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FINES will be charged if book is returned after the date stamped below. (5m 7&8 :2 e MECHANISM OF FORELIMB SKIN AND SKELETAL MUSCLE GLUCOSE UPTAKE DURING ESCHERICHIA COLI ENDOTOXIN SHOCK IN THE DOG BY Michael Donald Karlstad A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1982 ABSTRACT MECHANISM OF FORELIMB SKIN AND SKELETAL MUSCLE GLUCOSE UPTAKE DURING ESCHERICHIA COLI ENDOTOXIN SHOCK IN THE DOG By Michael Donald Karlstad This study was undertaken to test the hypotheses that glucose uptake by forelimb skeletal muscle and skin in- creases during endotoxin shock in the dog and that the mechanism for this pr0posed increase in glucose uptake is related to local tissue hypoxia. Mongrel dogs were anes- thetized with pentobarbital sodium and heparinized. The isolated, innervated, forelimb preparation perfused at ei- ther natural or constant blood flow was used. Shock was induced by i.v. infusion of 2 mg/kg E. 3211 endotoxin. Muscle and skin glucose uptake increased by 30 minutes of endotoxin shock and remained elevated in the natural flow study. Total forelimb blood flow decreased and the limb became severely hypoxic. In the constant flow study, glu- cose uptake by both muscle and skin was increased at 30 minutes of endotoxin shock but thereafter returned to con- trol. This group was neither ischemic or hypoxic. These data support the above hypotheses that glucose uptake by muscle and skin increases during endotoxin shock and that the mechanism for the increased glucose uptake is related to local tissue hypoxia. To my wife, Alice, and to my family, for their unfailing love and support ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my advisor Dr. Thomas E..Emerson, Jr., for the guidance and assistance he has provided in the pursuit of this research. To Dr. Ching-chung Chou and Dr. Leena Mela I also extend my appreciation for their advice and also for serving on my examination committee. Special thanks go to Dr. Richard M. Raymond for his never-ending advice and time during the course of this research and for his assistance in the preparation of this manuscript. Finally, I would like to express my thanks and appre- ciation to Ms. Irene Doroshko for her technical assistance and to William V. Stoffs who preformed the necessary chem- ical analyses. iii TABLE OF CONTENTS List Of Tables 0 O O O O O O O O O O O O O O O O O 0 List Of Figures. 0 O O O O O O O O O O O O O O O O 0 Chapter I. II. III. IV. INTRODUCTION 0 O O O O O O O O O I O O O O O SURVEY OF THE LITERATURE . . . . . . . . . . Structure of the Endotoxin Molecule. . . . . The Toxic Factor . . . . . . . . . . . . General Systemic Hemodynamic Effects of Endotoxin. . . . . . . . . . . . . . . . Transcapillary Fluid Fluxes duri 9 Shock The Role of Histamine in Endotoxin Shock Metabolism during Shock. . . . . . . . . ShOCk. O O O O O O O O O O O O O O O O O Hyperglycemia duri 9 End toxin S ock . . Hypoglycemia during Endotoxin Shock. . . . . Alterations in Blood Glucose during Endotoxin METHODS O O O O O O O O O O O O O O O O O O 0 Experimental Animals . . . . . . . . . . . . Forelimb Preparation . . . . . . . . . . . . Experimental Procedures. . . . . . . . . . . Group 1: Controlled Forelimb Temperature- Natural Flow . . . . . . . . . . . . . . . . Group 2: Uncontrolled Forelimb Temperature- Natural Flow . . . . . . . . . . . . . . . . Group 3: Controlled Forelimb Temperature— Constant Flow. . . . . . . . . . . . . . . . Chemical Analyses. . . . . . . . . . . . . . Calculations . . . . . . . . . . . . . . . . Details of Whole Blood Glucose Determination Principles of Operation. . . . . . . . . . . Statistical Analyses . . . . . . . . . . . . RESULTS. O O O O O O O O I O O O O O O O O 0 iv Page vi vii A U‘lb 15 18 19 21 24 32 32 33 35 35 35 35 37 38 38 39 46 Chapter V. VI. DISCUSSION . . . . . . . SUMMARY AND CONCLUSIONS. BIBLIOGRAPHY O O O O C O O O O O LIST OF TABLES Table Page 1. Changes in metabolic variables in the controlled forelimb temperature-natural flow group during E; 2211 endotoxin shock. . . . . . . . . . . . . . 52 2. Changes in metabolic variables in the uncontrolled forelimb temperature-natural flow group during E; gall endotoxin shock. . . . . . . . . . . . . . 55 3. Changes in metabolic variables in the controlled forelimb temperature-natural flow group in non-shocked, control animals . . . . . . . . . . . 63 4. Changes in metabolic variables in the uncontrolled forelimb temperature-natural flow group in non— shocked, control animals . . . . . . . . . . . . . 66 5. Changes in metabolic variables in the controlled forelimb temperature-constant flow group during E; gall endotoxin shock. . . . . . . . . . . . . . 80 6. Changes in metabolic variables in the controlled forelimb temperature-constant flow group in non-shocked, control animals . . . . . . . . . . . 88 vi Figure 1. LIST OF FIGURES Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncontrolled forelimb temperature groups during E; ggli endotoxin shock . . . . . . . . . Muscle metabolic and temperature variables in the controlled and uncontrolled forelimb temper— ature groups during E; 3311 endotoxin shock. . . Skin metabolic and temperature variables in the controlled and uncontrolled forelimb temperature groups during E; gall endotoxin shock. . . . . . Total forelimb metabolic and temperature vari- ables in the controlled and uncontrolled fore— limb temperature groups during E; coll endotoxin shock. . . . . . . . . . . . . . . . . . . . . . Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncontrolled forelimb temperature non—shocked control dogs . . . . . . . . . . . . . . . . . . Control muscle, skin and total forelimb meta— bolic and temperature variables in the con- trolled and uncontrolled forelimb temperature groups in non-shocked, control animals. . . . . . vii Page 42 45 48 51 59 61 Figure 1. LIST OF FIGURES Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncontrolled forelimb temperature groups during E; ggli_endotoxin shock . . . . . . . . . Muscle metabolic and temperature variables in the controlled and uncontrolled forelimb temper— ature groups during E; gall endotoxin shock. . . Skin metabolic and temperature variables in the controlled and uncontrolled forelimb temperature groups during §;_ggli_endotoxin shock. . . . . . Total forelimb metabolic and temperature vari- ables in the controlled and uncontrolled fore— limb temperature groups during E; 3311 endotoxin shock. . . . . . . . . . . . . . . . . . . . . . Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncontrolled forelimb temperature non-shocked control dogs . . . . . . . . . . . . . . . . . . Control muscle, skin and total forelimb meta— bolic and temperature variables in the con- trolled and uncontrolled forelimb temperature groups in non-shocked, control animals. . . . . . vii Page 42 45 48 51 59 61 CHAPTER I INTRODUCTION A characteristic feature of endotoxin shock in a vari- ety of animal species, including the dog (15), rat (7S), and subhuman primate (67), is the progressive development of severe hypoglycemia. This phenomenon appears to result from the combined effects of decreased glucose production by the liver (75) and an increase in peripheral glucose utilization. Although it is well recognized that there is an increase in peripheral glucose utilization after endo- toxin administration, the primary tissues involved in this response have not been completely defined. Hinshaw et a1 (67) have shown that the heart-lung system is not involved in this response. Raymond and Emerson (99) demonstrated that glucose uptake by the central nervous system decreases during endotoxin shock in the dog. Hinshaw and associates (lflfl) have observed an increase in glucose uptake by leuco- cytes from dogs given endotoxin, but it is improbable that such a small tissue mass could be totally responsible for the large increase in glucose utilization observed during endotoxin shock. Because skeletal muscle comprises a large percentage of total body mass and can utilize large quanti- ties of glucose via anaerobic glycolysis, as occurs during 1 2 low flow states, it is a prime candidate for the increase in peripheral glucose utilization. Recent work by Raymond et a1 (83) demonstrated that glucose uptake by the naturally perfused gracilis muscle increases during irreversible E; 3211 endotoxin shock in the dog. The mechanism for this increased glucose uptake was shown to be related to local tissue hypoxia secondary to muscle ischemia (83). Glucose uptake was also reported to increase in the dog hindlimb during mild endotoxemia (152). On the other hand, Furr et al (101) saw no change in glucose metabolism by the isolated forelimb during endo- toxin shock in the dog. Even though systemic arterial blood pressure and forelimb blood flow decreased markedly after endotoxin administration, the isolated forelimb never became hypoxic. This paradox, viz. lack of local forelimb hypoxia in the presence of severe forelimb ischemia, would implicate a reduced forelimb metabolic rate such that the severely reduced forelimb blood flow and decreased oxygen delivery to the forelimb was adequate to meet the metabolic needs of the tissue. Upon review of the protocols of the isolated gracilis muscle (83) and isolated forelimb studies (101), a major difference was that temperature of the isolated forelimb was not maintained at core temperature as it was in the isolated gracilis muscle and glucose uptake by the isolated forelimb could be related to the temperature of the iso- lated organ (Qlfl effect). Furthermore, a reduction in 3 isolated organ temperature could have also blunted the metabolic response of skin to endotoxin. Therefore the hypotheses to be tested are: 1) that glucose uptake by forelimb skeletal muscle and skin increases during endo- toxin shock in the dog, and 2) that the mechanism for this proposed increase in glucose uptake is related to local tissue hypoxia and/or tissue ischemia. CHAPTER II SURVEY OF THE LITERATURE Structure of the Endotoxin Molecule Bacterial endotoxin is a constituent of the outer cell wall of many gram-negative bacteria (1), which includes the common intestinal organism Escherichia coli. In fact E; £311, of the family Enterobacteriaceae, is the most common- 1y found facultative anaerobe in the fecal flora of many mammals (2). Endotoxins were detected in cell free fil- trates of autolyzed gram-negative cultures more than 108 years ago, indicating that these cells release endotoxin into the surrounding medium upon lysis or disintegration (3,4). The chemical composition of endotoxin has been elucidated through the efforts of numerous investigators (59). Specifically, endotoxin exists as a large macromole- cular complex composed of polysaccharides, lipids some amines, phosphorous and small quantities of proteins (57). The chemical structure and molecular arrangement of the various substituents of the cell wall of gram-negative bac- teria has recently been described as consisting of three distinct layers: a) an outer membrane; b) a layer of peptidoglycan (murein net); and c) an inner cytoplasmic membrane. Costerton et a1 (6) and Braun (7) give similar 4 5 descriptions of the cell wall in that it consists of two lipoprotein bilayer membranes separated by a periplasmic space. Based on chemical structure analysis, Braun (7), devised a model for the supermolecular structure of the rigid layer (murein—lipoprotein complex) of the cell wall of E; 3313. According to his data, approximately 250,000 lipoprotein molecules of known amino acid sequence are evenly distributed over the one-layered murein net (10). The murein net is made up of disaccharide units composed of N-acetyl—muramic acid linked by beta-1,4-glycosidic bonds to N-acetyl—glucosamine. These subunits are then cross— linked by short peptide bonds to form a large polysacchar— ide macromolecule (molecular weight 1-4 X 109 daltons) (7). The multi-layered cell wall of gram-negative bacteria has been attributed many functions including, protection of the organism in a wide range of environments, conferring rigidity to the microorganism, excluding certain toxic sub— stances, regulating the passage of ions, and binding spe- cific enzymes (6). The Toxic Factor For many years investigators have attempted to deter— mine which part of the lipopolysaccharide molecule of gram— negative bacteria was responsible for its vast array of pathophysiological effects. For example, endotoxin has been shown to inactivate complement (11), induce fever (12,13), initiate local and generalized Shwartzman react- ions (14), and deplete animals of their carbohydrate 6 reserves (14). Research during the last decade points to the lipid A molecule as being the toxic factor responsible for these pathophysiological alterations (2,16). Listed above are a few of the biological responses used to determine the relative potencies of different lipo- polysaccharides. For example, it has long been known that lipopolysaccharides can interact with complement in vitrg and cause its inactivation (11). This inactivation of com- plement by endotoxin has been shown to be a good qualititive measure of the endotoxicity of different lipopolysaccharide preparations (8,17). Bioassays such as this are routinely used to differentiate between active and inactive lipopoly— saccharides. The two major constituents of endotoxin were first dis- covered by Bovin, Mesrobeanu, and Mesrobeanu (18,19) in 1933. Extraction of endotoxin with trichloroacetic acid resulted in the isolation of a phosphorous-containing lipid and a degraded polysaccharide. Although the polysaccharide was neither immunogenic nor toxic, the lipid fraction ex— hibited residual toxicity (18). This finding suggested that specific components within the endotoxin molecule are responsible for its,toxic effects. Twelve years later, in 1945, Brinkley, Goebel, and Perlman attempted to identify a substructure within the endotoxin complex as being the toxic element (20). They split the endotoxin complex with alkaline and acidic hydro- lysis. Treatment with dilute acid led to a so-called, 7 non-toxic polysaccharide and a toxic lipoprotein, while treatment with alcholic alkali gave rise to a nontoxic protein and toxic lipopolysaccharide (20). The authors concluded from these experiments the existence of a toxic "T” factor which was neither protein nor polysaccharide. At the time, the authors did not realize the existence of lipid A in the complex. In the early fifties, Westphal and colleagues (21) claimed that lipid A was the biologically active component of the endotoxin molecule. Using mild acid hydrolysis, lipid A was precipitated from the parent endotoxin molecule and its biological properties investigated (21). The lipid A fraction exhibited one-tenth of the pyrogenic activity of the parent endotoxin molecule. Since the lipid A fraction no longer contained the polysaccharide carrier which makes it water soluble, it was assumed that the reduced solubili- ty of the isolated lipid A was responsible for its reduced pyrogenicity (8,22). However at the time, Westphal and colleagues could not present conclusive evidence in support of their theory i.e. that the lipid component is responsi— ble for the endotoxic activities of lipopolysaccharides. Meanwhile, Ribi, Landy, and coworkers (23) disagreed with Westphal's (21) theory that the lipid component is responsible for the endotoxic activities of lipopolysaccha- rides. Ribi et a1 (23) reasoned that since lipopolysaccha- ride preparations having minimal lipid A content were able to elicit potent endotoxic activity, the endotoxic 8 properties of lipopolysaccharides could not reside within .the lipid A complex. However, conclusive proof could not be presented since an entirely lipid-free endotoxin prepa- ration with full endotoxin activities could not be pro- duced. Such a preparation still does not exist. The question as to whether lipid A was primarily re- sponsible for the toxicity of the endotoxin molecule re- mained unanswered for nearly a decade until the discovery of so-called rough mutants that are defective in the syn— thesis of complete lipopolysaccharides. Subsequent inves- tigations using Salmonella minnesota R595, which contains only lipid A and a trisaccharide, 2—keto-3-deoxyoctonate (KDO), made the final answer possible (16). This lipid A- KDO complex was found to be as toxic as lipopolysaccharides from gram-negative bacteria (5). This finding suggested that the polysaccharide component of the lipopolysaccharide molecule is not essential for endotoxicity (16,24). Suc- cinylation did not diminish the toxicity of the lipid A-KDO complex (16-24). Finally, the lipid A-KDO complex was split with dilute acid to liberate free KDO units and water insoluble lipid A. In 1972, it was found that lipid A could be made soluble by complexing it with bovine serum albumin (24). Subsequent investigations with complexes of lipid A and bovine serum albumin revealed enhanced endotoxicity (25— 27). These experiments conclusively demonstrate that lipid A is the toxic component of the endotoxin complex. They 9 also demonstrated that the polysaccharide component is not specifically involved in endotoxicity. However, polysac— charides are important by acting as solubilizing agents allowing lipid A to interact with the host (8). General Systemic Hemodynamic Effects of Endotoxin Gram—negative septic shock is a serious clinical prob- lem with a high mortality rate in spite of significant advances in antimicrobial therapeuticsl(28). Recent re- ports have estimated between 70,000 to 100,000 persons a year die from septic shock in the United states (29). The effects of endotoxin on the host are extensive and extreme- ly complex, often leading to severe hemodynamic dysfunction and death. Circulatory shock of any etiology is characterized by lack of adequate tissue perfusion, which in itself causes tissue damage leading to further insufficiencies, setting up a vicious positive—feedback cycle resulting in complete circulatory deterioration and death. A decrease in tissue perfusion precipitates the development of tissue hypoxia which results in a variety of metabolic disorders at the cellular level (30). This aspect of circulatory shock will be discussed in a later section (see Metabolism during Shock). Cardiovascular function during endotoxin shock has been extensively studied in a variety of animal species, of which the canine is most popular. Hence, the largest body of knowledge about cardiovascular function during endotoxin l0 shock comes from the canine model on which the following discussion will be focused, with any exceptions noted. The overall hemodynamic response of the dog during lendotoxin shock is characterized by severe hypotension, hepatic portal hypertension, bradycardia, and an increased total peripheral resistance early in shock followed by a gradual decline towards or below control later in shock. The mechanism responsible for the paradoxical decrease in heart rate in the presence of severe hypotension has not been clearly established. It has been suggested by Trank and Visscher (31) and others (32,33) that this paradoxical cardiac response may be due in part to a resetting of arterial baroreceptors. A recent review by Hess et a1 (34) suggested that the second fall in arterial blood pressure during endotoxin shock is due to myocardial dysfunction. Whether myocardial dysfunction is due to a circulating myocardial depressant factor or a decrease in coronary perfusion is still contro- versial (35,36). It has also been postulated that the second fall in arterial blood pressure that occurs during the later phases of shock could be due to the progressive sequestration of blood in capacitance vessels and/or trans- capillary movement of fluid from the microvasculature into the surrounding tissues (37). This hypothesis will be discussed in a later section (see Transcapillary Fluid Fluxes during Shock). ll Endotoxin administered intravenously as a rapid bolus injection in the dog causes a decrease in systemic arterial blood pressure from approximately 150 mmHg to 50 mmHg within 2 to 5 minutes of injection. The initial fall in arterial blood pressure is followed by a compensatory rise in pressure which approaches near normal levels and lasts for approximately 20 to 60 minutes. Arterial pressure then declines progressively over a 4 to 6 hour period until death of the animal (38). The initial decline in arterial pressure has been attributed to a decrease in cardiac output subsequent to a decrease in venous return due to hepatosplanchnic pooling of blood (39,40). The classical studies of MacLean, Weil, Spink and Vis- scher (39,40) clearly demonstrated that the initial fall in arterial pressure after endotoxin administration in the dog is due to a reduced cardiac output subsequent to a decrease in venous return. Because arterial pressure is directly determined by cardiac output and total peripheral resis- tance, experiments were conducted to determine whether the initial drop in arterial pressure was due to a decrease in cardiac output or total peripheral resistance, or a combi- nation of the two. This was tested by experiments in which the total venous return of the dog was diverted into a reservoir from which it was pumped at a constant rate back to the right atrium (40). Since cardiac output is main- tained constant in this preparation, except for brief tran- sient changes due to accumulation or release of blood by 12 the lungs, a change in arterial pressure can occur only if total peripheral resistance changes. With this prepara— tion, no significant decline in arterial pressure was pro— duced by endotoxin for approximately 30 minutes, indicating that the initial fall in arterial pressure was not due to a decrease in total peripheral resistance. If total periph- eral resistance had fallen, arterial pressure would have also declined since cardiac output was maintained constant. However, this was not the case, therefore the initial fall in arterial pressure was due to a reduced cardiac output. This statement is supported by the fact that after adminis— tration of endotoxin, the volume of blood in the reservoir rapidly fell. Changes in the volume of blood in the reser— voir reflect changes in the inflow rate or venous return because the volume of blood in the reservoir is pumped out at a constant rate. Therefore, since cardiac output is actually a reflection of the total venous return, it was concluded that the initial fall in arterial pressure was due to a reduction in cardiac output subsequent to a de- crease in venous return, as evidenced by a decrease in the volume of blood in the reservoir (40). MacLean and Weil (39) and others (37,40,41) have ably demonstrated that the decrease in venous return in endotox- in shock in the dog is caused primarily by hepatosplanchnic pooling. MacLean and Neil (39) were also the first to note that after administration of endotoxin there was an imme- diate precipitious drop in arterial pressure and a 13 simultaneous rise in portal venous pressure. Extensive hemorrhagic congestion and edema of the liver and intes- tines were also noted. The fact that portal vein pressure was elevated allowed them to hypothesize that there must be an increase in resistance to outflow through the liver which would cause stasis and a loss of circulating blood volume. To answer this question, dogs were eviscerated and given endotoxin while arterial blood pressure was monitored (39). Numerous investigators have reported that when the hepatosplanchnic organs are removed or isolated from the circulation there is no immediate fall in arterial blood pressure or venous return (37,39). However, it should be pointed out that even though the initial drop in systemic arterial blood pressure can be prevented by removing the hepatosplanchnic organs (evisceration), a slow decline in arterial pressure is observed approximately 30 minutes after endotoxin administration, reaching lethal levels within 2.5 hours. Hinshaw et al (37) have also demon- strated that when cardiac output is held constant in either eviscerated or non-eviscerated dogs, a slow decline in systemic arterial blood pressure occurs. Hinshaw et a1 (37) indicated that the progressive fall in systemic arte- rial blood pressure observed in both ediscerated and non- eviscerated dogs in the presence of constant cardiac fill- ing was due to a gradual decline in total peripheral resis-' tance. These studies demonstrated that pooling of blood in the hepatosplanchnic organs is responsible for the decrease 14 in venous return and subsequent drop in cardiac output and arterial pressure after endotoxin administration. In summary, the initial fall in arterial blood pressure following endotoxin administration is due to a reduction in cardiac output subsequent to a decrease in venous return which is due to pooling of blood in the hepatosplanchnic organs. Transcapillary Fluid Fluxes during Shock As previously indicated, the initial fall in arterial pressure in the dog is due to a decrease in venous return subsequent to hepatosplanchnic pooling of blood (39). The possibility also exists that intravascular pooling of blood and/or extravascular pooling of fluid due to transcapillary fluid movement in sites other than the hepatosplanchnic organs may contribute to the decreasing cardiac output seen during both the early and late stages of endotoxin shock. The literature contains inconsistencies on the direction and location of transcapillary fluid movement during shock. Mellander and Lewis (42) reported a decrease in the pre- to postcapillary resistance ratio in muscle during hemorrhagic shock in the cat, resulting in a net movement of capillary fluid into muscle. Plasma volume, as determined by dye dilution techniques, has been shown to decrease during endotoxin shock in the dog, indicating net capillary fluid filtration (43). However, measurement of plasma volume during shock may be in error due to inadequate mixing of dye (44). 15 The studies of Weidner et a1 (45) and Hinshaw and Owens (46) demonstrated an increase in the pre- to postcapillary resistance ratio and a decrease in limb weight during endotoxin shock in the naturally perfused canine forelimb. The initial decrease in limb weight was largely attributed to a decrease in vascular volume subsequent to vasocon- striction, while the prolonged steady weight loss was due to extravascular fluid reabsorption subsequent to a fall in capillary hydrostatic pressure. Capillary hydrostatic. pressure would decrease during shock if the rise is post- capillary resistance was overwhelmed by the rise in precap- illary resistance and fall in aortic pressure. These stud- ies demonstrated that transcapillary fluid filtration or sequestration of blood in skin and/or skeletal muscle do not contribute to the decreasing venous return during endo- toxin shock in the dog.. The Role gf Histamine in Endotoxin Shock Various vasoactive substances such as catecholamines (47), serotonin (48), histamine (49), kinins (50), prosta- glandins (51), proteolytic enzymes (52) and beta—endorphins (53) are released during endotoxin shock. Of these hista— mine appears to play a dominant role in the early phase of endotoxin shock in the dog. Weil and Spink (41) recognized the similarities of the early effects of endotoxin to those seen in anaphylaxis. Their observation suggested the pos- sibility that histamine, which is known to be released during anaphylaxis, may also participate in endotoxin l6 shock. Plasma samples from the inferior vena cava upstream from the hepatic veins contained appreciable quantities of histamine (41). Kobald et a1 (54) demonstrated that hista- mine and other vasoactive polypeptides are present in high concentration in the venous blood draining the portal cir- culation of dogs shocked with endotoxin. Hinshaw et a1 (49) demonstrated a rise in unbound histamine and a de— crease in the bound form of histamine in whole blood within two minutes after endotoxin administration. Changes in whole blood and plasma histamine levels were explained on the basis that histamine is released from the bound form in whole blood (mast cells and basophils) and other tissue to the unbound form in plasma. Shayer (55) reported that endotoxin increased histidine carboxylase activity, thus accelerating the rate of histamine synthesis. Subsequent- ly, Hinshaw et al (56) demonstrated an accelerated conver- sion of histidine to histamine following endotoxin adminis— tration. Bauer et a1 (57) have shown that the hepatic veins contain smooth muscle sphincters, which are believed to be species-specific to the dog (58). Because apprecia- ble quantities of histamine were found in the venous blood draining the liver immediately after endotoxin administra- tion (41), combined with the fact that histamine acts primarily on smooth muscle and therefore on smooth muscle spincters located in the hepatic veins, it was suggested that histamine might be responsible for the rise in portal venous pressure and hepatosplanchnic pooling. Hinshaw et 17 a1 (59) demonstrated that pretreatment with the histamine- releasing agent 48/80 alters the typical response of endo- toxin, viz. portal vein pressure changed little and the rapid drop in systemic arterial pressure did not occur after endotoxin administration. It should to noted that injection of histamine or endotoxin causes an elevation of portal vein pressure and a substantial decrease in arterial pressure (59). Pretreatment with phenoxybenzamine, an alpha—blocker, also prevented the typical response to endo- toxin (60). However, depending on the concentration, phen- oxybenzamine has variable blocking properties on many a- gents such as histamine, epinephrine, norepinephrine, sero- tonin, and acetylcholine (61). To demonstrate that catecholamines were not responsible for the early rise in portal vein pressure, splanchnic neurotomy and/or adrenalectomy were performed on dogs (62). These interventions did not prevent the portal venous hy- pertension or systemic hypotension after endotoxin, sug- gesting that the early vascular response to endotoxin is not mediated by catecholamines. Furthermore, it has been demonstrated that catecholamines are released subsequent to the development of hypotension indicating that these agents probably do not play a role in the initial phase of endo- toxin shock (62). In summary, histamine induces a constriction of the hepatic veins with resultant venous pooling in the hepato— splanchnic organs, thus resulting in a decrease in venous 18 return, a reduced cardiac output, and a fall in systemic arterial blood pressure. Metabolism during Shock It has been known for many years that carbohydrate homeostasis is altered during circulatory shock in a vari- ety of animal species. As early as 1877, Claude Bernard reported increased blood glucose levels in dogs subjected to experimental hemorrhagic shock and also in man after accidental injury (63). In 1924, Menten and Manning (64) demonstrated that rabbits given endotoxins from gram-nega— tive bacteria developed a transient hyperglycemia followed by a progressive decline in arterial blood glucose concen— tration to hypoglycemic levels. The subsequent literature is replete with studies indicating that carbohydrate homeo— stasis is altered in a variety of pathogenic states includ- ing hemorrhagic (65), cardiogenic (66), bacteremic (67) and endotoxin shock (15). In 1946, Engel (68) described a variety of metabolic defects in various tissues due to hemorrhagic shock, and stated that ”from a biochemical standpoint there does not appear to be a great deal of difference between the various types of shock, for the common denominator in all is tissue anoxia." In 1972, a paper published by Lundsgaard-Hansen et a1 (69) supported Engel's basic premise; they stated that the decisive factor impairing cellular metabolism during endotoxin shock was the state of shock which it produces. A recent review on cellular metabolism during 19 shock by Schumer et a1 (30) clearly indicates that inade- quate tissue perfusion and the ensueing tissue hypoxia is the common denominator in all forms of shock. It is clear from the foregoing examples that a reduction in blood flow to the tissues has profound effects on the host by produc- ing disturbances in the normal aerobic metabolic pathways. It is the purpose of this thesis to review the litera— ture specifically dealing with carbohydrate metabolism during endotoxin shock and its pathophysiological conse- quences. However, since many of the metabolic consequences of endotoxin shock are similiar to various other forms of shock, studies done in these shock states will also be reviewed when the observations are applicable to endotoxin shock. Alterations in Blood Glucose during Endotoxin Shock Reviews by both Hinshaw (15) and Berry (70) on carbo- hydrate metabolism during circulatory shock stress a basic theme of hyperglycemia followed by a progressively develop- ing hypoglycemia. In 1970, Berk and associates (71) re- ported in an exhaustive study that a majority of dogs receiving various doses of E; 3311 endotoxin an early and transient hyperglycemia lasting for approximately one hour was observed, which was followed by a gradual decline in blood glucose levels. In the remainder of the dogs receiv- ing endotoxin, blood glucose levels declined immediately and remained low. They also noted that dogs having a more rapid and greater fall in blood glucose concentration 20 tended to die earlier than dogs exhibiting a gradual de— cline in blood glucose conCentration. Filkins et al (72) demonstrated that rats subjected to endotoxin or traumatic shock developed low levels of blood glucose within 5 hours. Hypoglycemia has also been report- ed in humans during septic (71) and cardiogenic shock (66). Groves et a1 (73) reported hypoglycemia during live E; 3311 bacteremic shock in the dog. Wolfe et a1 (74) reported that low doses of E. coli endotoxin administered to con— scious dogs induced a transient hyperglycemia reaching a peak within 15 minutes which was followed by a decrease in blood glucose concentration over the ensueing 4 hours of shock. Several studies have indicated that the dog seems to be more prone to developing hypoglycemia at a faster rate than primates, which generally exhibit hyperglycemia with hypoglycemia only occuring as a terminal event (67,71). However, Hinshaw's group (67) in recent studies observed an initial period of hyperglycemia followed by 4 to 15 hours of progressively developing hypoglycemia in baboons shocked with live E; 3311 bacteria. In summary, the net result of an intravenous bolus injection of purified endotoxin or administration of live E. coli bacteria in a variety of animal species, including —- the rat (75), rabbit (76), dog (15), and subhuman primate (67), is an early increase in blood glucose levels followed by a depletion of the animal's carbohydrate reserves and eventual hypoglycemia. 21 Hyperglycemia during Endotoxin Shock During the early phase of endotoxin shock blood glucose levels are elevated because of an increased mobilization of glucose by the liver via gluconeogenesis and glycogenolysis (75). The kidney is also theoretically capable of elevat- ing blood glucose levels via gluconeogenesis, however, Archer et a1 (77) reported the absence of a gluconeogenic role of the kidney during endotoxin Shock. Activation of the sympathoadrenal system in shock sub— sequent to a drop in systemic arterial pressure (62) causes the release of a variety of glucoregulatory hormones in— cluding epinephrine (47), glucagon (78), and the glucocort- icoids (79). It should be noted that secretion of insulin is relatively depressed during the hyperglycemic phase of shock because of sympathetic inhibition (80). Adrenalect- omy (81) or alpha-adrenergic blockade (82) will prevent the decrease in insulin secretion. Depressed plasma insulin levels during the early phase of endotoxin shock is impor- tant because insulin is a diabetogenic hormone i.e. it reduces blood glucose concentration. Consequently, there is no hormonal mechanism for counteracting the early rise in blood glucose levels after endotoxin administration. However, blood glucose levels do decrease during endotoxin shock and this decrease has been attributed to non-hormonal factors, viz. local tissue hypoxia (83) and the insulin- like activity of the endotoxin molecule (84) combined with a decrease in glucose production by the liver (75) and 22 kidney (77). These factors will be discussed in a later section (see Hypoglycemia during Endotoxin Shock). It is a well known fact that the breakdown of glycogen is initiated by the action of the enzyme of phosphorylase "a", which is specific for the breakdown of glycogen to yield glucose—l—phosphate (85). However, for this to occur phosphorylase "a" must be converted from its active form phosphorylase "b". The conversion of phosphorylase "b“ to phosphorylase "a" occurs as a result of stimulation of the enzyme adenylate cyclase by either epinephrine or glucagon. Through a series of enzymatic steps, adenylate cyclase eventually causes the conversion of phosphorylase "b" to phosphorylase "a" and the breakdown of glycogen. It has also been shown by Bitensky and associates (86) that endo— toxin stimulates the enzyme adenylate cyclase in mouse liver, which initiates the enzymatic steps necessary to cause activation of phosphorylase "a" and glycogenolysis. McCallum and Berry (87) demonstrated that endotoxin selec- tively inhibits glycogen synthase, the enzyme responsible for glycogen synthesis. These studies, plus the fact that hepatic glycogen stores are exhausted after endotoxin ad- ministration (73), lend strong support to the hypothesis that the initial phase of hyperglycemia is due, at least in part, to increased glycogenolysis. The formation of new glucose via gluconeogenesis has been shown to be affected biphasically during endotoxin shock (75). Characteristically, during the early phases of 23 irreversible endotoxin shock this process and the formation of new glucose is increased, however during the later stages of shock gluconeogenesis is depressed (72,75). The release of glucocorticoids (79), combined with the increase in gluconeogenic substrates such as lactate and alanine (72), have been shown to be responsible, at least in part, for the increase in gluconeogenesis during endotoxin shock. The glucocorticoids stimulate gluconeogenesis by stimulat- ing several enzymes in the liver to cause accelerated conversion of gluconeogenic substrates into glucose (85). The factors responsible for the depression of hepatic glu- coneogenesis during the later stages of endotoxin shock appear to be highly diversified and will be discussed in the following section (see Hypoglycemia during Endotoxin Shock).‘ Filkins et al (116) examined the relationship between fasted and fed rats and how enhanced insulin secretion is deleterious in endotoxin shock. Overnight fasting has been shown to markedly depress insulin levels as compared to the fed state. It was subsequentially shown that fed rats exhibited a greater sensitivity to endotoxin than overnight fasted rats. The fact that elevated insulin levels were sensitizing rats to endotoxin lead the authors to conclude that hyperinsulinemia was affecting the liver by preventing it from converting from a glycogenolytic organ to a gluco— neogenic organ. Whereas in the fasted state the liver is 24 already a gluconeogenic organ and is not as susceptible to gluconeogenic depression. In summary, the increase in blood glucose levels during the early phases of endotoxin shock is caused by an in- creased mobilization of glucose by the liver via gluconeo- genesis and glycogenolysis (75). Hypoglycemia during Endotoxin Shock Profound hypoglycemia during the intermediate and later stages of endotoxin shock is a well documented fact in-a variety of animal species. Hinshaw (15) recently reviewed the role of glucose in endotoxin shock and stated that ”hypoglycemia is not merely a terminal event but is corre- lated with the pathogenesis of shock.” Therefore, the mechanisms responsible for the shift from hyperglycemia to hypoglycemia during endotoxin shock are fundamentally im— portant to our understanding of its pathogenesis. A depletion of blood glucose levels during endotoxin shock could occur as the result of one or any combination of the following: 1) a loss of glucose through excretion, 2) a decrease in glucose production, and 3) an increase in glucose utilization. In shock, there is no evidence that glucosuria occurs (88). 'In cardiovascular collapse of any etiology renal shutdown is a feared complication. As to the second factor, decreased glucose production, Filkins et al (89) demonstrated a depression of gluconeogenesis as determined by in 3132 and in yitrg studies of the rat liver. They suggested that the depression appeared to be 25 caused by a mediated effect rather than a direct one, because hepatocytes incubated with endotoxin la glgaa showed no change in capacity for gluconeogenesis. Groves et a1 (73) reported impaired gluconeogenesis during live E; gall bacteremic shock in the dog and suggested that there appears to be a metabolic block between pyruvate and the formation of glucose in the gluconeogenic pathway. A study by Lanoue et a1 (90) demonstrated that the overall rate of gluconeogenesis in rat liver was impaired by endotoxemia and indicated that a decrease in glucose-6-phosphatase was the cause. Williamson's group (91) also demonstrated im- paired gluconeogenesis during endotoxin shock in the rat and suggested that there is a defect in the enzymatic step between fructose-1,6-diphosphate and fructose-G—phosphate, as evidenced by an accumulation of fructose-1,6-diphos- phate. Sufficient evidence therefore exists to document that in endotoxin shock glucose production is decreased and that this decrease is due essentially to a shutdown of gluconeo- genesis. However, the mechanisms responsible for the fail- ure of gluconeogenesis appear to be highly diversified. The third factor which can cause a decrease in blood glucose levels during shock is an increase in glucose utilization. Several investigators have established the contribution of the peripheral tissues to the development of hypoglycemia during shock by eliminating the liver, spleen, pancreas and the entire gastrointestinal tract from 26 the circulation (65,77,92). As early as 1944, Russel et al (65) reported rapid and profound hypoglycemia in eviscer- ated rats during hemorrhagic shock, which they attributed to inefficient metabolism of glucose via anaerobic glycoly- sis due to peripheral anoxia. More recently, Peyton et a1 (92) demonstrated similiar results during endotoxin shock in eviscerated dogs and attributed the decline in blood glucose levels to a predominance of anaerobic over aerobic metabolism of glucose. These studies (65,92) have demon— strated that peripheral carbohydrate utilization increases during shock and that the mechanism responsible for this increase is primarily due to the ineffecient metabolism of glucose via anaerobic glycolysis. A prominent feature of many pathological processes including sepsis is an increase in body temperature, which is often associated with an increased metabolic rate be- cause of the influence of temperature on chemical reactions (93). Dubois (94) pointed out that for every degree fahr- enheit increase in temperature there is a corresponding 7.2 percent increase in caloric expenditure. However, Roe and Kinney (95,96) demonstrated that the increased metabolic rate of septic patients was in excess to that predicted for an elevated temperature alone. Furthermore, a case study by Halmagyi et a1 (93) pointed out that some normothermic septic patients have elevated metabolic rates. Long et a1 (97) reported that the rate of glucose oxidation was more that doubled in septic patients. Hinshaw et a1 (98) 27 demonstrated that large quantities of glucose were needed to maintain constant blood glucose levels in endotoxin- shocked dogs and that exogenously administered glucose prevented death. It should be evident from the preceeding studies that there is an elevated metabolic rate associated with sepsis and that fever alone cannot account for the increase in energy requirements. Although it is well recognized that there is an in— crease in peripheral glucose utilization during endotoxin shock, the primary organs responsible for this increase have not been completely defined. Hinshaw et al (67) have demonstrated that neither the myocardium nor the lungs are sites of increased glucose utilization during endotoxin shock. Raymond and Emerson (99) demonstrated that glucose uptake by the central nervous system decreases during endo— toxin shock in the dog. Hinshaw and coworkers (100) demon- strated that glucose uptake by the leucocyte (WBC) mass increases during endotoxin shock in the dog. However, it is improbable that such a small tissue mass (WBC) could be totally responsible for the large increase in glucose uti- lization observed during endotoxin shock. Skeletal muscle has been considered a prime candidate for the increase in glucose utilization during endotoxin shock because it can utilize large quantities of glucose via anaerobic glycolysis, as occurs during low flow states. However, in 1978 a study by Furr et al (101) showed no change in glucose uptake by the isolated dog forelimb 28 during E; gall_endotoxin shock. On the other hand, recent work by Raymond et a1 (83) demonstrated that glucose uptake by the naturally perfused gracilis muscle increased during endotoxin shock in the dog. The mechanism for this increased glucose uptake was shown to be related to local tissue hypoxia secondary to muscle ischemia (83). At about the same time, Romanosky et al (102) reported that glucose uptake increased in the dog hindlimb during mild endotoxemia and that there was an increase in lactate and alanine concentra- tion in femoral blood draining the non-isolated hindlimb during moderate endotoxin shock. However this study should be viewed with skepticism since the hindlimb muscle was not vascularly isolated which makes it difficult to know the percent contribution of pure skeletal muscle to the observed changes. It is also difficult to explain the changes in calculated vascular resistance in this hindlimb preparation, which did not change during the first 30 minutes of shock, and then decreased substantially during the entire 4 hour shock period. This is surprising since both active and passive forces which accompany systemic hypotension would dictate an increase in vascular resistance in the hindlimb. Indeed, vascular resistance has been reported to increase substantially during endotoxin shock under conditions of natural and constant blood flow in dog forelimb (46,103), in skeletal muscle and skin of the dog forelimb (104), and in the gracilis muscle (hindlimb) (83,84). 29 This conclusion is not in agreement with work by Ray- mond et a1 (83) where it was reported that glucose uptake by the isolated gracilis muscle did not increase during endotoxin shock when blood flow to the muscle was main- tained constant, preventing the development of local tissue hypoxia. The lack of an increase in glucose uptake by gracilis muscle during endotoxin shock was attributed to the fact that local tissue hypoxia was prevented and that following an intravenous bolus injection of endotoxin the endotoxin molecules are rapidly cleared from the circula- tion by the reticuloendothelial system (105). It was also shown in a separate study, as indicated previously, that glucose uptake by the naturally perfused gracilis muscle increases during E; gall endotoxin shock and that the mechanism for this increased glucose uptake was related to local tissue hypoxia. However, while glucose uptake by the constant flow perfused gracilis muscle does not increase during endotoxin shock, gracilis muscle glucose uptake does increase in the constant flow perfused muscle during live g; gall bacteremic shock (84). This was believed to occur because the endotoxin molecules are released slowly as the live bacteria are killed and the reticuloendothelial system is unable to adequately clear the continually released endotoxin. Concerning the research of this thesis, we were some- what puzzled by the study of Furr et al (101), which showed no increase in glucose uptake by the naturally perfused dog 30 forelimb during endotoxin shock. Since the forelimb con- tains a substantial amount of skeletal muscle there is no reason to suspect that muscle in the forelimb is any dif- ferent in its response to metabolic stimuli than skeletal muscle from the hindlimb. This apparent discrepancy bet- ween glucose uptake by the isolated gracilis muscle and glucose uptake by the isolated forelimb preparation is the subject of this thesis research. Although systemic arterial blood pressure and forelimb blood flow decreased markedly after endotoxin administra- tion, the isolated forelimb never became hypoxic in the Furr et al (101) study. This paradox, viz. lack of local forelimb hypoxia in the presence of severe forelimb ische- mia, would implicated a reduced forelimb metabolic rate such that the severely reduced forelimb blood flow and decreased oxygen delivery to the forelimb was adequate to meet the metabolic needs of the tissue. Upon review of the protocols of the isolated gracilis muscle (83,84) and isolated forelimb studies (101), a major difference was that temperature of the isolated forelimb was not maintained at core temperature as it was in the isolated gracilis muscle. Therefore, glucose uptake by the isolated forelimb could be related to the temperature of the isolated organ (Q10 effect). Furthermore, a reduction in isolated organ temperature could have also blunted the metabolic response of skin to endotoxin. Therefore the hypotheses to be tested are: 1) that glucose uptake by 31 forelimb skeletal muscle and skin increases during endo- toxin shock in the dog; and 2) that the mechanism for this proposed increase in glucose uptake is related to local tissue hypoxia and/or tissue ischemia. CHAPTER III METHODS Experimental Animals Mongrel dogs of either sex weighing 2012 kg were fasted for 16 to 24 hours but allowed water ga libitum prior to use in this study. The animals were anesthetized with an intravenous infusion of pentobarbital sodium (30 mg/kg) and maintained at a surgical level of anesthesia with supple- mental doses of anesthetic (1 ml of a 50 mg/ml solution). ‘Surgical anesthesia was indicated by the loss of the wink reflex in response to touching the conjuctiva. The anesthetized animal was placed on a surgical table on its right side. The trachea was intubated with a cuffed endotrachial tube, and the animal ventilated with room air using a Harvard constant volume respirator. Positive end- expiratory pressure was employed to maintain acceptable arterial oxygen tension by submerging the end of the expir- atory line approximately 3 centimeters under water. Heparin sodium (10,000 U.S.P. units) was administered intravenously before the extracorporeal system was established to prevent clotting. Supplemental doses of heparin sodium (200 U.S.P. units) were given hourly. 32 33 Forelimb Preparation The isolated, innervated canine forelimb was employed as the test organ. The brachial artery, brachial vein, cephalic vein and forelimb nerves were isolated and kept intact after circumferentially sectioning the skin approxi— mately 3 centimeters above the elbow using a thermocautery. The remaining muscles and connective tissue were isolated and sectioned using a thermocautery. The humerus was cut with a bone saw and the ends of the marrow cavity packed with bone wax and capped with latex to prevent the exposed ends of the humerus from bleeding. The brachial and cephalic veins were retrogradely can- nulated with polyethylene (PE—320) tubing and their out— flows allowed to drain by gravity into a 600 ml glass reservoir. The reservoir was primed with either physiolog— ical saline or high molecular weight Dextran (79,000 dal- tons) prior to starting the extracorporeal circuit. Dex— tran, a volume expander, was used when arterial blood pressure was declining due to excessive surgical set—up time or blood loss. The level of blood in the reservoir was maintained constant using an electronic—liquid—level controller to prevent changes in total body blood volume during the course of the experiment. Blood was returned to the cannulated (PE—320) left external jugular vein by means of a Masterflex blood pump (model no. 7520-00). The reser- voir was placed on a combination stirrer—warmer to prevent 34 formed elements from accumulating on the bottom and to maintain blood temperature at core temperature, respective- ly. The forelimb stump was then bathed with mineral oil and covered with plastic wrap to prevent drying. After surgically isolating the forelimb, blood entered the forelimb only through the brachial artery and exited only through the brachial and cephalic veins. The median cubital vein, which represents the major anastomotic chan- nel between the brachial and cephalic veins, was ligated to ensure that brachial venous outflow was predominately from skeletal muscle and that cephalic venous outflow was pre- dominately from skin (106). Mean systemic arterial blood pressure was measured from the cannulated (PE-240) right femoral artery. The right femoral vein was cannulated (PE-240) for administration of endotoxin and/or saline and drugs. Pressures were recorded using Hewlett-Packard pressure transducers (model no. 1280) coupled to a 4-channe1 Hewlett-Packard direct writing re- corder (model no.77543). Temperature of the isolated forelimb, contralateral (intact) forelimb, and the animals core temperature was monitored with subcutaneously placed thermistor probes and a rectal probe, respectively. Temperatures were recorded using a Yellow Springs Tele-Thermometer (model no. 43TF). An electric heating pad was placed under the animal to maintain core temperature at control. .a. l . . . .. . a .. .w. ... .h . It I. t s... .. . .1. . n . w. .. . . . I v . E . i I I i i . . _ m u I .. q . . r . . . . w . . .. .. flu . . .u a . . 35 Experimental Procedures 95222 lg Controlled Forelimb Temperature-Natural Flow The surgical preparation of the isolated forelimb was as described above and provisions for temperature regula- tion were made. This was accomplished by wrapping the isolated forelimb in a temperature regulated water jacket and covering it with 4 towels and plastic wrap. Isolated forelimb temperature was then maintained at the same tem- perature as the contralateral (intact) forelimb, which was equal to the animals core temperature. This preparation was completed in 10 experimental animals and 6 control animals. 95222.31 Uncontrolled Forelimb Temperature-Natural Flow After surgically isolating the forelimb as described above, it was placed on the surgical table and exposed to ambient room temperature (23 degrees celcius) with no pro- vision for maintaining forelimb temperature at contralat- eral (intact) forelimb temperature. This preparation was completed in 8 experimental and 6 control animals. 93222 lg Controlled Forelimb Temperature~Constant Flow After surgically isolating the forelimb and equipping the forelimb for temperature maintenance as described above (Group 1), a Masterflex blood pump was interposed between the cannulated left femoral (PE-320) and right brachial artery (PE-320) for constant flow perfusion. Total fore- limb blood flow was set by adjusting the perfusion pump so that forelimb perfusion pressure approximated mean systemic 36 arterial pressure. Forelimb perfusion pressure was mea- sured from a needle tipped PE-90 catheter inserted into the perfusion tubing distal to the perfusion pump. Pressures were recorded as described previously. This preparation was completed in 4 experimental and 4 control animals. Chemical Analyses Simultaneous blood samples were collected anaerobically from the femoral artery pressure catheter and the brachial and cephalic venous outflow tubing. The following analyses were completed: 1) P02, PC02, and pH with a Radiometer Acid-Base analyzer; 2) glucose concentration with a Yellow Springs Glucose Analyzer; 3) hematocrit by microcentrifugation. Control systemic arterial blood pressure, muscle and skin blood flows and metabolic variables listed above were determined following a 15 to 30 minute stabilization per- iod. In the temperature controlled forelimb groups this: stabilization period allowed the temperature of the iso- lated forelimb to equilibrate with contralateral (intact) forelimb temperature. Shock was induced by a 5 minute intravenous infusion of 2 mg/kg purified E; gall endotoxin (LDl00; Lipopolysaccharide 8:8 127; Difco Co.) suspended in 20 ml of physiological saline. Hemodynamic and metabolic measurements were made before and at 30 minute intervals for 3.5 to 4 hours after shock induction or until death of the animal. Control animals received 20 m1 of physiologi- cal saline containing no endotoxin infused intravenously 37 over a 5 minute period. Otherwise, these animals were treated identically to the experimental animals. Calculations Forelimb skeletal muscle (MBF) and skin blood flows (SBF) were determined by timed collections of the brachial and cephalic venous outflows, respectively, using a grad- uated cylinder and stopwatch. Total forelimb blood flow (FBF) was obtained by adding muscle and skin blood flow. At the end of each experiment, muscle and skin were dis— sected from the forelimb and weighed so blood flow could be expressed on a weight basis. 1. MBF (ml/min/100gm) MBF (ml/min)/muscle wt. (gm) X 100 2. SEE (ml/min/l00gm) SBF (ml/min)/skin wt. (gm) X 100 3. FBF (ml/min/l00gm) (MBF (ml/min) + SBF (ml/min)) /forelimb wt. (gm) X 100 Forelimb skeletal muscle (MVR), skin (SVR), and total forelimb vascular resistances (FVR) were calculated by dividing forelimb perfusion pressure (FPP) by the appro— priate blood flow. 4. MVR (mmHg/ml/min/l00gm) FPP/MBF (ml/min/l00gm) 5. SVR (mmHg/ml/min/l00gm) FPP/SBF (ml/min/100gm) 6. FVR (mmHg/ml/min/l00gm) FPP/FBF (ml/min/100gm) Forelimb skeletal muscle (MGU) and skin glucose uptakes (SGU) were calculated as the product of the arterio-venous glucose difference and appropriate blood flow. Total fore- limb glucose uptake (FGU) was obtained by adding muscle and skin values. 38 7. MGU (mg/min/l00gm) = MBF (ml/min/100gm) X arterio- venous glucose difference (mg/ml) 8. SGU (mg/min/l00gm) = SBF (ml/min/l00gm) X arterio- venous glucose difference (mg/ml) Total forelimb venous POZ’ PC02 and pH was calculated by averaging the appropriate brachial and cephalic venous P02, PC02 and pH at each time period. Details al Whole Blood Glucose Determination Whole blood glucose concentration was determined with a Yellow Springs glucose analyzer (YSI model no. 23A). The YSI model 23A uses an oxidase enzyme hydrogen peroxide sensor which is highly specific for glucose. Glucose con— centration is determined with a sample of only 25 microli— ters, with no sample modification required. Glucose con- centration can be detected in the range of zero to 500 mg/dl with a sensitivity of l mg/dl. Principles al Operation The conversion of glucose and oxygen in the presence of glucose oxidase to form gluconic acid and hydrogen peroxide (Reaction 1) is the first reaction for the determination of glucose concentration. The platinum anode, which is part of the sensor probe, oxidizes a constant portion of the hydrogen peroxide (Reaction 2), formed in reaction 1. The current produced from reaction 2 is directly proportional to the glucose concentration in the sample. To complete the circuit, oxygen is reduced to water at the silver cathode (Reaction 3). 39 Reaction lg D-glucose + oxygen --—-§l353§E-2§i§353 ----- > gluconic acid + hydrogen peroxide Reaction 2: hydrogen peroxide ---------------- > 2 hydrogens + 2 electrons Reaction 2a 4 hydrogens + oxygen ----------------- > 2 waters + 4 electrons Statistical Analyses Data were analyzed using one- and two-way analysis of variance. One-way analysis of variance was used when com- paring uneven sample sizes, otherwise two-way analysis of variance was used. Uneven sample sizes were the result of animals dying during the course of the experiment. Means were compared using Duncans and Student-Newman-Keuls (SNK) tests. Duncans test, which is computationally identical to the SNK test except for the use of another table of criti- cal values, was used when the SNK test was unable to detect any significant difference between the means (107). The "Students T” test modified for unpaired replicates was used compare control, non-shocked animals to shocked animals of groups 1 and 3. A “P" value less than or equal to 0.05 was considered significant. CHAPTER IV RESULTS Hemodynamic changes and arterial glucose concentration of both the controlled and uncontrolled forelimb temperature shock groups are graphically illustrated in Figure 1. The numbers in parentheses at the top of Figures 1-12 and the numbers in the first line of Tables 1-6 represent the number of animals at each data point; the smaller numbers represent animals dying during the course of the experiment. In both groups, as shown in Figure 1, arterial blood pressure de- creased to approximately 55 mmHg by 30 minutes of shock (P<0.05) and remained below control for the duration of the experiment (P<0.05). Muscle, skin and total forelimb blood flows in both groups decreased to approximately the same level at 30 minutes of shock (P<0.05) and remained below control for the duration of the experiment (P<0.05). Mus- cle, skin and total forelimb vascular resistances were sig- nificantly increased throughout the shock period in both groups of animals (P<0.05). In both groups of animals, arterial glucose concentration increased at 30 minutes fol- lowing shock induction (P<0.05), then progressively de- creased for the duration of the experiment (P<0.05). This figure illustrates that the degree of systemic hypotension, 40 41 Figure 1. Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncon- trolled forelimb temperature groups during El gall endo— toxin shock. (n)=number of animals; ABP=mean systemic arte- rial blood pressure; BF=blood flow; R=vascu1ar resistances; PRU=peripheral resistance units expressed in mmHg/ml/min/l00gm; Art. Glc. Conc.=Arterial blood glucose concentration; a=P<0.05 relative to time zero; b=P<0.0l relative to time zero; Bars=SEM. 42 .H muswflm Angus—he . .1. . m . v . 1 a aisle/ml; ZAMJI/m/1. 111% mu. . .1\ 7.1% 11111 map 1......11fi1 1.1th .1|L\\........|..MM “airlimm11s. T111 £1-11. . 1X. 1113415 In sauna 1 1 on WHaHWNJTHxi/ --- ....HI --- .--- --- .--- .1 1.1.1.1.. 1.1 .../1.1.1.14 11 H H Rafi-I Tll. 1.22:2 611v... . . =1: 8 modvan. Q ....... o 38:: o .mlmizminmluarJT/n .wLJuTAmlIJuTI .-/ +6“ Qflfie A: E 8 6PT.. ...23 SSS—.282: .32.» as: 3:223"... 32; .u. I. .. . .m .— . i. w. I. (i .v. 1 . m1... . .. ta . a m .. .. . n .1 . .1 ,1 1.. ... u‘ . T ...I‘. .. ..ur in... W .... r u . . y .. .... . v ... 1.x. .. 1. . . T1 .. ..- ....fi..l .. a I t I .. 5 .. c g .... . ...... . ... 15.... 43 the reduced forelimb blood flows, the increased vascular resistances and the degree of hypoglycemia were similar in both controlled and uncontrolled forelimb temperature groups. Forelimb muscle glucose uptake, muscle venous P02 and core and isolated forelimb temperatures of both controlled and uncontrolled forelimb temperature shock groups are graphically illustrated in Figure 2. Following shock in- duction in the controlled forelimb temperature group, fore- limb muscle glucose uptake increased at 3% minutes following shock induction and remained elevated throughout the 4 hour experimental period (PE.GS). In the controlled forelimb tem- perature group, forelimb muscle venous PO2 decreased from 47 mmHg to 24 mmHg at 30 minutes of shock and remained depres- sed for the duration of the experiment (P<fl.01). In the uncontrolled forelimb temperature group, muscle venous P02 decreased to only 39 mmHg from a control value of 47 mmHg (P<0.05) at 36 minutes of shock and remained below control throughout the experiment (P<0.95). In the controlled forelimb temperature group, isolated forelimb temperature was not different from core temperature at any time before or during shock (P>a.05). However, isolated forelimb temperature did decrease slightly at 2 and 2.5 hours (P<6.05) and did increase slightly at 3 and 3.5 44 Figure 2. Muscle metabolic and temperature variables in the controlled and uncontrolled forelimb temperature groups during E; coli endotoxin shock. (n)=number of animals; a=P<fl.05 relative to time zero; b=P<@.01 relative to time zero; c=P<0.@5 comparing isolated forelimb temperature to the animal's core temperature at each time point; Bars=SEM. 45 u WIIIITITTI Winn-34933.....umuudm m.---m--..m.--.m..\m::m,/ Q as: 35358:: .32.” 0“".I I II J ‘m \\ E I 11113:: c . N a N p o q u d u u u 1 d 4 J on rigs: .. 0Q I ..- ii 1...; n ON ........---1..--...ii... . I. 1 m9 “8 van"- 5.o v11". .1 2. 3.0 v.1". sh J O M‘Olnmiclam IIIquIIuUiIILWOIOIMI \\ 1 . 1 A! E a Q 65".... N 5:.— SSEES .525 a: as: 9.32: 33...: mace W .E 53:: 38:3 33:8 46 hours (P<fl.@5) of shock compared to the control, preshock value. These temperature changes did not exceed Z.2 degrees celcius. Core temperature did not change significantly (P>fl.05) in either the temperature controlled or uncontrol— led temperature group (P>0.05). However, in the uncontrol- led forelimb temperature group, isolated forelimb tempera- ture decreased 3 degrees celcius on the average (P<0.05) by 30 minutes of shock and remained at this lowered level for the remainder of the experiment. Forelimb skin glucose uptake, skin venous P02 and core and isolated forelimb temperatures of both controlled and uncontrolled forelimb temperature shock groups are graphi— cally illustrated in Figure 3. Temperature data shown in Figures 3 and 4 are identical to those shown in Figure 2. After administration of endotoxin in the controlled forelimb temperature group, forelimb skin glucose uptake increased at 30 minutes following shock induction (P<0.05) and remained elevated throughout the experimental period (P<0.05) except at the fourth hour (P>fl.05). However, shock induction in the uncontrolled forelimb temperature group resulted in no change in forelimb skin glucose uptake for the entire 3.5 shock episode (P>0.05). In the controlled forelimb temper— ature group, forelimb skin venous PO2 decreased from 47 mmHg to 30 mmHg at 30 minutes of shock and remained depressed for the duration of the experiment (P<0.fll). In the uncontrol- led forelimb temperature group, skin venous PO decreased to 2 only 39 mmHg from a control value of 54 mmHg (P<fl.05) at 30 47 Figure 3. Skin metabolic and temperature variables in the controlled and uncontrolled forelimb temperature groups during E; coli endotoxin shock. (n)=number of animals; a=P<0.05 relative to time zero; b=P<fl.01 relative to time zero; c=P<0.fl5 comparing isolated forelimb temperature to the animal's core temperature at each time point; Bars=SEM. 48 Wannm.---W-uum.-.um%%.miu.wIIH T... m m ... ... 1.1 m :I1I./ 19 :3: 3.32532: .5231 111513: c 36 van. So v.1". mod van. 1/1 LL 1; .HI‘ .H .H .Hl. .7 a .3 E 3. 19 at: 3:323 .32.” T cauimx: allege“. .. map-await”--. unflmvlnwnufl l 68": .. mm 0? ON 0? Oh p E 1:11 a: .55 No.— «=23» 5: 5.3on 1111 me 32:: 38:3 2.5 49 minutes of shock and remained below control throughout the experiment (P<@.@5). Total forelimb glucose uptake, forelimb venous P02 and core and isolated forelimb temperature of both controlled and uncontrolled forelimb temperature shock groups are graphically illustrated in Figure 4. Following shock in— duction in the controlled forelimb temperature group, fore— limb glucose uptake increased 30 minutes after shock induc- tion (P<0.01) and remained elevated throughout the experi— mental period (P<0.05), except at 99 minutes following shock induction (P>0.05). In contrast, shock induction in the uncontrolled forelimb temperature group resulted in no change in total forelimb glucose uptake for the entire 3.5 hour shock episode (P>0.05). In the uncontrolled forelimb temperature group, forelimb venous P02 decreased to only 37 mmHg from a control value of 50 mmHg (P0.05). However, brachial venous PCO2 increased from a control value of 33.9 mmHg to 43 mmHg by 60 minutes of shock and remained elevated until 150 minutes of shock (P<0.05), after which it was not different from control (P>0.@5). Cephalic venous 50 Figure 4. Total forelimb metabolic and temperature vari— ables in the controlled and uncontrolled forelimb tempera— ture groups during E; ggli_endotoxin shock. (n)=number of animals; a=P<0.fl5 relative to time zero; b=P<0.@l relative to time zero; c=P<fl.05 comparing isolated forelimb tempera— ture to the animal's core temperature at each time point; Bars=SEM. 51 MIMITATAJ/m\w a ma: 3:95:32: .525 19 L L a a P u u q q . q u . J J on 9:52.: 1 1: allow: 1 . 3 .12: . cc .1 on 11111115 «.52: $5.22 modvau. . 56 v1.2 1 oh on Van. . o MITw'mellT \ mooF rm A5111 I F ug<—;= 38:5 22:2: 13 E 51 a1 6:"? u as: 332223 .323 x.VnI-—flto -- i X! HA — Fh .' ‘ ~ ‘1‘! O .o I. v — .. I . U I U ~ -‘ h IUH, A huhv ~ b. N! 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I . u NAEQV.~I 52 Amwmm uxmc co pmscfiuCOUv I I N 0.0+ 0.0+ 0.0+ 0.0M 1.0+ 0.0“ 0.0“ 1.0+ 0.1+ 001 maoam> 0.10 0.00 1.00 «0.00 0.00 10.00 «0.00 0.00 0.00 01100000 0.0M 0.0“ 0.0“ 0.0M 0.0H 0.0H 0.0“ 0.0“ 0.1“ N0011 000000 0.00 0.00 0.10 10.00 10.10 «0.00 10.00 0.00 0.00 10100010 0.1M 0.0H ~.~H 0.1“ 0.0“ 1.0M 1.0M 1.0M 0.1M N 0.00 0.10 0.00 0.00 0.00 1.00 .0.00 0.10 0.00 000 1011011< 1.1M 0.0M 1.0M 0.0M 0.0H 1.0M 1.0M 0.0M 0.0M N 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 00 101umuu< 0 1 1 0 0 01 01 01 01 100 100502 000 010 001 001 001 00 00 00 0 umumaaumm AGfiEV NEH? fiHXOuOCfim HOHUGOU .xoonm cwxouopao “Hoe .m wcfluav asouw 30am HonoumclousumuooEou nefiamuom meHouuaou oSu aw moanmfium> oaHonMumE cw compose .1 manna 53 .Amo.ovmv Howucoo scum .m.m H came mm wommmhaxo 01m modam> upopommfiw waucmofiwfidwfim mum £0053 wmsam> mmuoamw0 0.0M 1.0H 0.1M 0.1M 0.0M 1.0M 0.0M 0.0M 0.1+ 00.00 00.10 00.00 01.00 01.00 01.00 00.00 00.10 0.00 0100000000 00.0H 00.0H 00.0w 00.0H 00.0H 00.0H 00.0“ 00.0H 00.0H m0 00000> 000.1 001.1 010.1 010.1 000.1 000.1 011.1 000.1 00.1 00100000 00.0H 00.0H 00.0H 00.0H 00.0w 00.0H 00.0H 00.0H 10.0H 00 00000> 000.1 010.1 000.1 000 1 000.1 010.1 001.1 000.1 00.1 10100000 10.0w 00.0H 00.0H 00.0H 00.0H 00.0H 00.0H 00.0H 00.0w 001.1 010.1 010.1 000.1 000.1 000.1 000.1 000.1 00.1 00 10100000 000 010 001 001 001 00 00 00 0 000000000 Aaflev mafia GHKOHOUAHM HOHHQOU .10.00o00 1 01000 54 PCO2 was elevated at various times throughout the experi- mental period (P<0.flS). Arterial pH progressively decreased from a control value of 7.39 units to 7.19 units by 240 minutes of shock (P<0.05). Brachial venous pH and cephalic venous pH were also below control throughout the shock episode (P<fl.05). Hematocrit was increased by 3n minutes of shock (P<0.05) and remained above control for the duration of the shock episode (P<0.65). Table 2 shows changes in metabolic variables in the uncontrolled forelimb temperature-natural flow group during E. 3311 endotoxin shock. Following shock induction arterial P02 and arterial PCO were not different from control at any 2 point during the experiment (P>0.05). Brachial venous PCO2 was not different from control at any point during the shock episode (P>0.05) except at 90 minutes of shock when it was significantly elevated (P<0.05). Sixty minutes after the administration of endotoxin, cephalic venous PC02 increased (P<0.05) from a control value of 31.3 mmHg to 46.6 mmHg and remained elevated until 150 minutes of shock when it was not different from control (P>0.65). Arterial pH, brachial venous pH and cephalic venous pH all decreased immediately after shock induction (P<0I95) and remained below control for the duration of the shock episode (P<0.05). Hematocrit was elevated by 39 minutes of shock and remained elevated for the entire experimental period (P<fl.05). Hemodynamic variables and arterial glucose concentration of both control groups are shown graphically in Figure 5. "abiliflzPfllfl'LIii-HHTISF’EE'U Pa =- - HpUxhCh tad. FCLTUFHRUNIFD-yi ob £00 0.0% v...u.«.—-r¢. sufixCanus~_.i u ~1hu~ 90.6 ~ 2.0.. 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A m s & 0000.UV NM .4 H a~ 0.0.3 56 .m.m H 0005 mm cummwuaxm mum mmaam> .Amo.ovmv Houuaou 500m ucmummmaw >aunmowm0cwwm mum £0153 mmaam> mmuocmv« 0.0“ 0.0“ 0.0M 0.0M 0.0M 0.0M 0.0M 0.0+ 00.00 00.00 00.10 00.00 00.00 00.00 00.00 0.10 0100000000 00.00 00.0H 00.0H 00.0w 10.0H 00.0H 00.0w 00.00 00 000000 001.1 001.1 001.1 001.1 000.1 001.1 010.1 00.1 01100000 00.00 00.0H 00.0H 00.0H 00.0H 10.0H 00.0H 00.0w 00 00000> 011.1 011.1 001.1 011.1 001.1 011.1 000.1 00.1 10100000 00.00 00.0“ 00.00 00.00 00.0“ 00.0“ 00.00 00.00 000.1 000.1 000.1 000.1 000.1 000.1 010.1 00.1 00 10100000 010 001 001 001 00 00 00 0 000000000 Aswav mEHH CfixOuOVGm HOHHGOU .Aw.ucoov N anmH 57 In both groups, arterial blood pressure remained relatively constant, decreasing slightly during the last hour and the last 2 hours in the controlled and uncontrolled forelimb temperature groups, respectively (P<fl.05). Muscle, skin and total forelimb vascular resistances did not change signifi- cantly (P>0.05). Muscle vascular resistance in the uncon- trolled forelimb temperature group increased significantly by 1.5 hours of shock (P<0.05), while skin and total fore- limb vascular resistance did not change significantly in either group (P>0.05). This figure illustrates the stabil- ity of the experimental preparation and the similarities between the two groups as evidenced by stable hemodynamic variables and constant blood glucose levels for the duration of the four hour experimental period. Figure 6 shows muscle, skin and total forelimb glucose uptakes and venous P02, along with isolated forelimb and core temperature for both control, non-shocked groups. Glu- cose uptake did not change significantly in muscle, skin or total forelimb in either group (P>0.05). Muscle, skin and total forelimb venous P02 decreased slightly (P<fl.@5), but remained above 45 mmHg in both groups. In the controlled forelimb temperature group, isolated forelimb temperature was slightly lower than core temperature (0.2 degrees celcius) at several points during the experiment (P<@.@S). In the uncontrolled forelimb temperature group, core temper- ature gradually increased throughout the control episode (P<0.fl5). Isolated forelimb temperature was significantly 58 Figure 5. Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the controlled and uncon- trolled forelimb temperature non-shocked, control dogs. (n)=number of animals; a=P<0.05 relative to time zero; b=P<0.01 relative to time zero; c=P<0.ZS comparing isolated forelimb temperature to the animal's core temperature at each time point; Bars=SEM. 59 .m muswum 0.0 01.: Q d * q d a d * all d . /H . ....ll. 1.1.1:... 18v? ..||.. .....0 8 000V... 0 ...... o 33...: 0 10...... iii * 01 ...... I I I’llTCI-‘IvIl .9 .9". 09 ml: 3.33583.— ..8523 as: 332123.353“. 60 Figure 6. Control muscle, skin and total forelimb meta- bolic and temperature variables in the controlled and un— controlled forelimb temperature groups in non-shocked con- trol animals. (n)=number of animals; a=P<0.GS relative to time zero; b=P<0.fll relative to time zero; c=P<0.05 com— paring isolated forelimb temperature to the animal's core temperature at each time point; Bars=SEM. 61 .0---.. ..1.--..0.--.._0---0£0-10 h I h H ..I ILMD\ ...»..ILHIIIulanI III mH.II..IH IHHHUW 1.1%: .mih ..... «- Hun 0 [.m/ IIIH I¥ . mzmp 313528233528 10.13:: V 00.021“. 10.0 v.1“. . q u- q - q .1 q q d ...-£11.: .0111 . 21:31:: - 1...; 00.3.“. 93-..--. 1110.... Ti/MB1XTW [Ml-AWL /1\/1 muswfim 19”: I .02: 3.32th .3523 on mm 0? ON Q: .02: 232: Auoopv 1.11m... 32:... 33:5 62 different from core temperature between 1 and 4 hours of the control period (P>0.05). This temperature difference never exceeded 1 degree celcius and occured because core tempera- ture increased. An additional statistical analysis was made to compare the glucose uptake of the endotoxin-shocked animals and the non-shocked animals of the controlled forelimb temperature- natural flow groups. In this analysis, the “Students T" test was used to compare the control group to the shock group to determine whether there were any differences in the magnitude of the calculated glucose uptake between the groups at each data point. Results of this analysis indi— cate that the absolute values of glucose uptake by skeletal muscle, skin and total forelimb are not different between the two groups (P>@.05). However, as previously indicated (Figures 2-4), glucose uptake by skeletal muscle, skin and total forelimb does increase from control values during endotoxin shock in the temperature controlled-natural flow group (Figures 2—4) while there was no change in the non- shocked animals (Figure 6). .Table 3 shows changes in metabolic variables in the controlled forelimb temperatureenatural flow group in non- shocked, control animals. Arterial PO remained relatively 2 constant throughout the experimental period with no signi- ficant differences from control (P>0.65). Arterial PC02 was significantly different from control only at 180 minutes of shock (P<0.05), otherwise arterial Pcoizwas never different rgflIh-abaHu-IIIUII-dd-.~Ibmda,¢--ui-d nun-nu FI-th0v0u —erF-3.d.UF-Awnu awn-U Pu! ut..v~fi-.~h-I-U> ...uuth0L-VUU0: nhfi- mwflunlfluninsub IMH- MGHA~950N0 63 Amman axon so vmssfiucoov 0.1M 0.0M 0.0M 0.1M 0.1M 0.1M 0.1M 0.1M 0.1M 0000 00000> 1.00 0.10 0.00 0.00 1.00 0.00 1.00 0.00 0.00 01100000 0.00 0.1M 0.0“ 1.00 0.1H 0.1M 0.1M 0.1M 0.1M 0000 000000 0.00 0.00 0.00 1.00 1.10 0.10 0.00 0.00 0.10 10100000 0.00 0.1“ 1.0“ 0.10 0.00 0.10 0.10 1.1“ 0.00 0 0.00 0.00 00.00 0.00 0.00 0.00 0.00 1.00 0.00 000 10100000 0.00 0.00 1.00 0.0M 0.00 0.00 0.0M 0.0M 0.0M 0 0.11 0.01 1.01 0.01 0.01 1.00 0.00 0.10 0.00 00 10100000 0 0 0 0 0 0 0 0 0 100 000002 000 010 001 001 001 00 00 00 0 000000000 Aswav 0809 HOMUCOU .mamaficm Houuaoo .vwxoonmlco: c0 asouw acam Hmusumcimusumuoaawu naaawuom umHHonucoo 0:0 :0 moanm1um> ofiaonmuma n0 mmmcmnu .m manna 64 .m.m H came an vmmmmuaxw mum mmaam> .Am0.0va HOHUGOU EOHW ufimHQMMHfl 1AHuflMOfiMHfime mum £UH£3 m05Hm> mmu0fi0—uan 0.1M 0.1M 0.0M 0.0M 1.1M 0.0M 0.1M 0.1M 0.1“ 0.00 1.00 1.00 0.00 1.00 0.00 1.00 0.00 0.10 0100000000 00.00 00.0H 00.0H 00.0“ 00.0“ 00.0“ 00.0H 00.0“ 10.0H 00 0soa0> 00.1 00.1 00.1 00.1 00.1 10.1 00.1 00.1 00.1 01100000 00.0“ 00.00 00.00 00.0w 00.0“ 00.00 00.0H 00.00 10.0“ 00 00000> 00.1 00.1 00.1 00.1 00.1 00.1 00.1 00.1 10.1 10100000 00.00 00.00 00.0w 00.00 00.00 00.0“ 00.0w 00.0H 00.0“ 00.1 00.1 00.1 00.1 00.1 00.1 00.1 10.1 00.1 00 10100000 000 010 001 001 001 00 00 00 0 000000000 Aawev 0819 Houucoo .10.00000 0 01000 65 from control (P>0.@5). Brachial venous PC02 and cephalic venous PCO2 were not different from control at any point during the experimental period (P>G.05). .Arterial pH, brachial venous pH and cephalic venous pH did not change during the experimental period (P>0.05). Hematocrit re- mained at control levels throughout the experimental period (P>@.05). Table 4 shows changes in metabolic variables in the uncontrolled forelimb temperature-natural flow group in non- shocked, control animals. There were no changes in arterial P02, arterial PCO2 or cephalic venous PC02 during the entire experimental period (P>fl.05). Arterial pH and brachial venous pH did not change during the entire experimental protocol (P>6.95). Cephalic venous pH was not different from control during the entire experimental period (P>0.05) except at 155 minutes of shock when it was decreased slight- ly (P<fl.05). Hematocrit was not different from control during the entire experiment (P>fl.05). It should be noted that the data displayed on the left- hand side of Figures 1-4 titled "Shock, Controlled Temp." will be repeated on the left—hand side of Figures 7—10 retitled "Shock, Natural Flow". This was done so that a visual comparison between the controlled forelimb tempera- ture-natural flow and constant flow groups could be made. Hemodynamic changes and arterial glucose concentrations of both the natural and constant flow groups are graphically illustrated in Figure 7. In both groups, arterial blood 66 Amman uxw: no wmsawucoov 0.1M 1.0M 0.0“ 0.0“ 0.1M 0.1M 0.1M 0.0“ 0000 000000 0.00 0.00 0.00 1.00 0.00 0.10 0.00 0.10 01100000 0.00 1.1M 1.0M 0.0“ 0.1“ 0.10 0.1“ 0.1“ 0000 000000 0.00 0.00 0.10 0.00 0.00 0.10 0.10 1.00 10100000 1.0M 0.0M 0.0“ 0.0“ 0.1M 1.1M 0.0M 0.1M 0 1.00 0.10 0.00 1.00 0.00 0.10 0.00 0.10 000 10100000 1.10 0.1M 0.0“ 0.1“ 1.0M 0.00 0.0“ 0.0M 0 1.01 0.01 1.01 0.01 0.00 0.01 0.01 1.00 00 10100000 0 0 0 0 0 0 0 0 100 000002 - 010.. .- 001 , 001 001 00 00 00 0 000000000 Acwsv mefik HOHUCOU .meEHam Houucoo .wwxoosmlaoc :0 nsouw 30am HmMSumclmuoumumaEmu nsfiamuow vaHouucooca 0:0 60 moanmwum> oaaopmuwa :0 mwmcmnu .0 mange Phi. ‘F~fi\f\\ q ethfhl‘vht 67 .m.m H cmme mm vmmmouaxm mum mmsfim> Amo.ovmv Houucoo Eoum ucmummm0p haucmowmacwum mum 300:3 mm=Hm> mmuocmv« 0.0+ 0.0+ 0.0+ 1.0+ 0.0+ 0.0+ 000M 0.0M, 0.00 0.00 0.00 0.00 0.00 0.00 1.10 0.10 0100000000 00.0H 00.0H 00.0“ 00.0“ 00.0H 00.00 00.00 00.0H 00 000000 00.1 00.1 000.1 00.1 00.1 00.1 00.1 00.1 01100000 00.00 00.00 00.0H 00.0“ 10.0“ 00.00 00.00 00.0H 00 00000> 00.1 00.1 00.1 10.1 00.1 00.1 00.1 00.1 10100000 00.0w 10.0H 00.00 00.00 00.0“ 00.0“ 00.0“ 00.00 00.1 10.1 00.1 00.1 00.1 10.1 00.1 00.1 00 10100000 010 001 001 001 00 00 00 0 000000000 Acwfiv mafia HOHUGOU .Ap.ucoov e manna 68 pressure decreased to approximately 55 mmHg by 3G minutes of shock and remained below control for the duration of the experiment (P<0.05). Muscle, skin and total forelimb blood flows in the natural flow group decreased at 39 minutes of shock (P<0.05) and remained below control for the duration of the experiment (P<0.05). In the constant flow group, blood flows were maintained at control levels throughout the experimental protocol. Muscle, skin and total forelimb vascular resistances were significantly increased throughout the shock period (P0.05) in the natural flow group. In the constant flow group, muscle and forelimb vascular resis- tances did not increase until the third hour of shock (P<0.Z5) and remained elevated for the remainder of the experiment (P<fl.65). In both groups of animals, arterial glucose concentration increased (P<0.05) at 30 minutes fol— lowing shock induction (P<0.05); then progressively de— creased for the duration of the experiment (P<0.05). This figure illustrates that the degree of shock was similiar in both groups of animals. Forelimb muscle glucose uptake, muscle venous PO and 2 core and isolated forelimb temperatures of both natural and constant flow experiments are graphically illustrated in Figure 8. Following shock induction in the natural flow group, forelimb muscle glucose uptake increased at 30 min- utes of shock (P<fl.05) and remained elevated throughout the experimental period (P<0.95). In contrast, shock induction in the constant flow group resulted in an increased forelimb 69 Figure 7. Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the natural (free flow) and constant blood flow perfused isolated forelimb during E; coli endotoxin shock. (n)=number of animals; ABP=mean systemic arterial blood pressure; BF=blood flow; R=vascu1ar resistances; PRU=peripheral resistance units expressed in mmHg/ml/min/lflfigm; Art. Glc. Conc.=arteria1 blood glucose concentration; a=P<0.95 relative to time zero; b=P8: .352: .52; 71 muscle glucose uptake only at 30 minutes of shock (P<0.fll), after which it returned to values not different from control for the duration of the experiment (P>@.05). Forelimb mus- cle venous PO2 decreased (P<0.01) following shock induction and remained below control throughout the experiment (P<fl.01) in the natural flow group. However, forelimb mus- cle venous P02 was unaltered in the constant flow group (P>fl.05) throughout the 4 hour shock period. In both groups of animals, isolated forelimb temperature was never different from core temperature (P>0.05). In the natural flow group, isolated forelimb temperature was slightly below control at 2 and 2.5 hours (P<0.05) and slightly increased at 3 and 3.5 hours (P<0.05) when compared to their own control values. These temperature changes did not exceed 0.2 degrees celcius. Core temperature of the natural flow group was not significantly different from control (P>0.05). Subsequent temperature illustrations of Figures 9 and 16 are identical to that in Figure 8. Forelimb skin glucose uptake, skin venous P02 and core and isolated forelimb temperatures of both natural and con- stant flow experiments are graphically illustrated in Figure 9. Following shock induction in the natural flow group, forelimb skin glucose uptake increased at 30 minutes of shock (P<0.01) and remained elevated throughout the experi- mental period (P<0.05) except at the 4 hour reading (P>fl.05). In contrast, shock induction in the constant flow group resulted in an increased forelimb skin glucose uptake 72 Figure 8. Muscle metabolic and temperature variables in the natural (free flow) and constant blood flow perfused isolated forelimb during E; coli endotoxin shock. (n)=number of animals; a=P<0.@5 relative to time zero; b=P<@.@l relative to time zero; Bars=SEM. 713 .w muswflm 0.000: V 1 1 A 1 1 -1 1 1 fl 1 0 1 1 q 1 1 1 J 1 on ..---011: 0 - 0 o .008 -00 01501 \0LI0\0|0\0.|0\0 mimnnmvmmh. “0001-0.--- :00. 101.000 ..0.---1---.1 . ... .0. 0.. mmmmmm 02;: 01001: ...-31:10.--.0---..m.--..m.-..m- L r06 Van. ... Oh . 0.0.0 v00. .0 . 0 0 \\\\mnllumllll \0W 0* .IIIMillumolluflillmmllll sss m / w --..W 7T .130 0,00... 1400.00 0 . . 1 02:1. 0. 0000010 . 010012 a. .3 .. E E 19 1Q . 6:”..- N 2°: 12.15.30.183 >8: 12:22 .185 74 at only 30 minutes of shock (P<fl.fl5), after which it re- turned to values not different from control for the duration of the experiment (P<0.05). Forelimb skin venous PO2 de- creased (P<0.fll) following shock induction in the natural flow group. However, forelimb skin venous PO2 was unaltered in the constant flow group (P>0.05) throughout the 4 hour shock period. Total forelimb glucose uptake, total forelimb venous P02 and core and isolated forelimb temperatures of both the natural and constant flow experiments are graphically il- lustrated in Figure 10. Following shock induction in the natural flow group, total forelimb glucose uptake increased at 39 minutes of shock (P<0.01) and remained elevated throughout the experimental period (P<0.05) except at 1 hour following shock (P>0.05). In contrast, shock induction in the constant flow group resulted in an increased forelimb glucose uptake only at 30 minutes of shock (P<6.05), after which it returned to values not different from control for the duration of the experiment (P>fl.05). Forelimb venous PO2 decreased following shock induction (P<fl.fll) and remained below control throughout the experi- ment in the natural flow group. However, forelimb venous PO2 was not altered in the constant flow group (P>fl.05) through- out the 4 hour shock period. As previously mentioned the numbers in parentheses at the top of Figures 1-12 and the numbers in the first line of Tables 1-6 represent the number of animals at each data 75 Figure 9. Skin metabolic and temperature variables in the natural (free flow) and constant blood flow perfused iso- lated forelimb during E; ggli_endotoxin shock. (n)=number of animals; a=P<0.05 relative to time zero; b=P<0.01 relative to time zero; Bars=SEM. 76 .m muswfim on ma 0? ON mv Oh w v a u p u Answxzv a a 1 u . ...-2.1:: . .1118 . 4 . . - .mllldm/HJHXM/hlmku/ H\\W/H\mlmllmluwllm\m . _ . . 86 v1". 1 . 3.0 v1". . 1/1 ML l H H T . All A T71 . ./\./\7_ 7 k . g .3 R. .9 .0. .07.... n n .... 33. :25on .323 :3. 1:31: .323 3. at: a: .55 Ne.— ”.32”; 2.5 5.18. gnaw 32:5 132.5 2.5 77 Figure 10. Metabolic and temperature variables in the natural (free flow) and constant blood flow perfused iso- lated forelimb during E; coli endotoxin shock. (n)=number of animals; a=P<fl.05 relative to time zero; b=P<fl.01 rela- tive to time zero; Bars=SEM. 78 .oH muswfim 2 v a a 1 u 1 521:1. o n 1 o 1 1 1 1 1 1 1 1 1‘ I 1 1 1 1 1 1 1 1 d .. 0” Us...“ . . - 1 1:: l 18 . .. on . 31.5 . .u\.m/.mll.m\....ml.m\11\\. . me a: m\..wlm-\1..Im'm\\~i\1 / 2.2.: 12312 L Ed Van. .. on 36v? _ I. . m .. HIT. 71/ \ llmlT-TTW/ \ 1.8. H N 1% SEE . . . 1 3.11.3 122:1 22:12 a. .3 g .3 E .9 a. 611;... 33... 121323.323 33.. 125:: .328 79 point; the smaller numbers represent animals dying during the course of the experiment. Table 5 shows changes in metabolic variables in the controlled forelimb temperature-constant flow group during E; coli endotoxin shock. Control arterial PO2 was 80 mmHg and did not change significantly throughout the shocked period (P>B.ZS). Arterial PCOZ, brachial venous PC02 and cephalic venous PCO2 were not significantly different from control after the administration of endotoxin (P>0.05). Arterial pH, brachial venous pH and cephalic venous pH immediately decreased after the administration of endotoxin (P<0.05) and remained at this lowered level for the duration of the experiment (P<0.65). Hematocrit increased 30 minutes after shock induction (P<0.05) and remained elevated for the duration of the shock episode (P<0.05). Hemodynamic variables and arterial glucose concentration of two control non-shocked groups are shown graphically in Figure 11. In both groups of animals, arterial blood pres- sure remained relatively constant decreasing slightly during the last hour in the natural flow group (P<0.05). Muscle, skin and total forelimb blood flows decreased in the natural flow group at different times throughout the experiment (P<0.fl5) whereas, in the constant flow group, blood flows were unchanged (P>0.65). Vascular resistances gradually increased in both groups of animals (P<0.05). Muscle, skin and total forelimb glucose uptakes and venous P02, along with isolated forelimb and core 80 Awwma uxm: co vmscwucoov 1.1M 1.1M 1.11 1.1“ 1.11 1.1M 1.1M 1.1M 1.1M 1111 111111 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 11111111 1.11 1.11 1.11 1.1M 1.1M 1.1M 1.11 1.11 1.1M 1011 11111> 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 11111111 1.11 1.11 1.1M 1.11 1.11 1.11 1.11 1.11 1.1M 1 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 111 11111111 1.11 1.11 1.1“ 1.11 1.11 1.1M 1.11 1.11 1.1M 1 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 1.11 01 11111111 1 1 1 1 1 1 1 1 1 111 111112 111 111 111 111 111 11 11 11 1 111111111 -. 11111 1111 aonuovcm Houucou .xoonm saxouowam 1100 .m wcfiuav asouw 30am ucmumcoolmusumumaEmu nafiaouow coaaouucoo msu a1 moanmwuw> ofiaonmuma s1 mowcmnu .m canoe 81 .m.m H Emma mm wwmmeme mum mmafim> .Amo.ovmv Houucoo Eouw 1cowwwmflv 111C101m1cw1m 11m 301:3 mwsfim> mwuocmw# 1.1M 1.1M 1.1M 1.1M 1.1M 1.1M 1.1H 1.1M 1.1+ 11.11 11.11 11.11 11.11 11.11 11.11 11.11 11.11 1.11 1111111111 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11 11111> 111.1 111.1 111.1 111.1 111.1 111.1 111.1 111.1 11.1 11111111 11.1H 11.1H 11.1H 11.11 11.1H 11.1H 11.1w 11.1H 11.1H 11 11111> «11.1 111.1 111.1 111.1 111.1 111.1 111.1 111.1 11.1 11111111 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 11.1H 111.1 111.1 111.1 111.1 111.1 111.1 111.1 111.1 11.1 11 11111111 111 111 111 111 111 11 11 11 1 111111111 AcwEv mEHH cfixouowsm Houucou .11.11111 1 11111 82 Figure 11. Muscle, skin and total forelimb hemodynamics and arterial glucose concentration in the natural (free flow) and constant blood flow perfused isolated forelimb in non—shocked, control dogs. (n)=number of animals; ABP=mean systemic arterial blood pressure; BF=blood flow; R=vascu1ar resistances; PRU=peripheral resistance units expressed in mmHg/ml/min/lflflgm; Art. Glc. Conc.=arterial blood glucose concentration; a=P<0.05 relative to time zero; b=PB.05) but both increased during the experiment (P<6.01). An additional analysis of the data using the ”Students T" test was employed to determine whether there were any differences in the magnitude of the calculated glucose up- take by skeletal muscle, skin and total forelimb between the control animals and the shock animals of the controlled forelimb temperature-constant flow groups. Results of this ianalysis revealed no statistical differences (P>0.05) in the level of glucose uptake for skeletal muscle, skin and total forelimb between the groups at each time point. None of the metabolic variables listed in Table 6 chang— ed during the entire experimental protocol in the controlled forelimb temperature-constant flow non~shocked, control group (P>fl.05). It should be noted that while glucose concentration of muscle and skin venous blood was usually lower than arterial blood, occasionally forelimb venous glucose concentration 85 Figure 12. Control muscle, skin and total forelimb meta- bolic and temperature variables in the natural (free flow) and constant blood flow perfused isolated forelimb in non- shocked, control dogs. (n)=number of animals; a=P<fl.05 relative to time zero; b=P<fl.fll relative to time zero; c=P<0.ZS comparing isolated forelimb temperature to the animal's core temperature at each time point; Bars=SEM. 86 «1 n N p O H u u q u q q 1 1 1--.. .....evi LNI ".u" “ 0"- ---..."O I”W/l.M‘\oWI“-fl“k”.l”l \k kl.— 333 22523 .3523 ll.“ NH ouswfim 22.32: v n N p 0 q q 1 u q q 1 q u n O” 2:32.: 2: . 1119 I... . ...» .12: wl...1.-...m. ..--.m-.-..1--- .1--...1---.m.--.m L O? .. ON . 3255. ”VI/thflw. a . m1. «.... . ..II II II... 1 ... 32.: 36v... . 22:2: . O1 90.0Vau. c c 2;” 18v.-. 9 ....... . 33...: . l -...n\|..r ---. [MHWL uso; ... 3. . .... .0.”.— l N 23... 322—22 .3523 87 was higher than arterial blood concentration at one or two points during the shock period. Also, it should be noted that expressing blood flow on a 100 gram basis resulted in blood flow per 106 grams in muscle and skin appearing higher than total forelimb blood flow. This apparent paradox is due to the fact that bone weight was included in total forelimb weight, used to calcu- late total forelimb blood flow, but was not included with muscle or skin weight determinations or blood flow per 100 gram calculations. The same situation holds for calculated vascular resistance, which was obtained using blood flows per 100 grams of tissue. Therefore, resistance in the total forelimb appears to be higher than in forelimb muscle or skin. 88 Amman uxo: co umscfiuaoov 1.1M N.mH H.qH m.mH 1.mH o.mH ~.mH e.qH o.mH Noon msoam> o.om 1.1m o.~m 1.11 m.1m 1.11 m.om 1.1m 1.11 afiflmnamo 1.1H o.mH 1.1H o.mH 1.1M 0.1“ H.1H o.eH 0.1H Noom macaw> 1.01 m.1m m.1m m.mm 1.11 o.~m o.Hm m.1m c.1m Hmaaomum 1.mH 1.1H m.mH w.1H 1.1H n.1H 1.1M e.mH 1.1H 1 1.01 m.om 0.1m c.1m m.~m m.om 1.11 m.om w.om oom Hmfiumuu< 1.eH q.eH «.mH m.wH m.mH H.qH a.mH m.oH 1.1M N m.mm m.~m c.ew m.~m c.1m c.1m m.mm o.wa m.ma om Hmaumuu< e s q e e e e s e Any umnasz oqN oHN omfi omfi 011 cm on om o umumamumm Aafiav mafia HOHHCOU .mHmafiam Houuaoo .coxoosmnaoa ca maouw Bon uamumcoolmuoumquEmu nefiamuom woaaouucoo osu ca moHAMfium> owaoAMuoE ca momcmso .e canoe 89 .m.m H some on commouoxm mum mooao> .AMO.OVWV HOHHfiOU EOHM uflflHUMWfifi hHUfiNUHM-nfiwfiw OHM £0HS3 mUDHw> mUUOflmfifi 1.1“ w.mH «.mH m.mH o.qH e.eH e.eH m.qH m.qH 0.11 o.mm m.mm m.mm 1.1m w.mm 1.11 m.em m.mm uauuoumaom No.0“ No.oH mo.oH mo.oH No.0“ mc.oH No.0H Ho.oH No.oH ma msoam> sq.1 He.1 11.1 11.1 Nq.1 He.1 Ne.1 He.1 me.1 afiamnaoo Ho.oH mo.oH mo.oH mo.oH 1o.oH He.oH No.0H 1o.oH mo.oH ma maoam> cq.1 oe.1 1s.1 11.1 lq.1 ce.1 11.1 He.1 11.1 111somum No.oH mo.oH mo.cH mc.oH ~o.oH No.0“ No.0“ qo.oH No.oH 1e.1 me.1 me.1 Ne.1 Nq.1 Ne.1 Ne.1 Ne.1 ee.1 ma Ha1umuu< oqm cam cm“ on“ 011 cm oo 01 o HmumSmumm Aswav mawe HOHufiOU .Au.uaoov m magma CHAPTER V DISCUSSION The hypotheses tested by this research were that 1) glucose uptake by skeletal muscle and skin increases during .5; call endotoxin shock in the dog and 2) that the mech- anism for this proposed increase is related to local tissue hypoxia and/or tissue ischemia. The thesis postulated is that the hypoglycemia of endotoxin shock is, in part, due to an increased utilization of glucose by peripheral tis- sues, viz., skeletal muscle and skin. In this research the surgically isolated forelimb was used to study glucose uptake by skeletal muscle and skin during E; coli endotoxin shock. Three groups of animals, including appropriate controls and shocked animals, were studied. In groups 1 and 2 the isolated forelimb was perfused under natural flow (free flow) conditions in order to study the hemodynamic and metabolic response of the isolated forelimb during endotoxin shock. In the first group of animals isolated forelimb temperature was main- tained at the temperature of the contralateral intact fore- limb which was not different from core temperature. In group 2, the temperature of the isolated forelimb was uncontrolled and allowed to change independant of core 90 91 temperature. In the course of this research it was found that isolated forelimb temperature decreased approximately 3 degrees celcius within the first hour of the experiment if not maintained at core temperature. Furthermore, it was felt that this temperature drop may explain the contradic- tory data reported by Furr et al (101) where it was shown that glucose uptake by the isolated forelimb did not change during endotoxin shock. In their preparation the tempera- ture of the isolated forelimb was not maintained at core temperature and presumably decreased since it was neither reported nor equipped for temperature maintenance. In group 3 the isolated forelimb was perfused at a constant blood flow to alleviate the effects of low blood flow and the ensueing local tissue ischemia and tissue hypoxia that occurs during shock. Also, isolated forelimb temperature was maintained at core temperature in this group. The use of the isolated forelimb as a viable isolated organ has been well established by numerous investigators (104,196). As previously stated, ligation of the median cubital vein, which represents the major anastomotic chan— nel between the brachial and cephalic veins, ensured that brachial venous outflow was predominately from skeletal muscle and that cephalic venous outflow was predominately from skin (166). Therefore this technique allows one to study metabolic and hemodynamic variables in muscle and skin because venous drainage from either tissue can be 92 measured and sampled with a sufficient degree of accuracy. It was assumed that the contribution of bone blood flow was minimal and contributed little to the overall hemodynamic and metabolic response of the isolated forelimb, although we have no data to support this assumption. Results from this study clearly show that glucose up- take by forelimb skeletal muscle and skin increases during endotoxin shock in the dog provided forelimb temperature is maintained at core temperature, and that the mechanism for this proposed increase may be related to local tissue hypoxia and/or tissue ischemia (83). While this increase in forelimb skeletal muscle glucose uptake is similar to what occurs in gracilis muscle as previously reported by Raymond et a1 (83) during endotoxin shock and by others (102) during mild endotoxemia in the intact hindlimb, the increase in glucose uptake by skin during endotoxin shock has to our knowledge not been previously reported. It was also noted that glucose uptake by skin is greater than muscle during resting conditions and that the increase in glucose uptake is greater by skin than muscle during shock. It should be noted that in the analysis of the glucose uptake data using the ”Students T” test modified for un- paired replicates it was found that comparison of shocked animals to control animals of the controlled forelimb tem- perature-natural flow group (group 1) revealed no differ- ences at each time point between the control and the shocked animals. However when the data was analyzed with 93 the analysis of variance test, which takes into account the variability among individual animals and the variability occuring over time, it was found that glucose uptake does increase in skin and skeletal muscle when compared back to their own control values. Because of the variability among the animals in this study it was felt that the analysis of variance was a more sensitive test than the "Students T" test to ascertain changes in glucose uptake during shock. In this study it is not known whether the sex of the animal affected its response to shock and in particular its response to glucose metabolism. Previous studies from Dr. Emerson's laboratory have reported no differences in glucose uptake by the isolated gracilis muscle during control or shock states according to the sex of the animal (personal communication). In these studies metabolic and hemodynamic data were combined (83,84,195). In the present study the sex of the animal was not routinely recorded thereby pre- venting a detailed analysis of the data according to sex. The control experiments in natural flow preparations indicated that glucose uptake by forelimb skeletal muscle and skin does not increase over time. Evidence for the viability of the isolated forelimb preparation used in this study is noted from its metabolic and vascular integrity. The appearance of a positive arteriovenous PO2 difference, a negative arteriovenous PCO2 difference, and an increase in the arteriovenous pH difference are qualitatively similiar to results noted by others (106). To what extent the 94 increase in arteriovenous pH difference in the forelimb is due to carbon dioxide production or to lactate production is not known. Forelimb vascular resistance rose slightly with time, resulting in a gradual fall in limb blood flow. This results from a small fall in blood volume due to fluid loss (insensible, urine, and oozing) and perhaps from a lighten— ing of the anesthesia. By all usual measures the isolated forelimb preparation appeared to act like an intact limb. Data from the present study offers no explaination as to why glucose uptake by skin is greater during control and shock states, as compared to muscle. However, an earlier study from Dr. Emerson's laboratory (109) demonstrated an increase in glucose uptake by suprapubic subcutaneous adi- pose tissue during the first 2 and 2.5 hours of a 4 hour endotoxin shock protocol. It is possible that an increase in forelimb subcutaneous adipose tissue glucose uptake may have contributed to the "skin" glucose uptake during shock, but this study offers no data to support this possibility. It is of interest that the increase in glucose uptake in both forelimb skin and skeletal muscle reached a peak at 30 minutes of shock and then decreased, plateauing at a level still above control by approximately 1 and 1.5 hours, which roughly parallels the time course of glucose uptake observed in adipose tissue (109). Also, a partial explanation could be that even in the presence of oxygen, skin metabolizes most of the glucose only as far as to lactate (110-113). This phenomenon is unique to skin and has been attributed to 95 the limited ability of skin to convert pyruvate to acetyl— CoA and the very high activity of lactate dehydrogenase compared to other tissue (114). Also, the majority of metabolic requirements of skin are met through anaerobic glycolysis and a small percentage by the oxidative pentose pathway, while only negligible amounts of energy require- ments are provided by the Krebs cycle (115). It has been postulated by Fusaro and Johnson (115) that skin may play a multiple, time—dependent role in glucose homeostasis. In this scheme, glucose diffuses into the dermis during periods of hyperglycemia and drains into the lymphatic system and epidermis. While glucose in the lymphatics is carried back to the vascular system eventual— ly, that which enters the epidermal component of skin is converted to lactate which diffuses back into the blood and is transported to the liver for conversion to glycogen. As blood glucose concentration returns toward normal, the re- maining excess dermal glucose diffuses back into the blood and hence helps regulate plasma glucose concentration. Again, it is not known whether this glucose regulating mechanism is involved in the greater increase in glucose uptake by skin than skeletal muscle during shock. Also the so-called back diffusion of glucose may explain the higher concentration of glucose in forelimb venous than arterial blood which was sometimes observed by us, and also by Furr et al (101). 96 With reference to the Furr et al (101) paper, data from their study and from our uncontrolled forelimb temperature group (Group 2) are in agreement in that we also observed no increase in glucose uptake by the isolated forelimb during endotoxin shock. However, as observed in our controlled forelimb temperature study (Group 1), glucose uptake by both skeletal muscle and skin increases substantially during shock if temperature of the isolated forelimb is maintained at the temperature of the contralateral, non-isolated fore- limb, which turned out to be the same as core temperature of the animal. An explanation for no change in glucose uptake by the isolated forelimb during shock in the uncontrolled forelimb temperature group (Group 2) of this study and in the Furr et al (101) uncontrolled forelimb temperature study may be related to a decrease in isolated forelimb temperature below contralateral forelimb temperature. The lower temperature may have resulted in a decrease in the metabolic rate of this organ and hence a decrease in substrate and oxygen need. The decrease in metabolic requirements of the iso- lated forelimb is further substantiated by the fact that even though oxygen delivery to the forelimb in this study was severely decreased, the limbs only developed mild hypoxia (20% decrease in forelimb venous P92). The difference in forelimb skeletal muscle and skin glucose uptake between the temperature controlled and tem- perature uncontrolled experiments is likely due to the 97 3 degree celcius difference in contralateral and isolated forelimb temperature. To evaluate this, information is needed on the temperature coefficient (Q10) for the glucose uptake process under the experimental conditions of this study. Since such Q10 values are not known, it is not possible to ascertain whether or not the observed difference in glucose uptake between temperature controlled and temperature uncontrolled experiments is quantitatively equal to that due to a 3 degree celcius change. Also of importance in this study is that glucose uptake by forelimb muscle and skin is elevated only at about 30 minutes after shock induction when blood flow was maintained constant, similar to the gracilis muscle (83). This tran- sient increase in forelimb skeletal muscle and skin glucose uptake during the early phase of endotoxin shock can proba- bly be explained on the basis of the endotoxin molecule acting directly on the tissue to stimulate glucose uptake (84). However, by the time the 60 minute determination was made, all or most of the circulating endotoxin should have been cleared from the blood by the reticuloendothelial sys- tem and other tissue (105). Plasma levels of endotoxin have been shown to decrease rapidly within 1 hour of shock with only negliglible amounts of endotoxin detectable 2 hours after the administration of large doses of endotoxin (5 mg/kg) to dogs (108). This study did not specifically differentiate between the effects of local tissue hypoxia and local tissue 98 ischemia on glucose uptake during shock. However, Dr. Emerson's laboratory previously demonstrated the effects of local tissue hypoxia independent of local tissue ischemia by interposing an extracorporeal lung between the femoral arte- ry and the gracilis artery for local control of blood gases (83). Under non-ischemic, hypoxic conditions, gracilis muscle glucose uptake increased by 60 minutes and remained elevated throughout the experimental period. The effects of local tissue ischemia independent of tissue hypoxia were also investigated in the same study. This was accomplished by decreasing the gracilis muscle perfusion pump speed to induce,local tissue ischemia while an extracorporeal lung was ventilated with a gas mixture of 95% 02 and 5% CO2 to maintain arterial blood gases at hyperoxic levels supplying the gracilis muscle. Following the effects of local tissue ischemia and hyperoxia, gracilis muscle glucose uptake de- creased and remained depressed for the duration of the experimental protocol. It was thus concluded that local tissue hypoxia and not local tissue ischemia was responsible for the increased glucose uptake in gracilis muscle. It is reasonable to assume that the same conditions prevail in the present study and that local tissue hypoxia per se is pri- marily responsible for the increase in glucose uptake in forelimb skeletal muscle and skin although the definitive experiments have not been done. To reiterate, the present study demonstrates that glu- cose uptake increases substantially by both skeletal muscle 99 and skin in the isolated dog forelimb when temperature is maintained the same as the contralateral, non-isolated fore— limb. While these data provide no information relative to the role of this phenomenon in the hypoglycemia of endotoxin shock in the dog, the skeletal muscle data supports previous work in the gracilis muscle (83), and since skeletal muscle makes up a large percentage of total body mass, an increase in glucose uptake by skeletal muscle which presumably occurs generally throughout the body must contribute substantially to the development of hypoglycemia. Also, while the mass of skin is considerably less than that of muscle, the sustained increase in glucose uptake by skin during endotoxin shock may also contribute substantially to the progressive and pathologic depletion of blood glucose. Additionally, this study emphasizes the importance of maintaining isolated organ temperature at near body temper- ature, particulary during studies involving metabolism, since relatively moderate alterations in organ temperature can result in substantial alterations in the metabolic rate of that organ via the well known Ql0 effect. Also, although the observations reported by Furr et al (101) were no doubt correct and were verified by the present study, these data as applied to the pathophysiology of shock should be consid- ered artifactual, and hence not necessarily applicable be- cause of changes in isolated organ temperature independent of temperature changes in the intact animal. 100 In conclusion, data from the present study support the hypotheses that glucose uptake by forelimb skeletal muscle and skin increases during E; coli endotoxin shock in the dog and that the mechanism for this increase in glucose uptake is related to local tissue hypoxia and/or tissue ischemia. CHAPTER VI SUMMARY AND CONCLUSIONS The present study was designed to determine whether glucose uptake by forelimb skeletal muscle and skin in- creases during E; coli endotoxin shock in the dog and if the mechanism for this proposed increase in glucose uptake is related to local tissue hypoxia and/or tissue ischemia. The isolated, innervated, canine forelimb perfused at either natural or constant blood flow was used as the test organ in this study. In two groups of animals (Groups 1 and 3) the temperature of the isolated forelimb was maintained at the temperature of the contralateral (intact) forelimb, which was equal to the core temperature of the animal, by wrapping the isolated forelimb in a temperature regulated water jack- et. In another group of animals (Group 2) isolated forelimb temperature was allowed to change independant of core tem- perature. Shock was induced by a 5 minute intravenous infusion of 2 mg/kg E; gall endotoxin. The results of this study indicate that if the tempera- ture of the forelimb was maintained at the temperature of the contralateral forelimb, glucose uptake by the naturally perfused forelimb increases in skeletal muscle and skin during g; 2211 endotoxin shock in the dog and that the 101 102 mechanism for this increase is related to local tissue hypoxia and/or tissue ischemia. It was also noted that constant blood flow perfusion during endotoxin shock re- sulted in no change in isolated forelimb glucose uptake except at 30 minutes of shock induction. This transient increase in glucose uptake by forelimb skeletal muscle and skin was attributed to the ”insulin-like" effect of the endotoxin molecule acting directly on the tissue to stimu- late glucose uptake. It was also noted that when isolated forelimb tempera- ture was allowed to change independant of core temperature (Group 2) glucose uptake by forelimb skeletal muscle and skin did not change during endotoxin shock. A decrease in the metabolic rate of the isolated forelimb during endotoxin ‘shock was considered to be responsible for the reduction in substrate and oxygen need of the forelimb. Therefore these data emphasize the importance of maintaining temperature in isolated organ preparations during low blood flow states when metabolic variables are of importance. In conclusion, this study demonstrates that glucose uptake by forelimb skeletal muscle and skin increases during £2.2211 endotoxin shock in the dog and that the mechanism for this increase in glucose uptake is related to local tissue hypoxia and/or tissue ischemia. Furthermore, since skeletal muscle and skin makes up a large percentage of total body mass, an increase in glucose uptake by these 103 tissues, which presumably occurs throughout the body, must contribute substantially to the hypoglycemia of endotoxin shock. BIBLIOGRAPHY BIBLIOGRAPHY Roantree, R.T. The relationship of lipopolysaccharide structure to bacterial virulence. 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