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Therapeutic approaches to severe sepsis and septic shock

Fuhong Su

asbl Atomium - SABAM Beglium 2006

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P H Y S I O L O G Y L A B O R A T O R Y, I N T E N S I V E C A R E, E R A S M E H O S P T I A L

Therapeutic approaches to severe sepsis and septic shock

Fuhong Su

Promoter: Prof. Dr. Jean-Louis Vincent

A thesis submitted to Universite Libre De Bruxelles as the requirement for the degree of doctor of Biomedical Science

Academic year 2006-2007

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Table of contents

Summary 1

Declaration 2

Acknowledgements 3

Chapter 1: Introduction. 4

1.1 Introduction to sepsis, severe sepsis and septic shock. 4

1.1.1 History & terminology 4

1.1.2 Etiology 4

1.1.3 Pathophysiology 5

1.1.4 Epidemiology 5

1.1.5 Management of severe sepsis and septic shock 6

1.2 Current adjunctive therapies in septic shock. 6

1.3 Aims of the thesis. 7

1.4 References. 8

Chapter 2: Introduction of a reproducible ovine hyperdynamic septic shock model. 10

2.1 Introduction 10

2.1.1 Why choose sheep? 10

2.1.2 Advantages and disadvantages of sheep for an experimental septic shock model. 11

2.2 Methods 14

2.2.1 Instrumentation 14

2.2.2 Inducement of sepsis 16

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2.2.3 Variables measured & sampling 16

2.2.4 IL-6 measurements 17

2.2.5 Post-mortem study 18

2.2.6 Statistical methods 18

2.3 Results 19

2.3.1 General observations 19

2.3.2 Inflammatory variables 22

2.3.3 Hemodynamic variables 22

2.3.4 Organ dysfunction variables 24

2.3.5 Tissue perfusion variables 28

2.3.6 Histology 30

2.3.7 Outcome 32

2.4 Discussion 32

2.5 Summary 33

2.6 References 34

Chapter 3: Use of low tidal volume in septic shock may decrease severity of subsequent acute lung injury. 35

3.1 Introduction 35

3.2 Aims 35

3.3 Experimental protocol 36

3.4 Results 38

3.5 Discussion 46

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Chapter 4: Fever control in septic shock: beneficial or

harmful? 51

4.1 Introduction 51

4.2 Aims 52

4.3 Experimental protocol 52

4.4 Results 54

4.5 Discussion 57

4.6 Summary 60

4.7 References 61

Chapter 5: Euglycemic hyperinsulinemia in severe sepsis and septic shock. 65

5.1 Introduction 65

5.2 Aims 65

5.3 Experimental protocol 66

5.4 Results 67

5.5 Discussion 71

5.6 Summary 73

5.7 References 74

Chapter 6: Fluid resuscitation in severe sepsis and septic shock: albumin, hydroxyethyl starch, gelatin or Ringer’s lactate, does it really make a difference? 78

6.1 Introduction 78

6.2 Aims 78

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6.3 Experimental protocol 79

6.4 Results 80

6.5 Discussion 87

6.6 Summary 90

6.7 References 90

Chapter 7: Beneficial effects of ethyl pyruvate in septic shock. 96

7.1 Introduction 96

7.2 Aims 96

7.3 Experimental protocol 96

7.4 Results 97

7.5 Discussion 101

7.6 Summary 104

7.7 References 104

Chapter 8: Beneficial effects of alkaline phosphatase in septic shock. 107

8.1 Introduction 107

8.2 Aims 107

8.3 Experimental protocol 108

8.4 Results 109

8.5 Discussion 113

8.6 Summary 117

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Chapter 9: Conclusion. 121

Chapter 10: Remaining questions and future directions 122

10.1 Remaining questions 122

10.2 Future directions 122

10.3 References 126

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Summary

This thesis examines the role of different adjunctive therapies in an animal model of septic shock.

The thesis aims were to: 1) introduce a severe hyperdynamic septic shock animal model developed previously, which emulates the pathophysiologic derangements observed in patients with septic shock; 2) evaluate the efficacy of low tidal volume mechanical ventilation applied early during the course of sepsis; 3) challenge routine antipyretic treatment in septic shock; 4) study the influence of choice of fluid type during resuscitation; 5) investigate whether euglycemic hyperinsulinemia benefits septic shock in the acute phase; 6) test new therapies including ethyl pyruvate and alkaline phosphatase.

Our data demonstrate that compared with high tidal volume ventilation (12 ml/kg), low tidal volume ventilation (6 ml/kg), applied early after the onset of sepsis, might protect the lung from ventilator induce lung injury and improve outcome from sepsis; antipyretic interventions, including acetaminophen and external cooling are associated with lower circulating HSP70 concentrations, with harmful effects on respiratory function, with higher blood lactate concentrations, and with shortened survival times, our observations challenge routine antipyretic treatment in all critically ill patients; choice of fluid resuscitation from Ringer’s lactate, albumin, gelatin and HES has limited effect on outcome; euglycemic hyperinsulinemia decreased blood IL-6 concentrations but did not change hemodynamics or improve outcome; administration of EP may delay the development of hypotension and oliguria, and prolong survival; alkaline phosphatase administration can significantly improve gas exchange, decrease blood IL-6 levels, and prolong survival time.

The limitations of this septic shock model are discussed and ideas for future studies are

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Declaration

This work contains no material which has been accepted for the award of any other

degree or diploma in any university or other tertiary institution and, to the best of my

knowledge and belief, contains no material previously published or written by another

person, except where due reference is made in the text.

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Acknowledgements

Firstly, I would like to thank my supervisor

Prof. Jean-Louis Vincent

for his advice and guidance throughout my work. Without his support and encouragement this thesis would not have been possible.

I would also like to express my appreciation to all my collaborators

Dr. Qinghua Sun Dr. Peter Rogiers Dr. Nguyen Duc Nam Dr. Alejandro Bruhn Dr. Colin Verdant Dr. Zhen Wang

for their help and dedication during the experiments.

I am extremely grateful to

Dr. Karen Pickett

for her editorial assistance.

Additionally, I would like to thank Hassane Njimi for his statistical assistance.

Finally, I would like to thank my medical colleagues, technicians in the Intensive Care

Department, Erasme hospital, and my family and friends for their continued support and

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Chapter 1: Introduction.

1.1 Introduction to sepsis, severe sepsis, and septic shock.

1.1.1 History & terminology

In human history, infection has caused uncountable deaths: from the bubonic plague,

“Black Death” in medieval Europe to the recent Severe Acute Respiratory Syndrome (SARS) in Asia. As a sign of infection, fever was recognized and treated with herbal medicines by the Chinese Emperor, Shen Nong. This can be traced back as far as 2735 B.C. Sepsis, a term coined from a Greek word meaning “putrid”, was believed to be the reason for death when a wound came into contact with air and the process of putrefaction reached the blood (septicemia). It was not until the 19

th

century that the Austrian obstetrician, Semmelweis, and the British surgeon, Lister, introduced the concept of infection as a cause of sepsis [1]. Thereafter, the term sepsis was loosely defined and connected to bacterial infection. In 1991, the American College of Chest Physicians (ACCP) and the Society of Critical Care Medicine (SCCM) convened a “Consensus Conference,” and defined the systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic shock [2]. Ten years later, the European Society of Intensive Care Medicine (ESICM), ACCP, SCCM, the American Thoracic Society (ATS) and the Surgical Infection Society (SIS) reviewed these definitions and updated them, proposing also a new system for characterizing and staging the host response to infection:

Predisposition + Insult + Response + Organ dysfunction (PIRO) [3].

1.1.2 Etiology

The organisms involved in severe sepsis and septic shock are most often bacterial.

Although Gram-negative organisms were more commonly causative in the past, Gram-

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positive organisms are increasingly involved. The lung is the most common source of infection (40%), followed by the abdomen (20%), catheters and primary bacteremia (15%), and the urinary tract (10%) [4].

1.1.3 Pathophysiology

The systemic sepsis response starts with the recognition of invading organisms. Gram- positive and Gram-negative organisms, malarial parasites, fungi, endotoxin containing organisms, and other microbe proliferate and result in bacteremia and/or release toxins that stimulate the innate immune system, endothelial cells, and other cells. The immune cells, principally monocytes/macrophages and polymorphonulear neutrophils (PMNs), are able to recognize the pathogenic agents and their products: among the cell membrane receptors implicated are Toll-like receptors (TLR), intracellular signaling is activated, resulting in the activation of the transcriptional factor nuclear factor-kappa B activation and release a series of mediators including cytokines, lipid mediators, oxygen radicals and chemokines. These mediators include interleukin-1 (IL-1), IL-2, IL-6, IL-8, tumor necrosis factor (TNF-alpha), platelet activating factor (PAF), endorphins, eicosanoids, nitric oxide (NO), oxygen free radicals, high mobility group 1 (HMGB1), macrophage migration inhibitory factor (MIF), and others. These molecules can have profound effects on the cardiovascular system, kidneys, lungs, liver, central nervous system, and coagulation cascade. Furthermore, the expression of adhesion molecules on the vascular endothelium and circulating cells allows adhesion and migration of activated leukocytes into the subendothelial tissues. Alteration of the intercellular endothelial junction results in increased capillary permeability and generalized edema. Multi-organ failure including renal failure, myocardial dysfunction, acute respiratory distress syndrome (ARDS), hepatic failure, and disseminated intravascular coagulation may occur, ultimately resulting in death.

1.1.4 Epidemiology

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Data from epidemiological and health care economic surveys have estimated there to be 750,000 cases of severe sepsis annually in the USA, which will probably rise to over a million by the end of the current decade; at least 225,000 of these cases result in death, with the annual cost of treating severe sepsis in the USA estimated at $17 billion [5]. In the European Union, the estimated number of fatal cases is 150,000 annually, and the cost of treatment $ 6.7 billion [6].

In hospitalized patients, the morbidity and mortality of severe sepsis ranges from 0.2%- 8% and 20%-90% in different studies [7-9]. In ICU patients, 10-15% develop septic shock with a 50 to 60% mortality [6, 10-12].

Recently, some studies have reported a decreased mortality with some therapeutic approaches [13, 14]; however, these studies often exclude certain severe patients, which makes the interpretation of decreased mortality difficult.

1.1.5 Management of severe sepsis and septic shock

Management of the patient with septic shock involves three inseparable components:

antibiotics and source control (whenever indicated) to eliminate infection; fluid resuscitation and vasoactive agents to stabilize hemodynamics; and modulation of the sepsis response [15, 16]. In 2003, critical care and infectious disease experts representing 11 international organizations developed the Surviving Sepsis Campaign Guidelines for the management of severe sepsis and septic shock, representing an international effort to increase awareness and improve outcomes in severe sepsis [17]. These guidelines not only provide a comprehensive template for clinicians to follow but also provide an objective evaluation of the strength of evidence.

1.2 Current adjunctive therapies in septic shock.

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In the Surviving Sepsis Campaign guidelines [17], apart from early and appropriate antimicrobials, source control aimed to control infection, adjunctive therapies such as early goal-directed therapy, recombinant human activated protein C, corticosteroids, glucose control, and low tidal volume mechanical ventilation are recommended. These adjunctive therapies place early and aggressive management of severe sepsis and septic shock as integral to reducing sepsis mortality [18].

1.3 Aims of the thesis.

Despite the progress made in the development of the Surviving Sepsis Campaign guidelines [17], a lot of issues remain unclear regarding the management of sepsis. The aims of this thesis were therefore to investigate how adjunctive therapies might improve outcomes of sepsis and septic shock, in particular:

whether the early utilization of low tidal volume (6 ml/kg) ventilation (even before the development of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)) compared with high tidal volume (12 ml/kg) reduces subsequent lung injury in sepsis.

whether antipyretic treatment in sepsis influences outcome.

whether fluid type choice in fluid resuscitation influence outcome of septic shock.

whether tight glucose control benefits survival in the acute phase of sepsis and septic shock.

whether ethyl pyruvate administration has beneficial effects in septic shock.

whether alkaline phosphatase has beneficial effects in septic shock.

To fulfill aims, a reproducible, clinically relevant animal model of sepsis and septic

shock was needed.

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1.4 References.

1. Reinhart, K.B., F. Brunkhorst, F.M., Pathophysiology of sepsis and mutiple organ dysfunction. Fifth editioin ed, ed. M.P.A. Fink, E.Vincent, JL. Kochanek, P.M.

2004.

2. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med, 1992. 20(6): p. 864-74.

3. Murakami, K., et al., Activated protein C attenuates endotoxin-induced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood, 1996.

87(2 #0): p. 642-7.

4. Sise, M.J., et al., Serum oncotic pressure and oncotic-hydrostatic pressure differences in critically ill patients. Anesth Analg, 1982. 61(6): p. 496-8.

5. Nahum, A., et al., Effect of mechanical ventilation strategy on dissemination of intratracheally instilled Escherichia coli in dogs. Crit Care Med, 1997. 25(10): p.

1733-43.

6. Levy, M.M., et al., 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med, 2003. 31(4): p. 1250-6.

7. Vincent, J.L., et al., Sepsis in European intensive care units: results of the SOAP study. Crit Care Med, 2006. 34(2 #0): p. 344-53.

8. Angus, D.C., et al., Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med, 2001. 29(7): p.

1303-10.

9. Alberti, C., et al., Epidemiology of sepsis and infection in ICU patients from an international multicentre cohort study. Intensive Care Med, 2002. 28(2 #0): p.

108-21.

10. Martin, G.S., et al., The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med, 2003. 348(16): p. 1546-54.

11. Padkin, A., et al., Epidemiology of severe sepsis occurring in the first 24 hrs in intensive care units in England, Wales, and Northern Ireland. Crit Care Med, 2003. 31(9): p. 2332-8.

12. Weycker, D., et al., Long-term mortality and medical care charges in patients with severe sepsis. Crit Care Med, 2003. 31(9): p. 2316-23.

13. Brun-Buisson, C., et al., Epidemiology and outcome of acute lung injury in European intensive care units. Results from the ALIVE study. Intensive Care Med, 2004. 30(1): p. 51-61.

14. Finfer, S., et al., Adult-population incidence of severe sepsis in Australian and New Zealand intensive care units. Intensive Care Med, 2004. 30(4): p. 589-96.

15. Angus, D.C. and R.S. Wax, Epidemiology of sepsis: an update. Crit Care Med, 2001. 29(7 Suppl): p. S109-16.

16. Bernard, G.R., et al., Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med, 2001. 344(10): p. 699-709.

17. Rivers, E., et al., Early goal-directed therapy in the treatment of severe sepsis and

septic shock. N Engl J Med, 2001. 345(19): p. 1368-77.

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18. Vincent, J.L., Hemodynamic support in septic shock. Intensive Care Med, 2001.

27 Suppl 1: p. S80-92.

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Chapter 2: Introduction of a reproducible ovine hyperdynamic septic shock model.

2.1 Introduction

2.1.1 Why a sheep model?

The pursuit of an animal sepsis model is typically driven by the need to emulate the clinical situation in patients with sepsis and septic shock. Numerous groups have developed mice, rat, rabbit, dog, pig, sheep, and non-human primate’ sepsis and septic shock models. A variety of experimental conditions have been established that enable investigators to study the effects of septic shock and to assess the potential benefits of a wide spectrum of treatment options. However, translating these experimental findings into clinically applicable therapies has been challenging, suggesting the need for a more clinically relevant model and better understanding of the animal models being used [1]. A few groups have tried to define the characteristics of a clinically relevant sepsis model [2- 4]. According to these criteria, the ideal sepsis and septic shock animal model should possess the following characteristics:

o The injury should be toxic to the host that will be febrile, anorexic, weak and lethargic;

o Use outbred rather than inbred animals;

o Use both genders rather than one;

o Use a complex mixture of microbes rather than just one;

o Positive blood cultures should be acquired continuously in the study period;

o Long enough to allow the animal to respond to the insult;

o The model should be reproducible, standardized, versatile, and not expensive;

o Adjunctive treatments (e.g., mechanical ventilation, fluid resuscitation, antibiotics and vasoactive agents) should be added to the model;

o Clinically used physiological end points for monitoring should be available.

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To fulfill the above criteria some compromises are necessary, our lab developed a model of fecal peritonitis in sheep, in which we tested vessopressin [5], different combination of vessoactive agents [6], different cardiac output measurement methods [7]. This model is important because it involves cardiovascular similar to what is seen in human sepsis and septic shock.

2.1.2 Advantages and disadvantages of sheep for an experimental septic shock model.

As a docile ruminant animal, most physiologic variables in sheep are similar to humans (Tables 2.1, 2.2, 2.3). Sheep are also suitable for acute or chronic studies with their greater circulating volume, which is useful when multiple blood samples are needed to obtain cells or perform assays for hormones or other mediators. The pulmonary lymphatic anatomy in sheep has been extensively studied by Staub and colleagues in studies of lung microvascular permeability [4]. Pulmonary intravascular macrophages occupied 15.3% of the intravascular volume, and had 15.9 m2 of free surface available for contact with blood [8]. Macrophages resident in the pulmonary capillaries of sheep avidly remove injected particles from the circulating blood. The sheep cecal ligation and perforation (CLP) model is particularly useful in studying the effects of sepsis on systemic and pulmonary microvascular permeability [9, 10].

Disadvantages of this model include the expense, shortage of biological investigative

reagents, and the number of collaborators needed.

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Table 2,1 Hemodynamic variables in humans and sheep. Sheep data come from measurements of 20 healthy sheep. Results expressed as mean ± SD.

Variable (abbreviation) Unit

Normal range (Human)

Measured values in healthy

sheep

Systolic Blood Pressure (SBP) mmHg 100~140 118 ± 8 Diastolic Blood Pressure (DBP) mmHg 60~90 97 ± 8 Pulmonary Artery Systolic Pressure (PAS) mmHg 15~30 19 ± 5 Diastolic Pulmonary Artery Pressure (DPAP) mmHg 4~12 10 ± 3 Pulmonary Artery Occlusion Pressure (PAOP) mmHg 5~12 5 ± 1

Central Venous Pressure (CVP) mmHg 0~8 2 ± 2 Heart Rate (HR) beats/min 60~100 120 ± 14

Cardiac Output (CO) L/min 4~8 3.7 ± 0.7

Right Ventricular Ejection Fraction (RVEF) fraction 0.4~0.6 --- Intra-Abdominal Pressure (IAP) mmHg <10 1 ± 1

Intra-cranial Pressure (ICP) mmHg <10 ---

Table 2.2 Normal ranges of arterial blood gases in humans and sheep.

Arterial blood gas Human Sheep

Hemoglobin (g/dl)

Male: 13~18

Female: 12~16 8~16

pH 7.35~7.44 7.32~7.54

PaCO2 (mmHg) 35~45 33~45

PaO2 (mmHg) 70~100 ---

HCO3- (mmol/L) 21~28 20~25

SaO2 (arterial oxygen saturation) (%) 95~100% ---

K+ (mEq/L) 3.5~5.0 3.9~5.4

Na+ (mEq/L) 135~145 139~152

Ca2+(mg/dL) 8.5~10.5 11.5~12.8

Mg2+(mEq/L) 1.5~2.0 2.2~2.8

Cl- (mEq/L) 98~106 95~103

Glucose (mg/100ml) 70~110 40~90

Lactate (mmol/L) <1 mmol/L < 1 mmol/L

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Table 2.3 Normal cell count ranges in humans and sheep.

Cell count Human Sheep

White blood cell (WBC) (x109/ L ) 4.3~10.8 4 ~ 12.0 Neutrophil (NE) (x109/ L) 1.2~8.3 0.7 ~ 6.0 Lymphocyte (LY) (x109/ L ) 1~4.8 2.0 ~ 9.0 Monocyte (MO) (x109/ L ) 0.2~0.95 0.0 ~ 0,8 Eosinophil (EO) (x109/ L ) 0~0.4 0.0 ~ 1.0

Basophil (BA) (x109/ L) 0~0.3 0.0 ~ 0.3

Red blood cell (RBC) (x1012/L) 4.2~6.9 9.0 ~ 15.0 Hemotocrit (HCT) (%) Male: 45~62

Female:37~48

27.0 ~ 45.0

Mean corpuscular volume (MCV)(femtoliters) 76~100 28.0 ~ 40.0 Mean corpuscular hemoglobin (MCH) (pg) 27~32 8.0 ~ 12.0 Mean corpuscular hemoglobin concentration

(MCHC) (g/dL) 32~36

31.0 ~ 34.0

Red cell distribution width (RDW) 11.8~15.2 12.0 ~ 27.0 Reticulocytes (x109/ L ) 0.018~0.158 0.2 ~ 0.98 Platelet (PLT) (x109/ L ) 150~350 250 ~ 750

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2.2 Methods

Care and handling of the animals is in accordance with National Institute of Health guidelines (Institute of Laboratory Animal Resources). All of the study protocols are conducted in accordance with the guidelines established by the Institutional Review Board for animal care of the Free University of Brussels. Female sheep are fasted for 24 hours with free access to water prior to the experiment. With experience during the different protocols, we made some improvements to optimize the model: the changes are listed in table 2.4.

Table 2.4 Changes made in different protocols.

Low tidal volume protocol Temperature control protocol

Fluid type protocol Ethyl pyruvate protocol

Alkaline phosphatase protocol Euglycemic hyperinsulinemia

protocol Pre-medication xylazine

ketamine

midazolam ketamine Before intubation pancuronium bromide fentanyl

pancuronium bromide Sedation

muscular blockade

midazolam fentanyl pancuronium bromide

ketamine morphine midazolam pancuronium bromide Control digestive

congestion No Yes

Sterilized

surgical operation No Yes

Absolute baseline No Yes

Feces dose 0.5 gram/kg 1.5 gram/kg

Fluid type Ringer’s lactate Ringer’s lactate + HES (Voluven) Fluid resuscitation

titration goal Baseline PAOP CO, MAP and SvO2

2.2.1 Instrumentation

On the day of the experiment in the current model, animals are initially weighed,

premedicated with intramuscular midazolam (Dormicum, Roche, Attikis, Greece) (0.25

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mg/kg) and ketamine hydrochloride (Imalgine, Merial, Lyon, France) (20 mg/kg) and placed in the supine position. The cephalic vein is cannulated with a peripheral venous 18G catheter (Surflo I.V Catheter, Terumo, Belgium). Following intravenous administration of fentanyl (Fentanyl, Janssen, Berchem, Belgium) (30 g/kg) and pancuronium bromide (Pavulon, Organon, Oss, the Netherlands) (0.1 mg/kg), the trachea is intubated (Tracheal Tube, 8.0; Hi-Contour, Mallinckrodt Medical, Ireland). Mechanical ventilation is started in controlled volume mode (Servo ventilator 900 C, Siemens-Elema, Sweden) with a tidal volume of 9 ml/kg, a positive end-expiratory pressure (PEEP) of 5 cmH

2

O, an inspired oxygen fraction (FiO

2

) of 1, an inspiratory time/expiratory time (I/E) of 1:2, and a square wave pattern. Respiratory rate (RR) is adjusted to maintain end-tidal carbon dioxide pressure (PetCO

2

, 47210 A Capnometer, Boehlingen, Germany) between 35 and 45 mmHg. A 60 cm plastic tube (inner-diameter 1.8 cm) is inserted into the stomach to drain its content and prevent rumen distension. A Foley catheter (14F, Beiersdorf AG, Germany) is placed to measure urine output. The right femoral artery and vein are exposed under strictly sterile conditions. An arterial catheter (6F Vygon, Cirencester, UK) is introduced and connected to a pressure transducer (Edwards, Lifescience, CA, USA) zeroed at mid-thorax level. Through the femoral vein, an introducer is inserted and a 7F Swan-Ganz catheter (Edwards Life Sciences, Baxter, Irvine, CA, USA) is advanced into the pulmonary artery under monitoring of pressure waveforms. A midline laparotomy is performed and, after cecotomy, 1.5 g/kg body weight of feces is collected. The cecum is closed and the area around the cut is disinfected with iodine. An additional pouch suture is performed to prevent contamination and the cecum is returned to the abdominal cavity. A large plastic tube is inserted through the abdominal wall for later injection of feces. The abdomen is then closed in two layers. After the surgical preparation, animals are turned to the prone position and allowed to stabilize for 2 hours. At the end of this period, baseline measurements are performed.

All sheep are sedated with IV ketamine 10 mg/kg/h, morphine 0.5 mg/kg/h and

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mg/kg/h. Boluses of fentanyl 3 g/kg/h IV are administered if needed in order to prevent rise in heart rate and arterial pressure due to insufficient anesthesia. Controlled mechanical ventilation is adjusted to ensure normoxia (80 mmHg PaO

2

120 mmHg) and normocapnia (35 mmHg PaCO

2

45 mmHg) according to repeated blood gas analysis (ABL500 OSM3 Radiometer, Copenhagen, Denmark). Hemoglobin concentration and oxygen saturation are measured with an analyzer calibrated for animals (OSM3 Radiometer, Copenhagen, Denmark).

2.2.2 Inducement of sepsis

After baseline measurements, the feces are injected into the abdominal cavity followed by 120 ml of air to empty the tube. Core temperature is kept at a minimum of 36.5 ° C using heating pads. Hyperthermia is not treated. A 6% hydroxyethylstarch solution (HES) solution (MW: 130,000, degree of substitution 0.6, Voluven, Fresenius, Bad Homburg, Germany) and a Ringer’s lactate (RL) solution (volume ratio =1:1) are initially infused at a rate of 2 ml/kg/hour each. Fluid resuscitation is titrated to maintain mean arterial pressure (MAP) above 60 mmHg, cardiac filling pressures and cardiac output at least at baseline levels, and hemoconcentration (defined as a hemoglobin level increasing by more than 2 g/dl in one hour) throughout the experiment. Supplementary infusions (volume challenge) of RL 4 ml/kg + HES 4 ml/kg over 5 minutes are given if the cardiac output decreases by more than 10 % from baseline, or MAP falls below 60 mmHg (defined as hypotension). No antibiotics and no vasoactive agents are administered.

Animals are observed until spontaneous death or up to 30 hours after baseline.

2.2.3 Variables measured & Sampling

All monitored variables are recorded every 60 minutes. Heart rate, MAP, central venous

pressure (CVP), pulmonary arterial pressure (PAP), and pulmonary arterial occlusion

pressure (PAOP) measurements are referenced to mid-chest level and obtained at end

expiration (Sirecust 404 Siemens, Germany). Core temperature, cardiac output

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(Vigilance Baxter, Edwards Critical-Care, USA) and minute volume, plateau pressure, expiratory tidal volume, and PetCO

2

are continuously monitored. Body surface area is calculated from the equation BSA = 0.084 (body weight kg)

2/3

[11]. Cardiac index (CI) (l/min/m

2

), stroke volume index (SVI) (ml/min/m

2

), systemic vascular resistance index (SVRI) (dyne/sec/cm

5

), pulmonary vascular resistance index (PVRI) (dyne/sec/cm

5

), oxygen delivery (DO

2

) (ml/kg/min), and oxygen consumption (VO

2

) (ml/kg/min) are calculated using standard formulas.

Arterial samples are taken at baseline, and 1, 4, 8, 12, 16 hours after feces injection.

Three milliliters of blood are injected into a EDTA (K2) tube (VenojectII, Terumo European. Leuven Belgium) and saved on ice. After centrifugation at 3000 r/min for 10 minutes at 4°C, plasma is separated to an Eppendorf tube (Sigma-aldrich, Belgium) and stored at –70°C for future measurements.

2.2.4 IL-6 measurements

Enzyme Linked Immuno-sorbent Assay (ELISA): Mouse anti-ovine interleukin (IL)-6 monoclonal antibody (Serotec MCA1659, Kidlington, UK), rabbit anti-ovine IL-6 polyclonal antibody (Serotec AHP424) and sheep anti-rabbit HRP coupled (Serotec STAR64, Kidlington, UK) are used to measure IL-6 levels. In short, the monoclonal antibody is used as a coating antibody with a concentration of 1:200, diluted in phosphate-buffered saline (PBS) and incubated overnight on 96-well ELISA plates (Greiner) at 4°C. After discarding the coating solution, 250 l blocking buffer (PBS/ 1%

BSA) is added for 2 h at room temperature, and then rinsed three times with PBS/0.05%

Tween. A 50 l serum sample is diluted with 50l PBS / 1% bovine serum albumin [12],

placed in plate wells and incubated for one hour at room temperature. Because there is no

purified IL-6 protein available, one sample is randomly chosen and dilated to construct a

standard curve. The plates are then washed three times before adding detection

polyclonal antibody (HRP-Ab) and incubating for one hour at room temperature. After

being rinsed three times, the substrate for the conjugated HRP is added to the plate and

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allowed to react for 10 minutes. The optical density of the plate-wells is then read on an ELISA plate reader at 450 nm.

2.2.5 Post-mortem study

Specimens of lung, heart, liver, kidney, spleen, and small intestine are taken immediately after each sheep’s death. Samples are saved in 4% buffered formalin, routinely processed into paraffin blocks, sectioned at 4 m, and stained with hematoxylin and eosin (HE). A pathologist blinded to the study protocol examines the histology.

2.2.6 Statistical methods

Baseline characteristics of the animals are presented as mean ± SD and differences

among groups are compared with Student’s t-test (for two groups) or one-way ANOVA

(for more than two groups). The effect of the treatment is analyzed using either two-way

ANOVA (group and time) with repeated measurements (missing values are dealt with by

the last-observation-carried-forward method of imputation) or mixed-effects models

analysis with treatment group, time, and subjects nested in-group as factors. If significant,

each time point difference between group animals is compared with Student’s t-test (for

two groups) or one-way ANOVA (for more than two groups). Survival curves are

constructed using the Kaplan-Meier method and compared using the log-rank test

(without inter-cross) or Wilcoxon test (with inter-cross). Statistical tests are two-tailed

and a p-value less than 0.05 is considered statistically significant. All statistical analysis

are performed using JMP 5.0 statistical software (SAS Institute Inc, Cary, NC, USA).

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2.3 Results

Continuous blood sampling for bacterial culture shows both Gram-negative (Escherichia coli, Citrobacter freundii, Klebsiella oxytoca, and Enerobacter cloacae) and Gram- positive (Enterococcus facecalis) bacteremia after sepsis inducement.

2.3.1 General observations

These are presented according to the diagnostic criteria for sepsis defined in 2001 [13].

• Fever (core temperature >38.3 ° C)

The normal range of temperature in sheep is 38°C~39°C. Although there is no clear definition of fever and hypothermia in sheep, we observe that core temperatures can increase and decrease during the experimental period with induction of general anesthesia and a large amount of room temperature fluid resuscitation (Figure 2.1a). General anesthesia inducement generally decreases body temperature by about 2 ° C [14]. This could explain why the baseline temperature is lower than the normal range.

36 37 38 39 40 41

T1 T3 T5 T7 T9 T11 T13 T15 T17

Core body temperature (Centigrade)

Time (h)

33 34 35 36 37 38

T1 T3 T5 T7 T9 T11 T13 T15 T17

Core body temperature (Centigrade)

Time (h)

a b

(27)

• Hypothermia (core temperature <36 ° C)

In some animals, hypothermia is also observed (Figure 2.1 b). As in patients, this subgroup has worse outcomes, such as lower cardiac output, shorter survival time.

• Tachycardia (defined as heart rate > 100 beats/minute)

Two hours after feces injection, tachycardia occurs and the heart rate remains high during the experiment (Figure 2.2 a).

• Tachypnea

With muscular blockade and sedation, spontaneous breathing is completely inhibited during the experiment. However, to maintain the PaCO

2

in the range of 35~45 mmHg, the respiratory rate is increased slowly during the experiment (Figure 2.2 b).

60 80 100 120 140 160 180 200 220

T1 T3 T5 T7 T9 T11 T13 T15 T17

Heart rate (beats/min)

Time (h)

10 15 20 25

T1 T3 T5 T7 T9 T11 T13 T15 T17

Respiratory rate (breaths/min)

Time (h)

a b

Figure 2.2 Heart rate (a) and respiratory rate (b) in experimental period (N =10). Data

expressed as mean ± standard deviation.

(28)

• Significant edema or positive fluid balance (> 20 ml/kg over 24 hrs)

With aggressive fluid resuscitation, fluid accumulation (defined as total fluid intake – urine output) increases substantially during the experiment (Figure 2.3 a).

• Hyperglycemia (plasma glucose >120 mg/dL or 7.7 mmol/L) in the absence of diabetes

The normal range of arterial blood glucose concentrations for sheep is 40-90 mg/dl.

Arterial glucose concentrations increase greatly during the surgical operation period, which can be explained by stress, but remain in the normal range during the rest of the experiment (Figure 2.3 b). In some sheep, hypoglycemia (defined as < 40 mg/dl) develops in the terminal phase. Hyperglycemia occurs rarely.

40 60 80 100 120 140

T 0 T 4 T 8 T 12 T 15 T 19

Arterial glucose concentration (mg/dl)

Time (h)

0 5 10 15 20

T1 T3 T5 T7 T9 T11 T13 T15 T17

Fluid accumulation (Liter)

Time (h)

a b

Figure 2.3 Fluid accumulation (total fluid intake-urine output) (a) (N =10) and arterial glucose

concentrations (b) (N =53) during experiment. Data expressed as mean ± standard deviation.

(29)

2.3.2 Inflammatory variables

• Leukocytosis (WBC count >12,000 μL-1) In this model, leukocytosis never develops.

• Leukopenia (WBC count <4,000 μL-1)

Leukopenia is the universal phenomenon in this model (Figure 2.4 a). Inflammatory variables such as IL-6 (Figure 2.4 b) are also significantly increased.

2.3.3 Hemodynamic variables

• Arterial hypotension (SBP <90 mmHg, MAP <70, or an SBP decrease >40 mmHg in adults or <2 SD below normal for age)

Arterial hypotension develops after 10 ~ 12 hours (Figure 2.5 a & b).

Figure 2.4 White blood cell count showed leukopenia (a). Blood IL-6 concentration increases significantly in the experiment (N =10). Data expressed as mean ± standard deviation.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

T 1 T 3 T 5 T 7 T 9 T 11 T 13 T 15 T 17 T 20

Arterial IL-6 concentrations (Arbitrary unit)

Time (h)

0 1000 2000 3000 4000 5000 6000 7000 8000

T0 T2 T4 T6 T8 T10 T12 T14 T16 T19

White blood cell count (/ml)

Time (h)

a b

(30)

• SvO2 >70%

Baseline SvO2 is higher than 70% and remains high before decreasing after 13 hours (Figure 2.6 a).

• Cardiac index >3.5 L min-1 M2

60 70 80 90 100 110 120

T1 T3 T5 T7 T9 T11 T13 T15 T17

Mean arterial pressure (mmHg)

Time (h)

0 20 40 60 80 100 120 140 160

T1 T3 T5 T7 T9 T11 T13 T15 T17

Systolic blood pressure (mmHg)

Time (h)

a b

Figure 2.5 Systolic blood pressure (a) and mean arterial pressure (b) during the experiment (N

=10). Data expressed as mean ± standard deviation.

a b

Figure 2.6 SvO2 (a) and cardiac index (b) during the experiment (N =10). Data expressed as mean ± standard deviation.

40 50 60 70 80 90 100

T1 T3 T5 T7 T9 T11 T13 T15 T17

SVO2 (%)

Time (h)

4 5 6 7 8 9 10

T1 T3 T5 T7 T9 T11 T13 T15 T17

Cardiac index (L/min/m2)

Time (h)

(31)

Cardiac index (Figure 2.6 b) increases significantly after induction of sepsis and remains high during the experiments.

2.3.4 Organ dysfunction variables

• Arterial hypoxemia (PaO

2

/FiO

2

<300)

Arterial hypoxemia, as suggested by PaO

2

/FiO

2

< 300, develops after 8 ~ 10 hours and continues to decrease to less than 200 (Figure 2.7 a). MPAP increases initially and reaches a peak, then decreases sharply before slowly increasing again (Figure 2.7 b).

Mean airway pressure (Figure 2.7 c), pulmonary vescular resistance (Figure 2.7 d) and respiratory system resistance (Figure 2.7 e) increase slowly during the experiment.

Respiratory system compliance decreases sharply during the experiment (Figure 2.7 f).

(32)

50 100 150 200 250 300 350 400 450

PaO2/FiO2 (mmHg)

5 10 15 20 25

Mean airway pressure (cmH2O)

0 20 40 60 80 100 120

T1 T3 T5 T7 T9 T11 T13 T15 T17

Respiratory system resistance (cmH2O*sec/L)

Time (h)

15 20 25 30 35 40

Mean pulmonary arterial pressure (mmHg)

0 50 100 150 200 250 300 350 400

Pulmonary vascular resistance (dyne.sec.cm-5)

5 10 15 20 25

T1 T3 T5 T7 T9 T11 T13 T15 T17

Respiratory system compliance (L/cm2H2O)

Time (h)

a b

c d

e f

Figure 2.7 PaO

2

/FiO

2

(a), mean pulmonary arterial pressure (b), mean airway pressure (c),

pulmonary vascular resistance (d), respiratory system resistance (e) and respiratory system

(33)

• Acute oliguria (urine output <0.5 ml/kg/hr or < 45 ml for at least 2hrs)

Despite aggressive fluid resuscitation, acute oliguria and even anuria (defined as no urine output for at least 2 hrs) occur after 14~16 hours (Figure 2.8 a).

• Creatinine increase >0.5 mg/dL

Blood creatinine concentrations increase slowly at the terminal phase of the experiment (Figure 2.8 b).

• Coagulation abnormalities (INR >1.5 or APTT >60 secs)

PT (Figure 2.9 a) and APTT (Figure 2.9 b) increase significantly during the experiment.

While anti-thrombin concentration (Figure 2.9 c), D-dimer concentration (Figure 2.9 d), and protein C concentration (Figure 2.9 e) decrease substantially during the experiment.

a b

Figure 2.8 Urine output (a) and blood creatinine concentrations (b) during experiments (N =10).

Data expressed as mean ± standard deviation.

0 0.4 0.8 1.2 1.6 2 2.4 2.8

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T21

Blood creatinine concentrations (mg/dl)

Time (h)

-200 0 200 400 600 800 1000

T2 T4 T6 T8 T10 T12 T14 T16 T18

Urine output (ml/Kg)

Time (h)

(34)

Figure 2.9 PT(a), APTT(b), antithrombin III concentration (c), D-dimer concentration (d), protein C concentration (e) and platlet count (f) during the experiments (N =10). Data expressed as mean ± standard deviation.

a b

c d

e f

0 10 20 30 40 50 60

PT (sec)

0 50 100 150

APTT (sec)

0 10 20 30 40 50 60 70 80

Antithrombin concentration (%)

0 500 1000 1500 2000

D-dimer concentrations (mg/ml)

0 5 10 15 20 25 30 35 40

T0 T2 T4 T6 T8 T10 T12 T14 T16

Protein C concentration (%)

Time (h)

0 100 200 300 400 500

T0 T2 T4 T6 T8 T10 T12 T14 T16

Platelet counting (*1000/ml)

TIme (h)

(35)

• Ileus (absent bowel sounds)

Ileus develops after induction of sepsis.

• Thrombocytopenia (platelet count <100,000 / μ L)

Thrombocytopenia develops during the experiment (Figure 2.9 f).

• Hyperbilirubinemia (plasma total bilirubin > 4 mg/dL or 70 mmol/L)

We do not observe hyperbilirubinemia in this model, which suggests that only a mild liver lesion develops in this acute model.

2.3.5 Tissue perfusion variables

• Hyperlactatemia (>1 mmol/L)

0 2 4 6 8 10

T1 T3 T5 T7 T9 T11 T13 T15 T17

Arterial lactate (mEq/L)

Time (h)

Figure 2.10 Arterial lactate concentrations during the

experiment (N =10). Data expressed as mean ± standard

deviation.

(36)

Metabolic acidosis develops as suggested by increased arterial lactate concentrations (Figure 2.10).

• Decreased capillary refill or mottling

In this sheep model, we observe decreased capillary refill or mottling in the tongue area.

(37)

2.3.6 Histology

Multi-organ failure develops in our model, as suggested by hypotension, respiratory hypoxia, oliguria and anuria. Pathophysiological results show diffuse alveolar damage with alveolar flooding by proteinaceous fluid, neutrophil influx into the alveolar space, loss of alveolar epithelial cells, and deposition of hyaline membranes on the denuded basement membrane in the lung (Figure 2.11). Other findings include bacterial foci in the liver, neutrophil infiltration around the periportal vein area, and gut inflammation (Figure 2.12).

Figure 2.11 Bacterial foci (top left), alveolar thickening (top right), alveolar

edema (bottom left) and neutrophil infiltration (bottom right) in the lung.

(38)

Figure 2.12 Bacterial foci in the liver (top left), neutrophil infiltration in the

periportal area (top right), small artery thrombi in the kidney (bottom left) and

gut inflammation (bottom right).

(39)

2.3.7 Outcome

This model is a lethal model: the 24-hour mortality is 100% (Figure 2.13).

0 20 40 60 80 100

0 2 4 6 8 10 12 14 16 18 20 22 24

% sheep survival

Time (h)

Figure 2.13 Survival curve of sheep (N =10).

2.4 Discussion

This model attempts to reproduce the clinical condition as closely as possible. It

reproduces a laparotomy under anesthesia, and fecal peritonitis with prolonged

administration of sedation and analgesia. Fluid resuscitation is optimized according to

multiple clinical endpoints, including avoiding hypovolemia as suggested by

hemoconcentration or hypotension. Fever and tachycardia are associated with a

hyperdynamic pattern with arterial hypotension, elevated cardiac index, and decreased

systemic vascular resistance and lactic acidosis, all clinical features of human septic

shock. This model has been shown to reproduce a polybacterial peritonitis, which is

(40)

closely related to clinical peritonitis in humans, in which infection is mainly from the host. In the absence of antibiotic or vasopressor therapy, the mortality of our model is 100% after 24 hours. The evolution to death is related to multiple organ failure characterized by hypotension, oliguria, and respiratory failure.

2.5 Summary

This sheep model of septic shock due to fecal peritonitis emulates human septic shock

closely, and therefore is clinically relevant.

(41)

2.6 References

1. Matot, I. and C.L. Sprung, Definition of sepsis. Intensive Care Med, 2001. 27 Suppl 1: p. S3-9.

2. Deitch, E.A., Animal models of sepsis and shock: a review and lessons learned.

Shock, 1998. 9(1): p. 1-11.

3. Freise, H., U.B. Bruckner, and H.U. Spiegel, Animal models of sepsis. J Invest Surg, 2001. 14(4): p. 195-212.

4. Fink, M.P. and S.O. Heard, Laboratory models of sepsis and septic shock. J Surg Res, 1990. 49(2): p. 186-96.

5. Sun, Q., et al., Low-dose vasopressin in the treatment of septic shock in sheep.

Am J Respir Crit Care Med, 2003. 168(4): p. 481-6.

6. Sun, Q., et al., Optimal adrenergic support in septic shock due to peritonitis.

Anesthesiology, 2003. 98(4): p. 888-96.

7. Sun, Q., et al., Comparison of continuous thermodilution and bolus cardiac output measurements in septic shock. Intensive Care Med, 2002. 28(9): p. 1276- 80.

8. Warner, A.E., B.E. Barry, and J.D. Brain, Pulmonary intravascular macrophages in sheep. Morphology and function of a novel constituent of the mononuclear phagocyte system. Lab Invest, 1986. 55(3): p. 276-88.

9. Avila, A., et al., Peripheral lymph flow in sheep with bacterial peritonitis:

evidence for increased peripheral microvascular permeability accompanying systemic sepsis. Surgery, 1985. 97(6): p. 685-95.

10. Gnidec, A.G., et al., Ibuprofen reduces the progression of permeability edema in an animal model of hyperdynamic sepsis. J Appl Physiol, 1988. 65(3): p. 1024-32.

11. Dubois, E.F., The estimation of the surface area of the body. Basal metabolism in health and disease., ed. D. EF. 1936: Philadelphia: Lea and Febiger. 125-144.

12. Hafezi-Moghadam, A., et al., Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med, 2002. 8(5): p. 473-9.

13. Levy, M.M., et al., 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med, 2003. 31(4): p. 1250-6.

14. Matsukawa, T., et al., Heat flow and distribution during induction of general

anesthesia. Anesthesiology, 1995. 82(3): p. 662-73.

(42)

Chapter 3: Use of low tidal volume in septic shock may decrease severity of subsequent acute lung injury.

(This paper was published in Shock, 2004 Aug; 22(2): 145-50)

3.1 Introduction

In addition to its obvious lifesaving effects, mechanical ventilation can cause damage to the lungs (1), a phenomenon now referred to as ventilator-associated or ventilator- induced lung injury (VILI). Mechanisms implicated in VILI are barotrauma (2) and/or volutrauma, possibly leading to alveolar capillary wall stress and fracture (3, 4), and so- called atelectrauma, or biotrauma related to enhanced inflammation and surfactant dysfunction caused by repetitive opening and collapse of lung units (5). VILI may heighten pulmonary and systemic inflammation and thereby promote remote cellular apoptosis and organ failure (6, 7).

In established acute lung injury/acute respiratory distress syndrome (ALI/ARDS), animal studies and human studies showed that low tidal volume ventilation resulted in better outcomes than higher tidal volume ventilation (8, 9). These observations form the rationale for newer, ‘gentler’ approaches to managing mechanical ventilation in established lung injury (10). However, whether early application of a low tidal volume ventilation strategy can have beneficial effects before ALI develops is uncertain.

Sepsis may be the ideal setting to test the effects of low tidal volumes, since it is

associated with an intense inflammatory response and capillary leakage syndrome which

may cause lung dysfunction early. Mechanical ventilation is often required in septic

shock to improve gas exchange and reduce the work of breathing. Sepsis is the

predominant cause of ALI/ARDS and its presence is associated with a worse outcome

from these disease processes (11).

(43)

We tested whether the early application of low tidal volumes early in septic shock could limit the severity of the subsequent development of ALI. In an experimental sheep model of septic shock, we hypothesized that a ventilatory strategy using low tidal volumes applied early in the course of septic shock could have beneficial effects on lung function and hemodynamic status.

3.3 Experimental protocol

The experimental procedure is shown figure 3.1: Immediately following the surgical procedure, animals were alternately randomized to ventilation with low (6 ml/kg body weight) or high (12 ml/kg body weight) tidal volumes.

A PEEP of 10 cmH

2

O, plateau pressure at 10% of inspiratory time, I: E=1:2, and a square

wave pattern were maintained throughout the experiment in both groups. After the

surgical procedure, FiO

2

was adjusted to maintain PaO

2

between 80 and 140 mmHg. RR

was adjusted to maintain PaCO

2

between 35 and 45 mmHg, but was not increased when

the peak inspiratory pressure (PIP) reached 50 cmH

2

O, above which lung lesions occur in

sheep (12). After hemodynamic baseline stabilization, Ringer’s lactate was titrated to

maintain PAOP at baseline levels.

(44)

Anaesthesia

Surgical preparation

Sepsis inducement

Randomization

Low tidal volume Vt = 6 ml/kg/min

High tidal volume Vt = 12 ml/kg/min cephalic vein

cannulation Arterial catheterization Femoral vein catheterization Swan-Ganz, Foley catheter

Cecum perforation 0.5g Feces spillage

I:E = 1:2 PEEP = 10 cmH2O

Vt = 9 ml/Kg/min Square wave

FiO2 = 1

RR: PCO2 35~45mmHg I:E = 1:2 PEEP = 10 cmH2O Vt = 9 or 12 ml/Kg/min

Square wave

FiO2: PaO2 80~120mmhg RR: PCO2 35~45mmHg

Figure 3.1 Experimental procedure.

(45)

3.4 Results

There were no significant differences in baseline weight of the sheep in the two groups (25.0 ± 4.3 kg in high tidal volume group vs 27.9 ± 4.9 kg in low tidal volume group, P=0.25). The tidal volume was significantly lower and the respiratory rate was higher in the low tidal volume than higher tidal volume group (both P=0.002) (Figure 3.2 b and a).

The minute volume was also higher in the low tidal volume than in the high tidal volume group (P=0.002)(Figure 3.2 c), due to higher dead space; after 12 hours, the calculated dead space was 14% in the low tidal volume group compared to 7% in the high tidal volume group (P=0.018). Plateau pressure was only marginally higher in the high tidal volume than in the low tidal volume group (P=0.075) (Figure 3.2 d), but peak airway pressure and mean airway pressure were significantly higher in the high tidal volume than in the low tidal volume group (both P=0.042). No intrinsic PEEP was detected in either group. The thoraco-pulmonary compliance decreased early and remained lower in the low tidal volume than in the high tidal volume group (P=0.007) (Figure 3.3 a), whereas the respiratory system resistance remained stable in the low tidal volume group but increased significantly in the high tidal volume group (P=0.002) (Figure 3.2 c). The PaO2/FiO2 ratio tended to be lower in the low tidal volume than in the high tidal volume group but the difference did not reach statistical significance (P=0.06) (Figure 3.2 b).

PaCO2 increased in both groups toward the end of study. There was no significant difference in PaCO2 at any time between the groups (Figure 3.2 d).

MAP decreased over time in both groups (Figure 3.3 b) but the time to develop hypotension was longer in the low than in the high tidal volume group (18.1 ± 3.1 vs 12.0

± 2.8 hours, P<0.05). Stroke volume was lower in the high than in the low tidal volume

group (P= 0.028) but heart rate was also higher (P=0.002) (Figure 3.4 a), so that cardiac

index increased similarly in both groups (Figure 3.4 c). There were no significant

differences in systemic vascular resistance (Figure 3.4 d), pulmonary vascular resistance,

oxygen delivery (Figure 3.5 a), or oxygen consumption between the two groups. Blood

(46)

lactate concentration (Figure 3.5 c) increased and blood pH decreased similarly in both groups (Table 3.1).

To maintain PAOP at baseline, the amount of Ringer’s lactate infused was larger in the low tidal volume group than in the high tidal volume group (13.4 ± 2.1 vs. 10.6 ± 2.0 l, P= 0.013) (Figure 3.5 b), but the difference was due to the different survival times since there was no difference in fluid accumulation in the first 16 hours. Although there was no significant difference in urine output (P= 0.389) (Figure 3.5 b) between the two groups, the time to develop anuria was longer in the low than in the high tidal volume group (17.6

± 1.6 vs 14.1 ± 3.8 hours, P<0.05). The survival time was longer in the low than in the high tidal volume group (median 23 vs 17 hours, P< 0.05)(Figure 3.5 d).

The lung tissue wet/dry ratio was lower in the low tidal volume than in the high tidal

volume group (7.1 ±0.5 vs 9.1 ± 0.7, P< 0.05) (Figure 3.5 b). Lung histology revealed

infiltration by microorganisms, dilated microvessels, and alveolar thickening (Figure

3.6). Pulmonary neutrophil infiltration was greater in the high tidal volume group than in

the low tidal volume group (Figure 3.7).

(47)

Table 3.1 Evolution of measured variables with time in the two groups of animals.

Variables Groups

T0 T1 T4 T7 T10 T13 T16 T19

Temperature (°C) 6 ml/kg 38.6±0.4 38.8±0.3 39.2±0.5 39.5±0.7 39.5±0.6 39.2±0.9 39.1±1.4 39.9±1.1

12 ml/kg 38.5±1.2 38.7±1.3 39.1±1.7 39.6±1.7 39.7±1.7 39.5±1.7 38.7±2.0 -

Mean arterial blood pressure (mmHg) 6 ml/kg 105±18 107±20 110±24 97±25 95±24 87±24 79±17 61±8

12 ml/kg 90±18 94±19 102±26 96±23 89±33 67±31 69±31 -

Mean pulmonary arterial pressure (mmHg) 6 ml/kg 31±4 27±5 25±4 24±5 23±5 27±6 30±10 23±5

12 ml/kg 25±6 24±6 24±4 24±5 25±8 26±6 24±6 -

Cardiac index (l/min/kg) 6 ml/kg 2.0±0.4 1.6±0.4 1.8±0.4 2.4±0.6 2.8±0.5 2.8±0.8 3.2±0.6 3.8±1.8

12 ml/kg 2.1±0.6 1.9±0.6 2.2±0.7 2.7±0.8 2.9±0.9 3.0±1.6 3.0±1.0 - Systemic vascular resistance (dynes.cm.sec-5) 6 ml/kg 2029±383 2579±587 2358±936 1503±585 1117±266 1116±317 824±125 615±158

12 ml/kg 1905±799 2398±1298 1972±1004 1324±497 1597±961 1047±643 1102±839 - Pulmonary vascular resistance (dynes.cm.sec-5) 6 ml/kg 504±69 529±143 456±191 295±102 218±55 300±109 282±117 198±89

12 ml/kg 444±169 490±156 341±102 309±81 407±201 368±274 367±199 -

Arterial blood hemoglobin (g/dl) 6 ml/kg 16.6±1.9 17.0±2.2 17.3±3.1 16.3±3.1 15.5±2.4 16.2±2.3 15.9±2.1 13.6±3.3 12 ml/kg 14.3±2.2 14.6±2.2 16.4±2.6 16.5±3.1 17.2±3.3 14.6±1.2 14.4±1.1 - Arterial blood pH 6 ml/kg 7.40±0.06 7.43±0.06 7.41±0.06 7.33±0.09 7.30±0.06 7.26±0.13 7.19±0.21 7.25±0.02

12 ml/kg 7.44±0.03 7.47±0.03 7.41±0.08 7.35±0.06 7.31±0.09 7.25±0.14 7.24±0.10 -

PaCO2 (mmHg) 6 ml/kg 43.4±3.8 40.4±4.4 37.9±4.5 41.2±7.5 42.8±5.4 49.9±12.6 57.7±21.6 50.0±6.7

12 ml/kg 39.6±4.1 34.8±2.6 40.3±5.2 41.3±4.6 45.2±7.2 47.1±8.7 53.1±15.6 - Arterial blood lactate (mg/dl) 6 ml/kg 1.3±0.5 1.5±0.6 2.9±1.1 3.9±1.6 5.3±1.8 6.2±3.0 6.2±3.4 5.0±3.0

12 ml/kg 1.4±0.5 1.3±0.3 2.1±1.2 2.7±1.3 4.0±1.0 4.7±0.9 5.1±1.3 -

(48)

2 3 4 5 6 7 8

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

Minute volume (L/min)

Time (h)

* * * * * * * * * * * * * * * * * *

0 10 20 30 40 50

Respiratory rate (breaths/min)

* * * * * * * * * * * * * * * * * *

20 25 30 35 40 45 50

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

Plateau pressure (cmH2O)

TIme (h)

* * * * * * * * * * * * * * * * * *

150 200 250 300 350 400 450

Tidal volume (ml/breath)

a c

b d

Figure 3.2 Respiratory rate (a), minute volume (b), tidal volume (c) and plateau pressure (d)in

high tidal volume group (circle) and low tidal volume group (square) animals. * : p< 0.05.

(49)

30 40 50 60 70 80

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

PaCO2 (mmHg)

Time (h)

0 100 200 300 400

PaO2/FiO2 (mmHg)

* * * * * * * * * * * * * *

* * * * * * * * * * *

0 50 100 150 200

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

Respiratory system resistance (cmH2O/L/sec)

Time (h)

* * * * * * * * * *

5 10 15 20

Respiratory system compliance (ml/cm H2O)

a c

b d

Figure 3.3 Respiratory system compliance (a), resistance (b), PaO2/FiO2 (c)and arterial PaCO2 (d) concentrations in high tidal volume (circle) and in low tidal volume (square) group animals.

*: p < 0.05.

(50)

0 1 2 3 4 5 6

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

Cardiac index (L/min/Kg)

Time (h)

40 60 80 100 120 140

Mean arterial pressure (mmHg)

0 500 1000 1500 2000 2500 3000 3500

T1 T3 T5 T7 T9 T11 T13 T15 T17 T19 T22

Systemic vascular resistance (dyne*sec/cm5)

Time (h)

80 120 160 200 240

Heart rate (beats/min)

* * * * * * * * * *

a c

b d

Figure 3.4 Heart rate (a), mean arterial pressure (b), cardiac index (c) and systemic vascular resistance (d) in high tidal volume group (circle) and low tidal volume group (square) animals. *:

p< 0.05.

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