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CURRENT RESEARCH REVIEW Laboratory

Models of Sepsis and Septic Shock

MITCHELL P. FINK, M.D., FACS, AND STEPHEN 0. HEARD, M.D. Departments

of Surgery and Anesthesiology,

University

of Massachusetts Medical Center, Worcester, Massachusetts 01655

Submitted for publication August 4, 1989

features but occasionally addressing other aspects (e.g., metabolism and pulmonary function) as well.

INTRODUCTION

In this journal in 1980, Wichterman et al. published a review article entitled “Sepsis and Septic Shock-A Review of Laboratory Models and a Proposal” [ 11. This report introduced a new and useful animal model of sepsis: cecal ligation and puncture in the rat (see below). Moreover, these authors focused attention on the importance of using animal models in sepsis research that adequately replicate the features of the sepsis syndrome in humans. During the past decade, sepsis and related problems like the adult respiratory distress syndrome (ARDS) have been the subjects of intense investigation. As a result, there is improved understanding of the pathophysiological mechanisms underlying the derangements in organ system function and metabolism in the septic state. In addition, several new modes of therapy have been developed that are sufficiently promising to warrant evaluation in patients; some of these approaches may be introduced into clinical practice within a few years. Despite the considerable progress in this field, sepsis is still an important cause of mortality [2] and current therapy remains largely supportive. While new approaches for therapy ultimately require validation using well-controlled clinical trials, it is virtually always necessary to obtain preliminary data in animals before using experimental drugs or devices in humans. Furthermore, many studies designed to acquire information about pathophysiology can only be performed in animals because they require invasive monitoring (e.g., to measure pulmonary lymph flow) or utilize pharmacological interventions of no or uncertain benefit. Finally, sepsis, as a clinical entity, is very heterogeneous and clinical data are invariably confounded by the effects of age, coexisting diseases, and supportive therapy. To control for these confounding variables and thereby generate interpretable results, clinical studies require enormous sample sizes and are necessarily very expensive and time consuming. Thus, as in 1980, progress in sepsis research continues to depend upon studies using clinically relevant animal models. With no attempt to be exhaustive, the present report reviews some of these models, focusing upon their hemodynamic 0022-4804/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

TERMINOLOGY

Wichterman et al. [l] defined sepsis as “an acute infection wherein an animal is toxic (febrile, weak, anorexic, lethargic, etc.) because of invasive infection.” In 1982, Pepe et al. introduced the term sepsis syndrome, defining it as a “clinical picture of serious bacterial infection with a concurrent, deleterious systemic response” [3]. Both these definitions presume that the sepsis syndrome is a manifestation of an acute, serious bacterial infection. It is apparent, however, that serious fungal and even viral infections are often associated with clinical signs and symptoms of “sepsis” [4-61. Indeed, a subset of the group of patients that die with sepsis and multiple organ system failure have repeatedly negative blood and urine cultures and, at autopsy, have no or only minimal evidence of infection [ 7,8]. Thus, we propose the following as a working definition of the sepsis syndrome that is applicable to both human and laboratory studies: l

l

l

“Septic shock” is circulatory decompensation (i.e., hypotension) due to sepsis; it is often (but, not always) associated with bloodstream invasion by bacteria or fungi (i.e., bacteremia or fungemia, respectively).

186 Inc. reserved.

Sepsis is a constellation of clinical and laboratory findings indicative of a generalized inflammatory response that is otherwise unexplained and is accompanied by acute organ system dysfunction and is often, but not invariably, associated with the presence of a serious bacterial, fungal, or viral infection. Other causes of generalized inflammation (e.g., systemic lupus erythematosus in crisis, familial Mediterranean fever, etc.) are obviously excluded. Findings indicative of generalized inflammation include fever, tachycardia, tachypnea, low systemic vascular resistance, leukocytosis, thrombocytopenia, and altered mental status.

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MODELS

GENERAL REMARKS REGARDING CHOICE OF SPECIES

For studies of sepsis and septic shock, the choice of species is dictated by many factors, among which are local availability (pigs are presumably less expensive and more easily obtained in Iowa than New York City), the familiarity of the laboratory with a particular species, and cost. Because of the increasing activity of “animal rights” groups, another important consideration is the “visibility” of the species; thus, experiments utilizing dogs or primates are more likely to engender protests (and, perhaps, even illegal sabotage) than are experiments using mice or rats. Clearly, the most important considerations are the goals of the study and the contraints imposed by the experimental design. Thus, for example, in a study intended to assess the role of C5a des arg (a complement fragment) in sepsis-induced ARDS, the investigators were obligated to develop a primate model, because the study depended upon the use of an antibody to human C5a des arg that was cross-reactive with the equivalent protein in primates but not other species [9, lo]. Being inexpensive to purchase and maintain, small mammals (mice, rats, and guinea pigs) are desirable for many studies, particularly when a large number of conditions (e.g., graded doses of an investigational therapeutic agent) are being examined. Because it is economically feasible to use large sample sizes, mice, rats, and guinea pigs are widely utilized for studies having survival as a major endpoint. In addition, rats and guinea pigs are particularly suitable for studies examining organ function ex vivo, for example, by using the Langendorf preparation to examine cardiac function and metabolism or the isolated perfused liver preparation to examine hepatic function and metabolism. In addition, rats are ideal for certain specialized studies of microvascular function employing vital microscopy to study blood flow through capillaries in skeletal muscle, kidney, or mesentery [ 11, 121. Special characteristics (e.g., resistance to endotoxin or absence of the fifth component of complement) are available in certain strains of inbred mice (and, to a lesser extent, rats and guinea pigs). These special characteristics are important in some experiments [13-X]. Although it is possible to assess cardiac output and pulmonary artery pressure in rats, these variables are much more easily measured in larger animals (e.g., sheep, pigs, dogs, and primates). Because of their greater circulating volume, these larger species are useful when it is necessary or desirable to obtain multiple blood samples for obtaining cells or performing assays for hormones or other humoral mediators. Sheep are docile animals that are well suited to acute or chronic studies performed using unanesthetized animals. The pulmonary lymphatic anatomy in sheep has been extensively studied and the Staub lunglymph fistula preparation using this species is widely employed for studies of lung microvascular permeability [ 16251. Pigs are quite similar to humans with respect to renal,

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cardiovascular, and digestive anatomy and physiology [26]. Since pigs are relatively inexpensive to obtain and maintain, this species is becoming quite popular for physiological and pharmacological studies, including experiments in the area of sepsis and septic shock. Because of their obvious similarities to humans, nonhuman primates (usually baboons or cynomolgus monkeys) remain invaluable for some studies. These animals, however, are quite costly and working with them is often difficult. Thus, primates are reserved for key (often preclinical) experiments that typically require only a small sample size. ENDOTOXICOSIS

MODELS

Lipopolysaccharide Gram-negative bacteria contain within their cell wall a macromolecular glycolipid termed lipopolysaccharide (LPS) [27]. LPS comprises two components: the O-specific chain and the core. The O-specific chain is a polymer of oligosaccharides and accounts for the antigenic variability among species and strains of Gram-negative bacteria. The core is composed of an oligosaccharide covalently bound to a molecule lipid called lipid A. Most of the toxicity of LPS resides in the lipid A moiety. The structure of the core region is relatively constant across species and strains of Gram-negative bacteria. The terms LPS and endotoxin are not strictly synonomous; LPS refers to the purified glycolipid, whereas endotoxins contain small amounts of cell wall proteins, lipids, lipoproteins, and polysaccharides in addition to LPS. For the present discussion, however, the terms LPS and endotoxin will be used interchangeably and the term endotoxicosis will be used to denote the state induced by infusing LPS or endotoxin. Endotoxic shock denotes circulatory collapse (i.e., hypotension) due to overwhelming endotoxicosis. Clinical Relevance of Experimental

Endotoxicosis

In their review published a decade ago, Wichterman et al. were very dubious about the relevance of experimental endotoxic shock to the clinical problem of sepsis [ 11. Thus, these authors cautioned that “sepsis and endotoxin shock are different entities” and “extreme caution must be exercised in using experimental findings with endotoxin models as the basis for pharmacologic therapy in septic patients.” They concluded their report with this statement: “Although there are some similarities between sepsis and endotoxemia (e.g., both cause proteoiysis), the very differences in various parameters and hemodynamic responses between these two situations make the correlation of an endotoxin study to a septic model difficult.” Doubts about the clinical relevance of experimental endotoxicosis derive from several observations: 1.

In humans with compensated sepsis, cardiac output is typically elevated and systemic vascular resistance is usually abnormally low; this is the so-called “hyper-

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dynamic” state [2, 28-311. Low systemic vascular resistance is so characteristic of sepsis in humans that it is often used as a research criterion for establishing the diagnosis [3, 321. During episodes of septic shock, cardiac output tends to decrease into the normal range while systemic vascular resistance remains abnormally low [28]. The hemodynamic perturbations elicited by bolus injections of large doses of LPS into animals of many species are quite different from the above: cardiac output is generally low and systemic vascular resistance is typically normal or elevated [33-391. Gluconeogenesis is suppressed and hypoglycemia occurs in acute experimental endotoxicosis [40, 411, whereas the opposite occurs in sepsis [42,43]. According to some studies, measurements of circulating endotoxin correlate poorly with clinical findings, outcome, or microbiological status [44, 451. Mice infected with Mycobacterium tuberculosis [Bacillus Calmetle-Guerin (BCG)] are hypersensitive to LPS; yet, when these animals are infected with Salmonella typhimurium, death occurs at total body bacterial loads that are comparable to those associated with lethality due to S. typhimurium in normal mice [46]. Human volunteers rendered tolerant to LPS by repeated injections still manifest signs of fever and acute toxemia during infection with viable Gram-negative organisms [47]. C3H/HeJ mice are genetically very hyporesponsive to LPS, yet these inbred mice manifest increased susceptility to infection (and lethality) induced by certain species of Gram-negative bacteria 148,491. In a clinically relevant canine peritonitis model (discussed in greater detail later), experimental sepsis induced by Staphylococcus aureus induces cardiovascular perturbations that are the same as those induced by Escherichia coli [50]. Although circulating LPS is detectable only in dogs infected with the Gram-negative bacteria, mortality in this model is actually greater in dogs infected with S. aureus.

Despite these concerns, accumulating data support the idea that LPS is pathophysiologically important in (some cases of) human sepsis. Danner et al. recently reported results of a study wherein circulating concentrations of LPS were frequently measured in 110 critically ill patients, including 100 with proven or suspected sepsis (group I) and 10 with shock due to other causes (e.g., hemorrhage or adrenal insufficiency) (group II) [51]. LPS was detectable in the plasma of 43 and 10% of patients in groups I and II, respectively (P < .05). In another recent study, van Deventer et al. assayed blood samples for LPS at the onset of fever in 473 consecutive patients with elevated body temperature [52]. Nineteen (4%) of these patients subsequently met rigid criteria for the diagnosis of sepsis; endotoxemia was detectable in 15 of the 19 patients. Of the 454 febrile patients who did not meet criteria

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for sepsis, 16 had positive limulus assays for LPS (and 10 of these had culture-proven Gram-negative infections). Thus, detecting LPS in plasma predicted the development of sepsis with a sensitivity and specifity of 79 and 96%, respectively. Additional support for the idea that LPS is pathophysiologically important in human sepsis comes from recent studies using human volunteers. In normal subjects, very small doses of LPS (4 rig/kg) induce hemodynamic, hematological, and metabolic changes that are qualitatively similar to those observed in septic patients, including fever [53], tachycardia [53], elevated cardiac output [ 541, hypotension [ 541, decreased systemic vascular resistance [ 541, leukocytosis [53], lymphopenia [53], elevated circulating concentrations of “stress” hormones (corticotropin, cortisol, growth hormone, and catecholamines) [53], elevated oxygen consumption (VO,) [53], and widened alveolar-arterial oxygen tension gradient [55]. From the preceding discussion, it is apparent that the relationship between endotoxicosis and sepsis is complicated. It is likely that LPS is only one of several triggers capable of initiating pathophysiological events that lead to the signs, symptoms, laboratory abnormalities, and derangements in organ function characteristic of the sepsis syndrome. Although not a sine qua non of sepsis, LPS is almost certainly of fundamental importance in many patients with sepsis or septic shock. Thus, depending upon the hypothesis being tested, endotoxicosis in animals may be a reasonable paradigm for sepsis in humans, with the proviso that the animal model chosen must adequately replicate those features of the clinical syndrome that are the focus of the experiment. Advantages of Using Lipopolysaccharide LPS, being a stable and relatively pure compound, simplifies some aspects of experimental design. LPS is convenient to use, being stored in lyophilized form until use. In contrast, bacteria are typically stored frozen, grown in culture for 18-24 hr prior to use, washed several times to remove culture medium and solubilized bacterial products (e.g., endotoxins), roughly quantifiedusing nephilometry, and later accurately quantified by enumerating viable colonies on pour plates. Whereas doses of LPS are readily measured and controlled, doses of viable bacteria can generally be quantified accurately only retrospectively, rendering it more difficult to ensure the reproducibility of the septic challenge. Indeed, in sepsis models using fecal suspensions or bowel devascularization and perforation, the dose of infecting organisms is typically unknown. Models The simplest endotoxicosis traperitoneal or intravenous

models employ a large inbolus dose of LPS in the

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absence of any supportive therapy, such as intravascular volume resuscitation, intubation, and mechanical ventilation or inotropic agent infusion. These simple models, typically employing mice or rats and using mortality as the primary outcome variable, are well suited for preliminary pharmacological studies of new drugs or other therapeutic agents. For example, Proctor et al. made valid use of this approach in a study showing that lipid X (a biosynthetic precursor and competitive antagonist of lipid A) improves survival in C57BL/lO mice injected with an LDlOO dose of LPS [56]. In the past several years, numerous groups have described animal endotoxicosis preparations that successfully reproduce the hemodynamic profile of either compensated sepsis or septic shock in humans. Species that have been utilized include rats, rabbits, dogs, pigs, sheep, and nonhuman primates. These models generally fall into one or more of four categories. 1. Models that utilize small (sublethal) doses of LPS. When rabbits are injected with a large intravenous dose of LPS (5 mg/kg), cardiac output decreases and systemic vascular resistance increases [39]. However, if rabbits are injected with a much smaller intravenous dose of LPS (l-3 pg/kg), the animals manifest the hyperdynamic circulatory pattern characteristic of compensated human sepsis [57]. Similarly, in rats, large doses of LPS lead to a hypodynamic picture [33,38], whereas very small doses of LPS elicit a hyperdynamic response [58]. Within minutes of an intravenous bolus dose of LPS in the dog, pulmonary artery and portal venous pressures increase sharply; these changes are accompanied by a marked initial decline in cardiac output and mean arterial pressure [34,36,37,59,60]. Although tending to normalize during the secondary phase of the response, cardiac output is typically subnormal and systemic vascular resistance is supranormal in this “classic” canine model of endotoxic shock. If, however, a very low dose of LPS is infused continuously (250 rig/kg-min), the hemodynamic response is quite different [34]. Portal venous and pulmonary arterial pressures do not change and cardiac output is well maintained (even in the absence of resuscitation). As in human sepsis, mean arterial pressure and systemic vascular resistance decrease significantly. Low (sublethal) doses of LPS also elicit a hyperdynamic response in chronically instrumented unanesthetized sheep. Thus, Talke et al. reported that a low dose of LPS (0.75 /*g/kg) infused intravenously over 30 min elicits a polyphasic response in sheep, characterized by low cardiac output and peripheral vasoconstriction during the early phase (O-6 hr) and markedly elevated cardiac output during the delayed phase (6-15 hr) [24]. This observation was subsequently extended in a study examining the effects of infusing sheep with extremely low doses of LPS (6,9,12, or 24 rig/kg-hr) continuously for 24 hr [25]. When the three highest doses were employed, a vasodilated hyperdynamic state was produced during the period 6-12 hr after initiating the endotoxin infusion.

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In another study examining the effects of time on the ovine response to low doses of LPS, Demling et al. found that infusing sheep with LPS (1 pg/kg) every 12 hr for 5 days leads to the development of a hyperdynamic hypermetabolic state that persists for several days after the administration of endotoxin is discontinued [ 181. Clinicians recognize that the deleterious effects of sepsis on organ system function are often not manifest until the process has persisted for several days or even weeks; thus, it is likely that this ovine model of a persistent hypermetabolic state will prove to be valuable for a variety of studies addressing subacute pathophysiological events in the sepsis syndrome. 2. Models that provide aggressive resuscitation of intravascular volume. Patients in septic shock are generally infused with large volumes of an asanguineous isotonic crystalloid or colloid solution. In addition, patients are often treated with vasoactive drugs, like dopamine, dobutamine, and/or norepinephrine. Thus, our notions regarding the circulatory perturbations characteristic of human septic shock are based not upon observations made in untreated patients, but on data collected during and after resuscitation and, often, other hemodynamically significant interventions. In contrast, protocols for many animal models of endotoxic shock, particularly in the older literature, provide at most maintenance fluids; thus, the circulatory profile in these models reflects the “natural” hemodynamic pattern of endotoxicosis, but not the (clinically relevant) pattern resulting from the combined effects of endotoxicosis plus routine supportive therapy. Recently, several groups have described animal models of resuscitated endotoxic shock. Breslow et al. described a model using pentobarbital-anesthetized mechanically ventilated 45- to 50-kg pigs infused with LPS (50-200 yg/ kg over 40 min) and resuscitated with sufficient Ringer’s lactate to maintain pulmonary capillary wedge pressure at the baseline level [61]. This model satisfactorily reproduces the hemodynamic profile of human septic shock; cardiac output is well preserved, but mean arterial pressure and systemic vascular resistance decrease dramatically to about 50% of the baseline value from 1 to 3 hr after the infusion of LPS. Despite the marked decrease in mean arterial pressure, coronary perfusion is well maintained, a finding in agreement with human studies [62, 631. In our laboratory, we have made small modifications to the model of Breslow et al. [64-661. In particular, we utilize smaller pigs (13-20 kg); this is advantageous when performing studies with scarce or expensive agents that are administered on a per kilogram basis. In addition, our resuscitation protocol uses a fixed volume of crystalloid (1.0-1.2 ml/kg-min) rather than a volume titrated to pulmonary capillary wedge pressure. We employ this approach because myocardial performance is clearly impaired in sepsis [30, 50, 67, 681 and endotoxicosis [69711; thus, preload may need to be elevated to supranormal

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levels to maintain cardiac output. In addition, sepsis may lead to alterations in ventricular diastolic compliance [68]; thus, left ventricular end-diastolic and pulmonary capillary wedge pressures may be poor proxies for left ventricular end-diastolic volume, the relevant variable for assessing preload. Finally, central venous and pulmonary capillary wedge pressures are typically small numbers (say, O-20 Torr), and, using conventional catheter-transducer-amplifier systems, there is considerable imprecision (5-15%) in the measurement of these variables; thus, unless a blinded experimental design is utilized, investigator bias might influence results when resuscitation is guided by these somewhat “soft” endpoints. Several other models of resuscitated endotoxicosis warrant mentioning. Modig described another anesthetized porcine model that differs from the models just described in several ways [ 721. Perhaps the most important feature of this model is the use of a continuous infusion of a low dose (10 pg/kg-hr) of LPS. When the animals are resuscitated with dextran-70 to maintain a normal left atria1 pressure, cardiac output is well preserved while mean arterial pressure decreases approximately 10%. Depending upon the choice of resuscitation fluid (Ringer’s acetate versus dextran-70), pulmonary venous admixture increases progressively by four- to sixfold. Melchior et al. described a hyperdynamic endotoxic shock model in dogs anesthetized and paralyzed with flunitrazepam, fentanyl, and vecuronium [73]. In this model, graded doses of LPS are infused intravenously, starting with 0.3 mg/kg-hr for 20 min and increasing in steps (0.2 mg/kg-hr) every 10 min until mean arterial pressure declines to 60% of the baseline value. Colloid solution (polygeline) is infused continuously to maintain left ventricular end-diastolic pressure at 6-9 Torr. In this model, cardiac output increases substantially and systemic vascular resistance decreases by more than 60%. When norepinephrine is infused at a dose sufficient to restore normal mean arterial blood pressure, cardiac output increases to more than twice the baseline value. 3. Models that utilize a continuous infusion of LPS. Reasoning that endotoxins are probably released into the circulation over an extended period in most patients with sepsis, there has been interest for many years in the effects of prolonged continuous infusions of LPS in experimental animals [74, 751. Perhaps the most important model of this type is the one using rats described by Fish and Spitzer [76]. In this model, LPS is delivered by continuous intravenous infusion using an Alzet osmotic pump at a rate of 3 mg/kg-day. Compared to appropriate controls, animals infused with LPS manifest many of the features of compensated human sepsis, including hypermetabolism, anorexia, mild hypotension, leukocytosis, and hyperlactatemia [76]. Coronary perfusion is well maintained in this model [77], although myocardial performance is impaired [69]. Recently, Arita et al. described a similar small animal endotoxicosis model using guinea pigs [ 781. In this model,

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hypermetabolism is demonstrable when LPS is infused into the portal vein, but resting metabolic rate is unaffected when endotoxin is infused into the peritoneal cavity. A large animal model using a continuous infusion of LPS was described by Lee et aE. [71]. In this paradigm, chronically instrumented, unanesthetized pigs are infused with LPS (10 pg/kg-hr) using an osmotic minipump. Mortality at 48 hr is 33%. Survivors manifest tachycardia and a small (but significant) increase in cardiac output. In addition, as in human sepsis, myocardial systolic performance is impaired in this model when data are analyzed using a load-independent estimator of contractility. 4. Models using LPS injected intraperitoneally. We described an unanesthetized rabbit model of endotoxicosis, wherein animals are injected intraperitoneally 1618 hr prior to study with LPS (50-500 pg/kg) suspended in 100 ml of saline [79]. In this model, cardiac output increases about 30% and systemic vascular resistance decreases about 20%. As in the human syndrome, coronary blood flow increases significantly. In compensated human sepsis, splanchnic perfusion tends to increase [42]; similarly, in this rabbit model, portal venous and small intestinal blood flow increases significantly. SEPSIS MODELS

Wichterman et al. recognized four main approaches for producing sepsis in laboratory animals: (1) intravenously infusing live bacteria, (2) inducing peritonitis using a fecal or bacterial suspensions, (3) inducing soft tissue abscesses in extremities, and (4) partially destroying the normal barriers of the gastrointestinal tract [ 11. With some modification, this classification scheme remains useful today. Intravascular

Infusion

of Bacteria

Wichterman et al. [l] noted that “most patients are not challenged with . . . a massive bacterial load at one time, but rather . . . harbor a septic focus which is intermittently, but persistently, showering the body with bacteria.” Thus, these authors questioned the relevancy of animal models utilizing a “bolus” infusion of viable bacteria. Despite this criticism, numerous laboratories continue to utilize intravascular infusions of viable bacteria to induce sepsis in animals, and, in our view, many of these models remain very useful provided certain inherent limitations are recognized. In rats, the cardiovascular effects of intravenous viable E. coli are apparently quite dependent upon the experimental conditions employed. When pentobarbital-anesthetized rats are intravenously infused with 8-18 X 10’ bacteria per hour for 5 hr (approximate total dose: l-3 X 10” organisms/kg), cardiac output is initially unchanged, decreasing significantly after 3-4 hr [80]. In contrast, injection of pentobarbital-anesthetized rats with a bolus dose of live E. coli (1.5 X lOlo organisms/kg) results

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in a biphasic response: cardiac output is significantly elevated for about 90 min and then decreases back into the normal range [81]. Cryer et al. developed a similar model, using rats rendered cortically unconscious by precollicular brainstem transection, a technique that avoids the confounding hemodynamic effects of general anesthesia [ 111. In this model, rats manifest a hyperdynamic circulation for 2 hr after being injected intravenously over 5 min with viable E. coli (approximately 3 X 10’ organisms/kg). Cryer et al. have used this model effectively to study the effects of hyperdynamic sepsis on the microvasculature of several organs or tissues [ll, 121. Numerous laboratories utilize intravenous infusions of live bacteria to induce sepsis in primates. For many years, Hinshaw and colleagues have employed an antibiotictreated anesthetized baboon preparation to study the effects of various pharmacological agents on the response to a well-standardized 2-hr infusion of an LDloo dose of live E. coli [82-841. The primary endpoint in these studies has been mortality, although these authors have extensively documented consistent effects on many other parameters, including hemodynamics, circulating blood cells and hormones, and histopathology. It is our view that these studies are open to criticism because the effectiveness of adjunctive therapeutic agents (e.g., methylprednisolone) has been assessed in the absence of standard supportive measures, including adequate resuscitation of intravascular volume and mechanical ventilation. While mortality rate is certainly an unambiguous indicator of success or failure, using this endpoint may lead to erroneous conclusions when animals are denied standard supportive measures, including ICU-type nursing care, adequate resuscitation of intravascular volume, and mechanical ventilation, Given these concerns, it is not surprising that the remarkable efficacy of methylprednisolone in this model [82, 841 has not been borne out in wellcontrolled clinical trials [85-871. In 1982, Carroll and Snyder described another primate E. coli bacteremia model and used it to study the effect of intravenous fluid administration on systemic hemodynamics and oxygen metabolism in severe septic shock [88]. In this model using ketamine-anesthetized cynomolgus monkeys infused intravenously with a large dose of bacteria (9 X 1O1’ organisms/kg), cardiac output increases significantly over baseline when the animals are aggressively resuscitated with normal saline (1 ml/kgmin). This model, and variations of it used in our and other laboratories, adequately reproduces many of the features of severe septic shock in humans, including hyperlactatemia despite normal oxygen uptake [88], low systemic vascular resistance [9, 88, 891, decreased urine flow and glomerular filtration rate [89], elevated extravascular lung water [9], and arterial hypoxemia [9]. Recently, Tracey et al. described a similar primate model of resuscitated septic shock [go]. Developed by these authors to study the efficacy of anti-cachectin antibody F(ab’)z fragments, this model uses pentobarbital-anesthetized,

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spontaneously breathing baboons infused intravenously with an LDlm dose of viable E. coli and resuscitated with Ringer’s lactate to maintain a pulmonary capillary wedge pressure of 2-3 Torr. Cracker et al. at Ohio State University developed a porcine model of Gram-negative sepsis for studying the adult respiratory distress syndrome 191, 921. This paradigm utilizes pentobarbital-anesthetized immature swine continuously infused intravenously with viable bacteria. The hemodynamic and pulmonary changes observed in this model are dependent upon the organism infused [93]. Infusion of viable S. aureus (approximate 1 X 10’ organisms per 20 kg) results in only minimal changes. In contrast, profound hypodynamic shock accompanied by acute respiratory failure develops when animals are infused with E. coli or Pseudomonas aeruginosa, the cardiopulmonary effects being especially dramatic with the latter organism. Because the pulmonary derangements characteristic of this model are so striking, it has been employed extensively and effectively by both the Ohio State group [91951 and Sugerman and colleagues at the Medical College of Virginia [96-981 in studies of the pathophysiology and pharmacotherapy of sepsis-induced ARDS. This model represents an overwhelming insult and, like the baboon model of Hinshaw et al., resuscitation of intravascular volume is not part of the protocol. Thus, it is unclear whether the cardiopulmonary changes observed are relevant to the clinical problem of sepsis-induced ARDS, which typically (but not always) evolves more gradually. Not all models using an intravascular infusion of live bacteria are acute preparations characterized by cardiovascular collapse secondary to overwhelming bacteremia. Shaw and Wolfe described a chronically instrumented unanesthetized canine model wherein animals are infused intraarterially with viable E. coli and studied 24 hr later [99]. In this paradigm, the animals are aggressively resuscitated at the time sepsis is induced, the dose of bacteria being invariably lethal in the absence of adequate restoration of intravascular volume. However, with resuscitation, 85% of the dogs survive the protocol and, at the time of study, are hyperdynamic and hypermetabolic. In addition, dogs rendered septic according to this protocol manifest many of the hormonal perturbations typically observed in septic humans, including hyperinsulinemia and hyperglucagonemia. Although not widely utilized, this model warrants closer attention by investigators because it successfully mimics many of the features of clinical sepsis and avoids the confounding effects of anesthesia and surgical manipulation. Since purpose-bred canines are quite expensive, we wonder whether this model might be adaptable to other species, such as pigs or sheep. Peritonitis

Models Using a Defined Bacterial Inoculum

In 1980, Ahrenholz and Simmons showed that 24-hr mortality is 100% when viable E. coli (2 X lo8 organisms) suspended in saline are injected intraperitoneally into rats

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[loo]. However, when the same number of bacteria are implanted intraperitoneally in a bovine fibrin clot, early mortality is prevented, but the rats develop abscesses and lo-day mortality is 90%. Thus, fibrin delays the systemic absorption of the entrapped bacteria and promotes the development of a more local septic focus. Extrapolating from these data obtained in rats, Fink et al. developed a large animal model of hyperdynamic sepsis, using chronically instrumented dogs implanted intraperitoneally with a bovine fibrin clot containing live E. coli bacteria [ 1011. Natanson et al. subsequently modified this model slightly by using monitoring catheters placed percutaneously at the time of study, thereby obviating problems with catheter-related infections in longterm studies [68]. This model reproduces many features of sepsis in humans, including elevated cardiac output [67, 68, 1011, leukocytosis [68], and reversible left ventricular dilation and impaired systolic performance [67, 681. Unlike other peritonitis models using fecal implantation or cecal perforation/devascularization, the fibrin clot model allows the investigator to have complete control over the dose of bacteria [67, 68, 1011 and the type of organism implanted [5O]. Nakatani et al. developed a rat intraperitoneal abscess model that also permits control over type and number of infecting bacteria [ 1021. In this model, rats are implanted intraperitoneally with a pellet consisting of autoclaved rat feces, agar, and a known number of E. coli and/or Bacteroides fragilis. The model reproducibly leads to a dramatic increase in cardiac output coupled with a decrease in systemic vascular resistance. Blood lactate levels are elevated. This model has been quite useful for studying the effects of sepsis due to aerobic and anaerobic bacteria on intermediary metabolism [ 1031. Recently, Alexander et al. described another new small animal chronic peritonitis model that permits control over the type of infecting organism [ 1041. In this model, Alzet osmotic minipumps containing viable E. coli and S. aureus are implanted intraperitoneally into guinea pigs. Although apparently not a model of hypermetabolic sepsis, this paradigm results in significant lethality; with appropriate adjustment of the dose of bacteria, mortality is about 50% at Day 18 after inducing sepsis. Because mortality occurs quite late in this model and is often accompanied by pneumonia, this model reproduces key features of chronic sepsis in humans and represents a valuable addition to the armamentarium of researchers in this field. Cecal Ligation and Perforation Fecal Peritonitis

and Other Models of

The cecal ligation and perforation (CLP) model developed by Wichterman et al. [l] has been widely utilized in sepsis-related research. Although originally described in rats, this model has been extended successfully to other species, including mice [ 1051 and sheep [ 1061. As noted previously, mice are advantageous for immunological and

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other studies where the experimental design depends upon the availability of genetically homogeneous inbred strains [13, 141. The CLP model in sheep is particularly useful for studying the effects of sepsis (and the treatment thereof) on systemic and pulmonary microvascular permeability [ 20, 1071. The principal advantage of CLP models (irrespective of species) is their simplicity. Since sepsis is induced by a straightforward surgical procedure, there is no need to grow and quantitate bacteria or in other ways prepare the inoculum. Furthermore, these are models of sepsis due to peritoneal contamination with mixed flora in the presence of devitalized tissue and thus bear an obvious resemblance to clinical problems like perforated appendicitis and diverticulitis. However, this very similarity to the clinical situation represents a potential drawback, in that it is difficult to control the magnitude of the septic challenge. In studies using small animals (mice and rats), the problem of variability is easily overcome by increasing sample size; however, variability remains a problem in larger species. Nevertheless, at times the variation in the magnitude of the response can be turned to the investigator’s advantage, such as when physiological differences between “responders” and “nonresponders” are studied [ 1081. Some of the problems of controlling the magnitude of the septic challenge can be overcome by inoculating the peritoneal cavity with feces directly rather than allowing fecal material to leak from a disrupted viscus. For example, in the model described by Lang et al., peritonitis is induced in chronically instrumented rats by implanting a standardized inoculum of pooled fecal material [ 1091. This model leads to a sustained increase in VOz [ 1091 and cardiac output [ 1101. As in human sepsis, myocardial and total hepatic blood flow increase substantially [ 1101. Significant mortality occurs, with 76% of animals dying by 5 days after the onset of infection [ 1091. Soft Tissue Infection

Models

One of the earliest models of hyperdynamic sepsis is the canine model described by Albrecht and Clowes in 1964 [ill]. In this model, a soft tissue abscess is induced by injecting lo-20 ml of 10% calcium chloride solution into the muscles of the thigh. This procedure results in local tissue necrosis and spontaneous infection with a “great variety of pathogens and saprophytes.” Hermreck et al. subsequently described a similar model, wherein a cloth tape impregnated in feces is implanted into the soft tissues of the thigh, creating a local site of infection and a hyperdynamic circulatory state [ 1121. In 1981, Gahhos et al. compared three different models of sepsis using immature swine: (1) intravenous infusion of live E. coli, (2) intravenous infusion of live E. coli plus volume expansion with intravenous saline, and (3) intramuscular injection of viable E. coli 24 hr prior to study [ 1131. Cardiac output was elevated only in the intramuscular injection group, leading these authors to conclude

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that a “focus of infection” is necessary for the development of the hyperdynamic state. Recently, similar results were reported by Durkot and Wolfe, who compared the metabolic responses to infection in two groups of conscious guinea pigs: animals infused intravenously with a large dose of viable E. coli and animals infused subcutaneously with same dose of bacteria [114]. Systemic VOZ decreased in the former group, but increased in the latter group, suggesting that hypermetabolism requires the presence of localized inflammation and infection. This view, however, is almost certainly incorrect, since numerous investigators have documented elevated cardiac output and/or elevated systemic V02 in animal subjects [ll, 12, 18, 24, 25, 34, 57, 58, 66, 73, 76, 78, 81, 88, 991 and human volunteers [53, 541 injected intravascularly with purified LPS or viable bacteria. It is more probable that the metabolic and circulatory response to sepsis (and endotoxicosis) depends less upon the presence of a localized focus of infection and more upon the magnitude of the acute challenge and the adequacy of resuscitation (see above). One more soft tissue model of chronic hyperdynamic sepsis warrants mentioning. Described recently by MelaRiker et al., this rat model uses chronically instrumented unanesthetized animals that are injected subcutaneously every other day with viable E. coli and B. fragilis (10’ colony-forming units each) [ 1151. This protocol evidently leads to considerable variability in response and the authors described their results in terms of “moderately” and “severely” septic groups. In both groups, cardiac output is persistently increased for several days after the induction of sepsis. Mortality depends upon the strain of E. coli employed. This small animal model shares the advantages of the guinea pig model of Alexander et al. already cited [ 1041.

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will require a major commitment of resources, because it will require, in essence, the creation of an animal intensive care unit. Nevertheless, we believe that progress in sepsisrelated research would be substantially facilitated were such a model available. Even without such a model, progress will continue in this field. A wide variety of good animal models are already available to investigators. In the next decade, as new methods, such as the powerful tools of molecular biology, are applied to problems related to the sepsis syndrome, these models will be invaluable in improving our understanding of pathophysiology and in developing new and more effective approaches toward therapy. ACKNOWLEDGMENTS This work was supported in part by NIH Grant 1 R29 GM-37631-03. Penny Lucier assisted in the preparation of the manuscript.

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That there are so many models of sepsis and septic shock is tacit evidence that none of them are perfect. Although sepsis presents in many forms clinically, most clinicians would probably agree that virtually all severely septic patients manifest respiratory failure and ventilator dependence. Furthermore, failure of organs other than the lungs typically occurs days to weeks after the onset of the septic process. Although early deaths occur commonly in some situations (e.g., meningococcemia, pneumococcal bacteremia in asplenic individuals, Gram-negative bacteremia in the setting of profound granulocytopenia), most deaths due to sepsis occur after a protracted course in an intensive care unit. Thus, for certain important experiments, there is a need for an animal model of severe chronic sepsis characterized by these features: persistent hypermetabolism, low systemic vascular resistance, respiratory failure severe enough to require mechanical ventilation, late (nonpulmonary) organ system failure, and death. Obviously, creation of such a model

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