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The role of the polymorphonuclear leukocyte in the pathogenesis of the adult respiratory distress syndrome Thommasen, Harvey Victor 1985

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THE ROLE OF THE POLYMORPHONUCLEAR LEUKOCYTE IN THE PATHOGENESIS OF THE ADULT RESPIRATORY DISTRESS SYNDROME By HARVEY VICTOR THOMMASEN B.Sc, McGill University, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the,required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1985 © Harvey V. Thommasen, 1985 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of P A T H O L O G Y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date M a y 28, 1985 ABSTRACT This study was designed to follow up a chance observation in patients with an admission white blood c e l l (WBC) count showing an absolute lympho-cytosis and relative neutropenia that changed to a lymphopenia and neutro-philia within 24 hours. As 15 of the 20 patients were admitted following trauma, we examined this association further by reviewing charts of 69 patients who had sustained stab wounds to the chest and abdomen. A pros-pective study involving 40 patients in the Intensive Care Unit was also undertaken because of the related hypothesis that the Adult Respiratory Distress Syndrome (ARDS) is associated with sequestration of complement activated polymorphonuclear leukocytes (PMN) by the lung. These studies show that trauma is frequently associated with a lymphocytosis and relative neutropenia. In cases where ARDS did develop, the onset of respiratory failure was associated with a profound f a l l in the circulating PMN count. To test the hypothesis that these leukocyte changes were due to cate-cholamine release and sequestration of PMN within the pulmonary micro-vasculature, we studied the effects of epinephrine infusion, lowered cardiac output and complement activation on WBC uptake and release from the dog lung. The data show that pulmonary blood flow has a marked effect on the uptake and release of WBC by the lung but has no effect on differential counts. Epinephrine infusion increases circulating WBC counts but also does not alter differential counts. In contrast, activation of the complement cas-cade alters differential values by causing preferential sequestration of PMN. We conclude that trauma is frequently associated with a lymphocytosis and relative neutropenia and speculate that this phenomenon is due to a combination of catecholamine release and sequestration of PMN within pulmonary and systemic microvasculatures. The findings that a profound f a l l in PMN counts occurs prior to the onset of ARDS and after activation of the complement pathway with cobra venom factor support the hypothesis that complement activated PMN play a role in the pathogenesis of ARDS. These data also suggest that prospective leukocyte counts may be a useful predictor with respect to determining which patients w i l l develop this syndrome. - i v -TABLE OF CONTENTS Page ABSTRACT . i i TABLE OF CONTENTS , iv ABBREVIATIONS v i LIST OF TABLES ix LIST OF FIGURES xi ACKNOWLEDGEMENTS xv DEDICATION xv INTRODUCTION: ROLE OF THE POLYMORPHONUCLEAR LEUKOCYTE IN THE PATHOGENESIS OF THE ADULT RESPIRATORY DISTRESS SYNDROME 1 A. The Adult Respiratory Distress Syndrome 1 B. Polymorphonuclear Leukocytes and the Lung 7 C. Pulmonary Sequestration of Polymorphonuclear Leukocytes 10 D. Mechanisms of PMN Induced Lung Injury 15 E. Polymorphonuclear Leukocytes and the Adult Respiratory Distress Syndrome 17 F. Summary 20 SCOPE AND AIM OF THE PRESENT INVESTIGATION 22 CLINICAL STUDIES 26 A. Introduction 26 B. Materials and Methods 28 1. Patient Population 28 2. Criteria for ARDS 29 C. Statistical Analysis 30 -v-D. Results 31 1. Group 1 31 2. Group II 31 3. Group III 32 a. Trauma Patients 32 b. Sepsis Patients. 33 c. Miscellaneous Group with Multiple Risk Factors 34 d. Hematologic Measurements 34 e. Blood Gases.. 36 E. Discussion 37 ANIMAL STUDIES 71 A. Overview 71 B. Materials and Methods 74 1. Animals................... • 74 2. Surgical Procedure 74 3. Hematologic Measurements 77 4. Measurement of Regional Pulmonary Blood Flow 78 5. Complement Activity 79 6. Cobra Venom Factor 79 7. Experimental Procedures 80 a. Control 80 b. Transient Lowering of Pulmonary Blood Flow (PBF). 82 c. Prolonged Lowering of PBF 83 d. Cobra Venom Factor 84 e. Cobra Venom Factor Followed by Lowering of PBF 84 - v i -f. Lowering of PBF Followed by Cobra Venom Factor 85 g. Epinephrine Infusion and Cobra Venom Factor 86 C. Stat i s t i c a l Methods 86 D. Results 86 1. Control 86 2. Transient Lowering of PBF ....87 3. Prolonged Lowering of PBF 90 4. Cobra Venom Factor 91 5. Cobra Venom Factor Followed by Lowering of PBF 92 6. Lowering of PBF Followed by Cobra Venom Factor 93 7. Cobra Venom Factor and Epinephrine Infusion 94 E. Discussion 97 1. The Effect of Pulmonary Blood Flow on White Blood Cell Uptake and Release by the Dog Lung 97 2. The Effect of Complement Activation and Lowering Pulmonary Blood Flow on White Blood Cell Uptake and Release by the Dog Lung 103 3. The Effect of Epinephrine Infusion and Complement Activation on White Blood Cell Uptake and Release by the Dog Lung I l l 4. Conclusion * 114 REFERENCES ' 180 - v i i -ABBREVIATIONS Ac- Aorta ARBS Adult Respiratory Distress Syndrome A-V difference arterial-venous differences. BAND PMN nonsegmental polymorphonuclear leukocytes BP Blood pressure CGP •••• circulating granulocyte pool CO cardiac output CVF cobra venom factor Epi epinephrine F female Hct hematocrit ^2®2 hydrogen peroxide ICU intensive care unit IVC inferior vena cava LC lymphocyte M male MGP marginating granulocyte pool 0", superoxide anion OH* hydroxyl radical PBF pulmonary blood flow PLT platelet PMN polymorphonuclear leukocyte Pa systemic arterial blood pressure partial pressure of oxygen in arterial blood pulmonary artery blood pressure pulmonary arterial wedge pressure red blood c e l l right ventricle segmented polymorphonuclear leukocytes total blood granulocyte pool white blood c e l l - i x -LIST OF TABLES Page I. Etiologic Factors in the Adult Respiratory Distress Syndrome 2 II. Summary of Trauma Patient Information 40 III. Summary of Sepsis Patient Information 42 IV. Summary of Miscellaneous Group Patient Information 44 V. Risk Factors in Group III Patients..... 46 VI. Sensitivity, Specificity, Positive and Negative Predictive Values of a Fall in WBC to Predict ARDS 47 VII. Minimal Sampling Control Group Hematologic Data.. 115 VIII. Maximum Sampling Control Group Hematologic Data 116 IX. Hematologic Data for Transient Low Flow Experiments 118 X. Hematologic Data for Prolonged Low Flow Experiments 120 XI. Hematologic Data for CVF Experiments 121 XIIA. Hematologic Data for CVF and then Low Flow Experiments, F i r s t Run 123 XIIB. Hematologic Data for CVF and then Low Flow Experiments, Second Run 125 XIII. Fractional Uptake of WBC by the Lung for CVF and then Low Flow Experiments 127 XIV. Complement Protein Assay Results 128 XV. Arterial Blood Gas Values in CVF and then Low PBF Experiments 129 XVI. Hematologic Data for Epinephrine Infusion and CVF Experiments 130 XVII. Comparison of WBC Counts in the Five Sets of Experiments 132 XVIII. Comparison of RBC Counts in the Five Sets of Experiments 133 -x-XIX. Comparison of PMN Counts in the Five Sets of Experiments 134 XX. Comparison of Lymphocyte Counts in the Five Sets of Experiments... 135 XXI. Comparison of Hematocrit (Hct) in the Five Sets of Experiments.... 136 XXII. Comparison of Cardiac Output (CO) in the Five Sets of Experiments.137 XXIII. Comparison of Systemic Arterial Blood Pressure in the Five Sets of Experiments 138 XXIV. Comparison of Pulmonary Artery Blood Pressure in the Five Sets of Experiments 139 XXV. Comparison of Pulmonary Wedge Blood Pressure in the Five Sets of Experiments 140 XXVI. Comparison of Systemic Arterial PO^ in the Five sets of Experiments 141 -xi -LIST OF FIGURES Page 1. Normal Lung Parenchyma 3 2. Acute Alveolar Damage - Early ARDS 4 3. Exudative Phase of ARDS 5 4. Proliferative Phase of ARDS 6 5. Model of Production and Kinetics of PMN in Man 9 6. Shematic Representation of the Classical, Alternate and Common Pathways of Complement Activation 13 7. Model of PMN Mediated Lung Injury 19 8. Outline of Experimental Studies 25 9. Group I WBC and Differential Count Changes 48 10. Group I RBC, Hemoglobin, and Hematocrit Changes .48 11. Group II WBC and Differential Count Changes 50 12. Group II RBC, Hemoglobin, and Hematocrit Changes 50 13. Group II Patients with I n i t i a l Lymphocytosis and Neutropenia -WBC and Differential Count Changes 52 14. Group II Patients with I n i t i a l Lymphocytosis and Neutropenia - RBC, Hemoglobin and Hematocrit 52 15. Group III Trauma Patients WBC and RBC Changes 54 16. Group III Trauma Patients Lymphocyte and PMN Count Changes 54 17. Group III Trauma Patients Mature and Immature PMN Count Changes 56 18. Group III Sepsis Patients WBC and RBC Count Changes 56 19. Group III Sepsis Patients Lymphocyte and PMN Count Changes 58 20. Group III Sepsis Patients Mature and Immature PMN Count Changes 58 21. Group III Miscellaneous Patients WBC and RBC Count Changes 60 - x i i -22. Group III Miscellaneous Patients Lymphocyte and PMN Count Changes...60 23. Group III Miscellaneous Patients Mature and Immature PMN Count Changes 62 24. A Total WBC Count in Trauma Patients who Developed ARDS 63 24. B Total WBC Count in Trauma Patients who did not Develop ARDS ....63 25. A Total WBC Count in Sepsis Patients who Developed ARDS 65 25. B Total WBC Count in Sepsis Patients who did not Develop ARDS 65 26. A Total WBC Count in Miscellaneous Patients who Developed ARDS 67 26. B Total WBC Count in Miscellaneous Patients who did not Develop ARDS..67 27. Number of Blood Tests Ordered and Lowest Absolute Values Obtained for Patients who did and did not Develop ARDS 69 28. Outline of Animal Studies 73 29. Schematic Representation of the Canine Central Circulation .75 30. Outline of Mechanism of Action of Cobra Venom Factor 81 31. Systemic Blood Pressure and Cardiac Output in Control Dogs 142 32. Pulmonary Artery Blood Pressure, Wedge Pressure, and Partial Pressure of Oxygen in Arterial Blood of Control Dogs 142 33. Aorta and Right Ventricle WBC Counts in Control Dogs 144 34. Aorta and Right Ventricle RBC Counts in Control Dogs 144 35. Pulmonary Artery Blood Pressure During Transient Lowering of PBF....146 36. Systemic Blood Pressure During Transient Lowering of PBF 146 37. Cardiac Output During Transient Lowering of PBF 148 38. Aorta and Right Ventricle WBC Counts During Transient Lowering of PBF 148 39. PMN and Lymphocyte Count A-V Differences During Transient Lowering of PBF 150 - x i i i -40. WBC Uptake by the Lung During Transient Lowering of PBF 150 41. Aorta and Right Ventricle RBC Counts During Transient Lowering of PBF 152 42. Relationship Between PMN Uptake into the Lung and Pulmonary Blood Flow i 152 43. Aorta and Right Ventricle WBC Counts During Prolonged Lowering of PBF 154 44. Aorta and Right Ventricle WBC Counts in one Dog After Long Term Cardiac Arrest and Cardiac Massage 154 45. Aorta and Right Ventricle WBC Counts in Three Dogs who did not have Cardiac Arrest During Prolonged Lowering of PBF 156 46. Aorta and Right Ventricle WBC Counts in Three Dogs who had Cardiac Arrest During Prolonged Lowering of PBF 156 47. Aorta and Right Ventricle.WBC Counts in Two Dogs after Long Term Low Flow, Cardiac Arrest, and Cardiac Massage 158 48. Systemic Blood Pressure and Cardiac Output in Dogs who Received CVF 160 49. Pulmonary Artery Blood Pressure, Wedge Pressure, and Partial Pressure of Oxygen in Arterial Blood of Dogs who Received CVF 160 50. Aorta and Right Ventricle WBC Counts in Dogs who.Received CVF 162 51. Aorta and Right Ventricle RBC Counts in Dogs who Received CVF 162 52. Systemic Blood Pressure and Cardiac Output for Run I in Cobra Venom Factor Followed by Low PBF Dogs 164 53. Aorta and Right Ventricle WBC Counts for Run I in Cobra Venom Factor Followed by Low PBF Experiments 164 -xiv-54. Systemic Blood Pressure and Cardiac Output for Run II in CVF Followed by Low PBF Experiments 165 55. Aorta and Right Ventricle WBC Counts for Run II in CVF Followed by Low PBF Experiments '. 165 56. Comparison of Aorta and Right Ventricle WBC Counts for Run I and Run II in CVF Followed by Low PBF Experiments 168 57. Systemic Blood Pressure and Cardiac Output for Low PBF followed by CVF Experiments 170 58. Pulmonary Artery Blood Pressure, Wedge Pressure and Partial Pressure of Oxygen in Arterial Blood for Low PBF Followed by CVF Experiments.170 59. Aorta and Right Ventricle WBC Counts for Low PBF Followed by CVF Experiments 172 60. Aorta and Right Ventricle RBC Counts for Low PBF Followed by CVF Experiments 172 61. Systemic Blood Pressure and Cardiac Output after Epinephrine Infusion and CVF 174 62. Pulmonary Artery Blood Pressure, Wedge Pressure, and Partial Pressure of Oxygen in Arterial Blood After Epinephrine Infusion and CVF 174 63. Aorta and Right Ventricle WBC Counts after Epinephrine Infusion and CVF .176 64. Aorta and Right Ventricle RBC Counts after Epinephrine Infusion and CVF V 176 65. Aorta and Right Ventricle WBC Counts after Epinephrine Infusion, CVF, and Standardization to Control Hematocrit Levels 178 66. Aorta and Right Ventricle RBC Counts after Epinephrine Infusion, CVF, and Standardization to Control Hematocrit Levels 178 - xv-ACKNOWLEDGEMENTS First and foremost I wish to thank my supervisor Dr. J.C. Hogg for his patience, guidance, enthusiasm and friendship during the course of these s tudies. I also wish to thank Dr. J.L. Wright, Dr. D. Brooks, Dr. J. Fleetham, Dr. P. Pare, Dr. H. Goldberg, Dr. C.A. Goresky and Dr. P. Dodek for their interest and suggestions concerning the work presented in this thesis. I am indebted to the entire staff of the Pulmonary Research Laboratory, the Hematology Department, Record Keeping Department, and the Intensive Care Unit without whose help this thesis would not be possible. In particular, I wish to thank Ms. B.M. Martin, E.M. Baile and S. Lee for their technical expertise; Dr. W.J. Boyko and D. Johnson for their hematology expertise; Dr. D. Walker and Dr. W.C. Hulbert for their morphology expertise; Dr. J.A. Russel for his c r i t i c a l care medicine expertise; Dr. B.F. Mullen for his pathology expertise; Mr. Barry Wiggs for his assistance in s t a t i s t i c a l analysis; Mr. A. MacKenzie and Mrs. Carin Pihl for help in preparing figures; Dr. M. Glovsky for assaying complement levels; and Dr. J.S. G. Montaner and Dr. M. Quiroga for their help in reviewing medical charts and for allowing me to use data which they generated from these experiments. Last but not least, I wish to thank Miss Lee Kowk, Miss Sheree Corbett and Miss Joan Dixon for typing the various manuscripts and this entire thesis. I am also indebted to the Medical Research Council for financial support in the form of a two year studentship, The Canadian Heart Foundation -xvi-for financial support in the form of a Medical Scientist Scholarship, and the B.C. Lung Association for financial aid in the form of a bursary. Finally, I wish to thank the U.B.C. medical school for allowing me to take time out from my medical school studies to participate in these memorable experiments. - x v i i -DEDICATION "Wisdom is the principal thing; therefore get wisdom: and with a l l the getting get understanding" Prov. 4:17 This thesis is dedicated to my wife Carol whose continuing support and encouragement has contributed greatly to its preparation. - 1 -INTRODUCTION: ROLE OF THE POLYMORPHONUCLEAR LEUKOCYTE IN THE PATHOGENESIS OF  THE ADULT RESPIRATORY DISTRESS SYNDROME A. The Adult Respiratory Distress Syndrome The Adult Respiratory Distress Syndrome (ARDS) is a c l i n i c a l syndrome in which patients with no prior history of lung disease or left-sided heart-failure, suddenly present with severe dyspnea, tachypnea, hypoxemia, decreased lung compliance and bilateral diffuse pulmonary infiltrates after sustaining a variety of different insults (1, 2). It is a major cause of morbidity and mortality in c r i t i c a l l y i l l patients, affecting up to 150,000 persons per year in the United States (3). Estimations of the incidence of ARDS in admissions to Intensive Care Units range from 2-10%. The mortality rate associated with this syndrome ranges from 20-60% (4, 5). Table I l i s t s the disorders which have been associated with the development of ARDS (2, 6, 7). Major etiologic factors include a l l forms of septicemia, a l l forms of shock, trauma, aspiration and disseminated intravascular coagulation (2, 8, 9). Not surprisingly, there is usually overlap of these factors in many cases of ARDS. Al l etiologic factors share one thing in common, the potential to injure the alveolar-capillary membrane. Injury to the alveolar-capillary membrane causes an increase in pulmonary capillary permeability which triggers a characteristic lung response consisting of exudation and cellular proliferation (5, 6, 10). Figures 2-4 show the typical development of the lung lesion in ARDS. - 2 -TABLE I Etiologic Factors in the Adult Respiratory Distress Syndrome INFECTION 5. INGESTANTS Viral pneumonia Bacterial pneumonia Mycoplasmic pneumonia Gram negative sepsis Chemotherapeutic agents Heroin Paraquat SHOCK 6. ASPIRATION Traumatic Hemorrhagic Septic Gastric Contents Near-drowning TRAUMA 7. MICROEMBOLI Lung Contusion Nonthoracic trauma Fat emboli Air emboli Amniotic fluid emboli Massive blood transfusion Intravascular coagulation INHALANTS Oxygen Smoke Noxious gases and fumes 8. MISCELLANEOUS Acute pancreatitis Uremia Cardiopulmonary bypass Radiation Modified from Katzenstein and Aksin, 1982 (6). - 3 -Figure 1: Normal Lung Parenchyma. This light micrograph shows a terminal bronchiole (T8)> respiratory bronchioles (RB), alveolar ducts (AO), alveolar sacs (AS) and alveoli (A). Hematoxylin and eosin stain. Magnification = 63x - A -Figure 2: Acute Alveolar Damage - Early ARDS. This light micrograph of human lung parenchyma shows flooding of alv e o l i , capillary congestion, i n t e r s t i t i a l edema and some hemorrhage. Hematoxylin and eosin stain. Magnification = 63x. Abbreviations as in figure 1. - 5 -Figure 3: Exudative Phase of ARDS. This light micrograph of human lung parenchyma shows i n t e r s t i t i a l and intra-alveolar edema, intra-alveolar hemorrhage and prominent hyaline membranes (HM). Hematoxylin and eosin stain. Magnification » 63x. - 6 -Figure A: Proliferative Phase of ARDS. This lig h t micrograph of human lung parenchyma shows i n t e r s t i t i a l fibroblast proliferation, chronic inflammatory c e l l i n f i l t r a t e and Type II alveolar c e l l hyperplasia. Hematoxylin and eosin stain. Magnification • 63x. - 7 -Even though the pathology of ARDS has been f a i r l y well characterized, the pathogenesis of this disorder is s t i l l poorly understood. While one can appreciate how lung contusion, pulmonary infection and inhalation or aspiration of noxious substances can directly damage the alveolar-capillary membrane, the mechanisms responsible for the diffuse alveolar damage seen in ARDS associated with non-pulmonary disorders are not clear. Possible roles for various cellular, humoral, metabolic and neural mechanisms have been reviewed (5, 7, 11, 12). It is unlikely that any single mechanism can account for the lung injury seen in a l l patients who develop ARDS. One popular theory states that the polymorphonuclear leukocyte (PMN) plays a central role in the pathogenesis of ARDS. This hypothesis suggests that release of toxic products from large numbers of PMN sequestered within the pulmonary microvasculature contributes directly to the development of acute lung injury and subsequent pulmonary dysfunction (13-16). The purpose of this introduction w i l l be to c r i t i c a l l y examine this hypothesis by reviewing normal lung-PMN physiology, mechanisms by which the lung sequesters large numbers of granulocytes, ways in which PMN can produce lung injury and supporting c l i n i c a l data. B. Polymorphonuclear Leukocytes and the Lung In 1910, Andrews speculated that " i f the bone marrow is the birth-place of these cells (PMN) and the spleen is their ultimate tomb, while the blood is their means of transit, the lung may serve as a weekend at the seaside where they may recuperate their energies." (17). After 70 years of extensive research our understanding of the neutrophil lifecycle and its relationship to the lung remains basically unchanged from that elaborately - 8 -outlined by Andrews. That i s , PMN are derived from primitive precursors in the bone marrow; they enter into a mitotic pool where they develop into relatively mature cells which then migrate into a maturation-storage pool which is readily accessible to the circulation (18). The term, pool, is a conceptual one and does not necessarily imply that the various PMN forms are spatially separated from one another in the bone marrow (19). As shown in Figure 5, myelobasts, promyelocytes and myelocytes make up the mitotic pool whereas metamyelocytes, band cells and segmented PMN make up the maturation-storage pool of bone marrow neutrophils. In response to normal PMN u t i l i z a t i o n (18), complement activation (20), and hemorrhage (21), segmented PMN, band PMN and rarely metamyelocytes enter the blood stream to replace those leaving the circulation. Upon entering the blood stream, these cells immediately become distributed between marginating and circulating pools of PMN. The marginating pool consists of PMN which have temporarily dropped out of the axial stream (circulating pool) into a marginal stream located adjacent the endothelium of postcapillary venules in the systemic vascular bed (22, 23) and throughout the pulmonary microvasculature (23,24). Since marginated PMN can also drop back into the axial blood stream, one may assume that a dynamic equilibrium normally exists between the circulating and marginating cells and that together these pools constitute a single kinetic unit - the total blood granulocyte pool. The marginating granulocyte pool accounts for approximately 60% of the total granulocyte pool (25, 26). The lung is believed to be the major reservoir of marginated PMN (27, 28). One study has shown that the pulmonary marginated granulocyte pool in sheep stores up to 3 times the number of cells found in the circulating - 9 -Figure 5: A Model of the Production and Kinetics of PMN in man. TBGP = Total Blood Granulocyte Pool, CGP = Circulating Granulocyte Pool, MGP = Marginating Granulocyte Pool. (Modified from Wintrobe et a l , in C l i n i c a l Hematology, Philadelphia: Lea and Febriger, 1981, pp 209). - 10 -granulocyte pool ( 2 4 ) . Under normal conditions, the number of PMN entering the pulmonary marginating pool equals the number which reenter the circulat-ing pool ( 2 9 ) . Thus, under normal conditions, the pulmonary marginating pool is in a steady state. This steady state can be altered by increasing or decreasing pulmonary blood flow. Increasing blood flow by catecholamine administration flushes out marginated cells ( 3 0 , 3 1 ) . Under these conditions the concentration of leukocytes in the pulmonary artery is less than that in the pulmonary vein. On the other hand, when pulmonary blood flow is decreased by inflating a balloon catheter located in the inferior vena cava or by exhibiting a Valsalva maneouver, the concentration of PMN in the pulmonary artery is greater than that in the pulmonary vein ( 3 2 , 3 3 ) . Thus, i t appears that the lung may function to crudely regulate circulating leukocyte levels ( 2 7 , 3 4 ) . It should be reemphasized that the lung appears to function as a temporary storage site from which PMN are eventually released back into the circulating blood stream. One investigator found that in rabbits less than 1% of the PMNs leaving the total blood granulocyte pool do so by migrating into the alveoli ( 3 5 ) . The majority of PMN are permanently removed from the circulation by the liver and spleen ( 3 1 , 3 6 , 3 7 ) . C. Pulmonary Sequestration of Polymorphonuclear Leukocytes Macrophage-derived chemataxins, activated complement proteins and — low pulmonary blood flow produce sequestration of large numbers of granulocytes within the lung. Alveolar macrophages release PMN specific chemotaxins after hyper-oxia, bronchiolar lavage, and phagocytosis of heat k i l l e d Staphylococcus - 11 -aureus (38, 39, 40). The diffuse alveolar damage seen in the course of oxygen toxicity and perhaps sepsis, may therefore arise via macrophage orchestrated PMN sequestration, stimulation, and release of toxic oxygen radicals and granular enzymes (16, 41). Beginning as early as 1894, studies have shown that intravenous injection of bacterial proteins (42), 'hydrophillic colloids' (23), leukemic leukocytes (43), induction of anaphylaxis (44), trauma (45), and hemodialysis (46) a l l produce an acute neutropenia and/or sequestration of PMN within the lung. These diverse causes of acute neutropenia and intrapulmonary sequestration of PMN share the potential to activate the complement system. The complement system consists of a complex group of proteins which are present in plasma, cerebrospinal fl u i d and other body fluids (47). These proteins function to mediate various aspects of host defense and inflammation. There are two distinct pathways of complement activation, namely, the classical complement pathway and the alternate complement pathway which converge to form the common complement pathway (Figure 6). A total of 18 serum proteins make up the complement system (47-49): 5 proteins in the classical pathway (Clq, Clr, Cls, C4, C2), 3 proteins in the alternate pathway (B, D, P), 6 proteins in the common pathway (C3, C5, C6, C7, C8, C9), and 4 regulatory proteins (CI inhibitor, C3b inhibitor, C3b inhibitor accelerator, anaphylatoxin inactivator). The most important activators of the alternate md classical complement pathways include (47, 50, 51): - 12 -Alternate Pathway 1. Microbial polysaccharides - zymosan, endotoxin, teichoic acid 2. A r t i f i c a l membrane materials - hemodialysis, nylon-filter leukapheresis, cardiopulmonary bypass 3. Inactivated viruses and transformed mammalian cells 4. Helminthic and protozoan parasites 5. Toxins - cobra venom factor 6. Aggregated IgA, IgG - 13 -CLASSICAL PATHWAY a c t i v a t i n g l u b s t a n c * ALTHMATt PATHWAY Figure 6 : Schematic Representation of the Classical, Alternate, and Common Pathways of Complement Activation. (Modified from Mowat HZ: Current Topics in Pathology). - 14 -Classical Pathway 1. Antigen-antibody complexes ( IgM, IgG^, and IgG^) 2. Aggregated immunoglobulin 3. Bacterial endotoxins 4. Polyinosinic acid and DNA 5. Plasmin and trypsin Activation of either the classical or the alternate pathway triggers a cascading sequence of enzymatic conversion reactions whereby an activated complement protein catalyzes or facilitates enzymatic activity of a subsequent complement protein. Because the complement system is also intimately associated with other plasma protein systems, complement activation is often associated with blood coagulation, fibrinolysis and kallikrein-kinin formation (52). The final product of the common complement pathway is a large complex molecule which binds to biological membranes,and penetrates l i p i d bilayers creating a transmembrane channel into the interior of the c e l l . The membrane becomes very permeable to Na+ and H^ O, the c e l l swells, intracellular macromolecules diffuse out, and c e l l lysis occurs. In generating the final product of the common complement pathway, numerous small molecular weight products - anaphyla toxins - are generated, many of which have specific functions. Indeed, i t has been postulated that the most important function of complement activation is to generate these anaphylatoxins. C3a and C5a appear to be the most important anaphylatoxins (53). These small molecular weight products interact with membrane receptors and cause histamine release - 15 -from mast c e l l s , enhanced vascular permeability, smooth muscle contraction, and arteriolar constriction. They also f a c i l i t a t e PMN chemotaxis and immune complex adherence, enhance phagocytosis, and induce release of proteases and toxic oxygen metabolites (49, 50). Clearly, complement activation may lead to tissue injury. Infection, trauma, microemboli, pancreatitis (trypsin mediated), and cardiopulmonary bypass a l l have the potential to activate complement and in this way may cause ARDS. Both transient lowering of pulmonary blood flow and hemorrhagic shock are associated with sequestration of large numbers of PMN within the pulmonary microvasculature (29, 30, 32, 54). It is postulated that tissue damage could arise i f these cells were stimulated to release toxic products secondary to superimposed complement activation (32). These findings may shed some light on the mechanism by which shock predisposes to ARDS. D. Mechanisms of PMN Induced Lung Injury Theoretically, the PMN is the ideal candidate for causing lung injury. Its major function is that of a phagocyte to prevent or retard the intrusion of infectious agents and other foreign materials into the host environment (18). In carrying out this function, PMN release at least two groups of products which can cause tissue damage, namely, granular substances and toxic species of reduced oxygen (15, 16). Various chemotaxins mediate the response of PMN to pathologic stimuli. Major chemotaxins include bacterial factors, complement components, lymphokines, peptides, lipids including prostaglandins and leukotrienes, immunoglobulin fragments, fragments of fibrinogen and - 16 -collagen, macrophage products and c e l l surface antigen-antibody reactions (16). In response to these various chemotaxins, PMN adhere strongly to the vascular endothelium, migrate by the process of chemotaxis (55), and experience a respiratory burst, which consists of increased oxygen consumption, activation of the hexose monophosphate shunt and generation of reactive oxygen-derived free radicals and their metabolic products (15). By the process of phagocytosis (56), foreign material is engulfed and internalized within the phagosome, which in turn fuses with lysosomal granules that contain bactericidal substances and digestive enzymes to form a phagolysosome. The most important granular substances are elastase, collagenase, cathepsin, cationic protein, lactoferrin and lysozyme. In chronic Granulomatous Disease PMN do not undergo a normal respiratory burst. Bacterial k i l l i n g decreases under these conditions even though phagocytosis remains constant, suggesting that the generation of toxic oxygen metabolites is of major antimicrobial importance (15, 16). In vitro studies have shown that during phagocytosis PMN release proteases and oxygen metabolites into the external environment (56, 57). Investigators have proposed that tissue injury may result from either the direct effects of a pathologic agent or as a consequence of products released from inflammatory cells (58, 15). Studies have shown that toxic species of reduced oxygen damage fibroblasts, lung parenchymal c e l l s , endothelial c e l l s , erythrocytes, hyaluronic acid, and inactivate anti-proteases (15, 16). Elastase and collagenase digest glomerular basement membrane, articular cartilage, and elastin, while cathepsins D and E attack native proteins and basement membrane (16). These proteases also have the potential to amplify inflammatory processes by cleaving complement, - 17 -fibrinogen and Hageman factor (53). Both in vivo and in vitro studies have demonstrated that activation of granulocytes with complement derived chemotactic products, particularly C5a, produces cytotoxic effects on endothelial cells (59-61). Investigators have postulated that intravascular activation of the complement system plays a particularly important role in the pathogenesis of ARDS (13-15). According to this hypothesis, activation of either the classical or alternate comple-ment pathway results in the activation of the common complement pathway and the subsequent generation of C5a (Figure 7). Generation of C5a causes PMN to become 'sticky', to adhere to the pulmonary vascular endothelium and to release hydroxyl radicals (OH*), peroxide (^0^), superoxide anion (0^) and proteases which f a c i l i t a t e the i n i t i a l tissue damage by degrading basement membrane, elastin, and collagen. E. Polymorphonuclear Leukocytes and the Adult Respiratory Distress Syndrome Evidence supporting the hypothesis that sequestered, activated PMN are responsible for the lung lesion of ARDS is derived from autopsy studies, c l i n i c a l correlates, experimental animal models, and prospective c l i n i c a l s tudies. Autopsy studies have shown that increased numbers of PMN in pulmonary capillaries and/or microemboli consisting of aggregations of leukocytes, f i b r i n , and platelets are a frequent histologic finding in patients who have died during the acute stages of ARDS (10, 62, 63). In some c l i n i c a l situations, an association is seen between sequestration of large numbers of PMN and pulmonary dysfunction. PMN accumulation in the lung associated with respiratory distress is seen in - 18 -patients with acute nonlymphocytic leukemia within 10-48 hours of i n i t i a t i n g chemotherapy (64, 65). The c l i n i c a l picture is similar to that of ARDS with the patient presenting with acute onset of dyspnea, tachypnea, diffuse pul-monary rales, severe hypoxemia and bil a t e r a l pulmonary i n f i l t r a t e s . Hist-ology reveals blast c e l l aggregates and thrombi f i l l i n g and distending the pulmonary microvasculature, perivascular hemorrhage, i n t e r s t i t i a l edema and injury to endothelial cells and basement membrane. Other potential c l i n i c a l models of ARDS come from observations of patients who have undergone hemo-dialysis, nylon fiber leukapheresis and cardiopulmonary bypass (66-73). In a l l of these situations, plasma is exposed to polymeric extracorporeal per-fusion materials; complement activation occurs; PMN are sequestered; and acute lung injury and pulmonary dysfunction develop. Changes in pulmonary function are usually modest and include arterial hypoxemia, hypocapnea, decreased pulmonary diffusing capacity and increased small airway closing volume. Occassionally ARDS does develop presumably due to massive, prolonged complement activation and PMN stimulation (72-73). Numerous experimental animal models of ARDS have shown that the PMN plays a crucial role in effecting lung injury (74). Correlations exist between acute neutropenia or sequestration of PMN within the pulmonary microvasculature and the development of acute lung injury after infection (e.g. Pseudomonas bacteremia, pneumococcal bacteremia, E. c o l i septicemia [75-79]), traumatic and hemorrhagic shock (80-83), immune complex injury (84), pulmonary microembolization (85, 86), oxygen therapy (87-89), infusion of zymosan-activated plasma (90) or injection of cobra venom factor (61). As shown in Table I, many of these are major risk factors for ARDS. Complement activation occurs in many of these conditions (61, 76, 90, 91, 92, 93) but i t is not yet clear, whether complement activation alone can produce significant - n -Figure 7: Model of PMN Mediated Lung Injury. Schematic representation of endothelial c e l l injury and pulmonary dysfunction by polymorphonuclear leukocytes following intravascular complement activation and generation of hydrogen peroxide (H^O^), superoxide anion (0^) a n c* hydroxyl radicals (OH*) (Modified from Fantone JC and Ward PA; Am J Pathol 107:407, 1982). - 20 -lung injury or whether there must be pre-existing lung damage or accompanying release of other factors (94, 95). Pharmacologically induced depletion of circulating leukocytes prevents development of edematous lung injury in many of these models suggesting the PMN plays an essential part in effecting damage (61, 76, 84, 85, 86, 90, 96). Prospective c l i n i c a l studies have shown that the development of ARDS is associated with complement activation and elevated C5a plasma levels (97), transient leukopenia (98), and with sequestration of PMN within the pulmonary microvasculature (99). Circulating PMN from patients with ARDS are in an activated state showing increased chemotaxis (100), toxic granulation (98), and increased respiratory burst activity suggesting increased capacity to generate active oxygen metabolites (100). Patients developing ARDS have significantly higher levels of circulating lactoferrin and cationic proteins suggesting enhanced degranulation from granulocytes (101). Increased numbers of PMN as well as high levels of neutrophil elastase have also been found in bronchovascular lavage fluids (102, 103). These c l i n i c a l observations are consistent with findings from animal N studies, lending further support to the hypothesis that PMN mediated lung injury is indeed relevant to the pathophysiology of ARDS. F. Summary A major nonrespiratory function of the mammalian lung is that of a PMN reservoir. Within this reservoir, granulocytes are distributed between marginating and circulating pools. Under normal conditions these cells release l i t t l e , i f any, toxic metabolites. Situations which f a c i l i t a t e chemotactic release, activation of complement, or prolonged lowering of - 21 -pulmonary blood flow lead to sequestration of large numbers of PMN in the lungs. If these PMN are then stimulated to release toxic oxygen species, proteases or other metabolites, existing defense mechanisms are overwhelmed; and lung injury results. Anaphylatoxins generated by complement activation, humoral factors released from platelets or macrophages and activation of the kallikrein-kinin and coagulation systems may exacerbate damage to the alveolar-capillary membrane. Permeability of this membrane increases and i n t e r s t i t i a l and alveolar edema accumulates with subsequent pulmonary dys function. While there is l i t t l e doubt that this scenario holds true for some experimental models of acute lung injury, its applicability to ARDS is s t i l l controversial. Nevertheless, ARDS does arise under conditions f a c i l i t a t i n g chemotactic factor release from macrophages (e.g. hyperoxia), in situations where widespread activation of complement occurs (e.g. sepsis, trauma, microemboli), and in shock conditions where pulmonary blood flow is often lowered. Correlations exist between ARDS and activation of complement, acute neutropenia, sequestration of PMN and enhanced functional and metabolic activity of granulocytes. Although these findings suggest that PMN are an important factor in the pathogenesis of ARDS, its precise role remains to be determined. - 22 -SCOPE AND AM OF THE PRESENT INVESTIGATION If complement mediated sequestration and activation of PMN within the pulmonary microvasculature really is important in the pathogenesis of ARDS, one might expect to find that, there exists an association between intrapulmonary sequestration of PMN or acute neutropenia and the subsequent development of severe pulmonary dysfunction. In actual fact, the link between experimental observations and c l i n i c a l pathophysiology of ARDS remains to be made (16). Although Sibbald and associates have shown that labelled PMN do become sequestered within lungs of patients developing this syndrome (99), similar findings have been reported in healthy patients (31). Hammerschmidt et al have recently shown that there is a correlation between developing ARDS and in vivo complement activation as evidenced by elevated levels of C5a in plasma of patients developing this syndrome (97). Because complement activation is also associated with acute neutropenia and intrapulmonary sequestration, these findings suggest that changes in peripheral PMN count may also be seen in patients who develop this syndrome. An absolute lymphocytosis and relative neutropenia suggestive of complement mediated sequestration of PMN has been observed by the hematology personnel at St. Paul's Hospital. Two such cases illustrate this phenomena: CASE #1 A 19 year old gentleman was admitted to the Emergency Room at St. Paul's Hospital at 1010 hours after being in an industrial accident. The patient had been caught between two electric hatch doors (steel lids) on a ship. His head and chest were crushed b i l a t e r a l l y . The doors were then reopened causing the patient to f a l l 6 feet into a hatch. In emergency, he was unconscious and bleeding from both ears, as well as nose and throat. He vomitted blood 3-4 times. I n i t i a l blood pressure was 80/40, pulse 160, respirations were 32 breaths/minute. Substantial blood loss was suspected. He was transferred to Acute Care at 1320 hours but at 1500 hours he was rushed to surgery for a craniotomy and evacuation of an epidural hematoma. The patient eventually recovered and was discharged several months later. His i n i t i a l WBC on admission was 7.8 x 103 cells/mm3 with 4.9 x 103 lymph-ocytes/mm^ and 2.6 x 10 3 PMN/mm3 which was followed by an increase to 15.5 x 103 cells/mm3 with 2.0 x 103 lymphocytes/mm3 and 12.7 x 103 PMN/mm-3. - 23 -CASE #2 This 57 year old man was admitted to the Emergency Room approximately 1 hour after being struck by a motorvehicle while crossing a street. He was admitted with multiple injuries including pulmonary contusion and shock. His i n i t i a l WBC count was 7.6 x 103 cells/mm3 with 17% PMN and 80% lymphocytes. His blood pressure stabilized after infusion" of normal saline and 4 units of blood. Approximately 6 hours later, he became abruptly hypotensive and required emergency laparotomy to stop internal bleeding. Eleven hours postoperatively, his WBC count was 7.3 x 103 cells/mm3 with 83% PMN and 16% lymphocytes. The patient developed ARDS 20 hours later, required mechanical ventilation for 6 days and subsequently recovered over the course of the next few months. Both patients experienced severe trauma, bled substantially, became hypotensive and presented with an absolute lymphocytosis and relative neutropenia. One patient developed ARDS and the other did not. The following questions were asked: 1. Is the finding of a lymphocytosis and relative neutropenia after trauma real? 2. Is the neutropenia associated with the development of ARDS? 3. Can changes in systemic leukocyte counts be used to predict which patients wil l develop ARDS? 4. How does the i n i t i a l neutropenia arise? In particular, we were interested in determining whether or not i t is due to shock (low pulmonary blood flow) and/or tissue trauma (complement activation). Preliminary studies indicate that PMN can be sequestered in lungs by either mechanism (29,66,67). The principle objectives of this thesis w i l l be to characterize this phenomenon and to determine whether or not i t plays any role in the pathogenesis of ARDS. A review of the literature indicates that the phenomenon of transient lymphocytosis and relative neutropenia has not been f u l l y characterized. A lymphocytosis has been reported during severe emotional stress (104), after epinephrine administration (105,106) and with - 24 -exercise (107,108) but a neutrophilia is invariably present as well. A relative neutropenia could theoretically arise i f PMN were preferentially sequestered because of complement activation or because pulmonary blood flow is lowered. Thus, the phenomenon of a transient absolute lymphocytosis and relative neutropenia may reflect a catecholamine mediated leukocytosis with superimposed complement mediated sequestration of PMN within pulmonary and systemic microvasculatures. Figure 8 outlines the experimental studies which were undertaken to characterize the phenomenon of absolute lymphocytosis and relative neutropenia and to determine whether i t is important in the pathogenesis of ARDS. First, the hematology department at St. Paul's Hospital collected a l l cases showing an absolute lymphocytosis and relative neutropenia which came to their attention. After 20 cases were collected, charts were reviewed for various risk factors. After i t was shown that these leukocyte changes are associated with trauma, their frequency was characterized by choosing a standard type of trauma- stab wounds to the chest and abdomen. Also, at St. Paul's Hospital, 40 ICU patients with a high risk of developing ARDS were prospectively followed to see whether similar changes occurred in this patient population. In addition, the hypothesis that these leukocyte changes are due to complement mediated sequestration of PMN. within the pulmonary microvasculature superimposed on a catecholamine mediated leukocytosis was tested by developing an appropriate animal model. Cobra venom was used to activate the complement pathway while epinephrine was used to increase WBC counts. To rule out the possibility that these leukocyte changes are due to lowering of PBF, we also examined the effect of lowering PBF on the uptake and release of leukocytes from the dog lung. - 25 -INITIAL OBSERVATION: 'TRANSIENT LYlfHCOTOSIS AND fEUTROPENIA' COLLECT AND REVIEH 20 CASES - FEBRUARY 1977 TO FEBRUARY 1385 FINDING: ]S PATIENTS WITH TRAUMft PROSPECTIVE STUDY IMPORTANCE IN ARDS? 1. HIGH RISK PATIENTS - SEPTEM3ER 1382 TO SEPTEMBER 1983 - 10 PATIENTS RETROSPECTIVE STUDY FREQUENCY WITH TRAUMA? 1. STAB WOUND TO CHEST OR ABDOfEN - JANUARY 1377 TO JANUARY 1983 - Si PATIENTS FOE OF CATECHOLAMINES AND CCfPLEfENT ACTIVATION? ROUE OF LOW CARDIAC OUTPUT ASSOCIATED WITH SHOCK? CONTROL - 7 DOGS ANIMAL MODEL: DOG LOW PULMNARY BLOOD FLOW - 11 DOGS COBRA VOOI FACTOR - 5 DOGS COBRA wm FACTOR LOW FtJLMONARY BLOOD FLOW - 6 DOGS EPHOHRINE COBRA MENOM FACTOR - 6 DOGS Figure 8: Outline of Experimental Studies. These studies were undertaken to characterize the phenomenon of absolute lymphocytosis and relative neutropenia and to determine whether i t is important in the pathogenesis of ARCS. - 26 -CLINICAL STUDIES A. Introduction: It has been suggested that complement activated polymorphonculear leukocytes are responsible for the lung lesion of the adult respiratory distress syndrome. Hammerschmidt et a l . (97) have reported that measurements of complement proteins are of value in predicting the onset of this condition. As a number of experimental studies have shown that complement activation is associated with acute neutropenia and intra pulmonary sequestration of neutrophils (61,66,69,70,71,90) one might expect to see changes in the peripheral PMN count in patients who develop ARDS. This thesis work was designed to follow up a chance observation that 20 patients had an admission white c e l l count showing an absolute lymphocy-tosis and relative neutropenia that reversed to an absolute lymphopenia and absolute neutrophilia within 24 hours. As 15 of these 20 patients suffered severe trauma, including some stabbings, the records of 64 patients admitted following knife wounds to the chest and abdomen were reviewed to determine the frequency of a transzient lymphocytosis and relative neutropenia follow-ing this injury. At the same time, the white c e l l count and differential was followed prospectively in a third group of 40 c r i t i c a l l y i l l patients admitted to our ICU after severe trauma. These data show that trauma is frequently associated with a lymphocytosis and relative neutropenia and that this picture quickly reverses to a long-lasting relative lymphopenia and absolute neutrophilia within hours of admission to hospital. These leuko-cyte count changes were seen in both the patients who did and did not develop ARDS. They were of no value in predicting which patients would develop ARDS. - 27 -Instead, we found that the onset of ARDS was associated with an acute f a l l in both circulating PMN and lymphocyte counts. This finding suggests that frequent measurement of the peripheral WBC count may be useful in identifying those who w i l l develop ARDS. - 28 -B. Materials and Methods 1. Patient Population The f i r s t group of 20 patients (Group I) presented to the emergency room at St. Paul's Hospital between February 1977 and February 1983. These cases were collected by Dianne Johnson, a hematology technologist, because they a l l showed an absolute lymphocytosis and a relative neutropenia which changed to an absolute lymphopenia and absolute neutrophilia 2-24 hours later (109,110).* The patients ranged in age from 15-77 years (mean _+ SD: 43+19 years) and 6 were female while 14 were male. Five were admitted with congestive heart failure; myocardial infarction was confirmed in three and two showed evidence of severe myocardial ischemia. The remaining 15 patients suffered traumatic injuries - 2 gunshot wounds, 4 knife wounds, 2 injuries due to assault, 3 t r a f f i c accidents, one industrial accident and 3 fal l s from high places. * absolute lymphocytosis refers to a blood lymphocyte concentration of greater than 4,000 cells/mm3. relative neutropenia refers to a PMN differential value which is less than the lymphocyte differential value. absolute lymphopenia is defined as a blood lymphocyte concentration less than 1,500 cell/mm3. absolute neutrophilia refers to a blood PMN concentration of greater than 7.25 x 103 cells/mm3. - 29 -The second group of 64 patients (Group II) had sustained stab wounds to the chest or abdomen within 4 hours prior to their admission to St. Paul's Hospital. One hundred and twenty-one such patients were actually identified in this category during the six year period between January 1977 and January 1983 but only 64 met the cri t e r i a of having WBC counts and differential smear evaluations done on admission and repeated at least once in the next 24 hours. The patients ranged in age from 18 to 65 years (mean _+ SD: 32+11), 12 were female and 52 were male. A l l of them were eventually discharged. The third study was based on 40 patients (Group III) admitted to the St. Paul's Hospital ICU because they were at high risk of developing ARDS. These patients had one or more of the following risk factors on admission: 1) pulmonary contusion, 2) multiple major fractures, 3) multiple minor fractures, 4) multiple emergency transfusions, 5) prolonged hypotension, 6) aspiration of gastric contents or other noxious substance, 7) near drowning 8) inhalation of smoke, 9) sepsis syndrome, and 10) pancreatitis. With the exception of multiple minor fractures, smoke inhalation and aspiration of noxious substances, the risk factors studied and their definitions are identical to those used by Pepe et al (8). In the present study multiple minor fractures were defined as fractures of two or more of the fibula, radius, ulna, vertebra, ribs or skull. The category of aspiration of noxious substances was added to include a' patient who became unconscious and was found face down in a pool of fluid inside a railway o i l tanker. The category of smoke inhalation was added to include a patient who was overcome by smoke in a boarding house f i r e . Both of these patients were unconscious when admitted to the ICU. 2. Criteria for ARDS - 30 -The diagnosis was established on the basis of severe hypoxemia (PaO 75mmHg with an F l O ^ i O ^ ) , the presence of diffuse bi l a t e r a l i n f i l t r a t e s on the chest roentgenogram, a pulmonary wedge pressure less than 18 mmHg and exclusion of other diagnoses that might account for these findings (8). The diagnosis was established by physicians unaware of the results of the WBC measurements. 3. Data collection Total WBC and differential counts and platelet counts were done on admission and repeated at the time intervals indicated on the Figures. The blood gas tensions, vascular pressure measurements, chest roentgenographs and other intervestigations were obtained as indicated by the patients' course. As the measurements obtained for the study only required sampling from catheters inserted in the course of the patient's management, the Ethics Committee of St. Paul's Hospital ruled that informed consent was not required. C. Statistical Analysis A univariant analysis of variance (ANOVA) was used to determine whether or not the WBC or red blood c e l l (RBC) measurements changed following admission to hospital in the f i r s t two groups of patients. Whenever s i g n i f i -cant changes were detected, multiple comparisons were then performed to determine where the s t a t i s t i c a l differences existed with respect to the f i r s t , second and third blood samples (111). The data from patients admitted to the ICU and studied prospectively were analyzed for changes in leukocyte or RBC counts by comparing the measurements to those found on admission using a one-way analysis of - 31 -variance blocking on patients (112). Differences between individual time periods were found using Scheffe's Test on multiple contrasts (113). The association between WBC levels and ARDS was analyzed by chi-square using a Yates correction to account for low numbers and cells with zero entries (114). Specificity, sensitivity and predictive value of a f a l l in WBC count to low levels was calculated according to the method outlined by Galen and Gambino (115). A Kolmogoroff and Smirnoff test (114) was used to test for differences in the frequency of independent risk factors between groups. Student's t-test was used to examine the data for differences in the number of tests ordered in each group, for differences in the number of risk factors present per patient in each group and for differences in blood gas tensions. The mean values are given _+ (SE) standard error of the mean. D. Results 1. Group I Figure 9 shows that the patients presented with a lymphocytosis 3 3 that averaged 6.3 £ 0.7 x 10 cells/mm (58 + 2%) and a relative 3 3 neutropenia of 4.4 +_ 0.7 x 10 cells/mm (36 _+ 2%). Less than 24 hours 3 3 later the PMN counts were elevated to 11.0 _+ 1.1 x 10 cells/mm (84 3 2%) (P^ .001) while lymphocyte values were decreased to 1.4 _+ 0.2 x 10 3 cells/mm (12 + 2%) (P^ .001). Data from the third blood sample show that the PMN values remain elevated while lymphocyte values remain low. The total WBC count, RBC count, hemoglobin and hematocrit values did not change (p>.05) after admission to hospital (Figure 10). 2. Group II - 32 -The data from the 64 patients with stab wounds are shown in Figs. 11-14. Fig. 11 shows that WBC count became elevated (p<.01) on the second measurement and f e l l towards the i n i t i a l value on the third measurement. This was accompanied by a f a l l in the lymphocyte count (p< .001) and a persistent rise in PMN (p<.001) and band forms (p<.001). Fig. 12 shows that the RBC count, hemoglobin and hematocrit f e l l s lightly in these patients. While the group as a whole did not show an i n i t i a l absolute or relative lymphocytosis, 16 patients (25%) had leukocyte changes that were comparable to those seen in Group I (Figure 13,14). 3. Group III These 40 patients were divided into 3 groups: a group in which trauma was the major problem, a group in which sepsis was the major problem and a miscellaneous group. Table II, III and IV summarize the c l i n i c a l data for a l l 40 patients studied. Table V shows the number, sex, age and risk factors present in each group. When comparing patients with ARDS to patients without ARDS, we found that there was no difference in the kinds of risk factors present or the number of risk factors present per patient in any of the 3 study groups. a. Trauma Patients: There were 16 patients whose major problem was trauma. The four patients who developed ARDS consisted of three who f e l l from heights and one who was struck by a motor vehicle while 'crossing a street. The 12 patients who did not develop ARDS after trauma consisted of five who f e l l from heights, three who were struck by motor vehicles, three who were involved in motor vehicle accidents, and one who sustained multiple stab wounds. The risk factors present in this group (Table V) consisted of - 33 -6 with multiple major fractures, 10 with pulmonary contusion, 8 with multiple transfusions, 6 with multiple minor fractures, and 3 with a subdural hematoma. In addition, there was one incidence of near drowning in the patient who f e l l 180 feet into salt water and one incidence of prolonged hypotension. The hematologic data for those 11 patients who had total WBC and differential count data ordered on admission to hospital are shown in Figs. 15,16 and 17. Figure 15 shows that the mean + SE for the total RBC and WBC counts remain unchanged over the course of the experiments. On admission, 3 3 lymphocyte counts were 5.9 + 0.6 x 10 cells/mm (mean + SE) and PMN 3 3 counts were 7.0+ 1.0 x 10 cells/mm (Figure 16). These counts changed 3 3 3 3 to 1.5 + 0.3 x 10 lymphocytes/mm and 10.3 + 3.8 x 10 PMN/mm (p£ 0.05) within the f i r s t day; there were no further changes throughout the remainder of the hospital stay. Data from Fig. 17 shows that the increase in PMN was due to an increase in immature band forms suggesting release of cells from the bone marrow. Five patients presented with an i n i t i a l lymphocytosis and relative neutropenia which quickly reversed to a neutrophilia and relative lymphopenia. b. Sepsis Patients: There were 12 patients who f u l f i l l e d the cr i t e r i a of the sepsis syndrome as defined by Pepe et a l . (8). Three of these developed ARDS: two with gallstone ileus and one with ascending cholangitis. The nine patients with sepsis syndrome who did not develop ARDS included two with diagnosis of ascending cholangitis, five with peritonitis, one with a gangrenous l e f t leg and one with a group B streptococcal septicemia secondary to a toxic epidermal necrolysis. The hematologic data for those 10 patients who had total WBC and - 34 -differential counts ordered on admission to hospital are shown in figures 18-20. These data show that WBC and PMN counts tended to f a l l soon after admission to hospital, that the differential count showed a shift to the left (more band forms were counted), and that RBC counts changed l i t t l e over the 5 days studied. In contrast to the patients who had trauma, sepsis patients did not show an admission lymphocytosis. c. Miscellaneous Group with Multiple Risk Factors: The 12 patients in this group consisted of three who developed ARDS and nine who did not. The risk factors present (Table V) were not related to trauma or sepsis. The ARDS group consisted of two patients who aspirated gastric contents and one who had pancreatitis. The nine patients who did not develop ARDS consisted of one who received multiple transfusions, three with prolonged hypotension, one who aspirated gastric contents and one who aspirated noxious fluid while unconscious, three with drowning incidents, one with smoke inhalation and one with pancreatitis. The hematologic data for those 8 patients who had total WBC and differential counts ordered on admission to hospital are shown in Figures 21-23. These data show that total WBC and RBC counts change l i t t l e over the course of these experiments and that lymphocyte counts f e l l (p 0.05) while PMN counts rose after admission to hospital. Unlike the trauma patients, the i n i t i a l lymphocyte count in these patients was within normal range of -lymphocyte values. d. Hematologic: The s t a t i s t i c a l analysis of the number of WBC measurements per patient was 1 2 + 4 (mean+SD) for the non-ARDS group and 12 + 6 for the ARDS group. The measurements for the differential WBC counts numbered 9 + 3 per patient in .the ARDS group and 10 +_ 5 in the non-ARDS - 35 -group. The platelet count was measured 8 +_ 5 times in the ARDS group and 7 +_ 6 in the non-ARDS group. The differences between groups were not significant when compared by the student t-test for unpaired data (Fig. 26). Figure 24 shows the total WBC data from the 16 patients who suffered trauma. The four who developed ARDS (Fig. 24A) a l l showed a f a l l in the WBC count in relation to the onset of ARDS with three of the four f a l l i n g below the normal range. None of the 12 who did not develop ARDS (Fig. 24B) showed a f a l l below this lower limit. Figure 25 shows that a l l three patients with sepsis syndrome who developed ARDS (Fig. 25A) f e l l below the accepted normal limit in relation to the development of ARDS. While this also occurred in four patients who did not develop ARDS (Fig. 25B), the majority of patients without ARDS did not show this change. Figure 26 shows the data from the miscellaneous group of 12 patients where two of the three that developed ARDS (Fig. 26A) showed a f a l l below the normal range and the third was at the lower limit of normal. Those who did not develop ARDS (Fig. 26B) failed to show this change. The ten ARDS patients f e l l to significantly lower WBC counts 3 3 (2.8+1.6 x 10 cells/mm ) than did the 30 non-ARDS patients (8.7 + 6.3) (p^s .01) (Fig. 27). Chi-square analysis shows that there was an association between a f a l l in WBC count and the diagnosis of ARDS (pis, .01) when the entire group of 40 patients was considered. However, this was not true for the 12 patients with the sepsis syndrome where the WBC f e l l in both ARDS and non-ARDS patients. Nevertheless, Table VI shows that a f a l l in the total WBC count to low levels is a sensitive (80%) and specific (87%) test to determine which patients in this setting w i l l develop ARDS. It also shows that the predictive value of a positive test is 67% and the predictive value - 36 -of a negative test 93%. Analysis of the changes in individual c e l l counts during the periods of maximal f a l l shows that the change was due to a decrease in both PMN and lymphocytes and not to hemodilution. Toxic granulation in PMN was reported in 40% of a l l smears examined in the ARDS group and in 20% of a l l smears examined in the non-ARDS patients. While this difference was s t a t i s t i c a l l y significant for the entire ARDS group (p<0.01) i t was not for the group with the sepsis syndrome where both ARDS and non-ARDS patients showed a high incidence of toxic granulation. 3 A thrombocytopenia (< 100,000 platelets/mm ) was observed in six of ten ARDS patients and 16 of 28 non-ARDS patients. While the platelet counts in ARDS patients tended to f a l l to lower levels (81,000 + 51,000 3 platelets/mm ) as compared to non-ARDS patients (111,000 + 93,000 3 platelets/mm ), this difference was not significant (Fig. 27). e. Blood Gases: The ARDS group had a low Pa0 2 of 50 + 17 mmHg while on an FI0 2 > 0.5 as compared to 92 + 19 mmHg for the non-ARDS group. While this difference in PaO^ was s t a t i s t i c a l l y significant (pj£ .05), there was no difference between the groups with respect to the highest FIO received, its duration or the duration on an F l O ^ i O - S . The lowest VaO^ on an F l O ^ i . 0.5 followed (n=5) or coincided (n=4) with the minimal WBC count in nine out of the ten ARDS patients. In the single exception, the most severe hypoxemia coincided with a f a l l from 25,400 cells to 7,100 c e l l s . After this point, arterial oxygen tension 3 improved while the WBC count continued to f a l l to 4,500 cells/mm . The arrows in Figs. 24,25 and 26 show that ARDS was diagnosed after the f a l l in - 37 -WBC in five patients, at the same time as the f a l l in WBC in four cases and before the f a l l in WBC in 2 cases. E. Discussion ARDS is defined as a c l i n i c a l syndrome in which patients with no prior history of lung disease suddenly present with severe dyspnea, hypoxemia, diffuse bilateral pulmonary inf i l t r a t i o n s and ' s t i f f lungs (2,8). Major precipitating factors include sepsis, shock and trauma (9.116,117) which share the potential for massive complement activation (97,118,119). A current hypothesis suggests that hypoxemia is a result of damage to the pulmonary parenchyma by complement-activated PMN (12,120). Hammerschmidt et a l . (97) have reported a strong correlation between complement activation and the development of ARDS and suggested that measurement of C5a may be useful in predicting the onset of this syndrome. Since complement activation frequently produces neutropenia (66,69,70), one might expect to find a neutropenia occurring in patients who are at risk for developing ARDS. The data reported here show that a lymphocytosis and a relative neutropenia is a common finding immediately after non-surgical trauma. A transient lymphocytosis has been reported after administration of epinephrine (105,106) and strenuous physical exercise (107,108). Nelson et a l . (121) have also shown that lymphocyte counts f a l l 1-4 hours after maximal 17-hydroxysteroid levels have been reached with IV infusion. As both catecholamines and corticosteroid levels are li k e l y to be high in our patient population (122,123,124,125), i t seems possible that they might account for both i n i t i a l rise and subsequent f a l l in lymphocytes. It is - 38 -also possible that the catecholamines are responsible for the eventual increase in the PMN (26,126) that are shown in Figures 9,11,13,16,22. While the i n i t i a l neutropenia observed in a l l three groups is not a feature of catecholamine release, i t may result from complement-mediated sequestration of PMN within the systemic and pulmonary microvasculatures (93,119,118). A number of investigators (2,12,120,97,90) have speculated that complement activation is involved in the pathogenesis of ARDS. In this study, ARDS developed in one patient in Group I, none in Group II and three patients with trauma in Group III. We found no correlation between the lymphocytosis and neutropenia seen on admission and the subsequent development of ARDS. We conclude that trauma is frequently associated with a lymphocytosis and relative neutropenia which quickly reverses to a relative lymphopenia and absolute neutrophilia within hours of admission to hospital. This phenomenon appears to be a nonspecific response to trauma and other acute stress reactions. It has no predictive value with respect to the subsequent development of the adult respiratory distress syndrome. The source of the unusually large number of circulating lymphocytes seen immediately after trauma remains to be determined. On the other hand, the data reported here show that the development of ARDS was associated with a f a l l in circulating white blood cells (Table VI). Although this decrease occurred sli g h t l y after the onset of ARDS in two cases, more frequent WBC measurements may have shown a pre-diagnosis neutropen i a . The higher incidence of toxic granulation in the PMN of ARDS patients suggests that the PMN have been stimulated (127). This is - 39 -consistent with the hypothesis that complement activation of the PMN plays a role in the pathogenesis of ARDS. If the f a l l in leukocytes was solely due to complement mediated sequestration of PMN, one would have expected to see a preferential f a l l in PMN as compared to lymphocytes. However, the data on individual cells shows that the f a l l in circulating leukocyte count was due to a drop in both PMN and lymphocytes. While a lymphopenia commonly develops in c r i t i c a l l y i l l patients (122,124-128), i t cannot be attributed to complement activation. Therefore more studies w i l l be required to determine the precise reasons for the f a l l in both types of c e l l s . Fein and associates (9) have reported that there is no correlation between WBC counts and the development of ARDS following septicemia. Instead, they found that there was a correlation between thrombocytopenia and developing ARDS. Our results are consistent with theirs in that we showed that there was no correlation between the f a l l in WBC count and ARDS in sepsis patients (Fig. 25). It is of interest that a l l three septic patients developing ARDS showed a f a l l in the WBC count (Fig. 25A). Four of the nine patients who did not develop ARDS showed a f a l l in WBC below 4,200 3 cells/mm . However, our data on platelets is not consistent with those reported by Fein et a l . because i t shows that thrombocytopenia was of no value in predicting the onset of ARDS. These data therefore suggest that frequent measurements of the WBC count may be useful in predicting the onset of ARDS in patients who are at risk of developing this syndrome. TABLE II SUMMARY OF TRAUMA PATIENT INFORMATION GROUP NO. AGE AND SEX ADMISSION DIAGNOSIS ARDS RISK FACTORS MAJOR SURGERY OUTCOME ARDS 1 29-M 2 26-M Fall from 40 feet Fall from 80 feet Pulmonary contusion Multiple major fractures Multiple transfusions Transient hypotension Multiple minor fractures subdural hematoma Laparotomy Died 48 days Discharged 9.5 days 3 57-M 4 13-F Struck by motor vehicle while crossing street Fall from a 180 foot bridge into salt water Pulmonary contusion Multiple major fractures Multiple transfusions Transient hypotension Pulmonary contusion Near drowning Multiple transfusions Prolonged hypotension Laparotomy Laparotomy Thoracotomy Bilateral Burr holes Discharged 68 days Died 1.2 days Non ARDS 1 31-M 2 50-M Fall from 20 feet Struck by Motor vehicle while crossing street Pulmonary contusion Transient hypotension Pulmonary contusion Multiple transfusions Subdural hematoma Transient hypotension Laparotomy Transferred 6 months Laparotomy Died 2.5 days Table II (cont'd) 3 17-M 4 33-M 5 25-M 6 52-M 7 2 7-M 8 15-F 9 " 60-M 10 2 7-M 11 22-M 12 56-M Struck by motor vehicle while walking on side-walk. Motor vehicle accident Fall from 60 feet Motor vehicle accident Multiple stab wounds Fall from 40 feet Fall from 60 feet Fall from 20 feet Motor vehicle accident Struck by motor vehicle Pulmonary contusion Multiple major fractures Multiple transfusions Transient hypotension Multiple minor fractures Subdural hematoma Transient hypotension Multiple major fractures Multiple transfusions Transient hypotension Pulmonary contusion Transient hypotension Multiple transfusions Transient hypotension Multiple minor fractures Intercerebral hematoma Pulmonary contusion Multiple minor fractures Multiple major fractures Multiple transfusions Multiple minor fractures Transient hypotension Pulmonary contusion Pulmonary contusion Multiple major fractures Multiple minor fractures Open Discharged Guillotine 59 days amputation le f t leg below knee Craniotomy Died 0.7 days Laparotomy Discharged 109 days Died 2.1 days Thoracotomy Discharged 21 days Died 5.5 days Discharged 79 days Craniotomy Died 5.7 days Thoracotomy Discharged 22 days Laparotomy Died 15 days TABLE III SUMMARY OF SEPSIS PATIENT INFORMATION GROUP NO. AGE AND SEX ADMISSION DIAGNOSIS ARDS RISK FACTORS MAJOR SURGERY OUTCOME ARDS Non ARDS 72-M 73-M 80-F 36-M 42-M 55-F 83-F 69-M Ascending cholangitis Gallstone ileus with small bowel Gallstone ileus Sepsis syncrome Transient hypotension Sepsis syndrome Transient hypotension Sepsis syndrome Transient hypotension Fecal peritonitis Sepsis syndrome due to small bowel obstruction Gangrenous left leg Sepsis syndrome Prolonged hypotension Toxic epidermal necrolysis; Group B streptoccal septicemia Ascending cholangitis -Ascending cholangitis Sepsis syndrome Transient hypotension Sepsis syndrome Sepsis syndrome Laparotomy Died 15 days Laparotomy Died 9 days Laparotomy Died 4 days Laparotomy Discharged 47 days Amputation Transferred of 73 days gangrenous l e f t leg Discharged 14 days Laparotomy Discharged 25 days Laparotomy Discharged 33 days Table III (cont'd) 6 79-F 7 67-M 8 28-M Perforated Sepsis syndrome Laparotomy Died 8 days duodenal ulcer with C a n d i d a peritonitis Peritonitis due to Sepsis syndrome small bowel necros is Peritonitis due to Sepsis syndrome small bowel inflammation Peritonitis Sepsis syndrome Laparotomy Discharged 67 days Laparotomy Discharged 13 days Laparotomy Discharged 31 days I I TABLE IV SUMMARY OF MISCELLANEOUS GROUP PATIENT INFORMATION GROUP NO. AGE AND SEX ADMISSION DIAGNOSIS ARDS RISK FACTORS MAJOR SURGERY OUTCOME ARDS 50-M Aspiration secondary to cardiac arrest Aspiration Transient hypotension Died 16 days 21-M Aspiration secondary to drug intoxication Aspiration Transient hypotension Discharged 9 days 42-M Pancreatitis Pancreatitis Laparotomy Discharged 36 days NON ARDS 57-M 88-M 40-F 58-M 63-M Pancreatitis Pancreatitis Transient hypotension Ruptured abdominal Multiple transfusions aortic aneurysm Transient hypotension Uncontrolled atri a l f i b r i l l a t i o n and abdominal pain Prolonged hypotension Recurrent aspiration secondary to cerebellar hemorrhage Recurrent aspiration Prolonged hypotension Near drowning Near drowning Atrial f i b r i l l a t i o n Laparotomy Discharged 26 days Laparotomy Discharged 10 days Discharged 13 days Died 8 days Discharged 2 days Table IV (cont'd) 55-M Hypoxic coma due Near drowning Died 2 days to salt water Transient hypotension near drowning 44-M Smoke inhalation Smoke inhalation Discharged Atrial f i b r i l l a t i o n 2 days 32-M Hypoxic coma due Aspiration Discharged aspiration of Near drowning 4 days noxious material Transient hypotension 68-M Upper Gl hemorrhage Prolonged hypotension Laparotomy Discharged 57 days I TABLE V Risk Factors in Group III Patients NO. SEX Age (Mean + SD) RISK FACTORS M F (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) Trauma ARDS 4 3 1 31 + 19 3 2 1 3 1 0 1 0 0 0 NO ARDS 12 11 1 35 + 16 7 4 5 5 0 0 0 0 0 0 Sepsis ARDS 3 2 1 75 + 4 0 0 0 0 0 0 0 0 3 0 NO ARDS 9 6 3 54 + 21 0 0 0 0 0 0 0 0 9 0 Miscellaneous ARDS 3 3 0 38 + 15 0 0 0 0 0 2 0 0 0 1 NO ARDS 9 8 1 56 + 17 0 0 0 1 3 2 3 1 0 1 (1) Pulmonary contusion (2) multiple major fractures (3) multiple minor fractures (4) multiple emergency transfusions (5) prolonged hypotension (6) aspiration of gastric contents or noxious substances (7) near drowning (8) inhalation of smoke (9) sepsis syndrome (10) pancreatitis. There were no differences in the number of risk factors between those who developed ARDS and those who did not (see text for further explanation). TABLE VI Sensitivity, Specificity, Positive and Negative Predictive Values of a Fall in WBC to Predict ARDS POSITIVE NEGATIVE ARDS NO ARDS SENSITIVITY SPECIFICITY PREDICTIVE -PREDICTIVE VALUE VALUE Fall in WBC* 8 4 80% 87% 67% 93% No f a l l in WBC 2 26 * A f a l l in WBC was a decrease to less than 4,200 cells/mm3. The relationship between a f a l l in WBC and ARDS was significant (p .05). - 48 -LEGENDS Figure 9 Group I WBC and Differential Count Changes. The lymphocytosis and neutropenia seen on admission reverses rapidly by the next observation 11.0 +^2.3 hours later and remains unchanged. WBC -total white c e l l count, LC = lymphocyte, PMN = segmented polymorphonuclear c e l l s , BAND=immature PMN Figure 10 Group I RBC, Hemoglobin and Hematocrit Changes. These data show that the changes seen in Fig. a were not due to hemoconcentration or dilution. RBC CONCENTRATION (count x 106/mm*) im - * IO HEMOGLOBIN (gms/dl) ' y 1 HEMATOCRIT (percentage value) 10 ca £i 01 o> o 9 9 9 9 COUNT X 107mm3 - 50 -Figure 11 Group II WBC and Differential Count Changes. The changes observed in the lymphocytes (LC) and polymorphonuclear leukocytes (PMN) are similar to those in Group I. Figure 12 Group II RBC, Hemoglobin and Hematocrit Changes. These data show that the changes seen in Fig. 11 were not due to hemoconcentration or dilution. RBC CONCENTRATION (count x 106/mm3) z HEMOGLOBIN (gms/dl) HEMATOCRIT (percentage value) -* ro co * i cn o> -<i o o o p o o o I I I " i i i - 52 -Figure 13 Group II Patients with I n i t i a l Lymphocytosis and Neutropenia; WBC and Differential Count Changes. Figure 14 Group II Patients with I n i t i a l Lymphocytosis and Neutropenia; RBC, Hemoglobin and Hematocrit Changes. RBC CONCENTRATION O l k __ 05 — — i — O l = iiiiniiiiiiiiiiiiiiiiiiiiiiiiimin ^  -» ro (count x 10/mm3) Ul •k. HEMOGLOBIN (gms/dl) i o u ^ n o ) > g ( D i D O J i o u » u i HEMATOCRIT (percentage value) ro w -ik oi 0) "»j COUNT X 1 0 W ' ' i - 54 -Figure 15 Group III Trauma Patients WBC and RBC Changes. The numbers refer to the number of patients remaining at each day of the study. The data show no change in total WBC or RBC. Figure 16 Group III Trauma Patients Lymphocyte and PMN Count Changes. There was a relative lymphocytosis (LC) and neutropenia (PMN) on admission, followed by a f a l l in lymphocytes and rise in PMN so that the total WBC did not change. - S5-WBC •—• RBC • — • MEAN ± S E 20.0 T 18.0 § | 16.0 ° o 14.0 . f x X _ h 1 2 . 0 Z z O § ioo -O o o o 8 0 • 2 CD $ K 6.0 • 4.0 2.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 DAYS AFTER ADMISSION 4.0 4.5 5.0 PMN • — • LC • — • MEAN + SE 20.0 "E 18.0 E 16.0 o X 14.0 • (-z 12.0 -O o 10.0 , HI 8.0 o 6.0 4.0 2.0 -0.0 0.0 G.5 1.0 1.5 2.0 2.5 3.0 3.5 DAYS AFTER ADMISSION 4.0 4.5 5.0 - 56 -Figure 17 Group III Trauma Patients Mature and Immature PMN Count Changes. Figure 18 Group III Sepsis Patients WBC and RBC Count Changes. The numbers refer to the number of patients remaining at each day of the study. The data show that WBC count fal l s soon after admission to hospital and that RBC counts do not change. - 5 ? -SEG » BAND o-o MEAN + SE E E X 3 o o z 0_ 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION Reel'- : : : M E A N ± S E 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 D A Y S A F T E R A D M I S S I O N - 58 -Figure 19 Group III Sepsis Patients Lymphocyte and PMN Count Changes. There was an absolute neutrophilia and absolute lymphopenia on admission, followed by a f a l l in polymorphonuclear leukocytes and no change in lymphocyte counts. Figure 20 Group III Sepsis Patients Mature and Immature PMN Count Changes. - 51 -s E X h-z 3 o o LU O MEAN ± SE .0 0.5 1.0 1.5* 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION 10.0 9.0 E 8.0 E o 7.0 X 6.0 h-Z 3 5.0 o o 4.0 z 2 3.0 2.0 1.0 0.0 - 4 - . 10 10 SEG • — • BAND MEAN + SE 4-'° t-4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION - 60 -Figure 21 Group III Miscellaneous Patients WBC and RBC Count Changes. The numbers refer to the number of patients remaining at each day of the study. The data show that there was no change in RBC counts and a slight increase in WBC counts after admission to hospital. Figure 22 Group III Miscellaneous Patients LC and PMN Count Changes. PMN counts increase while lymphocyte counts decrease after admission to hospital. - 6| -F?BC M ^ A N r SE 20.0-, 18.0-" £ 16.0 E E "o "o 14.0-x x 12.0 i - h-3 3 10.0 O O *8 o o 8.0 O O CD CD § CC 6.0 4.0 2.0 0.0 •7- i . 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION PMN 20.0 18.0 16.0 -J E c 1 4.0 o 12.0 x 2 10.0 -8 8.0-1 LC MEAN ± SE LU o 6.0 -| 4.0 2.0 A 0.0 i 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION - 62 -e E o X h-Z r> o o 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 < > 8 SEG •—•• BANO MEAN ± SE 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 DAYS AFTER ADMISSION Figure 23 Group III Miscellaneous Patients Mature and Immature PMN Count Changes. Mature (SEG) and immature (BAND) polymorphonuclear leukocytes transiently increased after admission to hospital. - 63 -Figure 24A Total WBC Count in Trauma Patients who Developed ARDS. Arrow indicates time of diagnosis of ARDS. Numbers (1,2,3, etc.) indicate each individual case. * indicates presence of toxic granulation. + indicates patient died. Figure 24B Total WBC Count in Trauma Patients who did not develop ARDS. * indicates presence of toxic granulation. + indicates patient died. - 6* -25.0 J 0.0 0.75 1.50 2.25 3.0 3.75 4.5 5.25 6.0 6.75 7.5 DAYS AFTER ADMISSION f) n J 0.0 0.75 1.50 2.25 3.0 3.75 4.5 5.25 6.0 6.75 7.5 DAYS AFTER ADMISSION - 65 -Figure 25A Total WBC Count in Sepsis Patients who Developed ARDS. Symbols as in Figure 24. Figure 25B Total WBC Count in Sepsis Patients who did not develop ARDS. Symbols as in Figure 24. - 66 -0.0 0.75 1.50 2.25 3.0 3.75 4 .5 5.25 6.0 6.75 7.5 DAYS AFTER ADMISSION DAYS AFTER ADMISSION - 67 -Figure 26A Total WBC Count in Miscellaneous Patients who developed ARDS. Note that WBC Counts f e l l below the lower limit in two of three patients. Symbols as in Figure 24. Figure 26B Total WBC Count in Miscellaneous Patients who did not develop ARDS. Note that the WBC count did not f a l l below the lower limit in any of these patients. Symbols as in Figure 24. - 6* -0.0 0.75 1.5 2.25 3.0 3.75 4.5 5.25 6.0 6.75 7.5 DAYS AFTER ADMISSION 25.0 n 2.5 A 0 0 0!75 1.50 2.25 3!0 3!75 4.5 5.25 6.0 6i75 7.5 DAYS AFTER ADMISSION - 69 -Figure 27 Number of Blood Tests Ordered and Lowest Absolute Values Obtained for Patients who did and did not Develop ARDS. There was no difference between the ARDS and Non-ARDS groups with respect to the number of tests ordered. The lowest RBC and PLT values were not s t a t i s t i c a l l y different, however WBC and PMN f e l l to significantly lower levels in the ARDS group of patients. LOWEST ABSOLUTE VALUE - 71 -ANIMAL STUDIES A. Overview The results of the c l i n i c a l studies indicate that a lymphocytosis with relative neutropenia is a common finding after nonsurgical trauma and that the development of ARDS was associated with a marked f a l l in circulating white blood cells (WBC). The primary purpose of these animal experiments was to develop an experimental model for these c l i n i c a l observations. Our basic hypotheses are: 1) the lymphocytosis and relative neutropenia arises because elevated catecholamine levels increase both PMN and lymphocyte counts while trauma-associated activation of the complement system causes only PMN to be sequestered, 2) the acute f a l l in WBC associated with ARDS is due primarily to complement-mediated sequestration of PMN within the pulmonary microvasculature, and 3) the lowered pulmonary blood flow associated with some shock states contribute to these leukocyte changes by enhancing leukocyte sequestration within the lung. Several studies have shown that a large, dynamic leukocyte reservoir exists within the lung (27,30,31,34,129). This reservoir consists of cells that have temporarily dropped out of the axial stream into the marginal stream adjacent to the vascular endothelium (22). Since these marginated cells can also re-enter the axial stream, there must be a dynamic equilibrium between circulating and marginating leukocyte pools within the pulmonary vasculature. The i n i t i a l animal studies for this thesis were designed to determine whether or not both PMN and lymphocytes are taken up by the marginated pool during a period of low flow and whether they are returned to the circulation when flow is restored or increased. A recent study by - 72 -Martin et a l . examined the behavior of labelled polymorphonuclear leukocytes (PMN) during a single pass through the pulmonary circulation (29). These data showed that 80-90% of labelled PMN were delayed in a single pass through the lung and that this delay was inversely proportional to regional pulmonary blood flow. They also showed that decreasing total pulmonary blood flow by lowering cardiac output produced arterial-venous (A-V) leukocyte differences across the lung. Another object of this thesis was to determine i f activation of the alternate complement pathway with cobra venom factor (CVF) produces arterial-venous (A-V) leukocyte differences across the lung and whether this A-V difference can be influenced by changes in pulmonary blood flow. Indeed, other investigators have shown that activation of the alternate complement pathway is associated with acute neutropenia, permanent sequestration of PMN within the pulmonary microvasculature and increased permeability edema (61,66,69,70,71,90). In addition, the possible effect of WBC sequestration on gas exchange was followed by monitoring arterial blood gases, throughout each experiment. The study shows that large numbers of leukocytes become sequestered within the lung when pulmonary blood flow is low, and that an equivalent number of cells are released from the lung after flow is restored. Both PMN and lymphocytes were taken up by the lung when pulmonary blood flow was reduced, but the lung took up a greater number of PMN than lymphocytes. Activation of complement had no effect on cardiac output or RBC but was associated with a marked f a l l in PMN counts. Lowering of PBF after or prior to activation of complement appeared to enhance the complement-mediated uptake of PMN by the lung. Epinephrine infusion increased cardiac output, - 73 -ANIMAL MODEL: DOG N=40 COBRA VENOM FACTOR N=5 Figure 28 Outline of the Animal Studies. - 74 -white blood c e l l counts and red blood c e l l counts. B. Materials and Methods 1. Animals A total of 6 different sets of experiments were done on anesthetized mongrel dogs (Figure 28). The f i r s t set of control experiments was done on seven mongrel dogs weighing 24 +_ 6 kg (mean +_ SD) . The second set of experiments examined the effect of both a transient and a prolonged decrease in PBF on white blood c e l l uptake and release by the dog lung. Five mongrel dogs weighing 22 +_ 3 kg were used in the transient low PBF experiments; and six mongrel dogs weighing 25.5 + 6 kg were used in the prolonged low PBF experiments. The third set was designed to examine the effect of activating complement with cobra venom factor on leukocyte uptake into the dog lung. Five mongrel dogs weighing 29 + 9 kg were used in these studies. The fourth set was designed to examine the effect of activating complement and then lowering PBF on leukocyte uptake into the dog lung. Extravascular lung water and blood levels of complement protein were also measured. Six mongrel dogs weighing 20 +_ 3 kg were used in these studies. The f i f t h set examined the effect of lowering PBF and then activating complement on leukocyte uptake into the dog lung. Five mongrel dogs weighing 24 +_ 5 kg were used in these experiments. The sixth set examined the effect of epinephrine infusion and complement activation on leukocyte release and uptake into dog lungs. Six mongrel dogs weighing 19 +_ 4 kg were used in these experiments. 2. Surgical Procedure A l l animals were anesthetized using sodium pentobarbital (20-25 - 75 -Central Circulation of the Dog Pulmonary Artery — c o d i a c output — b lood pressure Right Atrium -CVF Right Ventricle — b l o o d sampling Inferior Vena Cava -~ — b a l l o o n catheter Aorta — b l o o d sampling Pulmonary QJ Circulation Pulmonary Vein < i l l Diaphragm j Femoral Vein — epinephrine Systemic Circulation Femoral Artery — b l o o d pressor* Figure 29 Schematic Representation of the Canine Central Circulation. The various catheterization sites are shown in this figure. - 76 -mg/kg), intubated and placed in the supine position, spontaneously breathing room a i r . Anaesthesia was maintained using intermittent injections of sodium pentabarbital. Via the right jugular vein and under fluoroscopic control, a blood sampling catheter was inserted into the right ventricle and a Swan-Ganz Special Animal Thermodilution Catheter (model 702-027-7 Fr, Edwards Laboratory, Santa Anna, California) was inserted into the pulmonary artery (Fig. 29). The right ventricular catheter was used to sample mixed venous blood, and the Swan-Ganz thermodilution catheter was used to measure cardiac output and pulmonary arterial blood pressure. Catheters were also placed into the aortic root via the left carotid artery to sample systemic blood and in the lef t femoral artery to measure systemic arterial blood pressure. To prevent excessive clot formation, a l l catheters were periodically flushed with heparinized saline (1000 units heparin:100 ml saline). In the experiments where PBF was lowered, the right femoral vein was cannulated with an Edwards Thrombectomy catheter (model 32 080-8/10F, liquid volume 8ml) placed into the inferior vena cava just above the diaphragm so that venous return could be reduced by inflating the balloon. In the last set of experiments a simple catheter was inserted into the right femoral vein. Through this catheter 2-20 micrograms/rain (adrenalin chloride solution 1:1,000-TM Parke, Davis & Company) epinephrine was infused using a Harvard pump for 20 minutes. In a l l dogs studied a single bolus of Heparin sodium (100 u/kg) was administered at the end of surgery. Pulmonary artery blood pressure (Ppa), Pulmonary wedge pressure (Pw) and Systemic blood pressure (Pa) were a l l recorded on an 8-channel Hewlett-Packard recorder (Model 9800) using Hewlett-Packard transducers (model 1290C) where the zero level of the transducer was referenced between - 77 -the mid-thorax and the back of the dog. Cardiac outputs were measured using a thermodilution technique (130). 3. Hematological Measurements Blood was sampled simultaneously from the right ventricle (RV) and aortic root (Ao) manually or with a rotating collecting device (see Experiment Procedures). Whenever samples were collected manually, catheters were f i r s t flushed with several mis of heparinized saline, then blood was drawn back into a 20 ml syringe. This syringe was removed and replaced by a 3 or 5 ml syringe into which 2 or 4 mis of blood, respectively, was drawn. Two ml was collected into dry 5 ml tubes that contained ethylenediaminetetraacetic acid (EDTA) and the remainder was kept for measuring complement protein levels. Leukocyte and erythrocyte counts for each blood sample were calculated using a Coulter Counter (model S). WBC differential values for individual c e l l types were obtained from smears made of each blood sample stained with Wright's stain and examined under, o i l with the compound microscope. The differential counts were determined by various observers who counted 100-400 leukocytes per smear depending on the particular experiments. The greatest source of error was in differentiating monocytes from large lymphocytes: The small numbers of monocytes, eosinophils and basophils was sr.ch that small errors in classification or in counting would lead to relatively large errors in results. For this reason, and because no consistent or significant changes were ever observed for these c e l l populations, the detailed results of these - 78 -data are not included in this paper. Instead, only the results obtained for PMN and lymphocytes are discussed. To minimize large interanimal variations, blood pressure, cardiac output, WBC, PMN, lymphocyte and erythrocyte values were expressed as a percent of control which was obtained by dividing a l l values by the i n i t i a l value (right ventricular values in the case of hematologic parameters) obtained during the control period. Raw hematologic data are presented separately in Table VII-XVI. 4. Measurement of Regional Pulmonary Blood Flow In order to measure the effect of complement activation on alveolar membrane permeability, ^ C r tagged red blood cells (RBC) were injected to mark the intravascular volume; and a blood sample was obtained 5 minutes later. The animal was then sacrificed by injecting a bolus of saturated KC1 into the aortic root. After the thorax was opened, the pulmonary vessels were tied by opening the pericardium, l i f t i n g up the heart, and placing a snare around the pulmonary artery and veins. The lungs were quickly removed, separated from the heart, inflated to 30 cm H^ O by constant air pressure and frozen in an insulated box over liquid nitrogen. The lungs were cut coronally into 2 cm slices from the posterior to anterior surface using a band saw. Each slice was placed on a slab of dry ice and divided into small samples which were put into preweighed s c i n t i l l a t i o n v i a l s , counted in a gamma counter (Beckman Model 7000), weighed, dried in an oven at 65°C until a l l the water had been removed and weighed again. These data were then used to calculate regional pulmonary blood flow and - 79 -extravascular lung water using a previously described technique in which extravascular water and the blood flow to each lung sample are expressed per gram of dry blood free tissue (131). 5. Complement Activity Plasma levels of complement components were determined in blood samples taken throughout the various experiments. The blood samples were placed into tubes containing EDTA in 1:10 dilution and were centrifuged at 600 g at 4°C for 20 minutes. The plasma was drawn off, quickly frozen to -70^C, coded and shipped in dry ice to the Kaiser Medical Center Immunology Laboratory, Los Angeles, California. The samples were used to determine CH^, and Cfi (132,133). The results were sent back to Vancouver where the code was broken. 6. Cobra Venom Factor (CVF) Lyophilyzed cobra venom, Naje naje, was purchased from the Ross Allen Reptile Institute, Silver Springs, Florida, and was purified by diethylaminoethyl (DEAE) cellulose chromatography followed by Sephadex G-100 chromatography instead of G-200 as described by Cochrane et al (134). Naja naja "cobra venom factor" (CVF) is a 144,000 dalton protein which interacts with the complement system by binding to a serum protein, Factor B. This interaction is in the form of a reversible, Mg + + dependent, stoichiometric complex (1:1) (Fig. 30). While in this bound state, Factor B is recognized and cleaved by a plasma serine esterase, D, yielding the active D , C convertase complex, CVFBb. CVFBb in turn, - 80 -acts to cleave and C,., thereby triggering activation of both the alternate and common complement pathways. The CVFBb complex slowly dissociates in an irreversible fashion. When this occurs, CVF is free to combine with another molecule of factor B resulting iii cleavage of more C^ and C 5 (153). Immunochemical studies indicate that CVF i s , in fact, cobra C^b (or a derivative) secreted by the snake's venom gland. CVF differs from mammalian C^b in that i t s complex with factor B is very stable (long t 1/2), i t is not attacked by the control protein, C^b inhibitor, and i t activates and consumes both C^  and C^ (153,154). The activity of CVF (64 units/ml) was determined by a method similar to that described by Cochrane et al (134). Briefly, 0.1 ml of dilutions of CVF were added to 0.25 ml of 1/25 diluted normal human serum (NHS) and 0 8 incubated for 20 min. at 37 C. Then 0.5 ml of 2.5 x 10 /ml sensitized sheep erythrocytes (EA) were added, plus 0.6 ml Veronal-buffered saline, and these were incubated for 30 min at 37°C. Finally 0.5 ml gel was added, the c e l l pellet centrifuged and the hemolysis assessed spectrophoto-metrically at 541 nm. The amount of CVF that inhibited hemolysis by 50% was considered 1 unit. 7. Experimental Procedures a. Control: The effect of simultaneously sampling blood from the right ventricle and aorta on systemic leukocyte counts and arterial-venous differences was determined in 7 control dogs. In 3 dogs these samples were - 81 -MECHANISM OF ACTION OF COBRA VENOM FACTOR CVF + B- •CVF,BB + BA C6 C7 C8 C9 C5-9 I N T R I N S I C DECAY OR C3B INHIBITOR ACCELERATOR C3B + Bi rt • C3B INHIBITOR I C3c * G D -P,(C3B)9,BB I I • I N T R I N S I C DECAY OR C3B INHIBITOR ACCELERATOR I P,C3B + Bi C3B INHIBITOR • P,C3c + C3D Figure 30 Outline of the Mechanism of Action of Cobra Venom Factor. - 82 -manually taken at 0,6,30,50,60,90,120 and 150 minutes. In the other 4 dogs blood samples were taken at least every 2 minutes (see Table VII and VIII) for 50 minutes, and also at 60,90,120 and 150 minutes. Systemic pulmonary arterial and pulmonary wedge pressures were continuously recorded; cardiac output was measured; and blood gases sampled at regular intervals during each experiment. b. Transient Lowering of Pulmonary Blood Flow: The effect of simultaneously sampling blood from the right ventricle and aorta on systemic leukocyte counts and arterial-venous differences while pulmonary blood flow was transiently lowered was examined in 5 experimental animals. Three 4-minute experimental runs were carried out for each of the five dogs. Each run consisted of five parts, namely; a control period (10 seconds), a period of rapid blood pressure decline (50 seconds) produced by IVC balloon inflation, a period of steady low blood pressure (90 seconds), a period of rapid blood pressure increase (40 seconds) caused by IVC balloon deflation, and a final control period (50 seconds). During the f i r s t run both blood pressures and cardiac outputs were measured. In the second run, blood samples were collected simultaneously from the right ventricle and aortic root every 10 seconds into dry 5ml EDTA vacutainer tubes using a rotating collecting device (29). Blood pressure was monitored throughout this run but i t was impractical to measure cardiac — output. A third run was therefore performed where both blood pressure and cardiac output were measured throughout the 4-minute interval. By performing runs before and after the blood collection run, we were able to determine the vascular response to balloon inflation and - 83 -deflation. The run in which the blood pressure pattern best matched the blood pressure pattern from the blood collection run (#2) was used to estimate cardiac output for the blood collection run. In the ideal situation, the time axis would have been the same for a l l experiments. However, the BP changes due to inflation and deflation of the IVC balloon did not always occur exactly at the same time or same rate. In order to relate the two CO runs (#1 and #3) to the blood sampling run (#2) for a l l the animals, run #2 from the f i r s t dog was ar b i t r a r i l y chosen as a standard reference run. The periods of time taken for each of the five parts of this run (see above) were used to adjust the corresponding parts of a l l other runs. This was done by multiplying the time spent in each of the five parts of the run by an appropriate constant so that they would a l l occupy the same number of units on the x axis. In this way we were able to collect BP, CO and A-V leukocyte count information in a manner that was suitable for s t a t i s t i c a l analysis. The WBC counts and differential values were used to calculate the number of PMN or lymphocytes in each of the 10-second blood samples. By subtracting pulmonary venous leukocyte counts from pulmonary arterial leukocyte counts, PMN and lymphocyte A-V differences were obtained. The number of WBC taken up or released by the lung was calculated using the raw data from each animal and multiplying A-V leukocyte differences for WBC by corresponding cardiac output values. The areas under these curves were measured to determine the net total number of WBC taken up and released from the lung during the experimental procedure. c. Prolonged Lowering of Pulmonary Blood Flow: The effect of simultaneous blood sampling from the right ventricle and aorta on systemic - 84 -leukocyte counts and arterial-venous differences while pulmonary blood flow was lowered for extended periods of time was examined in 6 dogs. One run was performed on each dog in which the IVC balloon was inflated for an extended period of time. During the run, systemic arterial blood pressure was monitored; and blood samples were drawn. Three dogs sustained cardiac arrests and died after 9.5, 19 and 57 minutes respectively, of lowered pulmonary blood flow. In the remaining 3 dogs, the runs were simply stopped after 25 minutes (2 animals) and 15 minutes (one animal). In a l l but one animal, blood samples were manually taken at one minute intervals for 5 minutes and then at 2 minute intervals for at least 10 minutes. The exception was the one dog who died after 9.5" minutes of lowered pulmonary blood flow. Systemic pulmonary arterial pressures were continuously recorded. d. Cobra Venom Factor: The effect of simultaneous blood sampling from the right ventricle and aorta on systemic leukocyte counts and arterial-venous differences after administering purified cobra venom factor was examined in 5 dogs. Blood samples were manually taken at least every 2 minutes (see Table XI) for 50 minutes, and also at 60,90,120 and 150 minutes. At 30 minutes 1 ml of purified cobra venom factor was injected in the right a t r i a l port of the Swan-Ganz catheter. Systemic, pulmonary arterial and pulmonary wedge pressures were continuously recorded. Cardiac output and arterial blood gas tensions were measured at regular times during ea ch exper iment. e. Cobra Venom Factor Followed by Lowering of Pulmonary Blood Flow: The effect of low PBF in concert with CVF on the uptake and release of WBC from the lung was determined by obtaining simultaneous samples from the RV - 85 -and aorta during 2 runs in each of 6 experimental animals. In the f i r s t run, these samples were taken manually every 30 seconds during a 4-minute control period, a 6-minute period when cardiac output was reduced by inflating the inferior vena cava (IVC) balloon catheter .and a 5-minute recovery period and the IVC balloon had been deflated. To determine the effect of prior complement activation on this maneuver, a second run was performed which was exactly the same as the f i r s t except that 1 ml of purified cobra venom factor was injected in the right a t r i a l port of the Swan-Ganz catheter at the 2-minute point of the i n i t i a l 4-minute control period. Systemic blood pressure was continuously recorded. CO and arterial blood gas tensions were measured at regular intervals during each run. After these two runs, the dog was monitored over a 2-hour period with blood samples being obtained in the same fashion every 30 minutes. Extravascular lung water and plasma levels of complement components were also measured. f. Lowering of PBF Followed by Cobra Venom Factor: The effect of simultaneous blood sampling from the right ventricle and aorta on systemic leukocyte counts and arterial-venous differences after lowering pulmonary blood flow and then injecting purified cobra venom factor was examined in 5 dogs. Blood samples were taken manually at least every 2 minutes for 50 minutes, and also at 60, 90, 120 and 150 minutes. At 25 minutes cardiac output was reduced by inflating the inferior vena cava (IVC) balloon catheter; at 30 minutes 1 ml of purified cobra venom factor was injected in the right a t r i a l post of the Swan-Ganz catheter; and at 35 minutes the IVC balloon was deflated. Systemic, pulmonary arterial and pulmonary wedge pressures were continuously recorded. Cardiac output and arterial blood gas tensions were measured at regular times during each experiment. - 86 -g. Epinephrine Infusion and Cobra Venom Factor: The effect of simultaneous blood sampling from the right ventricle and aorta on systemic leukocyte counts and arterial-venous differences after infusing epinephrine and then injecting purified cobra venom factor was examined in 6 experimental animals. Blood samples were taken manually at least every 2 minutes (see Table XVI) for 50 minutes, and also at 60, 90, 120 and 150 minutes. At 10 minutes, a 2-24 Mg/min (mean: 11.3 + 7 Mg/min) infusion of epinephrine was started and run continuously until 50 minutes. At 30 minutes, 1 ml of purified cobra venom factor was injected in the right a t r i a l port of the Swan-Ganz catheter. Systemic, pulmonary arterial and pulmonary wedge pressures were continuously recorded. Cardiac output and blood gases were measured at regular times during each experiment. C. Statistical Methods An analysis of variance using t-test on selected contrasts (111) was used to test for differences between control, and various study periods for each set of experiments. A multiple comparison s t a t i s t i c a l test was chosen because of the internal consistency of the data; that i s , each variable (cardiac output, blood pressure, WBC and RBC counts) always responded to balloon inflation and deflation, cobra venom factor, and epinephrine infusion in a consistent manner. A Student's t-test was used to examine the data for differences in hematologic or hemodynamic values at given times in different studies. The mean values are given +_ SE unless otherwise noted. D. Results 1. Control . The changes which occurred in blood pressures, cardiac output and - 87 -PO^  of arterial blood are shown in Figures 31 and 32. These data show that simultaneously sampling blood from the right ventricle and aorta has l i t t l e effect on these parameters. Although cardiac output f e l l to 76% +_ 6% of the control value( P^.05), pulmonary arterial blood pressure, pulmonary wedge pressure and systemic blood pressure were basically unchanged at the end of the 150 minute run (also see Tables XXII to XXVI). Table VII, VIII, XVII and Figure 33 show the right ventricle and aorta white blood c e l l (WBC) counts for the 7 experiments. Simultaneous sampling of blood from the right ventricle and aorta had l i t t l e effect on WBC counts during the f i r s t 50 minutes of the experimental run but thereafter there was a steady and significant increase in systemic WBC counts until the experiments were ended at 150 minutes. Analysis of the differential data (Table VIII, IX, XIX and XX) indicates that lymphocytes f e l l while polymorphonuclear counts increased over the course of these experiments. Consistently significant arterial-venous differences did not develop in these control animals. Figure 34 and Table VII, VIII, and XVIII show right ventricle and aorta red blood ce l l counts for a l l 7 experiments. In contrast to the WBC data, simultanous sampling of blood was not associated with marked changes in RBC counts. There was a tendency for RBC counts to f a l l during the rapid sampling periods (Table VIII, XVIII) which probably reflects hemodilution due to cardiac output measurements and catheter flushing with saline prior to each blood sampling. 2. Transient Lowering of Pulmonary Blood Flow The changes which occurred in blood pressure and cardiac output are - 88 -shown in Figs. 35,36 and 37. These data show that inflating the IVC balloon resulted in a decrease in pulmonary arterial blood pressure from 13 _+ 1 to 0.5 +_ 0.2 mm Hg with a return to control values when the balloon was deflated. Systemic blood pressure f e l l to 17 + 2% and rose to 79 +_ 5% of the control value with this manoeuver. Cardiac output f e l l to 21 + 2% of the control value in the low flow period, rose to 123 _+ 9% of the control value 40 sec after the balloon was released, and then f e l l to 80 +_ 3% of the control value at 4 minutes. Figure 38 and Table IX show the right ventricle and aorta leukocyte counts for the five experiments. Inflation of the IVC balloon was associated with a f a l l in right ventricle leukocyte counts to 75 _+ 3% control (p^O.OOl) while aorta leukocyte levels f e l l much more abruptly to 44 _+ 5% control level (p,£0.001). While aortic leukocyte counts f e l l below 95% control levels within 10 seconds of balloon inflation, an additional 30 seconds passed before right ventricle leukocyte counts had fallen to 95% of their control. With balloon deflation there was an immediate increase in both aorta and right ventricle leukocyte counts, with the rate of increase being much greater in the aortic blood. While right ventricle leukocyte levels gradually increased back towards control values, aorta leukocyte counts rose transiently to 127 _+ 6% (p^O.001) of the control value. In four of five animals both peak cardiac output and peak aortic leukocyte count levels occurred at the same time. Figure 39 shows the mean results of the differential counts done on blood smears obtained from a l l five experiments where PMN and lymphocyte A-V differences are plotted against time. These data show that a significant A-V leukocyte difference develops only when PBF is reduced by inflating the - 89 -IVC balloon and that i t is the differences in PMN counts which accounts for most of the leukocyte A-V difference. Lymphocytes constitute the next largest fraction of the leukocyte population but Fig. 39 shows that differences in lymphocyte numbers account for a relatively small portion of the total A-V difference. The data obtained by multiplying A-V differences by the cardiac output values (Fig. 40) show that there is a steady rate of leukocyte accumulation into the lung. Interestingly, the greatest rate of ce l l accumulation occurred in the 20-30 seconds after balloon inflation and deflation, that i s , when the pulmonary microvasculature was being derecruited and recruited respectively. A transient net expulsion of sequestered leukocytes occurred when pulmonary blood flow was fully restored. From Figure 40 the ratio of number of WBC released to the number of leukocytes taken up by the lung for individual experiments was calculated. Although there is some v a r i a b i l i t y between animals, the average shows that 99 +_ 7% of the cells taken up when flow was lowered were released after flow was restored. Figure 41 and Table IX show right ventricle and aorta erythrocyte counts for a l l five experiments. In contrast to the WBC data, lowering pulmonary blood flow has only a minor effect on the erythrocytes. Figure 42 shows the PMN difference across the lung plotted against total blood flow. The PMN difference was expressed as (RVs - AORTA^) %. Blood flow is expressed per gram of wet lung. RVc The data from a l l 5 dogs was plotted and then a computer derived line of best exponential f i t was plotted through the data. The line of best f i t was - 90 -estimated by a reiterative f i t t i n g procedure to an exponential function y=A+Be~CX (where A + B = 100). The data show that the A-V difference for PMN begins to increase when blood flow f a l l s below 7 ml/min/gm and sharply increases when blood flow f a l l s below 4ml/min/gm. 3. Prolonged Lowering of Pulmonary Blood Flow Figure 43 and Table X shows data from 5 experiments where balloon inflation was maintained for at least 15 minutes so that cardiac output remained low. These data show that there is a gradual diminution of the A-V leukocyte difference with both the right venticular and aortic leukocyte counts tending to rise from seven minutes onward. Figure 44 shows the data for a single experiment where the animal lived for more than 50 minutes. In this experiment the leukocyte A-V difference disappeared completely by 40 minutes and there was a definite increase in the WBC count in both right ventricular blood and aortic blood. The onset of cardiac arrest was associated with the development of a new large A-V difference while cardiac massage and IVC balloon deflation was associated with the appearance of large numbers of leukocytes within aortic blood, indicating leukocyte release from the lung. Figure 45 shows right ventricle (RV) and aorta (Ao) WBC counts in the 3 dogs who had their runs terminated without developing cardiac arrest. Figure 46 shows the mean RV and Ao leukocyte counts in the 3 dogs who developed cardiac arrest during the period of lowered pulmonary blood flow. These data show that in a l l 3 dogs cardiac *The subscript s refers to the samples obtained at various intervals during an experimental run whereas subscript c refers to the control RV value which was used in each experiment to standardize the data. - 91 -arrest was associated with the onset of new large A-V differences. In two of these animals cardiac massage was instituted and the mean RV and Ao leukocyte count changes are shown in Fig. 47. These data show that in both animals cardiac massage was associated with the appearance of large numbers of leukocytes within aortic blood, indicating leukocyte release from the lung. 4. Cobra Venom Factor The hematologic and hemodynamic changes which occurred after cobra venom injection are shown in Figure 48 to 51, Table XI and XVII and XVIII. Changes in blood pressure, pO^ and cardiac output were indistinguishable from those reported in the control group; namely, cardiac output f e l l to 75 _+ 13% of the control value towards the end of the experiment while pulmonary arte r i a l blood pressure, pulmonary wedge pressure and systemic blood pressure f e l l only slightly over the course of the experiment. The pO^ of arterial blood did not change significantly. Figure 50 and 51, and Table XI, XVII to XXI show the right ventricle and aorta leukocyte and erythrocyte counts for the 5 experiments. Prior to injection of CVF, both WBC and RBC values had fallen to about 90% of the control value. Injection of cobra venom factor resulted in an abrupt f a l l in aortic WBC counts to 55 +_ 13% of the control value (34 minutes) while RBC counts were unchanged (93 +_ 5% control value). The f a l l in WBC was due to loss of PMN from circulating blood. Aortic PMN counts f e l l to 48 +_ 15% of the control levels while lymphocyte counts were unchanged (92 +_ 21% of control). Slight but consistent A-V leukocyte differences were observed following injection of cobra venom factor indicating sequestration of PMN within the pulmonary microvasculature. - 92 -5". Cobra Venom Factor Followed by Lowering of Pulmonary Blood Flow The results in Tables XIIA, and Figures 52,53 show that during the f i r s t run systemic white blood c e l l count f e l l when blood flow was lowered and returned towards control values when blood flow was restored (Pfy .05). During the second run cobra venom factor caused systemic WBC to f a l l ( p i 0.05) without changing cardiac output (Figure 54,55 and Table XILB). Although there was not sufficient time for plateauing to occur after CVF was given, the data suggest that lowering cardiac output by inflating the IVC balloon enhances WBC arterial-venous differences (Figure 56). Immediately after blood flow was restored, WBC and PMN counts remained low but gradually returned to control values approximately two hours after CVF injection. Table XIII also shows that lowering the blood flow in the f i r s t run was associated with an increased uptake of WBC by the lung due mainly to an uptake of PMN. During run II, CVF injection was associated with a marked increase in uptake of PMN by the lung, even though there was no change in cardiac output. The fractional uptake of PMN was enhanced by reducing blood flow and remained high when blood flow was restored after CVF injection. Table XIV shows that CVF injection produced minimal changes in CH^Q, or Cg. Since the cobra venom factor forms a CVF B activated C3 convertase (134), i t should result in cleavage of C5, C6 and remaining components. The lack of an effect on CH^ 0 activity indicates less than 90% C3 fixation and shows a relatively small amount of complement activation. Measurement of C3 directly would have provided a better index of absolute alternative pathway activation but these measurements are d i f f i c u l t in the dog. - 93 -Table XV shows that neither lowering blood flow nor injection of CVF had any effect on gas exchange. The f a l l in PCO^ from 40 +_ 6 to 31 + 2 and the rise in P0 2 from 89 + 12 to 103 + 9 is attributed to mild hyperventilation in these anesthetized spontaneously breathing dogs. The pulmonary extravascular water measured at the end of the experiment was 3.42 +_ 0. 3 grams of water/gm of dry blood-free tissue which is well within the normal range. 6. Lowering of Pulmonary Blood Flow Followed by Cobra Venom Factor The changes which occurred in blood pressures, p0 2 and cardiac output with lowering of pulmonary blood flow, injection of cobra venom factor, and then deflation of the IVC balloon are shown in Figures 57,58 and Table XXII to XXVI. Figure 58 and Table XXVI shows that p0 2 rose from 85 _t 10 mmHg (mean _+ SE) to 122 +_ 20 mmHg after pulmonary blood flow was lowered, and then f e l l to 91 _+ 24 mmHg after CVF was injected (p .05). This changed l i t t l e over the course of the experiment. Inflation of the IVC balloon resulted in decreases in pulmonary arterial blood pressure, pulmonary wedge pressure and systemic blood pressure. Cardiac output f e l l from 97 + 16% of control to 14 + 9% control levels. After balloon deflations a l l values returned towards normal values. As in the control animals, cardiac output f e l l towards the end of the experiment to 71 _+ 17% control levels (p<.05). Figures 59 and 60 show the right ventricle and aorta leukocyte and erythrocyte counts for the 5 experiments. Inflation of the IVC balloon is associated with a f a l l in right ventricle leukocyte counts to 55 +_ 11% control while aorta leukocyte counts f e l l to 39 +_ 9% (p .05) control - 94 -values. Injection of CVF at 30 minutes produced an additional f a l l in RV and Ao WBC counts to 35 + 7% and 22 + 7% of control values (p£i.05). After balloon deflation there was a gradual increase in those values to levels significantly greater than control. After 4 minutes of low pulmonary blood flow, RBC counts showed a drop which was associated with the development of an A-V difference across the lung, suggesting that RBC as well as WBC became sequestered in the lung under these conditions. Injection of CVF did not significantly change RV RBC counts but was associated with a further decrease in Ao RBC counts. Deflation of the IVC balloon was associated with increases in both WBC and RBC counts. RBC counts rose to 92-95% control levels at the end of the experiments while WBC counts rose to 197-201% of control levels (p«jt.05). 7. Cobra Venom Factor and Epinephrine Infusion The changes which occurred in blood pressures, PO^  and cardiac output are shown in figures 61 and 62, and Table XXII to XXVI. Figure 62 shows that the PO^  rose from 78 + 6 ram Hg (mean +_ SE) to 106 + 4 mm Hg at 50 minutes before f a l l i n g back to 88 +_ 14 mm Hg at the end of the experimental run (150 minutes). These changes can be attributed to hyperventilation in anesthetized spontaneously breathing dogs which have received intravenous epinephrine. Cardiac output increased to 160 + 17% of the control value soon after epinephrine infusion began and stayed at about this level until epinephrine was discontinued at 50 minutes (p .01) (Figure 61). At the end of the experimental run cardiac output had fallen to 69 _+ 4% of the control value (p£.05). Similarily, pulmonary artery blood pressure and pulmonary wedge pressure increased with epinephrine infusion and f e l l below control levels after epinephrine infusion was discontinued - 95 -(Fig. 61,62). Epinephrine infusion had no effect on systemic blood pressure. Injecting cobra venom factor at 30 minutes had no significant effect on PC^' cardiac output or blood pressures when compared to control experiments. Figure 63,64 and Table XVI (mean + SD), XVII to XXI show the right ventricle and aorta leukocyte and erythrocyte counts for the 6 experiments. Epinephrine infusion was associated with an increase in both aorta and right ventricle leukocyte and erythrocyte counts. For example aortic WBC counts rose from 92 +_ 2% of the control value at 10 minutes to 113 +_ 9% of the control value at 24 minutes. Similarly, the aorta RBC count rose from 96 _+ 2% of the control value at 10 minutes to 113 +_ 3% of the control value at 24 minu tes. Interestingly, Fig. 65 and 66 show that when WBC and RBC are corrected for hematocrit, epinephrine infusion is not associated with an increase in WBC counts. This implies that epinephrine is either hemoconcentrating the blood or causing both RBC and WBC to enter the circulating blood stream - perhaps via contraction of the spleen. On the other hand, cobra venom injection is associated with a preferential f a l l in both aorta and right ventricle WBC counts but not with fa l l s in RBC counts. For example, Ao WBC counts f e l l from 109 + 10% of the control value to 58 + 12% of the control value after injection of cobra venom factor whereas Ao RBC counts f e l l from 112 + 3% to 108 + 3% of the control value during the.... same time interval. Analysis of differential count (Table XVI,XIX,XX) confirms that the f a l l in WBC is due primarily to a f a l l in PMN and not lymphocytes. Significant arterial-venous differences suggestive of release of cells from the pulmonary marginating pool after epinephrine was infused - 96 -could not be detected. Similarily, significant arterial-venous differences suggestive of sequestration of leukocytes within the pulmonary microvasculatures after cobra venom factor injection could not be detected. Tables XVII to XXVI compare hematologic and hemodynamic values at various key points in 5 studies which had similar protocols, namely, the minimal and maximal sampling control groups, the cobra venom factor group, the epinephrine plus cobra venom factor, and the low flow plus cobra venom factor group. These data show that WBC, RBC, PMN and HCT tended to f a l l over the course of the i n i t i a l control periods. Infusion of epinephrine increased WBC, RBC, PMN, HCT, CO and pulmonary pressures. Lowering PBF for 10 minutes by IVC balloon inflation resulted in decreases in WBC, RBC, PMN, LC, CO and a l l blood pressures measured. Injection of CVF was associated with a decrease in WBC and PMN which was independent of any changes in cardiac output. The combination of lowering PBF and giving CVF was somewhat additive with respect to WBC and PMN. For example, lowering PBF caused aortic WBC to f a l l 52% from 91% of control value to 39% of control value. Injection of CVF caused an additional 44% f a l l from 39% to 22% control value. Similar findings were reported in the fourth set of experiments where dogs served as their own controls. In these experiments, lowering PBF resulted in a 49% decrease in aortic WBC counts, while CVF plus low flow resulted in 82% decrease in aortic WBC counts (Figure 56). In a l l experiments, CO f e l l significantly by 150 minutes. Similarly, WBC and PMN counts increased significantly by 150 minutes. Animals which received CVF at normal or increased cardiac output tended to - 97 -have the smallest increases in leukocyte counts. In contrast, the animals which received CVF at low cardiac outputs showed the largest increases in leukocyte counts at the end of the experimental run. The p0 2 of arterial blood showed minimal changes. The only significant changes were seen after the IVC balloon was inflated (85 +_ 10% to 122 + 20% control levels) and after CVF was injected (122 + 20% to 91 + i 24% control levels). E. Discuss ion The effect of pulmonary blood flow and/or complement activation with cobra venom factor on leukocyte and erythrocyte uptake and release by the dog lung was investigated in 40 anaesthetized, spontaneously breathing, heparinized mongrel dogs. Arterial blood gases, systemic and pulmonary blood pressures and cardiac output were also monitored in these animals. In one set of experiments, plasma levels of complement proteins and extravascular lung water was also measured. Extravascular lung water was measured to determine whether or not there was increased vascular permeability following injection of cobra venom factor and sequestration of PMN within the lungs. 1. The Effect of PBF on WBC Uptake and Release by the Dog Lung The results for the control animals indicate that simultaneous blood sampling from the right ventricle and aorta of anaesthetized, spontaneously breathing dogs has l i t t l e effect on mean systemic or pulmonary blood pressure (Figures 31, 32). Pentobarbital sodium consistently lowered mean arterial blood pressure immediately after injection, for a very short duration only. Arterial pO^  did not change significantly over the course of these experiments. The cardiac output decreased after the f i r s t hour to - 98 -77% control levels at the end of the experimental run (150 minutes) (p 0.5). Similar hemodynamic findings have been reported by others (135, 136, 137). In control dogs, large and consistent arterial-venous leukocyte or erythrocyte differences could not be demonstrated (Figures 33 and 34). When present, they could usually be attributed to inadequate removal of diluted blood in the catheter following the flush with heparinized saline prior to blood collection. Hemodiluted samples were readily recognized because a l l values on the hemogram would be proportionately lowered. There was a tendency for systemic WBC and RBC to f a l l slightly over the f i r s t 30 minutes. Thereafter, RBC values remained fa i r l y constant while WBC counts rose to significantly higher than control levels. This apparent hemodilution seen in the f i r s t 30 minutes appears to result, at least in part, from pooling of erythrocytes and leukocytes in the spleen and pulmonary microvasculature (135,138,139). Our frequent measuring of cardiac output probably contributes to the hemodilution as well because we inject 10-15 ml of cold dextrose solution each time we take this measurement. Hemorrhage is a well known cause of leukocytosis (21, 140). If the end-experiment leukocytosis seen in these control animals is due to hemorrhage, i t is not related to the amount of blood loss because similar end-experiment leukocyte values were reported for the minimal sampling (n=3) and maximal sampling (n=4) control animals (Table XVII). A leukocytosis is also a consistent finding after intravascular complement activation (93,141,142,143). Although its mechanism is poorly understood, the third complement component (C^) appears to be involved (20,144). As complement can be activated after tissue trauma (93), i t is possible that there was - 99 -enough surgical trauma that small but significant amounts of leukocyte mobilizing factor was generated from activated (20). Stress associated corticosteroid release could also contribute to this leukocytosis by inhibiting raargination and by recruitment of bone marrow reserves (212). On the other hand, this leukocytosis may be partly factitious. Gilmore has shown that white blood c e l l counts can f a l l to 47% and 41% of preanaesthetic levels one and two hours, respectively, after anaesthetic dosages of pentobarbitol sodium (30 mg/kg plus supplementary doses) (135). By four hours white c e l l counts were 79% preanaes thetic levels and 167% 1 hour post-anaesthetic levels. In other words, pentobarbital sodium can by i t s e l f cause WBC to f a l l significantly and i t takes between 2-4 hours before WBC recover to preanaesthetic levels. We did not take preanaesthetic blood samples and therefore are unable to confirm or deny these findings. In four separate studies, we examined the effect of lowering pulmonary blood flow on leukocyte A-V differences across the lung and found that reducing pulmonary blood flow caused leukocytes to be sequestered within the lung (Figures 38, 43, 53, 59). In addition, the data show that restoration of PBF by IVC balloon deflation or by cardiac massage results in a net expulsion of leukocytes from the pulmonary vasculature (Figures 40, 44, 53). The fact that mean right ventricle leukocyte numbers f e l l below 90% control level 30-40 seconds after a similar mean aorta leukocyte number f a l l (Fig. 38) suggests that the lung is the most important leukocyte sequestering organ under conditions of low cardiac output. The decreasing right ventricle leukocyte count probably reflects both the rapidly decreasing aorta leukocyte count and the occurrence of sequestration within other highly vascular organs such as the liver and spleen (34,36). Recent - 100 -studies from our laboratory have shown that the mean transit time of ^ m T c - l a b e l l e d erythrocytes from the right atrium to aortic root is approximately 8 seconds with normal cardiac output and increases to approximately 16 seconds under conditions of lowered pulmonary blood flow (145). We did not offset aortic samples to take into account lengthened transit time from RV to aorta during low flow because transit times were not measured in these studies. Nevertheless, Figure 39 shows that offsetting aortic samples by 10-20 seconds would not have significantly changed our results. Although both PMN and lymphocytes became sequestered within the pulmonary vasculature during periods of low flow, PMN retention is much greater than lymphocyte retention (Fig. 39). This fact does not necessarily mean they are preferentially selected. If they were, one would expect to be able to see a f a l l in the percentage of PMN in the differential counts of aortic blood. A change in differential count is never observed (Table IX,X,XIIA). This failure to demonstrate significant changes in leukocyte differential counts might reflect the large errors which are inherent in doing differential counts (146). However, i t may be that the differences in PMN and lymphocyte retention are simply due to the fact that there are more PMN than lymphocytes and not that they are handled differently by the lung. The data on pulmonary blood pressure (Fig. 35) show that substantial portions of the lungs must have been under zone 2 conditions after IVC balloon inflation. Perlo et a l . (147) have shown that the ratio of WBC to RBC is greater in zone 2 than in zone 3 in isolated perfused lungs. They suggest that a capillary bed under zone 2 conditions serves as a more efficient leukocyte f i l t e r than one under zone 3 conditions because vessels - 101 -are less distended. Bagge has studied the behaviour' of granulocytes under conditions of microvascular flow (148). He found that normal granulocytes are "highly deformable, but slowly deforming c e l l s , which can temporarily retard or stop capillary blood flow". Presumably, the physical properties of lymphocytes are such that they too could become trapped in the pulmonary microvasculature under conditions of low blood flow or low perfusion pressure (149). A major difference between our study and that of Perlo et al was that they found an excessive accumulation of lymphocytes under zone 2 conditions while our data suggest that PMN are retained in greater numbers than lymphocytes. On the other hand, both Wilson, R a t l i f f et a l . (150,151) and Michel et a l . (82) have examined the lung in hemorrhagic shock and found that there were leukocyte plugs consisting of large numbers of granulocytes. It is also possible that under conditions of lowered pulmonary blood flow, large numbers of leukocytes become marginated within the larger vessels of the pulmonary microvasculature (23,24). Recently, Staub et a l . have reported that large numbers of neutrophils marginate along the walls of small arteries but not in small veins in normal sheep (24). While i t is possible that large numbers of leukocytes accumulate on the arteriolar side of the pulmonary vasculature during a period of reduced flow and that restoration of the cardiac output provides the energy for expulsion of these cells through the microvasculature, more experiments w i l l be needed to examine this point. Lowering pulmonary blood flow consistently caused the right ventricular blood to have a slightly lower hematocrit, hemoglobin and RBC number than aortic blood. The differences in a l l values observed during IVC - 102 -balloon inflation were small and can be attributed to Starling forces where lowered systemic hydrostatic pressure causes fluid to shift from the extravascular compartment to dilute the systemic venous blood. It should be pointed out, however, that during the peak low flow period, right ventricular f i l l i n g pressures were very low and as a result the amount of blood collected per right ventricle EDTA vacutainer tube was often less than lml. Although EDTA does have a relatively large margin for dilutional errors, a volume of blood which is greater than +_ 20% of the optimal quantity w i l l distort c e l l indices (152). In particular, excessive EDTA w i l l result in red c e l l shrinkage or cessation leading to changes in c e l l volume and artifactual reduction in hematocrit values. Even i f this were a factor, the minimal changes observed show that the changes in leukocyte counts seen with balloon inflation and deflation are not due to hemoconcentration or hemodilution. In the experiments where low blood flow is maintained for extended periods of time (Fig. 43) two things are apparent. One is that the systemic white count (RV) is increasing which implies that WBC are being added from other pools. The second is that a new equilibrium is eventually being reached across the pulmonary circulation because A-V differences tend to diminish (Fig. 43) or even disappear (Fig. 44) i f the lung remains in a low flow state long enough. The fact that the marginated pool of WBC in the lung is saturated at a new steady state is suggested by the fact that large numbers of WBC were observed in aortic blood whenever cardiac massage was carried out after the heart had stopped implying leukocyte release from the pulmonary microvasculature (Fig. 43,46). These data support the concept that a dynamic equilibrium exists - 103 -between the circulating and marginating leukocyte pools in the lung. This equilibrium is affected by blood flow because lowering pulmonary blood flow causes leukocytes, particularly PMN, to accumulate within the lung while increasing blood flow causes leukocytes to be released from the lung. Such findings may be important with respect to the pathogenesis of the Adult Respiratory Distress Syndrome (ARDS). As shock frequently preceeds the development of ARDS (6,8,10) i t s role might well be that of lowering pulmonary blood flow resulting in the accumulation of large numbers of PMN within the pulmonary microvasculature. Subsequent activation of the complement cascade by sepsis or tissue trauma might then stimulate these cells to release toxic oxygen metabolites and proteases. 2. The Effect of Complement Activation and Lowering PBF on WBC Uptake and Release by the Dog Lung The second major object of these experimental studies was to determine i f activation of the alternate complement pathway with cobra venom factor (CVF) produces arterial-venous (A-V) leukocyte differences across the lung, and whether this sequestration of cells could be influenced by changes in pulmonary blood flow. Cobra venom factor (CVF) is used classically to investigate the pathophysiologic effect of brisk activation of the complement cascade in the circulation (155). The hemodynamic and hematologic changes produced by CVF varies according to the route administered, dosage, and the species studied. In the rabbit (2 - 2.5 kg), IV injection of CVF induces transient decreases in mean arterial blood pressure, circulating PMN and platelet counts (156-158). There is no dose relationship between CVF and blood pressure; rather, doses above 300 units produce decreases of 20-50%, while - 104 -those below 300 units are ineffective in producing hemodynamic changes. The maximal % f a l l in BP is observed within 10 minutes, after which i t slowly returns to preinjection levels within 100 minutes of CVF injection. The injection of 300 units of CVF into rabbits with normal complement levels results in the disappearance of 40-60% of the circulating platelets within 5 minutes (156). Similar changes in PMN counts have been documented after intravenous injection of 0.6 - 1.0 ml of purified CVF (157). Both the hematologic and hemodynamic changes require the presence of C^ and occur in rabbits genetically deficient in the sixth component of complement -implying the terminal complement components are not required for these changes. The hypotensive effects of CVF appear to be mediated via the generation of histamine-releasing agents or anaphylatoxins because i t can be blocked by the histamine R^-receptor antagonist burimamide. Alternate pathway complement activation in rabbits is also associated with progressive hypoxemia and tachypnea, and pulmonary vascular plugging by aggregates of degenerating granulocytes with i n t e r s t i t i a l edema and endothelial injury (66,67,90). The arterial hypoxemia is attributed to alveo-capillary block resulting from leukocyte microemboli. In sheep intravenous injection of 200 +_ 46 u/kg CVF causes the rapid onset of a transient period of complement activation. Changes that coincide with this period include a decrease in circulating PMN and platelet counts, and leukocyte trapping in pulmonary vessels and interstitium (159). Pulmonary arterial blood pressure and pulmonary lymph flow tend to increase, arterial p0^ tends to decrease, while cardiac output, pulmonary blood flow, mean pulmonary arterial wedge pressure, and pCO^ do not change significantly. Such changes resemble those reported in sheep after infusion - 105 -of Zymosan activated plasma (ZAP) (67,160-162) or endotoxin (79,96,163,164) - both potent activators of the complement system. The decrease in p02> dynamic compliance and. functional residual capacity and the increase in pulmonary vascular permeability and airway resistance are changes which appear to be linked to granulocyte sequestration (160,163,96,67,164). The increase in Ppa, and pulmonary lymph flow are not dependent on PMN pulmonary sequestration (160,96). The increase in Ppa is the result of thromboxane synthesis - probably by injured endothelial cells (162,165). With ZAP, the decrease in WBC is due exclusively to decreases in circulating granulocyte counts whereas with endotoxin the decrease in circulatory WBC counts is due to sequestration of both PMN and LC within the pulmonary microvasculature. The site of PMN sequestration also varies within the lung. With ZAP, PMN accumulate and marginate in alveolar capillaries, small arteries and veins, while with endotoxin PMN are sequestered primarily in the capillary level (161,79). The dog resembles the rabbit in that IV injection of a large amount of CVF results in a rapid decrease in mean arterial blood pressure (166). The small amount of CVF used in our experiments (1 ml:64 units) did not produce any significant hemodynamic changes. However, intravenous injection of this amount of CVF was associated with marked decreases in circulating PMN counts. Within 4 minutes circulating PMN counts f e l l to approximately 50% control levels while lymphocyte counts were unchanged from control levels (Tables XIX and XX). The presence of small but consistent A-V differences across the lung during normal flow suggests PMN were sequestered in the pulmonary microcirculation. Lowering pulmonary blood flow enhanced these A-V leukocyte differences in an additive fashion, (Table XVII) - 106 -suggesting that sequestration of cells occurs by a different mechanism. In the experiment where complement protein levels were assayed, we were unable to demonstrate significant complement activation (Table XIV). This is not suprising considering the fact that we used such a small dose of CVF. The CH^Q test is insensitive and has a wide normal range. Decreases in individual complement components to 1/2 or less of the normal concentration may have l i t t l e or no effect on the results of the test (50). Similar limitations exist when assaying individual complement proteins such as C 0 and C,. A number of investigators have noted that Z o granulocytopenia and granulocyte aggregometry are more sensitive than hemolytic assays in demonstrating complement activation (167-169). Much data supports the hypothesis that C^a is responsible for the pulmonary leukostasis seen after activation of the complement system. In v i t r o studies have shown that C^a causes PMN activation, aggregation and adherence to endothelial cells (19,170,59,60). Intravenous injection of C^a into rabbits causes a pronounced neutropenia due to leukoaggregation (171,94). Other studies have shown that coincident with the development of profound neutropenia following complement activation is the appearance of chemotactic activity in the serum. This chemotactic activity is immunochemically related to C^a in that antibody to human C^a was shown to block chemotaxis (61). Whether or not activation of the complement system with subsequent release of C5a produces increases in vascular permeability to protein and pulmonary edema is controversial. While most studies indicate that there is an increase in vascular permeability after complement activation (61,67,76,90,141,159,172,173), other studies have shown no evidence for - 107 -increased vascular permeability unless there was concomittant alveolar hypoxia or prostaglandin E infusion (94,172,173,174). T i l l and Ward believe that failure to observe changes in lung vascular permeability may simply reflect the fact that increases in permeability are shortlived and can easily be missed (141). Their studies indicate that at 4 hours post CVF injection, lung vascular permeability changes in CVF-treated rats had returned to normal levels. This, in turn probably reflects the short h a l f - l i f e of intravascular C5a (approx. 3 minutes in rabbits) (175,176,141). The rapid clearance of C5a may be the result of the interaction of C5a with PMN or may be due to rapid inactivation by chemotactic factor inactivator (141). In the one study reported here, no significant changes in extravascular water was demonstrated 2 hours after intravenous injection of 64 units of CVF (1 ml). Because the dosage of CVF was so low, i t is likely that any increase in vascular permeability would have cleared long before 2 hours post injection. The fact that CVF injection caused VO^ to f a l l from 122 +_ 20 to 91 +_ 24 mmHg in the experiments where blood flow was lowered before CVF injection, indicates CVF can under the right circumstances decrease arterial PO^ • The i n i t i a l increase in from 85 _+ 10 mmHg to 122 +_ 20 mmHg can be attributed to decreased pulmonary blood flow and an associated reduction in intrapulmonary right to left shunt (177 ,178)(Figure 58). Two important questions arise concerning the use of CVF to activate complement in heparinized, anaesthetized dogs: 1) To what extent does heparin inhibit activation of the complement system by CVF? - 108 -2) Could phospholipase - a known contaminant of CVF - be responsible for any of the effects of CVF? It has been known for over 50 years, that heparin can inhibit activation of the complement system (179). This inhibitory effect appears to be reversible, time dependent and is a function of the size of the complex 'sugar' and the content of carboxyl groups (180,181). It is independent of the anticoagulant activity of heparin. Both the alternate and classical pathways are affected (182,183). Heparin regulates classical complement pathway activation by interfering with CI binding to immune complexes (184), inhibiting CI q s interaction with C4 and C2 (185), inhibiting C2 binding to C4 (186) and by interfering with the formation of the terminal C5b-C7 complex (187). The predominant mechanism of heparin's action on the classical pathway, however, appears to be that mediated through interaction with and potentiation of CI inhibitor function (188,189,190). Heparin regulates alternate complement pathway activation by interfering with binding of B to C3b (191-193) and by augmenting the cofactor role of C3b inhibitor accelerator in the inactivation of bound C3b (194,195). The concentrations of heparin required are similar to those required for its anti-thrombin cofactor activity (192). The biological importance of each inhibiting effect is controversial and d i f f i c u l t to assess. Although the-predominant mechanism of heparins action on the classical pathway appears to be that mediated through interaction with and potentiation of Cl inhibitor function, other investigators have been unable to demonstrate heparin mediated enhancement - 109 -of Cl-Cl inhibitor complex formation (196) and in fact, one investigator has shown that heparin can inhibit Cl-inhibitor function (197). Both Rent et a l , and Logue have shown that in certain circumstances heparin appears to have a biphasic effect upon alternative pathway complement activation, enhancing reactions at low concentrations and inhibiting at high concentrations (198,199). Despite heparins impressive an ti-complementary potential, CVF should s t i l l be able to activate complement in its presence. Heparin's inhibitory activity appears to l i e primarily in its a b i l i t y to inhibit the classical pathway and the early stages of the alternate pathway. CVF bypasses these major sites of heparin inhibition by directly cleaving C3 and C5, thereby generating ana phyla toxins (C3a, C5a) which have potent PMN adhering, agglutinating and activating properties (60,200) (Figure 30). Phospholipase is a known contaminant of Naja naja 'cobra venom factor' (CVF) which has been purified by sequential ion exchange and gel f i l t r a t i o n chromatography. Recent studies have shown that small (0.4%) but biologically significant amounts of phospholipase affect systems designed to assess the anticomplementary ac t i v i t y of CVF (155,156,201,202). The biologic significance of phospholipase A^  lies in the fact that i t perturbs ce l l membrane structure and thus potentially alters c e l l function (203,204). Complete removal of this enzyme's acyl hydrolase activity necessitates treatment of the CVF with p-bromophenacyl bromide (BPB), an irreversible modifier of the histidine residue in the active site of phospholipase A^ (155,205). Because the CVF used in our experiments was not treated in this manner, i t seems likely that small amounts of - 110 -contaminating phospholipase k^ were present. The effect, i f any, that this contaminant might have had on our results cannot be determined. Shaw and co-workers have demonstrated that contaminating phospholipase k^ is responsible for the acute neutrophil-associated lung injury seen in rabbits receiving transtracheally injected CVF (155). On the other hand, T i l l and his co-workers have shown that intravenous infusion of CVF with or without contaminating phospholipase produces lung injury in rats, whereas infusion of purified phospholipase k^ failed to produce evidence of lung injury (61). Contaminating phospholipase also has no effect on complement mediated hypotension and thrombocytopenia when CVF was injected intravenously into rabbits (155). Thus, i t would appear that when CVF is injected intravenously, phospholipase A^-mediated changes are minimal. There is no evidence to suggest that phospholipase k^ is responsible for the acute neutropenia seen in the dogs after injection of CVF. Presumably, this acute neutropenia reflects sequestration of WBC within the pulmonary microvasculature. A crude indicator of leukocyte sequestration is the product of mean cardiac output (liters/minute) and mean A-V difference (WBC counts/liter) across the lung for any given time interval. In the experiments where CVF was injected and cardiac output was carefully measured (CVF alone, CVF plus epinephrine, low flow plus CVF), leukocyte sequestration increased 2.9-3.0 fold. In terms of absolute numbers of sequestered c e l l s , there is an increase from control levels of 8 8 3.2-7.2 x 10 cells/minute to 13-21 x 10 cells/minute after CVF was injected (Fig. 60). In the experiments where CVF was injected after CO was lowered, CVF had a slight additional effect on net WBC sequestration within g the lung. That i s , control period sequestration was approximately 4 x 10 - I l l -cells/min increasing to 11 x 10 cells/min when CO was lowered, and to 13 g x 10 cells/min after CVF was injected. This suggests that up to 10% of the cells in the total blood granulocyte pool (0.7 x 10 cells/kg) are being sequestered per minute in the lung when CO is lowered and CVF is injected. 3. The Effect of Epinephrine Infusion and Complement Activation on WBC Uptake and Release by the Dog Lung It is well known that epinephrine, regardless of the mode of administration produces an immediate and absolute increase in circulating WBC counts (206-213). Both PMN and lymphocyte counts increase, with the latter tending to increase to relatively higher levels. This epinephrine associated leukocytosis has been attributed to: 1) release from the spleen (208,209,214) 2) release from the bone marrow (215,216) 3) release from lymphatics (210,213) 4) release from the lung (34,30) 5) dilation of quiescent capillaries (208,209) 6) decreased adhesivity between leukocytes and the endothelium (217-219) 7) re-entry into the blood stream of cells adhered to vessel walls (demargination) secondary to an increased cardiac output (23,26). The relative importance of these mechanisms has not yet been determined. The increase in WBC concentration is apparently not due to hemoconcentration (208,220,221,222). The purpose of the last set of experiments was to produce an absolute lymphocytosis and relative neutropenia by giving epinephrine and then cobra venom factor. We expected to see an i n i t i a l - 112 -increase in both lymphocytes and polymorphonuclear leukocytes, followed by a complement-mediated sequestration of PMN within the pulmonary and systemic raicrovasculatures. Thus, theoretically, this experiment should have produced a lymphocytosis and relative neutropenia similar to that observed in the c l i n i c a l studies. We found that administration of 2-24 micrograms/min (11 _+ 3) of epinephrine hydrochloride was associated with an increase in WBC and that the lymphocyte increase was relatively greater than the PMN increase (Tables XVI,XIX,XX). Injection of CVF was associated with a preferential f a l l in PMN counts from 110% of control to approximately 50% of control levels while lymphocyte counts changed minimally. But, because the f a l l in PMN never exceeded the increase in lymphocyte counts we were unable to demonstrate a relative lymphocytosis, let alone an absolute lymphocytosis and relative neutropenia. Nevertheless, i t would s t i l l seem possible to produce an absolute lymphocytosis and relative neutropenia i f dogs with higher i n i t i a l lymphocyte counts were chosen and i f larger doses of CVF were administered. Significant A-V differences showing leukocyte release from the lung could not be demonstrated after epinephrine infusion even though cardiac output rose to 170 +_ 34% of control values (60). It has been shown that high levels of epinephrine decrease PMN adherence in vitro (217-219). In addition, Ambrus (34,223), Bierman and associates (212), Hamilton and Horvath (220,222) have a l l demonstrated significant release of leukocytes from the pulmonary microcirculation after IV infusion of epinephrine. Our sampling technique was similar to that used in their experiments. However, these investigators used dosages of 100 to 500 micrograms and infused i t rapidly over several minutes. The highest dose we administered over 1 - 113 -minute was 24 micrograms which is less than l/10th the dosage given dogs in the other studies (34,223,220,222). Perhaps our inability to show A-V leukocyte differences across the lung was because epinephrine levels were not high enough to modify leukocyte adherence to the pulmonary endothelium. During hemorrhagic shock in dogs, the maximum concentration of endogenously released epinephrine is around 20-30 micrograms per l i t e r (224,225). This level can be achieved by infusing 20 to 35 micrograms/minute (226,227). Thus, i t would appear that the dosages used in our experiments (2-24/minute) are more physiological than those used by Bierman and associates (100-300 micro grams/minute), Ambrus (200-400 micrograms/minute) or Hamilton and Horvath (500 micro grams/minute ). On the other hand, i n a b i l i t y to show leukocyte A-V differences might simply reflect "crude" sampling technique. One can never be sure that the blood sampled at the aortic root truly represents values which would have been attained had the blood sampled at the right ventricle passed through the lung. In addition, i t becomes increasingly d i f f i c u l t to show A-V differences across the lung when pulmonary blood flow is increased. For example, A-V leukocyte differences were readily demonstrable when cobra venom, factor was given during low blood flow. Slight, but s t a t i s t i c a l l y significant A-V leukocyte differences could s t i l l be demonstrated when there was normal blood flow. Under increased blood flow conditions, no A-V leukocyte differences were demonstrable. Epinephrine also increased RBC, Hematocrit, cardiac output, mean pulmonary artery pressure, heart rate and pulmonary wedge pressure. Mean systemic blood pressure and the P0„ of arterial blood did not change - 114 -significantly over the course of these experiments. These findings have been reported previously (228-232). 4. Conclusion In summary, these animal studies have shown that there is dynamic equilibrium between marginating and circulating pools of leukocytes within the dog lung. This equilibrium is markedly affected by pulmonary blood flow. Decreasing pulmonary blood flow results in sequestration of leukocytes in the lung while increasing pulmonary blood flow results in these cells being released back into the systemic circulation. Intravenous injection of cobra venom factor (1 ml: 64 units) is associated with the preferential sequestration of PMN into systemic and pulmonary microvasculatures. This sequestration is independent of pulmonary blood flow. The combination of complement activation and lowering pulmonary blood flow has an additive effect on leukocyte sequestration within the dog lung. We were unable to demonstrate significant release of cells from the lung after epinephrine infusion even though cardiac output rose to 170% control value. Clearcut changes indicative of hypoxia or increased pulmonary edema were also not demonstrated. Such findings suggest that transient sequestration of PMN cannot by i t s e l f cause ARDS. This does not rule out the possibility that massive and continuous activation of complement and PMN plays an integral part in the pathogenesis of this tragic disorder. - 115 -TABLE VII MINIMAL SAMPLING CONTROL GROUP HEMATOLOGIC DATA TIME WBC PMN LC RBC HCT* (min) (103/mm3) (103/mm3) (103/mm3) (106/mm3) (%) 0.0 RV 5.8 + 1.3* 4.0 + 0.6 1.1 + 0.6 5.8 + 0.2 38.1 + 2.9 Ao 5.6+1.1 4.1+0.4 1.1+0.8 5.7+0.4 37.6+1.8 6.0 RV 6.2 + 2.0 4.3 + 1.5 1.3 + 0.4 5.5 + 0.4 37.8 + 3.5 Ao 6.3+2.3 4.5+1.9 1.1+0.7 5.6+0.5 37.9+3.0 30.0 RV 6.3 + 2.0 4.8 + 1.2 0.9 + 0.3 5.5 + 0.3 38.4 + 2.1 Ao 6.2+1.5 4.5+1.3 1.1+0.7 5.6+0.3 38.5+2.3 50.0 RV 6.9 + 0.8 5.3 + 0.7 1.0 + 0.2 5.5 + 0.4 38.1 + 3.7 Ao 6.6+0.6 5.2+0.2 0.9+0.5 5.4+0.4 37.4+4.4 60.0 RV 7.1 + 0.3 5.6 + 0.7 1.0 + 0.7 5.6 + 0.4 38.1 + 4.9 Ao 7.1+0.4 5.7+0.5 0.8+0.2 5.6+0.5 38.8+5.3 90.0 RV 7.4+0.5 6.3+0.9 0.6+0.2 5.4+0.6 37.5+4.3 Ao 7.1+0.9 5.9+1.2 0.7+0.3 5.5+0.6 38.9+7.6 120 RV 8.9+1.1 7.4+1.5 0.6+0.3 5.5+0.6 37.8+5.2 Ao 8.5+1.2 7.1+1.4 0.7+0.2 5.4+0.5 37.1+6.0 150 RV 9.2+1.6 7.8+1.6 0.6+0.3 5.4+0.3 37.7+3.8 Ao 9.3+1.7 7.9+1.7 0.7+0.1 5.5+0.5 38.2+5.2 * mean + SD for n=2 dogs because of technical d i f f i c u l t i e s . - 116 -TABLE VIII MAXIMUM SAMPLING CONTROL GROUP HEMATOLOGIC DATA TIME WBC PMN LC RBC HCT (min) (103/mm3) (103/mm3) (103//mm3) (106/nim3) (%) 0.0 RV 6.8 + 2 6 5 .3 + 2 3 0. 9 + 0 5 5 .8 + 0. 8 38.5 + 5 .4 Ao 7.2 + 3. 0 5 .7 + 2 .6 1 0 + 0. 3 5 .9 + 0. 4 39.7 + 4. 3 2.0 RV 7.0 + 2 9 5 .5 + 2 6 0 8 + 0 5 5 .9 + 0. 6 39.4 + 5 .4 Ao 6.9 + 3. 0 5 .5 + 2 .5 0 .8 + 0. 3 5 .8 + 0 .5 38.1 + 4 .6 4.0 RV 7.0 + 3. 0 5 .0 + 2 0 1 .0 + 0 4 5 .7 + 0. 7 37.7 + 6 .9 Ao 6.5 + 3. 0 0 .8 + 2 .5 0 .8 + 0. 2 5 .5 + 0 .8 36.6 + 5 .6 6.0 RV 6.8 + 2. 9 5 .4 + 2. 5 0. 9 + 0. 3 5 .8 + 0. 7 38.6 + 5 .5 Ao 6.7 + 3. 3 5. 4 + 2 .6 0 .8 + 0. 2 5 .5 + 0 .9 36.4 + 6. 3 8.0 RV 6.6 + 3. 0 5 .1 + 2 2 0. 7 + 0. 2 5 .7 + 0. 7 37.5 + 6 .9 Ao 6.0 + 2 .6 4 .9 + 2. 2 0 .6 + 0 .1 5 .1 + 1. 2 33.9 + 8 .6 10.0 RV 6.4 + 2. 9 5 .0 + 2 1 0. 8 + 0. 1 5 .5 + 0. 7 36.7 + 6 .7 Ao 6.4 + 3 .7 5. 3 + 3. 0 0 .6 + 0. 2 5. 3 + 1. 4 34.9 +11 .5 12.0 RV 6.5 + 3. 6 5 .1 + 2. 6 0. 8 + 0. 4 5 .5 + 0. 7 36.2 + 7 .5 Ao 6.3 + 3 .8 5 .1 + 2 .9 0 .8 + 0. 4 5. 0 + 1 .5 33.7 +10 .1 14.0 RV 6.5 + 3. 3 5 .0 + 2. 4 0. 8 + 0. 2 5 .7 + 0. 6 37.5 + 6 .4 Ao 6.2 + 3 .6 4 .8 + 2 .5 0 .7 + 0. 3 5. 3 + 1. 3 34.9 +10. 3 16.0 RV 6.3 + 3. 3 5 .0 2. 4 0. 8 + 0. 4 5 .5 + 0. 7 36.6 + 6 .8 Ao 6.1 + 3 .7 4 .9 + 2 .8 0 .7 + 0. 2 5. 0 + 1 .6 33.3 +10 .9 18.0 RV 6.4 + 3. 3 5 .1 + 2. 4 0. 8 + 0. 3 5 .6 + 0. 7 37.0 + 6 .2 Ao 6.4 + 3. 2 5. 2 + 2 .5 0 .7 + 0. 3 5 .5 + 0 .8 36.2 + 5 .7 20.0 RV 6.4 + 3. 3 5 .4 + 2. 8 0. 6 + 0. 2 5 .5 + 0. 6 36.3 + 6 .8 Ao 6.2 + 3. 0 4 .9 + 2. 3 0 .7 + 0 .1 5. 3 + 0 .6 35.4 + 4. 4 22.0 RV 6.6 + 3. 2 5 .4 + 2. 4 0. 6 + 0. 3 5 .5 + 0. 7 36.5 + 6 .2 Ao 6.2 + 3 .5 5. 0 + 2 .8 0 .7 + 0. 2 5. 2 + 1. 2 34.6 + 8. 0 24.0 RV 6.4 + 3. 4 5 .2 + 2. 4 0. 6 + 0. 3 5 .3 + 0. 7 35.4 + 6 .9 Ao 6.4 + 3 .5 5 1 + 2 .6 0 .7 + 0. 3 5. 2 + 1. 0 34.6 + 7. 0 26.0 RV 6.5 + 3. 2 5 .2 + 2. 6 0. 7 + 0. 2 5 .4 + 0. 7 36.0 + 5 .7 Ao 6.4 + 3. 3 5 1 + 2. 4 0 .7 + 0. 2 5. 3 + 0. 8 35.4 + 5 .7 28.0 RV 6.5 + 3. 3 5 .4 + 2. 6 0. 6 + 0. 1 5 .4 + 0. 7 35.7 + 6 1 Ao 6.4 + 3. 1 5. 3 + 2. 4 0 6 + 0. 1 5. 3 + 0. 8 36.1 + 6. 3 30.0 RV 6.6 + 3. 4 5 .4 + 2. 7 0. 6 + 0. 2 5 3 + 0. 7 35.3 + 6 4 Ao 6.3 + 3 5 5 1 + 2 6 0 7 + 0. 3 4 9 + 1. 4 32.7-+ 9. 1 32.0 RV 6.7 + 3. 4 5 .6 + 2. 6 0. 5 + 6. 1 5 3 + 0. 7 34.8 + 6. i Ao 6.8 + 3. 4 5 5 + 2. 3 0. 7 + 0. 2 5. 4 + 0. 7 35. 2 + 5. 0 34.0 RV 6.9 + 3. 5 5 .8 + 2. 8 0. 6 + 0. 2 5 4 + 0. 7 35.8 + 5 7 Ao 6.8 + 3 5 5 7 + 2. 8 0. 6 + 0. 1 5. 4 + 0. 7 35. 2 + 5. 1 36.0 RV 6.9 + 3. 5 5 .8 + 3. 1 0. 6 + 0. 2 5 3 + 0. 7 34.6 + 6. 3 Ao 7.1 + 3 5 5 8 + 2. 9 0 6 + 0. 2 5. 3 + 0. 7 35.8 + 5. 8 38.0 RV 7.0 + 3. 5 5 .6 + 2. 8 0. 7 + 0. 3 5 4 + 0. 6 35.4 + 5. 2 Ao 7.1 + 3 5 5 8 + 2 7 0. 6 + 0. 2 5. 3 + 0. 7 35. 2 + 4. 7 40.0 RV 7.1 + 3. 7 6 0 + 3. 2 0. 6 + 0. 2 5 3 + 0. 6 34.6 + 5. 9 Ao 7.0 + 3. 8 5 7 + 3. 0 0. 7 + 0. 2 5. 2 + 0. 6 34.1 + 4. 5 42.0 RV 7.2 + 3. 7 6 .0 + 3. 0 0. 6 + 0. 3 5 3 + 0. 6 35.3 + 5 1 Ao 7.0 + 3 6 5 8 + 2 9 0 6 + 0. 3 5. 2 + 0. 7 34.5 + 5. 3 - 117 -Table VIII (Cone'd) TIME WBC PMN LC RBC HCT 44.0 RV Ao 46.0 RV Ao 48.0 RV Ao 50.0 RV Ao 60.0 RV Ao 90.0 RV Ao 120.0 RV Ao 150.0 RV Ao 7.1 + 4.0 7.3 + 4.1 7.3 + 4.0 7.2 + 3.8 7.3 + 4.0 7.1 + 3.8 7.5 + 4.1 7.0 + 3.8 7.8 + 4.2 8.0 + 4.2 10.2+4.7 10.4 + 4.4 11.4 + 5.1 11.7 + 5.0 12. 3 + 6.0 12.6 + 5.5 5.7 + 3.1 6.1 + 3.3 6.1 + 3.2 5.8 + 3.1 6.2 + 3.5 6.0 + 3.3 6.3 + 3.4 5.9 + 3.1 7.7 + 3.5 6.8 + 3.6 8.9 + 4.0 8.9 + 4.0 8.1 + 3.2 10.1 + 4.2 10.7 + 5.6 11.3 + 5.6 0.6 + 0.4 0.6 + 0.3 0.7 + 0.3 0.7 + 0.3 0.6 + 0.1 0.5 + 0.4 0.6 + 0.3 0.5 +0.1 0.5 + 0.3 0.6 +0.1 0.4 +0.1 0.7 + 0.2 0.6 + 0.2 0.6 + 0.3 0.6 + 0.3 0.5 + 0.4 5.3 + 0.6 5.3 + 0.7 5.3 + 0.6 5.3 + 0.8 5.2 + 0.6 5.2 + 0.8 5.3 + 0.6 5.2 + 0.8 5.5 + 0.7 5.4 + 0.8 5.8 + 0.6 5.8 + 0.6 5.8 + 0.6 5.8+0.6 5.9 + 0.6 5.8 + 0.6 35.0 + 5.7 34.8 + 5.0 36.1 + 6.3 34.6 +5.6 34.4 + 6.2 34.6 +5.7 35.0 + 5.4 33.9 + 6.0 36.7 + 7.0 36.0 +5.9 39.6 + 6.6 38.6 +5.6 38.7 + 5.0 38.7 + 5.3 39.1 + 3.8 38.4 + 5.0 - 118 -TABLE IX HEMATOLOGIC DATA FOR TRANSIENT LOW FLOW EXPERIMENTS TIME WBC PMN LC RBC (min) (103/mm3) (103/mm3) (103/mm3) (106/mm3) 0.00 RV 8.8 + 4.3 6.6 + 3.4 1.3 + 0.7 6.1 + 1 .0 Ao 8.8 + 4.3 6.8 + 3.6 1.1 + 0.4 6.1 + 1.0 0.17 RV 8.9 + 4.2 6.8 + 3.6 1.1 + 0.5 6.2 + 1 .0 Ao 8.8 + 4.3 6.7 + 3.4 1.1 + 0.8 6.1 + 1 .1 BALLOON INFLATION 0.33 RV 8.8 + 4.2 7.0 + 3.9 1.1 + 0.6 6.2 + 1 .0 Ao 8.4 + 4.0 6. 2 + 3.1 1.2 + 0.9 6.1 + 1 .1 0.50 RV 8.9 + 4.1 7.2 + 3.6 1.1 + 0.7 6.2 + 1 .0 Ao 7.6 + 3.7 6.0 + 2.7 1.1 + 0.8 6.2 + 1 .1 0.67 RV 8.9 + 4.2 7.1 + 3.6 1.0 + 0.6 6.2 + 1 .0 Ao 7.5 + 3.8 5.8 + 2.9 0.9 + 0.5 6.2 + 1 .1 0.83 RV 8.8 + 4.4 7.1 + 3.8 1.1 + 0.6 6.1 + 1 .1 Ao 6.8 + 3.9 5.3 + 3.0 0.9 + 0.5 6.1 + 1 .0 1.00 RV 8.6 + 4.6 7.1 + 4.0 0.9 + 0.6 6.1 + 1 .1 Ao 5.7 + 3.4 4.6 + 2.7 0.6 + 0.4 6.1 + 1 .1 1.17 RV 7.6 + 3.4 6.2 + 3.1 0.8 + 0.4 6.0 + 1 .1 Ao 5.1 + 2.3 4.1 + 2.0 0.6 + 0.2 6.1 + 1 .1 1.33 RV 7.2 + 3.2 5.6 + 2.7 1.1 + 0.7 5.9 + 1 .1 Ao 4.8 + 2.1 3.6 + 1.7 0.8 + 0.5 6.1 + 1 .1 1.50 RV 6.9 + 3.1 5.6 + 2.7 0.7 + 0.3 5.9 + 1 .1 Ao 4.5 + 1.9 3.5 + 1.8 0.5 + 0.2 6.0 + 1 .1 1.67 RV 6.7 + 3.0 5.4 + 2.6 0.9 + 0.5 6.0 + 1 .2 Ao 4.3 + 1.7 3.5 + 1.7 0.5 + 0.2 6.0 + 1 .1 1.83 RV 6.5 + 2.9 5.2 + 2.3 0.8 + 0.5 5.9 + 1 .1 Ao 3.8 + 1.6 3.0 + 1.6 0.5 + 0.2 6.1 + 1 .0 2.00 RV 6.7 + 2.9 5.4 + 2.6 0.7 + 0.4 5.8 + 1 .0 Ao 4.1 + 2.0 3.3 + 1.8 0.5 + 0.3 5.9 + 1 .1 2.17 RV 6.7 + 3.0 ' 5.4 + 2.7 0.7 + 0. 3 5.8 + 1 .0 Ao 4.0 + 2.2 3.3 + 1.9 0.4 + 0.3 5.9 + 1 .1 2.33 RV 6.8 + 3.3 5.5 + 2.8 1.0 + 0.7 5.8 + 1 .0 Ao 4.0 + 2.4 3.1 + 2.0 0.05+ 0.4 6.3 + 1 .0 2.50 RV 6.7 + 3. 3 5.4 + 2.7 0.8 + 0.4 5.7 + 1 .1 Ao 4.0 + 2.5 3.3 + 2.1 0.4 + 0.3 6.0 + 1 .1 - 119 -Table IX cont'd. BALLOON DEFLATION TIME WBC PMN LC RBC 2.67* RV 8.4 + 3.3 6.6 + 2.1 1.2 + 0.9 5.9 + 1.0 Ao 5.9 + 4.4 4.0 + 3.3 0.7 + 0.7 6.2 + 1.3 2.83* RV 8.4 + 2.8 6.9 + 2.1 0.8 + 0.4 6.0 + 0.9 Ao 6.4 + 4.7 5.3 + 3.8 0.7 + 0.4 6.3 + 1.2 3.00* RV 8.8 + 2.0 7.0 + 1.5 1.1 + 0.5 6.4 + 1.1 Ao 8.4 + 4.4 6.9 + 3.7 0.9 + 0.5 6.3 + 1.3 3.17* RV 8.6 + 2.5 6.9 + 1.9 1.0 + 0.6 6.2 + 1.0 Ao 12.7 + 3.9 10.5 + 4.0 1.3 0.6 6.5 + 1.2 3. 33* RV 9.2 + 2.3 7.4 + 1.8 1.0 + 0.6 6.3 + 1.0 Ao 11.3 + 3.7 9.0 + 3.1 1.3 + 0.3 6.5 + 1.1 3.50 RV 8.2 + 3.8 6.5 + 3.2 1.0 + 0.6 6.4 + 1.1 Ao 8.8 + 4.0 6.9 + 3.6 1.1 + 0.6 6.5 + 1.1 3.67 RV 8.6 + 3.5 6.8 + 3.2 1.1 + 0.4 6.5 + 1.2 Ao 9.1 + 4.1 7. 2 + 3.6 1.2 + 0.6 6.5 + 1.1 3.83* RV 10.2 + 3.0 8.2 + 2.4 1.1 + 0.6 6.4 + 1.3 Ao 10.2 + 3.2 8.2 + 2.7 1.3 + 0.5 6.6 + 1.1 * mean + SD for n=4 dogs because in one animal smears were spoiled at times indicated. - 120 -TABLE X HEMATOLOGIC DATA FOR PROLONGED LOW FLOW EXPERIMENTS TIME WBC PMN LC (min) (103/mm3) (103/mm3) (103/mm3) 0.0 RV 4.7 + 2.3 3.7 + 2.3 0.7 + 0.4 Ao 4.8 + 2.1 4.0 + 2.3 0.6 + 0.3 1.0 RV 4.7 + 2.3 3.8 + 2.3 0.7 + 0.3 Ao 4.8 + 2.4 3.9 + 2.6 0.6 + 0.2 BALLOON INFLATION 0.0 RV 4.3 + 2.1 3.5 + 2.0 0.6 + 0.3 Ao 2.7 + 1.3 2.2 + 1.4 0.4 + 0.1 1.0 RV 3.8 + 1.7 3.2 + 1.8 0.5 + 0.1 Ao 2.7 + 1.7 2. 3 + 1.4 0.3 + 0.1 2.0 RV 3.9 + 1.6 3.3 + 1.7 0.5 + 0. 2 Ao 2.9 + 1.5 2.4 + 1.5 0.4 + 0.1 3.0 RV 3.9 + 1.5 3.2 + 1.6 0.6 + 0.2 Ao 3.1 + 1.6 2.6 + 1.5 0.4 + 0.1 4.0 RV 4.0 + 1.4 3.3 + 1.5 0.5 + 0. 2 Ao 3.2 + 1.6 2.5 + 1.5 0.4 + 0.1 5.0* RV 4.0 + 1.3 3.4 + 1.3 0.4 + 0.1 Ao 2.8 + 1.6 2.6 + 1.7 0.4 + 0.1 7.0 RV 4.0 + 1.3 3. 3 + 1.5 0.5 + 0.2 Ao 3.1 + 1.5 2.6 + 1.5 0.4 + 0.1 9.0 RV 4.0 + 1.0 3.3 + 1.3 0.4 + 0.1 Ao 3.4 + 1.3 2.8 + 1.4 0.5 + 0.1 11.0 RV 4.0 + 0.9 3.4 + 0.9 0.5 + 0.2 Ao 3.5 + 1.2 3.1 + 1.4 0.4 + 0.2 13.0 RV 4.1 + 1.2 3.4 + 1.3 0.6 + 0.3 Ao 3.8 + 1.6 3.2 + 1.6 0.5 + 0.2 15.0 RV 4.5 + 1.5 3.7 + 1.6 0.6 + 0.3 Ao 4.1 + 1.5 3.3 + 1.4 0.4 + 0.2 * Mean + SD for n=4 dogs. - 121 -TABLE XI HEMATOLOGIC DATA FOR CVF EXPERIMENTS Time WBC PMN LC RBC HCT (min) (l6J/wm3) (IO 3/™ 3) (lO^Jmm3) ( lWmm 3 ) (%) 0.0 *RV 10.4 + 2.0 8.3 + 2.5 1. 5 + 0.7 5.2 + 0.8 36.6 + 4.7 Ao 11.0 + 1.1 8.3 + 1.3 1. 9 + 0.6 5.4 + 0.6 37.9 + 3.5 2.0 RV 10.1 + 2.5 7.9 + 2.4 1. 4 + 0.9 ,5.5 + 0.6 38.2 + 3.6 Ao 10.1 + 2.3 7.7 + 2.1 1. 6 + 1.0 5.4 + 0.6 37.8 + 3.7 4.0 RV 10.0 + 2.5 8.1 + 2.2 1. 3 + 0.7 5.5 + 0.7 37.8 + 4.0 Ao 10.0 + 2.2 7.6 + 2.1 1. 6 + 1.1 5.4 + 0.7 37.2 + 3.8 6.0 RV 9.9 + 2.5 7.8 + 2.4 1. 4 + 0.7 5.5 + 0.6 38.9 + 3.7 Ao 9.8 + 2.4 7.5 T 2.0 1. 5 + 1.0 5.5 + 0.5 38.4 + 3.4 8.0 RV 9.7 + 2.6 7.5 + 2.4 1. 3 + 0.9 5.4 + 0.6 38.1 + 3.8 Ao 8.8 + 2.4 6.9 + 2.5 1. 3 + 0.7 5.0 + 1.0 35.0 + 6.6 10.0 RV 9.6 + 2.6 7.6 + 2.5 1. 2 + 0.8 5.4 + 0.6 37.5 + 4.1 Ao 9.4 + 2.3 7.2 + 2.2 1. 4 + 0.7 5.3 + 0.6 36.9 + 3.8 12.0 RV 9.8 + 2.6 7.7 + 2.7 1. 1 + 0.7 5.5 + 0.6 37.1 + 4.0 Ao 9.5 + 2.6 7.6 + 2.4 1. 2 + 0.6 5.3 + 0.6 37.0 + 3.6 14.0 RV 9.6 + 2.6 7.4 + 2.5 1. 2 + 0.9 5.5 + 0.6 38.3 + 3.5 Ao 9.2 + 2.4 7.1 + 2.3 1. 4 + 0.9 5.3 + 0.6 36.7 + 4.1 16.0 RV 9.5 + 2.5 7.4 + 2.5 1. 5 + 1.0 5.4 + 0.6 37.6 + 3.9 Ao 9.4 + 2.5 7.2 + 2.1 1. 4 + 0.8 5.3 + 0.6 37.2 + 4.3 18.0 RV 9.6 2.7 7.4 + 2.4 1. 5 + 0.7 5.4 + 0.6 37.9 3.8 Ao 9.5 + 2.5 7.2 + 2.3 1. 3 + 1.1 5.4 + 0.5 37.9 + 2.9 20.0 RV 9.2 + 2.5 7.2 + 2.4 1. 1 + 0.8 5.3 + 0.6 37.3 + 3.8 Ao 9.2 + 2.6 7.1 + 2.2 1. 2 + 0.9 5.2 + 0.5 36.5 + 3.3 22.0 RV 9.4 + 2.4 7.3 + 2.2 1. 4 + 0.9 5.4 + 0.6 37.2 + 3.7 Ao 9.2 + 2.4 6.9 + 2.1 1. 5 + 0.9 5.3 + 0.5 36.4 + 3.1 24.0 RV 9.3 + 2.6 7.0 + 2.5 1. 4 + 0.9 5.2 + 0.6 36.6 + 3.9 Ao 9.0 + 2.6 6.9 + 2.6 1. 4 + 0.9 5.3 + 0.5 36.6 + 3.5 26.0 RV 9.2 + 2.4 7.1 + 2.4 1. 3 + 0.7 5.3 + 0.6 36.9 + 4.1 Ao 9.2 + 2.5 7.0 + 2.2 1. 4 + 0.8 5.3 + 0.6 36.8 + 3.8 28.0 RV 9.1 + 2.3 7.0 + 2.6 1. 3 + 0.8 5.2 + 0.6 36.6 + 4.0 Ao 8.9 +- 2.4 6.7 + 2.3 1. 3 + 0.8 5.2 + 0.7 36.1 + 4.8 INJECT BOLUS OF COBRA VENOM FACTOR 30. 0 RV 8.9 + 2.6 6.7 + 2.5 1. 4 + 0.7 5.2 + 0.6 36.3 + 4.0 "Ao "8.3 + 2.5 6.4 + 2.4 1. 2 + 0.5 4.9 + 1.0 34.2 + 6.3 30.3 RV 8.9 + 2.4 7.0 + 2.4 1. 4 + 0.7 5.3 + 0.5 37.6 + 2.9 Ao 8.4 + 2.4 6.3 + 1.9 1. 4 + 0.6 5.2 + 0.7 36.4 + 4. 2 30.7 RV 8.3 + 2.5 6. 2 + 2.3 1. 4 + 0.8 5.4 + 0.4 37.5 + 2.9 Ao 7.6 + 2.4 5.8 + 2.2 1. 2 + 0.6 5.2 + 0.5 36.7 + 3.0 - 122 -Table XI (cont'd) T I M E W B C P M N L C R B C H C T 31.0* RV 8.1 + 2.7 6.2 + 2.4 1. 2 + 0.6 5.5 + 0.4 38.3 + 2.5 Ao 8.0 + 2.5 6.3 + 2.3 1 .1 + 0.7 5.4 + 0.4 37.8 + 2.4 31.3 RV 7.4 + 2.4 - 5.7 + 2.2 1. 0 + 0.5 5.4 + 0.5 37.6 + 3.0 Ao 6.8 + 2.4 5.2 + 2.1 1 .1 + 0.4 5.3 + 0.5 36.9 + 2.8 31.7 RV 6.9 + 2.6 5.0 + 2.3 1. 4 + 0.8 5.4 + 0.5 37.6 + 3.0 Ao 6.7 + 2.7 5.0 + 2.5 1 .3 + 0.7 5.2 + 0.5 36.1 + 2.9 32.0** RV 5.9 + 3.0 4.4 + 2.4 1 .1 + 0.6 5.4 + 0.5 37.5 + 3.2 Ao 5.5 + 2.7 3.8 + 1.9 1 .0 + 0.5 5.3 + 0.5 36.9 + 3.4 34.0 RV 6.3 + 2.3 4.4 + 2.4 1. 4 + 1.2 5.3 + 0.6 36.9 + 4.1 Ao 5.8 + 2.4 4.1 + 2.4 1 .3 + 0.8 5.2 + 0.6 36.5 + 3.6 36.0 RV 6.0 + 2.4 4.3 + 2.4 1. 3 + 1.0 5.2 + 0.6 36.1 + 3.9 Ao 6.1 + 2.4 4.4 + 2.5 1 .2 + 0.7 5.1 + 0.6 35.8 + 3.8 38.0 RV 6.5 + 2.5 4.8 + 2.5 1. 3 + 0.8 5.2 + 0.6 36.7 + 3.7 Ao 6.4 + 2. 2 4.8 + 2.2 1 .0 + 0.7 5.1 + 0.6 36.0 + 4.0 40.0 RV 7.0 + 2.5 5.2 + 2.3 1. 2 + 0.7 5.1 + 0.7 35.5 + 4.3 Ao 6.4 + 2.7 4.8 + 2.8 1 .2 + 0.7 4.9 + 0.8 34.5 + 5.5 42.0 RV 7.2 + 2.5 5.6 + 2.7 1 .1 + 0.6 5.2 + 0.6 36.5 + 4.0 Ao 7.1 + 2.6 5.4 + 2.7 1 .1 + 0.5 5.2 + 0.7 36.3 + 4.3 44.0 RV 7.2 + 2.7 5.4 + 2.5 1 .1 + 0.6 5.0 + 0.6 35.3 + 3.6 Ao 7.3 + 2.5 5.6 + 2.5 1 .1 0.6 5.1 + 0.7 35.9 + 4.8 46.0 RV 7.5 + 2.8 6.1 + 2.8 1 .1 + 0.6 5.1 + 0.6 36.2 + 3.7 Ao 7.6 + 2.6 6.0 + 2.5 1 .1 + 0.5 5.1 + 0.6 36.7 + 5.3 48.0* RV 7.1 + 2.2 5.1 + 1.9 1. 3 + 0.9 5.1 + 0.6 35.8 + 3.7 Ao 7.1 + 2.2 5.4 + 2.2 1 .2 + 0.8 4.9 + 0.7 34.8 + 4.5 50.0 RV 8.4 + 2.5 6.5 + 2.4 1. 0 + 0.5 5.2 + 0.5 36.2 + 3.4 Ao 8.4 + 2.4 6.8 + 2.4 1 .2 + 0.7 5.1 + 0.5 36.0 + 3.4 60.0 RV 8.9 + 3.2 7.0 + 3.2 1 1 + 0.7 5.2 + 0.7 36.5 + 4.2 Ao 9.2 + 2.8 7.3 + 3.2 1 .1 + 0.7 5.2 + 0.6 36.2 + 4.1 90.0 RV 10.8 + 3.4 8.6 + 3.4 1. 2 + 0.8 5.1 + 0.6 36.0 + 4.0 Ao 11.0 + 3.7 9.1 + 3.8 1 2 + 0.7 5.1 + 0.6 36.1 + 4.0 120* RV 13.7 + 3.7 11.8 + 3.2 0. 8 + 0.5 5.5 + 0.4 38.6 + 3.1 Ao 14.0 + 4.6 12.0 + 4.2 1 0 + 0.4 5.6 + 0.5 38.8 + 3.3 150 RV 13.9 + 3.4 11.9 + 3.3 1. 1 + 0.5 5.2 + 0.6 36.3 + 4.2 Ao 13.9 + 3.2 11.8 + 3.6 1 2 + 0.8 5.4 + 0.6 36.2 + 4.9 * mean _+ SD for n = 4 dogs ** mean +_ SD for n = 3 dogs - 123 -TABLE XIIA HEMATOLOGIC DATA FOR CVF AND THEN LFOW FLOW EXPERIMENTS, FIRST RUN TIME WBC PMN LC (min) (103/mm3) (103/mm3) (103/mm3) 0.0 RV 8.3 + 9.0 6.4 + 8.1 1.2 + 0.5 Ao 8.0 + 7.8 5.8 + 7.0 1.3 + 0.6 0.5 RV 8.3 + 8.7 5.4 + 6.2 1.8 + 1.0 Ao 8.2 + 8.7 5.9 + 7.2 1.6 + 1.0 1.0 RV 8.4 + 9.1 6.5 + 8.1 1.4 + 0.9 Ao 8.2 + 8.3 5.8 + 6.8 1.5 + 0.8 1.5 RV 8.5 + 9.3 6.3 + 7.7 1.6 + 0.9 Ao 8.2 + 8.3 6.2 + 7.6 1.6 + 0.6 2.0 RV 8.4 + 9.2 6.4 + 8.2 1.5 + 0.8 Ao 8.4 + 8.4 6.2 + 7.2 1.6 + 0.8 2.5 RV 9.4 +10.0 7.3 + 8.9 1.7 + 1.1 Ao* 7.8 + 7.7 5.9 + 6.8 1.4 + 0.7 3.0* RV 8.9 +10.0 6.9 + 8.4 1.4 + 1.1 Ao 8.9 + 9.8 6.8 + 8.5 1.4 + 0.8 3.5* RV 8.6 + 9.1 5.8 + 7.3 2.1 + 1.4 Ao 8.6 + 8.5 6.2 + 7.7 1.9 + 0.5 4.0 RV 8.0 + 8.1 5.7 + 6.8 2.0 + 1.5 Ao 8.0 + 7.9 5.6 + 6.4 1.8 + 1.2 BALLOON INFLATION 4.5 RV 7.1 + 7.1 5.2 + 6.5 1.5 + 0.7 Ao 5.4 + 4.7 3.8 + 4.2 1.4 + 1.5 5.0 RV 6.5 + 6.1 4.9 + 5.5 1.3 + 0.7 Ao* 5.0 + 5.2 4.0 + 5.1 0.8 + 0.3 5.5** RV 7.5 + 7.0 6.2 + 6.3 1.0 + 0.5 Ao 5.8 + 3.9 4.5 + 3.4 1. 2 + 1.3 6.0** RV 7.4 + 7.1 5.7 + 6.1 1.2 + 0.8 Ao 4.8 + 4.4 3.7 + 3.8 0.9 + 0.6 6.5* RV 7.0 + 6.2 2.9 + 0,7 1.1 + 0.6 Ao 4.1 + 3.9 3. 2 + 3.3 0.6 + 0.2 7.0* RV 6.8 + 6.4' 6.3 + 6.3 1.7 + 0.4 Ao 3.5 + 3.5 2.8 + 3.3 0.6 + 0.3 7.5** RV 7.4 + 7.6 5.7 + 6.3 1.2 + 0.3 Ao 5.7 + 3.7 4.3 + 3.6 1.0 + 0.3 8.0** RV 8.3 + 6.9 6.6 + 6.6 1.5 + 0.6 Ao 4.7 + 4.5 3.8 + 4.0 0.6 + 0.2 8.5 RV 6.6 + 5.9 5.0 + 5.5 1.2 + 0.5 Ao 4.1 + 3.8 3.2 + 3.6 0.7 + 0.5 9.0* RV 6.9 + 6.8 5.7 + 6.4 1.8 + 0.6 Ao 3.7 + 3.7 2.9 + 3.6 0.7 + 0.5 - 124 -Table XIIA (cont'd) TIME WBC PMN LC 9.5** RV 7.7 + 7.3 5.9 + 6.0 1.3 + 0.9 Ao 8.0 +10.6 6.1 + 8.6 1.6 + 1.9 10.0* RV 7.5 + 6.2 5.7 + 5.2 1.4 + 0.7 Ao 5.8 + 5.6 4.5 + 4.9 1.0 + 0.7 BALLOON DEFLATION 10.5 RV* 9.9 +10.1 7.1 + 8.1 1.9 + 1.3 Ao ** 6.1 + 1.0 3.9 + 0.7 1.6 + 1.0 11.0 RV 7.4 + 6.4 5.2 + 5.2 1.8 + 1.2 Ao* 4.4 + 0.9 2.8 + 0.7 1.1 + 0.6 11.5 RV** 10.0 +10.2 7.1 + 8.9 2.0 + 0.8 Ao *** 4.0 + 0.9 2.3 + 0.3 1.5 + 0.4 12.0 RV** 10.0 +9.8 8.2 + 8.5 1.3 + 0.9 Ao *** 5.0 + 1.9 3.7 + 1.8 1.0 + 0.5 12.5* RV 9.6 + 9.3 7.4 + 8.6 1.6 + 1.0 Ao 9.7 + 8.9 7.4 + 8.2 2.1 + 1.2 13.0 RV 8.6 + 8.8 6.3 + 7.4 1.8 + 1.1 Ao 8.2 + 8.0 6.1 + r 7.3 1.6 + 0.7 13.5 RV* 9.3 + 9.2 7.2 + 3.9 1.6 + 0.4 Ao** 4.9 + 1.5 2.7 + 0.6 1.9 + 0.8 14. 0* RV 9.2 + 9.3 6.7 + 7.1 1.4 + 0.7 Ao 9.0 + 8.8 7.1 + 8.3 1.4 + 0.8 14.5 RV 8.4 + 8.1 6.3 + 6.5 1.7 + 0.7 Ao 7.2 + 5.4 5.3 + 4.7 1.6 + 0.9 15.0 RV* 9.1 + 8.7 6.8 + 6.8 1.7 + 1.0 Ao 7.9 + 7.0 6.2 + 6.6 1.4 + 1.7 * Mean + SD for n=5 dogs ** Mean + SD for n=4 dogs *** Mean _+ SD for n=3 dogs - 125 -TABLE XIIB HEMATOLOGIC DATA FOR CVF AND THEN LOW FLOW EXPERIMENTS, SECOND RUN TIME WBC PMN LC (min) (103/mm3) (103/mm3) (103/mm3) 0.0 RV 9.9 + 6.3 7.7 + 4.6 1.7 + 1.2 Ao 9.7 + 5.5 7.9 + 4.6 1.5 + 0.9 0.5 RV 10.0 + 6.2 8.2 + 5.3 1.3 + 0.8 Ao 9.3 + 5.5 7.7 + 4.6 1.2 + 0.9 1.0* RV 10.7 + 6.3 9.0 + 5.1 1.2 + 0.5 Ao 9.4 + 6.1 7.8 + 5.0 1.0 + 0.7 1.5 RV 9.8 + 5.8 8.4 + 5.0 1.0 + 0.5 Ao* 9.1 + 5.1 7.5 + 4.5 1.0 + 0.6 2.0 RV 9.9 + 5.9 8.3 + 4.8 1.2 + 0.8 Ao* 7.2 + 2.7 5.9 + 2.4 1.0 + 0.6 INJECT BOLUS OF COBRA VENOM FACTOR 2.5 RV 9.3 + 5.6 7.7 + 5.0 1.3 + 1.1 Ao* 6.7 + 2.3 4.8 + 1.6 1.2 + 0.8 3.0 RV 7.0 + 3.5 5.6 + 2.9 1.1 + 0.5 Ao 5.2 + 1.8 3.5 + 1.7 1.5 + 1.3 3.5 RV 5.6 + 2.4 4.5 + 2.1 1.0 + 0.5 Ao 4.1 + 1.9 2.9 + 1.6 1.1 + 0.5 4.0* RV 4.5 + 2.2 3.5 + 1.5 0.9 + 0.7 Ao 3.3 + 1.4 2. 2 + 1.3 1.0 + 0.7 BALLOON INFLATION 4.5 RV 4.0 + 1.9 2.9 + 1.3 1.0 + 0.6 Ao 2. 2 + 1.1 1.5 + 0.8 0.6 + 0.4 5.0 RV 3.4 + 1.4 2.3 + 1.0 0.9 + 0.5 Ao 1.9 + 1.0 1.2 + 0.8 0.6 + 0.3 5.5 RV 3.3 + 1.5 2.1 + 0.9 1.0 + 0.6 Ao 1.7 + 1.0 1.1 + 0.7 0.6 + 0.3 6.0** RV 3.1 +1.7 2.1 + 1.2 0.8 + 0.6 Ao 1.4 + 0.9 0.9 + 0.7 0.5 + 0.3 6.5* RV 3.5 + 1.6 2.3 + 1.2 1.0 + 0.7 Ao 1.6 + 1.0 1.1 + 0.8 0.5 + 0.4 7.0** RV 4.1 + 1.2 2.7 + 1.0 1.1 + 0.3 Ao 2.0+0.9 1.1 + 0.7 0.8 + 0.3 7.5 RV 3.6 + 1.6 2.6 + 1.3* 1.0 + 0.7 Ao 1.8 + 0.9 1.1 + 0.6* 0.7 + 0.7 8.0 RV 3.8 + 2.0 2.6 + 1.5 0.9 + 0.5 Ao 2.0 + 1.0 1.1 + 0.9 0.5 + 0.3 8.5** RV 2.4 + 1.3 1.7 + 1.4 0.6 + 0.4 Ao 1.7 + 0.9 1.0 + 0.5 0.6 + 0.5 - 126 -Table XITB (cont'd) TIME WBC PMN LC 9.0* RV 4.9 + 2.1 3.6 + 1.5 1.0 + 0,7 Ao 2.4 + 1.4 1.5 + 0.9 0.7 + 0.6 9.5** RV 4.6 + 2.3 3.1 + 1.9 1.4 + 0.8 Ao 2.7 + 1.3 1.8 + 1.4 0.8 + 0.4 10.0* RV 4.2 + 2.6 3.1 + 1.9 0.8 + 0.5 Ao 2.0 + 1.1 1.2 + 0.9 0.7 + 0.2 BALLOON DEFLATION 10.5 RV** 6.1 + 6.1 4.3 + 3.6 1.0 + 1.3 Ao* 4.6 + 4.2 2.8 + 2.7 1.5 + 1.4 11.0 RV* 5.0 + 2.6 3.0 + 1.7** 1.0 + 0.6 Ao* 3.5 + 1.6 2.0 + 1.4 1.3 + 0.4 11.5 RV 4.4 + 2.5 2.8 + 1.6 1.4 + 1.1 Ao 3.9 + 2.7 2.7 + 2.4 1.0 + 0.5 12.0*** RV 3.6 + 2.7 2.4 + 2.3 0.9 + 0.5 Ao 3.7 + 3.7 2.0 + 2.7 1.5 + 0.9 12.5 RV 4.7 + 2.8 2.9 + 1.6 1.5 + 1.2 Ao 4.1 + 2.8 2.7 + 2.4 1.1 + 0.5 13.0 RV 4.7 + 2.8 3.3 + 2.3 1.1 + 0.7 Ao 4.3 + 2.8 2.7 + 2.0 1.3 + 0.8 13.5* RV 5.5 + 2.6 3.7 + 1.9 1.4 + 0.9 Ao 5.0 + 3.0 3.4 + 2.1 1.2 + 1.0 30** RV 8.5 + 5.0 6.7 + 4.2 1.3 + 0.7 Ao 6.5 + 2.6 5.1 + 2.0 1.0 + 0. 3 60 RV** 10.0 + 5.9 8.0 + 4.4 1.5 + 1.1 Ao* 8.6 + 4.4 6.6 + 3.6 1.4 + 0.7 90* RV 12.2 + 6.9 9.7 + 5.2 1.9 + 1.4 Ao 11.3 + 5.5 9.2 + 4.5 1.5 + 1.0 120 RV*** 17.3 + 8.4 15.4 + 8.3 2.2 + 1.4 Ao* 12.4 + 6.0 10.4 + 5.4 1.4 + 0.7 * Mean + SD for n=5 dogs ** Mean +_ SD for n=4 dogs *** Mean + SD for n=3 dogs - 127 -" TABLE XIII FRACTIONAL UPTAKE OF WBC BY THE LUNG FOR CVF AND THEN  LOW FLOW EXPERIMENTS (RV-AO) x 100 RV RUN I RUN II WBC -0.8 + 1.8 4.90 + 1.4 Control Period PMN 2.1 + 2.7 7.2 + 2.2 CO. 2.56 + 0.18 2.61 + 0.19 WBC -0.6 + 1.9 18.5 + 3.7 *** CVF injection PMN -2.1 + 3.1 30.2 + 4.4 *** (in run II only) CO. 2.67 + .17 2.37 + 0.15 WBC 31.8 + 3.8 * 48.3 + 1.7 *** Low PBF Period PMN 31.5 + 3. 2 * 54.7 + 2.0 *** (in run I and run II) CO. 0.64 + 0.14 0.56 + 0.13 WBC 1.2 + 1.8 14.9 + 3.1 ** Recovery Period PMN 0.1 + 3.8 20.1 + 4.7 *** CO. 2.03+ 0.12 1.70 + 0.42 * = p .01 (comparing control period to other periods in each run) ** = p .01 (comparing values between Run I and Run II) *** = p .001 (comparing values between Run I and Run II) - 128 -TABLE XIV COMPLEMENT PROTEIN ASSAY RESULTS RUN I RUN II Control C2 1584 + 236 1443 + 61 Period C6 23430 + 2382 22100 + 1422 CH50 92.8 + 5.6 78.1 + 3.3 CVF C2 1831 + 167 1286 + 41 injection (in C6 2135 7 + 1312 184 88 + 2342 run II) CH50 100 + 6.4 79.7 + 3.9 Recovery C2 1690 + 137 1233 + 45 Period C6 21040 + 1417 20940 + 2093 CH50 89 + 6 84 + 4.6 - 129 -TABLE XV ARTERIAL BLOOD GAS VALUES IN CVF AND THEN LOW PBF EXPERIMENTS Period 1 Period 2 Period 3 pH 7.32 + .02 7.27 +.08 7.30 +.06 pC02 (mmHg) 40 + 6 38 + 8 31 + 2 p0 2 (mmHg) 89 + 12 92 + 10 103 + 9 Sat. (%) 95 + 3 94 + 2 97 + 1 Period 1 = start of run 1 Period 2 = start of run 2 Period 3 = after cobra venom injection - 130 -TABLE XVI HEMATOLOGIC DATA FOR EPINEPHRINE INFUSION AND CVF EXPERIMENTS TIME WBC PMN LC RBC HCT (min) (103/mm3) (103/mm3) (103/mm3) (106/mm3) (%) 0.0 RV 9.1 + 1.9 6.9 + 3.6 1.4 + 0.4 5.9 + 0.6 40.5 + 5.5 Ao 9.2 + 4.1 6.8 + 3.8 1.6 + 0.4 5.8 + 0.7 39.9 + 5.8 2.0 RV 9.0 + 3.8 6.6 + 3.4 1.3 + 0.6 5.9 + 0.6 39.9 + 6.4 Ao 8.8 + 3.6 6.5 + 2.8 1.3 + 0.4 5.9 + 0.6 39.7 + 5.4 4.0 RV 8.8 + 3.7 6.4 + 3.1 1.6 + 0.6 6.0 + 0.6 40.0 + 5.7 Ao 8.7 + 3.8 6.2 + 3.1 1.7 + 0.6 5.8 + 0.6 39.4 + 5.3 6.0 RV 8.7 + 3.6 6.6 + 3.3 1.3 + 0.4 5.8 + 0.7 39.5 + 5.9 Ao 8.2 + 3.6 6.3 + 2.9 1.2 + 0.4 5.6 + 0.5 38.0 + 5.3 8.0 RV 8.1 + 3.7 5.9 + 3.2 1.5 + 0.5 5.5 + 0.6 37.6 + 5.7 Ao 8.6 + 4.1 7.4 + 3.5 1.1 + 0.4 5.7 + 0.7 39.1 + 6.0 10.0 RV 8.3 + 3.6 6.3 + 3.2 1.4 + 0.5 5.7 + 0.7 38.8 + 6.1 Ao 8.4 + 3.8 6.2 + 3.2 1.4 + 0.6 5.7 + 0.7 38.6 + 6.1 START EPINEPHRINE INFUS ION 10.5 RV 8.2 + 3.5 6.0 + 2.7 1.5 + 0.5 5.6 + 0.7 38.3 + 5.9 Ao 8.5 + 3.8 6.4 + 3.1 1.3 + 0.5 5.7 + 0.7 38.9 + 6.0 11.0 RV 8.3 + 3.4 6.2 + 3.2 1.5 + 0.5 5.7 + 0.8 38.9 + 6.9 Ao 8.2 + 3.5 6.1 + 3.2 1.2 + 0.4 5.7 + 0.8 38.5 + 6.4 11.3 RV 8.4 + 3.6 6.2 + 2.8 1.4 + 0.5 5.7 + 0.8 39.2 + 6.8 Ao 8.0 + 3.3 6.2 + 3.0 1.2 + 0.4 5.9 + 0.9 40.4 + 6.9 11.7 RV 8.6 + 3.4 6.4 + 3.1 1.4 + 0.5 5.9 + 0.7 40.4 + 6.3 Ao 8.5 + 3.6 6.2 + 2.8 1.4 + 0.6 5.9 + 0.8 40.0 + 6.4 12.0 RV 8.9 + 3.4 6.7 + 3.1 1.3 + 0.4 6.1 + 0.6 41.8 + 5.7 Ao 8.8 + 3.4 6.4 + 3.2 1.5 + 0.4 6.1 + 0.6 41.5 + 5.5 14.0 RV 9.6 + 3.9 6.7 + 3.7 1.8 + 0.3 6.6 + 0.5 44.7 + 4.7 Ao 9.6 + 3.3 6.8 + 2.9 1.6 + 0.4 6.8 + 0.6 46.2 + 4.7 16.0 RV 9.7 + 3.2 7.1 + 3.2 1.6 + 0.5 6.9 + 0.6 46.9 + 5.2 Ao 9.6 + 3.2 6.8 + 2.9 1.9 + 0.6 6.8 + 0.6 46.6 + 5.3 18.0 RV 10.0 + 3.4 7.3 + 3.0 1.7 + 0.6 6.9 + 0.7 46.8 + 5.7 Ao 9.8 + 3.6 7.2 + 2.9 1.7 + 0.6 6.9 + 0.6 46.0 + 5.4 20.0 RV 10.0 + 3.5 7.3 + 3.2 1.9 + 0.6 6.8 + 0.6 46.4 + 5.5 Ao 9.9 + 3.6 7.1 + 2.9 1.9 + 0.6 6.7 + 0.7 46.2 + 5.5 22. 0 RV 9.9 + 3.6 7.2 + 3.0 1.7 + 0.6 6.6 + 0.6 44. 2 + 4.9 Ao 9.9 + 3.6 7.0 + 2.6 1.9 + 0.8 6.8 + 0.6 46.1 + 5.4 24.0 RV 10.0 + 3.7 7.0 + 3.1 2.0 + 0.5 6.7 + 0.7 45.8 + 6.0 Ao 10.1 + 3.8 7.3 + 3.1 2.0 + 0.8 6.7 + 0.6 45.4 + 5.1 26.0 RV 10.1 + 3.8 7.4 + 3.1 1.8 + 0.4 6.7 + 0.6 45.9 + 5.8 Ao 10.0 + 3.9 7.4 + 3. 2 1.7 + 0.6 6.7 + 0.6 45.5 + 5.3 28.0 RV 10.0 + 3.7 7.4 + 2.7 1.6 + 0.7 6.6 + 0.6 44.9 + 5.5 Ao 9.8 + 4.0 7.3 + 3.3 1.7 + 0.6 6.6 + 0.6 45.4 + 5.5 - 131 -Table XVI (cont'd) INJECT BOLUS OF COBRA VENOM FACTOR TIME WBC PMN LC RBC HCT 30.0 RV 9.4 + 3.6 6.9 + 2.8 1.6 + 0.9 6.4 + 0.6 .43.6 + 5.7 Ao 9.4 + 3.7 7.0 + 2.7 1.6 + 0.6 6.4 + 0.7 43.9 + 6.3 30.3 RV 9.2 + 3.6 6.8 + 2.9 1.7 + 0.6 6.5 + 0.7 44.4 + 6.1 Ao 9.6 + 3.2 7.0 + 2.2 1.9 + 0.7 6.5 + 0.8 44.5 + 7.3 30.7 RV 8.5 + 3.5 6.1 + 2.5 1.5 + 0.4 6.5 + 0.8 44.3 + 6.5 Ao 8.2 + 3.9 5.6 + 2.6 1.6 + 0.7 6.4 + 0.8 43.9 + 6.4 31.0 RV 7.7 + 3.7 5.5 + 2.8 1.5 + 0.8 6.5 + 0.8 44.1 + 6.9 Ao 7.6 + 4.2 5.3 + 3.2 1.7 + 0.7 6.5 + 0.8 44.4 + 6.5 31.3 RV 8.2 + 3.7 5.6 + 2.6 1.8 + 0.7 6.5 + 0.8 44.6 + 7.2 Ao 7.1 + 4.2 4.8 + 3.2 1.6 + 0.6 6.5 + 0.7 44.4 + 6.3 31.7 RV 6.8 + 4.0 4.6 + 3.0 1.6 + 0.7 6.5 + 0.7 44.3 + 6.4 Ao 6.7 + 4.6 4.7 + 3.6 1.5 + 0.8 6.5 + 0.7 44.5 + 6.3 32.0 RV 6.4 + 4.1 4.4 + 3.3 1.4 + 0.5 6.4 + 0.7 43.9 + 6.3 Ao 6.2 + 4.2 3.9 + 3.0 1.8 + 0.9 6.5 + 0.7 44.2 + 6.3 34.0 RV 5.8 + 4.2 3.7 + 2.9 1.6 + 0.9 6.4 + 0.8 43.7 + 6.8 Ao 5.5 + 4.2 3.7 + 3.4 1.4 + 0.5 6.4 + 0.7 43.4 + 6.5 36.0 RV 6.2 + 4.6 4.1 + 3.5 1.3 + 0.4 6.5 + 0.8 44.9 + 6.2 Ao 6.0 + 4.6 3.9 + 3.6 1.5 + 0.5 6.4 + 0.9 43.6 + 7.6 38.0 RV 6.3 + 4.1 4.4 + 3.4 1.4 + 0.5 6.3 + 0.9 42.8 + 7.6 Ao 6.1 + 4.4 4.4 + 3.6 1.3 + 0.7 6.5 + 0.7 4.4.1 + 6.5 40.0 RV 6.6 + 4.0 4.9 + 3.5 1.2 + 0.8 6.0 + 0.9 41.5 + 7.7 Ao 6.6 + 4.1 4.6 + 3.4 1.4 + 0.5 6.1 + 0.9 41.8 + 7.6 42.0 RV 7.2 + 3.8 5.3 + 3.4 1.3 + 0.3 6.2 + 0.9 42. 2 + 7.3 Ao 7.0 + 4.2 5.3 + 3.6 1.2 + 0.3 6.2 + 0.9 42.2 + 7.0 44.0 RV 8.2 + 4.1 6.0 + 3.0 1.6 + 0.8 6.4 + 0.7 43.3 + 5.6 Ao 8.1 + 3.9 5.9 + 3.1 1.6 + 0.7 6.3 + 0.7 43.5 + 5.5 46.0 RV 8.6 + 3.8 6.7 + 3.1 1.4 + 0.5 6.3 + 0.8 43.1 + 6.2 Ao 8.2 + 3.8 6.2 + 3.3 1.3 + 0.4 6.3 + 0.8 43.1 + 6.3 48.0 RV 8.9 + 4.1 7.0 + 3.6 1.3 + 0.5 6.2 + 0.8 42.4 + 6.3 Ao 9.0 + 4.3 7.0 + 3.7 1.5 + 0.5 6.2 + 0.7 42.1 + 5.7 50.0 RV 9.5 + 4.3 7.3 + 3.8 1.6 + 0. 3 6.2 + 0.8 42.4 + 6.6 Ao 9.2 + 4.0 7.1 + 3.6 1.5 + 0.4 6.1 + 0.8 41.7 + 6.1 STOP EPINEPHRINE INFUSION 60 RV 8.4 + 3.6 7.0 + 3.2 1.0 + 0.2 5.7 + 0.8 39. 2 + 6.6 Ao 8.4 + 3.6 6.8 + 3.5 1.1 + 0.4 5.7 + 0.9 39.4 + 7.0 90.0 RV 10.1 + 6.0 8.4 + 5.5 1.1 + 0.4 5.2 + 0.8 35.5 + 6.7 Ao 10.5 + 7.1 8.8 + 6.3 1.0 + 0. 3 5.2 + 0.8 35.7 + 6.9 120 RV 12.0 + 6.6 10.1 + 6.3 1.1 + 0.3 5.2 + 0.9 36.0 + 7.3 Ao 12.0 + 7.2 10.5 + 6.7 0.8 + 0.4 5.2 + 0.4 35.7 + 7.6 150 RV 12.7 + 7.0 10.7 + 6.1 1.2 + 0.8 5.2 + 0.8 36.1 + 6.5 Ao 12.4 + 7.6 10.8 + 7.3 1.0 + 0.3 5.3 + 0.8 35.8 + 6.2 * mean _+ SD for n = 4 dogs because of technical d i f f i c u l t y . - 132 -TABLE XVII COMPARISON OF WBC COUNTS IN THE FIVE SETS OF EXPERIMENTS WBC (% Control) CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n = 6 n = 5 6.0 RV 9 9 + 8 9 8 + 1 9 5 + 3 9 8 + 4 Ao 96+11 9 7 + 3 9 1 + 4 9 7 + 6 10.0 RV 93+12 9 5 + 2 9 1 + 4 9 6 + 6 Ao 89+19 9 3 + 2 9 2 + 4 9 4 + 4 EPINEPHRINE 24.0 RV 91+16 9 1 + 4 112+24 89+ 9 Ao 92+18 8 9 + 5 113 +23 9 1 + 8 Lower f1ow 28.0 RV 94+14 9 0 + 3 112+24 55+11 Ao 92+12 8 8 + 5 109 +24 3 9 + 9 CVF CVF CVF 30.0-30.5 RV 94+14 8 7 + 4 102+18 53+ 9 Ao 89 + 16 83 + 6 101 + 20 36 + 5 34.0 RV 98+14 60+11 61+29 35+ 7 Ao 99 +18 55 + 13 58+28 2 2 + 7 Restore Flow 36.0 RV 99+15 58+11 63+29 4 8 + 9 Ao 102 +17 59 + 11 63 +29 49 + 19 38.0 RV 99 +15 63 + 10 67 + 24 66 + 12 Ao 102 + 17 63 + 8 65 + 26 69 + 12 60.0 RV 112 +30 87 + 12 94 +24 86 + 12 Ao 116 + 33 92 + 14 94 + 24 89 + 15 150.00 RV 184 + 66 140 + 17 136 + 37 201 + 28 Ao 193 + 70 140 +18 131+40 197 + 32 - 133 -RBC (% TABLE XVIII COMPARISON OF RBC COUNTS IN THE FIVE SETS OF EXPERIMENTS Contr ol) " CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n = 6 • n = 5 6.0 RV 100 + 3 99 + 2 98 + 4 99 + 1 Ao 95 + 10 98 + 1 95 + 6 98 + 3 10.0 RV 96 + 7 100 + 7 95 + 4 95 + 1 Ao 91 + 20 96 + 2 96 + 5 95 + 3 EPINEPHRINE 24.0 RV 92+12 9 4 + 4 1 1 3 + 9 9 3 + 2 Ao 90 + 13 94 + 4 113 + 8 92 + 4 Lower Flow 28.0 RV 93+10 9 4 + 4 1 1 1 + 8 8 7 + 8 Ao 92 + 7 93 + 5 112 + 7 83 + 8 CVF CVF CVF 30.0-30.5 RV 92+11 9 6 + 3 1 0 9 + 7 8 7 + 5 Ao 85 + 18 9 3 + 5 1 0 9 + 8 7 8 + 2 34.0 Rv 9 3 + 9 9 4 + 5 1 0 8 + 6 8 4 + 3 Ao 9 3 + 7 9 3 + 4 1 0 8 + 6 7 8 + 7 Restore Flow 36.0 RV 9 1 + 1 1 9 3 + 5 107 + 7 94+18 Ao 9 2 + 8 9 2 + 3 1 0 8 + 6 94+20 38.0 RV 9 3 + 8 9 4 + 4 1 0 6 + 7 99 + 17 Ao 9 2 + 8 9 3 + 3 1 0 7 + 6 97+16 60.0 RV 95+10 9 3 + 3 9 6 + 9 8 2 + 9 Ao 94 + 8 93 + 3 97 + 9 83 + 11 150.0 RV 102+10 9 3 + 4 8 8 + 8 92 + 14 Ao 101 + 5 9 2 + 6 8 8 + 9 95+12 - 134 -TABLE XIX COMPARISON OF PMN COUNTS IN THE FIVE SETS OF EXPERIMENTS TOTAL PMN (% Control) CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n - 4 n = 5 n = 6 n = 5  6.0 RV 104 + 13 9 8 + 7 9 6 + 6 1 0 1 + 7 Ao 104 + 16 95 + 8 92 + 7 96 + 4 10.0 RV 98+20 9 5 + 7 9 0 + 6 9 9 + 8 Ao 98+18 9 1 + 3 8 9 + 5 9 3 + 6 EPINEPHRINE 24.0 RV 102+19 8 8 + 7 104+25 91+11 Ao 100 +26 8 5 + 9 107 +24 8 9 + 9 Lower Flow 28.0 RV 104+23 87+7 114+24 58 + 11 Ao 105 +25 8 4 + 6 108+21 3 7 + 8 CVF CVF CVF 30.0-30.5 RV 103 +18 8 3 + 8 104 +20 54 + 11 Ao 96 + 16 80 + 10 105 + 19 40 + 8 34.0 RV 111 +21 52 + 16 51+26 3 3 + 7 Ao 111+31 48 + 15 48 +28 20 + "7 Restore Flow 36.0 RV 110+25 51+16 55+30 4 1 + 6 Ao 113 +30 52 + 16 52 +30 41 + 17 38.0 RV 108 +23 58+13 61 + 28 63 + 12 Ao 115 +30 59 + 12 60 +28 63 + 13 60.0 RV 150+ 15 86+13 104+33 88 + 16 Ao 135 +54 90 + 18 99+32 9 1 + 8 150.0 RV 222 +104 151 + 12 154 + 49 220 + 30 Ao 235 +111 149 + 9 149 + 48 212 + 35 - 135 -TABLE XX COMPARISON OF LYMPHOCYTE COUNTS IN FIVE SETS OF EXPERIMENTS LYMPHOCYTE (% Control) CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n =- 6 n = 5 6.0 RV 108+42 112+17 92+16 90+55 Ao 99 + 49 107 +21 84 + 18 107 + 39 10.0 RV 108+67 90+21 96+17 68+36 Ao 74+39 109 +22 93 + 40 91 + 41 EPINEPHRINE 24.0 RV 76 + 43 102 + 38 141 + 33 63 + 33 Ao 97 + 55 102 + 9 139 + 39 86 + 43 Lower Flow 28.0 RV 69 + 25 102 + 30 117 + 48 44 + 13 Ao 78+54 97+7 115 +35 26+14 CVF CVF CVF 30.0-30.5 RV 69 + 25 106 + 20 110 +35 48 + 18 Ao 104 + 54 101 + 33 115 +29 25 + 11 34.0 RV 73 +39 97 + 24 105 +37 60 + 30 Ao 72 +39 92 + 21 98 +17 40 + 17 Restore Flow 36.0 RV 74 + 42 92 + 15 86 + 13 114 + 61 Ao 77 +38 92 + 19 103 + 15 107 + 62 38.0 RV 91+66 97+17 96+16 97+31 Ao 78 +34 76+10 83+26 . 125 + 28 60.0 RV 49 +28 77 + 18 70 +16 65 + 20 Ao 74 + 24 84 + 20 84 + 32 74 + 31 150.0 RV 67 +35 87 + 24 82 +41 89 + 50 Ao 53 + 14 102 + 71 68 + 19 114 + 30 - 136 -TABLE XXI COMPARISON OF HEMATOCRIT (HCT) IN THE FIVE SETS OF EXPERIMENTS HCT (% Control) Time (min) 6.0 RV Ao CONTROL n = 4 100 + 3 95 + 10 CVF n = 5 9 9 + 1 98 + 2 EPINEPHRINE + CVF LOW FLOW + CVF n = 6 n = 5 9 8 + 4 9 6 + 6 101 + 8 9 7 + 4 10.0 RV Ao 9 5 + 7 90 + 19 9 5 + 2 9 4 + 2 9 6 + 4 9 5 + 4 95 + 94 + 3 3 EPINEPHRINE 24.0 RV Ao 92 + 11 90 + 13 9 3 + 3 9 4 + 2 113 + 9 113 + 8 92 + 91 + 3 4 Lower Flow 28.0 RV Ao 93 + 9 9 1 + 7 9 3 + 3 9 2 + 5 111 + 8 113 + 7 86 + 7 8 2 + 8 CVF CVF CVF 30.0-30.5 RV Ao 92 + 10 85 + 17 9 3 + 3 87 + 11 108 + 8 109 + 6 86 + 78 + 4 4 34.0 RV Ao 9 3 + 8 92 + 7 9 4 + 4 9 3 + 3 108 + 7 107 + 6 85 + 78 + 3 6 Restore Flow 36.0 RV Ao 90 + 10 9 2 + 8 9 2 + 4 9 1 + 4 107 + 7 107 + ! 95 + 18 94 + 21 38.0 RV Ao 9 2 + 8 9 2 + 8 9 3 + 3 9 2 + 4 106 + 106 + 100 + 17 99 + 17 60.0 RV Ao 93 + 8 9 5 + 8 9 3 + 3 9 2 + 3 97 + 9 9 7 + 9 82 + 10 83 + 11 150.0 RV Ao 102 + 9 100 + 5 8 9 + 8 9 2 + 6 8 9 + 8 8 8 + 8 92 + 14 95 + 12 - 137 -TABLE XXII COMPARISON OF CARDIAC OUTPUT IN THE FIVE SETS OF EXPERIMENTS CARDIAC OUTPUT (% Control) CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n = 6 n = 5 2.00-10.0 104+ 7 96+16 104+ 7 1 0 1 + 9 EPINEPHRINE 20.0-22.0 94 + 6 109 + 15 170 + 34 97 + 16 Low FIow 25.0-29.0 9 4 + 5 108+17 163+54 14+ 9 CVF CVF CVF 34.0-36.0 93+10 100+15 162+40 20+15 Restore Flow 60.0 8 3 + 4 83+13 141 +53 86 + 18 150.0 70 + 17 75 +13 69+10 71 + 17 - 138 -TABLE XXIII COMPARISON OF SYSTEMIC ARTERIAL BLOOD PRESSURE (Pa) IN THE FIVE SETS OF EXPERIMENTS Pa (% Control) Time (min) 5.0-10.0 24.0 25.0-30.0 32.0 34.0-37.0 60.0 150.0 CONTROL CVF n = 4 n = 5 9 7 + 5 9 9 + 4 9 6 + 7 9 8 + 4 9 5 + 7 9 6 + 9 96 + 11 9 5 + 7 EPINEPHRINE + CVF n = 6  102 + 4 EPINEPHRINE 101 + 14 CVF 97 + 10 96 + 12 95 + 15 91 + 11 CVF 97 + 11 9 7 + 9 9 3 + 7 9 5 + 7 LOW FLOW + CVF n = 5 100 + 22 97 + 2 Lower Flow 30 + 11 CVF Restore Flow 75 + 16 8 7 + 8 9 2 + 6 - 139 -TABLE XXIV COMPARISON OF PULMONARY ARTERY BLOOD PRESSURE (Ppa) IN FIVE SETS OF EXPERIMENTS Ppa _(% Control)  CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n = 6 n = 5 5.0-10.0 36 + 6 37 + 10 28 + 2 3 5 + 9 EPINEPHRINE 24.0 32 + 6 32 + 7 35 + 7 33 + 6 Low FI ow 25.0-30.0 CVF CVF 1 2 + 6 CVF 32.0 31 + 7 30 + 6 33 + 3 Restore Flow 34.0-37.0 30 + 9 29 + 8 31 + 8 3 6 + 7 60.0 29 + 9 32 + 11 26 + 5 30 + 6 150.0 29 + 5 30 + 8 21 + 7 27 + 8 - 140 -TABLE XXV COMPARISON OF PULMONARY WEDGE BLOOD PRESSURE (Pw) IN THE FIVE SETS OF EXPERIMENTS (mmHg) Pw CONTROL CVF EPINEPHRINE + CVF LOW FLOW + CVF Time (min) n = 4 n = 5 n = 6 n = 5 2.0-10.0 17+ 5 20 + 7 17 + 3 16 + 5 EPINEPHRINE 24.0 1 5 + 4 1 4 + 8 2 2 + 5 1 5 + 6 Low Flow 25.0-30.0 CVF CVF CVF 32.0 1 4 + 5 1 6 + 4 2 2 + 4 Restore Flow 34.0-37.0 1 4 + 5 1 4 + 6 1 9 + 6 2 0 + 4 60.0 1 4 + 6 1 5 + 5 1 5 + 3 1 5 + 4 150.0 1 1 + 6 1 4 + 4 1 1 + 4 1 2 + 6 - 141 -TABLE XXVI COMPARISON OF SYSTEMIC ARTERIAL P0 9 IN THE FIVE SETS OF EXPERIMENTS POo (mmHg) Time (min) 6.0 16.0 26.0 31.0 36.0-42.0 60.0 150.0 CONTROL n = 4 99 + 23 117 + 51 102 + 16 98 + 25 CVF n = 5 107 + 11 102 + 21 100 + 9 103 + 30 111 + 26 CVF 98 + 18 97 + 23 111 + 31 EPINEPHRINE + CVF LOW FLOW + CVF n = 6 86 + 16 CVF 88 + 12 9 4 + 5 88 + 31 n = 5 78 + 13 EPINEPHRINE 87 + 19 8 1 + 9 85 + 10 Low FI ow 122 + 20 CVF 91 + 24 Restore Flow 9 3 + 9 98 + 28 104 + 22 - 142 -Figure 31 Systemic Blood Pressure and Cardiac Output in Control Dogs. The mean (+SE) systemic blood pressure and mean (j+SE) cardiac output for 7 control dogs is shown. Simultaneous blood sampling from the right ventricle and aorta had no effect on systemic blood pressure but cardiac output f e l l over the course of the experimental run. Figure 32 Pulmonary Artery Blood Pressure, Wedge Pressure, and Partial Pressure of Oxygen in Arterial Blood of Control Dogs. Mean (+ SE) Ppa, mean (+ SE) Pw, and mean (+ SE) P0 2 are shown. Simultaneously sampling blood from the right ventricle and aorta had no effect on pulmonary art e r i a l blood pressure, pulmonary wedge pressure or P0„. - It3 -O oc t-z o u 2 0 0 . 0 1 8 0 . 0 1 6 0 . 0 140.0-1 120 .0 1 0 0 . 0 8 0 . 0 6 0 . 0 40.0-1 2 0 . 0 0.0-1 P a - e — • C O - * N: 4 * N : 7 (mean + SE) S s — ^ - l W ' f ^ ^ - T - 1 " ' ** *~ I l l I V 0.0 7.5 15.0 22 .5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 60 .0 9 0 . 0 1 2 0 . 0 1 5 0 . 0 TIME(min) 1 5 0 . 0 p O , -Ppa-Pw-N: 4 k N : 7 (mean + SE) 0 .0 7T5 15.0 2275 30 .0 37 .5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 120 .0 1 5 0 . 0 TIME (min) - 144 -Figure 33 Aorta and Right Ventricle WBC Counts in Control Dogs. The mean (+ SE) WBC count for RV and Ao are shown for seven dogs. Simultaneous sampling of blood from the right ventricle and aorta had l i t t l e affect on WBC during the f i r s t 50 minutes but thereafter WBC counts increased significantly. Consistant arterial venous differences could not be demonstrated in these experiments. Figure 34 Aorta and Right Ventricle RBC Counts in Control Dogs. The mean (+ SE) RBC counts in RV and Ao are shown for seven dogs. These data show that simultaneous sampling of blood from the aorta and right ventricle has l i t t l e effect on red blood c e l l (RBC) counts during the course of these experiments. - 145 -c o o * O U O CQ 5 0 .0 -0 0 7.5 15 .0 2 2 . 5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 1 2 0 . 0 1 5 0 . 0 TIME (min) e o o O O O co cc 200.0- , 1 8 0 . 0 1 6 0 . 0 1 4 0 . 0 -1 2 0 . 0 1 0 0 . 0 8 0 . 0 -6 0 . 0 4 0 . 0 20 .0-0.0-RV-Ao-0.0 III TIME (min) N : 4 * N : 7 (mean ± SE) IV - 146 -Figure 35 Pulmonary Artery Blood Pressure during Transient Lowering of PBF. Mean (+ SE) Ppa is shown for five dogs. With balloon inflation there is an i n i t i a l rapid f a l l , followed by a more gradual decline in blood pressure. Balloon deflation is associated with a return in pulmonary arterial blood pressure to c on tr o l . Figure 36 Systemic Blood Pressure During Transient Lowering of PBF. Mean (+ SE) Pa is shown for five dogs. With balloon inflation there is an i n i t i a l rapid f a l l , followed by a more gradual decline in blood pressure values. Balloon deflation is associated with an increase in blood pressure back towards control. - m -IVC Balloon inflation 0 l4 0:8 1 ^ 1 ^ 2.0 2l4f 2:8 3 ^ N=5 (MEAN+SE) 3.6 4 .0 TIME (min) Sfiwixf" - 148 -Figure 37 Cardiac Output During Transient Lowering of PBF. Mean (+ SE) CO is shown for five dogs. Balloon inflation is associated with an i n i t i a l rapid f a l l , followed by a very gradual decline in cardiac output. After balloon deflation there is a transient increase in cardiac output to levels significantly greater than control before CO f e l l to control levels. Figure 38 Aorta and Right Ventricle WBC Counts During Transient Lowering of PBF. Mean (+ SE) WBC counts for RV and Ao are shown for five dogs. Large A-V leukocyte differences develop following balloon inflation and balloon deflation. These data show that during low flow conditions leukocytes are sequestered within the pulmonary raicrovasculature and that expulsion of sequestered leukocytes occurs after balloon deflation. - IH? -N=5 (MEAN±SE) 0 l 4 0 T 8 TT2 1T6 2:0 2.44 2T8 3T2 316 4.0 b a l l o o n . . . |VC b a l l o o n IVC i n f l a t i o n TIME (min) donation 1 4 0 1 2 6 1 1 2 -2 9 & e Z 8 4 * *» § 5 6 o OD 2 6 14-II o!of 0:4 0:8 I V C b a l l o o n inf l a t i o n ' ^ -* -?-2--7,...T - Y 111 N = 5 ( M E A N i S E ) 1.2 1.6 2.0 2.4f 2.8 3.2 3.6 4.0 TIME (min) I V C b a l l o o n d e f l a t i o n - 150 -Figure 39 PMN and Lymphocyte Count A-V Differences During Transient Lowering of PBF. These data show that PMN constitute the largest fraction of the A-V difference which develops after balloon inflation and also after balloon deflation. These data also show that lymphocytes are taken up into the lung and released from the lung following balloon inflation and deflation, respectively. n=5 (mean +_ SE) except after 3 minutes where n=4 (mean +_ SE). Figure 40 WBC Uptake into the Lung During Transient Lowering of PBF. Mean (_+ SE) WBC counts/min. are shown for five dogs. Note that leukocytes continue to be sequestered within the pulmonary microvasculature 20-30 seconds after balloon deflation. Calculation of the areas under these curves using the raw data for each of the animals revealed that 99 _+ 7% (mean _+ SE) of the cells taken up when flow was lowered were released when flow was res tored. - 151 ~ o o 55 44 33 x > 22 O C T si ~ 0 z o H -11 Z UI H -22 UI cc * -33 -44 -55-I o: PMN - • - - • LYMPHOCYTE-o-o AT ^ II in 0.4 0.8 IVC balloon intlation 1.2 1.6 2.0 2.4f TIME (min) N = 5 (MEANtSE) 2 . 8 IVC'bailoon deflation 3.6 4.0 6.0-4.2-2.4-0.6 c -1.2-c E -3.0-o -4.8 X O C O -6.6 -8.4--10.2 N = 5 (MEANtSE) IVC balloon - inflation TIME (min) IVC balloon deflation - 152 -Figure 41 Aorta and Right Ventricle RBC Counts During Transient Lowering of PBF. Mean (+ SE) RBC Counts for the RV and Ao are shown for five dogs. These data show that neither RV nor Ao erythrocyte counts are affected markedly by inflating or deflating the IVC balloon. In contrast to WBC, RBC counts in the RV become lower than corresponding Ao counts following balloon infla t i o n . Figure 42 Relationship Between PMN Uptake into the Lung and PBF. PMN A-V differences are plotted against total PBF per gram of wet lung for the five transient lowering of PBF experiments. A computer derived line _+ SD of best f i t is plotted through the data points. These data show that the A-V difference for PMN begins to increase when blood flow f a l l s below 7 ml/min/gm. - 153-140-. 126-1 = 112 o c o o o o o ffl cc 98 84 •S 70-1 56 42 281 14-1 infl 1 I-II III rbot.. lation gA o.a 1:2 i : e 2:0 2 . * IV R V -A o -N = 5 (MEAN4.SE) TIME min) *»ta»»" PMN s-AORTA^O/ L RVC !] '0 SO 45 40 35 30 25 20 15 10 5 0 UNLABELLED PMN RETENTION •r I ' i f 4 N=5 (MEAN!SO) 6 * 8 10 i l h 14 16 i d BLOOD FLOW ( ML/MIN/GM) mJ 20 - 154 -Figure 43 Aortic and Right Ventricle WBC Counts During Prolonged Lowering of PBF. Mean (+SE) WBC counts in RV and Ao are shown for five dogs. Large A-V leukocyte differences develop following balloon inflation but these do increase with time. Figure 44 Aortic and Right Ventricle WBC Counts in One Dog after Long Term Low Flow, Cardiac Arrest and Cardiac Massage. The AV difference tend to disappear with time suggesting that a new steady state has been reached. The fact that both RV and Ao counts rise above control levels implies that cells are being added from other sites. A cardiac arrest at about 54 minutes was associated with uptake of cells by the lung and cardiac massage was associated with the appearance of large numbers of leukocytes in aortic blood. The slight rise in mean system blood pressure between 27 and 47 minutes was due to a slight change in position of the IVC balloon (see test for further information). - 155 -o o (0 e a o o U •s 125.0-1 112.5-100.0- b 87.5-75.0 -62.5 -50.0-37.5-25.0 -12.5 -0.0 -N=5 ( M E A N t S E ) -2.0 f-0.3 1.4 3.1 IVC balloon inflation t r 4.8 6.5 8.2 9.9 11.6 13.3 15.0 TIME (min) E E \ rt o X (0 c 3 o u o ffl i •< (D 3 o CD •o ? 3 o.o-i -5 0 f 1.5 8.0 14.5 21.0 27.5 34.0 40.5 47.0 53.5 f 60.0 IVC ba l loon Card iac inf lat ion massage TIME (min) - 156 -Figure 45 Aortic and Right Ventricle WBC Counts in Three Dogs who did not have Cardiac Arrest During Prolonged Lowering of PBF. Mean (+ SE) and Ao WBC counts are shown for 3 dogs while the IVC balloon catheter was inflated. Figure 46 Aortic and Right Ventricle WBC Counts in Three Dogs who had Cardiac Arrest During Prolonged Lowering of PBF. Mean (+ SE) WBC counts in RV and Ao are shown for 3 dogs while IVC balloon catheter was inflated. The data show that the appearance of large arterial-venous differences was associated with the development of cardiac arrest. - 15?-TIME (min) - 158 -Figure 47 Aortic and Right Ventricle WBC Counts in Two Dogs after Long Term Low Flow, Cardiac Arrest and Cardiac Massage. Mean (_+ SE) WBC in RV and Ao are shown for the two dogs. The data show that large numbers of WBC are released from the pulmonary microvasculature after cardiac massage was performed. - 159 -1.0 A o . o - | _ , F , , , , , , , , -7.0 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 TIME (min) - 160 -Figure 48 Systemic Blood Pressure and Cardiac Output in Dogs which Received CVF. Mean (+ SE) Pa and CO are shown for five dogs. As in the control group of animals, CO f e l l after the f i r s t hour (75 _+ 13%) while Pa showed only minimal changes (91 _+ 11%). Figure 49 Pulmonary Artery Blood Pressure, Wedge Pressure and Partial Pressure of Oxygen in Arterial Blood of Dogs who Received CVF. Mean (_+ SE) Ppa, Pw, and PO2 for five dogs is shown. As in the control group of animals, there was no significant changes in any of these values over the course of these experiments. [Ppa and PW values are, in fact, expressed as cmH^ O and should be multiplied by 0.761 to get mmHg values]. - UI -2 0 0 . 0 180 .0 1 6 0 . 0 140 .0 £ 120 .0 cc £ 100 .0 o ° 80 .0 J T 60.0 -j 40.0 20 .0 -I 0.0 CVF 1 I I | I co-N: 5 * N : 4 0-° 7.5 15.0 22 .5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 1 2 0 . 0 1 5 0 . 0 TIME (min) cn X E E L U CC 3 CO CO L U CC 0. 0.0 7.5 15.0 22 .5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 120 .0 1 5 0 . 0 TIME (min) - 162 -Figure 50 Aortic and Right Ventricle WBC Counts in Dogs who Received CVF. Mean (+ SE) WBC counts in RV and Ao are shown for five dogs. The data show that injection of CVF is associated with an abrupt f a l l in RV and Ao leukocyte counts. Aortic values f e l l from 88 ± 5% of the control value to 55 +_ 13% of the control value within five minutes of injecting the CVF. Small, but consistant A-V differences developed suggesting leukocyte sequestration within the pulmonary microvasculature. After 34 minutes, WBC counts rose to levels which were significantly greater than control. Figure 51 Aortic and Right Ventricle RBC Counts in Dogs who Received CVF. Mean (+ SE) RBC counts in RV and Ao is shown for five dogs. As in the control experiments, RBC counts f e l l slightly during the rapid sampling period. The data also show that injection of CVF is not associated with any significant changes in either the RV or Ao RBC counts. - 1 63 -o u 3 O O o ffl 5 0.0 7.5 1 5 . 0 2 2 . 5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 1 2 0 . 0 1 5 0 . TIME (min) 2 0 0 . 0 180 .0 160 .0 £ 140 .0 c o ° 120 .0 l_ 100 .0 z O 8 0 . 0 J o g 6 0 . 0 J rr 4 0 . 0 20 .0 0.0 0.0 Ill A o - 4 k N: 5 (mean ± SE) IV 7.5 15.0 22 .5 30 .0 37 .5 45 .0 52.5 6 0 . 0 9 0 . 0 120 .0 1 5 0 . 0 TIME (min) - 164 -Figure 52 Systemic Blood Pressure and Cardiac Output for Run I in Cobra Venom Factor Followed by Low PBF Dogs. Mean (+ SE) CO and BP are shown for 6 dogs. With balloon inflation there is a f a l l in both CO and BP. Balloon deflation is associated with a return to control l e v l e l s . Figure 53 Aortic and Right Ventricle WBC Counts for Run I in Cobra Venom Factor Followed by Low PBF Dogs. Mean (+ SE) WBC counts in RV and Ao are shown for six dogs. Large arterial-venous differences, indicative of leukocyte sequestration within the pulmonary microvasculature develop under low flow conditions. * indicates times at which the IVC balloon slipped and systemic blood pressure transiently increased to control levels. - 165 -X E E a 03 150.0. 135.0-120.0 105.0-90.0 75.0-60.0 45.0-I 30.0 15.0 0.0 1 I BALLOON ON - i BALLOON OFF II \ / \ / \ \ / . " r " in A IV .0 2.0 4.0 6.0 8.0 10.0 12.0 14 TIME (min) r5.0 C O - * - - - * BP-*—• -4.5 -4.0 3.5 3.0 2.5 2.0 H.5 1.0 0.5 0.0 0 N: 6 (mean + SE) O O 3 5* BALLOON OFF C o u »-z 3 O u o 03 5 RV-o-A o - « -N: 6 (mean ± SE) 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 TIME (min) - 166 -Figure 54 Systemic Blood Pressure and Cardiac Output for Run II in CVF Followed by Low PBF Experiments. Mean (+ SE) CO and BP are given for six dogs. The data show that injection of CVF had no effect on CO and BP. With balloon inflation there is a f a l l in both CO and BP. Balloon deflation is associated with a return to control levels. Figure 55 Aorta and Right Ventricle WBC Counts for Run II in CVF Followed by Low PBF Experiments. Mean (+ SE) WBC counts in Ao and RV are shown for six dogs. These data show that injection of cobra venom factor was associated with both a f a l l in RV and Ao leukocyte counts and the development of arterial-venous (A-V) differences. Inflation of the IVC balloon appears to enhance the A-V difference. - 1 6 ? -B A L L O O N C V F O N 0) z E E a ca 1 5 0 . 0 1 3 5 . 0 1 2 0 . 0 1 0 5 . 0 -9 0 . 0 7 5 . 0 6 0 . 0 4 5 . 0 -30 .0 -1 5 . 0 0 .0 B A L L O O N O F F ML - I 1 i n A ,1 IV CO-BP- • N : 6 (mean + SE) 5 .0 1-4-5 4 . 0 3 .5 3 .0 2 .5 2 .0 1.5 1.0 0 .5 hO.O ^ ( I )( l X 0 .0 2 .0 4 . 0 6 .0 8 .0 10 .0 1 2 . 0 14 .0 6 0 . 0 9 0 . 0 1 2 0 . 0 t E N O TIME(min) O F R U N O O 3 3 - 168 -Figure 56 Comparison of Aorta and Right Ventricle WBC Counts for Run I and Run II in CVF Followed by Low PBF Experiments. Mean WBC counts for Run I and Run II are shown for six dogs. The data show that injecting cobra venom factor (Run II) caused both RV and Ao counts to f a l l to levels which were lower than those achieved during Run I. - 169 -n 1 1 1 1 1 1 I I I I I l I ' I I I I I 0 .0 2 .0 4 . 0 6 .0 8 .0 1 0 . 0 1 2 . 0 1 4 . 0 3 0 . 0 6 0 . 0 9 0 . 0 1 2 0 . 0 TIME (min) - 170 -Figure 57 Systemic Blood Pressure and Cardiac Output for Low PBF Followed by CVF Experiments. Mean (+ SE) Pa and CO are shown for five dogs. The data show that IVC balloon inflation was associated with a f a l l in both CO and Pa and that IVC balloon deflation was associated with an increase in these values. At the end of the experiment CO had fallen to 71 +_ 8% of the control value. Injecting cobra venom factor had no significant effect on any of these variables. Figure 58 Pulmonary Artery Blood Pressure, Wedge Pressure and Partial Pressure of Oxygen in Arterial Blood for Low PBF Followed by CVF Experiments. Mean (_+ SE) Ppa, Pw and FO^ are shown for five dogs. The data show that balloon inflation was associated with a f a l l in Ppa, PW and consistent increase in PO^. Injecting cobra venom factor had l i t t l e effect on cardiac output pressure, i t did cause P0 2 to f a l l from 122 + 9 mmHg to 91 + 11 mmHg. IVC balloon inflation was associated with an increase in Ppa and PW back to control levels. It had no effect on systemic arterial PO^. [Ppa and PW values are, in fact, expressed as cmH„0 and should be multiplied by 0.761 to get mmHg values.] - 171 -O cc t-z o o <# 2 0 0 . 0 1 8 0 . 0 -J 160 .0 140 .0 120 .0 -I 1 0 0 . 0 8 0 . 0 . 6 0 . 0 -4 0 . 0 -2 0 . 0 . 0 .0 I IVC B A l l O O N I N F L A T I O N I CVF \ •r~H \ \ \ \ I V C B A L L O O N D E F L A T I O N \ CO-P a - • - — • N : 5 (mean ± SE) II in IV 0 .0 7.5 15 .0 2 2 . 5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 1 2 0 . 0 1 5 0 . 0 TIME (min) o> z E E CO U l cc 0. .0 7 .5 15 .0 2 2 . 5 3 0 . 0 3 7 . 5 4 5 . 0 5 2 . 5 6 0 . 0 9 0 . 0 1 2 0 . 0 1 5 0 . 0 TIME (min) - 172 -Figure 59 Aorta and Right Ventricle WBC Counts for Low PBF Followed by CVF Experiments. Mean (+ SE) WBC counts for RV and Ao are shown for five dogs. The data show that large arterial-venous differences, indicative of leukocyte sequestration within the pulmonary microvasculature, develop after IVC balloon inflation at 25 minutes. Injection of CVF at 30 minutes causes an additional f a l l in RV and Ao white blood c e l l counts. After balloon deflation at 35 minutes there is a gradual increase in those values to levels significantly greater than control. Figure 60 Aorta and Right Ventricle RBC Counts for Low PBF Followed by CVF Experiments. Mean (+ SE) RBC Counts for RV and Ao are shown for five dogs. The data show that prolonged IVC balloon inflation causes RBC sequestration within the pulmonary microvasculature. Injection of cobra venom factor has minimal effects on RBC counts. IVC balloon deflation was associated with a transient increase in systemic RBC counts presumably due to release from sequestration sites. p b RBC COUNT (% c o n t r o l ^ ^ ^ o ° o o WBC COUNT <% c >ntrol) t - t • « s pb - 174 -Figure 61 Systemic Blood Pressure and Cardiac Output after Epinephrine Infusion and CVF. Mean (+ SE) Pa and CO are shown for six dogs. The data show that infusion of epinephrine was associated with an increase in CO which returned to control levels after epinephrine infusion was discontinued. At the end of the experiment CO continued to f a l l to 69 +_ 4% of the control value. Epinephrine infusion did not significantly change systemic blood pressure. Injecting cobra venom factor had no significant effect on either of these variables. Figure 62 Pulmonary Artery Blood Pressure, Wedge Pressure and Partial Pressure of Oxygen in Arterial Blood after Epinephrine Infusion and CVF. Mean (_+ SE) Ppa, Pw and P O 2 are shown for six dogs. The data show that infusion of epinephrine was associated with slight increases in Ppa and Pw and that these a l l returned to control levels after epinephrine infusion was discontinued. PO did not change in any s t a t i s t i c a l l y significant manner. - 175-200.0-j 180.0-160.0-140.0-120.0-1 100.0 80.0 60.0-1 40.0-20.0-1 S T A R T E P I C V F S T O P E P I 0.0-1 1 C O - * - - -Pa - *—-•-...I - • I" N: 6 (mean + ± SE) I l l f . — 1-1 - -±-i I V 0.0 7.5 15.0 22.5 30.0 37.5 45.0 52.5 60.0 90.0 120.0 150.0 TIME (min) START IPI 150 135 120 105 90 75 60 45 30 15 0 0 0 0 0-1 0 0- | .0-0- | C V f • se-l l STOP _ EPI pO,-• Ppa- * -P w - o -III I V N: 6 (meant SE) 0.0 7.5 15.0 22.5 30.0 37.5 45.0 52.5 60.0 90.0 120.0 150.0 TIME (min) - 176 -Figure 63 Aorta and Right Ventricle WBC Counts after Epinephrine Infusion and CVF. Mean (+ SE) WBC Counts for RV and Ao are shown for six dogs. The data show that epinephrine infusion was associated with an increase in both RV and Ao WBC counts and that injection of cobra venom factor (CVF) was associated with a f a l l in both RV and Ao WBC counts. CVF did not cause any change in the A-V difference. Figure 64 Aorta and Right Ventricle RBC Counts after Epinephrine Infusion and CVF. Mean (+ SE) RBC Counts are shown for six dogs. The data show that epinephrine infusion was associated with an increase in both RV and Ao RBC counts. Injection of cobra venom factor (CVF) had no effect on RBC counts. RBC COUNT (% c o n t r o l ) WBC COUNT (% c o n t r o l ) CO O cn ui ro In p ro o *• o CO o oa O o p ro p o o> o 00 p b b b b b b b b b b ro O p b "TJ > 20 to O o o> o 0 3 o o o I O p o p OB O b b b b b b b b b z m 3 5" c < < - 178 -Figure 65 Aorta and Right Ventricle WBC Counts after Epinephrine Infusion, CVF and Standardization to Control Hematocrit Levels. Mean (+ SE) WBC counts for RV and Ao are shown for six dogs. The data show that while there was no increase in WBC counts after epinephrine infusion, injection of cobra venom factor (CVF) was s t i l l associated with a f a l l in both RV and Ao WBC counts. Figure 66 Aorta and Right Ventricle RBC Counts after Epinephrine Infusion, CVF and Standardization to Control Hematocrit Levels. Mean (+ SE) RBC counts for RV and Ao are shown for six dogs. The data show that RBC counts did not change after infusion of epinephrine nor after injecting cobra venom factor. CORRECTED RBC COUNT (% cont ro l ) o b to o 4* o 0) o CO o o p to p p CO o CO p b b b b b b b b b IO o o CORRECTED WBC COUNT (% con t ro l ) o I O p *. o O ) p CO o o p b b b b b b to o p cn o CO o b b b - 180 -REFERENCES 1. Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distress in adults. Lancet 1967; 2:391-23. 2. Hudson, L.D. ed. Adult respiratory distress syndrome. Semn Respir Med 1981; 2:99-174. 3. Lung program, National Heart and Lung Institute. Respiratory Diseases: Task Force Report on Problems, Research Approaches, Needs. Washington, D.C: Government Printing Office. 1972 : 171 (DHEW Publication no. (NIH) 73-432), pp. 165-180. 4. Hopewell PC, Murray JF. 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