu mnficwmucoo .oow>ow wqfianoE one e ogswfim 47 48 compensating widefield 10x eyepiece (cat. no. 77857) and ocular micrometer. The photo tube was placed on a Nikon S-KB microscope with Koehler illumination and moveable stage.. The microsc0pe was also equipped with 4x, 10x, and 40x objectives (Fig. 8). Easily discernable counts were obtained for subsequent stereologic examinations (Fig. 9). Weibel (1963) defined point counting as the simplest stereologic-method for determining relative volumes of tissue components from histologic sections. Displacement of the microscope's mechanical stage enabled sampling of individual sections. To eliminate bias, a systematic sampling procedure was employed. This was accomplished by subdividing each section into nine units, numbered as in Fig. 10. Each unit was further divided into equal upper and lower halves. Such segmentation avoided overlapping of fields evaluated during the point counting procedures. Pulmonary airways, ranging from tertiary bronchi through terminal bronchioles, were initially counted. The first peripheral section was moved into the upper half of unit area one and focused in the 4x objective. When any portion of a single end-point (42 total end-points) fell partially or wholly within an airway lumen, a "hit" was recorded. Next, pulmonary arteries, arterioles, veins, and venules were recorded. The section, remaining within the upper half of unit area one, was refocused in the 10x 49 Figure 8 Complete microscopic system employed in the stereologic analysis of the serial histologic sections. Figure 9 Figure 10 Arbitrary division was made of each histologic section into nine unit areas . Close-up view of the projection head and stereo- logic test system. Note the test point "hits" within the lumen of the centrally positioned airway. 50 I O 2 S I 3 . I l L Figure 8 Figure 10 Figure 9 51 objective and luminal "hits" were counted. Ingaddition, the length of these pulmonary vessels was recorded. The screen was scanned and any vessel lying within the test area was included. Vessels intersected by the left and upper edges of the squared test area were counted. Those intersected by the right and lower edges were not counted. Finally, the relative volume of ink-perfused capillaries was recorded. The section was refocused in the 40x objec- tive and the number of ink—filled capillary "hits" was counted. The same section was manuvered to the upper halves of unit areas two and three and subsequent counts recorded. After recording counts from the first peripheral section, the film strip was advanced six sections and the counting process repeated. The number of "hits" from the upper halves of unit areas four, five, and six were re- Corded. After advancing another six sections, the upper halves of unit areas seven, eight, and nine were counted. Sampling every sixth section was continued by positioning Wi thin the lower half of unit area one and sequentially evaluating the lower halves of unit areas two and three. After advancing another six sections, the lower halves of unit areas four, five, and six were evaluated. The lower halves of unit areas seven, eight, and nine were finally recorded on the next sixth section. After another six Secztion advancement, the repetitive evaluations were 52 continued by returning to the upper half of unit area one. This sampling of every sixth section with rotating unit areas continued until stimulated and control lobes had been counted. The point counting procedure readily determined relative volumes of the organ's tissue components. Rela- tive volumes were defined as number of test points falling on the respective components (airways, vessels, and capil- laries). For example, a test system, containing a total of PT test points, yielded PK test points counted as falling within component K. The quotient of PK and PT defined the relative volume V of that component within the specimen: Vvk = PK/PT' Therefore, the volume fraction of a component, contained in a unit volume of an organ, equalled the number of test points falling on profiles of that component divided by the total number of test points. Thus, the volume fractions of pulmonary airways, blood and/or ink perfused pulmonary arteries, arterioles, veins, and venules and ink perfused alveolar capillaries were determined. Respective volume fractions were evalu- ated for both experimental and control lobes. In addition, the length of all pre- and postcapillary vessels was evaluated. Counts of vessel profiles were made within each test area observed. Vessel length (L) was defined as two times the average profile counts (P) divided by the 53 area of the test square L = 3%. The length dimensions were recorded as cm/cms. Further evaluation of the volume fractions required stereologic examinations of ink perfused, non-stimulated (control) animals. Following previously described surgical preparation, biologic Pelikan ink (volume approximated pulmonary blood volume) was delivered into the external jugular vein. After carbachol-induced cardiac arrest and lung formalin-fixation, the lungs' surface color patterns were assessed. After serial sectioning the left and right middle lobes from hilus to periphery, the volume fractions of vessels, capillaries, and airways were recorded. Pre- and postcapillary vessel lengths were also determined. Final assessment of the control and stimulated preparations demanded conversion of the volume fractions to absolute values. Therefore, total middle lobe volumes were then measured by water displacement. The middle lobes of the remaining control and stimulated lungs (Tenney and Remmers, 1963, reported equal lung volumes in animals of similar weights) were excised at the hilus and individually placed in a 500ml volumetric flask. The flask was sealed with a two-hole rubber stopper. One inlet contained a one milliteter pipette and the other a hollow glass rod. Poly- ethylene tubing connected the rod to a syringe containing 20 percent formalin. The flask was initially filled with formalin until the fluid reached the 0.5 mark on the S4 pipette. The procedure was then repeated after placing each middle lobe into the flask. Differences between the initial formalin volume and that required with each middle lobe represented the lobar volumes. Mean volumes of the left and right middle lobes from the stimulated and control groups were then determined. Linking the respective volume and length fractions to the lobar volumes yielded absolute volumes and lengths. Cats Ten cats, weighing approximately 3.6-4.0kg were studied. Six parasympathetic and four sympathetic evalua- tions were made. Surgical procedures employed and pressure and volume measurements recorded were similar to those previously described. Therefore, only technical differ- ences are presented below to elucidate specific procedures used. Initial sodium pentobarbital anesthesis (32mg/kg) was administered intraparitoneally rather than intra- venously. However, maintenance of pr0per anesthetic dosage often required secondary injection via 26 gauge needle within the cephalic vein. The right external jugu- lar vein was catheterized with a 50cmm 4F radio-opaque Lehman-ventriculo-graphic cardiac catheter. Smaller vessel diameters relative to the dog and potential vaso- spasm (peculiar to cats during catheterizations) 55 necessitated use of the smaller catheter. Left ventricular cannulation also required the smaller 4F cardiac catheter. To maintain small catheter patency the animals were heparinized immediately following insertion of the intra- venous catheter. The heparin solution (2.5-4.0mg/kg) was directly infused into the venous catheter. In addition, during sympathetic stimulations, atropine (2mg/kg) was delivered intravenously to insure pure sympathetic responses and to eliminate excessive salivation (frequent response to sodium pentobarbital). Tracheal cannulation was performed with a three- sixteenth inch diameter polyethylene catheter. Its free end was adapted to a glass T-tube which presented, in series, the volumetric-pressure transducer and Wright respirometer. Parasympathetic Stimulations As with dogs, the cervical portion of the feline vagus nerve was bound with the sympathetic chain within a common connective tissue sheath. Contrary to the dog, it was feasible to grossly separate the two nerve trunks allowing stimulation of one component independent of the other. Using fine scissors, the common nerve trunk sheath was incised and the fascial connection between the nerves was carefully severed. Vagus nerve identification was 56 readily made for its diameter always exceeded that of the sympathetic chain. Final verification was determined by the bradycardia induced by subsequent nerve stimulation. After isolating a five to six cm segment, the left vagus nerve was severed. The distal stump was positioned around the electrode for subsequent electrical stimulations. During three, one minute left vagal stimulations (2-10v/ 2.5-5msec/10-20cps), pressure and volume measurements were recorded. Subsequent Pelikan ink infusion and lung fixa- tion paralleled previously defined methods. Sympathetic Stimulations According to Daly and Hebb (1972), mammalian sympathetic outflow originated from Tl-T5 ganglia, the stellate ganglion,-and the middle cervical ganglion. Duke and Stedeford (1960) described pulmonary sympathetic fibers after stellate ganglion stimulation in cats. Thus, after grossly isolating the left cervical sympathetic trunk, additional isolation of either the left middle cervical ganglion (sometimes termed posterior cervical ganglion) or stellate ganglion was necessary. Since the former proved inconsistent in size and location, stellate ganglion stimulation was performed. Lying immedi- ately caudal to the first rib, ganglion stimulation in «:losed-chest cats demanded additional dissection. 57 Initially, the sternohyoid and sternothyroid muscles had been midcervically separated along the midline for tracheal and intravascular cannulations. The separation was extended to the sternal manubrium. Surgical retractors, applied to the muscles' medial surfaces, enabled lateral retraction of the cervical muscle mass. In addition, two six and one-half inch straight hemostats, interspaced slightly, were secured along the cranial third of the origins of the left pectoantibrachialis and pectoralis major muscles. After muscular incision between the hemo- stats, the retractor was positioned to include the severed thoracic muscles. Brief electrical stimulation of the isolated left sympathetic nerve trunk always produced dilation of the left pupil. Thus, sympathetic nerve fibers were conclusively identified. Following midcervical sectioning, the left sympathetic chain was traced caudally to its union with the stellate ganglion. The ganglion, lying dorsal to the subclavian artery, was readily identi- fied and subsequent tachycardia following electrical stimulation (2-8v/2.Smsec/18-30cps) verified sympathetic motor activation. Gross and Microscopic Evaluations Surface color patterns, determined by ink perfusion into the pulmonary circulation, were subjectively assessed 58 as previously described. Serial, histologic sectioning from hilus to periphery again enabled stereologic evaluations. Rabbits Eight, white, New Zealand rabbits, weighing approxi- mately 3.0-3.5kg were studied. Five parasympathetic and three sympathetic evaluations were made. Specific varia- tions from previously defined surgical procedures are discussed below. Sodium pentobarbital (32mg/kg) was intravenously delivered via the marginal ear vein. Contrary to the cat, the external jugular vein was catheterized with a 50cm, 5F Lehman-ventriculo-graphic cardiac catheter. Absence of inherent vasospasm enabled use of the slightly larger catheter. The left ventricle was again catheterized with the 4P cardiac catheter, which again required animal heparinization (2.5-4.0mg/kg). Atropine (2mg/kg) was also administered intravenously during sympathetic stimulations. Parasympathetic Stimulations In rabbits, the cervical vagus nerve was independent of the sympathetic chain, lying immediately lateral to the carotid artery. After mid-cervical transsection, the left vagal distal stump was electrically stimulated (2v/2.5msec/ 30cps). Pelikan ink infusion and lung fixation followed the pressure and volume measurements. 59 Sympathetic Stimulations Stimulation of pulmonary sympathetic fibers required activation of the inferior cervical ganglion (rabbits lack stellate ganglia). The left ganglion, positioned immedi- ately caudal to the first rib and dorsal to the subslavian artery, was reached by employing the previously described dissection techniques. Again, subsequent tachycardia and increased left ventricular systolic pressure after electri- cal stimulation (5v/2.5msec/l8cps) verified sympathetic activity. Gross and Microscopic Evaluations Surface color patterns were subjectively assessed and stereologic evaluations were performed as previously described. RESULTS AND DISCUSSION Fluctuations in pulmonary arterial pressures initi- ally dépicted the pulmonary vascular response to unilateral sympathetic or vagal stimulations. Active versus passive vascular reactivity was assessed by concomitant measure- ments of left ventricular pressure and the minute ventila- tion and tracheal pressure responses to the stimulations. That is, the sources of passive pulmonary vascular activity, namely cardiomotor and bronchomotor tone, were monitored. Secondary, intravascular injections of biologic Pelikan ink served to isolate the site(s) within the pulmonary circulation responsible for any changes in pulmonary vascu- lar pressures. Observations of the gross lungs established surface ink perfusion patterns. Stereologic evaluations made from the serial-sectioned middle lobes enabled final correlation between the ink's distributions and pulmonary vascular pressures in accordance with the nerve stimula= tions. For clarification, presentations of these results will be presented according to species. Dog Table 1 presents average values for the pressure and volume parameters in the control, parasympathetic, and 60 61 .nofiumfisswpm :oflamcww Hmow>Hoo oawufla umoH u mouZA ”cowpmHSEwum Hmwm> pmoa u m>g vcm noesHo> Hmwflp u 9> ”zonoscoum zyoumufimmou n m maoflumflfiuao> opscfia u m> “chammoum Hmonomnu u me noumh Hume: u mm ”whammona pmfisoflnuco> umoH u m>q monommoum Hmfihouhm Anacosasm umoa u m on em ma HH Aefie\maueohnv m os.e OH.4 OH.N oo.~ flefia\fiv m> +GH ma OH NH flammabv my +~AH MSH ++mm mm flefie\mpaonv mm +man SH\H4H fimov HH\SNH ++fiNmV m\4a “SSS a\aHH “messy e>q flofiv 4H\m~ «mefiv mH\m~ ++fiofiv OH\NN Enemy NH\mN ammsev mq Hoypcou .mow onu :H whouosmhmm oesfio> was whammona :o mqoflumasaflum o>hoc ofianopsm mo muoommm .H oases 62 sympathetic trials. Each animal served as its own control and stimulation induced changes were assessed for statis- tical significance by the paired t-test (Lewis, 1966). The lungs' surface color patterns, determined by the ink's intravascular distribution are represented in Fig. 11 and 14. Average perfusion percentages for the five vagal and five sympathetic preparations are graphi- cally presented in Figs. 12 and 13. Black, ink-perfused areas and light, non-perfused regions were contrasted. Representative photomicrographs from the serial-sectioned middle lobes are also shown in Figs. 15 and 16. Table 2 presents the stereologically determined absolute length and volumetric data from control and stimulated animals. In addition to the mean values, each lobar evaluation was divided into hilar, middle, and peripheral portions. These approximately equal segments consisted of composite samples from the respective lobar regions. For example, the hilar evaluation represented the mean lobar value obtained when sampling only from the hilar segment. Thus, contrasting these segmentally determined lobar means served to elucidate any differences observed between the grand lobar mean values. The control and experimental mean values were statistically analyzed (Appendix D) by an analysis of covariance (Ostle, 1970) and Stapelton's (1972) procedure for comparing indi- vidual means. 63 .:0fiuhom Huhogmfihom m ”cowupom magmas u z “cowunom HmHH: u I .wonflfiyowas monam> umnoa :woz« .momocunohmm cw mossflo> HanoH kuoe+ om.oH mH.o Na.a 4H.o HG.OH mm.m a mm.m 04.0 mm.» Ho.m Ho.OH mm.o z ulhhhm 11mph» .IIpMEM _||HMhm .Inppww .Inpwwm m uuH4.m oomo.o uu~u.h uumo.m uuAH.m 68mm.a oasfio> SASHHHQSU Ho.H 4m.o mo.H N~.H Nm.N ow.H a ma. mm.H 4m.~ 4H.N Hm. mm.H 2 No.“ A .H Hm.m mm.m ”m.m MH.H m uueA.H uumm.H uuo~.m uuom.~ uu~m.m oomo.H masfio> Hommo> mo.~ma NN.NmH ”5.45H oo.mea Hm.am~ Hm.mNN A mo.AGH mo.m4H m4.mmH o~.omH mm.uq~ om.mmH z IbphpMH .Impwpww .Inwwppw .prwrrw .nwmwwmn .Inpwpww m som.nmfl amA.H4H aem.wwfi smN.emH amm.o4N son.mofi gamma; Hmmmm> ou.o wm.o om.o Hm.o NH.H mu.o A ma.o Ho.o em.o mm.o OH.N mm.H z E E E IE LE IE.“ 3 uumo.H 6645.0 ouch.o uuHA.o boom.H «UUON.H mesfio> smzAfl< hummNU fiuuwfiv fiuummg abommv Anaemmv +m66m~v ugmflm puma pgmflm Smog pamfim Smog :ofiumasawpm cofiumaaawum Abouwfiseflum-:ozv UABEEHSQESm Smog flamm> puma Hosoeoo .m::H wow on“ GM mcofipmsam>o owmoaoohoum .N oflnme 64 Figure 11 Representative dog lungs after unilateral vagal. stimulation. Note the complete bilateral ink distribution into both left and right sides. The figure's right column shows the left and right lateral surfaces while the left column shows the ventral and dorsal surfaces respectively. 65 Figure 11 66 Figure 12 Average lobar perfusion percentages after left vagal stimulation in the dog. percentage of lungs' surface showing ink perfusion (black color) Cr cranial lobe Ca caudal lobe M middle lobe I intermediate lobe Figure 13 Average lobar perfusion percentagesoafter left middle cervical ganglion stimulation in the dog. percentage black As in the vagal preparations, each lobe appears predominately black. 67 CrMCal Cr M Ca left lung right lung Figure 12 I I I I I I I I I I I I I I I I III I I I I I I I I I I I I I I I I I I I I I I . I I I I I I I I I I I I I I I b D D i D IIIIIIIIIIIIIIIIIIIII . . . , . .I.I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I OW I I a I I I I I I Cr MCa I CrMCa right lung left lung Figure 13 68 Individual control and experimental pressure and volume measurements are listed in Appendix A. Perfusion percentages for each of the stimulated and control animals are also presented in Appendix A. In addition, the stereo- logic count totals, which determined the relative length and volumetric fractions for the experimental and control animals are listed in Appendix A. Parasympathetic Studies Mean pulmonary arterial and left ventricular pres- sures decreased significantly after left vagal stimulation (Table 1). As shown (Fig. 17), the pressure deflections‘ occurred concomitantly. This suggested a passive pulmon- ary vascular response to decreased cardiac output induced by the vagally-mediated bradycardia (Table 1). Active pulmonary vasodilation and/or opening of arteriovenous shunts, the alternate explanations of decreased pulmonary arterial pressure, were not supported. Such responses under conditions of constant pulmonary inflow, would gen- erate a secondary increase in return to the left heart causing a rise in left ventricular pressure. However, pulmonary flow was diminished as cardiac output fell, thus, the secondary activity was not expected. The virtually even bilateral ink perfusions (Fig. 11 and 12) also failed to establish unilateral vagally-induced vasodilation. In addition, the stereologic 69 measurements defined comparable volumes in the left and right middle lobes occuppied by the vessels and ink-filled alveolar capillaries (Table 2). Contrasting the control and stimulated animal vascular measurements presented fur- ther evidence of a passive pulmonary vasculature. After vagal stimulation, the total left and right middle lobar volumes (determined by water displacement) maintained the 10cc difference observed in the control animals. Thus, the differences, shown in Table 2, were attributed to this size differential only as evidenced by the statistical insignificance. Rushmer (1961) reported decreased heart rate and atrial contractility with diminishing cardiac output, after vagal stimulation in mammals. During the present'investiga- tion, the ink injection created a proportionately similar venous return in the control and stimulated animals. Thus, the vagally-induced bradycardia suggested a reduced cardiac output relative to the control preparation. Accordingly, the ink-filled capillaries demonstrated a slight bilateral volume decline, especially in the peripheral segments, of the stimulated animals (Table 2 and Appendix A).v However, the decrease was not significant which suggested a small reduction in cardiac output after the stimulation. Nadel, Colebatch, and Olsen (1964) recorded con- striction of terminal bronchioles after vagal stimulation in dogs and cats. Bronchiolar constriction caused increases 70 in airway resistance and alveolar volumes (attributed to greater difficulty in expiration). Respiratory bronchiole and alveolar duct smooth muscle was unaffected by vagal activation. In addition, Karczewski and Widdicombe (1968) recorded only slight decreases in lung compliance after vagal stimulation in rabbits. Thus, vagal efferent fibers allegedly acted on large conduction airways and not the smaller divisions responsible for lung compliance. Staub (1966) defined extra-and intra-alveolar vessels in relating airway mechanics and pulmonary vascu- lar resistance. The larger.extra-alveolar vessels experienced tractional forces from the surrounding lung parenchyma during normal respiratory movements. With lung expansion (inspiration) the vessels were elongated and widened while expiration passively reduced the vessel dimensions. The intra-alveolar vessels (muscular arteries, arterioles, capillaries, and venules), associated directly with the alveolar septal framework, were flattened or "squeezed" during rises in alveolar pressure. In addi- tion, Simon, Linde, Miller, and O"Reilly (1961) recorded rises in pulmonary vascular resistance after increased or decreased lung volumes. During volume increases, intra- alveolar vessel caliber was reduced, while decreased lung volumes tended to collapse the extra-alveolar vessels. Rodbard (1966) suggested an increased resistance to capillary flow during vagal stimulation. Bronchiolar 71 constriction created an increased alveolar pressure which passively compressed the contiguous alveolar capillaries. Staub, however, suggested that alveolar surface tension would reduce capillary compression with increased alveolar pressures. The alveolar walls were defined as flat sur- faces with a sharp curvature at the corners or wall junctions. When alveolar pressure exceeded capillary pressure, capillaries within the flat walls were compressed while those at the wall junctions were protected by surface tension acting across the curved corners. Earlier, Mead, Whittenberger, and Radford (1957) reported surfactant induced fluctuations in alveolar surface tension during the respiratory cycle. Surface tension progressively rose during inspiration as surfactant activity concomintantly declined. At end-inspiration peak surface tension was reached, which coupled with elastic fiber recoil, activated the expiratory phase. During expiration, the surfactant molecules demonstrated increased activity which lead to the minimal surface tension at end-expiration. Thus, surface tension may be triggered to counteract vagally-induced alveolar expansion thereby limiting passive capillary compression. During the present unilateral vagal stimulations, lobar volumes occupied by the conductive airways (tertiary bronchi through terminal bronchioles) were not significantly decreased in the ipsilateral lung (Table 2). Tracheal 72 pressure and minute ventilation also failed to substantially change after stimulation (Table 1). This data, however, may have masked an ipsilateral bronchiolar constriction. An increased downstream resistance would tend to expand volumes of the bronchi and primary bronchioles in the stimulated lung, thereby offsetting the diminished terminal bronchiolar volumes. That is, the stereologically measured volume would be unchanged. Nadel, et al. also reported a shifting of ventilation away from the constricted lung into the contra- lateral side. Increased ventilation would create airway volume expansion to approximate the volume of the ipsi- lateral lung. The unaltered tracheal pressure and minute volumes suggested that a ventilation shift toward the non- stimulated lung would adequately compensate for any active bronchomotor response to vagal stimulation. DuBois (1964) described normal bronchiolar smooth muscle tone in mammalian lung maintained by tonic vagal discharge. Such tone eliminated undue airway expansion which prevented an excessively large anatomic dead space and its attending increase in tidal volume and work of breathing. Therefore, the significant decrease in tidal volume after vagal stimulation (Table 1) suggested a decreased anatomic dead space. A unilateral airway con- striction would account for the decreased dead space, or a possible vagal reflex response may have been initiated by increased flow through the contralateral airways. 73 The alternate hypothesis of essentially unaltered bronchomotor tone after vagal stimulation also merits dis- cussion. The unchanged airway volumes and ventilation parameters coupled with the decreased tidal volume may have suggested moderate ipsilateral bronchiolar contraction. That is, dead space within the stimulated lung was diminished without causing major shifts in air flow resistance or distri- bution. Whatever the airway response was to vagal stimula- tion, it ultimately failed to elicit substantial changes in pulmonary vascular perfusion. That is, minimal vagal control passively exhibited via decreased cardiac output, was recorded in the canine pulmonary vasculature. Sympathetic Studies Left middle cervical ganglion stimulation yielded an increased mean left ventricular pressure while mean left pulmonary arterial pressure was unchanged (Table 1 and Fig. 17). Sarnoff, Gilmore, and Wallace (1965) reported an increased heart rate and force of-ventriCular contrac- tion after sympathetic stimulation. However, pulmonary arterial pressure remained nearly unchanged after threefold increases in cardiac output. In addition, Pace, Keefe, Armour, and Randal (1969) demonstrated two to five fold increases in pulmonary arterial flow after stellate ganglion stimulation in dogs. Pulmonary arterial pressure remained 74 Figure 14 The dog lungs after left middle cervical ganglion stimulation. As shown in the vagal preparations, thorough bilateral ink distribution is demonstrated. The lateral surfaces (figure's right column) and the lung's ventral and dorsal aspects (left column) display the ink's surface perfusion patterns. 75 Figure 14 76 Figure 15 Histologic section from dog lung after vagal stimulation. Note the right angled branching pattern of the arterial and precapillary-sized vessels. The thorough ink perfusion, noted previously on the lung's surface, is depicted in the pulmonary microvasculature. 225x. Figure 16 Section from the hilar region of left middle lobe after left sympathetic stimulation in the dog. A bronchus (lower right corner) is shown branching into the successive bronchiolar tree. Note the- ink-filled pulmonary vessels adjacent to the air- ways. The bronchial vessels, located within the bronchial adventitia also appear ink perfused. 90X. Figure 16 78 unchanged or rose slightly. The authors attributed the unaltered pressure to ample pulmonary vascular reserve capacity and/or vessel compliance. Figures 13 and 14 and Table 2 show complete bi- lateral ink distribution and insignificant differences in vascular length and volumes after unilateral sympathetic stimulation. Again, the consistent 10cc interval separated the left and right lungs. The sympathetically stimulated animals possessed smaller total lobar volumes because their body weights were less, i.e. 16 lbs. versus 20 lbs. mean body weights (Appendix A). If vessel compliance had enabled accommodation of the increased flow during stimulation, greater filling would have been expected in both left and right lungs of the stimulated animals. Comparable venous return, estab- lished by the proportionally similar ink injections in the control and stimulated animals, coupled with sympa- thetically induced increases in heart rate (Table l) and ventricular contractility insured the elevated cardiac output during stimulation. Ingram, et al. (1968) recorded a decrease in pulmonary arterial compliance with an attend- ant increase in pulse wave velocity (pulmonary arterial pressure was unchanged) after sympathetic stimulation in isolated dog lungs. In addition, Aarseth, et a1. (1971) reported decreases in pulmonary blood volume (occurring largely in the venous compartment) after sympathetic 79 stimulation in isolated dog lungs. Active decreases in venous compliance were suggested. Such alterations in the arterial and venous beds would account for similar vascular compartment volumes in the control and stimulated animals. However, the comparable volumes in left and right lungs of the stimulated animals requires further discussion. Normal pulmonary arterial compliance would tend to create slight "pooling" with an increased flow in the right (control) lung. The less compliant, stimulated lung also accepted an increased flow. However, it appeared to rapidly pass across its conductive vascular bed without greatly in- creasing vessel volume. Previously, Franklin, VanCitters, and Rushmer (1962) demonstrated precise phasic balance between outputs of the left and right ventricles. For example, an increase in right ventricular stroke volume increased stroke volume of the subsequent left ventricular beat. In addition, Morkin, Collins, Goldman, and Fishman (1965) reported that the flow pulse generated by the right ventricle was transmitted across the pulmonary circuit in approximately 0.10 seconds and was propelled into the left atrium at the precise moment of rapid ventricular filling. Thus, during sympa- thetic stimulation the ventricular synchrony would be upset without the reported increase in pulse wave trans- mission rate (induced by pulmonary arterial stiffening) across the pulmonary circulation. The stiffening of the 80 Figure 17 Physiologic pressure measurements during control and stimulation periods in the dog. The accompany- ing abbreviations represented the following: MEAN PAP: mean pulmonary arterial pressure LVP: left ventricular pressure TP and RATE: tracheal pressure and respira- tion frequency Upper record depicts changes during left vagal stimulatiOn (darker portion of time line). Note the bradycardia and decreased pulmonary arterial and left ventricular pressures. Tracheal pressure and respiratory frequency were virtually unchanged. Lower record represents the pressure responses to left middle cervical ganglion stimulation. Mean pulmonary arterial pressure remained steady while left ventricular pressure was increased. Again, tracheal pressure and respiratory frequency were unchanged. ,Hw-‘w 81 ' ' 1' 1‘ '1 1'1: . .. ..11. ...... WW“ 1111.11.11 1 Lb 111111W1U1uu1111114111111111111 no rm 2 :5 . nun m 200 m 1111111111111111111111111111111111111111 111111111111111111 1111 1W‘1T1‘1WT11 “11131 "on MY! 111'“ J 1‘1 '11 " ,,,,,,, 111 ' 1 1.“. , ,. 111,111; 11’ 11111 111111111 1/1 '1 1 11 11 1’ 11'1“ * 11 111 11 11 W 111'111111111111111111.1131111 ' ' - no I """ {mat "[1 o Inn up zoo .. 11111111111111111111111111111111111111111111111111111111111111111111111111" 11 1111111111 1111111\\\\\\\\\\\\\\11\ 11111111\111\\\\\111\11\\\1\\\\1 11111111\\\\\\\\\\1\\\‘\\1\\1\\\\1\\\11‘11\\\11 LVP inuun' Figure 17 82 large conductive vessels in the stimulated lung, coupled with the right lung's normal distensibility, would also enable greater filling of the intra-alveolar vessels on the stimu- lated side. That is, the increased pulmonary flow would distend the larger extra-alveolar vessels in the control (right) lung, while the stimulated side's decreased compli- ance would induce greater filling of the smaller intra- alveolar vessels. Thus, the increased flow would "pool" in the larger pulmonary vessels of the control lung while diminished compliance on the stimulated side would prevent such pooling in its extra-alveolar vasculature. Hence, a greater proportion of right ventricular output would traverse the stimulated side with a tendency to increase intra-alveolar vessel perfusion. Further assessment of sympathetically-induced decreases in vascular compliance requires evaluation of pulmonary flow distribtuion during normal and altered right ventricular outputs. West, Glazier, Hughes, and Maloney (1968), employing oxygen labelled gas inhalation methods, demonstrated that blood flow decreased steadily from the base to the apex of the normal upright human lung. This distribution was accounted for by the relative magnitude of pulmonary arterial, venous, and alveolar pressures across the lung. Thus, the lung was divided into apical, middle, and basal zones. In the apical zone, arterial pressure was less than alveolar presSure and there was no 83 flow. That is, the alveolar capillaries collapsed when alveolar pressure exceeded perfusion pressure. Arterial pressure steadily increased down the middle zone as more vascular units were opened coupled with distension of those already perfused. Therefore, the intra-alveolar vessels behaved as Starling resistors, that is, collapsible tubes surrounded by a pressure chamber with flow determined by arterial minus alveolar pressures. In the bottom zone, venous pressure exceeded alveolar pressure and flow was determined by the arterial-venous pressure difference. The increase in flow down the zone was attributed to gravi- tational forces which created a rise in transmural pressure leading to vessel distension and the opening of additional channels. Additional observations of dogs in the supine position demonstrated approximately equal apical and basal flows while flow within the dependent (dorsal) channels exceeded that through the ventral lung aspect. Increases in right ventricular output, e.g. via exercise, without concomitant pulmonary arterial pressure elevations were previously described. West et al. reported virtually equal apical and basal flow distribution during exercise in upright subjects. Permutt, Caldini, Maseri, Palmer, Sasamori, and Zierler (1969) reported that changes in pulmonary blood volume produced by alterations in pres- sure or flow were due to recruitment or derecruitment of parallel units differing only in their critical Opening 84 pressures- Using isolated dog lungs, end-tidal PC02 was measured while left atrial pressure was elevated and pulmon— ary blood flow and alveolar pressure were held constant. As left atrial pressure rose, both pulmonary arterial pres- sure and end-tidal Pcoz were elevated. Thus, alveolar dead space was decreased which suggested recruitment of previously unperfused alveolar capillaries rather than distension of channels already open. The same results were also obtained when left atrial and alveolar pressures were held constant and pulmonary arterial pressure was elevated by increasing flow into the isolated lungs. The authors concluded that the small intra-alveolar vessels were essentially indise tensible and the number of parallel units perfused was determined by inflow pressures. In addition, Engelberg and DuBois (1959) reported greater compliance of the arterial and venous beds than of the capillaries in isolated rabbit lungs. The lungs were suspended in a plethysmograph and passively ventilated with 100 percent N20. A motor-driven pump delivered a constant volume inflow of Kreb-Ringer's solution into the cannulated pulmonary artery. Equilibration of the N20 gas ‘with the perfusate occurred in the capillaries which afforded removal of gas molecules from the lung. Hence, lung volume decreased which in turn decreased pressure within the plethysmograph. Prior calibration of plethysmograph pres- .sure changes with known volume flow rates enabled 85 calculation of capillary flow from the measured decreases in plethysmograph pressure. Thus, pulmonary arterial compliance was determined by measuring flow rates entering and leaving the arterial tree. Pulmonary venous compliance was similarly recorded by retrograde perfusion through the cannulated left atrium. Thus, increased flow into the pulmonary vasculature appeared to distend the arterial and venous beds while the less compliant intra-alveolar vessels prompted perfusion of previously closed capillary networks. Accordingly, Engelberg and DuBois reported that 60-70 percent of in- creased pulmonary blood volume with elevated cardiac output was accommodated in the small intra-alveolar vessels. Contrasting the stereologic capillary compartment measurements in the control and stimulated animals (Table 2 and Appendix A) demonstrated bilateral increases in the volumes occuppied by ink-filled capillaries in the stimu- lated animals. Since the volume increase was not statisti- cally significant, this suggested thorough pulmonary perfusion in the control preparations (ink volumes were proportionally similar in the control and experimental trialS). The lung volumes occupied by the arteries and veins were virtually equal in the control and stimulated animals. Therefore, the ultimate unilateral sympathetic effect appeared to be a stiffening of the larger vessels allowing greater capillary perfusion and more rapid transit across the entire vascular bed. 86 A second alternative, namely entrance of ink into the pulmonary circulation via the bronchial arteries must be considered. Daly and Hebb (1966e) failed to establish ana- tomic evidence of bronchial-pulmonary artery (precapillary) anastomoses in rabbit, dog, and cat. Evidence was presented, however, of capillary communications between the bronchial and pulmonary vascular systems chiefly in the respiratory bronchiole region. The extent of blood flow (maximal bronchial arterial flow represented one to two percent of cardiac output) through these connections remains unknown. However, the speculated direction of flow suggested a bronchial to pulmonary direction in conjunction with normal pressure gradients. Drainage of bronchial flow was accom- plished by three potential channels. Bronchial veins drained the primary arterial divisions, bronchopulmonary veins drained smaller order divisions into the pulmonary veins, and the pulmonary veins also drained bronchial blood that had reached the alveolar capillaries. The relative flow magnitudes within the channels remains unknown. Aviado (1965c) reported a direct relation between aortic pressure and bronchial arterial flow, that is, increased transmural pressure passively reduced arterial resistance allowing an increased flow in isolated dog lungs. Sympathetic stimulation appeared to elicit bronchial venous constriction as evidenced by decreased venous flow after stimulation. However, the author emphasized further 87 evidence was required to establish the extent of bronchial flow and its control. In the present investigation, ink may have entered the pulmonary capillary bed via the bronchial circulation. However, as suggested by Daly and Hebb, the low average bronchial arterial flow would not be expected to signifi- cantly alter pulmonary vascular resistance and hence pulmon- ary perfusion. The rapid cessation of cardiac activity via carbachol administration also suggested a limitation of bronchopulmonary ink flow. Intravascular injections of epinephrine characteristi- cally promoted bronchodilation in mammalian lung (Koelle, 1970b). Konzett and Hebb (1949), however, reported the bronchodilator potential of norepinephrine as one-tenth to one-fifth that of epinephrine. In addition, the broncho- dilator effects were dependent upon a pre-existent state of tonus usually established by prior injections of hista- mine or acetylcholine. According to Koelle (1970b) the neurohumoral transmitter released at the postganglionic sympathetic terminal was norepinephrine. Thus, broncho- dilator response to the present sympathetic stimulations was not anticipated, that is, the atrOpine administration blocked vagal tone which further diminished the slight dila- tor potential of norepinephrine. Table 1 shows essentially unchanged minute ventila- tion and tidal volumes after left middle cervical ganglion 88 stimulation. In addition, lung volumes occupied by the con- ductive airways of the left (stimulated) lung were unaltered relative to the right (control) side (Table 2). However, tracheal pressure substantially increased, and thus, coupled with unchanged minute volumes, suggested an increased work of breathing. The lack of increased tidal volumes reflected a consistent anatomic dead space. Conductive airway volumes in the non-stimulated animals were also virtually equal to those in the stimulated preparations. Thus, tracheal pressure rose without altering airway volumes or flow rates. As discussed above, intra-alveolar vessel volume, namely capillary volume, tended to increase bilaterally after stimulation. The increased tracheal pressure might denote a passive response to decreased transmural pressures within the alveoli. Obviously, tracheal pressure elevation failed to diminish pulmonary vascular perfusion. Therefore, the reverse phenomenon of vascular perfusion affecting tracheal pressure is a possibility. However, alveolar pressure was not monitored, thus, conclusive evidence of the purported passive activity was not obtained. cat Average values of pressure and volume measurements in the control, vagal, and sympathetic preparatiOns are listed in Table 3. A sequential review of the Pelikan ink injections is again presented by Figs. 18-23. Table 4 89 .cofiumHSEHum Hmmm> “wofi n m>q .cofiuwasaflpm :oflamamw oumHHoum n mum Ho..v m++ mo. v a+ .nmoe pocfiehouop AHHmoflconpooHo on» paw whammohn uflfiopmmfiv\oH:mmoum ufifioumxme am am he om naumogn\flev F> mN 0N . wH. AH nefie\m;umopnv m mm.H em.H mw.o om.o fi:«e\fiv me OH m CH HH flonauv as ++oNH «ma +am mNH flawe\moamnv mm +flowv e~\oafi flHaV HN\HNH ++flocv OH\HHH ”may HN\¢mH nmmaev m>q +fio~V oH\¢N nmfiv ~H\o~ ++fivflv OH\HN «mafiv NH\H~ Amazed mq Hagueou .pmo may :H muouoEmamQ oesao> paw whammohm :o mcowumassfium o>hoz ofianousm mo muoommm .m oHan 90 .cofipuom Hanogmwhom n m ”cofiwaom mappfie n z ”nowuhoa mafia: u m .muxop oomv mo. v mm ponwfiuopns ohm mosHm> cwozg .momoaucoumm cw moESHo> Honofi Hmuoe+ o~.~ mw.H mo.H HN.H ww.~ me.~ a eo.~ wa.H ¢S.H m~.H wa.~ BN.N 2 uuo~.~ uuma.fi muona.fi muuHm.H monao.~ muuom.N oesflo> symfififiamu. a¢.o mm.o om.o em.o va.o mm.o a om.o mm.o Nw.o ca.o em.H mm.o z Ilbwhp Ilhhhb IIhMLb. IIHMhb Ilbhhb. _Ilbbhp m uumm.o oomm.o uuom.o uumm.o uumo.o oomA.o oesflo> Hommo> om.»oa mm.qa mm.me av.om om.o~H H¢.¢OH a oo.mm N~.ao mN.om om.ov ma.oo~ 5N.ow z m“.Nm mm._m mH.mn .MH.m_ H_.mm mH.mm z ama.Hm eHm.mo mame.oe mame.m¢ maem.wOH sHH.mw gumeog Hommm> am.o mH.o OH.o ofi.o mm.o efi.o a me.o am.o N~.o Hm.o me.o Nv.o 2 .m.m m_.D “H.D MH.o mn.p mm.o m oomm.o oomm.o uueH.o uuHN.o «uow¢.o ouom.o oszflo> smz~fi< muuoav AUUAU AUUOHV nuumv +fiuoHHV fiuumv ugmfim “was “swam “was “swam puma :ofiumHsawum :ofiumassfium fivoumHDEHpm-nozv uflpmsommasm omen Hamm> “mag Houoeou .mMGSH you on» :fi mcoflHMSHm>o ofimoaoohoum .e ofinns 91 includes the stereologic measurements of absolute pulmonary vascular lengths and volumes. Statistical analysis of the recorded data again involved the paired t-test and an analysis of covariance (Appendix D). The individual physio- logic measurements, perfusion percentages, and stereologic count totals are listed in Appendix B. Parasympathetic Studies Table 3 and Fig. 24 define the characteristic cardiac and pulmonary vascular response to left vagal stimulation. Decreased pulmonary arterial pressure again reflected the passive response to a reduced cardiac output (pulmonary flow). The even bilateral ink distributions (Figs. 18 and 19) also negated active pulmonary vasomotor response to unilateral vagal activation. Stereologic evaluations of the middle lobar vascular compartments also indicated a passive pulmonary vascular bed (Table 4). After vagal stimu- lation, pulmonary arterial and venous lengths in the stimulated animals were less than their counterparts in the control preparations. The control animals' right lung demonstrated significantly greater vessel lengths than either lung of the stimulated animals. In addition, the right control vessel lengths were slightly greater (not signifi- cantly) than those of the left control side. Consequently, vessels of the left control lung were longer, but not 92 Figure 18 The cat lungs after left vagal stimulation. As demonstrated in the dog, complete bilateral ink distribution is depicted on the lungs' lateral surfaces (figure's right column) and its ventral and dorsal aspects (left column). 93 Figure 18 94 Figure 19 Average lobar perfusion percentages after left vagal stimulation in the cat. percentage of lungs' surface showing ink perfusion (black color) Cr cranial lobe Ca caudal lobe M middle lobe I intermediate lobe Figure 20 Average lobar perfusion percentages after left stellate ganglion stimulation in the cat. percentage black As in the vagal preparation, each lobe appears predominately black. I IIIII-Id o. a. o o o I a o I. O o 0 0.0.0.0.... OOOOOOIIo-o.oonuo n IDIDI} .- I 9 D o I. tutu-oo- , loo-IIIIIIOII-IOII11-o‘u Donate-Io. local-IIIl-lo-nilihblE 95 CrMCaI Cr M Ca left lung right lung Figure 19 . D o Ono-Cococo-co ...... OI. 0.0.0.0...tf. 100 P Cr MCal CrMCa ht lung "9 left lung Figure 20 96 significantly, than those of either side in the stimulated preparations“ Thus, an uneven flow distribution was sug- gested in the control animals. That is, greater flow through the right lung tended to lengthen its conductive vessels. The volumes occupied by the arteries and veins were also slightly greater in the right lung. However, both the length and volume measurements in the right side were not significantly greater than those on the left. In addition, the lobar volumes occupied by ink-filled capil- laries were virtually equal in the control animals' left and right sides. Therefore, this suggested tendency toward greater perfusion through the right control lung was not substantiated. The bilaterally equal ink-filled capillary com- partments in the control animals were also significantly greater than their counterparts in the stimulated prepara- tions. Thus, capillary perfusion was markedly diminished through both lungs after unilateral vagal stimulation. Analysis of the hilar, middle, and peripheral lobar portions suggested ink congregation within the hilar capillary com- partments of both lungs. The bilaterally equal capillary compartmental volumes and the attendant hilar ink concentra- tion conclusively demonstrated a passive pulmonary vascular response to the reduced pulmonary flow (cardiac output). Table 3 also depicts insignificant variations in tracheal pressure, minute ventilation, and tidal volumes 97 after the unilateral stimulations. The stereologically measured airway compartments were also bilaterally similar after left vagal activation. The slight decline in tidal volume failed to establish a substantially reduced anatomic dead space after stimulation. Therefore, a bronchoconstric- tor response was minimal or nonexistent. Thus, the possible passive pulmonary vascular response to an altered broncho- motor tone was not observed in the cat. Sympathetic Studies Left stellate ganglion stimulation created increases in mean left ventricular and pulmonary arterial pressures (Table 3). The increased pulmonary arterial pressure appeared to reflect an increased flow rather than increased resistance. That is, the even bilateral ink distribution (Figs. 20 and 21) after unilateral sympathetic activation established essentially equal pulmonary vascular perfusion. Evaluation of the absolute vascular compartmental volumes and lengths (Table 4) and the respective volume fractions (Appendix B) again suggested probable sympathetic moderation of pulmonary vascular compliance. Virtually equal volumes occupied by the vessels and ink-filled capil- laries, found bilaterally in the control and stimulated animals, established the unilateral decrease in vessel compliance after stimulation. As discussed for the dog, the increased pulmonary flow, rather than distending the 98 Figure 21 Thorough ink perfusion into the left and right lungs is shown after left stellate ganglion stimulation in the cat. The dorsal and ventral surfaces (upper half of figure) and the lateral aspects (figure's lower half) display the pre- dominately black colorization. Again, this pattern parallels the vagal preparations. 99 Figure 21 100 Figure 22 Histologic section from cat lung after vagal stimulation. In the center of the figure a longitudinally sectioned pulmonary artery and its right-angled precapillary vessels are shown. Note the thorough ink distribution within the pulmonary vasculature. 225x. Figure 23 This figure depicts a higher magnification of the ink-filled feline pulmonary microcirculation. At the left a pulmonary arteriole feeds its right- angled precapillary vessels which, in turn, perfuse the alveolar capillary network. The extensiveness of this network is shown in the center of the figure. 900x. 7!! 5" r.‘ ,; Figure 22 Figure 23 102 vasculature of the stimulated animals, tended to more rapidly traverse the left lung's pulmonary circuit in accordance with the diminished compliance after left sympathetic activa- tion. Concurrently, less ink was permitted to collect in the normally distensible right lung as the left sides' flow rate accounted for greater proportions of the increased right ventricular output. The normal (control) two to three cm size variation between the left and right middle lobes appeared consistently after sympathetic stimulation. Thus, the volume fractions may furnish additional evidence of vascular stiffening in the left or stimulated lung (Appendix B). Volumes occupied by the large vessels tended to decrease in both lungs of the stimulated animals. Portions of the lobar volumes con- sisting of ink-filled capillaries appeared constant in the left sides of the control and stimulated animals while the capillary component of the right sides diminished slightly in the stimulated preparations. Therefore, sympathetic- activation again tended to shift ink away from the conduc- tion vessels into the exchange vascular compartment of the left (stimulated) lung. However, this tendency toward increased capillary perfusion was not statistically signifi- cant which again suggested thorough ink perfusion in the control preparations. The comparable airway volumes in left and right lungs of the control and stimulated animals (Table 4), coupled 103 Figure 24 Physiologic pressure measurements during‘control and stimulation periods in the cat. The accom- panying abbreviations represented the following: MEAN PAP: mean pulmonary arterial pressure PAP: pulmonary arterial pressure LVP: left ventricular pressure TP and RATE: tracheal pressure and respira- tion frequency Upper record represents changes during left vagal stimulation (darker portion of time line). Note the bradycardia and decreased pulmonary arterial and left ventricular pressures. Tracheal pressure and respiratory frequency were virtually unchanged. Lower record depicts the pressure responses to left stellate ganglion stimulation. Pulmonary arterial pressure was steady while left ventricular pressure rose during the stimulation. The aberrant vascular pressure and EKG tracings represent extra-ventricular systole, resulting from ventricular cannulation. Again, respiratory parameters were unchanged. 104 25 W °[1 MEAN PAP 200 1 ‘1 ‘1 ,1 1 ‘ 1 ...... “11111111111111 7P1"! RATE 1 31W Figure 24 105 with the unaltered ventilation patterns (Table 3) after stimulation, negated changes in bronchomotor tone. Hence, any consequent passive pulmonary vascular response was also eliminated. Rabbit Average values of pressure and volume measurements in the control, vagal, and sympathetic trials are listed in Table 5. Figures 25-30 present a review of the Pelikan ink injections. Table 6 includes the stereologic measurements of absolute pulmonary vascular lengths and volumes. Statis- tical analysis of the recorded data was again performed with the paired t-test and an analysis of covariance (Appendix D). The individual physiologic measurements, perfusion percent- ages, and stereologic count totals are listed in Appendix C. Parasympathetic Studies The characteristic decline in mean left ventricular and pulmonary arterial pressures after vagal stimulation is shown in Table 5 and Fig. 31. Passive pulmonary vascular reactivity to decreased cardiac output was again suggested. That is, the essentially even bilateral ink distribution (Figs. 25 and 26) negated an active pulmonary vasomotor response. In addition, the stereologically determined vascular lengths and volumes (Table 6) indicated cardio- dynamic control of pulmonary perfusion. 106 .cofiumfinewum Hmmm> umoH u m>q .coflumfizsfium cofiawcmm HmUw>noo Howhomcfi u muuH Ho. v a++ mo. v m+ .nmoa confiEuouop AHHmownonuuoHo onu paw enammonm ofifiopmmfiw\ousmmogm ufifioumxme mm om +H¢ ow hapmopn\fiev H> mm Ho co mm mcue\mgomopnv m mm.H mw.~ oe.~ ou.~ Aefie\fiv me w a mH 0H AONmeUV my me OHN ++mmH NGH heme\momonv m: +A¢ov o¢\aw showy Nm\wa +h~mv GH\mm _ flood 5N\mm Amazed a>q fimmv mH\m~ fiNNV HH\©N +fimHU w\NN «flofiv NH\mN Ammeav a4 Hahunou .ufinnmh on“ :« muouoswhmm oESHo> paw whammopm co maoflumassfium o>hoc UHEonopsm mo muuommm .m manmh 107 .:0wpwom Hmaonmwuom u m mqofiuuom ofiwwwe u z ”cofluhom Mafia: n m .mpxop oomv mo. v mm .wocwahowcs mum monaw> cmoze .momogucoumm cw moESHo> Honoa Hmpoe+ ~m.~ mm.~ we.» wH.~ oH.v H~.m a a¢.m an. Na.m am.~ mm.m ma.~ z m_.m mm.“ H_.m mm.“ “m.m Hm.“ m ova~.m uumm.~ muume.m muuee.~ muu~m.m muuom.~ mesfio> shmfiflammo Nm.o N¢.o am.o Ne.o Ho.H va.o a mm.o m¢.o mN.H Na.o oe.H ow.o z .Inmmwp nlnmwp nlhbhh. nlhhnb. 11thh. IImHhH : uumm.o uum¢.o UUHo.H uu~o.o uuaH.H uumm.o mssfio> Hmmmo> em.~a aq.~a m~.Hm Hm.Ha me.w~H wm.mo~ a H~.mm om.mo mm.vm MH.ma Ha.w~H me.mm z 1bhhhh. Imbhmm. Ikhhhba IMMhMb IMNLbhH. _|mmppn z 55¢.mm som.eo maha.em mamm.ma me~¢.m~fl meao.om summon Hommm> m~.o m~.o em.o -.c Hm.o ¢~.o a mo.o mm.o vm.o mm.o m~.o om.o z Ilhbhb IIthp .Ilmwhp .Ilbwwp IIMbLb. Ilhhhb. : comm.o uume.o oomm.o uumm.o oohe.o «ouem.o me=Ho> swzpa< flUUOHV fiuuAV fiuuNHV fiuumv nuuHHU +fluumv gamma “mos “swam puma “swam “you cowumasafium cowumasafium mpoumaseflum-:ozv ufioogomaesm “mag Hmwm> Smog Hoppeoo .m::H panama on“ ca maowuwSHm>o owmoHoohoum .o canny 108 According to Weibel's (1964) stereologic measuree ments in the human lung, the total capillary length is 5.4 x 106 meters. Length measurements of the larger pulmonary vessels have not been recorded in the human nor any other species. Therefore, the present length measurements must be evaluated in respect to Weibel's capillary data. Such comparison yielded an acceptable correlation between pulmon- ary blood vessel length measurements in the human and those in the proportionally smaller rabbit. Vessel lengths of the left and right control lungs were significantly greater than their counterparts in the stimulated preparations. Thus, the bilateral response sug- gested a passive vessel shortening in response to the decreased cardiac output. However, anatomic volumes of the ink-filled capillaries demonstrated only slight bilateral diminution (greatest in the peripheral segments) after vagal activation. The left and right pulmonary arterial and venous compartmental volumes were also slightly reduced after stimulation. As in the capillary compartment, the peripheral portions displayed the greatest volumetric reductions. Therefore, the vagally induced decrease in cardiac output was evident but not sufficient in magnitude to significantly diminish mean lobar vessel or capillary filling. The changes in lung ventilation and airway capacities after stimulation paralleled those described in the canine 109 Figure 25 The rabbit lungs after left vagal stimulation. As shown in the dog and cat, thorough ink perfusion is demonstrated on the surfaces of both left and right lungs. The figure's left column displays the dorsal and ventral surfaces while the lateral lung aspects are shown in the right column. 110 Figure 25 111 Figure 26 Average lobar perfusion percentages after left vagal stimulation in the rabbit. percentages of lungs' surface showing ink perfusion (black color) Cr cranial lobe Ca cadual lobe M middle lobe I intermediate lobe Figure 27 Average lobar perfusion percentages after left inferior cervical ganglion stimulation in the rabbit. percentage black As in the vagal preparations, each lobe appears predominately black. o a o o o n o o o o ago-cocoa o cacao-coo outdooaqoocuouououonnd-"onan-How..0.0.10.5 00.0001... o {dun-Mound”- . o — n"ammuwmh""nun””nunufiHmwuflmhuvwflufibflu”“unwnuuuunuuvvvfifl.u.n.3.u~.u.nuunuununuuuuwn.....u.u.u.n.uuuuun.. ' .‘........."..I..-l.-.. . .H."’".”.”...“..."U".”.".'." ........ . ..........C.UH.“IuO“.".”.".".”'I....I."I.I.I..'.-"'".".”.".". .... 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The virtually unaltered tracheal pressure, minute ventialtion, and airway compart- mental volumes were demonstrated in conjunction with the substantially reduced tidal volumes (Tables 5 and 6). Thus, the degree of bronchoconstriction elicited after vagal activation was again inconsequential in passively creating ipsilateral changes in pulmonary vascular perfusion. In both stimulated and control animals vessel lengths of the right side exceeded those on the left. In addition, the volumes occupied by ink-filled capillaries were substantially less on the left side in both the control and vagally stimulated animals. Thus, as discussed in the cat parasympathetic studies, a greater flow through the right lung appeared to expand its conductive vessels and alveolar capillary compartments. The vessel compartmental volumes, however, failed to display the right to left dif- ferences. Hence, the unequal flow patterns changed vessel lengths without substantially effecting vessel capacity. That is, the increased flow through the right side was accommodated by the small intra-alveolar vessels. The diminished volume of the left side's ink-filled capillary compartment also concurred with Engelberg and DuBois' con- clusion that significant changes in pulmonary blood volume were primarily felt in the small intrapulmonary vessels. The present statistical analysis eliminated existing size differences between the left and right lungs. That is, 114 the greater flow through the right lung was not accounted for by the right side's natural size advantage. The animals were secured in the supine position which also eliminated biased gravitation effects favoring an increased right lung perfusion. Vagal stimulation also failed to alter the right side's predominance. Therefore, physical and mechani- cal effects were not portrayed as precursors to the uneven flow distribution. Thus, the present studies suggested greater perfusion of the right lung in rabbits. Further experimental evidence would be required to establish this as an inherent tendency. Sympathetic Studies Left inferior cervical ganglion stimulation produced an increased mean left ventricular pressure while mean left pulmonary arterial pressure remained virtually unchanged (Table 5 and Fig. 31). That increased cardiac output failed to elevate pulmonary arterial pressure again re- flected ample pulmonary reserve capacity. The even bilat- eral ink distribution (Figs. 27 and 28) after unilateral sympathetic activation established essentially equal per- fusion increases in the left and right lungs. As discussed in the parasympathetic studies, the control animals demonstrated greater vessel lengths and anatomic capillary volumes on the right side. Evaluations of the stereologic measurements (Table 6 and Appendix C) 115 Figure 28 Even bilateral ink distribution appears after left sympathetic stimulation in the rabbit. The figure's upper half displays the dorsal and ventral views while the lower half depicts the lateral sur- faces. This surface perfusion pattern is comparable to the vagal preparations. 116 Figure 28 117 Figure 29 Histologic section from rabbit lung after vagal stimulation. Note the thorough ink-filling on the left and the absence of ink on the right. This microscopic picture is representative of relatively light areas on the lungs' surface. Active precapillary arteriolar constriction is not present as suggested by the staggered micro- vasculature filling. 225x. Figure 30 A muscular pulmonary artery, located in the middle segment of the rabbit's middle lobe, is shown. The relatively thick tunica media is characteristic of the rabbit pulmonary vasculature. The patent, ink-filled lumen and the adjacent ink-perfused microvasculature suggest minimal constrictor acti- vity after sympathetic stimulation. 225x. 9 2 e r u g .1 F Figure 30 119 after sympathetic stimulation, however, further emphasized an even flow diStribution in the stimulated animals. That is, the left and right lung vessel lengths and ink-filled capillary volumes were not significantly different. Thus, the most complete decline in vascular compliance was present. The increased pulmonary flow, rather than distending the vasculature of the stimulated animals and maintaining the right lungs' dominance, appeared to preferentially traverse the left pulmonary circuit. Therefore, less ink was per- mitted to collect in the distensible vasculature of the right lung as the left side's flow accounted for greater proportions of the increased right ventricular output. The increased flow was distributed to the intra-alveolar vascu- lature as evidenced by the virtually equal anatomic capillary volumes on the left and right sides. That is, unilateral vascular stiffening after sympathetic stimulation enabled perfusion of previously closed alveolar capillary networks. Thus, the pattern of greater capillary filling in the right side of the control animals was eliminated in the stimulated preparations. The comparable airway volumes in left and right lungs of the control and stimulated animals (Table 6), coupled with the unaltered ventilation patterns (Table 5) after stimula- tion, negated substantial changes in bronchomotor tone. However, tidal volumes tended to increase which suggested a possible anatomic dead space expansion (bronchiolar dilation) 120 Figure 31 Physiologic pressure measurements during control and stimulation periods in the rabbit. Legend: PAP: pulmonary arterial pressure LVP: left ventricular pressure TP and RATE: tracheal pressure and respira- tion frequency Upper record depicts pressure changes after left vagal stimulation. Note the characteristic pul- monary and left ventricular pressure responses. Tracheal pressure and respiratory frequency were virtually unchanged. Lower record represents the pressure responses to left inferior cervical ganglion stimulation. Again, the characteristic vascular responses are shown as were previously demonstrated in the dog and cat. The respiratory parameters were also unchanged. 121 no mu 1 1 1:1: 11m» zoo up LVP "mam _, 1.91 111WWWWMWWWV1W11W1111WWWM1 1T'GNIRATI . Figure 31 122 after sympathetic activation. The volume increases were not significant and furthermore, substantial alterations in vascular perfusion were not demonstrated in the ipsilateral lung. Thus, a consequent pulmonary vascular response to slight bronchomotor tone alteration was also eliminated. GENERAL DISCUSSION OF TECHNIQUE The majority of previous investigations suggesting active nervous control of the pulmonary circulation were performed with isolated, artifically ventilated and per- fused lungs. The elimination of normal physiologic condi- tions with these procedures was obvious. The present studies, employing intact animal pre— parations, more accurately approximated the natural physio- logic condition. However, aberrations from the normal state were included. First, Sharpless (1970) reported that sodium pentobarbital anesthesia depressed the central and peripheral nervous systems. In addition, respiration rate and systemic blood pressure were slightly diminished. Goldberg, Linde, Gaal, Momma, Takahashi, and Sarna (1968) reported minimal pulmonary circulatory response to pentobarbital in intact dogs. This state of general depression was equally felt by the control and stimulated animals. Thus, owing to the absence of direct barbiturate action on the pulmonary vascu- lar bed, the electric nerve stimulations represented the essential variable in the experimental preparations. The vagal stimulations in the dog also required prior intravenous injections of adrenergic blocking agents. According to Nickerson (1970), phenoxybenzamine, the 123 124 alpha-receptor blocker, caused little change in systemic blood pressure with moderate increases in cardiac output and slight decreases in total peripheral resistance in normal laboratory animals. Minor shifts of blood volume from the pulmonary to systemic vascular bed, due to a greater reduc- tion of tone in the latter, were also reported. However, direct actions on the pulmonary vasculature were not demon- strated. In addition, Nickerson reported reduction in heart rate and mild systemic blood pressure decreases after injec- tions of propranolol, the beta-receptor blocker. The car- diac depression was considered minimal in the resting animal, but more profound under stressful conditions. Again, a direct effect on the pulmonary circulation was not reported. Thus, the combined adrengic receptor blockade failed to estab- lish critical pulmonary vascular reactivity while antago- nizing its sympathetic input during the vagal stimulations. Sympathetic activation in the dog, cat, and rabbit were also performed in conjunction with administration of atropine, an acetylcholine antagonist. According to Innes and Nickerson (1970), atropine inhibited the actions of acetylcholine on structures innervated by postganglionic Cholinergic nerves while transmission at the autonomic gan- glia or skeletal neuromuscular junctions was unaffected. In addition, atropine produced mild heart rate increases without accompanying changes in blood pressure or cardiac output in laboratory animals. Its direct effect on systemic and 125 pulmonary vessels was also minimal. The authors attributed the slight cardiovascular effects to sparse parasympathetic innervatiOn of most vascular beds. Thus, the effects of sympathetic stimulation on the pulmonary vasculature were observed without initial vascular reactivity to atropine. Finally, the intraventricular carbachol injections, 1 which produced an immediate cardiac arrest and thereby 1 enabled ink retention within the pulmonary circulation, required further discussion. Access of this parasympae thomimetic agent to the pulmonary circulation was not con- trolled. That is, a single left ventricular contraction, after carbachol injection, suggested its presence in the pulmonary vascular bed, via the bronchial-pulmonary capil- lary anastomoses. The potential amount and concentration within the pulmonary system appeared extremely small and its possible effects would have arisen bilaterally. Thus, inter- ference with interpretation of the nervous stimulation effects remained minimal. SUMMARY AND CONCLUSIONS The effects of vagal and sympathetic stimulation on the pulmonary circulation were observed in closed-chest dogs, cats, and rabbits. Pulmonary arterial and left ventricular catheterization provided records of the respec- tive stimulation-induced pressure responses. Concomitant measurements of minute ventilation and tracheal pressure were also recorded. Thus, the sources of passive pulmon- ary vascular activity, namely cardiomotor and bronchomotor tone, were monitored. Secondary biologic Pelikan ink injections served to isolate the site(s) within the pulmon- ary vasculature responsible for any changes in pulmonary vascular pressures. Observations of ink perfusion patterns on the gross lung's surface and stereologic evaluations of the serial-sectioned middle lobes enabled final correlation between pulmonary flow distribution and the nerve stimula- tions. Unilateral vagal stimulation produced bilateral decreases in pulmonary vascular perfusion. Pulmonary arte- rial pressure and tidal volumes were also diminished. Thus, the pulmonary vascular bed passively responded to the vagally-induced decline in cardiac output. Because minute ventilation and tracheal pressure were essentially unchanged, 126 127 the moderate bronchoconstriction (tidal volume decreased) contributed little to the passive response. This indirect vagal control of the pulmonary circulation was most evident in the cat. The regulatory mechanism was less profound in the dog and rabbit. The unilateral sympathetic ganglion stimulations produced ipsilateral decreases in pulmonary vascular com- pliance. The sympathetically-induced elevation in cardiac output failed to distend the pulmonary vascular tree while I!) » alveolar capillary perfusion tended to increase. Pulmonary arterial pressure and ventilation patterns were virtually unchanged. 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APPENDICES APPENDIX A (DOG) 137 AH m4 OH OH .m 4H OH 44 m4 .4 m4 4 m4 mH .m wN NN 44 m .N OH 4 m 4H .4 Acmmsuv my 444 QNH 4m Nm .m 444 NmH N4 44 .4 444 mOH co OHH .m N54 omH km ow .N OHN 444 44 44 .H mcfis\mpmonv a: flowv OH\m4H AmaV mH\mMH flm4v OH\ow ammv OH\OOH .m menu OH\omH flukv OH\mmH fi~4v OH\ma Ammu OH\OOH .4 “may OH\mNH ANSV OH\mHH Awkv m\m44 Ammv OH\mkH .m “may OH\444 nmwv o~\om4 OA\OOH moH\m~H .N 544V mH\o~H mm4v OH\ma flo4v m\mk 4mmv m\044 .H mmmeev m>q anV OH\o~ anV OH\4N “44V ~H\mm momv NH\mN .m away OH\ON flmHV OH\4N Away OH\om Ammv wfi\mm .4 m4NV om\mm fi4NV m~\o4 fimHV NH\NN momv NH\4N .m AmHV OH\NN fimfiv OH\mH Amflv OH\4H fimHv w\o~ .N away mH\m~ fl~HU m4\m~ fiNHV m\m4 Away m\oN .H Ammaev mg Hocuzoo Among monummoum paw moESHo> Hmswfl>wqu .:04444:Efipm :04Hmcmm 4464>4mu 646445 pmoa u muuzq .aowuaasswum Hmwm> puma u m>44 138 444 444 444 444 .4 444 444 444 444 .4 44 44 444 444 .4 444 444 444 444 .4 44 444 444 444 .4 44444444444 4> 44 44 4 4 .4 44 44 44 44 .4 44 44 44 44 .4 44 44 44 44 .4 44 44 44 44 .4 4444444444444 4 44.4 44.4 44. 44.4 .4 44.4 44.4 44.4 44.4 .4 44.4 44.4 44.4 44.4 .4 44.4 44.4 44.4 44.4 .4 44.4 44.4 44.4 44.4 .4 4444444 m> 444424 4444444 44>4 4444444 woscfiunou--mmomv monsmmohm paw mmEsHo> Hmswfi>4wnH 139 Lobar Perfusion Percentages (Dogs) (Percent black) Cranial Middle Caudal Intermed. L. R. L. R. L. R. R. Control 1. (20)* 100% 95% 100% 100% 95% 95% 90% 2. (20) 95 95 95 95 100 100 100 LVS 1. (20) 100 100 100 95 100 100 100 2. (20) 90 so 90 30 85 95 50 3. (20) 100 95 100 95 100 100 100 4. (25) 100 100 100 100 100 100 100 5. (15) 100 100 95 95 100 100 100 LMCGS 1. (15) 100 95 100 95 95 100 75 2. (15) 100 95 100 95 100 100 85 5. (20) 100 100 100 100 100 100 100 4. (15) 9o 85 9o 95 80 90 so 5. (15) 100 95 100 100 100 100 100 *Dog body weights (lbs.). Stereologic Count Totals (Dogs) 140 (Middle Lobes) Total Lobar Volumes (Water Displacement) Left Lobe Right Lobe Control 25cc 35cc Parasympathetic l. 25 35 2. 25 35 3. 20 30 Mean 23cc 33cc Sympathetic l. 15 25 2. 20 30 3. 20 30 Mean 18cc 28cc Stereologic Control Vagal Sympathetic C°unt5 L. R. L. R. L. R. Sections Observed 236 217 259 270 221 184 T°tal 652 602 706 739 634 529 Counts Total Possible 27,384 25,284 29,652 31,038 26,628 22,218 "Hits" 141 ammo. 4444. 4444. 4444. 4444. 4444. 44444444 444.44 444.44 444.44 444.44 444 44 444 44 4444:: 44444444 444 444.4 444.4 444 . 444.4 444.4 :4444: 444 444 444 444 444 444 444444 44:44> 44904 :44: 4444. 4444. 4444. 4444. 4444. 4444. 44444444 444.4 444.4 444.4 444.4 444.4 444.4 4444:: 44444444 444 444 444 444 444 444 44444: 444 444 444 444 444 444 444444 :044404 444ogm4444 4444. 4444. 4444. 4444. 4444. 4444. 44444444 444.4 444.4 444.44 444.44 444.4 444.4 44444: 44444444 444 444 444 444 444 444 4444:: 444 444 444 444 444 444 444444 4444444 44444: 4444. 4444. 4444. 4444. 4444. 4444. 44444444 444.4 444 4 444 44 444.4 444 4 444 4 :4444: 44444444 444 444 444 444 444 444 4444:: 444. 444 . 444 444 444 444 444444 4444444 4444: .4 .4 .4 .4 .4 .4 uwuonummexm 4444:4495444444 4044:00 mmnsq mom :4 mqoquMHm oESHo> 443444 142 M444. 4444 4 . 44m.44 444.44 4444. 44 . 4 444. 444.44 44 4 . 444 4 444. 444.44 444 4 . 444 4 4 444.4 444 44 444. 44 444.4 444 44 4444444 OCR. M a :WHH: hm comm 4Nmo. o :4443: 4 . 4444. 44444 444 444 4 44 . 4 444 444.4 44 4 . 44s4m> 44 o 444 44 444.4 444 4 4 4 4442 444 4 444.4 444. 444 444 444. 4 4 ace 4 044044 44 44 4444: 4 444 . 444 444 . = 44444444 comm memo. N 2444:: 4 . 4444. 44444 444 444 4 444. 4444. 4044404 0 4 . 4 4 444 4mm 4 444 444.4 .444 44444444 ON . vON WW5 : .m: GHQHmmOQ 4444 4444. 4 44444: 4 4 . 4444. 44444 444 444 4 444.4 4444. 44444 4 444 444 444. 4 444.4 4444. 44 . . 44 44444: 444 44 4 444 444 4 44 4 444 444.4 444 4 444 44444444 NHN mow : .3: mHflflmmom .m HHN :mHHI: 04 .4 .m 444500 .44444444m 0444 .4 .4 4044404 44444 . 44448444444 .4 4044400 mmcz4 mo 4 a 4 440440444 05:40> Ho mmo> mw44 4 143 nqv oesfio)\num:mq« ewN.N mmahm omm.m onu.~ washm mno.m moawmopm ado: VHN.H ”Ho N «mo.fi mea.H wwo H m¢u.H muasou mflfimogm mmm vmo amp con Noe mmo mucsou monaa> umnoq cam: oo~.~ mmv.m ovH.~ mom.~ ovu.~ Hmo.m moflfimoum mam: can mos mmv mvm Nmm was muasoo mafimoum owH mom cum #am owH mHN muasou newphom Hmumamflumm oae.~ mm~.m vum.~ oeo.m «HB.N mwm.~ moHHmoum :wmz mmv one qu own «mm cum mammou oHfimoum oma OHN cum 5mm vow NNN mussou :owupom mewflz oo~.~ mom.m umm.~ NHH.m VHB.N ~wn.a moHHMOhm 2mm: mmm omo mom woo Hmm ohm mpasou oHfimonm moa “Hm new mmH NHN HHN munsoo nowuhom thHm 0“ .4 Ox DA 0“ CA uwuosuwmsxm uwqumeexmmpwm aopucou mans; moo :H «msumcmq m>fiumfiom Hammo> owumq 144 mmmm. NNN.N ovv.5N mnmm. mNm wNo.o wmm.wN NOMN. vmo Nmo.N m MNN.NN MMNN. u . m o. comm N mam MN NNON. mNN.N mmqm. ooN NNN.mN N omH N vacaw BHONO NON. NQH. mceN. Noe vwm.uN NON m Nm¢.m Nom.m HwN. mOON. Nmo :mua QOH hoe 0N N wwm. .m: oH.puwpm comm» HIII N wmm.m wwom. ammom 0 ¢ 0 H V. a. fl 0mm.N onm N vmm.w omum. mous> munsmm H no .w mam omH N mmH. Hmno 0H cm. v m a: N NH 5 a : Hem. MVN. HN :mHH OH N N v .m. .uumu mmN. ouN Nv.N . wand m mac. ONO. N mowN. wmmom mwo.h mme. th m Awomaw GO m me m N ¢H . .mm . mom . Nuuo “:50: ca H m V N N m 0 man. HHmH. VON va.m Hmhoaa NH N oON. Nwm. Mum N H OH Hm N N m a a ma. mN. NN : pwm own .m H omH. : m 059 . «N m N HnNm m a mmm. omH. . mom UH . mmH H Wmm.w No mmuwm o“ 0 HM .— mflummfikm4 NMW.H Newmw GOfiwhom 500 .m on . me N u Ham N mp no v.2 H o .- .uonummsx A Nm: mHNuumum may nfimmo mm .m :WHH m muzmm: . :o o How A way 9:00 on pmHN: macs; mo . D m ”E: H m . U H C M H 145 OH OH .O N O OH OH .O NH OH NH OH .O O O O OH .N OH O O O .H HONmsuO OO OOH ONH .O OOH OOH OO NN .O OOH OOH OO ONH .O OOH OOH OO ONH .N ONH OOH ONH OOH .H HOHO\OOOOOO Om . . OH\ONH ON\OOH .O HOOO NO\OOH OO\OOH O\OHH OH\ONH .O HOOO ON\OOH OH\OO OH\OOH OH\OOH .O HHHHOOO\OOH ON\OOH OH\NOH ON\OOH .N HNOO OH\OOH OH\OHH OHNOO OO\OO .H HOmeeO O>H ‘ NHNON NH\ON .O HONO OH\ON NH\ON O\OH OH\ON .O HNHO NH\OH OHNOH OH\ON NHNON .O HNNO OH\ON OH\NN OH\ON NH\ON .N HNNO OHNON NH\OH ONON .OHNNN .H HOmseO OH HOOHOOO annoy monsmmmnm wqw Oma=Ho> Hmswfi>wvcH 146 .:0HumH:EHpm :oHszmm oumHHopm umoH u mmmq .coHumHserm Hamm> ummH u m>qg HN NO .m on NO Nm mm .O OO OO NO OO .O OO OO NO NO .N NN NO Nm Ow .H HOOOOOO\HEO O> OH NH .m OO Nv HN OH .v ON NN HN ON .m ON NN mH OH .N OH OH OH NH .H HOHE\OOOOOOOO O mm. ON. .m ON.H mN.H OO. OO. .O OO.N OO.H mN.H mO. .m OH.H OO.H OO. mm. .N OO.H OO.H Om. mm. .H HOHONHO OO «mmmq Hoppsou «m>H Houpaou wosaHuqou--HumoV mowsmmo9m Onm mossHo> HOSOH>HOOH Lobar Perfusion Percentages (Cats) (Percent black) 147 'Cranial ‘Middle Caudal Intermed. L. R. L. R. L. R. R.- Centto1 . 1. (8)* 95% 100% 95% 95% 90% 100% 90% 2. (8) 80 100 95 95 95 95 90 LVS 1. (9) 100 100 75 75 100 100 100 2. (8) 100 100 100 100 100 100 100 3. (8) 100 100 100 100 100 100 100 4. (9) 100 100 100 100 100 100 100 5. (9) 95 95 100 95 90 75 70 L868 1. (8) 100 100 95 95 100 95 85 2. (8) 9O 95 90 85 95 95 95 3. (9) 100 95 95 95 95 100 95 4. (8) 95 95 85 95 90 95 95 *Cat body weights (lbs.). Stereologic Count Totals (Cats) 148 (Middle Lobes) Total Lobar Volumes (Water Displacement) Left Lobe Right Lobe Control 9cc 11cc Parasympathetic 1. 11 2. 10 3. 10 Mean 8cc 10cc Sympathetic 1. 10 2. 10 3. 10 Mean 7cc 10cc Stereologic Control Vagal Sympathetic C°unts L. R. L. R. L. R. seCti°ns 159 195 163 150 159 Observed Total Counts 439 555 388 435 456 Total Possible 18,438 23,310 16,296 21,084 18,270 19,152 ”Hits" 149 Nva. Hmeo. NOH.OH OOH . OOO.H ONN.OH OOOOH NONO. H . OOO ONO N OON.OH OOO OO . OOO NOO ON OH0.0N OO NOO O . OOO.O :oHooO ONO H H O OO OOO OOO OON : OHO: OHOHOOOO HNOO. OOO :OOHO: OOO.O MWWOO OOHO. mo OOOOOO . S H OOH OO NO O NHO.N OOHO. OOH O ONN OOO O OOH OOHOUOOO HH OQH Hm O. .3: QHDflmmom ONOO. OOOO OOH :OOHO: ON0.0 . OHN . OOOOOO wwo. o . GOH mm 5 . :3 mm. :2. O . 32%: OOH OOH OOO.N NOO OOH OOH NON : OOO: OHOHOOOO NOOO. NOH =OOHO= o No . do mm .5 ...... a”... a... . SE H NOH HNH OO O NHO.N HOOO OOH OHO OHN O O OOHHUOOO mNH OMH HOW 2 “ME: OHQHWWOQ o wVH :mpfim-O a .H mpzsou uHOOOOOOsNO O .H . OOHOOOO OOHH: oHuonpmnskmmpmm m .H Hopuaou manna umu :H maoHuumum oesHo> Na SHH< 150 mNmO. NmH.mH emmo. OOH.H ONN.OH NOOO. OOO mHO.H OOO.HN mmOO. OOO OOH.H OON.OH NOOO. O . OH . HOO . NOO OO H O.ON O O wmm mmo N we mH :oHpu , OOOO. OOO OOO. :OuHm . Ohm H O. QHDHW oom.O NNOO. One . mom OON OOO.O HOOO. =OOHO= OOH OON OOO.O OHOO. .IIII. Opesoo . NN . mo: OOH OOH OOO O OO HO> OOOOH OOH NON WHO.N mmwm. cam: OH NO O omho. H ow mmm :muH cowHUMHm H .3: QHQH NHm OO0.0 OHmO. :mpHm: OOH OOO OOO.N OOOO. co Opqsou VVH mNO wvoao VHmH. “whom HwHO Q OOH ONO “OO.N “MOW. O HOOO QVH HOOH H O G OOOO. OOH OOO :OOHO: OOHOUOOO ONO.O OOOO. NOH HOHOOOO NOO ONH.O OOOO. =OOHO= OOH OOO OOO.O ONOO. Opcsou NVH NNM wHVOm HNho. nowuhom 0 mOH mOm NHO.N mmmm. HOOO: ONH mOm OHN O :oHuum .m omH Nmm :muwI: mHmH hm . OO .OOOO A H Uflum . :WHME: . cummfikm m .4 manneu . fl UfipmgmeEXWNHmm m .4 OHHHOQ HNHH: HOHHGOU mmasq uwu :H . mcoHpomum oESHo> Hemmo> om RNA 151 mOO OEOHOO\OOOOOOO OHNMO HOOMO OHOMN OOO.N OO0.0 OON.O OHOmopm amoz OOO H HON H OON H OOO OOH.N ONO.H “zoom oHOmoum OOO OOO NOO OOO OOO OOO mucsou mosz> Hm404 cam: OOH.O NH0.0 OOO.N OOO.N OO0.0 HO0.0 mmHHmon saw: ONO HNO OOO OON OOO OOO mucnou QHHmopm OOH OOH OOH OHH OOH OOH munsou :oHuhom Hmpoggfihom NO0.0 OO0.0 ONN.N OOO.N OHN.O NH0.0 mmHHmonm :woz NOO OOO NOO OOO HOO HNO mucsoo oHOwoum OOH OOH OOH OOH OOH NOH muasou :oHpnom OHOOHZ OO0.0 HON.O OOO.N OOO.N OO0.0 NO0.0 moHOmonm :moz OHO OOO OOO HNO OOO OOO mpcsoo oHOmonm OOH NOH OOH ONH OOH OOH maqsou nofluuom OOHOm .m .4 .O .4 .m .4 uwuoaummexm oOuoaummsxmmumm Houpnoo mmcs4 pmu :O «mgumao4 o>OuwHom Hommo> omum4 152 HOHN. NOON. OONH. ONOH. ONON. OOON. OOHOUOOO NOH.OH ONN.OH OOO.HN OON.OH OHO.ON OOO.OH :OOHO: OHOOOOOO OON.O OOO.O NON.O OOO.N HOO.O OHN.O :OOOO: OOO OOO NOO OOO OOO OOO OOOOOO mosHm> Omno4 cmoz OONN. ONOO. OOOH. OOOH. OHOW. OHNN. OOOOUOOO OOO O OOO O OOO O OOO.O NHO N OOO.O :OOOO: OHOOOOOO ONO.H OOO.H OOO.H NNN OOO.N OOO.H :OOOO: OOH OOH OOH OHH OOH OOH Opssou :oHpuom HmuogmHhom NOON. OOON. HOOO. OOOH. NOON. NNON. OOOOUOOO ONO.O OOO.O OOO N OO0.0 OOO N ONH.O zmuHO: OHOHOOOO OOO.H OOO.H NON.H HOO OHO.H OOO.H :OOOO= OOH OOH OOH OOH OOH NOH OOOOOO OOOOOOO OHOOO: OONN. ONON. OOOH. OHOH. ONHN. OHNN. OOOOUOOO ON0.0 ONH.O OOO.O OHO.O NHO.N OHN.O :OOOO: OHOHOOOO OOO.H NNO.H OOO.H OOO OOO.H OOO.H :OOOO: OOH NOH OOH ONH OOH OOH Opasou :oOOOOO OOHHO .O .O .O .O .O .O owuocpmmENm UHuo4ummENmmumm Hopucou mm::4 pmu :H macauownm oESHo> OHOHHOQOU OoHHHm-4:H APPENDIX c (RABBIT) 153 OH ON .O OH NN .O O O OH O .O N O NH NH .N O OH OH HH .H HONOEUO OH OO OHN .O OOH OON .O OON OHN OOH OOH .O OON OON OHH OOH .N OON ONN OOH OOH .H HOHENOOOOOO Om HOOO ONON HOOO OHNOO .O HOOO OHNONH HNOO OONOOH .O HHNO OONNO HNOO OONOO HONO OONONH HNNO ONNONH .O HOOO ONNOO HOOO ONNON HNOO OHNOO HOOO ONNON .N HOOO NONOO HNOO ONNOO HOOO NONON HOOO OONOO .H HOmasO O>O HOHO ONNH HOHO ONON .O HNHO ONON HNHO ONON .O HOOO ONNNO HNNO OHNON HOHO OHNON HOHO ONON .O HNNO NHNON HONO OHNON HNHO OHNON HOHO OHNOO .N HONO OHNOO HONO NHNON HOHO ONNN HONO OHNON .H HOOEEO OOO «OOOHO HOOOOOO OO>O HOOOOOO HOHOOOOO mopsmmonm OOO mmssHo> HOOOH>HOOH .coHumHserm :oHHmamO HmoO>pou Hoflhomcfi ummH u mmuH4 .conmHseHum Hamm> pon u m>4« 154 N.HO OO .O O.NO N.OO .O O.ON O.OO O.NO O.OO .O O.NN O.OO O.OO OO .N H.NN O.ON O.OO H.OO .H HOOOOOONHEO P> ON NN .O ON NO .O OO NO OO OO .O OO OO OO ON .N OO NO OO OO .H HOOENOOOOOOOO O OO.O OO.O .O OO.N OO.O .O OO.H OO.N OO.H ON.H .O OO.N OO.H OO.N OO.N .N ON.H NN.H OO.H OO.H .H HOHENHO m> OOOOHO HOOOOOO OO>O HOOOOOO wondflucou--nuwnnmmv mmpSwmoOm Ocm moenHo> HOSOH>HO:H 155 Lobar Perfusion Percantages (Rabbits) (Percent black) Cranial Middle Caudal Intermed. L. R. L. R. L. R. R. Control 1. (7)* 95% 90% 90% 90% 90% 90% 80% 2. (7) 100 100 100 100 100 100 100 LVS 1. (8) 60 50 55 10 95 50 50 z. (8) 95 80 7o 75 7s 90 9o 3. (3) 100 100 70 100 100 100 95 4. (8) 80 95 7o 70 90 60 85 5. (8) 95 90 7s 75 95 80 80 LICGS 1. (7) 95 9o 90 9o 80 80 9o 2. (7) 95 9o 90 9o 95 95 80 3. (7) 85 9o 90 95 9o 95 9o *Rabbit body weights (lbs.). 156 'Stereologic Count Totals (Rabbits) (Middle Lobes) Total Lobar Volumes (Water Displacement) Left Lobe Right Lobe Control 9cc 11cc Parasympathetic 1. 9 12 2. 9 11 3. 9 12 Mean 9cc 12cc Sympathetic 1. 6 9 2. 7 10 3. 7 10 Mean 7cc 10cc Stereologic Control Vagal Sympathetic C°unts L. R. L. R. L. R. Sections Observed 120 133 160 196 141 120 T°tal 338 368 354 522 387 330 Counts Total Possible 14,196 15,456 14,868 21,924 16,254 13,860 "Hits" 157 “OOO. ONOO O . OON OH OON.OH OOOO. HO . NOO NNM H OOO OOO.OH NOOO. OOO NOO OH OH .OOH :OHOUOOO O . OOO NNO . .O: OHOOOOOO NMMO OOOO. OOO :OOOO: ONH O OHO.O MWMO. OONO. Oo>HO Ouasou maH o . > MM 0 HHH ONH ONH WMO.O WMMOO ONNO. O O OOO: NOH OO ONO OON.O OOO OOOOOOOO OON . ONH WOO : .O: OHOOOOOO NOOOO OONO. HH mmpwm: NON OHO.O WMWO. OOOO. :OHOOO ucsou HHH MMO NNO O NN0.0 OONO. OOOO. . O HOOOOOHOOO H OOH OON OOH O OHO. : HOH NOO O .OOH OOOOOOO w o MNH W”? O .m: OHQMWMOQ ONO ON . HH :OOOO OOO O NO O : OOO OHO.O HHNO. O . Ocaou NOO OOO.N HOO NN . OOOOOOO OOH ONO OO0.0 NO OHO . OHOOOz ONH NNH OON ONH O O ONH HNO MOO O OOH OOOOOOOO NN.H mm = .m: @Hflflmmom om NOH :muvflmu .4 mud: . owuonpmmax .m . :oO ou O OH O .O .OOOO OOHOO .umnummexmmumm .4 . Hohunou mmc54 uH .npmm cw OOOOpomOm mazHo> N O3OO< 158 OOOO. OOOO. OONO. OOOO. OOOH. ONOH. OOOOOOOO OOO OH OON OH ONO.HN OOO.OH OOO.OH OOH.OH =OOOO= OHOOOOOO NHO HHH.H OOO.H NOO.H NOO.H NOO.H :OOHO: OOO NOO NNO OOO OOO OOO Opcsou mosHm> hmno4 mam: NHOO. OOOO. OOOO. OOOO. NHOO. OHOO. OOOOOOOO NOO O OHO O ONH.O OOO.O OOH.O OON.O :OOOO: OHOOOOOO OOH ONO ONN NOH ONO NOO :OOOO: HHH ONH NOH OO ONH OHH OOOOOO :owpuom Hanonmwhom NOOO. OHOO. OOOH. OOOO. ONOH. OOOO. OOOOOOOO NO0.0 OH0.0 OH0.0 NN0.0 OOH.O OHO.O =OOOO: OHOOOOOO ONN OOO OOO ONO NOO OOO :OOOO: HHH ONH OOH HOH ONH NHH OOOOOO OOOOOOO OHOOOz HOOO. OOOO. NOOO. OHOO. OHOO. NHOH. OOOOOOOO OOO.O OHO.O OOO.N OOO.O ONH.O OOO.O :OOOO: OHOOOOOO OOO OOO OOO HHO HNO OOO =OOOO: OOH ONH NNH ONH NNH NOH OOOOOO OOOOOOO OOHOO .O .O .O .O .O .H uHuonumaENm oOuonquENmmumm Houucou mO::4 OOOOOO :O mcofiuomnm oasHo> Hommo> owpw4 159 H4v oesHo>\4uO:m4« OO0.0 ONN.O OON.O OO0.0 OON.O ON0.0 OOHOmoum :moz HOH.H OOO.H OON.H ONH.H OON.H NOO.H Oucsou OHHOOOO OOO NOO NNO OOO OOO OOO mp::ou mmsHm> Om404 :Ooz NOO.N OOH.O HO0.0 OON.O OON.O OO0.0 moHOmohm :mmz ONO HOO OOO NOO OOO NOO mucoou OHOmoum HHH ONH NOH Om ONH OHH mundou cowuhom Hmuonawuom OH0.0 OON.O NOH.O HON.O OON.O HO0.0 mmHOmopm cam: OOO OOO NOO OOO OOO NOO muasou OHOmoum HHH ONH OOH HOH ONH NHH mucsou :oOuuom OHOOOZ OON.O ONN.O ON0.0 OO0.0 OON.O OO0.0 moHOmoOm :mmz OOO OHO ONO OOO OOO OOO munnou mHOmonm OOH ONH NNH ONH NNH NOH muanou :ofiuuom HOHOm .m .4 .m .4 .m .4 ofiuogummezm uOuoaqustmumm Houucou mmaz4 uwpnmm :O «mnuwco4 o>OumHom Hommm> owhm4 160 HNNO. OO0.0 HOO.OH OOOO. OOO OON.O ONO.OH HNON. NOO ONO. NNO.HN OHNN. ONON. NNO O OOO. NOO.O OOO.NH OOOO. OOO.H OHOO. OOO OOO.O HHH OHO.O ONO OH OONO. OOO.H ONON. OOO OOH.O ONH MMH.O NN ”OO.OH q o :m HH wcvm. NV“ H COMM mm “HI: OOHHUNH NOO.O OOO O OONO. HOOOOOO BHOOH Hmmm. mm OOHOm :mHHmm HHH OHO.O OOO. NOOO. OOOHO Oucmo: HOH.N OOHO. ONH H WWWMW. > umno u ONH OHO.O O OON.H 4 sum: M meON mwN. VHH :WHH GOH ovm. me Num. .3: 0 .Humh m V we mm mo Hhm. womm. HVH H COMO :mHHmQ OOH H OHO.O OOO.O OOOO. :oOpOo Ouamo: meOH cmwN. MNH H VHO. m Hth U ONH OOO.N OOO.O 4QOOO mOHON mowN. NHH H :m#H COH Q . NNH OOO. .O: O .OOOO m OOO.O OOOO. Hnfimmom uHuoa .4 ONH H NMH.O ONN mwwwmm O U. a o .- OOENO . NNO H OOOm OOOOOOO sou O HOO.“ OHOOO UMHQ o NOH :WHH :OH .2 49 4 .: .uo One» : m mum OO HOOO OOO .m :m mom O 8%.. Houuao 4 oHuuom o u HOHH: mmaz4 annm mzoa m H 3 o H o m GH 0 ovum o U o 5M H APPENDIX D ANALYSIS OF COVARIANCE TABLES FOR DOG, CAT, AND RABBIT 161 O0.00 OO NHO Hmuou ON.N N O0.0H O0.0H O O mucoEHOOOp :Hnuwz H0.0 OO.H O O0.0 O0.0H OO NHO mucoEwmmhu macaw "oEsHo> NOOHHOQOO NO.NN O0.00 NHO Hmuou OO.N N OO.NH OO.NH O O mpcmsumopu :OAHHz NH.O H0.0 O O0.0 O0.0H O0.00 NHO OOOOEOOOOO macaw ”oEsHo> Hommo> OH.OH O0.00 NHO Hmpop NO. N ON.O ON.O O O Opaospmogp :OOOOz O0.0 NO. O HO.H O0.0 O0.00 NHO Oucmsumoup Ocoem “camco4 Hommm> OO.NH O0.0H NHO Hmpop NO.H N ON.OH ON.OH O O OOOOEOOOOO :HOsz O0.0 H0.0 O OO.H NO.N O0.0H NHO OO:OEOOOOO ansm "mossHo> Nm39H< m m: MO xxm -NNO NNO Nxm xxm :oflumwnm> mo oohsom NNNXOV HmoHan mucmHum>ou mo mOmNHmc NumHHflmOo NO. OO.H OH mHmuou ONO. N HO. HO. O O mucoEummhp cwnpwz N0.0 OOO. O OH. OO. OO.H OH Oucospmouu macaw ”oasHo> Hommo> O0.0 O0.0 OH mHmuou OOH. N ON. ON. O O mangumopp cwauwz ONO.O NOO. O OO.H ON.O O0.0 OH mudmfipmohu mnosm vauwco4 Hommo> OO. HO.H OH Hmuou NNO. N OH. OH. O O mpnospmmhu :Onufiz NN.H NOO. O OH. HN. HO.H OH mucoEpmonu macaw "moEsHo> NOBOO< m mz MO xxm -NNO NNO NXO xxm :oOmeOm> mo mousom JIINH OOO HmmHnt oocmwhm>ou mo mNmNHmc NNOHHOQOO gumnm4 Hommo> "NHmsoocmquewm OH04 chonHom map “may uaowwwnoo OOO Em H Hmeflam OoumHssfipm mo OczH unmwp n O: HOEOOO OmpmHseflum mo OnsH umoH n O: Hmeficm Honuaou mo OGSH H4OON u N: Hmsficm Houuaoo wo OcsH pmoH u H1 .mcmos HODOO>OO:O :mozuon moucmnommfiw mummEoo op OmngHnmumm ohm: OHO>OOHGO ouaowflmqoo .ommoaom Omscflucoo--mHmOOh ufiuonumgexmmhmm uwu 164 .umscwpcou mflmzfimn xanHflmwu HO.H mN.H mm.o~ Hmpou Hmo. 5 mm. mm. o o mucoEummHu cflnuwz mH.H mmo. m mm. ev. an.H mm.om mudmsummhp macaw "mossao> Hommo> mm.~ mo.m m~.o~ Hmpou «no. O NH. NH. o o mangpwohu :ngwz «mm.v~ omm. m NN.H Nv.N mo.m mm.o~ mucoEummnu macaw ":uwcmq Hammo> em. NN.H m~.o~ Hmpou coo. 5 cc. ow. o o mucoEumonu :ngwz NH.O oao. m mo. mo. ON.H mm.o~ mucospmohp macaw "mmezao> zMSHH< m m: mw xxm -xzm xxm %Xm xxm coflumfium> o monsom NNNXmM m Ammanmh mucmfium>oo mo mfimwac znmaaflmwu Aumcog Hmmmo> ”zamsomcmuHSEwm vac: mcwzoaaom 0gp Hana ucowwmcou Omm aw H Hmecm woumasefipm mo MGSH pawwp u v: Hmeficm wmpmHsEflum mo mama umma u m: Hmafiqm Howaqou mo mesa pamfih u N: Hmsficm Houpcou mo m::~ umma u H: .mcmoe Hmswfl>fiwafi cmmspon mmuqmummme mammfiou ou wonmwfinmumo who: mam>uopcw mucmwwmnou .mmmogum wmscwuaou--mamfihe vaponammexmmhmm panama 166 m~.mm O0.00 New prou ON.N N mm.mH mm.mH o o mangumouu canvas No.o Hm.H m Om.v Nv.- O0.00 NOO mummEuwmuu macaw "mmesHo> NNOHHMQOU OO.NN om.mo New Hmuou OO.H N mm.oH mm.oH o o mucmsumohp :fiauwz O0.0 OOO. m mm.~ NO.NH om.mo New muzoEumoup macaw “mmESHo> Homm0> om.wH ~m.mm NOO Hauou mm.H N HN.O HN.O o o munmepmmhu afinuwz mv.o omo. m OO.H mw.m mm.mm New mummEumopp macaw ”mnumcoq Hommm> mo.¢ mm.mN NOO Hmpou OON. N OO.H OO.H O o muqoepmouu OOOOH: mN.o ONc. m HN. OO.N mm.m~ NOO mpamEpmohp macaw "woesHo> xmzpfi< m m: mu xxm -zzm xxm xxm XXm aofluwfihm> mo muuzom NANXmM mmoanmh ounmfinm>ou mo mwmzamc NHOHHHQOU mm. mH.m mN.oN Haqu omc. N mm. mm. o o mucosumohu :anfiz O0.0 OOO. O OH. mm. OH.O ON.ON OOOOOOOOOO anam “moE:Ho> Hommm> OO.H NN.m mN.o~ Hmuou mmo. N mo. me. o o mucospmmuu cwnuwz NH.O NHO. m mo. om.H NN.m mm.o~ mangumoNu macaw "mnuwwoq Hommo> me. om.H mm.oN Hmpou wee. N Om. em. o o mucospmohu canvas Nm.o mac. m co. HH. mm.H m~.om mucoEpmohu macaw ”mmESHo> Nmzpfi< m m: mv me -Nxm Axm Nxm KXm :oHumHhm> o mopsom MANMMM . . w Ammanmh ounmwum>ou mo mfimxamc NMOHHHmwu NN.H OO.m m~.om Hmuou Omo. N mm. mm. o o mucoEumopu :HAuHB OO.N mOH. m mO. mm. mO.m m~.om mucoEummhu macaw .ummasHo> Hmmmm> oN.m NO.w m~.om Hmuou Nmo. N OO. OO. o o mucoEpmmhu :HSHHS wo.m OHN. m mo. om.m NO.w m~.om mpamsummuu macaw "mapwnoq Hommm> NO. Hm.o mN.cN Hmuoa Omo. N HO. HO. o o munosumohu :anHz ON.O NHO. O OO. OO. 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