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The effects of crystalloid resuscitation on oxygen extration in whole body and gut during endotoxemia Gow, Kenneth William 1997

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THE EFFECTS OF CRYSTALLOID RESUSCITATION O N OXYGEN EXTRACTION IN WHOLE BODY A N D G U T DURING ENDOTOXEMIA  by  K E N N E T H WILLIAM G O W B.Sc, The University of Manitoba, 1991 B.Sc.(Med), The University of Manitoba, 1991 M.D., The University of Manitoba, 1991 FRCS(C), The University of British Columbia, 1997  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE , .(SURGERY ) in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF SURGERY We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1997 © Kenneth William Gow, 1997  In  presenting  degree freely  this  at, t h e available  copying  of  department publication,  thesis  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or of  reference  thesis by  this  for  his thesis  and  scholarly  or  her  for  The  University  Vancouver;  Date  D E - 6 (2/88)  SuC&gRY  of of  British  Columbia  Canada  QGTQfeEf*  \ Z \ 9 9"7 •  further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  Department  study.. I  requirements  It not  be  that  the  Library  permission  granted  is  for  by  an shall for  the  understpbdthat be  allowed  advanced  without  make  it  extensive  head  of  copying my  my or  written  ABSTRACT  Many investigators advocate aggressive fluid therapy in sepsis, yet changes in the microcirculation may make fluid counterproductive. Sepsis is characterized by a generalized "leak" in capillaries which may promote interstitial edema which in turn, may decrease diffusion of oxygen, increase the distance from capillaries to cells, and alter capillary density. Further, fluid administration' may result in capillary hemodilution. Therefore, the author's hypothesis was that crystalloid resuscitation will impair the ability of tissue to extract oxygen. Four groups (n=8) of anesthetized pigs received either normal saline infusion (48 ml-kg-l-hr" ) or no saline, and E coli endotoxin (50 mg/kg i.v.) or no endotoxin. 1  Whole body and gut oxygen delivery and consumption were measured during progressive hemorrhage. Dual line regression analysis was used to determine the onset of ischemia  (DO2C)  and oxygen extraction ratio (ERc). At onset of ischemia,  gut was removed to determine degree of interstitial volume and the capillary hematocrit. With use of radiolabelled microspheres as a marker of blood flow, the gut blood flow transit time was determined. Endotoxin significantly decreased ERc for the whole body (0.82+0.06 to 0.55±0.08, p < 0.05) and gut (0.77 ± 0.07 to 0.52 ± 0.06, p < 0.05). Saline resuscitation also significantly decreased ERc in the control pigs for the whole body (0.82 ± 0.06 to 0.62 ± 0.08, p < 0.05) and gut (0.77 ± 0.07 to 0.67 ± 0.06, p < 0.05) but did not significantly change the already decreased ERc in the endotoxin treated pigs. Morphometric techniques revealed that saline resuscitation increased gut interstitial volume (p < 0.05), and lead to arterial hemodilution (p < 0.05) but not  ii  capillary hemodilution (p > 0.05). Using radiolabeled microspheres, saline was shown to increase the relative dispersion of blood flow transit times from 0.33 ± 0.08 to 0.72 ± 0.53 (p < 0.05). Thus, saline resuscitation impairs tissue oxygen extraction possibly due to interstitial edema or increased heterogeneity of microvascular blood flow. After endotoxin infusion, where ERc is already decreased, saline resuscitation has a lesser effect. Therefore, the author questions the use of aggressive crystalloid resuscitation for treatment of sepsis in humans.  iii  TABLE OF CONTENTS  Abstract Table of Contents List of Tables List of Figures Acknowledgments Chapter 1.  ii iv vii viii x  INTRODUCTION  1  1.1  OVERVIEW  1  1.2  SEPSIS A N D SYSTEMIC INFLAMMATORY RESPONSE  1.3  SYNDROME (SIRS)  3  1.2.1 Definitions  3  MEDIATORS OF SEPTIC TOXICITY  6  1.3.1 Mediation of septic toxicity  '  1.3.2 Bacterial endotoxin  1.4  6 7  1.3.3 Host defenses  10  1.3.4 Inflammatory Mediators  13  1.3.4.1 Peptide cytokines  13  1.3.4.2 Platelet activating factor  15  1.3.4.3 Eicosanoids  15  1.3.4.4 Nitric oxide  15  1.3.4.5 Oxygen radicals  16  1.3.5 Ischemia and Tissue Injury  17  SEPSIS A N D TISSUE ISCHEMIA  18  1.4.1 Central hemodynamic changes in sepsis  18  1.4.2 Microvascular changes in sepsis  19  1.4.2.1 Alterations in erythrocyte deformability  19  1.4.2.2 Viscosity alterations  21  1.4.2.3 Increased vascular permeability and interstitial edema 21  iv  1.4.2.4 Alterations in microvascular flow and capillary heterogeneity 1.5  1.6  1.7  22  SPLANCHNIC ISCHEMIA IN CRITICAL ILLNESS  26  1.5.1  Protective mucosal barrier  26  1.5.2  Splanchnic Metabolic Model of O 2 control  26  1.5.3  Counter-current shunting (CCS)  28  1.5.4  Gut Ischemia during Septic Shock  30  AEROBIC A N D ANAEROBIC METABOLISM  31  1.6.1  Aerobic metabolism  31  1.6.2  Anaerobic metabolism  34  1.6.3  Oxygen Consumption and Delivery  34  1.6.4  Oxygen Extraction Ratio  37  1.6.5  Oxygen Consumption and Delivery in Sepsis  37  1.6.6  Oxygen Extraction in Sepsis  37  T R E A T M E N T OF SEPSIS  41  1.7.1  Treatment Options  41  1.7.2  Augmentation of Oxygen Delivery  43  1.7.3  Fluid resuscitation in septic shock - crystalloids and colloids  44  1.7.4  Diffusion vs. Filtration  46  1.7.5  Starling's Law of the Capillary  47  1.7.6  Tonometry  1.7.7  Lactate  52  Chapter 2.  HYPOTHESIS  54  Chapter 3.  OBJECTIVES OF THE THESIS  55  Chapter 4.  RESEARCH P L A N  57  T H E PORCINE MODEL OF SEPTIC SHOCK  57  4.1  '  v  49  4.2  MORPHOMETRIC ANALYSIS OF GUT TISSUE  59  4.3  INFRA-SCAN ANALYSIS OF G U T TISSUE  59  4.4  DETERMINATION OF G U T CAPILLARY HEMATOCRIT  61  4.5  RELATIVE DISPERSION OF BLOOD TRANSIT TIMES  63  4.6  TONOMETRY  64  Chapter 5.  EFFECT OF CRYSTALLOID RESUSCITATION O N WHOLE BODY A N D G U T IN ENDOTOXEMIA  65  5.1  INTRODUCTION  65  5.2  METHODS  68  5.2.1  Experimental Design and Protocol  68  5.2.2  Surgery Preparation and Instrumentation  69  5.2.3  Hemodynamic Measurements and Calculations  73  5.2.4  Transit Times  74  5.2.5  Interstitial Volume  75  5.2.6  Capillary Hematocrit  77  5.2.7  Ileal Tonometry  77  5.2.8  Statistical Analysis  78  5.3  5.4  RESULTS  80  5.3.1  Effect of endotoxin  80  5.3.2  Effect of Fluid Resuscitation  81  DISCUSSION  99  Chapter 6.  CONCLUSIONS  106  Chapter 7.  SUMMARY: CLINICAL RELEVANCE, FUTURE DIRECTIONS  108  REFERENCES  115  vi  LIST OF TABLES  Table 1.  Onset of Ischemia and Extraction Ratio  90  Table 2.  Arterial and Capillary Hematocrit  91  Table 3.  Arterial and Portal V e i n Leukocyte Counts  92  Table 4.  Intestinal p H i at Baseline and Onset of Ischemia  93  Table 5.  Arterial Lactate Values  94  Table 6.  Onset of Rise of Arterial Lactate  95  Table 7.  Oxygen Transport Variables  96  Table 8.  H e m o d y n a m i c Variables  97  Table 9.  C l i n i c a l Variables  98  vii  LIST OF FIGURES  Figure 1.  Sepsis and SIRS  4  Figure 2.  Mediators of Septic Toxicity  8  Figure 3.  Endotoxin (Lipopolysaccharide (LPS))  9  Figure 4.  Host defenses  12  Figure 5.  Toxic Mediators  14  Figure 6.  Toxicity  20  Figure 7.  Capillary heterogeneity  24  Figure 8.  Metabolic model of O 2 control in the gut  27  Figure 9.  Countercurrent Shunting (CCS)  29  Figure 10.  Gut as "Motor of Multiple Organ Dysfunction"  32  Figure 11.  Aerobic Metabolism  33  Figure 12.  Anaerobic Metabolism  35  Figure 13.  Relationship between oxygen delivery and consumption  36  Figure 14.  Oxygen Extraction Ratio  38  Figure 15  Relationship between oxygen delivery and consumption in sepsis  39  Figure 16.  Oxygen Extraction Ratio during sepsis  40  Figure 17.  Treatment options  42  Figure 18.  Starling's Forces of the Capillary  48  Figure 19.  Tonometric Analysis  51 -  Figure 20.  Morphometric Analysis  60  Figure 21.  Infra-scan Analysis  62  viii  Figure 22.  Protocol Timeline  70  Figure 23.  Surgery and Instrumentation  72  Figure 24.  Processing jejunum for morphometric analysis  76  Figure 25.  An example of C»2 Consumption and Delivery Curve  79  Figure 26.  Whole Body Ability to Extract Oxygen  84  Figure 27.  Gut Ability to Extract Oxygen  85  Figure 28.  Whole Body Onset of Ischemia  86  Figure 29.  Gut Onset of Ischemia  87  Figure 30.  Relative dispersion of Gut Blood Transit Times  88  Figure 31.  Interstitial Volume in Gut  89  ix  ACKNOWLEDGMENTS  I dedicate this thesis to my mother and father, May and Stephen Gow, who have always helped and encouraged me throughout my years of study. To my brother; James Gow whose determination has always been a standard against which I set myself. I would like to thank my supervisors, Drs. Terry Phang and Keith Walley, for their constant guidance and support. It is because of their example that I have learnt so much. I also thank the other members of my Supervisory Committee, Drs. John MacFarlane, York Hsiang, and Mike Allard, who have  provided much  encouragement and constructive criticism as my experiments and thesis developed. I am grateful for the thoughts and technical assistance of Drs. Blair RudstonBrown, Oliver Bathe, Robert Granger, Suzy Tebbutt-Speirs, Christopher Goddard, Diane Minshall, Lynn Carter, Dean English, Lubos Bohunek, Betty Poon, and Peter Whitehead without whom this project could not have been completed.  Chapter 1 INTRODUCTION  1.1  OVERVIEW  Sepsis is an all too frequent occurrence in critically ill patients . Overall, the cost to health care is staggering (1) with an estimated cost of $5 to $10 billion in the US annually. Sepsis will likely be a problem for clinicians not only now but for the foreseeable future (2). The increasing incidence of septic shock continues to be associated with a mortality rates ranging from 40% to 60% despite advances in critical care medicine. Clinicians have long sought the optimal treatment for patients suffering from sepsis. However, sepsis, by its very nature is a systemic illness whereby many of the toxic effects are the result of the patient's own immune system as it mounts a defense to the infection.  The immune system reaction to infection focuses on  various aspects of the microorganism but the lipopolysaccharide moiety known as endotoxin has been best described in literature. The immune system through its various leukocytes mounts a defense to the organisms. This defense is in turn mediated by numerous cytokines which have beneficial effects as well as side effects. While the immune system overall is beneficial in its role, if the effects are allowed to go unhindered, the immune system mediators will themselves lead to significant illness. Various toxicity effects occur from these cytokines such that further damage may ensue. Ultimately, the damage done to cells occurs at a microvascular level with ischemia as the final end-point.  1  While sepsis is a systemic insult, the splanchnic circulation is considered to be particularly affected. There are various reasons why the gut is predisposed to injury. Two explanations are the high metabolic demands which make the gut susceptible to minor deficits and the countercurrent organization of the vessels which leads to possible shunting from arterioles to venules in times on decreased flow.  While  ischemia in any tissue is potentially harmful, ischemia to the gut is particularly concerning. With the high volume of bacteria that reside in the gut, damage to the mucosal protective layer may result in further bacterial load to the host. Since this translocation of bacteria may ultimately lead to systemic toxicity, the gut has been termed the "Motor of Multiple Organ Failure" (3). The toxicity that leads to multiple organ dysfunction is ultimately due to tissue ischemia. Ischemia may be defined by the decrease in blood flow. In turn, ischemia results in hypoxia which may be described as the lack of oxygen for aerobic metabolism.  This results in anaerobic metabolism which is a less efficient  mechanism for energy production and leads to metabolic byproducts. Techniques have been described which allow investigators to determine the onset of tissue ischemia and the tissue's ability to extract oxygen. These studies allow one to determine the potential benefit of various treatments of sepsis. Treatment of sepsis may be aimed at any step of the pathway from initial insult to the final endpoint of tissue ischemia. While new techniques such as blocking endotoxin or modulating the immune system are being studied (4), time honored therapeutic interventions such as antibiotics and fluid resuscitation are considered the mainstay of therapy (2). While the use of antibiotics is intuitive to  2  treat the infection, the use of fluid therapy is less than straight forward. Fluid therapy is thought to mediate its benefit by augmentation of filling pressures, replacement of intravascular deficits, decreased viscosity of blood, and a more uniform distribution of blood flow to tissues which is known as capillary blood flow homogeneity. However, since the septic state may be described as a generalized leak in the capillaries, the potential benefit of crystalloid resuscitation is questionable (5). The potential side effects from fluid resuscitation include interstitial edema, hemodilution, and a less uniform distribution of blood flow to tissues which is known as capillary blood flow heterogeneity.  Therefore, while some consider  aggressive crystalloid resuscitation as essential in the resuscitation of septic patients, physiologic changes that accompany sepsis may not only lead to a limited benefit but could make fluid resuscitation detrimental. In this thesis, the effect of crystalloid resuscitation is studied on the main outcome variable, oxygen extraction in a porcine model of endotoxemia.  The  literature pertaining to these topics has been reviewed and is discussed in detail below.  1.2  SEPSIS AND SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS)  1.2.1  Definitions  The following definitions were established by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (ACCP/SCCM) in 1991 (6) (Figure 1).  3  Figure 1.  Sepsis and SIRS.  This pictogram illustrates the differences between the terms described by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (ACCP/SCCM).  4  Systemic inflammatory response syndrome (SIRS) - Describes a continuous  process, and describes an abnormal host response that is characterized by a generalized activation of the inflammatory reaction in organs remote from the initial insult. When the process is due to infection, the terms sepsis and SIRS are synonymous. SIRS can be seen following a wide variety of insults and includes, but is not limited to, more than one of the following clinical manifestations: (1) a body temperature > 38°c or < 36°c; (2) a HR >90 bpm; (3) tachypnea, with a RR > 20 bpm, or hyperventilation, as indicated by a PaC02 < 32 mmHg; and (4) an alteration in the WBC, being either > 12,000/mm3/ or <4,000/mm3, or the presence of more than 10% immature neutrophils. Infection - is a microbial phenomenon  characterized by the presence of  microorganisms or their invasion of normally sterile host tissue by those organisms. Bacteremia - presence of viable bacteria in the blood. The presence of viruses,  fungi, parasites, and other pathogens in the blood should be described in a similar manner. Septicemia - has been defined in the past as the presence of microorganisms  or their toxins in the blood. However, this term has been used clinically and in the medical literature in a number of ways leading to confusion and difficulty in interpreting data. Therefore, it was recommended to abandon this term. Sepsis - the systemic inflammatory response to infection. In association with  infection, manifestations of sepsis are the same as those previously defined for SIRS.  5  Severe sepsis - sepsis associated with organ dysfunction, hypoperfusion abnormality, or sepsis-induced hypotension. Hypoperfusion abnormalities include lactic acidosis, oliguria, and acute alteration of mental status.  Sepsis-induced  hypotension is defined by the presence of a systolic blood pressure of less than 90 mmHg or its reduction by 40 mmHg or more from baseline in the absence of other causes for hypotension. Septic shock - a subset of severe sepsis and is defined as sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction. Multiple organ dysfunction syndrome (MODS) - the previous term multiple  organ failure has been replaced with this term.  The main change is the use of  "dysfunction" used in place of the term "failure" to represent a continuum  of  physiologic derangements. This can evolve in the absence of an untreated focus of invasive infection and can be reproduced experimentally by the infusion of a diverse spectrum of endogenously derived mediators of inflammation.  The term  syndrome refers to a pattern of multiple and progressive symptoms and signs that are thought to be related.  1.3  MEDIATORS OF SEPTIC TOXICITY  1.3.1  Mediation of septic toxicity  The body's immune system is paramount in importance in defending against infections. There are various mechanisms at work which comprise a fine balance of stimulatory and inhibitory effects. While for the most part, success results in the  6  host surviving, the mediators of defense may have toxic effects themselves. The various mechanisms important for immune defense have been shown to affect host tissues as well. The end result of these toxic effects is ischemia at the microvascular level (7) (Figure 2).  1.3.2  Bacterial Endotoxin  While various aspects of bacteria are potentially toxic, the best described has been that of endotoxin.  Gram-negative bacteria contain within their cell wall a  macromolecular glycolipid termed lipopolysaccharide (LPS) (Figure 3). It comprises two components:  the O-specific chain and the core.  The O-specific chain is a  polymer of oligosaccharides and accounts for the antigenic variability among species and strains of bacteria. The core is composed of an oligosaccharide covalently bound to a molecule lipid called lipid A . Most of the toxicity of LPS resides in the lipid A moiety.  The structure of the core region is relatively constant across species and  strains of gram-negative bacteria. The terms LPS and endotoxin are not strictly synonymous as LPS refers to the purified glycolipid whereas endotoxins contain small amounts of cell wall proteins, lipids, lipoproteins, and polysaccharides i n addition to LPS (8). There are several proposed mechanisms by which endotoxin effects damage. Endotoxin has been shown to activate complement, the fibrinolytic  pathway,  neutrophils, and other mediator systems (9) (10). Furthermore, endotoxin has been shown to stimulate macrophages to release cytokines, including tumor necrosis factor (TNF) and interleukin-1 (IL-1), which, in turn, amplify the systemic response  7  F i g u r e 2.  M e d i a t o r s of Septic Toxicity.  This figure illustrates the overall mediation of the toxicity involved with sepsis. It starts at the interaction between bacteria and the host-defense mechanisms.  This interaction is meditated by  various mediators leading to various toxicity's ultimately leading to ischemia.  8  Figure 3.  Endotoxin.  This figure illustrates endotoxin (lipopolysaccharide) as part of the cell wall of a gram negative bacteria. Endotoxin is comprised of two components: the O-specific chain and the core. The O-specific chain is a polymer of oligosaccharides and accounts for the antigenic variability among species and strains of bacteria. The core is composed of an oligosaccharide covalently bound to a molecule lipid called lipid A. Most of the toxicity of LPS resides in the lipid A moiety.  9  to endotoxin by stimulating neutrophils, endothelial cell, platelets, and the release of other cytokines, eicosanoids, platelet activating  factor  (PAF), endorphins,  endothelial relaxing factor, and a variety of other plasma and cellular mediators (11) (10) (12). Some have speculated that endotoxin  leads to mucosal hypoxia as a  mechanism of damage. However, VanderMeer et al (13) showed that there is significant mucosal acidosis despite absence of mucosal hypoxia. Other mechanisms by which LPS may cause mucosal acidosis include uncoupling  of  oxidative  phosphorylation (14) (15), inhibiting mitochondrial respiration (16) (17) (18) (17), decreasing availability of substrates (19), increasing metabolism, or decreasing the clearance of H  1.3.3  +  or C02-  Host Defenses  Baker and Huynh (7) divides host defenses into nonspecific and specific factors.  Nonspecific factors include  complement,  hemolytic  factors,  and  skin,  mucus  phagocytosis.  membranes,  as well  Phagocytosis  as  involves  interactions with a number of other systems, particularly the complement system. The specific immune system is responsible for recognizing bacteria and creating antibodies to help opsonize them.  The specific immune system is a delicately  balanced system with cells that have both stimulatory and inhibitory (or regulatory) function. While, for the most part, the immune system is important in preventing infection, the host's response to injury is often worse than the injury itself and can develop into a malignant, uncontrolled SIRS (7). This occurrence represents a state  10  of disseminated activation of the host's inflammatory systems. While any of the cells of immunity may be involved (Figure 4), it is thought that the macrophage is central to the development of this process (20). Macrophages play a crucial role i n regulating the immune response after injury (21). They reside at strategic positions throughout the body, armed to eliminate foreign and infective agents. It is thought that it is the excessive, unregulated, prolonged stimulation of the macrophage i n conjunction with other leukocytes and the endothelial system that leads to a vicious cascade of inflammatory mediator release (3).  The macrophage is capable of  producing directly toxic substances in addition to numerous  cytokines and  mediators that act synergistically to augment the inflammatory response (7). The various substances including TNF, interleukins, PAF, arachidonic acid metabolites, and nitric oxide are discussed below.  Further, a number of investigators have  studied the role of neutrophils in the development of SIRS and MODS. Much like the macrophage, it is thought that unregulated activation of neutrophils and the endothelial system are important factors (22) (23). The neutrophils are thought to adhere to the endothelium, creating a microenvironment derived  proteases and  concentrations.  toxic-reactive  in which neutrophil-  oxygen species are present  at  high  These toxic products can directly injury the endothelial lining,  leading to intercellular gap formation, altered vascular permeability, and further neutrophil  migration  (24).  Together, the interaction  among macrophages,  neutrophils, and the endothelial system augments the inflammatory cascade and results in bystander organ failure (7).  11  Macrophages  B-cells  Neutrophils T-cells  Figure 4.  Host Defenses.  This figure illustrates the different leukocytes thought to be important in immunity.  12  1.3.4  Inflammatory Mediators  There are a multitude of toxic mediators thought to have importance i n mediating the damages seen in SIRS and sepsis including T N F , IL-1, P A F , prostaglandins, thromboxanes, leukotrienes, nitric oxide, O 2 radicals, interferon, and complement (Figure 5).  1.3.4.1  Peptide cytokines  Some degree of inflammatory response is due to the biological effects and balance of peptide cytokines (22). Both tumor necrosis factor (TNF) and interleukin1 (IL-1) are important mediators in the pathogenesis of SIRS and MODS.  These  peptides are synthesized in nearly all organs containing phagocytes and blood mononuclear cells in response to a large number of stimuli including endotoxin, complement, eicosanoids, interferon, other interleukins, and viral antigens. The mechanism of action is thought to be the induction of subsequent effector molecules of SIRS. Infusion of either TNF or IL-1 will lead to lung injury characterized by increased permeability and neutrophil  sequestration (22).  T N F also causes  neutrophil degranulation and superoxide production (22). It appears that TNF and IL-1 act synergistically in inducing shock (25), prostaglandin synthesis, and neutrophil chemotaxis (22). Inhibition of either TNF or IL-1 independently has been shown to prevent lung injury, shock, and death following endotoxin or E. coli administration (22).  13  Interleukins Interferon TNF  ^  ° i radicals  ^^OQ Complement 0«Q^ O * ©®© Leukotrienes  ^  A  ©  P%  O O O Q ° 0  r% Thromboxanes  Nitric oxide ° PAF Prostaglandins  F i g u r e 5.  Inflammatory Mediators.  This figure illustrates the various mediators thought to be important in the development of toxicity in sepsis.  14  1.3.4.2  Platelet activating factor  Platelet activating factor (PAF) is a labile ether lipid first seen to be released by platelets in the presence of antigen and leukocytes.  Cipolle et al (22) points to  several reasons why PAF is considered an important mediator in SIRS. Infusion of PAF mimics many of the effects of sepsis and endotoxin. Further, P A F is produced during endotoxemia in animals and severe sepsis.  Finally, several structurally  different antagonists to PAF have been reported to inhibit  endotoxin-induced  hypotension, lung injury, and mortality.  1.3.4.3  Eicosanoids  Eicosanoids are derivatives of the substrate arachidonic acid (AA), a cell membrane phospholipid. They include prostaglandins (PG), thromboxane (Tx), and leukotrienes (LT). A variety of inflammatory stimuli can directly or indirectly activate phospholipase leading to release of A A from phospholipid pools. Once released, A A is oxidatively metabolized via two major  pathways leading to  production of the eicosanoids. These derivatives all have varying functions which are beyond the scope of this thesis.  1.3.4.4  Nitric oxide  Nitric  oxide (NO) is formed  via arginine  metabolism  endothelium appears to be the most important source (22).  and vascular  Endotoxin has been  shown to stimulate N O formation in vitro (26). The vasodilation and vascular unresponsiveness that occur during sepsis and endotoxemia appear to be mediated  15  largely via the L-arginine-NO pathway (27).  Inhibition of the L-arginine-NO  pathway blocks TNF-mediated hypotension in rats (28).  While N O may have  negative effects, some argue that N O may in fact serve an important role. N O may be protective by inhibiting platelet aggregation and vasodilation, thereby preserving blood flow to important vascular beds to maintain critical oxygen delivery (22). Therefore, inhibiting N O may be detrimental during sepsis in that blood flow to already ischemic areas could be further reduced.  1.3.4.5  Oxygen radicals  Under normal  physiologic conditions, 80%-90% of cellular  oxygen is  consumed at the level of the mitochondrial respiratory chain and is directly reduced to water via the cytochrome oxidase complex (22).  In pathologic conditions,  fundamental oxygen ( C>2) can be reduced one electron at a time, giving rise to a 3  cascade of activated oxygen species. The most important species is a free radical, the superoxide anion ( O 2 ) . Active oxygen metabolites may react to a variety of biologic substrates. They promote D N A scission and base modification, inactivate plasma proteins, cross-link membrane proteins, and most importantly,  induce  lipid  peroxidation in the membrane which may lead to disorganization of its structure and lead to cell death (29). Oxygen free radicals are especially important during ischemia-reperfusion type injuries (22). During the reperfusion phase, severe tissue injury occurs following massive production of oxygen free radicals generated by several mechanisms.  16  1.3.5  Ischemia and Tissue Injury  The effects described above lead to an alteration in the microvascular flow of. blood to tissue the mechanisms of which are described later.  Therefore, the  inflammatory mediators ultimately results in ischemia as the final endpoint of SIRS and sepsis. Ischemia not only precipitates cellular ATP degradation and impairs energy-requiring cellular function, but it may also activate processes that lead to an increase in oxygen-derived, free-radical production, which  can augment or  accelerate tissue injury (30). Ischemia also promotes the formation of xanthine oxidase from xanthine dehydrogenase.  Furthermore, there is activation of cellular proteases which  converts the xanthine dehydrogenase by proteolysis to xanthine oxidase which uses molecular oxygen as its electron acceptor leading to the generation of superoxide anion and/or hydrogen peroxide (31). Circulating levels of xanthine oxidase have been demonstrated in patients with ARDS (32). It has been hypothesized that liver or gut tissue releases xanthine oxidase, thereby allowing it to serve as a toxic, oxygen-metabolite-generating system in the lung capillary bed (33). Furthermore, inflammation may be part of the ischemic process or may serve to augment and accelerate the ischemic process. Toxic oxygen metabolites, formed by xanthine oxidase during ischemia, may increase capillary permeability and increase the influx of neutrophils. The arrival of neutrophils and their subsequent activation by factors released from injured cells are likely to augment tissue injury. Local inflammation may also accelerate the ischemic process by causing granulocyte plugging and tissue edema, leading to a further impairment of oxygen delivery (31).  17  1.4  SEPSIS A N D TISSUE ISCHEMIA  The hemodynamic changes that are described in sepsis may be divided into central and microvascular vascular changes (34).  1.4.1  Central hemodynamic changes in sepsis  With regards to the central effects in sepsis, there is an initial stage during which homeostasis is maintained involving  various changes including  both  tachycardia, tachypnea, and fever (12). Ultimately, homeostatic mechanisms fail and signs of circulatory shock ensue and is described as having both a hyperdynamic and a preterminal hypodynamic phase. The hyperdynamic phase of septic shock is typically seen and consists of a high cardiac output, low pulmonary artery occlusion pressure (PAOP), and a low peripheral vascular resistance (12). The hypodynamic stage is manifested by a low cardiac output and an increased systemic vascular resistance. Despite the increased cardiac output, many investigators have described a global myocardial dysfunction in septic shock (35) (36) (37). The increased cardiac output reflects the increase in heart rate while the stroke volume is usually low (35) (38). Plotted on a Frank-Starling curve against the PAOP, the left ventricular stroke work index was typically reflective of left ventricular  failure  (12) (39) (40).  Furthermore, the magnitude of the myocardial depression correlates with survival as patients capable of maintaining their cardiac output by increasing their heart rate and left ventricular end-diastolic volume are more likely to survive (12). There have been several substances that have been implicated in contributing to this  18  myocardial depression (41) including TNF, low-molecular-weight  water-soluble  molecules, and lipid-soluble substances (42) (43) (44). The presence of these depressant factors has been shown to correlate with the severity of cardiac dysfunction and systemic hypoperfusion (45).  1.4.2  Microvascular changes in sepsis  While initial investigations focused on measurable hemodynamic changes, more recent studies have emphasized the role of the microcirculatory changes i n sepsis (46) (47). deformability, development  The changes described include: alterations  viscosity  alterations,  increased  vascular  in erythrocyte  permeability  of interstitial edema, and changes in microvascular  development of capillary heterogeneity.  with  flow with  A l l these changes will go to alter the  overall microcirculatory flow of blood and thereby alter the delivery of oxygen from capillaries to cells (Figure 6).  1.4.2.1 Alterations in erythrocyte deformability  The normal state of deformability is the ability of the red cell to alter its biconcave, discoid shape to allow passage through a capillary smaller in diameter than itself (46). Driessen et al (48) emphasized the importance of normal erythrocyte deformability for the maintenance of adequate perfusion of the microcirculation, especially when perfusion pressure is reduced. They further explained that red cell deformability depends on the viscoelastic properties of the cell membrane, the viscosity of the cytoplasm, and the surface area/volume ratio, all of which may  19  Sludging  ARegulation  Edema  1 Permeability  Microthrombi  Figure 6.  Toxicity.  This figure illustrates the various microcirculatory effects described in sepsis. This includes alteration in autoregulation in the vessels with the ellipses representing the vascular tone, leukocyte sludging and microthrombi formation which leads to vascular obstruction, and increased capillary permeability which leads to interstitial edema as depicted by the shaded area surrounding the cell.  20  change during shock.  When erythrocyte deformability  is decreased, the time  required for red cell passage through capillaries is prolonged and the cells may themselves impede blood flow and may lead to an impaired ability to extract oxygen as has been demonstrated by Powell et al (49) in septic humans. Furthermore, the increased rigidity of red cells promotes arteriovenous shunting of blood, which further decreases microcirculatory flow during sepsis (50).  1.4.2.2 Viscosity alterations  Blood viscosity is an important  determinant  of microcirculatory  flow.  According to Poiseuille's equation, blood flow varies directly with vessel radius to the fourth power and inversely with the length of blood vessels and blood viscosity. Chien (51) studied the rheological factors in the microcirculation during low-flow states and concluded that venular blood viscosity increases due to a decrease i n shear stress. The result of increased venular viscosity would be increased postcapillary resistance leading to diminished blood flow and increased transcapillary leakage (51).  1.4.2.3  Increased vascular permeability and interstitial edema  Sepsis has often been characterized as a diffuse increase in microvascular permeability (46) (52) (12). Solomon and Hinshaw (53) showed that endotoxin increases capillary permeability in the skin and muscle tissues independently from changes in hydrostatic and colloid pressures, thereby implying the mechanism to be an alteration in the cell membrane structure.  21  According to Starling's Forces, an  increase vascular permeability will predispose to interstitial edema. Hersch et al (54) studied the histologic and ultrastructural changes in a rat model of hyperdynamic sepsis and showed that despite a preservation of hemodynamic variables, there was a widespread degree of lesions from sepsis including interstitial and intracellular edema. Interstitial edema may in turn lead to an impaired diffusion of oxygen from vessels to cells (55) (34).  Heughan et al (56) showed that saline loading will lead to  interstitial edema and this was associated with a decrease in tissue oxygenation. N o t only can interstitial fluid itself limit the diffusion of oxygen but interstitial edema i n sepsis is known to result in an increased distance from cells to capillaries (57). Knisely et al (58) have theorized that such an increased distance will significantly limit the diffusion of oxygen. Other possible changes promoting edema formation include protein leakage (59), separation of tight junctions between endothelial cells (60), dysfunctional rather than destructive changes of vascular endothelium (61), and release of vasoactive agents (62). Finally, tissue edema formation may compress capillaries and thereby limit oxygen delivery (34).  1.4.2.4  Alterations in microvascular flow and capillary heterogeneity  The ability of tissue to alter blood flow to actively metabolizing tissue is of paramount importance in survival.  The organism adjusts oxygen extraction i n  response to changes in oxygen delivery through a balance between vasoconstrictor tone among organ systems and local metabolic vasodilation within tissues (63). However, various components required for normal adaptation to oxygen demands are altered in sepsis including development of vascular obstruction, development of  22  arteriovenous shunts/and endothelial damage. All these effects lead to an inability to provide normal homogenous blood distribution and thereby leads to a deleterious heterogenous blood flow (Figure 7). Various components of blood may lead to obstruction of vascular flow. Knisely et al (64) studied patients and animals and found large rigid erythrocyte aggregates in all vessels, and concentrated "sludge" in plugged vessels which lead to obstructed blood flow.  Clotting factors may also be activated leading to  disseminated intravascular coagulation (DIC) during sepsis (65) whereby fibrin and microthrombosis are deposited in vessels. Finally leukocytes have been shown to cause capillary obstruction during sepsis (65) (41) (66) which not only contribute to vascular obstruction but also has effects on red cell deformability and capillary crosssectional area (65). Various studies have shown the occurrence of shunts in various tissue beds in sepsis. Cronenwett and Lindenauer (67) used microspheres to demonstrate arteriovenous shunting of blood in the septic canine. Further, Archie (68) described arteriovenous shunting in a cecal ligation shock model occurring in the splanchnic and renal circulation. Whether these changes represent the effects on the changes of red cell deformability (see above) or represent true anatomical arteriovenous shunts is unclear. While previously, the endothelial layer was thought of as only a passive tissue, more recent work has described its importance in modulating vascular tone, controlling local blood flow, influencing the rates of leakage of fluids and plasma proteins, modulating the accumulation and extravasation of leukocytes into tissue,  23  Homogenous distribution  F i g u r e 7.  Heterogeneous distribution  Capillary Heterogeneity.  Homogeneous distribution leads to adequate bloodflowto the cells. Heterogeneous distribution leads to maldistribution of blood flow to the cells.  24  and influencing leukocyte activation (69). Therefore, damage to the endothelium and the underlying smooth muscle from hypoxia, inflammation,  complement  activation, O 2 radical production, and lipid peroxidation, all postulated in the sepsis syndrome, would interfere with the matching of O 2 delivery and metabolic needs by interfering with vascular smooth muscle tone and possible interactions between the endothelium and the smooth muscle (55) (70). Schumacker and Samsel theorize that alteration in endothelial function leads to the loss of autoregulation of the microvasculature which in turn results in the inability of tissue to adjust to reduced oxygen delivery (63) (71). Indeed, sepsis typically is described as having a decreased systemic vascular resistance (72) which reflects the decreased arteriolar and venular tone (36). Dantzker (55)  explains that a loss of autoregulatory ability could explain  several of the features seen in sepsis.  The effects explained above, all point to  changes that will affect the ability to adapt to increased oxygen demands. Delivery of oxygen to tissue is ideally done by both an adequate and a homogenous distribution of capillaries. However, Drazenovic et al (73) found endotoxemia in a canine model reduced capillary density in mucosal villi and crypts. Further, Lam et al (57) using intravital microscopy to analyze changes in muscle capillaries in a rat model of sepsis not only found a decreased density of perfused capillaries, but also an increase in capillary heterogeneity, an increase in the inter-capillary distance, an increase i n capillary red blood cell velocity, and a decrease in the peak hyperemic response. Thus, while homogenous capillary flow promotes oxygen extraction, all the microcirculatory changes described will result in a heterogenous flow of blood  25  w h i c h may impair oxygen diffusion and extraction by tissue (74) (75) (76) (63) (77) (78) (79).  1.5  SPLANCHNIC ISCHEMIA IN CRITICAL ILLNESS  1.5.1  Protective mucosal barrier A n intact mucosal layer is important to act as a defense against infection.  It  has been shown that an intact mucosal layer maintains an effective barrier e v e n during states of immunosuppression (80).  This implies that the cell-mediated  i m m u n i t y provided by the intestinal wall's lymphocytes, macrophages, and the Peyer's patches, and the mesenteric l y m p h nodes serves a secondary or supportive role to the epithelium.  W h i l e the mucosa is an important barrier, the gut is  susceptible to ischemia during sepsis or endotoxemia due to the manner that the blood flow is organized.  1.5.2  Splanchnic Metabolic Model of O2 Control Metabolic demand theory i n the splanchnic circulation proposes that the  intestines control local microvascular smooth muscle tone, based o n the p r e v a i l i n g tissue PO2,  and independent of nervous or humoral influences. (81). There appears  to be two separate microvascular mechanisms by w h i c h the intestine can regulate the rate of oxygen delivery (82) (Figure 8). First, local arteriolar tone governs the amount of total blood flow that is available to the i n d i v i d u a l capillary beds.  Control  of the precapillary sphincters modulates the number of capillaries that are perfused w i t h i n a capillary bed, thereby effecting changes i n the surface area and the capillary-  26  feedback  Figure 8 .  Metabolic model of O 2 control in the gut.  This figure illustrates theoretical model of oxygen control at the microvascular level in the gut. Local arteriolar tone governs total blood flow.  Small and moderate decreases in oxygen or build-up of  metabolites leads to alteration in the tone of precapillary sphincters.  27  to-cell diffusion distance (81). Second, w i t h small and moderate decreases i n oxygen delivery, the m a i n compensatory mechanism  for maintaining a stable oxygen  consumption are the precapillary sphincters (83).  This mechanism  causes a n  increase i n capillary surface area and results i n increased oxygen extraction. w i t h larger decreases i n oxygen delivery does the arteriolar resistance  Only  decrease,  thereby increasing blood flow to the tissue (84). However, despite this fine control (55), there is a loss of autoregulatory ability i n sepsis resulting i n a reduced capillary density i n mucosal v i l l i and crypts (73). In fact, the reduction i n splanchnic blood volume is disproportionately greater than that seen i n other tissue beds (85).  1.5.3  Counter-current shunting (CCS) The microcirculation of the intestinal villus is arranged i n a counter-current  system of arterioles  and venules  which  is designed  to improve  absorption.  However, the countercurrent shunting has been hypothesized as another reason for the susceptibility of the mucosa to ischemia during sepsis (82) (86) (55) (87) (88). T h i s theory proposes that since the vessels are arranged i n a villus w i t h both the arteriole and the venule side by side, O2 may diffuse from the arteriole straight across to the venule without the villus ever benefiting from the O2, thereby effectively s h u n t i n g oxygen away from the villus (Figure 9). Furthermore, it has been s h o w n that this effect is more exaggerated during times of decreased blood flows (89).  28  Villus Arteriole  Figure 9.  Venule  Counter-current shunting (CCS).  This figure illustrates theoretical concept of counter-current shunting in the intestinal villus.  It  describes how oxygen may shunt across from the arterioles to the venules due to the vessels being arranged side by side.  29  1.5.4  Gut Ischemia during Septic Shock W h i l e the mucosal barrier serves a particularly important role i n p r e v e n t i n g  infection, its o w n fine microvascular balance places it at risk i n sepsis (90). Fink et al (52) showed that even w h e n the hemodynamic variables were maintained i n sepsis, there are significant levels of gut mucosal acidosis. Furthermore, the h i g h metabolic demands of the intestinal surface places the gut at further risk d u r i n g times of limited oxygen supply (91) (92) (93) (94). Since the gut is susceptible to the effects of sepsis, the protective mucosal barrier is thereby placed i n jeopardy (91) (95) (96). Salzman et al (97) have shown that endotoxin infusion leads to increased intestinal permeability to macromolecular hydrophilic solutes. injected  endotoxin i n healthy  humans  Similarly, O'Dwyer et al (98)  and found  increased permeability  to  nonmetabolizable sugars. Bacterial translocation is the process by w h i c h microorganisms migrate across the mucosal barrier and invade the host. Rush et al i n a series of studies (99) h a v e s h o w n that shock i n animal models and humans  leads to the development of  bacteremia and endotoxemia. Further, they have shown that most of the bacteria found i n the blood were enteric organisms and by using radiolabeled E. coli and demonstrating them escaping from the gastrointestinal tract and into the blood stream, conclude that bacterial translocation is a true phenomenon.  Deitch et al  (100) had similar conclusions i n a mouse model and further determined that the mechanism  may be v i a increased gut permeability v i a activation of xanthine  oxidase leading to development of oxygen free radicals (101). M a i n o u s et al (102) studied the route of bacteremia and determined that bacterial translocation is  30  primarily v i a portal blood as opposed to mesenteric lymphatics and occurs i n a dose dependent fashion to the degree of insult. It has been theorized that injury to the gut w i t h the resultant bacteremia and endotoxemia  may  perpetuate  a continuous  cycle of injury  leading  to  the  development of multiple organ dysfunction syndrome (85). Hence, the term, the gut as the "motor of multiple organ dysfunction" (3) (Figure 10).  1.6.  AEROBIC A N D ANAEROBIC METABOLISM  1.6.1  Aerobic metabolism The routine means to derive energy by cells is by aerobic metabolism. Energy  is derived from amino acids, glucose, and fatty acids v i a the Kreb's (Tricarboxylic Acid) Cycle and oxidative phosphorylation. The Kreb's cycle is a series of controlled oxidation-reduction reactions during w h i c h the energy, released from the transfer of electrons, is captured. This involves the reduction of N A D + to N A D H , a shuttling of the electrons from N A D H into the mitochondria, transferring the electrons along a series of electron-carrier enzymes, and the capture of energy i n the high-energy phosphate bonds of adenosine triphosphate (ATP). Oxygen is the terminal electron acceptor i n this scheme and sufficient amounts  are required if optimal use of  substrate for the generation of A T P is to continue. W h e n oxygen is not present i n adequate amounts, the organism relies on the less efficient anaerobic m e t a b o l i s m w h i c h generates less energy per substrate when compared to aerobic metabolism (55) (Figure 11).  31  o  Figure 10.  2  Bacteria Endotoxin  ^  Gut as "Motor of Multiple Organ Dysfunction".  It has been theorized that injury to the gut with the resultant bacteremia and endotoxemia may perpetuate a continuous cycle of injury leading to the development of multiple organ dysfunction syndrome. Hence, the term, the gut as the "motor of multiple organ dysfunction"  32  Figure 11.  Aerobic Metabolism.  Energy is derived from amino acids, glucose, and fatty acids via the Tricarboxylic Acid Cycle (TCA) and oxidative phosphorylation. The TCA cycle is a series of controlled oxidation-reduction reactions during which the energy, released from the transfer of electrons, is captured. reduction of N A D  +  This involves the  to NADH, a shuttling of the electrons from NADH into the mitochondria,  transferring the electrons along a series of electron-carrier enzymes, and the capture of energy in the high-energy phosphate bonds of adenosine triphosphate (ATP). acceptor.  33  Oxygen is the terminal electron  1.6.2  Anaerobic metabolism  When oxygen is not available, ATP is generated by glycolysis where pyruvate is the terminal electron acceptor.  While the aerobic pathway results in the  generation of 36 mmol of ATP per mol of glucose, the anaerobic pathway results i n only 2 mmol of ATP per mol of glucose. Not only does this produce much less energy per mol of substrate, but the pyruvate is reduced to lactate which may then contribute to the development of metabolic acidosis (55) (Figure 12).  1.6.3  Oxygen Consumption and Delivery  Investigators have developed techniques whereby the oxygen consumption and delivery variables can be quantified to allow determination of the onset of ischemia and ability to extract oxygen. The technique of decreasing oxygen delivery by progressive hemorrhage and following the effects on oxygen consumption have been described (103) (104) (105). The ability of tissue to utilize O 2 during shock can be determined by plotting the relationship between oxygen consumption ( V O 2 ) and oxygen delivery  (DO2).  With progressive decrease in oxygen delivery, the tissue  compensates by increasing the amount of oxygen extracted. If the tissue is able to extract enough oxygen then aerobic metabolism is maintained. However, there is a point when O 2 delivery falls below sufficient O 2 requirements after which anaerobic metabolism ensues. This critical point is considered the onset of ischemia (Figure 13).  34  (D02c)  Glucose  Glycogen  Glyceraldehyde 3 - P 4 ADP 4 ATP  1  ^**  Lactate  — NAD NADH  Pyruvate  F i g u r e 12.  Anaerobic Metabolism.  Anaerobic metabolism of ATP from glycolysis. When oxygen is not available, ATP is generated by glycolysis where pyruvate is the terminal electron acceptor. Not only does this produce much less energy per mol of substrate, but the pyruvate may be reduced to lactate possibly contributing to the development of metabolic acidosis  35  O Consumption z  (V0 ) 2  Onset of ischemia  (D0 c) 2  0 Delivery 2  (D0 ) 2  Figure 13.  Relationship between Oxygen Delivery and Consumption.  With progressive decrease in oxygen delivery, the tissue compensates by increasing the amount of oxygen extracted. If the tissue is able to extract enough oxygen then aerobic metabolism is maintained. However, there is a point when O2 delivery falls below sufficient O2 requirements after which anaerobic metabolism develops. This critical point is the onset of ischemia (DO2C).  36  1.6.4  Oxygen Extraction Ratio The ability to extract oxygen is calculated as the oxygen consumption at the  onset of tissue ischemia ( V 0 2 c ) divided b y the oxygen delivery at the onset of tissue ischemia ( D 0 2 c ) . This can also be plotted against oxygen delivery to represent the increases i n the amount of oxygen extracted as oxygen is delivered (Figure 14). T h e ability to extract oxygen is based o n three major factors:  adequate oxygen delivery,  unimpeded oxygen diffusion, and the ability to utilize oxygen.  1.6.5  Oxygen Consumption and Delivery in Sepsis Studies (106) (107) (108) (109) have shown two m a i n changes i n the oxygen  consumption and delivery curves. First, the oxygen consumption at baseline is at a higher level, representing the increased overall metabolic rate.  Secondly, tissues  have been noted to have an earlier onset of ischemia (Figure 15). This earlier  E)02C  may be due to the second major change w i t h sepsis, that of an impaired ability to extract oxygen b y the tissue.  1.6.6  Oxygen Extraction in Sepsis A s w i t h studies into the onset of ischemia, the ability to extract oxygen has  also been documented to be impaired (106) (107) (108) (109). This can be documented w i t h a lower oxygen extraction ratio curve for the same oxygen delivery (Figure 16). Dantzker suggested three possible reasons for an impaired oxygen extraction and utilization. generation  First, the apparent utilization of anaerobic mechanisms  for A T P  despite high D O 2 may suggest an abnormality of the cells' ability to  37  0 Delivery (D0 ) 2  2  F i g u r e 14.  O x y g e n Extraction Ratio.  With progressive decrease in oxygen delivery, the tissue compensates by increasing the amount of oxygen extracted. If the tissue is able to extract enough oxygen then aerobic metabolism is maintained. However, there is a point when O2 delivery falls below sufficient O2 requirements after which anaerobic metabolism develops. This critical point is the onset of ischemia (DO2C).  38  Figure 15.  Relationship between Oxygen Delivery and Consumption in Sepsis.  There are two main changes in the oxygen consumption and delivery curves. The oxygen consumption a t baseline is at a higher level and tissues have an earlier onset of ischemia.  39  F i g u r e 16.  O x y g e n Extraction Ratio i n Sepsis.  During sepsis, the ability to extract oxygen has been shown to be impaired.  40  utilize the oxygen that is delivered to it. Indeed, Hersch et al (54) i n a study on the, histologic and ultrastructural changes i n a rat model of hyperdynamic sepsis showed widespread mitochondrial destruction. Second, the finding of a high mixed v e n o u s PO2 i n the face of tissue hypoxia may indicate a degree of shunting of blood a r o u n d metabolizing tissue. Finally, there may be an impaired ability of oxygen to diffuse from the systemic capillaries to the cells requiring oxygen due to interstitial edema.  1.7  T R E A T M E N T OF SEPSIS  1.7.1  Treatment Options Treatment of sepsis may be focused at any of the levels of septic induced  toxicity (Figure 17). Certainly the prevention of infection w o u l d be the best strategy. Next w o u l d be to treat infection early to limit the effects by either early drainage of infections or treatment w i t h antibiotics (12) (2). N e w treatments include attempting to block the effects of endotoxin w i t h antibodies and i m m u n o m o d u l a t i o n  of the  host defenses. However these new techniques have meet w i t h conflicting results. Indeed, attempting to modulate the immune  system is complicated at best as one  needs to balance the advantages and disadvantages of the immune system. However, all too frequently, patients present far along their course of SIRS. Therefore, the treatments clinicians are left w i t h are an attempt to i m p r o v e oxygen delivery (47) and maintain  adequate arterial perfusion pressure.  T w o general  options are available, including inotropic support and fluid resuscitation.  41  Immunomodulate  F i g u r e 17.  Treatment Options.  This figure illustrates the various treatment options currently being investigated. advocated regimens include prevention, antibiotics, and augmenting oxygen delivery. investigated include immunomodulation and blocking the mediators of toxicity.  42  The currently Areas being  1.7.2  Augmentation of Oxygen Delivery Oxygen delivery is determined by the following equation: D O 2 = Oxygen content x Cardiac output where Oxygen content = Hgb x 1.39 x O 2 sat + 0.003 x P a 0 2 F r o m this, one may see that oxygen delivery is dependent on h e m o g l o b i n ,  oxygen saturation, and cardiac output.  Increases i n any or all of these variable w i l l  go to increase the oxygen delivery (47). To increase hemoglobin, one may transfuse packed red blood cells to increase the hemoglobin concentration (110). This is usually done if the hemoglobin is <100 g/1  Many  (111).  studies  showed  no  benefit  i n oxygen consumption  when  transfusing patients above these levels (112) (113). To increase oxygen saturation, one may increase the oxygen concentration that is given. In many situations, this may require intubation and ventilation to maximize the F i 0 2 - A second benefit of intubation and paralysis is a reduction i n the w o r k of breathing.  Hussain and Roussos (114) showed that early use of  mechanical ventilation has been shown beneficial i n the management  of septic  shock. To  increase  cardiac  output,  both  Vasopressors are used to try to augment contractility  (47).  Of the  current  vasopressors cardiac output  inotropes,  P-agonists  and  fluids  are  used.  by increasing cardiac including  dopamine,  dobutamine, norepinephrine, and epinephrine have been used most often. A l l but dobutamine  also have  a-adrenergic  action  43  that  increases  uneven  arteriolar  vasoconstriction Dobutamine  has  and both  may pi  intensify and  P2  microcirculatory actions;  the  flow  latter  maldistributions. relaxes  previously  vasoconstricted arterioles and may improve small vessel blood flow i n peripheral tissues (115). Vasodilators play a limited role i n the care of septic patients since hypotension is invariably already present (7).  Overall, the clinical efficacy and  optimal therapy for pressor treatment has not been w e l l documented i n prospective randomized trials. Studies have described important side effects that w o u l d caution against their use i n any situation including increased overall metabolic rate and systemic oxygen demands (116). Further, Ruiz et al report an increased mortality w i t h the use of inotropes i n such situations (117). Therefore, since the benefits of vasoactive agents are at present unclear, augmentation of cardiac output is primarily achieved by the administration of fluid resuscitation.  1.7.3  Fluid resuscitation in septic shock For many clinicians, fluid resuscitation is one of the prime therapies for the  treatment of patients i n septic shock (118) (2). The rationale for use of fluids has centered  on  re-establishing  normal  hemodynamic  variables  including  the  augmenting of filling pressures (2) and replacing relative and absolute intravascular volume deficits (2) (119) (120) (121) to augment cardiac function (122). F l u i d therapy in septic shock is thought to increase venous return and cardiac output. W h e n fluid therapy is associated w i t h an increase i n DO2 i n patients w i t h lactic acidosis, systemic oxygen consumption VO2 increases and lactic acid levels decrease (123) (124).  Other investigators  have  proposed benefits  44  at a microvascular  level.  H e m o d i l u t i o n of blood has been shown to promote survival i n critically i l l patients (111). The potential benefits include a decreased viscosity of blood (125) leading to an increased velocity of red cells (126) and possibly i m p r o v e d entry of erythrocytes into channels w i t h smaller diameter, thereby decreasing the heterogeneity of blood flow (126) (79). While  both crystalloid and colloidal  solutions  are  frequently  used  for  resuscitation, the choice between these two fluids is controversial. Crystalloids h a v e been generally advocated i n view of availability, the lack, of potentially infectious complications, and overall costs. However, since only about 1/3 to 1/4 of the saline solution infused remains i n the vascular space, large volumes are required i n order to achieve therapeutic goals (127). Furthermore, w i t h a significant amount going to the interstitial space, some have raised concerns regarding the development interstitial edema (128).  of  W i t h these concerns, many have looked to the use of  colloids for resuscitation. Colloid fluids may be advantageous because the sustained increases i n plasma colloid osmotic pressure (COP) by these fluids w i l l aid i n the retention of fluid i n the intravascular space (129). Further, evidence shows that sepsis is associated w i t h reprioritization of hepatic protein synthesis w i t h decreased albumin production w h i c h contributes to a decreased colloid oncotic pressure (130). Demling et al (131) suggest that it is the hypoproteinemia w h i c h may be responsible for the early edema i n soft tissues w i t h sepsis. O n the other hand, the maintenance of an oncotic gradient by colloid infusion may be difficult i n systemic areas i n w h i c h microvascular permeability is increased.  Further, there is concern that egress of  larger molecules into the extravascular space may also increase the colloid oncotic  45  pressure in the interstitium, thereby limiting the effects of the colloid and in fact, may worsen the severity of the interstitial edema and hinder its resorption (127). In view of this complex issue, there is no clear agreement as to the ideal fluid to use in sepsis. Possible side-effects of crystalloid resuscitation include hemodilution that may lead to compromise of systemic oxygen delivery (132) (118), pulmonary edema (133) (5) (134) (135) (47) or interstitial edema secondary to the increased permeability (136), and hyperchloremic acidosis due to a large volume of saline required for stabilization (137). Indeed, the diffusion of oxygen from red blood cell to the tissue is dependent on surface area for diffusion and the length of the pathway from red cell to mitochondria, both functions of microvascular control, and the diffusion characteristics of oxygen in the tissue (55). Therefore, the effect of tissue edema or inflammation on the diffusing characteristics of the tissue may be significant (55). Further, while some argue that hemodilution leads to a decreased hematocrit which may reduce viscosity and improve blood flow and may thereby be beneficial for some organs like the heart and brain, other organs accommodate poorly to the decrease in oxygen carrying capacity (55).  1. 7.4 Diffusion vs filtration Diffusion refers to movement of substances in either direction across the capillary.  Furthermore, diffusion rate depends on various factors including  solubility of the substance in the tissues, the temperature, and the surface area available; it is inversely related to molecular size and the distance over which  46  diffusion occurs. Filtration refers to the net movement of fluid out of the capillaries and is governed by the Starling law of the capillary.  1.7.5  Starling's Law of the Capillary F l u i d balance between the intravascular and interstitial fluid compartments is  determined by the forces operative i n the Starling law of the capillary (Figure 18): Qf = K f { P - P i - 8 ( 7 i c - 7 t i ) } c  i n w h i c h Qf is the total flow of fluid across the capillary membrane; K f is the f l u i d filtration coefficient; P  c  is the capillary hydrostatic pressure; P i is the interstitial  hydrostatic pressure; 8 is the reflection coefficient; Ttc is the capillary colloid osmotic pressure;  and K{ is the interstitial colloid osmotic pressure (138) (see below for  details). The interaction of these four Starling forces determines fluid, flux between interstitial and intravascular compartments. dominant  Capillary hydrostatic pressure is the  d r i v i n g force favoring fluid filtration across the capillaries into  interstitium.  the  Interstitial hydrostatic pressure is usually negative, but can become  positive if large amounts of edema fluid accumulate.  The plasma colloid osmotic  pressure is the only force acting to retain fluid w i t h i n the intravascular space.  In  contrast, interstitial colloid osmotic pressure favors fluid retention i n the interstitial space and may be diluted by the accumulation of protein-sparse  edema  fluid.  Increases i n interstitial hydrostatic pressure and reductions i n interstitial colloid osmotic pressure serve to limit edema formation (137).  47  Q = K ( P - Pj - 8 [ 7 T - 7X;]) f  F i g u r e 18.  f  c  C  S t a r l i n g ' s Forces o f the C a p i l l a r y .  The Starling's Forces determines the fluid flux between interstitial and intravascular compartments. Qf is the total flow of fluid across the capillary membrane; Kf is the fluid filtration coefficient; P is c  the capillary hydrostatic pressure; Pj is the interstitial hydrostatic pressure; 8 is the reflection coefficient; 7t is the capillary colloid osmotic pressure; and TCJ is the interstitial colloid osmotic c  pressure.  48  The fluid filtration coefficient represents the net amount of fluid crossing the capillary membrane for a given level of Starling's forces.. This value varies w i t h changes i n the surface area of the functional microcirculation at any one time. Dilation of arterioles, and especially dilation of precapillary sphincters, can increase the number of functional capillaries. Normally, only a fraction of capillaries exhibit blood flow at any one time. The reflection coefficient is a measure of the ability of the capillary membrane protein.  A membrane  to exclude large particles and limit the movement  of  completely impermeable to protein w o u l d demonstrate a  reflection coefficient of 1. The average reflection coefficient for systemic capillaries is approximately 0.9 and for pulmonary capillaries is approximately 0.7 (139) (140). Under conditions of increased capillary permeability, the reflection coefficient may decrease to 0.4 (141), thereby favoring flow of fluid into the interstitium. The colloid osmotic pressure of plasma is determined  by the number  particles i n solution that are impermeable to the capillary membrane.  of  A l b u m i n is  responsible for approximately 80% of the plasma osmotic pressure (142).  The  n o r m a l plasma colloid osmotic pressure is 21-25 m m H g , and i n a critical care population ranges from 18-20 m m H g (143) w h i c h w i l l also tend to favor flow of fluid into the interstitium.  1.7.6  Tonometry A s discussed earlier, the splanchnic circulation is particularly vulnerable i n  sepsis. Since some investigators have shown that the gut becomes ischemic earlier than whole body (86), many have sought a mechanism by w h i c h one may detect  49  changes early enough to prevent mucosal injury. The use of tonometry has been a n evolving one. Bergofsky (144) estimated PO2 and P C O 2 i n gallbladders and u r i n a r y bladders tonometrically by instilling the organs w i t h saline and after e q u i l i b r i u m , measuring P O 2 and P C O 2 i n the intraluminal fluid. Dawson et al (145) carried this idea into use i n the intestinal tract w i t h small bowel. Then, rather then placing fluid w i t h i n the lumen, Kivisaari and N i i n i k o s k i (146) measured P O 2 and P C O 2 i n tissues by determining the partial pressures of O 2 and C O 2 i n saline  contained  w i t h i n a Silastic tube w h i c h was permeable to these gases. Fiddian-Green et al (147) proposed the idea that tonometry could be used to estimate intramucosal p H (pHi) w i t h the idea that H C O 3 " concentrations i n tissue and arterial blood are sufficiently similar to permit substituting the latter value into the  Henderson-Hasselbalch  equation (Figure 19). Finally, Antonsson et al (148) validated this technique by using microelectrodes to directly measure the ileal p H i and correlating these values to those derived by tonometry during endotoxemia and mesenteric occlusion i n pigs. Clinicians have studied the role of tonometry recently and have  showed  some encouraging results (149) (150) (151). Gutierrez et al (152) have s h o w n that tailoring therapy to p H i improved overall outcome patients.  i n a group of critically i l l  M a y n a r d et al (153) showed that gastric tonometry was the most reliable  indicator of adequacy of tissue oxygenation i n a group of critically i l l patients. Finally, studies have shown such a good correlation to oxygenation of tissue that tonometry may be used to determine the onset of anaerobic metabolism (154) (155).  50  Figure 19.  Tonometric Analysis.  This figure illustrates the manner that the tonometer is used to determine the mucosal pH (pHi). The pCC>2 of the mucosal wall equilibrates across the semipermeable membrane with the saline in the balloon. This can then be inserted into the Henderson-Hasselbalch equation using the arterial HCO3" to determine the pHi.  51  1.7.7  Lactate Several investigators have used lactate as a marker for anaerobic m e t a b o l i s m  (74) (106). W h e n there is insufficient oxygen to sustain oxidative phosphorylation, increased glycolysis is called upon to maintain tissue A T P levels. D u r i n g glycolysis, pyruvate acts as the terminal electron acceptor and leads to the production of lactate (55). However, Danztker (55) points out that there may be some confounding factors which  may  make  straightforward.  the  interpretation  of  serum  lactate  levels  less  Serum lactate levels reflect balance i n production and  than the  metabolism of lactate. In sepsis there are several reasons for increased production of lactate (156) including inflammatory mediators, cytokines, and other vasoactive substances that impair vasomotor tone, increase microvascular permeability, and facilitate aggregation of leukocytes and platelets. Capillary leakage causes decreased circulating blood v o l u m e and cardiac output that is further impaired by the direct effects of sepsis on ventricular function. Ultimately these changes lead to a fall i n perfusion and ischemia w i t h the resultant anaerobic metabolism. H o w e v e r , other factors may either raise or lower the level of lactate.  Factors raising the levels  include a decreased clearance due to decreased perfusion to the liver and kidneys, the m a i n sites of metabolism of lactate (157) (156), and a decrease i n the activity of pyruvate dehydrogenase i n sepsis (156). Factors leading to an underestimation of anaerobic metabolism include some tissue only producing lactate once all other alternatives to oxidative phosphorylation are exhausted, and as tissue p H falls, glycolytic flux is inhibited thereby providing a feedback on the level of acidosis (55).  52  Therefore, since lactate levels i n the blood reflect the balance of all these complex effects, lactate levels are difficult values to interpret i n sepsis (149) (158).  53  Chapter 2 HYPOTHESIS  Sepsis has been characterized by increased capillary permeability. In v i e w of k n o w n changes w i t h respect to the Starling's forces at the capillary level, one w o u l d expect crystalloid resuscitation to result i n a significant degree of interstitial edema. Interstitial edema may i n turn impair oxygen diffusion from capillary to cells and thereby limit the ability of tissue to extract oxygen. Furthermore, interstitial edema along w i t h endothelial edema may worsen an already heterogeneous distribution of capillaries and thereby further perfusion  impair the  w i t h oxygen demands.  ability of tissue to properly  Finally, h e m o d i l u t i o n  match  alone may lead to a  decrease i n the capillary hematocrit such that the delivery of oxygen w i l l be lowered. Therefore, my hypothesis is that crystalloid resuscitation w i l l impair the ability of tissue to extract oxygen i n endotoxemia.  54  Chapter 3 OBJECTIVES OF THE THESIS  The use of fluid resuscitation i n sepsis is a generally accepted therapeutic maneuver.  However, i n studying the physiological implications of fluid therapy,  many questions arise as to the potential disadvantages of fluid resuscitation. In this thesis, an attempt is made to address several important questions as to the effects of crystalloid fluids i n an endotoxemic porcine model of sepsis.  Specifically, the following questions w i l l be addressed: 1.  Does crystalloid resuscitation impair the ability to extract oxygen? In the clinical scenario, the use of crystalloids has generally been the first l i n e  of therapy and often result i n improved clinical parameters.  However, measuring  the oxygen extraction may be a more important parameter as it w i l l assess tissue ability to adapt to sepsis.  To elaborate on the effects of crystalloid resuscitation, several potential m e c h a n i s m s of action were studied: 2.  Does crystalloid resuscitation lead to interstitial edema? In view of the described generalized leak i n sepsis, one w o u l d anticipate that  aggressive fluid loading w i l l lead to edema and thereby prevent oxygen extraction by tissue. Since it is difficult i n the clinical scenario to quantify the degree of interstitial edema, our experiments w i l l examine this possibility.  55  3.  Does crystalloid resuscitation lead to capillary blood flow heterogeneity? While some argue that fluid resuscitation reduces blood viscosity and allows  for improved flow of blood, others argue that there may be endothelial and interstitial edema which may prevent flow of blood and thereby increase an already capillary heterogeneity and thereby impair oxygen extraction. 4.  Does crystalloid resuscitation lead to decreased capillary hematocrit? While crystalloid resuscitation leads to hemodilution at a large vessel level,  there have been no papers documenting the capillary red blood cell concentration. This is a possible side effect of fluids which may impair the overall concentration of oxygen delivered to tissues. Using morphometric techniques, one may determine these values.  56  Chapter 4 RESEARCH PLAN  4.1  THE PORCINE MODEL OF SEPTIC SHOCK Various considerations for the model of septic shock included the animal to  be used and the type of sepsis produced.  Pigs are quite similar to humans  with  respect to renal, cardiovascular, and digestive anatomy and physiology (159) and therefore serve as a useful experimental animal. W i t h regards to the septic m o d e l , various types have been described including intravascular infusion of bacteria, peritonitis by bacterial i n o c u l u m , cecal ligation and perforation, or infusion of endotoxin. W e chose the later for several reasons. qua n o n of sepsis, is considered fundamental  Endotoxin, although not a sine  i n the development  of sepsis (8).  Further, endotoxin is a stable and relatively pure compound w h i c h simplifies some aspects of experimental design.  In contrast, bacteria are typically stored frozen,  grown i n culture and washed several times to remove  culture m e d i u m  and  solubilized bacterial products, and later require quantification (8). Finally, C a i n and Curtis point out that endotoxin may provide a better experimental  model of  hyperdynamic sepsis i n acute animal experiments than live bacteria as it m o r e closely matches changes i n oxygen consumption w i t h a rise by 15% to 20% above the critical D O 2 (160). Our study also examined the role of fluid resuscitation i n the endotoxemic model. W h i l e fluid administration i n the clinical setting is not standardized, we needed a method of administration w h i c h was consistent and rational.  57  A fixed  volume of crystalloid has been described by Fink and Heard (8). A fixed v o l u m e as opposed to one titrated to hemodynamic variables has several advantages.  First,  myocardial performance is impaired by endotoxemia, thus preload may need to be elevated to supranormal levels to maintain cardiac output.  Second, sepsis may lead  to alterations i n ventricular diastolic compliance, thus, left ventricular end-diastolic and pulmonary capillary wedge pressures may be poor estimates for left v e n t r i c u l a r end-diastolic volume, the relevant variable for assessing preload.  Finally, central  venous and pulmonary capillary wedge pressures are typically small numbers, and using conventional catheter-transducer-amplifier  systems, there is considerable  imprecision (5-15%) (8) i n the measurement of these variables; thus, it w o u l d lead to imprecise titration of fluid administration. A porcine model of endotoxemia i n w h i c h hemodynamic stability is achieved by an aggressive fluid resuscitation protocol has been previously described (52) (161). The m o d e l adequately reproduces several features of septic shock i n  humans  including profound systemic arterial hypotension and l o w SVRI (96). Further, the volumes of fluid used were 25 c c / k g / h r for maintenance  and 48 c c / k g / h r for  resuscitation. This model has been used i n recent studies of sepsis i n our laboratory (74). Thus, we used a similar porcine model of endotoxemia w i t h fluid resuscitation being crystalloid administered as a continuous infusion at the same fixed rates.  58  4.2  MORPHOMETRIC ANALYSIS OF GUT TISSUE M o r p h o m e t r y is the measurement  of structure.  It's aim is often to answer  questions such as "how many" and "how large" something is. W h i l e this is often the goal of morphometry, it is only w i t h stereology that one is able to answer these questions. Stereology is the three-dimensional interpretation of flat images by the criteria of geometric probability. It can also be defined as extrapolation from twodimensional to three-dimensional space. Stereology is practiced by measuring and counting profiles i n sections (Figure 20). For most stereological problems, this can be done by superimposing grids of lines or squares and circles over an image.  One may adjust sampling w i t h i n the  i n d i v i d u a l animal to a specific level of acceptable error.  Various formulae  are  available (162) to allow determination of numbers of counts to limit error rates. Counting involves laying the grid on the area of interest and then counting the grid intersections w h i c h hit the component of interest.  The number of points that h i t  profiles of the component, divided by the total number of test points equals the volume  fraction of that component  i n the entire specimen.  W e apply these  techniques i n the determination of the gut interstitial v o l u m e and the capillary hematocrit.  4.3  INFRA-SCAN ANALYSIS OF G U T TISSUE The infra-scan method  of tissue  analysis is a recent  development  in  microscopic analysis. It utilizes a microscope attached to a camera w h i c h sends  59  Capillaries  F i g u r e 20.  Slide to count  Morphometric Analysis.  Morphometry attempts to determine the number of a certain area interest, in this case capillaries measuring and counting profiles in sections  60  images through to a computer.  The investigator is able to choose the image of  interest and to select the area i n question by use of a cursor. The imaging software (Infrascan, R i c h m o n d B.C.) w i l l analyze the entire field for pixels w i t h  similar  characteristics as the one chosen. The software w i l l then quantify the total images matching the area of interest and determine the fraction area that it represents of the total field (Figure 21). By using visual inspection, the automated analysis may be confirmed.  Various areas of interest may be looked at. W e were interested i n the  area fraction of interstitial space as this could be used to estimate the degree of interstitial edema that was present within the gut.  4.4  DETERMINATION OF GUT CAPILLARY HEMATOCRIT W h i l e the effects of fluid loading result i n h e m o d i l u t i o n at an arterial and  venous level, the hematocrit i n the microvessels may very w e l l be different (55). Indeed, some investigators believe that the ability of the microvascular hematocrit to change independent of the systemic hematocrit i n response to alterations i n the microenvironment  may account for some of the conflicting observations  during alterations i n systemic hematocrit (55).  made  Therefore, since changes i n the  systemic hematocrit may not reflect the results at a capillary level, we sought to determine  this  by morphometric  technique.  Using  previously  established  techniques of morphometric analysis (163) (164), random samples w i l l be cut, fixed, and stained for cutting and processing for photography under electron microscope.  a transmission  Point counting technique w i l l be used to determine  61  the  Microscopic Appearance  F i g u r e 21.  Infrascan Analysis  Infra-scan A n a l y s i s .  Infrascan is a technique whereby the slide is placed on a microscope attached to a camera which feeds the image to a computer. The investigator chooses the image of interest and select the area in question. The imaging software analyzes the entire field for pixels with similar characteristics as the one chosen. The software will then quantify the total images matching the area of interest and determine the fraction area that it represents of the total field.  62  relative volume fraction occupied by erythrocyte and total capillary v o l u m e , thereby determining the capillary hematocrit.  4.5  RELATIVE DISPERSION OF RED BLOOD CELL TRANSIT TIME A major determinant of the transport of oxygen from blood to tissue is the  time spent b y the red blood cell passing through the vessels, called the red blood cell transit time. The red cell transit time i n a given region is dependent on the blood volume and blood flow i n that region, both of w h i c h may vary. The blood v o l u m e and blood flow may each be determined as per studies performed on the l u n g by Hogg et al (163) and the heart by A l l a r d et al (164). Regional blood v o l u m e w i l l be determined using Technetium (^ Tc) m  labelled red blood cells and regional blood  flow w i l l be measured using a reference flow technique and a left atrial injection of 15 urn microspheres labelled w i t h Strontium (85Sr).  The different radioactive  signals can be used to quantify red cells and microspheres. Since the microspheres are also removed w i t h a reference flow technique, one may correlate the counts of microspheres w i t h the flow of these microspheres. Red blood cell transit time for the entire portion of tissue was determined by the quotient of blood v o l u m e and blood flow.  By analyzing the red blood cell transit times i n various segments of  intestine, one may determine the average transit time and the variability i n these values.  The relative dispersion or coefficient of variation is the quotient of the  standard deviation and the mean.  A s such, it provides one w i t h a measure of the  heterogeneity of the microcirculation i n question.  63  4.6  TONOMETRY Fiddian-Green et al ( 1 4 7 ) introduced the concept that tonometry could be used  to estimate intramucosal p H (pHi) w i t h the idea that H C O 3 " concentrations i n tissue and arterial blood are sufficiently similar to permit substituting the latter value i n t o the Henderson-Hasselbalch equation  (Figure 1 9 ) . Antonsson et al ( 1 4 8 )  have  validated this technique by using microelectrodes to directly measure the ileal p H i and correlating these values to those derived by tonometry during endotoxemia and mesenteric occlusion i n pigs. The gastrointestinal tonometer consists of a saline-filled silicone balloon that is placed i n the gut lumen. The silicone balloon is highly permeable to oxygen and carbon dioxide. N o r m a l saline is injected into the ileal tonometer balloon and left to equilibrate for 4 0 minutes. A t the end of the equilibration period, the saline w i l l be removed under anaerobic conditions.  The first milliliter of saline removed is  discarded, as this is not i n direct contact w i t h the balloon. The C O 2 content of the subsequent v o l u m e of saline removed, w h i c h reflects the mucosal C O 2 , w i l l be measured. Simultaneously, the arterial bicarbonate (assumed to be equal to the bicarbonate content of the gut mucosa) w i l l be measured. U s i n g a conversion table, measured PCO2 w i l l be transposed to a steady state PCO2 ( P C C ^ s s ) , depending on the exact duration of equilibration. Gut mucosal p H w i l l be calculated by substituting the PCO2SS and the simultaneously measured arterial bicarbonate i n the HendersonHasselbalch equation (ie: p H i = 6 . 1 + Log{(HC03)/Pcc>2}).  64  Chapter 5 EFFECT OF CRYSTALLOID RESUSCITATION O N WHOLE BODY A N D GUT IN ENDOTOXEMIA  5.1  INTRODUCTION In addition to antibiotic therapy, fluid resuscitation is a key component of the  management  of sepsis and septic shock (2). The goal of fluid resuscitation is to  restore intravascular v o l u m e to maintain an adequate blood pressure and cardiac output.  Restoring a normal cardiac output requires intravascular replacement of  third space losses, compensation for venous pooling of blood, and a sufficiently h i g h left ventricular filling pressure to compensate for decreased ventricular contractility during sepsis (72). Importantly, even a normal cardiac output may not necessarily be adequate during sepsis k>ecause sepsis is accompanied by impaired tissue oxygen extraction (55) (74) (106) (12). That is, higher oxygen delivery (equals cardiac output times arterial oxygen content) is required to prevent evidence of unmet  oxygen  demand (76) (12), including decreasing oxygen consumption (74) (106), rising lactate levels (74) (106), l o w gastric intramucosal p H (152), and organ system dysfunction (55) (76) (12). The causes of impaired tissue oxygen extraction d u r i n g sepsis have not been fully elucidated.  However, impaired oxygen uptake, due to mismatching of  oxygen delivery to demand (74) (78) and due to impaired oxygen diffusion  from  erythrocytes  roles.  to  tissue  mitochondria  (165), appear  to  play  important  M i s m a t c h i n g of oxygen delivery to demand and inadequate oxygen diffusion  are  due to several microvascular p h e n o m e n o n ; capillary plugging w i t h leukocytes and  65  other debris (41), capillary endothelial edema and tissue edema (54), and  altered  microvascular flow (74) (57). While normal response to impaired oxygen delivery is met  with  a homogeneous  capillary recruitment,  the  microvascular  associated w i t h sepsis lead to a heterogeneous capillary distribution (74) (57).  changes While  some argue that microvascular flow distribution and tissue oxygen extraction may be i m p r o v e d by fluid resuscitation (125), others argue that fluid resuscitation lead  to interstitial  microvascular Considering  and  flow the  endothelial  and  multiple  edema  lead to a higher effects  of  (136) w h i c h degree of  sepsis and  fluid  may  further  capillary  may  impair  heterogeneity.  resuscitation  on  the  intravascular volume, on cardiac function, on the microvascular distribution of oxygen delivery and on tissue oxygen demand, fluid resuscitation i n sepsis is a complex intervention.  O u r goal was to determine whether fluid resuscitation i n  sepsis impairs tissue oxygen extraction, possibly due to interstitial edema and altered microvascular flow distribution (126) (78). The splanchnic circulation has been thought to be particularly susceptible to ischemic injury (52) (86) (90).  This susceptibility has been attributed to specific  dysregulation of organ perfusion (90), to increased overall oxygen requirements by the metabolically active splanchnic organs (86), and to the countercurrent flow i n the intestinal v i l l i whereby there is shunting of oxygen away from the tips of the villi  (55) (87) (89).  Furthermore,  it has been proposed  that ischemia  of  the  splanchnic circulation may predispose to decreased mucosal integrity and lead to bacterial  translocation  w h i c h may  then  66  lead to multiple  organ  dysfunction  syndrome (166) (52).  Thus, we were particularly interested i n intestinal oxygen  extraction and the splanchnic circulation. Most describe sepsis as characterized by an increased capillary permeability (136) such that tissues are more prone to develop interstitial edema (136) (54). T o further determine whether interstitial edema occurs i n tissue i n endotoxemia, we determined the volume of interstitial space by morphometric techniques. Some investigators have suggested that microvascular flow may be i m p r o v e d w i t h hemodilution by reducing hematocrits (111) (125) leading to a decrease i n blood viscosity (125) thereby increasing blood velocity and favoring entry into channels w i t h smaller diameters (126). However, h e m o d i l u t i o n also may lead to a decrease in the oxygen carrying capacity of blood w i t h a resulting decrease i n oxygen delivery at a microvascular level.  Further, since hematocrits have only been determined at  a large vessel level, this may not reflect the effects of h e m o d i l u t i o n at a tissue l e v e l (55). Therefore, to further elucidate the possible effects of fluid resuscitation, we sought to determine the capillary hematocrits by morphometric analysis. Accordingly, we sought to determine whether crystalloid resuscitation i n a porcine model of septic shock w o u l d impair oxygen extraction capacity of the w h o l e body and specifically of the splanchnic bed. To better understand the mechanism of this effect we measured the dispersion of blood flow transit times i n the gut, the volume fraction of interstitium, and the gut capillary hematocrit.  67  5.2  METHODS  5.2.1  Experimental Design and Protocol The experimental design was similar to that performed previously i n o u r  laboratory (74). Sample size was calculated by analyzing the results of a p r e l i m i n a r y study using a sample size of 8 animals per group i n endotoxic and control groups. To determine  a difference i n critical D O 2 of 4 m l 0 2 / k g . m i n using a standard  deviation of 2 m l 0 2 / k g . m i n , required four groups  of eight animals, for an alpha =  0.05 and beta = 0.10 ("N" Calculator for Macintosh, v 0.9, Compuserve #70721,3243). Therefore, a size of 8 animals per group were used as a reasonable estimate of required sample size. D u r i n g instrumentation  (see below), surgical preparation, and stabilization,  all animals received 0.9% S o d i u m Chloride solution (Baxter, Toronto, O N ) infused via the left external jugular catheter at 25 ml'kg~l*hr~l. F o l l o w i n g this, the a n i m a l s were randomized to one of four groups:  Control / F l u i d (n=8), C o n t r o l / N o fluid  (n=8), E n d o t o x i n / F l u i d (n=8), and E n d o t o x i n / N o - f l u i d (n=8) groups.  Endotoxin  groups received E . coli endotoxin 50 | i g / k g (0111:B4, Sigma, St. Louis, M O ) i n 60 m l n o r m a l saline over 30 minutes immediately after the baseline data set.  Control  groups received an infusion of 60 m l normal saline without endotoxin.  Fluid  groups received an infusion of normal saline at 48 ml*kg~l*hr~l from the baseline measurement set until the end of the experiment. any further  saline infusion.  N o - f l u i d groups d i d not receive  F o l l o w i n g randomization to respective  groups, progressive hemorrhage  was undertaken  at 3 m l per minute  treatment using a  constant withdrawal p u m p from the left carotid catheter u n t i l the animal died. 68  Prior to death, w h e n whole body oxygen consumption had fallen by 25%, suggesting that oxygen delivery was inadequate to meet demand, we infused radiolabeled red blood cells, injected radiolabeled microspheres (see below), and rapidly excised the previously prepared segment of jejunum (Figure 22).  5.2.2  Surgical Preparation and Instrumentation This study was approved by the A n i m a l Care Committee of the University of  British C o l u m b i a and conforms to N a t i o n a l Institute of Health (NIH) standards for animal experimentation.  Thirty-two pigs, weighing 25.7 ± 2.8 kg, were  fasted  overnight and then sedated w i t h 0.5 m g / k g m i d a z o l a m i.m. (Hofman la Roche, Mississauga, O N ) .  Thirty minutes  later the  animals  were anesthetized  using  ketamine 500 m g i.m. ( M T C Pharm, Cambridge, O N ) followed by thiopentol 125 to 250 m g i.v. (Abbott, Montreal, PQ) titrated to effect. throughout  Anesthesia was m a i n t a i n e d  the experiment using ketamine 5 ml*kg"l*hr"l i.v. infusion and 0.5%  inspired isoflurane (Anaquest, Mississauga, O N ) . To avoid changes i n whole body oxygen demand, skeletal muscle  relaxation was maintained  with  intravenous  pancuronium bromide infusion (Organon, Scarborough, O N ) at 6 m g / h r , titrated to effect. A tracheostomy was performed and an 8.0 m m endotracheal tube (Portex, Wilmington,  M A ) was inserted  experimentation  and  secured.  D u r i n g instrumentation  the animals" were mechanically ventilated  and  (Harvard Apparatus  dual phase control respirator pump, model 613, M i l l s , M A ) w i t h 30% oxygen. A l o w compliance catheter was inserted into the right carotid artery for arterial pressure  69  Endotoxi n or Sham Infusion Surgical Prep.  Stablizel  1 hr4  Orrin [  WB Ischemia (Gut harvest) Hemorrhage  i  3cc/rrin  t  TREATMENT GROUPS: NonRuid resuscitated: Occ/kg.hr (n=8)  ^p1:  25cc/kg.hr  Qp2:  25ccVkg.hr (s)  Ruid resuscitated: 48cc/kg.hr (n=8)  ^pa  25^hr(E)  NonRuid resuscitated: 0cc/kg.hr (n=8)  O p 4-  25cdkg.hr (£)  (s)  Ruid resuscitated: 48cc/kg.hr  (n=8)  \^ : y : •  Figure 22.  Protocol Timeline.  This figure illustrates the protocol for all four groups of animals. endotoxin infusion.  70  S = saline sham infusion, E =  measurement and arterial blood sampling (Figure 23). A catheter was inserted into the left external jugular vein for saline infusion and administration of medications. A pulmonary artery catheter (Criticath model DSP5105H, Ohmeda M e d i c a l Devices, Oxnard, C A ) was placed via the right external jugular vein for measurement of right atrial pressure, pulmonary artery occlusion pressure, sampling, and for cardiac output  measurement  for mixed venous  blood  i n triplicate ( T h e r m o d i l u t i o n  Cardiac Output monitor Edwards M o d e l 9250, Baxter Health Care, Irvine, C A ) . A catheter was inserted into the left carotid artery for hemorrhage. A n anterolateral  thoracotomy was performed through the fifth  or sixth  intercostal space on the left. The pericardium was entered and a catheter inserted and secured i n the left atrial appendage. This catheter was used for injection of radiolabelled red blood cells and radioactive microspheres for gut blood volume and flow measurements, respectively. Through a m i d l i n e laparotomy, the pancreaticoduodenal v e i n at the second part of the duodenum and the superior rectal vein at the promontory of the sacrum were tied off. F o l l o w i n g this, the splenic artery and vein were tied off to prevent autotransfusion  and a catheter to sample portal vein blood was inserted v i a the  splenic v e i n stump.  Ligation of these vessels ensured that the  gastrointestinal  circulation was isolated such that venous drainage passed through the portal v e i n . Portal venous flow was measured by placement of a 1.5 cm ultrasonic flow probe connected to a Transonic T201 Ultrasonic Blood F l o w Meter (Transonic Inc., Ithaca, N Y ) around the portal vein. the gastric secretions.  A n orogastric tube was inserted to allow drainage of  A tonometric catheter (Tonometries, Inc., 373 Plantation  71  72  Street, Worcester, MA) was inserted into the lumen of the ileum 30 cm proximal to the ileocecal valve through a small wound in the antimesenteric border and held i n place with a suture.  Thirty cm distal to the duodenal-jejunal junction a 60 cm  length of jejunum was isolated for later resection for morphometric Umbilical tapes were brought around the mesentery  analysis.  to allow ligation of the  vasculature following injection of radiolabelled microspheres (see below). mesentery was otherwise not disrupted.  The  The abdomen was loosely closed to  facilitate later removal of the jejunal segment.  5.2.3  Hemodynamic Measurements and Calculations We measured arterial, mixed venous, and portal vein p H , Pc0 ' 2  (ABL30, Radiometer, Copenhagen, Helsinki), C»2 content Instrumentation  (IL 482  a n <  ^ ^0  2  co-oximeter,  Laboratories, Lexington, MA) and lactate concentration (YSI  2300STAT lactate analyzer, Yellow Springs Instruments, Yellow Springs, OH) at baseline and every 20 minutes during progressive hemorrhage.  Heart rate, mean  arterial pressure, pulmonary artery pressure, central venous pressure, pulmonary capillary occlusion pressure, cardiac output, portal vein flows, and whole body oxygen consumption using a metabolic cart (mean of 5 measures at 1 minute intervals, MBM-1000, Deltatrac, Helsinki, Finland) were also recorded at 20 minute intervals. A complete blood count (Coulter counter, Coulter, Miami Lakes, FL) and tonometer measurements was measured every 40 minutes (see below). Blood oxygen content was calculated as hemoglobin x 1.39 x blood oxygen saturation + 0.003 x PO2. Whole body oxygen delivery (WB-DO2)  73  w  a  s  calculated as  cardiac output m u l t i p l i e d by arterial oxygen content.  Gut oxygen c o n s u m p t i o n  (Gut-VC>2) was calculated as portal vein flow m u l t i p l i e d by the difference arterial and portal venous  oxygen content.  between  Gut oxygen delivery (Gut-DC^) was  calculated as portal v e i n flow m u l t i p l i e d by arterial oxygen content.  The oxygen  extraction ratio for both whole body (WB-ER) and gut (Gut-ER) was calculated as oxygen consumption  d i v i d e d by oxygen delivery.  F r o m the  multiple  oxygen  delivery - oxygen consumption points obtained during progressive hemorrhage the whole body and gut critical oxygen extraction ratio was determined using Samsel and Schumacker's dual-line regression analysis (167).  5.2.4  Transit times Gut blood flow and blood volume were determined i n the isolated segments  of gut after the lumenal contents were removed and after the tissue had been fixed for 24 hours i n 6% phosphate buffered gluteraldehyde (163). Technetium  (99mj ) c  labelled red blood cells were injected  Prior to r e m o v a l , into the  appendage and allowed to distribute i n the vascular compartment  left atrial  for 10 minutes.  15 | i m microspheres labelled w i t h Strontium (85Sr) were then injected rapidly (10 seconds) into the left atrium.  A t the time of microsphere injection, blood was  w i t h d r a w n from the left common carotid artery at 10 m l / m i n u t e for 2 minutes into weighed vials for  later  radiation  counting.  Next, the  arterial  and  venous  vasculature of the gut segment were simultaneously cross clamped and the segment of gut was rapidly excised and immersed i n 6% phosphate buffered  gluteraldehyde.  The 60 cm segments of gut were divided into 30-two cm pieces and each placed into  74  preweighed vials containing 6% phosphate buffered gluteraldehyde. Each segment was weighed and counted using a Beckman 8000 gamma counter for 3 m i n u t e s (164). The blood v o l u m e of each 2 cm piece of gut was calculated as counts per minute of each piece divided by counts per minute per m l of the reference blood sample. Blood flow to each 2 cm piece of gut was calculated as microsphere counts per minute from that piece of gut divided by microsphere counts per minute per m 1 of the reference arterial withdrawal sample. Average transit time for each piece of gut was calculated as blood volume divided by blood flow giving units of time (163). F o l l o w i n g calculation of i n d i v i d u a l transit times, a distribution of transit times for all of the pieces of gut taken together was then determined for each gut segment.  5.2.5  Interstitial Volume Jejunum from 5 animals i n each group was randomly selected (Figure 24).  F r o m each of these animals, six of the thirty 2 cm sections of jejunum  were  randomly selected and the tissue was embedded i n glycol methacrylate, sectioned at 2 jxm and stained using methenamine  silver.  Slides were coded so that the  morphometric analysis was blinded. The fraction of bowel w a l l that was interstitial space was quantitated at 400X using an automated image analysis system (Infrascan, R i c h m o n d B.C.) for 5 random fields on each slide.  O n each slide an area of  interstitial space was identified manually. The analysis program then identified the fractional area on the slide that had matching characteristics. In each case the area fraction of interstitial space identified by the analysis package was confirmed by v i s u a l inspection.  75  60 cm Jejunum  V  Figure 24.  V  V  V  V  V  Processing jejunal specimen for morphometric analysis.  Jejunum from 5 animals in each group was randomly selected. From each of these animals, six of the thirty 2 cm sections of jejunum were randomly selected. A portion of each piece selected was embedded in glycol methacrylate.  Interstitial volume was determined for various areas while  hematocrit was determined by analyzing 5 capillaries/block.  76  capillary  5.2.6  Capillary Hematocrit Jejunum from 5 animals i n each group was randomly selected. F r o m each of  these animals, six of the thirty 2 cm sections of jejunum were randomly selected and fixed overnight i n 2.5% gluteraldehyde i n 0.1M cacodylate buffer.  The tissue was  postfixed for 2 hours i n 1% osmium tetroxide, then stained en bloc for 1 hour i n 5% aqueous uranyl acetate and embedded i n Effapoxy resin. from  1 |im  toludine  blue-stained  section, were  T h i n sections, selected  cut on  a Reichert  Ultracut  ultramicrotome, mounted on 200 mesh copper grids, and stained w i t h lead citrate. Five capillaries (<10 Jim) per section (30 capillaries per jejunal photographed  segment) were  at 5000X magnification on a Zeiss 10CR transmission  electron  microscope. Capillary plasma and erythrocyte volume fractions were determined by point-counting using a 400 point grid. The capillary hematocrit was calculated as the quotient of erythrocyte volume fraction and total capillary volume fraction.  5.2.7  Ileal Tonometry The gastrointestinal tonometer consists of a saline-filled silicone balloon that  is placed i n the gut lumen. The silicone balloon is highly permeable to oxygen and carbon dioxide. N o r m a l saline (5.0 m l , at room temperature) was injected into the ileal tonometer  balloon and was left for 40 m i n . A t the end of the equilibration  period, the saline was removed under anaerobic conditions. The first milliliter of saline removed was discarded, as this was not i n direct contact w i t h the b a l l o o n (since the tonometer tube has a residual volume of one milliliter). The C O 2 content of the subsequent volume of saline removed, w h i c h reflects the mucosal CO2, was  77  measured  using the  A B L Blood  Gas Analyzer.  Simultaneously,  the  arterial  bicarbonate - assumed to be equal to the bicarbonate content of the gut mucosa - was measured. U s i n g a conversion table supplied by the manufacturer  (Tonometries  Inc.), measured PCO2 was transposed to a steady state PCO2 (PCO2SS), depending o n the exact duration of equilibration. Gut mucosal p H was calculated by substituting the PCO2SS and the simultaneously measured arterial bicarbonate i n the H e n d e r s o n Hasselbalch equation (ie: p H i = 6.1 + Log{(HC03)/PCC»2}). The use of tonometry to measure changes i n ileal intramucosal p H induced by endotoxin infusion i n pigs has previously been validated (148).  5.2.8  Statistical Analysis The relationship  between V O 2 and D O 2 for whole body and gut  was  determined for each animal by finding two best-fit linear regression lines (167) f r o m a plot of V O 2 and D O 2 . The critical oxygen extraction ratio, ERc, was determined at the point of intersection of the two lines (Figure 25).  The relationship  between  lactate and D O 2 for whole body and gut was determined for each animal by f i n d i n g two best-fit linear regression lines as above from a plot of lactate and D O 2 . T o determine  whether  endotoxemia,  intravascular  volume  we used a two-way A N O V A  endotoxin taking p < 0.05 as significant. moment  expansion  altered  ERc  during  testing for effect of v o l u m e  W e calculated the mean  and  (|i), second  (a ) and relative dispersion (o7|4,) of the distribution of gut blood flow 2  transit times using standard formulas (74). W e also used a 2 w a y A N O V A to test for  78  Critical Extraction Ratio  0.0 L 0.0  i  5.0  I  10.0  I  15.0  i  i  i  i  i  20.0  25.0  30.0  35.0  40.0  D0  2  (micymin.kg)  F i g u r e 25.  A n e x a m p l e o f O2 C o n s u m p t i o n a n d D e l i v e r y C u r v e .  Typical whole body oxygen consumption (VO2) and delivery (DO2) data points, measured during progressive hemorrhage, are illustrated. During progressive hemorrhage aerobic metabolism maintains oxygen consumption at approximately a constant value (7.60 ml C»2/min.kg) until a critical point is reached (10.00 ml 02/min.kg), after which oxygen consumption falls indicating anaerobic metabolism. Dual regression lines are fit to the data points. The intersection of the two lines identifies the critical oxygen extraction ratio (=7.60 / 10.00 = 0.76).  79  differences due to fluid resuscitation and endotoxin i n the relative dispersion of gut blood flow and i n the area fraction of interstitial space. A one-way A N O V A  was  used to test for effect of saline resuscitation on arterial and capillary hematocrit. W h e n a significant difference was found we used a sequentially rejective Bonferroni test procedure to identify i n d i v i d u a l differences.  Results are presented as mean ±  standard deviation throughout the text, tables, and figures.  5.3  RESULTS  5.3.1  Effect of Endotoxin Endotoxin infusion significantly reduced the critical oxygen extraction ratio i n  the whole body (Endotoxin 0.55 ± 0.08 versus Control 0.82 ± 0.06, p < 0.05) (Figure 26) (Table 1) and gut (Endotoxin 0.52 ± 0.05 versus Control 0.77 ± 0.07, p < 0.05) (Figure 27) (Table 1). Endotoxin infusion resulted i n an earlier onset of ischemia i n w h o l e body (Endotoxin 12.2 ± 3.8 versus Control 9.1 + 1.7 m l 0 2 / m i n . k g , p < 0.05) (Figure 28) (Table 1) but not i n gut (Endotoxin 24.3 ± 9.0 versus C o n t r o l 25.3 ± 14.8 m l 0 2 / m i n . k g , p > 0.05) (Figure 29) (Table 1). Relative dispersion of R B C flow transit times between segments of jejunum was not affected by endotoxin (Figure 30). T h i s measurement  of blood flow  distribution  is not  a measure  of the  distribution as it includes transit time through all jejunal w a l l vessels.  capillary Endotoxin  had no significant effect on the volume fraction of interstitium (p > 0.05) (Figure 31). Endotoxin d i d not affect arterial hematocrit (p > 0.05) or capillary hematocrit (p > 0.05) (Table 2).  Endotoxin significantly decreased the arterial and portal v e i n  leukocyte counts compared to controls at onset of ischemia (Endotoxin arterial 2.2 ±  80  0.5, portal 2.3 ± 0.8 w b c / m m versus Control arterial 25.0 ± 11.7, portal 25.2 ± 12.1 3  w b c / m m , p < 0.05) (Table 3). Endotoxin reduced the p H i as determined by 3  tonometry at onset of ischemia (Endotoxin 6.91 ± 0.15 versus Control 7.06 ± 0.10, p < 0.05) (Table 4). Endotoxin did not change the arterial lactate values compared to controls (p > 0.05) (Table 5). Endotoxin also had no effect on the onset of rise of lactate compared to controls (p > 0.05) (Table 6). Initial oxygen transport (Table 7) and hemodynamic (Table 8) variables did not differ between the Endotoxin and Control groups (p > 0.05).  At the critical  oxygen delivery the Endotoxin groups had lower mean arterial blood pressure (Endotoxin 44 ± 14 versus Control 59 ± 11 mmHg, p < 0.05), lower systemic vascular resistance (Endotoxin 516 + 376 versus Control 1651 + 408 dynes^sec'l'cm"^, p < 0.05), and higher pulmonary artery occlusion pressure (Endotoxin 7.7 ± 2.5 versus Control 4.3 + 1.8 mmHg, p < 0.05). In addition the Endotoxin groups survived for a shorter period of hemorrhage (Endotoxin 128 ± 43 versus Control 202 + 60 minutes, p < 0.05) (Table 9).  5.3.2  Effect of Fluid Resuscitation  Fluid resuscitation significantly reduced the critical oxygen extraction ratio i n the whole body (Fluid 0.62 ± 0.08 versus No-Fluid 0.82 ± 0.06, p < 0.05) ) (Figure 26) (Table 1) and gut (Fluid 0.67 ± 0.06 versus No-Fluid 0.77 ± 0.07, p < 0.05) ) (Figure 27) (Table 1). Fluid resuscitation did not alter the onset of ischemia in whole body (Figure 28) or gut (Figure 29) (p > 0.05) (Table 1). Fluid resuscitation significantly increased the relative dispersion of RBC flow transit times in both the control (Fluid  81  0.81 ±0.59 versus No-fluid 0.36 ± 0.11, p < 0.05) and the endotoxin groups (Fluid 0.60 ± 0.46 versus No-fluid 0.31 ± 0.04, p < 0.05) (Figure 30). Fluid resuscitation significantly increased the area fraction of interstitial space to the same extent in both the Endotoxin (Fluid 63 + 20% versus No-Fluid 50 ± 20%, p < 0.05) and Control groups (Fluid 70 ± 21% versus No-Fluid 45 ± 19%, p < 0.05) (Figure 31). Arterial hematocrit at the onset of ischemia decreased compared to baseline with fluid resuscitation in Control (Ischemic 16 ± 4% versus Baseline 22 ± 3%, p < 0.05) and Endotoxin (Ischemic 20 ± 4 versus Baseline 23 ± 3%, p < 0.05) groups (Table 2). However, capillary hematocrit at the onset of ischemia was unchanged from baseline by fluid resuscitation in Control and Endotoxemic animals (p > 0.05) (Table 2). Fluid resuscitation decreased the arterial and portal vein leukocyte counts in the control groups at onset of ischemia (Fluid arterial 14.9 + 10.5, portal 14.9 + 9.9 wbc/mm versus Non-fluid arterial 25.0 ± 11.7, portal 25.2 ± 12.1 wbc/mm , p < 0.05) 3  3  (Table 3). Fluid resuscitation did not alter the arterial or portal vein leukocyte counts in the endotoxemic groups at onset of ischemia (p > 0.05) (Table 3). Fluid had no effect on the pHi as determined by tonometry at onset of ischemia when compared to non-fluid groups (p > 0.05) (Table 4). Fluid did not change the arterial lactate values when compared to non-fluid groups (p > 0.05) (Table 5). Fluid also had no effect on the onset of rise of lactate compared to non-fluid groups (p > 0.05) (Table 6). The initial oxygen transport (Table 7) and hemodynamic (Table 8) variables did not differ between the Fluid and No-fluid groups (p > 0.05). At the critical oxygen delivery the Fluid groups had a higher mean arterial pressure (Fluid 77 ± 12  82  versus No-fluid 59 ± 11 mmHg, p < 0.05), higher cardiac output (Fluid 5.7 ± 2.7 versus No-fluid 2.8 ± 0.7 L/min, p < 0.05), lower systemic vascular resistance (Fluid 1100 ± 600 versus No-fluid 1650 ± 410 dynes*sec~l*cm~5, p < 0.05), higher pulmonary artery occlusion pressure (Fluid 6.9 ± 1.1 versus No-fluid 4.3 ± 1.8 mmHg), and a lower hemoglobin (55 ± 11 versus No-Fluid 83.6 ±13.0 g/1, p < 0.05). The fluid resuscitated groups had a greater time of survival after onset of hemorrhage (368 ± 105 minutes) compared to the non-fluid resuscitated group (202 + 60 minutes, p < 0.05) (Table 9).  83  1.00  p  0.90  .  0.80 • Whole Body " Extraction 0.60 • 0  R a t i o  7  •I  .  0  II  0.82 ± 0.06  0.50  0.62 ± 0.08  0.40 0.30 0.20 0.10 0.00  Non-Fluid  Fuid  ii  Si  0.55 ±0.08  0.48 ±0.04  Non-fluid  Fluid  Endotoxin  Control Groups  F i g u r e 26.  W h o l e B o d y A b i l i t y to E x t r a c t O x y g e n .  The whole body critical oxygen extraction ratio for all four groups is illustrated.  Fluid-resuscitation  reduced the critical oxygen extraction ratio in the Control groups but not significantly in the Endotoxin groups. Endotoxin reduced the critical oxygen extraction ratio ( A N O V A , p expressed as mean ± SD.  84  <0.001). Values are  1.00 0.90 0.80 Gut Extraction Ratio  0.70 0.60 0.50  :i i  0.77 ±0.07  0.30 0.20  *  0.67 ± 0.06  0.40  ;i  0.52 ±0.05 Non-fluid  0.10 0.00  Fluid  Non-fluid  0.52 ±0.09 Fluid  Endotoxin  Control Groups  Figure 27.  Gut Ability to Extract Oxygen.  The gut critical oxygen extraction ratio for all four groups is illustrated.  Like the whole body, fluid-  resuscitation reduced the gut critical oxygen extraction ratio in the Control groups but not in the Endotoxin groups. Endotoxin reduced the gut critical oxygen extraction ratio ( A N O V A , p Values are expressed as mean ± SD.  85  <0.001).  25  20 Whole Body Critical D 0 15 (ml0 /min.kg)  • •  12.2 ± 3 . 8  10.2 ± 2 . 2  2  2  15.3 ± 3 . 1  r  .9.1 ± 1 . 7  10  •  a  si Non-fluid  Fluid  Non-fluid  Fluid  Endotoxin  Control Groups  F i g u r e 28.  W h o l e B o d y Onset of Ischemia.  Critical DO2 is not altered by fluid resuscitation in the whole body in nonseptic or the septic groups. However the critical DO2 is higher in the pooled septic group compared to the pooled nonseptic group.  86  Gut Critical D 0 (mlO^min.kg)  2  50  r  45  -  40  -  35  -  30  -  25 20 15 10 5 0  • 25.3 + 14.8 24.3 ± 9.0 • 20.5±6.8  • 22.9 ± 11.7  •I"  ••• :l :I :• Non-fluid Fluid Control  Non-fluid Fluid Endotoxin Groups  F i g u r e 29.  G u t Onset of Ischemia.  Critical DO2 is not altered by fluid resuscitation in the gut in nonseptic or the septic groups.  87  1.60 0.81 ± 0 . 5 9 1.40  r  1.20  Relative dispersion  0.60 ± 0.46  1.00 0.80 0.60 0.40  0.36 ± 0 . 1 1  X  0.31 ± 0.04  0.20 0.00  Nonfluid  Fluid  Nonfluid  Control  Fluid Endotoxin  Groups  F i g u r e 30.  Relative dispersions o f gut b l o o d transit times.  The average relative dispersion of total gut blood flow transit times in thirty ~2g segments of jejunum is shown for the four experimental groups. Fluid resuscitation significantly increased relative dispersion (ANOVA, p < 0.05). Values are expressed as mean ± SD.  88  100 „  70±21%  90  i  80  % Interstitial Space 60 7  0  63±20%  50±20%  45±19%  50 40 30  3  20 10 0  N6nfluid  Fluid  Control  Figure 31.  Nonfluid Groups  'Fluid  Endotoxin  Interstitial Volume in Gut.  Area of interstitial space is illustrated for the four groups. Fluid resuscitation significantly increases the area of interstitial space (p > 0.05) however, endotoxin has no significant effect ( A N O V A , p > 0.05). Values are expressed as mean ± SD.  89  Table 1. Onset of Ischemia and Extraction Ratios CONTROL Non-Resuscitated Fluid-Resuscitated (n=8) (n=8) WB D 0 vs WB V 0 Onset of Ischemia - WB D0 c (ml0 /min.kg of WB wt) Critical V 0 - WB VO c (ml0 /min.kg of WB wt) 0 Extraction Ratio WB ERc (=WBV0 c/WBD0 c) 0 Extraction Ratio WB ERmax (0 ER at final measurement) GUT D 0 vs GUT V 0 Onset of Ischemia - GutD0 c (ml0 /min.kg of gut wt) Critical V O - Gut VO c (ml0 /min.kg of gut wt) 0 Extraction Ratio Gut ERc (=GutV0 c/GutD0 c) 0 Extraction Ratio Gut ERmax (0 ER at final measurement) WB D 0 vs GUT V 0 Onset of Ischemia - GutD0 c (ml0 /min.kg of gut wt) Critical V O - Gut VO c (ml0 /min.kg of gut wt) 2  ENDOTOXIN Non-Resuscitated Fluid-Resuscitated (n=8)  (n=8)  2  2  9.1 + 1 . 7  1 0 . 3 ± 2.2  1 2 . 2 ± 3.8+  1 5 . 3 + 3.2+  7.5 ± 1 . 3  6.3 ± 1.3  6 . 5 + 1.5  7.4 ± 1 . 2  0.82 + 0.06  0.62 ± 0 . 0 8 *  0.55 ± 0 . 0 8 t  0.49 ± 0.04+  0.86 ± 0 . 0 3  0.77 ± 0.08*  0.74 + 0.10  0.81 + 0 . 1 1 +  25.3 ± 14.8  2 0 . 5 ± 6.8  2 4 . 3 + 9.0  22.9 ± 11.7  19.6 ± 12.0  14.0 ± 5.6  12.9 + 6.0  11.8 ± 5.2  0.77 + 0.07  0.67 ± 0.06*  0.87 + 0.03  0.72 + 0 . 0 8 *  0.68 + 0.07  10.6 ± 4.4  1 3 . 6 ± 6.1  1 3 . 5 + 7.1  15.3 ± 5 . 9  16.7 ± 11.7  1 4 . 0 ± 5.1  10.2 ± 5.7  1 4 . 0 ± 6.2  2  2  z  2  2  2  2  2  2  2  2  2  2  z  z  2  2  2  0.52 ±  0.05t  0.52 + 0.09+  2  2  0.65 +  0.08t  2  2  2  2  2  z  z  2  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups t indicates difference between the control and endotoxin groups (p<0.05)  Arterial and Capillary Hematocrit  Arterial Hct(%) @ Arterial Hct(%) @ Capillary Hct(%) Baseline Onset of Ischemia Onset oMschemia CONTROL Non-Resus. Fluid-Resus. ENDOTOXIN Non-Resus. Fluid-Resus.  23.0 ± 3.2 22.0 ± 3.3  25.0 ± 3.5 16.1 ± 3.5*+  27.5 ± 8.6 29.2 ± 14.3  24.0 ± 3.5 22.6 ± 2.6  24.4 ± 4.2 19.8 ± 3.8**+  25.3 ± 7.7 25.3 ± 5.3  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between Arterial Hematocrit at Baseline and at Onset of Ischemia (p<0.05)  Table 3. Arterial and Portal Vein Leukocyte Counts  CONTROL Non-Resus. Fluid-Resus. ENDOTOXIN Non-Resus. Fluid-Resus.  Arterial WBC Baseline  Arterial WBC Critical  Portal WBC Baseline  Portal WBC Critical  13.0 ± 3.8 10.4 ± 7.4  25.0 ± 11.7* 14.9 + 10.5*¥  12.0 ± 4.2 8.8 ± 6.6  25.2 ± 12.1** 14.9 ± 9.9**¥  10.1 ± 3.6 10.5 ± 7.9  2.2 + 0.5*t 1.7 ± 0.9*+  9.0 ± 3.3 9.6 ± 7.3  2.3 + 0.8**+ 2.0 ± 1.4**+  WBC are expressed as leukocytes X 10 /L * indicates difference between Baseline and Critical Arterial WBC (p<0.05) ** indicates difference between Baseline and Critical Portal WBC (p<0.05) + indicates difference between control and endotoxin groups (p<0.05) ¥ indicates difference between Non-Resuscitated and Fluid-Resuscitated (p<0.05) 9  Table 4. Intestinal p H i at Baseline and Onset of Ischemia  CONTROL Non-Resus. Fluid-Resus. ENDOTOXIN Non-Resus. Fluid-Resus.  pHi at Baseline  pHi at Onset of Ischemia  7.25 ± 0.09 7.19 ± 0.09  7.06 ± 0.10* 7.07 + 0.11*  7.21 + 0.10 7.12 ± 0.26  6.91 ± 0.15*+ 6.90 + 0.24*+  * indicates difference between pHi at Baseline and Onset of Ischemia (p< 0.05) + indicates difference between p H i between control and endotoxin (p < 0.05)  Table 5. Arterial Lactate Values Art. Lactate CONTROL Non-Resus. Initial Critical Fluid-Resus. Initial Critical ENDOTOXIN Non-Resus. Initial Critical Fluid-Resus. Initial Critical  2.51 ± 1.06 3.94 ± 1.24 1.76 ± 0.70 2.41 ± 0.58*  1.93 ± 0.63 3.20 ± 0.97 1.53 + 0.50 3.07 ± 1.07  Lactate is expressed as mmol/L * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between the control and endotoxin groups (p<0.05)  Table 6. Onset of Rise of Arterial Lactate CONTROL Non-Resuscitated Fluid-Resuscitated (n=8) (n=8) WB D O vs Arterial Lactate Onset of Ischemia - WB D0 c (ml0 /min.kg of WB wt)  ENDOTOXIN Non-Resuscitated Fluid-Resuscitated (n=8) (n=8)  z  2  13.1 ± 3.3  12.0 ± 4.7  14.9 ± 5.7  12.3 ± 0.8  2  Ul  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between the control and endotoxin groups (p<0.05)  Table 7. Oxygen Transport Variables Pa0 (mmHg)  PaCO (mmHg)  Arterial pH  (g/D  ArtO content (g o2)  Temp (°C)  151.0 ± 20.1 134.6 ± 19.5  29.9 ± 2.9 32.1 ± 4.4  7.46 ± 0.05 7.40 ± 0.06  77.3 ± 10.0 83.6 ± 13.0  103.5 ± 12.3 109.7 ± 16.0  37.0 ± 0.8 38.4 ± 0.5  137.4 ± 31.9 109.4 ± 38.6  31.2 + 3.9 36.0 ± 11.0  7.41 ± 0.05 7.28 ± 0.08*  73.9 ± 9.8 55.0 ± 11.2*  98.3 ±11.9 68.6 +16.1*  36.9 ± 0.7 37.9 ± 0.8  159.0 ± 30.3 107.2 ± 35.8  31.2 + 6.1 35.3 ± 9.9  7.45 ± 0.05 7.30 ± 0.07  78.9 ± 11.1 83.4 ± 17.5  107.0 ± 14.5 105.2 ± 27.5  38.0 ± 1.2 38.7 ± 0.7  129.7 ± 36.7 74.3 ± 19.9**+  33.5 + 4.3 43.8 ± 5.8**  7.43 ± 0.05 79.1 + 13.5 102.91 ± 17.2 7.21 ± 0.08**+ 67.9 ± 10.1**+ 76.5 ± 15.6**  37.2 + 0.5 38.5 ± 0.5  2  CONTROL Non-Resus. Initial Critical Fluid-Resus. Initial Critical ENDOTOXIN Non-Resus. Initial Critical Fluid-Resus. Initial Critical  z  Art. Hgb  z  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between the control and endotoxin groups (p<0.05)  Table 8. Hemodynamic Variables  CONTROL Non-Resus. Initial Critical Fluid-Resus. Initial Critical ENDOTOXIN Non-Resus. Initial Critical Fluid-Resus. Initial Critical  Wedge Pressure (mmHg)  Mean Art.Pres. (mmHg)  Cardiac Output (L/min)  Sys.Vasc.Res. (dynes/sec/cm )  Portal Flow (L/min)  6.17 ± 2.36 4.32 ± 1.78  77.2 ± 13.8 59.2 ± 10.6  7.79 ± 1.93 2.76 ± 0.68  784.30 ± 256.95 1651.76 ± 407.98  0.75 ± 0.17 0.32 + 0.21  7.94 ± 1.12 6.89 ± 1.10*  83.3 ± 10.4 76.7 ± 11.6*  8.23 ± 3.60 5.74 ± 2.67*  882.37 ± 407.10 1183.38 ± 604.19*  0.86 ± 0.22 0.66 ± 0.32*  8.19 ± 2.37 7.71 ± 2.45+  75.7 ± 19.1 43.5 ± 13.9+  6.65 ± 2.45 3.64 ± 1.07  833.16 ± 483.85 515.90 ± 375.47+  0.70 ± 0.24 0.32 ± 0.14  9.13 ±1.74 9.49 + 1.91+  83.5 ± 13.9 49.3 ± 9.6+  6.78 + 1.93 5.90 ± 2.11**  920.24 ± 412.09 380.14 ± 415.89+  0.73 ± 0.26 0.53 ± 0.35  5  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between the control and endotoxin groups (p<0.05)  Table 9. Clinical Variables  CONTROL Non-Resuscitated Fluid-Resuscitated ENDOTOXIN Non-Resuscitated Fluid-Resuscitated 0 0  Animal wts (kg)  Total fluid (cc/kg)  Total urine (cc/hr)  Time of survival (min)  25.4 ± 1.8 27.6 ± 3.9  69 ± 23 353 ± 80*  59 ± 3 0 267 ± 160*  202 + 60 368 +105*  24.3 + 1.5 25.8 ± 2.9  89 ± 18 161 ± 61**+  145 + 93 178 ± 115t  128 ± 43+ 171 ± 81+  * indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in control groups ** indicates difference between Non-fluid and Fluid-resuscitated groups (p<0.05) in endotoxin groups + indicates difference between the control and endotoxin groups (p<0.05)  5.4  DISCUSSION The major  finding from our study is that fluid resuscitation significantly  impairs whole body O 2 extraction i n control pigs but d i d not significantly change the already decreased O 2 extraction i n the endotoxin treated animals. Impairment of O 2 extraction is associated w i t h increased interstitial edema and increased heterogeneity of transit times but not to altered capillary hematocrit. Sepsis has substantial effects on the microcirculation that may account for impaired tissue oxygen extraction (74) (126).  The host defense response against  invading microorganisms can cause the development of the systemic i n f l a m m a t o r y response syndrome (SIRS) (6).  Endotoxin and other bacterial products  trigger  macrophages and other inflammatory cells to release TNF-oc, IL-1, IL-6 and m a n y other pro-inflammatory mediators (166) (6). These endogenous mediators of the septic inflammatory  response have important  effects on the  microvasculature  including leukocyte slowing and microthrombi (168) (52) (4i) that may lead to decreased capillary flows, increased capillary permeability - that is, "leaky capillaries" (52) (12) w i t h the resultant interstitial edema (136) and, an alteration i n vascular tone (76) (74) (169). Altered microcirculatory regulation of blood flow may lead to mismatching of oxygen supply to demand resulting i n impaired tissue oxygen extraction (78). L a m et al. demonstrate that after cecal ligation and puncture i n rats, there was a 36% reduction i n perfused capillary density w i t h an increase i n heterogeneity spatial distribution of perfused capillaries (57).  of the  These results are similar to the  observations by H u m e r et al. that the relative dispersion of gut capillary blood flow  99  increases after endotoxin infusion i n pigs and this was associated w i t h i m p a i r e d oxygen extraction (74).  Some time ago, H o n i g and Odoroff suggested that the  dispersion of capillary transit times w o u l d have an impact on oxygen exchange by the capillary bed (170). A subsequent theoretical analysis suggested that an increased dispersion of capillary transit times w o u l d impair oxygen exchange by increasing mismatch between oxygen supply and demand w i t h i n small regions of a capillary bed (78).  Conversely, Morff has suggested that increased capillary recruitment  improves tissue oxygenation i n rat cremaster muscle (171). A number of investigators have suggested that to improve microvascular flow, reduced hematocrits may be useful (111) (125). H e m o d i l u t i o n decreases blood viscosity, may improve capillary red blood cell flux (125), and may improve blood flow distribution w i t h i n the capillary bed (125) (126). V a n der L i n d e n et al found that h e m o d i l u t i o n to a hematocrit of 20 or 30% using colloid infusion resulting i n an  increased  critical oxygen extraction ratio during  compared to a hematocrit  of 40% (125).  heterogeneity  of microvascular  hemodilution  (126).  flow  in  Tyml rat  However, while some  has  skeletal  progressive  hemorrhage  demonstrated muscle  argue that  is  that  reduced  microvascular  the by flow  distribution and tissue oxygen extraction may be i m p r o v e d by fluid resuscitation, others argue that fluid resuscitation may lead to interstitial and endothelial edema (136) w h i c h may further impair microvascular flow and lead to a higher degree of capillary heterogeneity.  Therefore,  our hypothesis was that not only  would  endotoxemia increase capillary heterogeneity, but that fluid resuscitation may also increase capillary heterogeneity and thereby impair oxygen extraction.  100  A s per the hypothesis, the author found that fluid resuscitation resulted i n impaired O 2 extraction associated increased heterogeneity  of transit times. I n  addition, fluid resuscitation resulted i n increased interstitial edema.  Therefore, a  possible mechanism for impaired O 2 extraction associated w i t h fluid resuscitation is increased heterogeneity of transit times possibly resulting from decreased capillary recruitment secondary to altered morphology of the capillary bed due to interstitial edema. Since sepsis is characterized by a generalized increase i n capillary permeability (52) (12), tissues are predisposed to development of interstitial edema (136). Since crystalloid resuscitation is k n o w n to redistribute according to Starling's forces, one w o u l d anticipate that interstitial edema w o u l d be more prone to occur i n sepsis. Further, an increased O 2 diffusion distance associated w i t h interstitial edema could by itself result i n decreased interstitial O 2 tension (56), but whether O 2 uptake is affected by interstitial edema is unsupported i n studies by others (172). A key new observation i n the current  study is that fluid  resuscitation  significantly increases the relative dispersion of blood flow transit times throughout the gut w a l l . This may indicate an increased relative dispersion of blood flow transit times i n the capillary bed as w e l l as w i t h i n the larger arterioles and venules w i t h i n the gut w a l l .  Conceivably, the resultant  decrease i n capillary diameter due to  endothelial edema may impair microvascular blood flow or contribute to leukocyte retention and plugging w i t h i n the capillary bed (164). In addition, red blood cell rheology may be altered i n the septic state (49) and conceivably could be altered by fluid resuscitation resulting i n more heterogeneous microvascular flow.  101  As a  result, impaired oxygen extraction may be accounted for by mismatching of oxygen supply to demand.  Thus, the potential detrimental effects of fluid resuscitation  appear to contribute to overall impaired oxygen extraction i n the current set of experiments. In our previous study (74) impaired O 2 extraction i n endotoxin pigs was associated w i t h increased heterogeneity of transit times. In that study endotoxin pigs also received fluid resuscitation whereas controls d i d not.  Similarly, i n this study  impaired O 2 extraction associated w i t h increased heterogeneity of transit times was observed i n endotoxic pigs w h i c h received fluid resuscitation. But impaired O 2 extraction i n endotoxic pigs was not associated w i t h increased heterogeneity  of  transit times without fluid resuscitation. Therefore, increased heterogeneity  of  transit times is not the sole mechanism of impaired O 2 extraction i n endotoxemia. Similarly, interstitial edema was increased i n association w i t h fluid resuscitation rather than w i t h endotoxemia. Therefore, while interstitial edema w i t h increased heterogeneity of transit times could result i n impaired O 2 extraction from f l u i d resuscitation this is not sufficient to account for impaired O 2 extraction f r o m endotoxemia. One of the side-effects of massive saline infusion is the development  of a  metabolic acidosis (137). Acidosis is k n o w n to alter the oxygen dissociation curve by shifting the curve to the right.  This w o u l d lead to a higher P50 and therefore  reduced affinity of hemoglobin for oxygen;  a  thus hemoglobin releases oxygen at  higher P O 2 , w h i c h w o u l d tend to raise tissue P O 2 . Therefore, w h i l e saline i n f u s i o n  102  may have shifted the dissociation curve, it w o u l d have done so i n favor of improved  tissue  oxygenation. This  would  not  explain  the  impaired oxygen  extraction that was found. Capillary transit times are more closely related to O2 extraction than transit times i n the whole gut wall. Capillary transit times was not measured i n this study but i n a study by H u m e r et al (74), capillary transit times accounted for about onehalf of whole gut w a l l transit times. In that study altered heterogeneity of capillary transit times associated w i t h endotoxemia and fluid resuscitation was paralleled by altered heterogeneity i n whole gut w a l l transit times. W h i l e  it is possible that  increased heterogeneity of capillary transit times w i t h unchanged heterogeneity of whole gut w a l l transit times could occur i n endotoxic pigs w h i c h d i d not receive fluid resuscitation, one does not have basis for suggesting that this possibility could be a mechanism of impaired O2 extraction i n endotoxic non-fluid  resuscitated  animals. Interestingly, the author notes that although arterial hematocrit decreased as anticipated w i t h saline resuscitation, microvascular hematocrit determined u s i n g morphometric measurements  was not altered by saline resuscitation.  Thus, the  putative beneficial effect of crystalloid resuscitation on blood viscosity may not extend to the microvasculature. Furthermore, h e m o d i l u t i o n does not explain the decreased oxygen extraction seen w i t h fluid resuscitation. H o w e v e r , it is i m p o r t a n t to point out that the technique of determining capillary hematocrits only i n c l u d e d those vessels that had erythrocytes present. A s mentioned, there is heterogeneity of blood flow w h i c h implies that there w i l l be some vessels w h i c h were not perfused at  103  all and therefore, these w o u l d not have been counted as part of the m o r p h o m e t r i c analysis. The leukocyte counts i n the endotoxemic groups showed a dramatic decrease compared to controls. Since the effect was seen i n not only the arterial but also the portal vein samples, one w o u l d anticipate not only systemic but splanchnic trapping of leukocytes.  Indeed Barroso-Aranda et al (168) showed that endotoxemia  is  associated w i t h activation of polymorphonuclear leukocytes such that it results i n microvascular  trapping.  They further  have  shown  that  obstruction  occurs  exclusively at the capillary level without involvement of arterioles or venules and suggest that leukocyte capillary plugging may be an important contributor i n the fatal outcome  after endotoxin administration.  Indeed capillary plugging may  explain the heterogeneity i n capillary blood flow described by some  investigators  (168) (74) (57) w h i c h is thought to lead to the impaired oxygen extraction by tissue (78). Tonometry has been used by many as an indicator of mucosal ischemia (148) (150) (151) (152). O u r results show that tonometry can be used to indicate changes i n mucosal acidosis and showed that endotoxemic animals have a larger degree of mucosal acidosis at onset of ischemia. Furthermore, some have used tonometry to indicate onset of ischemia (154) (155). W h i l e the author attempted to perform d u a l line regression between p H i values and oxygen delivery, there were not enough p H i data points to obtain useful numbers to determine onset of ischemia. W h i l e m o r e data points w o u l d have been ideal, the short period of time of the entire experiment w o u l d have required a shortening of the time interval w h i c h w o u l d perhaps not  104  allow enough time to adequately equilibrate the gases w i t h the saline i n  the  tonometric balloon. W h i l e many investigators use lactate as a marker of onset of ischemia (74) (106), others consider lactate is a difficult value to interpret (55). W h i l e the arterial lactate levels are higher i n at onset of ischemia, there appears to be too m u c h variability to make these differences significant. Further, our values show that rise i n lactate as determined by a dual line regression w i t h whole body oxygen delivery corresponds to the onset of ischemia as determined w i t h a dual line regression between whole body oxygen delivery and consumption.  The only group not to  correspond was that of the control non-fluid group. The reasons for this difference are not clear. Possible reasons include an aerobic source of lactate production, early anaerobic production by some tissues, or an inability to metabolize lactate i n this group (156). While  current  recommendations  include early  and  aggressive  use  of  crystalloids i n sepsis, the data suggests that saline resuscitation does not improve the oxygen extraction defect seen i n sepsis. increased interstitial edema  Indeed, the saline was s h o w n to lead to  and a maldistribution of blood flow w h i c h  may  contribute to the oxygen extraction defect. Therefore, the author questions the use of crystalloid resuscitation for treatment of sepsis i n humans.  105  Chapter 6 CONCLUSIONS  A review of the current literature on the microvascular changes associated w i t h sepsis and endotoxemia lead us to our hypothesis that crystalloid resuscitation w o u l d impair the ability of tissue to extract oxygen. significant  impairment  W e found that there is a  i n oxygen extraction capabilities w i t h the  addition of  crystalloid resuscitation w h i c h w o u l d therefore support our hypothesis.  Potential  reasons for this impairment were investigated. First, we studied the potential role of the described increase i n capillary permeability leading to interstitial edema. W e found an increase i n interstitial volume by use of morphometric analysis.  This  w o u l d support the theory that interstitial edema may be a reason for the i m p a i r e d ability to extract oxygen.  Further, alterations i n capillary distribution has been  theorized as a potential reason for decreased oxygen extraction i n shock states. W e found there was an increase i n blood flow heterogeneity by use of microspheres as markers of blood flow. This effect was seen not only i n endotoxemia but w i t h f l u i d resuscitation. Therefore, an alteration i n the capillary flow either by interstitial fluid hindering capillary flow or by fluid impairing endothelial control may be possible. Finally, some reports suggest that fluid resuscitation may lead to h e m o d i l u t i o n at a capillary level w h i c h may thereby decrease oxygen delivery to tissue beds.  We  found that w h i l e arterial hematocrit is significantly reduced w i t h saline loading, capillary hematocrit was not altered compared to baseline arterial values. Therefore, an alteration i n capillary hematocrit w o u l d not explain the  106  decreased  oxygen  extraction found for the gut and whole body.  Thus, while aggressive and early  saline resuscitation is considered by many to be a fundamental  therapeutic  maneuver in sepsis, our study has shown that this therapy leads to significant impairment in oxygen extraction and thereby may prove detrimental.  107  Chapter 7 SUMMARY: CLINICAL RELEVANCE, FUTURE DIRECTIONS  The ideal therapy for sepsis has still to be defined. There are numerous levels at w h i c h therapy may be initiated as was described earlier. W e sought to determine the effects of saline resuscitation i n our model and found a significant i m p a i r m e n t i n oxygen extraction.  W e based our findings on the likely development  interstitial edema as w e l l as an impairment leading to capillary heterogeneity.  of  i n the microvascular distribution  We w o u l d anticipate that if these findings were  to be similar to what one sees clinically, that saline resuscitation may worsen a n already critical situation. There are several areas of possible further study.  Areas  w o u l d include further studies i n the current project, logical follow-up studies, and finally likely future endeavors w h i c h may prove beneficial i n the therapy of sepsis. First, there may be other areas of investigations i n the current study w h i c h may shed further light into the mechanisms of impaired oxygen extraction.  While  we have shown interstitial edema and heterogeneity of total gut blood flow, there are other aspects w h i c h have not yet been studied.  W h i l e we have looked at the  total gut blood flow transit times, we have not yet subdivided these into large, m e d i u m , and small vessel as per H u m e r et al (74).  This may give  information as to w h i c h vessels are contributing to the heterogeneity. while we have mechanisms  demonstrated  heterogeneity  i n transit  further  Furthermore,  times, there are  for impaired oxygen extraction w h i c h might be investigated.  other As  described earlier, leukocytes have been shown, to cause capillary obstruction d u r i n g  108  sepsis  (65) (41) (66) w h i c h  may contribute  to  vascular  obstruction.  Using  morphometric techniques, one may attempt to quantify the number of leukocytes present i n capillaries to determine whether this is a contributing factor i n the impaired oxygen extraction i n endotoxin and saline resuscitated animals.  Further,  not only has development of capillary heterogeneity been shown to decrease oxygen extraction, but an alteration i n the capillary density may also be involved (57). A l s o w i t h morphometric  techniques,  the number  of capillaries may be quantified.  Another technique involves the use of intravital microscopy as has been used to study the microvasculature by others (126) (173). One w o u l d expect that increased interstitial edema w o u l d lead to decreased number of open capillaries and therefore a decreased capillary density (34). Finally, some investigators have theorized that the inability to utilize oxygen may be an important factor i n the oxygen extraction problem  seen  in  sepsis.  Mechanisms  include  uncoupling  oxidative  phosphorylation (14) (15), inhibition of mitochondrial respiration (16) (17) (18) (17) and  mitochondrial  destruction  (54).  One  may  look  at  the  possibility of  mitochondrial destruction as a contributing factor to impaired oxygen extraction. Hersch et al (54) utilized electron microscopy to analyze mitochondrial features and describe significant mitochondrial destruction i n a sheep model of sepsis. W h i l e a l l these studies w o u l d add further information to our current study, they go n o further i n suggesting what other therapies may be more successful i n i m p r o v i n g oxygen extraction i n sepsis. Follow-up studies based on our current investigation includes using the same model w i t h different types of fluid resuscitation. The aim w o u l d be to preserve the  109  beneficial effects of fluid resuscitation but to avoid the side-effects demonstrated i n our current study. Possible fluid include other crystalloids and the colloids. A s for crystalloids, two other common fluids used include Ringers Lactate and hypertonic saline. W i t h respects to Ringers Lactate (RL), advantages of its use w o u l d include less development of hyperchloremic acidosis i n view of the decreased concentration of sodium chloride as w e l l as the buffering action of sodium lactate. However, since lactate requires conversion by the liver to pyruvate and then to bicarbonate to act as a buffer, there may be concern during sepsis where the liver has been shown to have decreased function that lactate may accumulate (174). The other potential crystalloid to study w o u l d be hypertonic saline. Because s o d i u m is primarily an extracellular ion, infusion of hypertonic saline w o u l d be expected to expand the extracellular fluid  (ECF) space by a greater  amount  than  the  volume  infused,  because  theoretically, water should enter the E C F from the intracellular fluid (128).  Further,  there may be potential positive inotropic effects as w e l l as systemic and p u l m o n a r y vasodilation (128). Horton and Walker (175) have studied hypertonic saline (HS) i n a canine model of endotoxemia. reduces the total volume  They have shown that the addition of H S to R L  of R L required to maintain  hemodynamic  Further, the net fluid gain was five times less than w i t h R L alone.  variables.  This w o u l d  potentially limit the degree of interstitial edema that accompanies most crystalloid fluid resuscitation as occurred i n our study. Despite the increased chloride content, it has not been shown to lead to hyperchloremic acidosis as does normal  saline  (174). However, if H S is rapidly infused, it may precipitate pontine m y e l i n o l y s i s (174) (128).  While  other crystalloids may be a potential area of investigation,  110  another area w i t h great potential is the use of colloids. Sepsis has been characterized by a diffuse increase i n microvascular permeability (46) (52) (12). Thus, the use of colloids w h i c h have been shown to stay i n the intravascular space for longer durations (118) w o u l d seem obvious. A study i n v o l v i n g i n v i v o microscopy and surface oxygen partial pressure electrodes by Funk et al (173) was performed  to  compare crystalloids to colloids. They showed that colloids had i m p r o v e d capillary perfusion and tissue oxygenation compared to crystalloid resuscitation.  Further,  sepsis also is accompanied by a reprioritization of hepatic protein synthesis w i t h decreased a l b u m i n production w h i c h leads to a decreased colloid oncotic pressure (130). This w o u l d , according to Starling's forces, add to the leak of fluid out of capillaries leading to pulmonary edema (176) (177) (178) (179) and interstitial edema (180) (178). M o r i s a k i et al (181) showed that w h e n compared to crystalloid, colloid therapy decreases the progression of extrapulmonary tissue injury i n septic sheep and based this on the preservation of microvascular surface area for tissue O 2 exchange. The different types of colloids include albumin and hydroxyethyl starch. Some important properties of albumin include antioxidant activity, b i n d i n g free fatty acids and endotoxin (182) (128). Because heat treating a l b u m i n at 60°C for 10 hours inactivates hepatitis virus and other infectious agents, there is no concern regarding infections w i t h this colloid (118). Adverse reactions to a l b u m i n are rare (137). Intravenous  administered albumin distributes initially to the intravascular  space, but gradually redistributes to the interstitial space. Its intravascular half-life is 16 hours, longer than crystalloids and similar to endogeneous  albumin  (128).  H y d r o x y e t h y l starches (HES) are synthetic colloid derivatives from corn starch. T h e  111  agent is prepared by incorporating hydroxyethyl ether into the glucose residues of amylopectin (174). They have been shown to effectively expand plasma v o l u m e by 70% at 3 hours and 40% by 12 hours after infusion (174). Z i k r i a et al (183) h a v e suggested that one of the beneficial roles of H E S is to alter the microvascular permeability of an ischemic limb, thereby reducing the development of interstitial edema and eventual  compression ischemia. Hydroxyethyl starches have  been  s h o w n to have anaphylactic reactions i n 0.8% as well as decreases i n factor VIII activity and prolongation of partial thromboplastin time (137) however there h a v e been no increased frequency of bleeding reported. immunosuppressive  Concerns about the possible  effect of hydroxyethyl starch have  also been raised (184).  Higher molecular weight particles of hydroxyethyl starch are deposited i n  the  reticuloendothelial system and may affect phagocytic function. H o w e v e r , i n a study by Shatney and Chaudry (185) demonstrated that reticuloendothelial clearance rates of labeled l i p i d emulsions were unchanged after hydroxyethyl starch, and similarly, mortality rates from peritonitis were not altered i n animals receiving hydroxyethyl starch.  Pentastarch is a modification of the hetastarch formulation as it is has a  lower average molecular weight, a more homogeneous  size of particles, and less  hydroxyethyl substitution. These changes allow for a more predictable excretion of pentastarch compared w i t h hetastarch.  Its volume-expanding effect lasts 12 hours.  Because of its higher colloid oncotic pressure (about 40 m m Hg), it has a greater degree of volume expansion (1.5 times the volume infused) than either 5% albumin or 6% hetastarch (128). Further, it appears to have less effect on coagulation t h a n hetastarch.  112  Thus, w i t h the potential benefits of colloids over crystalloids, and w i t h the advantages of pentaspan over some other colloids, it places pentaspan as the most rationale fluid to study.  One may study the effects i n a similar experiment  as  described w i t h use of pentaspan as the resuscitative fluid and study the oxygen extraction at onset of ischemia. Further, by studying the interstitial v o l u m e as an indicator of interstitial volume, one may determine  whether  colloids i m p r o v e  oxygen delivery by limiting interstitial edema. W h i l e the use of fluid resuscitation to augment oxygen delivery is directed a relatively phase of SIRS, others have studied novel approaches at earlier stages i n the inflammatory response. nonspecific.  These therapies may be d i v i d e d into specific and  Specific therapies include monoclonal antibody derived against gram-  negative bacterial endotoxin has been tested i n septic patients i n a multicenter, prospective, randomized, double blind fashion  (186).  A d m i n i s t r a t i o n of this  antibody to I C U patients w i t h suspected gram-negative sepsis early i n their septic course resulted i n a significant reduction i n the mortality i n the group w h o p r o v e d to be bacteremic on blood culture.  However, w h e n looking at the entire  group,  there was no significant reduction i n mortality. P o l y m y x i n B is another c o m p o u n d w h i c h has the property of efficiently binding endotoxin (187). Further, a p o l y m y x i n B-impregnated filter is being developed as part of hemofiltration systems to chelate circulating endotoxin (182). Trials are also underway on monoclonal  antibodies  designed to interfere w i t h neutrophil adhesion (anti-CD18 or CD11), IL-1 receptor, T N F , and P A F (22).  M u c h work needs to be done not only on its potential  effectiveness but the timing, potential adverse effects, and the specificity of therapy.  113  Less specific therapies  include  eicosanoid  inhibitors.  Increased  activity of  phospholipase A 2 , an important enzyme i n the release of arachidonic acid f r o m membrane  phospholipids, is correlated w i t h the severity of septic shock (188).  Blocking the synthesis or activity of these substances results i n increased s u r v i v a l i n experimental models (189). Other nonspecific therapies being studied include free radical scavengers, xanthine oxidase inhibitors, and superoxide dismutase to name a few (182). Whether  the  crystalloids, colloids, or the  novel  approaches  potential i n reducing the o v e r w h e l m i n g mortality rates remains  will  have  to be seen.  However, it is unlikely that any one therapy w i l l be the "magic bullet" that everyone seeks.  114  REFERENCES  1. Rackow E C , Astiz M E . Mechanisms and Management of Septic Shock. Crit Care C l i n 1993;9:219-237. 2. Parrillo JE, Parker M M , Natanson C , et al. Septic Shock i n H u m a n s : Advances i n the Understanding of Pathogenesis, Cardiovascular Dysfunction, and Therapy. A n n Int M e d 1990;113:227-242. 3. Carrico DJ, Meakins JL, Marshall JC, et al. M u l t i p l e organ failure syndrome. A n n Surg 1986;121:196-208. 4. Light R B . Sepsis Syndrome. In: H a l l JB S G , W o o d L D H , ed. Critical Care. N e w York: M c G r a w H i l l , 1992:645-655. 5.  Principles of  Ketai L . F l u i d Loading i n Septic Shock. A n n Int M e d 1990;113:991.  6. Bone R C , Balk R A , Cerra FB, et al. Definitions for Sepsis and Organ Failure and Guidelines for the Use of Innovative Therapies i n Sepsis. Chest 1992;101:16441655. 7. Baker C C , H u y n h T. Sepsis i n the Critically 111 Patient. C u r r Probl Surg 1995;32:1018-1083. 8. Fink M P , Heard SO. Current Research Review: Laboratory Models of Sepsis and Septic Shock. J Surg Res 1990;49:186-196. 9. Morrison D C , Ryan JL. Endotoxins and disease mechanisms. A n n u Rev M e d 1987;38:417-432. 10. M o r r i s o n D C , Ulevitch RJ. The effects of bacterial endotoxins mediation systems. A m J Pathol 1987;93:527-618. 11. Dinarello C. The proinflammatory cytokines interleukin-1 and necrosis factor and treatment of septic shock. J Infect Dis 1991;163:1177-1184.  on host  tumor  12. Tuchschmidt J, Obltas D , Fried J C Oxygen consumption i n sepsis and septic shock. Crit Care M e d 1991;19:664-671. 13. VanderMeer TJ, W a n g H , Fink M P . Endotoxemia causes ileal m u c o s a l acidosis i n the absence of mucosal hypoxia i n a normodynamic porcine model of septic shock. Crit Care M e d 1995;23:1217-1226. 14. Spitzer J A , Deaciuc IV. Effect of endotoxicosis and sepsis on intracellular calcium homeostasis i n rat liver. Circ Shock 1986;18:81-93.  115  15. Tavakoli H , M e l a L . Alterations of mitochondrial metabolism and protein concentrations i n subacute septicemia. Infect Immun 1982;38:536-541. 16. Stadler J, Billiar TR, C u r r a n R D . Effect of exogenous and endogenous nitric oxide on mitochondrial respiration i n rat hepatocytes. A m J Physiol 1991;260:C910C916. 17. Astiz M , Rackow E C , W e i l M H , Schumer W . Early Impairment of Oxidative Metabolism and Energy Production i n Severe Sepsis. Circ Shock 1988;26:311-320. 18. Simonson SG, Welty-Wolf K , H u a n g Y - C T , et al. Altered M i t o c h o n d r i a l Redox Responses i n G r a m Negative Septic Shock i n Primates. Circ Shock 1994;43:3443. 19. Souba W W , Herskowitz K , Klimberg V S . The effect of endotoxemia on gut glutamine metabolism. A n n Surg 1990;21:543-551. 20. Deitch E A . M u l t i p l e organ failure: therapy. A n n Surg 1992;216:117-134.  sepsis  pathophysiology and potential  21. Unanue ER, A l l e n P M . The basis for the immunoregulatory macrophages and other accessory cells. Science 1987;236:551-557. 22. Cipolle M D , Pasquale M D , Cerra FB. Secondary Organ Dysfunction: Clinical perspective to Molecular Mediators. Crit Care C l i n 1993;9:261-298.  and future  role of From  23. Heflin A C , Jr., Brigham K L . Prevention by Granulocyte Depletion of Increased Vascular Permeability of Sheep L u n g following Endotoxemia. J C l i n Invest 1981;68:1253-1260. 24. M i l e s k i W J . Sepsis: what it is and how to recognize it. Surg C l i n N o r t h A m 1991;71:749-763. 25. Okusawa S, Gelfand J A , Ikejima T, et al. Interleukin-1 induces a shock-like state i n rabbits. Synergism w i t h tumor necrosis factor and the effect of cyclooxygenase inhibition. J C l i n Invest 1988;81:1162-1170. 26. F l e m i n g I, Gray G A , Julou-Schaeffer G , et al. Incubation w i t h e n d o t o x i n activates the L-arginine pathway i n vascular tissue. Biochem Biophys Res C o m m u n 1990;171:562-570. 27. Julou-Schaeffer G , Gray G A , Parratt JR, et al. Activation of the L-arginine pathway is i n v o l v e d i n vascular hyporeactivity induced by endotoxin. J Cardiovasc Pharmacol 1991;17:S207-S212. 28. K i l b o u r n R G , Gross SS, Jubran A , et al. NG-methyl-L-arginine inhibits t u m o r necrosis factor-induced hypotension: Implications for the i n v o l v e m e n t of nitric oxide. Proc N a t l A c a d Sci U S A 1990;87:3629-3635. 116  29. Deby C , Goutier R. N e w perspective on the biochemistry of superoxide a n i o n and the efficiency of superoxide dismutase. Biochem Pharmacol 1990;39:399-405. 30. M c C o r d J M . Oxygen-derived free radicals i n postischemic tissue injury. N Engl J M e d 1985;312:159-163. 31. G r u m C M . Tissue oxygenation i n l o w flow states and during hypoxemia. Crit Care M e d 1993;21:S44-S49. 32. G r u m C M , Ragsdale R A , Ketai L H . Plasma xanthine patients w i t h A R D S . J Crit Care 1987;2:22-26.  oxidase activity i n  33. B a l d w i n SR, Simon. R H , G r u m C M , Ketai L H , Boxer L A , D e v a l l LJ. Oxidant activity i n expired breath of patients w i t h adult respiratory distress syndrome. Lancet 1986;1:11-14. 34. Thijs L G , Schneider A J , Groeneveld A B J . The haemodynamics shock. Intensive Care M e d 1990;16(Suppl 3):S182-S186.  of septic  35. Rackow E C , Kaufman BS, Falk JL, et al. H e m o d y n a m i c response to f l u i d repletion i n patients w i t h septic shock: Evidence for early depression of cardiac performance. Circ Shock 1987;22:11-22. 36. Ellrodt A G , Riedinger M S , K i m c h i A , et al. Left ventricular performance i n septic shock: Reversible segmental and global abnormalities. A m Heart J 1985;110:402-409. 37. Parker M M , McCarthy K E , Ognibene FP, Parrillo JE. Right V e n t r i c u l a r Dysfunction and Dilation, Similar to Left Ventricular Changes, Characterize the Cardiac Depression of Septic Shock i n Humans. Chest 1990;97:126-131. 38. ' Nishijma H , W e i l M H , Shubin H , et al. H e m o d y n a m i c and metabolic studies on shock associated w i t h gram-negative bacteremia. Medicine 1973;52:287-294. 39. Jafri S, Lavine S, Field B, et al. Left ventricular diastolic function i n sepsis. Crit Care M e d 1990;18:709-714. 40. Ognibene FP, Parker M M , Natanson C , et al. Depressed left v e n t r i c u l a r performance: response to volume infusion i n patients w i t h sepsis and septic shock.. Chest 1988;93:903-910. 41. Goddard C M , A l l a r d M F , Hogg JC, Walley K R . Myocardial m o r p h o m e t r i c changes related to decreased contractility after endotoxin. A m J Physiol 1996;270(4 Pt 2):H1446-H1452. 42. Baumgartner J, M c C u t c h a n J, Melle G , et al. Prevention of G r a m - N e g a t i v e shock and death i n surgical patients by, antibody of endotoxin core glycolipid. Lancet 1985;2:54-63. 117  43. Natanson C , Eichenholz P, Danner R, et al. Endotoxin and tumor necrosis factor challenges i n dogs simulate the cardiovascular profile of h u m a n septic shock. J Exp M e d 1989;169:823-832. 44. Parrillo JE, Burch C , Shelhamer J H , et al. A circulating myocardial depressant substance i n humans w i t h septic shock. J C l i n Invest 1985;76:1539-1553. 45. Reily J, C u n n i o n R, B u r c h - W h i t m a n C, et al. A circulating myocardial depressant substance is associated w i t h cardiac dysfunction and peripheral hypoperfusion i n patients w i t h septic shock. Chest 1989;95:1072-1080. 46. H i n s h a w L B . Sepsis/septic shock: participation of the microcirculation: abbreviated review. Crit Care M e d 1996;24:1072-1078.  an  47. Shoemaker W C , A p p e l P L , K r a m H B , Bishop M , A b r a h a m E. H e m o d y n a m i c and Oxygen Transport M o n i t o r i n g To Titrate Therapy i n Septic Shock. N e w Horizons 1993;1:145-159. 48. Driessen G K , Haist C W M , H e i d t m a n n H , et al. Effect of reduced red cell "deformability" on flow velocity i n capillaries of rat mesentery. Pflugers A r c h 1980;388:75-78. 49. P o w e l l RJ, Machiedo G W , Rush BF, Jr. Decreased R e d Blood C e l l Deformability and Impaired Oxygen Utilization during H u m a n Sepsis. A m Surg 1993;59:65-68. 50. Bellary S, A r d e n W W , Schwartz R W , et al. Effect of lipopolysaccharide, leukocytes and monoclonal anti-lipid A antibodies on erythrocyte membrane elastance. Shock 1995;3:132-136. 51. C h i e n S. Rheology i n the microcirculation i n normal and l o w flow states. Circ Shock 1982;8:71-80. 52. Fink M P , C o h n S M , Lee P C , et al. Effect of lipopolysaccharide on intestinal intramucosal hydrogen ion concentration i n pigs: Evidence of gut ischemia i n a normodynamic model of septic shock. Crit Care M e d 1989;17:641-646. 53. Solomon L A , H i n s h a w L B . Effect of endotoxin on isogravimetric capillary pressure i n the forelimb. A m J Physiol 1968;214:443-447. 54. Hersch M , Gnidec A A , Bersten A D , Troster M , Rutledge FS, Sibbald W J . Histologic and ultrastructural changes i n n o n p u l m o n a r y organs d u r i n g early hyperdynamic sepsis. Surgery 1990;107:397-410. 55. Dantzker D . Oxygen Delivery and Utilization 1989;5:81-98.  118  i n Sepsis. Crit Care C l i n  56. H e u g h a n C , N i i n i s o s k i J, H u n t T K . Effect of Excessive Infusion of Saline Solution on Tissue Oxygen Transport. Surg Gynecol Obstet 1972;135:257-260. 57. L a m C , T y m l K , M a r t i n C , Sibbald W . Microvascular perfusion is impaired i n a rat model of normotensive sepsis. J C l i n Invest 1994;94:2077-2083. 58. Knisely M H , Reneau D D , Binely D F . The development and use of equations for predicting the limits on the oxygen supply to the cells of l i v i n g tissues and organs. Angiology 1969;20(Suppl.ll):l. 59. Schutzer K - M , Larsson A , Risberg B, et al. L u n g protein leakage i n feline septic shock. A m Rev. Respir Dis 1993;147:1380. 60. Timesvari P, A b r a h a m GS, Spur C P , et al. Escherichia coli 0111 B4 lipopolysaccharide given intracisternally induces blood-brain barrier opening during experimental neonatal meningitis i n piglets. Pediatr Res 1993;34:182-186. 61. Voss B L , DeBault L E , Blick K E , et al. Sequential renal alterations i n septic shock i n the primate. Circ Shock 1991;33:142-155. 62. Bradley JR, W i l k s D , Rubenstein D . The vascular endothelium shock. J infect 1994;28:1-10.  i n septic  63. Schumacker PT, Samsel R W . Oxygen Delivery and Uptake by Peripheral Tissues: Physiology and Pathophysiology. Crit Care C l i n 1989;5:255-269. 64. Knisely M H , Cowley R A , Hawthorne I, et al. Experimental and separation of hypovolemic and septic shock. Angiology 1970;21:728-744.  clinical  65. Astiz M E , DeGent G E , L i n R Y , et al. Microvascular function and rheologic changes i n hyperdynamic sepsis. Crit Care M e d 1995;23:265-271. 66. Schmid-Schonbein G W . Capillary plugging by granulocytes and the no-reflow phenomenon i n the microcirculation. Federation Proc 1987;46:2397-2401. 67. Cronenwett JL, Lindenauer S M . Direct measurement of arteriovenous anastomotic blood flow i n the septic canine hindlimb. Surgery 1979;85:275-282. 68. Archie JP. Anatomic arterial-venous shunting i n endotoxic and septic shock in dogs. A n n Surg 1977;186:171-176. 69. K r z a n o w s k i JJ- Vascular endothelium i n sepsis and endotoxemia. J Fla M e d Assoc 1994;81:119-122. 70. W y l a m M E , Samsel R W , Umans JG, Mitchell R W , Leff A R , Schumacker PT. Endotoxin In V i v o Impairs Endothelium-dependent Relaxation of Canine Arteries In Vitro. A m Rev Respir Dis 1990;142:1263-1267.  119  71.  Samsel R W , Schumacker  FT. Systemic hemorrhage  augments local O 2  extraction i n canine intestine. J A p p l Physiol 1994;77:2291-2298. 72. Parker M M , Shelhamer J H , Bacharach SL, et al. Profound but Reversible Myocardial Depression i n Patients w i t h Septic Shock. A n n Int M e d 1984;100:483-490. 73. Drazenovic R, Samsel R W , W y l a m M E , Doerschuk C M , Schumaker PT. Regulation of perfused capillary density i n canine intestinal mucosa, d u r i n g endotoxemia. J A p p l Physiol 1992;72:259-265. 74. H u m e r M F , Phang PT, Friesen BP, A l l a r d M F , Goddard C M , Walley K R . Heterogeneity of gut capillary transit times and impaired gut oxygen extraction i n endotoxemic pigs. J A p p l Physiol 1996;81:895-904. 75. Herbertson M J , Werner H A , Russell J A , Iversen K , Walley K R . M y o c a r d i a l oxygen extraction ratio is decreased during endotoxemia i n pigs. J A p p l P h y s i o l 1995;79:479-486. 76. Gutierrez G . Regional blood flow and oxygen transport: Implications for the therapy of the septic patient. Crit Care M e d 1993;21:1263-1264. 77. N e l s o n D P , Samsel R W , W o o d L D H , Schumacker PT. Pathological supply dependence of systemic and intestinal O 2 uptake during endotoxemia. J A p p l Physiol 1988;64:2410-2419. 78. Walley K R . Heterogeneity of oxygen delivery impairs oxygen extraction by peripheral tissues: theory. J A p p l Physiol 1996;81:885-894. 79. Z h a n g H , Vincent J-L. Oxygen Extraction Is Altered by Endotoxin D u r i n g Tamponade-Induced Stagnant H y p o x i a i n the Dog. Circ Shock 1993;40:168-176. 80. Maddaus M A , Wells C L , Simmons R L . Role of cell-mediated i m m u n i t y i n preventing the translocation of intestinal bacteria. Surg F o r u m 1986;37:107-109. 81. Granger H J , N y h o f R A . Dynamics of intestinal oxygenation: between oxygen supply and uptake. A m J Physiol 1982;243:G91-G96.  Interactions  82. Shepherd A P . Metabolic control on intestinal oxygenation and blood flow. Federation Proc 1982;41:2084-2089. 83. Granger H J , Shepherd A P , Jr. Intrinsic microvascular control of tissue oxygen delivery. Microvasc Res 1973;5:49-72. 84. Granger H J , N o r r i s C P . Intrinsic regulation of intestinal oxygenation i n the anesthetized dog. A m J Physiol 1980;238:H836-H843.  120  85. Mythen M G , Webb AR. The role of gut mucosal hypoperfusion in the pathogenesis of post-operative organ dysfunction. Intensive Care Med 1994;20:203209. 86. Nelson DP, King CE, Dodd SL, Schumacker PT, Cain SM. Systemic and intestinal limits of O 2 extraction in the dog. J Appl Physiol 1987;63:387-394. 87. Schacterle RD, Adams JM, Ribando RJ. A Theoretical Model of Gas Transport between Arterioles and Tissue. Microvasc Res 1991;41:210-228. 88. Falk A , Redfors S, Myrvold H , Haglund U . Small Intestinal Mucosal Lesions in Feline Septic Shock: A Study on the Pathogenesis. Circ Shock 1985;17:327-337. 89. Shepherd AP, Kiel JW. A model of countercurrent shunting of oxygen in the intestinal villus. A m J Physiol 1992;262:H1136-H1142. J  90.  '  Vallett B, Lund N , Curtis SE, Kelly D, Cain SM. Gut and muscle tissue P O 2 in  endotoxemic dogs during shock and resuscitation. J Appl Physiol 1994;76:793-800. 91. Wilmore DW, Smith RJ, ODwyer ST, Jacobs DO, Ziegler TR, Wang X-D. The gut: A central organ after surgical stress. Surgery 1988;104:917-923. 92. Takala J, Ruokonen E. Oxygen transport in septic shock. Schweiz med Wschr 1992;122:776-779. 93. Ruokonen E, Takala J, Kari A , Saxen H , Mertsola J, Hansen EJ. Regional blood flow and oxygen transport in septic shock. Crit Care Med 1993;21:1296-1303. 94. Dahn MS, Lange P, Lobdell K, Hans B, Jacobs LA, Mitchell RA. Splanchnic and total body oxygen consumption differences in septic and injured patients. Surgery 1987;101:69-80. 95. Bulkley GB, Kvietys PR, Parks DA, Perry M A , Granger D N . Relationship of Blood Flow and Oxygen Consumption to Ischemic Injury in the Canine Small Intestine. Gastroenterology 1985;89:852-857. 96. Fink MP, Antonsson JB, Wang H , Rothschild HR. Increased Intestinal Permeability in Endotoxic Pigs. Arch Surg 1991;126:211-218. 97. Salzman A l , Wang H , Wollert S, et al. Endotoxin-induced ileal mucosal hyperpermeability in pigs: role of tissue acidosis. A m J Physiol 1994;266:G633-G646. 98. O'Dwyer ST, Michie HR, Ziegler TR, Revhaug A, Smith RJ, Wilmore DW. A Single Dose of Endotoxin Increases Intestinal Permeability in Healthy Humans. Arch Surg 1988;123:1459-1464.  121  99. Rush BF, Jr., Sori AJ, Murphy TF, Smith S, Flanagan JJ, Jr., Machiedo G W . Endotoxemia and Bacteremia During Hemorrhagic Shock: The Link Between Trauma and Sepsis? Ann Surg 1988;207:549-554. 100. Deitch EA, Berg R, Specian R. Endotoxin Promotes the Translocation of Bacteria From the Gut. Arch Surg 1987;122:185-190. 101. Deitch EA, Specian RD, Berg RD. Endotoxin-induced bacterial translocation and mucosal permeability: Role of xanthine oxidase, complement activation, and macrophage products. Crit Care Med 1991;19:785-791. 102. Mainous MR, Tso P, Berg RD, Deitch EA. Studies of the Route, Magnitude, and Time Course of Bacterial Translocation in a Model of Systemic Inflammation. Arch Surg 1991;126:33-37. 103. Schumacker PT, Cain SM. The concept of a critical oxygen delivery. Intensive Care Med 1987;13:223-229. 104. Leach R M , Treacher DF. The Relationship Between Oxygen Delivery and Consumption. Dis Mon 1994;40:306-368. 105. Stein JC, Ellsworth ML. Capillary oxygen transport during severe hypoxia: role of hemoglobin oxygen affinity. J Appl Physiol 1993;75:1601-1607. 106. Nelson DP, Beyer C, Samsel RW, Wood LDH, Schumacker PT. Pathological supply dependence of O 2 uptake during bacteremia in dogs. J Appl Physiol 1987;63:1487-1492. 107. Samsel RW, Nelson DP, Sanders W M , Wood LDH, Schumacker PT. Effect of endotoxin on systemic and skeletal muscle O 2 extraction. J Appl Physiol 1988;65:1377-1382. 108. Vincent J-L. Oxygen transport in severe sepsis. Acta Anaesthesiol Scand 1993;37(Suppl 98):29-31. 109. Kreymann G, Grosser S, Buggisch P, Gottschall C, Matthaei S, Greten H . Oxygen consumption and resting metabolic rate in sepsis, sepsis syndrome, and septic shock. Crit Care Med 1993;21:1012-1019. 110. Steffes CP, Bender JS, Levison M A . Blood transfusion consumption in surgical sepsis. Crit Care Med 1991;19:512-517.  and  oxygen  111. Czer LSC, Shoemaker WC. Optimal hematocrit value in critically ill postoperative patients. Surg Gynecol Obstet 1978;117:363-368.  122  112. Conrad S, Dietrich K , Herbert C , et al. Effect of red cell transfusions on oxygen consumption following fluid resuscitation i n septic shock. Circ Shock 1990;31:419420. 113. Fortune J, Feustel P, Saif J, et al. Influence of hematocrit on cardiopulmonary function after acute hemorrhage. J Trauma 1987;27:243-249. 114. Hussain S N A , Roussos C . Distribution of respiratory muscle and organ blood flow during endotoxic shock i n dogs. J A p p l Physiol 1985;59:1802-1810. 115. Shoemaker W C , A p p e l P L , K r a m H B . Oxygen transport measurements to evaluate tissue perfusion and titrate therapy. Crit Care M e d 1991;19:672-688. 116. Ruttimann Y , Schutz Y , Jequier E, et al. Thermogenic and metabolic effects of dopamine i n healthy men. Crit Care M e d 1991;19:1030-1036. 117. R u i z C , W e i l M H , Carlson R. Treatment of circulatory shock w i t h dopamine. J A M A 1979;242:165-168. 118. Haupt M T , Kaufman BS, Carlson R W . F l u i d resuscitation i n patients w i t h increased vascular permeability. Crit Care C l i n 1992;8:341-353. 119. MacLean L D , W e i l M H . Hypotension i n drugs produced by Escherichia coli endotoxin. Circ Res 1956;4:546. 120. MacLean L D , W e i l M H , Spink W W , et al. Canine intestinal and liver weight changes induced by E. C o l i endotoxin. Proc Soc Exp M e d 1956;92:602. 121. Teule GJJ, Hollander W D , Bronsveld W , et al. Effect of v o l u m e loading and dopamine on hemodynamics and red-cell redistribution i n canine endotoxin shock. Circ Shock 1983;10:41. 122. W e i l M H , Nishijma 1978;64:920-927.  H . Cardiac output i n bacterial shock. A m J M e d  123. Astiz M E , Rackow EC, Falk JL, Kaufman BS, W e i l M H . Oxygen delivery and consumption i n patients w i t h hyperdynamic septic shock. Crit Care M e d 1987;15:2628. 124. Kaufman BS, Rackow E C , Falk JL. The Relationship Between Oxygen Delivery and C o n s u m p t i o n during F l u i d Resuscitation of H y p o v o l e m i c and Septic Shock. Chest 1984;85:336-340. 125. v a n der L i n d e n P, Gilbart E, Paques P, S i m o n C , Vincent J-L. Influence of hematocrit on tissue O 2 extraction capabilities during acute hemorrhage. A m J Physiol 1993;264:H1942-H1947.  123  126. T y m l K . Heterogeneity of microvascular flow i n rat skeletal muscle is reduced by contraction and by hemodilution. Int J Microcirc C l i n Exp 1991;10:75-86. 127. Vincent JL. The 1991;Suppl XXVL104-111.  Colloid-Crystalloid  Controversy.  Klin  Wochenschr  128. Griffel M I , Kaufman BS. Pharmacology of Colloids and Crystalloids. Crit Care C l i n 1992;8:235-253. 129. Carlson R W , Rattan S, Haupt M T . F l u i d resuscitation increased permeability. Anesthesiol Rev 1990;17:14.  i n conditions of  130. Spagna G , Siegel J, B r o w n G , et al. Reprioritization of hepatic plasma protein i n trauma and sepsis. A r c h Surg 1985;120:187-197. 131. D e m l i n g R H , W o n g C , Wenger H . Effect of Endotoxin on the Integrity of the Peripheral (Soft Tissue) Microcirculation. Circ Shock 1984;12:191-202. 132. Rackow E C , Falk JL, Fein I A , et al. F l u i d resuscitation i n circulatory shock: a comparison of the cardiorespiratory effects of albumin, hetastarch, and saline solutions i n patients w i t h hypovolemic and septic shock. Crit Care M e d 1983;ll:839850. 133. K a r z a i W , Reilly J M , Hoffman W D , et al. H e m o d y n a m i c effects of dopamine, norepinephrine, and fluids i n a dog model of sepsis. A m J Physiol 1995;268:H692H702. 134. D ' O r i o V , Mendes P, Carlier P, Fatemi M , Marcelle R. L u n g fluid dynamics and supply dependency of oxygen uptake during experimental endotoxic shock and volume resuscitation. Crit Care M e d 1991;19:955-962. 135. M u l l i n s RJ, Tahamont M V , Bell D R , M a l i k A B . Effect of fluid resuscitation from endotoxin shock on lung transvascular fluid and protein exchange. A m J Physiol 1991;260:H1415-H1423. 136. 633.  E l l m a n H . Capillary permeability i n septic patients. Crit Care M e d 1984;12:629-  137. Astiz M E , Galera-Santiago A , Rackow E C . Intravascular V o l u m e and F l u i d Therapy for Severe Sepsis. N e w Horizons 1993;1:127-136. 138. Starling E H . O n the absorption of fluids from the connective tissue spaces. J Physiol (Lond) 1896;9:312-326. 139. Taylor A E , Garrl C A . Estimation of equivalent pore radii of p u l m o n a r y capillary and alveolar membranes. A m J Physiol 1970;218:1133-1141.  124  140. Wittmers L E , Barlett M , Johnson J A . Estimation of capillary permeability coefficient of insulin i n various tissues of the rabbit. Microvasc Res 1976;11:67-74. 141. Brigham K , Bowers R E , Haynes J. Increased sheep l u n g vascular permeability caused by E . coli endotoxin. Circ Res 1979;45:292-300. 142.  Tullis JL. A l b u m i n . J A M A 1977;237:355-360.  143. W e i l M H , Michaels S, P u r i V , et al. The stat laboratory. A m J C l i n Pathol 1981;76:34-42. 144.  Bergofsky E H . Determination  tonometers:  of tissue O 2 tensions  by h o l l o w visceral  effect of breathing enriched O 2 mixtures. J C l i n Invest 1964;43:193-200.  145. D a w s o n A M , Trenchard D , G u z A . Small bowel tonometry: assessment of small gut mucosal oxygen tension i n dog and man. Nature L o n d 1965;206:943-944. 146. Kivisaari J, N i i n i k o s k i J. Use of Silastic tube and capillary sampling technique i n the measurement of tissue P O 2 and P C O 2 . A m J Surg 1973;125:623-627. 147.  Fiddian-Green R G , Pittenger G , Whitehouse W M . Back-diffusion of C O 2 and  its influence on the intramucosal p H i n gastric mucosa. J Surg Res 1982;33:39-48. 148. Antonsson JB, Boyle C C , Kruithoff K L , et al. V a l i d a t i o n of tonometric measurement of gut intramural p H during endotoxemia and mesenteric occlusion in pigs. A m J Physiol 1990;259:G519-G523. 149. Fiddian-Green R G , H a g l u n d U , Gutierrez G , Shoemaker W C . Goals for the resuscitation of shock. Crit Care M e d 1993;21:S25-S31. 150. Groeneveld A B J , K o l k m a n JJ. Splanchnic Tonometry: A Review Physiology, Methodology, and Clinical Applications. J Crit Care 1994;9:198-210.  of  151. Clark C H , Gutierrez G . Gastric Intramucosal p H : A N o n i n v a s i v e M e t h o d for the Indirect Measurement of Tissue Oxygenation. A m J Crit Care 1992;2:53-60. 152. Gutierrez G , Palizas F, Doglio G , et al. Gastric intramucosal p H as a therapeutic index of tissue oxygenation i n critically i l l patients. Lancet 1992;339:195199. 153. M a y n a r d N , Bihari D , Beale R, et al. Assessment of Splanchnic Oxygenation by Gastric Tonometry i n Patients W i t h Acute Circulatory Failure. J A M A 1993;1993:1203-1210. 154. Schlichtig R, Bowles S A . Distinguishing between aerobic and anaerobic appearance of dissolved C O 2 i n intestine during l o w flow. J A p p l P h y s i o l 1994;76:2443-2451.  125  155. Gutierrez G , Bismar H , Dantzker D R , Silva N . C o m p a r i s o n of gastric intramucosal p H w i t h measures of oxygen transport and consumption i n critically ill patients. Crit Care M e d 1992;20:451-457. 156. 245.  Stacpoole P W . Lactic Acidosis. Endocrinol Metab C l i n N o r t h A m 1993;22:221-  157. L u p o M A , Cefalu W T , Pardridge W M . Kinetics of lactate transport into rat liver i n v i v o . Metabolism 1990;38:374-377. 158. Ronco JJ, Fenwick JC, Tweeddale M G , et al. Identification of the Critical Oxygen Delivery for Anaerobic Metabolism i n Critically 111 Septic and Nonseptic Humans. J A M A 1993;270:1724-1730. 159.  Dodds WJ. The p i g model for biomedical research. Fed Proc 1982;41:247-250.  160. C a i n S M , Curtis SE. Experimental models dependency. Crit Care M e d 1991;19:603-612.  of pathologic oxygen supply  161. Breslow M J , M i l l e r C F , Parker SD, W a l m a n A T , Traystman RJ. Effect of vasopressors on organ blood flow during endotoxin shock i n pigs. A m J P h y s i o l 1987;252:H291-H300. 162. Elias H , H y d e D M . A n elementary microscopy). A m J Anat 1980;159:412-461.  introduction to stereology  (quantitative  163. H o g g JC, M c L e a n T, M a r t i n B A , Wiggs B. Erythrocyte transit and n e u t r o p h i l concentration i n the dog lung. J A p p l Physiol 1988;65:1217-1225. 164. A l l a r d M F , K a m i m u r a C T , English D R , H e n n i n g SL, Wiggs BR. R e g i o n a l myocardial capillary erythrocyte transit time i n the normal resting heart. Circ Res 1993;72:187-193. 165. Schumacker PT, Samsel R W . Analysis of oxygen delivery and relationships i n the K r o g h tissue model. J A p p l Physiol 1989;67:1234-1244.  uptake  166. Beal A L , Cerra FB. M u l t i p l e Organ Failure Syndrome i n the 1990s: Systemic Inflammatory Response and Organ Dysfunction. J A M A 1994;271:226-233. 167.  Samsel R W , Schumacker PT. Determination of the critical O 2 delivery f r o m  experimental data: sensitivity to error. J A p p l Physiol 1988;64:2074-2082. 168. Barroso-Aranda J, Schmid-Schonbein G W , Zweifach B W , M a t h i s o n JC. Polymorphonuclear neutrophil contribution to induced tolerance to bacterial lipopolysaccharide. Circ Res 1991;69:1196-1206. 169. Piiper J, Haab P. Oxygen supply and uptake i n tissue models w i t h u n e q u a l distribution of blood flow and shunt. Respir Physiol 1991;84:261-271. 126  170. H o n i g C R , Odoroff C L . Calculated dispersion of capillary transit significance for oxygen exchange. A m J Physiol 1981;240:H199-H208.  times:  171. Morff RJ. Contribution of capillary recruitment to regulation oxygenation i n rat cremaster muscle. Microvasc Res 1988;36:150-161.  tissue  of  172. Ostgaard G , Reed R K . Interstitial fluid accumulation does not influence oxygen uptake i n the rabbit small intestine. Acta Anaesthesiol Scand 1995;39:167-173. 173. F u n k W , Baldinger V . Microcirculatory Perfusion d u r i n g V o l u m e Therapy: A comparative Study U s i n g Crystalloid or C o l l o i d i n A w a k e A n i m a l s . Anesthesiology 1995;82:975-982. 174. Wagner B K J , D ' A m e l i o L F . Pharmacologic and clinical considerations i n selecting crystalloid, colloidal, and oxygen-carrying resuscitation fluids, part 1. C l i n P h a r m 1993;12:335-346. 175. H o r t o n JW, Walker PB. Small-volume Hypertonic Saline Resuscitation from Canine Endotoxin Shock. A n n Surg 1991;214:64-73.  Dextran  176. G u y t o n A C , Lindsey A W . Effect of elevated left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circ Res 1959;7:73-84. 177. Zarins C K , Rice C L , Peters K M , et al. L y m p h and p u l m o n a r y response to isobaric reduction i n plasma oncotic pressure. Circ Res 1978;43:925-930. 178. Kramer G C , Harms B A , Bodai BI, et al. Effects of hypoproteinemia and increased vascular pressure on lung fluid balance i n sheep. J A p p l P h y s i o l 1983;55:1514-1522. 179. Rackow E C , W e i l M H , M a c N e i l A , et al. Effect of crystalloid and colloid fluids on extravascular l u n g water i n hypoproteinemic dogs. J A p p l Physiol 1987;42:24212425. 180. D e m l i n g R H , Harms B, Kramer G . Acute versus sustained h y p o p r o t e i n e m i a and post-traumatic pulmonary edema. Surgery 1982;92:79-86. 181. M o r i s a k i H , Bloos F, Keys J, M a r t i n C, Neal A , Sibbald W J . Compared w i t h crystalloid, colloid therapy slows progression of extrapulmonary tissue injury i n septic sheep. J A p p l Physiol 1994;77:1507-1518. 182. D e m l i n g R, LaLonde C, Saldinger P, Knox J. Multiple-Organ Dysfunction i n the Surgical Patient: Pathophysiology, Prevention, and Treatment. C u r r Prob Surg 1993;30:345-424.  127  183. Z i k r i a B A , Subbarao C , O z M C , et al. Macromolecules reduce a b n o r m a l microvascular permeability i n rat limb ischemia-reperfusion injury. Crit Care M e d 1989;17:1306-1309. 184. M a u e r P H , Bernadinelli B. Immunologic studies w i t h hydroxyethyl starch - a proposed plasma expander. Transfusion 1968;8:265-268. 185. Shatney C H , Chaudry I H . Hydroxyethyl starch administration does not depress reticuloendothelial function or increase mortality from sepsis. Circ Shock 1984;13:21-25. 186. Ziegler EJ, Fisher CJ, Jr.,, Sprung C L , et al. Treatment of Gram-negative bacteremic and septic shock w i t h H A - 1 A h u m a n monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. N Engl J M e d 1991;324:429-436. 187. Munster A , W i n c h u r c h R, Thupari J , . et al. Reversal of postburn immunosuppression w i t h l o w dose p o l y m y x i n B. J Trauma 1986;26:995-1000. 188. Vadas P, Pruzanski W , Stefanski E, et al. Pathogenesis of hypotension i n septic shock: Correlation of circulatory phospholipase A 2 levels w i t h circulatory collapse. Crit Care M e d 1988;16:1-7. 189. 12.  Lefer A . Significance of lipid mediators i n shock states. Circ Shock 1989;27:3-  128  

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