CORONARY CIRCULATORY RESERVE

N NORMOTENSIVE HYPERDYNAMIC SEPSIS

by

Frank Bloos

Graduate Program

in

MedicaI Siophysics

Submitted in partial fulfillment

of the requirements for the degree of

Doctor of Phiiosoplip

Faculty of Graduate S tudies

The University of Western Ontario

London, Ontario

Nov. 1998

O Frank Bloos 1998

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Abstract

Sepsis is a syndrome describing a malignant inflamrnatory response to an infection

leading to an alteration of the cardiovascular system including myocardial depression.

loss of vasomotor control. and microcirculatory dysfunction. Most studies concluded that

myocardiai ischernia is not the cause of rnyocardial depression in sepsis since coronary

blood flow is elevated in this syndrome. However. these studies did not take into account

the important feature of the coronary circulation which has to adjust quickly to changes in

myocardial O2 demand. This feature is supported by the coronary circulatory reserve

consisting of a blood flow and an O-, extraction (02E) reserve. In the first experiment a

sharn group and sheep rendered septic by cecal ligation and perforation underwent a

hypoxia trial to challenge and exhaust the coronary circulation. The conclusion of this

study was that sepsis did not completely abolish but significantly depressed both

components of coronary circulatory reserve. As it is suspected that sepsis lefi-shifis the

O2 dissociation curve (ODC) and. thereby. depresses hemoglobin O2 unloading? a

retrospective reconstruction of ODCs was undertaken and their effect on the systemic and

coronary circulation was analyzed. The p50 was significant Iower in septic sheep than in

sharn sheep. The lefi-shift of the ODC in CLP sheep was associated with a depression in

maximum systemic 02E while this was not seen in the coronary circulation. This is rnost

likely due to the convergence of different ODCs at lower pOzs where OIE happens in the

coronary circulation. To further analyze the effects of the blood's Oz carrying capacity on

the coronary circulatory reserve. a study was designed to compare septic sheep with two

different hemoglobin levels (70 g/l by isovolemic hemodilution; 120 gA by blood

transfusion) during the hypoxia trial. Blood transfusion was not able to restore coronary

circulatory reserve. However. hemodilution imposed significant alterations on coronary

blood flow including a maldistribution of intramyocardial blood flow suggesting that

maintaining a high hemoglobin in sepsis is beneficial in terms of the coronary circulation.

Keywords: sepsis, sheep. cecal ligation and perforation, coronary circulation. O2

transport, O7 dissociation curve- hypoxia. hemodilution. blood transfusion

... 111

Dedcated to

MY

parents

Acknowledgments

The animal experiments. which are presented in this thesis. would not have been

possible without the continuous support of Mrs. A. Neal and Mrs. M. Pitt-Hyde. Their

experience as animal health technicians ensured the quality of the studies. provided

solutions to the many technical problems typical of animal research. and ensured the

keeping of the ethics protocols.

I am also very grateful to Mr. D. Gravelle. Department of Nuclear Medicine. He

worked on the gamma-canera and his expertise was of great importance to obtain

accurate organ blood flows by the radiolabeled microspheres technique. Dr. C. Martin

developed the spreadsheet that calculated organ blood flows from the raw data.

I would like to thank the members of the advisory comrnittee Dr. Roach and Dr.

Boughner. They took care. that three years of animal work were cornposed into the

scientific plot presented in this thesis.

Finally. the progress of this thesis was accompanied by the tight supervision of Dr. C.

Ellis and Dr. W.J. Sibbald. It \vas their cornprehensive scientific advice and their

knowledge in organizing animal experiments which ensured the completion of the

experiments as well as tliis thesis.

Table of contents

CERTIFICATE OF EXAMINATION ........................................................................... II

.............................................................................................. ACKNO WLEDGMENTS V

............................................................................................. TABLE OF CONTENTS VI

LIST OF FIGURES .................................................................................................... VI11

LIST OF TABLES ......................................................................................................... X

LIST OF APPENDICES .............................................................................................. XI

................................................................................. CHAPTER 1 . INTRODUCTION 1 1.1 Sepsis - A historicai review ...................................................................... 1

.......................................................................... 1.2 Pathophysiology of sepsis 4 ....................................................... 1.3 Regulation of myocardial O? delivery 9

1.4 Animal rnodek of sepsis ......................................................................... 16 1.5 Overall goals and hypotheses- .................................................................. 18

... CHAPTER 2 . CORONARY CIRCULATORY RESERVE DURING HYPOXIA 19 2.1 Introduction .............................................................................................. 19 3 ? -.- 77 Methods ................................................................................................... .--

...................................................................................................... 3.3 Results 31 2 -4 Discussion ................................................................................................ 40

CHAPTER 3 -THE O2 DISSOCIATION CURVE lN SEPSIS ..................... ... .... 50 3.1 Introduction .............................................................................................. 50 3 -2 Methods .................................................................................................... 54 3 -3 Results ...................................................................................................... 56 3 -4 Discussion ................................................................................................ 61

CHAPTER 4 . CORONARY CIRCULATORY RESERVE DURING MODEST ANEMIA .................................................................................................. 66

.............................................................................................. 4.1 Introduction 66 .................................................................................................... 4.2 Methods 68

4.3 Results ..................................................................................................... -73 4.4 Discussion ............................................................................................... -83

CHAPTER 5 . DISCUSSION ...................................................................................... 89 . . ........................................................................... 5.1 Tissue hypoxia in sepsis 92 5.3 Limitations to the animal mode1 .............................................................. 94 5.3 Future Work ............................................................................................. 95

............................................................................................... 5.4 Conclusion 98

.................................................................................................................. APPENDIX 99

............................................................................................................ REFERENCES 105

VITA .................... ..... ..................................................................................... 120

vii

Lis t of figures

...................................................................................... Fig . 1 : Host response to endotoxin 5

................................................................. Fig . 2: Regulation of myocardial O? availability 9

Fig . 3: Autoregdation f pressure-flow-relationship) of coronary blood flow .................... 10

7' .......................................................... Fig . 4: Study design of the hypoxia trial ................ .. -3

. . Fig . 5: The concept of cntical O? delivery ........................... .... ................................... 25

', - Fig . 6: Heart work and coronary blood flow during progressive hypoxia ......................... 3s

........................................... . Fig 7: Coronary circulatory reserve in sham and CLP sheep 37

....................... Fig . 8: Redistribution of cardiac output during hypoxia ....................... .. 38

.......................................................... . Fig 9: Effects of hypoxia on regional O? delivery 39

................................................................. . Fig 10: The O? dissociation cuve in humans 1

Fig . 1 1 : Effect of the hemoglobin O? saturation on the CO? Bohr effect in sheep ............ 56

Fig . 12: Reconstruction of the OZ dissociation curve for one sharn sheep as an example.59

Fig 13: The p50 and the Hill coefficient n in CLP and sharn sheep ................................. 59

................... Fig . 14: Relationship between maximum systemic O? extraction versus p50 60

Fig . 15: Study design of the hemodilution/blood transfusion trial .................................... 69

Fig . 16: Hernoglobin IeveIs and arterial O? content before and after

Fig . 17: Changes of systemic O7 extraction and V 0 7 over the change of arterial O2

content dunng the hypoxia trial ................................................................................. 77

Fig . 18: Changes of heart work and myocardial O? consumption over the change of

arterial 0 2 content ...................................................................................................... 78

Fig . 19: Left-ventricular endocardia1:epicardial blood flow ratios .................................... 78

Fig . 20: Changes of myocardial O2 delivery and myocardial OzE over the change of

...................................................................................................... arterial 0 2 content 79

Fig. 21 : Linear regession between artenal and coronary sinus POr. ................................ 80

Fig. 22: Myocardial O î consurnption versus coronary blood flow. ....................... .....8 I - Fig. 23: Percent change of organ QO? - during hypoxia at different hemoglobin levels .... 82

Fig. 74: Proposed pathophysiology describing the loss of coronary circulatory reserve in

List of tables

Table 1 : Definition of sepsis ................................................................................................ 3

Table 2: Summary of studies investigating the effect of septic shock on myocardial Oz . . .

avadability .................................................................................................................. 13

Table 3 : Summary of studies investigating the effecr of normotensive sepsis on

myocardial O1 availabiiity ......................................................................................... 14

............................................................ TabIe 4: Normal organ blood flows in aduit sheep 28

Table 5: Effects of cecal liearion and perforation (CLP) on systemic circulation ............. 32 1- ................................................... Table 6: Effects of hypoxia on systemic hemodynamics 32

Table 7: Effects of hypoxia on systemic O2 transport ................... .. ................................ 34

.............. Table 8: Effects of hypoxia on rnyocardial O? transport .................... ... J

Table 9: Results of the regression malysis for the COI Bohr effect ................................. 56

Table 10: Artenal blood gas analysis during hypoxia ....................................................... 58

Table 1 1 : Effects of different hemoglobin levels on systemic hemodynamics before and

afier C LP .................................................................................................................... 74

Table 12: Effects of anemia and hypoxia on systemic O2 transport .................................. 75

List of appendices

APPENDIX 1 ABBREVIATIONS ............................................................................... 99

APPENDiX II CALCULATED PARAMETERS ...................................................... 101

APPENDIX III ETHICS APPROVAL .................................................................... 102

Chapter 1 - Introduction

1.1 Sepsis - A historical review

Sepsis is a medical t e m that has been introduced not only with the development of

intensive care medicine but lias been used for centuries. It's meming, however. was

subject to many changes even during the last years. In order to understand the

comprehensive literature available about this syndrome. it is important to be aware of the

development of this term.

The word 'sepsis' is a denvative fiom the Greek word septikos meaning putnd

(Buttenvorths -Medical Dict ionq. 1965). It was believed that the contact of air with a

wound led to putreikation of this wound and finally of the whole body ('septicemia').

This complication was feared by many surgeons until the late 19th century and was a

common cause of death after surgery.

A more modem view of sepsis began to develop in the 1860's. Ignaz Philip

Semmelweis ( 18 18- 1865). obstetrician at the General Hospital in Viema. faced the

problem that the mortality of women afier delivery reached 30% which was a very high

mortality rate compared to other hospitals. The cause of death was the so-called puerperal

tever. Semmelweis discovered that the students and physicians. who attended the

pregnant women. frequently spent time in the post-mortem room. He postulated that

those physicians c h e d 'decomposed animal matter' from the dead bodies to the

pregnant women. After Semmelweis forced every person examining a pregnant woman to

disinfect their hands. mortality dropped dramatically in his hospital. Despite his obvious

success. his work was not appreciated by his colleagues. He became il1 over his constant

fight for professional recognition and died in a psychiatnc institution ( 162).

It was Joseph Lister ( 1827- 19 12). surgeon at the Glasgow Royal Infirmary. who was

able to coni-im Semmelweis' thinking. Lister discovered that suppuration in wounds was

caused by rnicroscopic organisms. In 186 1. he started to propagate so-called antiseptic

procedures by using carbolic acid to disinfect instruments as well as the surgeon's hands.

He demonstrated tliat the antiseptic procedures dramatically decreased the incidence of

septicemia. His work is regarded as one of the most important inventions of modem

surgery (1 62)-

Although the introduction of antiseptic procedures was a major breakthrough in the

development of modem medicine. there \vas always a certain rate of patients dying of

severe infection afier surgery. In the 1930's. it was discovered that those patients suffered

trom profound hypotension and the term 'septic shock' was introduced (63). At this tirne,

the so-called rnicroscopic orgmisms were identified. in 1935. the most frequent

pathogens of sepsis were pneumococcus. group A streptococcus. and staphylococcus

aureus (63) . Since antibiotic treaunent was not available. rnortality was very hi"&. These

data demonstrate the tight association between sepsis and bacteremia which is a concept

that has s w i v e d untii today. Wlien efficient antibiotic treatment was introduced.

rnortality of sepsis dropped until the 1960s (63). Despite the simultaneous development

of intensive care medicine. the onset of septic shock remained a frequent cause of death.

Since septic shock seemed to be unresponsive to antibiotic treatment alone. it became

clear that sepsis was more than just a severe infection.

In 1967. Ashbough and coworkers described a clinical pattern. which they called adulr

wspiratory clisrress syndrome (ARDS). consisting of severe dyspnea. loss of lung

cornpliance and diffuse alveolar infiltration (9). At first. the etiology of this syndrome

and its relationship to sepsis was unknown but very soon it was discovered that ARDS

and sepsis were a frequent combination in an intensive care patient (10). It was believed

that ARDS was directly caused by bacteremia and that the respiratory failure would cause

failure of other organ systems due to hypoxemia finally leading to death ( 129). However,

Bell and coworkers discovered that death in patients with ARDS could not be attributed

to hypoxemia and that failure of non-pulmonary organs independently accompanied

ARDS (1 9). The mechanisms. which were believed to be responsible for the development

of ARDS afier bacteremia seen as leukocyte activation. release of lysosomal enzymes,

endothelial ce11 damage etc(129). were in tact the result of a systemic inflammation.

Therefore. in the 1980s sepsis was defined as invasion of microorganisms andlor their

toxins into the bloodstream together with the host response to this invasion (30). It was

proposed that the ef'fects of an autodestructive inflammation as symptom of the host

response finally resulted in multiple oqan failure where A R D S was its pulmonary

manifestation (5 1 ).

In the following years. it h a been realized that a sepsis-like syndrome cannot o d y be

induced by severe infection but by several other impacts such as severe trauma. massive

blood transfusion. bums. pancreatitis etc. As sepsis was supposed to describe the

spstemic response to i&wion. the new term -Systemic Inilammatory Response

Syndrome' (SIRS) was introduced (3) . In order to clarify the large numbers of different

t e m s and definitions in this area. a set of definitions has been laid down by the

,4CCP/SCCM consensus conference (Table 1 ).

Term Definition

Bacterernia The presence of viable bactena in the blood

Systemic Inflarnrnatory The systemic inflarnmatory response to a variety

Response Syndrome of severe clinical insults

Sepsis Systemic response to infection

Severe Sepsis Sepsis associated with organ dysfùnction,

hypoperfùsion- hypotension

Septic Shock Sepsis with hypotension with the presence of

perfusion abnorn~alities despite adequate fluid

resuscitation

Table 1 : Definit ion o f sepsis Modified from the ACCP/SCCM Consensus Conference Cornmittee, Crit. Care Med. 1992 (3).

1.2 PathophysioIogy of sepsis

The importance in the understanding of the pathophysiology of sepsis lies in the host

response to the infection. Since injection of endotoxin is a common mode1 of septic

shock. the host response to endotoxin has been well described (Fig. 1). The presence of

endotoxin in the blood activates several cascades including the humoral and cellular

immune system. the coagulation cascade as well as endothelial cells.

Cvtokines: An important pathway is the activation of monocytes which then release

the so-called primaq cytokines tumor necrosis factor a (TNF-a) and interleukin-1 p (IL-

I p). These two mediators are synergistic in releasing a magnitude of s e c o n d q cytokines

like platelet activating factor PAF. IL-?. IL-6. IL-8. IL- 1 O. and eicosanoids. Secondary

mediators rnay induce the expression of tissue factor which then activates intravascular

coagulation. The activation of cytokines plays an important role in the pathogenesis of

sepsis and developrnent of organ dyshnction. TNF-a is known to inflict darnage to the

liver (179). the lung (60). and the myocardium (58. 63). Furthermore. persistence of high

levels of TNF-a and IL-6 are correlating with a poor outcome in septic shock patients

( 147).

Endathelid cells: Septic shock is associated with an increased expression of the

inducible nitric oxide-synthase (NOS ) (98). This enzyme mediates the s ynthesis of the

endothelium derived relaxant factor nitric oxicle @JO) from the amino acid L-arginin. NO

mediates a vasodilation via relaxation of the arteriolar smooth muscle cells (140). The

excessive drop of the systemic vascular resistance is a cliaractenstic feature of septic

shock (143).

Endothelial cells exposed to endotoxin express adhesion molecules leading to a rolling

of leukocytes on the wall of postcapillary venules (168). Normally. leukocyte rolling is

necessary for the emigration into the extravasciilar space. However. the uncontrolled

interaction between Ieukocytes and endothelial celis during sepsis causes a massive

liberation of oxidative substances (respiratory burst) which cause endothelial ce11 edema.

cell damage and subsequently Ioss of the capillary wall integrity ( 1 89).

expression of tissue factor

activation of the \

activation of monocvtes

coagulation 1 cascade \ \ E T disserninated bradykinin ' ' intravascular I l lA.6.8 coagulation

I PAF. + clotüng of 1 eicosanoids Wood in capillaries

1 I

activation of the endotheliurn

i expression of adhesion- molecules + leukocyte adhesion , respiratory burst -, endothelial- ceII damage

vasodilatation 1 1

myocardial L Induction of depression NO-syn thase

+ vasodilation

Fig. 1 : Host response to endotoxin. Activation of the different humoral and cellular cascades by endotoxin leading to the hemodynamic changes (bo Id test) typical of septic shock.

Coagzilation: The extrinsic coagulation cascade is normally activated by the release of

tissue factor. which is located on the membrane of tissue cells. In septic shock. tissue

factor is also found on monocytes and sndothelial cells. thus. changing the

anticoagulatory properties of the vesse1 wall to a procoagulatory surface. At the same

time. there is a lack of the physiological substances inhibiting coagulation accompanied

by an activation of fibrinolysis. The simultaneous activation of coagulation and

fibrinoiysis induces a disseminated intravascular coagulation followed by the generation

of intracapillary microthrombi ( 167).

This complex host response causes charactenstic hemodynarnic changes including

alterations of the systemic. regional. and microregional circulation.

Although septic shock is associated with a hyperdynamic state and. thus. an increased

cardiac output. alterations of cardiac function c m be demonstrated early in sepsis. Both

systolic and diastolic functions are disturbed. Myocardial contractility is depressed early

afier induction of sepsis in animal experiments (1 3 1). A decreased ejection fraction as a

sign of an altered contractility c m be dernonstrated in septic patients (144). As left

ventricular contractility decreases. a diastolic dilation of the left ventricle with an

increase of end-diastolic volume c m be observed by which an adequate stroke volume is

rnaintained. This compensatory dilation is frequently not observed in patients dying of

sepsis (46). In the clinical setting, a right-shifi of the Frank-Starhg-curve is observed

requiring greater cardiac fiiling pressures than in the healthy patient to achieve a similar

stroke volume ( 13 7).

The pathogenesis of mocardial dysfùnction in sspsis is not completely understood

and probably includes multiple hctors such as myocardium depressant substances as well

as tissue hypoxia. Since semm of septic patients depresses contractility of isolated

beating cardiomyocytes. the presence of the so-called myocardium depressant substances

(MDS) has been postulated (146). In particular TNF-u seems to negatively act on cardiac

hnction (104). TNF-a. interleukin-lj3. and sndotoxin are known to induce the NO-

Synthase expressed on cardiac cells. and NO depresses cardiac contractility (62).

Whether activation of the NO-synthase is the only mode of action of MDS is not known.

yet.

Tissue hypoxia is a fundamental pathomechanism of multiple organ failure in sepsis

(159). Therefore. it seems likely that the heart shares this injury. The literature

conceming this issue is inrroduced in chapter 1 2.

1.2.2 Circulation in sepsr's

The induction of the rnediator cascade dernonstrated in Fig. 1 results in an impairment

of al1 leveis of the circulation: the systemic. regional. and microregional level. Beside the

negative effect on cardiac function. sepsis involves a profound vasodilation. As

mentioned above. the induction of the iNOS during sepsis results in a massive release of

NO. As N O directly causes arterioiar vasodilation. vessels become hyporeactive to

receptor mediated vasoconstrictors ( l l S) . As this leads to a massive reduction cardiac

afierioad. blood pressure drops. Addi tionall y. vasodilation causes perïpheral blood

pooling, thus. reducing cardiac preload.

ï h e arteriolar vasodilation due to the release of NO also affects the regional

mechanisms important for the regulation of organ perfusion (118). The loss of

autoregulation of blood flow is always associated with the risk of organ ischemia since

blood flow regulation is an important mechanism in rnaintaining an adequate tissue O2

delivery. Loss of the vascular tonus in intestinal arteries causes an impairment of gpt

perfùsion (186). In the regional circulation of the liver. the coupling between blood flow

in the hepatic artery and the portal vein is depressed in endotoxic shock (12). The

coronary circulation rnay also share in this injury. As this concept is the main topic of this

thesis. the c o r o n q circulation in sepsis will be introduced separately in chapter 1.3.

The microcirculation also participates in the septic injury. This is caused by interaction

between leukocytes and endothehl cells as well as by capillary occlusion due to

intravascular coagulation (Fig. 1). The liberation of proteases and oxidative substances

from activated leukocytes induces endothelial ce11 damage with intracellular edema and

loss of the integrity of the vesse1 wall (168). This causes capillary leakage and loss of

tluid into the extravascular space (1 89). Additionally. erythrocyte deformability is

reduced in sepsis due to alteration of red ce11 membrane integriry (149). Reduction of

capillary lumen by endothelial ce11 edema. microthrombozation. and loss of red blood ce11

deformability rnay result in occlusion of capillaries. By using intravital microscopy in

septic animals, an increased distance of perfused capillaries could be demonstrated in

several organs like the gut mucosa (56). the skeletal muscle (107). and the diaphragm

(27). An increased fiequency of capillaries not sl-iowing any red blood ce11 flow was

observed. as well (107). The decrease of capillarity in sepsis is seen clinically as a drop in

0 2 extraction.

In surnmary. sepsis induces a relative (bIood pooling by peripheral vasodilation) and

absolute (capillary leakage) tluid deficit, thus reducing cardiac preload. Myocardial

function is depressed. Sepsis also impairs vascular reactivity. thus. reducing blood

pressure and impairing regional blood flow regulation. Additionally. sepsis reduces

capillary surface area resulting in a depression of O2 extraction. As sepsis severely

interacts with a11 pathways to maintain cellular 0 2 delivery. tissue hypoxia becomes a

substantial part in the pathophysiology of sepsis since this disease is frequently

complicated by the multiple organ dysfunction syndrome (MODS). Although the

development of MODS is most likely rnultifactorial. tissue hypoxia is believed to play an

important role in the onset of this syndrome (1 11). The severity of MODS is directly

associated with mortality of intensive care patients ( 1 17).

1.3 Regulation of myocardial O2 delivery

3 1 Mjocardial Availability in Health

The myocardiurn is an exclusively aerobic organ. Unlike other organs. lack of oxygen

cannot be covered sufficiently by anaerobic glycolysis. Even short-time ischemia will

produce myocardial dysfunction (-stumed myocardiurn') (109). As oxygen is a limiting

substrate. two mechanisms ensure the adequacy of coronary perfusion: autoregulation

and metabolic regulation (Fig. 2).

Sire of t-egrrlurian:

change in pertùsion prcssurc change in rnyocardial O- need

Fig. 3: Reguiation of myocardial O2 availability. blecl~anisms supporting changes in perfiisioii pressure (autoregulation) and changes in rnyocardial O? demand (rnetabolic regulation). Botli inecliaiiisms partly rely on the same site of regulation. 02E: O2 extraction. CBF: coronary blood tlow.

A utoregtdurion of Coronary Blood Flow

Autoregulation describes the ability of the c o r o n q vasculature to maintain blood

flow independent of arterial perfiision pressure ( 177). At rest. the autoregulatory range of

the heart includes coronary perfiision pressures between 60 and 130 mrnHg. Thus. when

outside these limits. coronary blood tlow becomes dependent on coronary pressure.

The site of autoregulation could be located in arterioles with a diarneter Iess than

LOO p m (99). The mechanism that supports autoregulation is not clear. The myogenic

theory States that changes in perfusion pressure affect the artenolar wall inducing

vasoconstriction and vasodilation. respectively (66). According to the rnetabolic theory,

changes of coronary perfusion pressure alter tissue p 0 2 . which then rnediates a change in

coronary bIood flow (33).

Autoregulation as well as metabolic regulation (Fig. 2) are supported by changes in

coronary blood flow. When coronary blood flow increases due to changes in rnyocardial

O? demand (see below). the autoregulatory range is smaller than at rest (Fig. 3 ) . When

coronary arterioles are maximally dilated. autoregulation is lost. As the O7 demand

differs within the heart. the autoregulation curve retlects an average range. Since the

subendocardium has a greater O. consumption than the subepicardiurn. the blood flow to

the subendocardium is also greater and the autoregulatory range smaller than in the

subepicardium (89).

O 50 100 150 coronary perfusion pressure [mmHg]

Fig. 3: Autoregulation (pressure-flow-relationship) of coronal blood flow This figure shows the autoregulatory range of the Iieart at rest (B) and the relationsitip at maximal coronary vasodilation (D). The distance between tliese two lines is the coronary flow reserve at a civen perfusion pressure. Pressure flow-relatioiisliip of the subepicardium (A) and of the - subendocardiurn (B). Modified from (88).

O2 demand refiects the tissue's rnetabolic requirements for oxygen. An increase in O-

demand c m be measured by an increase in O? consurnption. The fraction of oxygen

consumed from the oxygen delivered is called Oz extraction. Myocardial O? delivery is

closely regulated by the O? demand of the hean and c m be quickly adjusted. This system

can be quantified by the metabolic reserve of the lieart. An incrense in myocardial Oz

demand and. thus. qocard ia l O2 consumption may be covered by an increase in

coronary blood flow and/or an incrense in myocardiat O? extraction. Therefore. the

metabolic reserve of the heart consists of hvo components: the blood tlow resenre and the

O? extraction reserve. As myocardial O? extraction is already high at rest - about 70% -

inetabolic regulation relies rnainly on changes in coronary blocd flow to maintain

myocardial Or consumption.

The metabolic coupiing between cardiac myocytes and the coronary vasculature is

realized by the release of mediators. Whenever there is an increase in the rnyocardial O?

demand, the cardiac celfs release substances which result in coronary vasodilation within

20 S. thus causing an increase in coronary bload tlow. Adenosine is the most important

mediator of metabolic coupling but prostacyclines c m also mediate an increase in

coronary blood flow (57). The possible increase in coronary blood flow (coronary flow

reserve) is limited by maximal coronary vasodilation ( Fig. 3) and depends also on the

perfusion pressure (88). If necessary. this increase in blood flow has to be supported by a

redistribution of cardiac output From non-vital orgnns whicli c m maintain their Or

demand by an increase in O? estraction (4).

Due to the high myocardial O2 extraction at rest. the importance of myocardial O2

extraction reserve has been much discussed in the literature. Feigl et al. (59) argued that

myocardial Oz extraction increases only when coronary flow reserve is exhausted. This

concept was supported by isolated heart preparations. However. intravital microscopy of

the heart shows that more capillaries c m be recruited during hypoxia ( 1 19) suggesting

that O1 extraction c m be increased beyond the value at rest. Furthemore, the coronary

vasculature is subjected to a-receptor activity which partly diminishes the abiiity of

coronary vasodilation. Under those condition. myocardial Oz demand is also covered by

an increase in 0 2 extraction even within the range of coronary flow reserve (15). Thus,

myocardial Or extraction reserve is an importing mechanism in supporting metabolic

reserve.

As tissue ischemia is a suspected mechanism for the development of multiple organ

dysfunction in sepsis (159). many investigators have assessed the importance of a

depressed myocardial 0 2 delivery as a possible cause for cardiac failure in sepsis. Since it

is difikult to study the regional circulation of the heart in humans. most studies

addressing myocardial O2 availability in sepsis have been conducted on anirnals. Since

the first description of the effects of endotoxin on coronary vessels in 1972 (38). our

understanding of sepsis has changed. As a result. many different animal models have

been used to describe this syndrome. For the purpose of this chapter. it is important to

differentiate between sepsis rnodels with and without hypotension.

Coronary Perfirsion in Sepiic Shock

Most of these studies have used animal models in which sepsis was induced by a bolus

injection of endotoxin which produced pro found hypotension. A surnmary of these

studies is shown in Table 2.

Since a decrease in coronary blood flow rvas associated with a depression in cardiac

contractility. it was concluded that endotoxemic shock induces cardiac hypoperfusion

(29. 54. 102). However. it was argued that myocardial 0 2 demand falls in endotoxic

sliock due to the drop in afterload (54). This wouid mean tliat the lower coronary blood

ilow would be adequate to maintain rnyocardial O? demand. Blood flow distribution in

the heart is nonuniform in septic sliock. While sorne areas in the heart were supplied with

a very high blood flow. other areas were hardly perfused at al1 (74). This could lead to

focal ischernia. In fact. histological damage in the heart of endotoxernic dogs was

observed (1 02).

Clinical studies on septic patients have been undertaken by Cunnion et al. as well as

by Dhainaut et al. (47. 52). They have reported an increase in coronary blood flow in

patients with septic shock. It was argued that myocardial dysfimction in those patients

was not due to ischemia since no myocardial lactate production was observed. Yet,

Dhainaut and coworkers concluded that the low rnyocardial Oz extraction in patients with

septic shock may result in cardiac ischemia (52). This does not contradict Cunnion's

findings since it is difficult to employ myocardial lactate metabolism as a marker of

cardiac ischemia (47).

Cho (35)

Bohs et al. (37)

Kleinman et al. (98)

Cumion et al. (45)

Goldfarb et al. (69)

DtOrio et al. (52)

Dhainaut et al. (50)

Groeneveld et al. (70)

Study design Res u 1 ts

lsolated hearts fiom

endotoxemic dogs

Endotoxemic dogs

Endotoxemic dogs

Septic shock patients

Endotoxemic pigs

Endotoxemic dogs

Septic shock patients

Endotoxemic dogs

Endotoxin constricts coronary artenes

Myocardial dysfunction may be

associated cvith cardiac ischemia.

Endotoxemia is associated with non-

uniform perfùsion and histological

damage of the heart.

Global myocardial ischemia is not the

cause of myocardial depression.

Myocardial dysfunction may be due to

relative hypoperfusion of the

subendocardium.

Coronary blood flow is sufficient to

maintain myocardial Oz demand.

Myocardial hypoxia may have

participated in cardiac depression.

Increased heterogeneity of coronary

blood flow may involve focal

ischemia.

Table 2: Surnmary of studies investigating the effect of septic sliock on myocardial O- availability.

Coronar y Perfusion in Norrnorensive Sepsis

These studies try to mimic the development of sepsis in the human situation by using a

chronic animal mode1 and/or trying to avoid hypotension by adequate fluid resuscitation.

Table 3 shows a surnrnq of the cited literature. Al1 studies foiuid that coronary blood

tlow was increased in normotensive sepsis seconda- to c o r o n q vasodilation. However.

this increase in coronary btood tlow was not necessarily accompanied by a similar

increase in heart work (64. 65). Fish and coworkers found that the septic myocardium

uses more Or per unit work than the healthy heart. thereby suggesting that the efficiency

of the heart is depressed in this syndrome (64). nius. the high coronary blood flow in

sepsis may be necessary to maintain an eIevated O? demand of the hem. The increased

coronary blood flow in sepsis is supported by a redistribution of blood flow from

splanchnic organs to the heart ( 108).

Lang et al. ( 1 03)

Fish et al. (60)

Fish et al. (61)

Lee et al. (1 05)

Study design Results

Rats with intra- Sepsis alters myocardial efficiency.

peritoneal inoculum

Chronic endotoxemia Myocardium less efficient.

in rats

Chronic endotoxernia hcreased coronary blood flow without

in rats increase in heart work.

Chronic endotoxemia The septic myocardium is

in pigs. overperfused.

Table 3: Summary of studies investigating the effect of normotensive sepsis on myocardial O3 availability.

As stated above. the adequacy of coronary perfusion is not only described by the

absolute value of blood flow but by the capacity of the coronary vasculature to modulate

blood flow according to the changinç demands of the myocardium. However, rnetabolic

coupling and coronary flow reserve have not been described in sepsis. As metabolic

regdation depends on arteriolar hinction, failure of these vessels to respond to

physiologie stimuli has been reported in several organs during sepsis (1 18. 123).

Coronary vessels of septic organs showed an altered response to mediators important for

the regulation of coronary blood tlow ( 105. 154. 180). Alteration of coronary blood fl ow

reserve in sepsis seems to be likely but has not been proven yet. Similarly. it has been

reported that myocardiai O2 extraction was depressed in sepsis (47. 52). Microcirculatory

dysfùnction has been demonstrated in several organs during sepsis (27. 56. 107). As

sepsis is a systemic event it seems Iikely that the Iieart shares in these injuries. However.

myocardial O? extraction reserve has also not been investigated in this disease.

1.4 ,4nimal models of sepsis

Most of the work uncovering the compticated pathophysiologica~ interactions in sepsis

has been undertaken in animal experiments. Bot11 acute models as well as chronically

instmmented models of sepsis are available. Acute models usually consist of intravenous

infusion of viable bacteria (156). their toxins or one of the mediators such as tumor

necrosis factor (58) known to play a role in the pathophysiology of sepsis.

Endotoxic shock is the most frequently applied model to sirnulate sepsis in animais.

Endotoxin from E. coli or other gram negative bacteria is injected intravenously as a

bolus- Endotoxic shock develops within minutes and the effects are strictly dose

dependent (61). This mode1 has man- advantages. Endotoxin can be easily obtained by

several suppliers and is cheap. The model is widely used and almost every aspect of

sepsis has been investigated by administering endotoxin. Since the onset of sepsis is

defined by the start of the endotoxin inîùsion. the time-course of treatment protocols may

be exactly defined. However. the sudden onset of severe sepsis in the endotoxin model

does not correlate with the clinical situation. Furthemore. endotoxin induces splanchnic

blood pooling leading to severe hypovolemia and hypotension (76). Thus. studies

investigating the circulatory effects of sepsis may be confounded by a combination of

effects such as septic and hypovolemic shock. To avoid sudden hypovolemia and to

simulate the clinical setting more closely. Fisli and coworkers described a rat model

where endotoxin was slowly inhsed over 6 hours (65) . This protocol allowed a sufficient

t'luid resuscitation during the study period.

Beside the controlled administration of fluid. chronically instrumented models allow

for a setup where sepsis may develop tkorn a focal infection corresponding to the

deveIopment of sepsis in patients. Pentonitis is a commonly chosen site of infection for

the purpose of stwdying sepsis. In dogs. the implantation of an intraperitoneal fibrin-clot

containing bacteria was successfully applied for inducing sepsis (1 3 1).

Another technique for the induction of sepsis by experimentall y generating peritonitis

is cecal ligation and perforation (CLP), whicli is the method chosen for the studies

outlined in this thesis. CLP was tirst described in rats by Wichterman and coworkers

(184) but was used iater on otlier animals such as sheep. CLP produces a necrotic

inflammatory focus which is then followed by pentonitis. M e r 24 hours of

contamination. sheep start to show a hyperdynamic septic profile with a high cardiac

output. a Iow systemic vascular resistance but normal blood pressure when sheep are

sufficientl y fluid resusci tated. 48 hours afler C LP. sheep demonstrate the cornpiete

hyperdynamic profile of sepsis (23. 14. 1241. This animal mode1 does not need

vasopressor support and does not include symptorns of severe malpefision such as

lactate acidosis. Therefore. the CLP rnodei in sheep represents an early stage of sepsis

(97) and. according to the ACCPISCCM definitions (3) shown in Table 1. does not

sirnulate severe sepsis or septic shock. However. circulatory abnormalities are already

demonstrable in this early stage and are of importance for the further deveiopment of this

disease ( 1 18).

1.5 Overall goals and hypotheses

Based on the literature reviewed above. there is a lack o f knowledge about the

coronary circulatory reserve in sepsis as weil as physiologie parameters determining the

coronary circulatory reserve in sepsis. The goals of this thesis were

1. to describe the coronary flow resenre and the myocardial O7 extraction resenre in

an animal madel of sepsis.

2. to describe the interaction between the O? dissociation c u v e and the coronary

circulatory reserve.

3 to describe the interaction between mild anemia and the coronary circulatory

reserve.

1. to compare the sepsis induced regionai alterations of the coronary circulation to the

systemic circuiation.

ft was hypothesized that sepsis depresses the coronary circulatory reserve by reducing

both the coronary flow as well as the myocardial 0, extraction reserve. It was Further

hypothesized that alterations of the blood's O? carrying properties in terms of the

position of the O2 dissociation curve as well as the hemoglobin concentration affect the

coronary circulatory reserve. and finally that the circulatory injury typically seen in other

organs durinp sepsis is shared by the c o r o n q circulation.

Chapter 2 - Coronary circulatory reserve during hypoxia

2.1 Introduction

As described in chapter L. sepsis is characterized by significant abnormalities in the

circulatory control of tissue Oz delivery. These abnorrnalities include microcirculatory

dysfunction (107). hyporesponsiveness of arterial resistance vessels (1 18) and depressed

myocardial hnction (46). By Ieading to an imbalance between tissue O7 availability and

O? demands. these circulatory abnomahies may lead to an ischemic tissue injury and

thereby contribute to the pathogenesis of the multiple organ dysfunction syndrome in

sepsis.

As sepsis is a hyperdynamic syndrome. where the h e m produces a hi& cardiac

output, rnyocardid O7 demands are elevated because of the sustained increase in cardiac

work load (145). Therefore. this organ rnay be particularly vulnerable to the circulatory

effects of this syndrome. However. studies investigating the septic coronary circulation at

rest did not find evidence of cardiac ischernia (Table 2 and Table 3). Coronary blood flow

was increased in septic patients and no evidence of rnyocardial lactate production was

observed (47.52).

While coronary perfusion at rest rnay be sufficient in sepsis. these studies did not

investigate the ability of the coronary circulation to sufficiently adjust to changes in

myocardial O? dernand. As demonstrated in chapter 1.3. the coronary circulatory reserve

consists of a blood flow reserve and an 0- extraction reserve. lncreasing coronary bLood

tlow is the primary means of augmenting myocardial 0: delivery when Oz demands are

elevated (182). whiie increasing O? extraction is an important ancillary mode (15). In

sepsis. however. there is reason to believe that both of these components may be

depressed in the ability to appropriately match an increase of myocardial Or demands.

When cardiac output was esperirnentally depressed in hyperdynamic sepsis by

applying a positive end-expiratory pressure (PEEP) of 20 cm HzO, septic sheep

demonstrated a significantly lower redistribution of blood flow from the splanchnic to the

coronary circulation than sheep of the control group (22). Additionaily. the response of

coronaxy vessels to mediators important for the regulation of coronary btood flow are

altered in animal models of sepsis. Adenosine. which usually mediates an increase in

coronary blood flow. did not have a significant effect in endotoxin-treated rats. It was

concluded that sublethal endotoxemia causes an uncoupling of flow from regulation by

cardiac metaboIism ( 154). Furthermore. endothelium-dependent and independent

vasorno tor responses are altered during endotoxemia ( 1 3 0. 142).

When the myocardium of septic sheep was examined for ultrastructural changes, a

patchy injury pattern was observed where necrotic cells were located directly next to

normal cells (83). Such an injury pattern would be suggestive of a microcirculatory

dysfunction since depression of blood flow in Iarger vessels would result in larger areas

of necrotic ceils. Both Cunnion (17) and Dhainaut (52) have suggested that the coronary

circulation during sepsis shares in the typical microcirculatory injury characterized by a

Iow O2 extraction. Thus. these observations support indirect evidence that the flow

reserve as well as the O2 extraction reserve of the coronary circulation might be

depressed in sepsis.

Despite such arguments. categorical evidence that the metabolic regdation of the

coronary circulation is significantly disnirbed in hyperdynarnic sepsis is lacking for a

number of reasons. First. there are few reports that have simultaneously measured both

the blood flow and 0- extraction components of the coronary circulation in relevant

models of sepsis. Second. studies assessing the myocardium during sepsis were

investigating the coronary circulation at rest instead of challenging the coronary

circulation by a sudden increase in myocardial O2 demand. Third. conclusions from these

studies have been confounded by issues that rnay independently alter the coronary

circulation. These cofounders inciude hypotension and the use of vasoactive or anesthetic

agents. As listed in Table 2. the majority of studies investigating coronary blood flow in

sepsis were undertaken in patients or animals witli septic sliock. Although the coronary

circulation is autoregulated. the low perfusion pressure in septic shock might not generate

a sufficiently high coronary blood tlow since myocardial 0, demand is increased in

sepsis (145). Thus. severe hypotension may alter myocardial tissue perfusion. The

treatment of septic patients with vasoactive agents such as the a-mimetic dmg

norepinephrine (47. 52) may also affect myocardial perfusion. ai-receptors mediate a

vasoconstriction in the coronary circulation (15). As this limits coronary vasodilation.

norepinephnne diminishes coronary tlow reserve ( 13). Finally. animal experiments

studying coronary blood flow in sepsis were undertaken in generzl anesthesia. However.

anesthetic agents are known to induce coronary vasodilation (40). Thus. generd

anesthesia wiIl also affect the response of the coronary circulation to the impact of sepsis.

These considerations necessitate the need for a study that investigates the coronary

circulatory reserve in an akvake animal mode1 of normotensive sepsis.

2.1.1 Goals and izypot/lesrs

The goal of this study was to describe the c o r o n q circulatory reserve by using acute

hypoxia in an awake sheep mode1 of normotensive hyperdynarnic sepsis. A second goal

was to compare these changes to the systemic hemodynamic response induced by

hypoxia. It was hypothesized that sepsis in mature sheep would lirnit the extent to which

both myocardial blood flow and O2 extraction were increased in response to an

intervention. acute hypoxia. which depresses O2 delivery and increases myocardial Or

demand. It was also hypothesized tiiat septic sheep would demonstrate an insufficient

increase in systemic O2 extraction as a symptom of a generalized microcirculatory

dysfunction.

2.2 Methods

Sixteen mature Suffolk sheep weighing 37-70 kg (average 48.9 kg) were incIuded in

this study. Experirnents were undertaken on male sheep only to avoid bias of the data due

to pregnancy. Following one week of acclimatization within the laboratory. sheep

undenvent instrumentation. The study animals were anesthetized with 5% halothane and

100% O? (5-6 I/min) via mask. afier which the trachea was intubated and the sheep were

ventilated with 100% oxygen. Before instrumentation. 1O"its of penicillin were given

intrarnuscularly and 500 mg chloramphenicol were administered intravenousiy. Through

a Iefi postero-lateral thoracotomy. a saline-filled silastic catheter (0.125 inch OD: Dow

Corning, Midland. MI. USA) was inserted into the Iefi atrium. secured and extenorized.

The coronary sinus was retrogradely camulated via the Iiemiazygous vein with similar

grade tubing. Correct placement of this coronas sinus catheter was confinned during

surgery by demonstrating that its 0. saturation was Iess than 30%. Using a direct

cutdown technique. saline-filled silastic catheters (0.125 inch OD) were then placed into

the lefi femoral and carotid arteries. while the Ieft external jugular vein was cannulated

with a No. 8 French introducer (Cordis. Miami. FL). Afier recovery. the sheep were

piaced in rnetabolic cages and were allowed tiee access to food and water. Ringer's

lactate. 4 mllkgh. was administered post-operatively to inaintain adequate hydration.

Xnalgesia was provided with meperidine. 100 mg admixed with each 1 O00 ml of Ringer's

Lactate. Catheter patency was maintained by intermittent flushes with heparinized saline

( 1.000 U heparid500 ml saline). Sheep were given three days to recover from the

instrumentation.

2.2. I Ekperimen tui Pro toc of

The study design is siiown in Fis. 4. Tliree days after the initial surgery. a 7 French

Swan Ganz catheter (mode1 93- 13 I : American Edwards Laboratories. Santa Ana CA)

was flow-directed into the pulmonary artery through the jugular vein introducer. A

baseline, pre-Iaparotomy study was then performed with the sheep conscious. Systemic

arterial and pulmonary artery pressures and cardiac output were measured. Blood was

simultaneously obtained from the carotid artery. central vein and the coronary sinus to

measure blood gases. hemoglo bin and Iactate.

1 Pre-laparotomy study

[ Instrumentation ]

Cecal ligation and perforation;' I sham laparotomie

Baseline (38 tirs study

- C

l i?srrtlloid inliston ro ntninruin lcji a»-kif pressure on pr~-/ctpc~ro~om~ levels

Fig. 4: Study design of the iiyposia trial

Afier this non-septic study. the sheep were randomly allocated to undergo either a

SHAM laparotomy (n=8) or cecal ligation and perforation (CLP. n=8). Under general

anesthesia. a partial omentectomy was performed in the CLP group. The cecum kvas then

located. devascularized. ligared and the tip incised. SHAM sheep undenvent general

anesthesia of a similar duration as the CLP sheep and the abdominal wall. including the

peritoneum. \-as opened. In the SHAM sheep. the cecum was not manipulated. The

wound was closed in two Iayers witli 2-0 coated vicryl ties. Finally. a tracheostomy was

performed in both groups and a 39 Fr. low pressure cuffed tracheostomy tube (Shiley.

USA) was inserted into the trachea. The tracheostorny was then connected to a system

providing humidified. warmed air.

As previously described (61). CLP leads to a panperitonitis and polymicrobial

bacteremia within two days. Thro~ighout the time between laparotomy and study 48 hours

later. an infusion of Ringer's lactate was titrated to maintain left atrial pressures (LAP) at

non-septic baseline level. Analgesia was continued as previously detailed, and increased

if the sheep showed discomfort.

Forty-eight hours afier either CLP or SHAM Iaparotomy. we repeated al1

measurements as described for the non-sep tic study. Additionally. radioactive

microspheres were injected to allow the later calculation of orçan blood flows. while

coronary sinus blood was obtained to calculate myocardial 0 2 consumption and lactate

consumption. This rneasurement also served as a baseline measurement for the follocving

hypoxia trial.

Progressive hypoxia \vas used to challenge the coronary circulatory reserve since

hypoxia increases myocardial O? need as the h e m has to increase its work Ioad to

maintain whole body O2 delive-. Sheep were comected to a semi-open system to lower

the inspired Ot fraction (Fioz) by rnising room air with nitrogen (Bird Corp. 0 2 blender.

PaIm Springs. USA). Using a polarographic O2 monitor (model 5570. Ventronics. USA),

the FiOz was reduced through up to four successive stages to a lowest FiOz of 0.08 being

adjusted to achieve a similar decrease in arterial Oz content (Ca02) between stages. From

pilot studies. we determined that the lowest Fi02 that could be generally tolerated in this

unanesthetized model approximated 0.08.

Hemoglobin and arterial 0: sat~~rations were rneasured at and in between each stage to

calculate the Cao?. Twenty minutes of equilibration time was allowed at each

expenmentai level before measurements were repeated as described in the 48 hour

baseline study. including the injection of another set of radioactive microspheres. After

defining the upper level of myocardial Oz consumption. as above. the sheep were

euthanized with an overdose of pentobarbital and organs were harvested for gamma

counting to calculate orsan blood flows.

S ystemic and pulmonary pressures were recorded with a 2-charnel monitor (model

78353A, Hewlett Packard, USA) and were referenced to the sheep's lefi atrium. Cardiac

outputs were measured in triplicate by the thermodilution technique using a cardiac

output computer (model 9570A. Hewlett Packard. USA). The cardiac output was indexed

to the body surface area and heart work was estimated as the product of cardiac index and

mean aortic pressure.

BIood Cas samples were stored on ice prior to analysis with an ABL-3 blood gas

analyzer (Radiometer Copenhagen. Denmark. During the experiment. the hemoglobin

and the O2 saturations were rneasured by a CO-oximeter (OSbl-11 Hemoximeter.

Radiometer. Copenhagen. Denmark). Subsequently. hemoglobin Ievels were c o n f m e d

by a Coulter Ce11 Counter (mode1 5. Burlington. Canada) adjusted to sheep blood. Lactate

was measured by a Greiner G-400 Chernistry Analyzer (Switzerland). Caiculations for

hemodynamics and O2 transport related parameters are listed in the appendix II of this

thesis.

Determining the critical 0: delive/?:

When systemic O? delivery (QO:) drops. whole-body O? consumption is kept constant

by increasing O2 extraction. When ma..imum O7 extraction is reached. a further decline

in Q02 resuits in a drop of O2 consurnption (Fi-. 5). The critical systemic QOz describes

that point where systemic O2 consumption becornes dependent on Q02 and may be used

to delineate the onset of tissue ischemia ( 157).

critical 02-delivery

p hysiologic 02-delivery

Fig. 5: The concept of critical O? delivery O2 consumption is independent from clianges in 0: delive? by increasing O? extraction. When rnaxirnum O? extraction is reached. O? consumption becornes dependent on O2 delivery when 0 2

deiivery drops below the critical O2 delivery.

When systemic QO2 drops beyond the critical QOz. arterial lactate levels increase as a

sign of tissue ischemia. The critical systemic QOz was calculated by using the

relationship between arterial lactate levels and the calculated systemic QOz (128). As the

standard deviation of lactate measurements at baseline \vas 0.45 rnrnol/l. an increase of

more than &vice the baseline standard deviation (0.9 mrnol/l) was considered abnormal

and therefore consistent with a QO? level which identified the onset of supply-

dependency of systemic O2 consumption.

In the animal experiment. two golden standards are available to measure organ

blood Flows: flow probes and the microsphere technique. Flow probes have to be

surgically placed at the vessel of interest and allow constant monitoring of the blood flow

in this vessel. The microsphere technique only a1Iows blood flow rneasurement at a

certain point in time. On the other hand. this technique gives data for every desired

regional circulation. Even the blood flow distribution within one organ may be andyzed.

The microspheres are labeled either with color or with radioactive isotopes for the

purpose of detection. The microsphere technique with radiolabeling was chosen to

rneasure organ blood tlows in this study. To measure blood flows at different time points

in the same animal. spheres labeled with different isotopes having a different primary

photo peak rnay be used.

Radioactive Iabeled rnicrospheres with a diameter of 15 Fm have to be injected into

the left atrium or lefi ventricle to reach al1 regional circulations including the coronary

arteries. The spheres are camed with the blood stream and are trapped in the

microcirculation. The number of spheres trapped inside an organ is directly proportional

to the blood flow to this organ at the time of injection. At the sarne time of rnicrosphere

injection, a reference blood sample is taken from a large artery with a withdrawal purnp.

The blood sampIe works as an artificial reference organ with a Iinown blood flow. At the

end of the study, organs have to be harvested for further processing (84).

The technique for analyzing tissue samples afier repeated injections with differently

labeled microspheres have been described in detail by Levine et al. (1 11). The

radioactivity of tissue samples is measured by a gamma carnera where the energy

window is set to the primary photo peak of eacli isotope. Then. the background radiation

is subtracted in each window. The gamma counter output contains the so-called sample

matrix with counts for each of the chosen energy windows. Since the energy spectra of al1

isotopes overlap. each isotope contributes counts to each window. To obtain the

contribution of each isotope to each window. a spectral analysis has to be performed.

First, a sampte of each isotope is counted in the gamma canera by itself. The result

contains the counts of each isotope in each window and is called the reference matrix.

The reference matrix is norrnalized by dividing each row by the nurnber of counts in the

primary photopeak window and is then transposed. The relationship between the sarnple

matrix SM and the normalized transposed reference matrix RI.- is described by the

following equation:

where i represents the pnmary photopeak windows and j the isotopes. The answer

matrix A k f contains the counts in each window due to the isotope with the prirnary

pho topeak in this window. Therefore. for the purpose of blood flow calculation. AM has

to be derived. The solution is given by the so-called stripping technique (42):

A M , = (RW' x Fy, x RRM, ) -' x RM,, x W, x SMr Eqn. 2

The weighting factor for each isotope calculates to wi = l/Si\.l. The weighting matrix

W,, is formed where the weights associated with each isotope are situated on the principal

diagonal and where al1 off-diagonal positions are zero. The answer matrix is calculated

for each tissue sample as well as for the reference blood sample. The organ blood flow

for each time point may then be calculated as follows:

prunp speech coirnts of spheres in orgnn Eqn. 3 organ bloodflow=

corlnts qf'spherrs in refërence hlood

where organ blood tiow and pump speed are expressed in rnlhin. Organ blood flows

should be indexed to the organ weight.

The radiolabeled microsphere technique is a well established technique to measure

organ blood flows. When two differently labeled sets of spheres are injected. an accuracy

of 10% can be achieved (53) . Similar values were obtained in sheep. Hales observed a

mean difference of 7.2% afier duplicate measurement of blood flows. In the same study,

normal values for blood flow to several organs have been determined in sheep (Table 4).

Organ

Organ blood flow

[mlimin/100 g tissue]

S keletai muscle

Kidneys

Spleen

Heart

Rumen

Srnall &ut

Large gut

Brain

Table 4: Normal organ blood flows in adult sheep Data were obtained by Hales and are expressed as mean * sem: modified from (79).

Appliccftion of'ihe rnicrosphere teclmique iu the sheep mode2

39 In this study. latex spheres with a diameter of 15 prn labeled with either %c, Zn. 85 95 Sr. Nb. or ""ce obtained from New England Nuclear (Dupont Canada Inc.,

Mississauga. Canada) were used. Afier mixing the spheres for five minutes with a Vortex

Mixer (Model 58233. Scientific Products. Evanston. USA). an amount equivalent to

approximately 25 to 30 pCi \vas injected into the left atrium. Using an

infusiodwithdrawal pump (Harvard Apparatus Co.. USA). sarnpling of the reference

blood from the carotid artery and the femoral artery (10 ml/min) was started during

injection of the spheres and was continued for 90 seconds after the injection.

Afier sacrifice. the heart was obtained. the left and right atrium were removed from the

heart and discarded. The ventricles were counted together to represent coronary blood

flow. In case of the liver. diaphragm. small and large gut. random sarnples were taken

while the kidneys. gallbladder and pancreas were processed as whole organs. For

measurement of blood ffow to the brain. gray and white matter were separated and gray

matter was used for fùnher analysis only. The gastrocnemius muscle was obtained to

represent skeletal muscle blood flow. Al1 tissues were cut in 1 cm long pieces and placed

on a Petri dish. After drying for 77 Iiours in a lieater (Biological Safety Cabinet. Nuaire.

Plymouth, USA). tissue was pIaced into plastic tubes. Tissue and the reference blood

samples were then counted in a multichannel Automatic Gamma Counter System, Series

1 185 (Scarle Analytic Inc.. Des Plaines, IL). The raw data were read into Lotus 1-14

(Lotus Development Corp.) spreadsheet where the Eqn. 2 and Eqn. 3 had been

implemented. Al1 organ blood flows are expressed as ml/min/100g wet weight of tissue.

Fractional blood Bows are expressed as percent of cardiac output per 1 OOg tissue.

The quality of the blood tlow measurement was assessed by comparison of blood

flows to lefi and nght kidney using linear regression. Afier pooling the measurements of

al1 five stages. the dope of the regression line between lefi and right kidney blood flow in

both goups was not different from 1 . R2 was 0.9 1 for the S HAM group and 0.8 1 for the

CLP group.

To address the primary study objective. the Ca02 was experimentaily lowered to a

level which defined the limits of the heart's metabolic Oz reserve. which would be

exceeded in this unanesthetized sheep mode1 if either of two critena were met: [1] a

decrease in rnyocardial O2 consumption. Xccording to Walley. sequentially lowering the

Caol identifies a level of myocardial-QO? where myocardial O? consumption cannot be

maintained (178): [2] the onset of circulatory failure. As the sheep were awake during the

hypoxia trial. the onset of circulatory failure. defined as the abrupt occurrence of

hypotension or agitation. was also taken as evidence that the metabolic O- 7 reserve was

exceeded and the expenment was stopped at this stage. Data reported as defining the

limits of the heart's metabolic O2 reserve are from the stage just preceding the

identification of either of these tvio criteria.

The data were analyzed by analysis of variance by using a two factor design with

repeated measurements on one factor by using SPSS/PC+ 4.0 (SPSS Inc., Chicago.

USA). Further analysis was performed according to Winer (1 85). Separate anaiysis for

each group was oniy performed if the interaction term was statistically significant. The

Tukey test was used as a post-hoc examination for painvise comparisons. A p-value of

less than 0.05 was considered to be statistically significant. Data describing the metabolic

reserve of the heart were summarized at baseline and when study endpoints were reached.

2.3 Results

Table 5 lists hemodynamic parameters before and 48 hours after CLP or SHAM

laparotomy. There was no difference in the observed parameters at the pre-laparotomy

study (Pre). 48 hours Iater. the mean aortic and tefi atrial pressures were unchanged

cornpared to the pre-laparotomy study in both groups. The CLP sheep received

83.3i0.6 rnl/kg/day of Ringer's lactate to maintain end-diastolic filling pressures which

were comparable with pre-laparotorny levels. while the SHAM anirnals required

64.0*0.5 ml/kg/day (p<O.Oj). During the 48 hour study. the CLP group demonstrated a

higher cardiac index and systemic Q02. but lower systemic vascular resistance and

systemic Or extraction compared to the SHAM group. At post mortem examination

following the final stage of study. al1 CLP sherp were found to have a necrotic cecum.

ascites and peritonitis. wliile the SHAM sheep Iiad no evidence of intraabdominal

pathology.

Seven of eight SHAiM sheep and sis of eight CLP sheep completed al1 study stages of

progressive hypoxia. The rernaining sheep were euthanized because of the onset of

circulatory failure. A plateau or depression in myocardial Oz consumption was detected

in four SHAi i and six CLP slieep.

Excluding data frorn the study in animals where the sxperiment was terminated. the

mean FiOl was sequentially lowered to a final value of 10%. At this time. the mean aortic

blood pressure and lefi atrial pressure rernained unchanged from the baseline 48 hrs study

group in both groups (Table 6).

Mean aortic pressure

[mm Hg]

Cardiac index

[Z/.'rn i d m y

Heart rate

[hecrtsh in]

Lefi atrial pressure

[172rn Hg]

S ystemic vascular resistance

[ctvnr sec/crn'/m~]

O2 delivery

[n7102h in/'m']

O2 consumption

[mlO_l/'min/rn']

O? extraction

SHAM Grorrp (n=8) CLP Group (n=8)

Pre 48 hrs P re 48 hrs

Table 5: Effects of cecal ligation and perforation (CLP) on systernic circuIation. Parameters are expressed as mean 2 sem. *: p~O.05. *+: p<O.O 1 between pre-laparotomy (pre) and 48 Iirs study. #: p<0.05. ##: p 4 . O 1 benveen sham and CLP group.

sham

CLP

sham

CLP

mean aortic pressure sharn

r m m w CLP

cardiac index sham

heart rate sham

[b e « t s / w CLP

stroke volume index s h m

[mt/rrly CLP

lefi atrial pressure sharn

h m H g 1 CLP

SVR sham

[~(vne - sec/cmj/m'] C LP

Baseline Hypoxial Hypoxiat Hypoxia3 Hypoxia4

Table 6: Effects of hypoxia on systemic Iiemodynainics Parameters are expressed as rnean f sem. Only the systemic vascular resistance (SVR) showed a significant interaction term in the analysis of variance. All the otl~er siçnificances were obtained on the basis of significant main effects. *: ~ ~ 0 . 0 5 between stages. -T: ~ ~ 0 . 0 5 to the baseline (48 Iirs) study. f f : pC0.05. ##: peO.0 I betweeii sham and CLP group.

A significant group effect indicated that a higher cardiac index diRerentiated the CLP

from the SHAM study group during al1 experimental stages. In contrast to the CLP group.

the S HAM group demonstrated a progressive decline in systemic vascular resistance

throughout the study. The systemic Q02 began to faIl during the second stage of hypoxia.

when the Caol in both Sroups approximated 90 mlO&-rd (Table 7). Although the mean

systemic Or consumption was iunaltered tliroughout the various stages of depressed

systemic O2 delivery. arterial lactate levels rose significantly compared to baseiine values

dunng the final stage of hypoxia in both shidy groups. A significant elevation in arterial

lactate levels occurred at a higher systemic Q02 in the CLP goup (5271% rnI/rnin/m2)

cornpared to the SHAM group (357119 ml/min/m2. p<O.Oj). Simultaneously. the highest

systemic 0, extraction achieved in the SHAM group (0.7310.03) was significantly

greater than was demonstrated in the CLP group (O.54*0.03: p<0.05).

arterial p02 sham

[ m m w CLP

Cao-, sharn

[dOl;mZ] CLP

0: delivery sharn

[rn102/m i n i d ] CL P

0. consumption sharn

[m107/min/m2] CCLP

0: extraction sham

CLP

arterial lactate sharn

[mrnoZi~ CLP

Baseline Hyponia 1 Hyponia2 Hypoxia3 Hypoxia4

Table 7: Effects of Iiyposia on systemic O2 transport Parameters are expressed as mean i; sem. The analysis of variance revealed a significant interaction term for arteriai O? content (Cao?) and O? extraction. All the other significant p- values were obtained on tlie basis ofsignificant main effects. *: p<0.05 between stages. t: p<0.05 to tlie baseline 48 hrs study. #: pcO.05 between shaiti and CLP group.

2.3.2 Effects of lrypoxia on the nryocnrdicim

Fig. 6 demonstrates coronary blood flow and the blood pressure x cardiac output

product. an estimate of heart work. rose in parallel with the progressive decline in Cao?.

Although heart work was signiticantly greater in the CLP compared to SHAM sheep

during the final experimental stage. a progressive increase in myocardiai blood flow was

similar between the study groups during al1 experimental stages. The increase in

myocardial blood flow was accornpanied by a paraIlel decrease in coronary vascular

resistance (Table 8). Finally. throughout the experimental stages, changes in myocardial

lactate metabolism were not dissimilar betw-een the two study groups.

Heart work [mm Hg- l/mn/m7

Coronary biood flow [ml/min/l OOg]

O Sham CLP

u I

O 20 J O 60 X O 100 120 140 O 70 J O 60 80 IO0 120 140

arterial 0 2 content (m/O: ml] arterial 0 2 content [rnlo~~rnij

Fig. 6: Heart work and coronary blood flow during progressive hypoxia Parameters are expressed as mean sem. *: p<0.05 between stages. #: pC0.05 between sham and CLP group. Analysis of variance revealed a significant group effect (p4.O 1. dashed Iines) for heart work.

Table 4 lists rnyocardial Oz utilization variables at baseline and during the

experirnental stage where these study endpoints were identified. Completing the final of

the four stages of hypoxia or the inability to maintain myocardial Oz consumption was

identified at a Cao2 of 56.9*4.2 mlOl/rnl in the SHAM sheep and at 79.S7.2 (p<O.Oj) in

the CLP group. In the SHAM group. this Caoz tended to be Iower than the Caoz where

supply-dependency of systemic O2 consumption was identified (72.8I7.3 m102/ml:

p=O.O6). In CLP sheep. however. the Cao2 where we identified the study endpoints was

not significantl y di fferent from the level where suppl y-dependency of systemic O?

consumption (72.0*6.7 rnlOz/ml: n.s.) was identified. With the definition of study

endpoints, myocardial O? estraction had increased significantly in the SHAM group, but

not in the CLP group (Table 8).

arterial O2 content

[ml O2/mlj

heart work

[rnrnHg.l/min/m~

coronary blood flow

[ml/midlOOg]

coronary vascular resistance

[mm Hg-min- IOOg/mU

myocardial O2 delivery

[ml/min/l OOg]

myocardial O2 consumption

[ml/min/l O O g j

myocardial Oz extraction

myoc. Iactate consumption

[rnrnoZ/'mirv'l OOg]

SHA M Gro clp CLP Group

Baseline study endpoint Baseline study endpoint

Table 8: Effects of hyposia on myocardid 0 2 transport Parameters are expressed as mean 2 sem. *: p<O.O5. **: p<O.Ol Betsveen the 48 hrs baseline study and study endpoint. #: p<0.05. i f#: pCO.0 1 benveen sham and CLP çroup.

Fie. 7 shows the two components of the metabolic OS reserve of the heart. coronary

blood flow and myocardial O2 extraction. expressed as percent change fkom baseline to

study endpoints. The median of the coronary flow reserve approximated 100% in the

CLP group. less than in the SHAM group (ca. 300%. p<0.05). The maximum increase in

myocardial O? extraction to definition of study endpoints was not different from zero in

the CLP group. but reached 10% in the SHAiM group.

Coronary blood flow reserve [*;o of baselinrj

O 1

sham CLP

Myocardial Or extraction reserve [ " O ot'briselinel

-30 ' 1

sham CLP

Fis. 7: Coronary circulatory reserve in sliarn and CLP slieep The increase of coronary blood flow and niyocardial O2 extraction from baseline to the study endpoint is shown in percent. Data are displayed ris Box-Plots with 3%. 25%. 50%. 75%. 95% percentiles. *: peO.05. percent increase different from O: 8: pcO.05 benveen groups by Mann- Whitney Test.

Redistribution of cardiac output to the h e m is described by calculating fractional

blood flows. In both groups. FractionaI blood HOWS to the heart were increased during

severe hypoxia (Fig. 8). Although there was a tendency that sham sheep received a higher

portion of cardiac output during Iiypoxia than CLP shrep. no statistical difference was

observed. Fractional blood tlow to pancreas and srnall gut decreased in the sham group

while CLP sheep did not show a change in fractional blood tlows to these organs.

Fractional blood ff ows [% change from baseline to maximal myocardial VOz]

U Sham mi CLP

Fig. 8: Redistribution ofcardiac output during Iiypoxia This figure shows changes in fractional blood tlow (blood flow as a fraction of cardiac output) from baseline to srudy endpoints. Data are expressed as mean * sem. *: p<O.Oj. **: p<O.O I : change in fractional blood flow differeiit of O .

2.3.3 Effects of hypoxia on tioir-cardioc circrrlntions

Fig. 9 lists organ QOz at baseline and when study endpoints were achieved- At the

begiming of the 48 hour study. Q 0 2 to the smali gut and liver was greater in the CLP

nroup than in the SHAM group. rvhile an opposite pattern was noted in pancreatic QOr. CL

When the systemic QOz was depressed. QO? fell to al1 of the brain. kidney. gallbladder.

rumen and small and large gut. During the experimental stage where the study endpoints

were identified. however. Q 0 2 to small gut and liver remained higher in the CLP sheep

compared to the SHAM sheep.

707 CLP

Fi?. 9: Effects of hypoxia on regionai O2 delivery Data are summarized ar baseline and when study endpoints chancterizing the exhaustion of myocardial metabolic reserve ivere reached. Data are given as mean + SEM. Significances: *: ~ ~ 0 . 0 5 . +*: p<O.O 1 between stages. è: p<O.Oj. ##: p<O.O I SHAM vs. CLP.

2.4 Discussion

While myocardial O2 demands may be elevated in sepsis as a consequence of an

eievated heart work, there is evidence that the metabolic regulation of myocardial Oz

availability may be simultaneously depressed ( 142. 154). However. as outlined in chapter

1 A.2. the majority of data which lias evaluated the adequacy of the coronary circulation

to augment rnyocardial Oz delivery in sepsis remains inferential. This study therefore

measured and compared changes in both myocardial blood flows and O2 extraction

between septic and healthy control sheep when artenal 0 2 content was lowered through

four stages to both increase heart work and depress convective O? transport. At the

whole-body level. this mode1 of hyperdynarnic sepsis confirmed the elevated critical

systemic Q02 and depressed capacity to rna.imally extract O1 which has been reported in

other septic models (135). New information tiom this experiment is the demonstration

that myocardial metabolic 0 2 reserve is also depressed in hyperdynarnic sepsis as the

arterial O2 content which identified an inability to sustain myocardial O2 consurnption

was greater in the CLP compared to the SHAM study group. This finding rnay have been

related to an impaired ability to increase coronary blood flow and myocardial Oz

extraction. respectively. in the septic animals.

There rernains debate about whether circulatory abnormaiities demonstrated at the

b e l of individual organs in sepsis (56. 135) rnay be generalized to the coronary

circulation (5). Some of these abnomalities include hyporesponsiveness of arteriolar

resistance vessels (1 18). a lesion which inay explain the abnomal distribution in blood

flows reported in this syndrome ( 1 OS). The microcirculation is also altered in sepsis (56,

1 35). as increased endotlief i d pem~eability and decreased capiIlarity may limit both

convective and diffusive Q 0 2 . thereby restricting the ability to extract O?. Despite such

evidence for abnomalities in the metabolic control of tissue O2 dernands in sepsis,

rxisting data remain inconclusive about whether sirnilar abnormalities may be

rreneralized to the coronary circulation. For example. both Cunnion and Dhainaut Y

reported that coronary blood tlow is elevated in sepsis. generally in proportion to a

sirnultaneous increase in hean work (47. 52). Tliese data have been taken as irnplicit

evidence that the coronary circulation adequately couples QOz to changing rnyocardial

Oz demands in sepsis (47). In contrast to these clinical studies. animal models of sepsis

have reported depressed vasodilation to adenosine ( 154) and impaired endothelium-

dependent relaxation ( 105. 1 42).

Discrepancies in existing data about the circulatory control of the metaboIic Oz

capabilities of the coronary circulation may be explained by differences in the

experimental design of these various studies. For example. shock andor the simultaneous

administration of exogenous sympathornimetics characterize clinical reports of the

coronary circulation in sepsis (47. 52)- -et both can modify myocardial-QO? independent

of any direct effect of sepsis itself. M e n animal models are used to study the regional

circulations in sepsis. other factors may confound data interpretation. for example. the

use of anesthetic agents which depress coronary vasoreactivity independent of the effects

of sepsis (40).

Myocardial Oz demands are not only elevated in clinical sepsis. but whole-body O2

demands are constantly changing. The objective of this study. therefore. was to determine

the capacity of the coronary circulation's rnetabolic Oz reserve in this syndrome.

Specifically. we wanted to determine the ability of the coronary circulation to respond to

an abrupt increase in O2 demands by augmenting both myocardial blood flows and. to a

lesser extent. O2 extraction (183). To address concems about previous experiments, an

awalce animal model of hyperdynamic sepsis. in which systemic hypotension was not a

confounding issue. kvas employed. In tliis esperiment. cecal ligation and perforation in

sheep caused generalized perironitis and resiilted in a hyperdynarnic circulatory state. In a

pattern typical of septic patients. the CLP slieep necessitated a greater volume of

parenteral fluid replacement than the SHAM sheep to rnaintain end diastolic filling

pressures comparable to preoperative levels. thereby to support development of a

hyperdynamic circulatory state over the 48 Iiours following CLP (24).

As ernployed by other investigators (2 1. 128. 155. 178). inspired O7 concentrations

were subsequently lowered to sequentially lower systemic QOz. Throughout up to four

progressively lower levels of arterial Oz content. we measured blood flotv and Oz

extraction in both the myocardium and wliole body to determine the metabolic 0- 7 reserve

at both these levels of the circulation. in septic compared to control sheep. When

metabolic Oz reserve is exceeded. at either the whole body level or in individual organs.

O2 consurnption falls with tùrther decreases in the QO,. This point where Oz

consumption becomes supply-dependent is referred to as the "critical Q02". and

demarcates the onset of ischernia as shown in studies at both the whole body (138. 178)

and individual organ ( 1 3 5. 1 78) level.

Determining a critical QOi in the coronary circulation is not supported by the same

degree of empirical background as exists in studies of the systemic circulation. Similar to

the approach at the whole-body Ievel. however. near-maximum coronary circulatory

compensation would exist at a point just preceding definition that myocardial Oz

demands exceed O? delivery. Thus. the heart's metabolic Oz resewe should be exceeded

with the appearance of either of the two following criteria, first a decline in myocardial

0, consumption when O2 delivery was acutely depressed and second, the onset of

circulatory failure. In support of the former concept. Walley and CO-workers

demonstrated using hypoxia as the intervention that an arterial O2 content could be

demonstrated where myocardial O? consurnption plateaued or Fe11 (178). Beyond this

level. these investigators demonstrated that fiirther depressions in arterial O- 7 content were

accompanied by depressed ventricular contractility since 0- demands had exceeded Oz

delivery and myocardial ischemia had supervened. This concept of identieing a point

where myocardial O? consumption either plateaus or falls when arterial O2 contents are

sequentially lowered has also been supported by experiments in an isolated h e m

preparation. where the ma.ximal increase in myocardial Oz consumption identified

ischemia. as confirmed by the onset of myocardial lactate production (1 14). The onset of

circulatory failure may also be taken as evidence that the heart's rnetabolic O2 reserve has

been exceeded (21). In one of the SHAM and sis of the CLP sheep. an acute onset of

sudden respiratory distress and Iowered arterial pressures with the institution of the fourth

experimental stage were identified. k t this time. individual experiments were

immediately stopped and the stage just preceding this \vas taken to represent the point of

maximum ci rcula to~ compensation. With this approach. the 'critical' myocardial arterial

O? content identified in Our control sheep was similar to the value reported by Bernstein

in ventilated lambs, where the ability to maintain myocardiai O7 consumption was

exceeded below an arterid Oz content of 56. l* 1 -5 rnlOz/ml (2 1 ).

2.4.1 Tite effects of hypo-uia on systrmic iremodyrzuinics and O7 delivery

The normal physiological adaptation to hypoxia involves al1 levels of the circulation.

An elevated cardiac output serves to augment systernic QO?. This hyperdynamic response

is accompanied by a decrease in the tone of arterial resistance vessels (103)- which

fàcilitates an increase in organ blood flows. Additionalty. Q02 is redistributed from the

splanchnic to the core circulation to support organs with a limited O? extraction reserve.

like the heart. At the level of the microcirculation. capillary recruitrnent during acute

hypoxia decreases diffusion distances and thereby facilitates an increase in Oz extraction

( r 59).

While these mechanisms were observed in the control group. the systemic circulatory

response to hypoxia in the CLP slieep was substantialIy different. As is anticipated in

septic models (22. 24), the CLP sheep demonstrated a higher cardiac output and

depressed systemic vascular resistaiice at the baseline study cornpared to the SHAM

sheep. With acute hypoxia. a stight depression in the systemic resistance was

insignificant. albeit remaining lower than the maximal depression which was noted in the

SHAM sheep. This rnight indicate that systemic vasodilation was near-maximal in the

CLP sheep during the baseline septic study- perhaps the consequence of septic-associated

release of vasodilating mediators (153) or altered reactivity to endotheliurn-derived

relaxing factor in this syndrome ( 1 18). Furthemore. Our septic mode1 is consistent with

data from other animal esperiments (135). whicli have demonstrated that the systemic

metabolic 0, reserve is depressed in this syndrome because the critical Q02 is elevated

and the ability to maxirnally extract O? is depressed.

2.4.2 Effects of itypoxia on myocarninl O? delivery

In the myocardium. the sequential depression in arterial O? content was accompanied

by coronary vasodilation in bot11 study groups and by a simultaneous increase in coronary

blood flows. Unlike the concliisions from previous studies (105)- these data indicate that

the metabolic 'coupling' of myocardial O? demands and deiivery remained intact in this

septic model. Overall. however. the CLP group reached study endpoints at a higher

arterial O? content than was demonstrated in the S H M animals. It might be argued that

any differences in coronary blood flow at study endpoints may be artificial due to a

different degree of hypoxia. However, study endpoints were chosen for a cornmon

behavior of the coronary circulation as myocardial O2 consumption started to drop or

circulatory failure developed when arterial O2 contend was dropped below study

endpoints. We. therefore. believe this is evidence that the metabolic O2 reserve of the

coronary circulation was depressed in the septic compared to the control groups. n iere

are two considerations to explain a depressed myocardial metabolic O7 reserve. frrst, an

inability to maximally augment local blood flows and second. an inability to rnavirnally

increase myocardial O? extraction.

DurÏng the baseline study- no difference in myocardial blood flows between the

groups was detected . It was previously reported that rnyocardial blood flows in this

septic mode1 are elevated. generally in proportion to an increase in myocardial work (23).

a finding confirmed in other animal (108) and c h i c a l (47. 52) studies. With the

definition of study endpoints. however. the increase in myocardiai blood flow in the

septic group was less than was identified in the SHAM group. although the absolute levei

was not significantly dissirnilar between the two study groups at this time. The failure to

detect a statistical significance in the baseline elevation in the CLP group was probably

due to the experimental setup which was not designed to Find differences in coronary

blood flow under resting conditions.

Myocardial blood flow reserve. which can be defined as the difference between

baseline and study endpoint. was less in the CLP sheep compared to SHAM sheep. This

observation could be explained by one of two possibilities. first. an effect of sepsis to

require that baseline myocardial blood flows are increased and second. inability to

increase myocardial blood flows îùrther tlian kvas demonstrated in the CLP sheep in this

experiment. The study design using an intact and awake animal model does not allow us

to conclude whether definition of study endpoints in the SHAM sheep identified a point

where coronary vasodilation. and subsequent increases in myocardial blood flow, was

maximal as it might have been demonstrated in an isolated h e m preparation. Therefore,

it cannot be concluded whether the eievation in myocardial blood flows in the CLP sheep

with definition of study endpoints should have been greater than was demonstrated.

However. the nearly threefold difference in the ability to augment rnyocardial blood

tlows in the control versus the septic sheep should be a significant discriminating Factor

between these two groups. This concept is supported by the fact that coronary circulatory

reserve was exhausted at a greater arterial 0, content in CLP sheep than in sham sheep.

Consideration for this would include an impairment to Further vasodilate andor

redistribute blood Aows frorn non-vital circulations to the myocardium in the septic state.

To support an increase in myocardial blood tlows with a depression in artenal Oz

contents. the cardiac output in the SHAM sheep was redistributed from the small gut and

pancreas to the myocardium. This response requires a simultaneous increase in the gut's

O? extraction to maintain this orgai's O2 availability. If the increase in myocardial blood

flows in the CLP sheep was depressed. an inability to maximally redistribute Q02 Iiom

the nonvital to vital circulations. as demonstrated in this experiment and other reports

(22) may be a significant issue. Other animal models of acute injury. for example

tollowing burns. have also concluded that a depression in the ability to redirect QO- 7 f rom

the _rut and liver's circulations rnay contribute to impaired myocardial blood flow reserve

(35).

We also found that myocardial O? extraction failed to increase in the CLP sheep to the

same extent noted in the SHAM sheep at the arterial O? content level where myocardial

circulatory compensation to hypoxia had been exceeded. Although myocardiai

extraction is normally high at rest. a significant increase is possible when myocardial Cl2

demands acutely increase (49). Tlius. myocardial O7 extraction c m make an important

contribution to the heart's metabolic O2 reserve ( 1 82). Our observation is consistent with

Nelson and coworker's demonstration that a depressed ability to rnaxirnally increase gut

O-, extraction reduced this organ's tolerance to depressed Q02's in endotoxemic animals

( 1 35) . Previously. both clinical (47. 52) and animal (74) studies have only inferred that

O? extraction in the myocardial circulation is depressed in sepsis. Data from this

experiment are the most direct evidence that the systemic microcircu~atory dysfunction.

which contributes to the O3 extraction defect in hyperdynamic sepsis. is shared by the

coronary circulation. There are many potential causes for depressed microcirculatory

fùnction in sepsis which have been outlined in chapter 1. including capillary obstruction

by endothelid edema leukocytes and/or red ce11 plugging.

2.4.3 Meth odological corzsicierntions

It k v a s intended to use an animal mode1 of sepsis which allows the study of the

c o r o n q circulation without the administration of anesthetic or vasoactive agents. The

cecal ligation and perforation in sheep is a well established animal mode1 of

hyperdynarnic normotensive sepsis when an appropriate fluid resuscitation is applied (see

chapter 1.4). The L M representing the lefi ventricuIar preload was successfully

maintained at the pre-Iaparotomy level in both groups by infusion of Ringer's lactate.

Under these conditions. CLP sheep developed a hemodynamic pattern with a high cardiac

output. Iow systemic vascular resistance. and low systemic O2 extraction which is

typically seen in patients with sepsis (143. 145). As the post-mortem examination in CLP

sheep showed signs of peritonitis. this study demonstrated a focal infection with a

systemic hemodynamic response. thus successfùlly simulatine human sepsis.

In sham sheep. a significant increase of systemic vascular resistance was observed

from the pre-laparotomy snidy to the 48 Ius study. Vasoconstriction may be a sign of

hypovolemia or cardiac failure. However. other hemodynamic parameters do not support

this notion since blood pressure. cardiac indes. and cardiac filling pressures remained in

the normal range. 4 s systemic vascular resistance is a calculated parameter, its significant

increase rnay be due to the small drop in cardiac index and non-significant increase in

inean blood pressure 48 hours after the pre-laparotomy study (Table 5)

Cntical O2 delivery was determined by the increase of lactate levels as it has been

described before (128). However. sepsis may interfere with lactate metabolism

independent of tissue hypoxia. Lactate levels have been described to be elevated during

endotoxemia despite a Lack of signs of tissue hypoxia (95). Significant release of lactate

has been seen clinically in patients with ARDS or multiple organ dysfunction syndrome

(34. 55) . However, the CLP tnodel in sheep simulates an early stage of sepsis without

signs of organ dysfùnction. Thus. a major release of lactate independent of tissue hypoxia

rnay not play a sizgificant role in this rnodel. Additionally. lactate levels had to increase

by 0.9 mmoVl (twice the standard deviation of lactate levets at room air) above the

individual lactate Ievei at room air before O? delivery was considered to be critical. This

procedure rnay control for differences in the baseline lactate levels between sham and

CLP animals.

C o r o n q blood tlow as well as al1 other organ blood îlows were rneasured by the

microsphere technique. A major determinant of error in blood flow measurement by

using rnicrospheres is the nurnber of spheres in the sampled tissue. Dole et al.

demonstrated that the error of flow rneasurement is 10%. if the sample contains at least

400 microspheres (53). Thus. a sufficient number of spheres (about 10') has to be

injected systemically to obtain a sufficiently high number of microspheres in each organ

of interest. In this study. an amount of microspheres with a total radioactivity of 25-

30 pCi were injected. In the previous sheep studies. this amount of radioactivity was

consistent with sufficiently high tissue counts (22. 125. 15 1 ). Although such an amount

of spheres rnay be injected several times during an experiment. no detrirnental

hemodynamic effects were obsenred (79). Even in small animals. the microsphere

technique has been applied without detectable hemodynamic side-effects ( 170).

The main goal of this study was to assess the coronary circulatory reserve by using

progressive hypoxia. Two criteria were defined where coronary circulatory reserve was

rxhausted: A drop in myocardial 0 2 cons~imption and the onset of circulatoxy failure.

respectively. It is possible the two criteria used to identic where the heart's metabolic 0 2

reserve was exceeded may have underestimated tàilure of circulatory compensation to the

depressed Q02. The two criteria used. where four of the eight studied SHAM animals had

not shown a depression in myocardial O2 consurnption by the end of the final

experimental stage. In contrast. ail but two of the eight CLP sheep demonstrated a plateau

or depression in myocardial O2 consumption before the final experimental stage. In this

context, therefore, concem for linderestimation of the maximal increase in either

myocardial blood flows andor 0 2 extraction in the coronary circulation would have been

an issue prirnarily in the SHAiM groiip. therefore sewing only to minimize differences in

the myocardial metabolic O2 reserve demonstrated between the septic and control snidy

proups. However. it cannot ruled out that the circulatory failure. which also was

considered as stirdy endpoint. is not only due to the failure of the heartrs metabolic control

but also due to mechanisms independent from myocardial O7 delivery. This could include

severe vasodilation followed by profound hypotension. However. these data support that

vasodilation of peripheral arteries must be aimost mmimal in septic animals without

applying liypoxia since these animals were unable to tiirther dilate the artenes when the

Cao-, was dropped.

Other experiments (1 78) have used changes in transmyocardial lactate metabolism to

identi@ that this organ's metabolic O2 reserve is e'rceeded. When calculating myocardial

lactate consumption or myocardial lactate extraction in this study. large standard errors

were observed. This is likely because the hypoxic stress in an intact animal model

increases artenal lactate concentrations substantialiy and myocardial lactate uptake is

determined by arterial lactate levels. In this circumstance- calculating myocardial lactate

uptake becomes an insensitive method for the detection of rnyocardial ischemia (72).

2.4.4 Conclusion

The main goal of this study was to describe the coronary circulatory reserve in sepsis.

Metabolic 'coupling' between Oz delivery and O? demand in the myocardium remained

demonstrable in this animal model of hyperdynarnic sepsis. However. the extent to which

the septic coronary circulation was able to further adjust to changes in rnyocardial O7

availability was depressed. Restrictions to the ability to increase both myocardial blood

flow and O2 extraction rnay explain why the septic myocardium reached a point where its

O2 consumption was not sustainable at a çreater arterial O2 content compared to healthy

controls. In this context. these data are consistent with concepts regardin; the effect of

sepsis on the systemic circulation and in the gut. Thus. sepsis infringes on the coronary

circulatory reserve available ta match increases in myocardial Oz availability with O2

demands. by virtue of its hypermetabolic profile.

The second goal of this study was to compare the aiterations of the regional coronary

circulation to the systemic changes. This study conIirmed previous findings that the

capacity to rnavirnally extract 0 2 is depressed in sepsis and that septic animals have a

peater cntical O? delivery than non-septic animals. The Oz extraction defect is believed

to be due to a microcirculatory injury. Septic sheep demonstrated both a depression in the

myocardial as well as in the systemic O2 extraction reserve. Thus. this study gives

rvidence that the coronary circulation shares in the microcirculatory injury seen in other

organs in sepsis.

Chapter 3 - The O2 dissociation curve in sepsis

3-1 Introduction

Sepsis affects Or transport by causing circulato~ dysfimction and depressing O2

extraction mainly by redocins capillary surface area available for difision. ïhus. sepsis

is a disease reducing cellular Oz availability. On tlie other hand. sepsis increases Oz

demand by induction of a hyperdynarnic state. The mismatch between O2 delivery and 0 2

demand is a characteristic feature of septic shock. [n Chapter 2 it was demonstrated that

depression of the Oz extraction reserve can be seen systemically as well as in the regional

circulation of the heart. Alteration of 0: extraction in septic shock is usually attributed to

microcirculatory faiiure due to leukocyte adhesion and endotheliai ce11 edema (Fig. 1).

However. Or extraction is also limited when tlie afinity of hemoglobin to 0 2 is

increased. Other diseases that induce a mismatch between O. delivery and O2 demand.

i.e. chronic lung disease or anemia. are accompanied by a right-shifi in the Ot

dissociation curve (ODC) to improve O2 unloading at the capillary level (122). Only little

knowledge is available about the ODC in sepsis and possible effects on Oz transport in

this disease.

3.1.1 The dissociatiot~ cttrve

The OZ dissociation curve (ODC) describes tlie relationship between the partial

pressure of O2 (pOz) in the serurn and the percentage of hernoglobin saturated with 0 2

(SO?). It. therefore. describes the ability of hemoglobin to bind O?. A decrease of the

affinity of hemoglobin to O2 is seen as a right-shift of the ODC. while the ODC shifts to

the left when the 0.-hemoglobin affinity increases (Fig. 10). The position of the ODC

affrcts Oz transport since a hi& affinity eases the loading of hemoglobin with O2 and?

lience. increases arterial O2 content. The unioading of Cl2. on the other hand. is impeded

and O2 extraction may be depressed by a left-shified ODC.

The position of the ODC highly depends on environmental factors such as pH and

temperature. Intracellular molecuIes like 2.3-diphosphoglycerate (2.3-DPG) rnay be used

to regulate the position of the ODC. The p50. wliich is the p 0 2 at a hemoglobin Oz

saturation of 50%. describes the position of the ODC. ODCs are usually drawn at

standard conditions to ensure comparability. In sheep. standard ODCs are drawn at a pH

of 7.40 and temperature of 38.0 "C ( 1 32). Thus. p 0 2 values obtained under different

conditions have to be corrected. The effect of temperature on the ODC is expressed as

ApOdAternp. An increase in temperature right-shifts the ODC.

Fis. 10: The O2 dissociation cunle in Iiumans The figure shows a physiologic Iiuman 0: dissociation curve (bold line) with an example of a IeFt- and a right-shified ODC. The hatched lines mark the different p50s depending on the position of the ODC.

The effect of pH on the ODC is called Bohr effect and is expressed by the DiIl's ratio

ALog pOi/ApH. K-ions cause the hemoglobin rnolscule to release O2 more easily. Thus.

acidosis right-shifis the ODC. The Bohr effect has been studied by equilibrating the blood

with different p C 0 2 s (CO2 Bohr effect) since respiratory changes are often cause of

alterations of the acid-base status in the clinical setting. However. changing the pH by

altering the pC02 does not only shifi the ODC by changing the H'-concentration.

Additionally. CO2 binds to hernoglobin due to carbarnate formation which directly affects

the affinity of hemoglobin to Oz. Thus. a change in pH witliout a change in pCOz (fixed

Bohr effect) would right-sl-iift the ODC to a lesser degree. The t k e d Bohr effect is tested

by adding sodium hydroxide or hypochloric acid to the blood. In vivo. a change in pH

independent of respiratory changes is reflected in the base excess (BE). The BE can be

used to estimate the fixed Bohr effect. The total Bohr effect may be calculated by using

the fo llowing equation:

rotai Bohr rffecf = CO? Bohr + k x BE Eqn. 4

where k is a constant. The Dill's ratio as well as k have been established for the human

blood gas analysis by Severinghaus (160). However. these data cannot be simply

transferred for calculating a standard ODC in sheep since these animals have a lower

buffer capacity (70) and a more intluential CO2 Bohr effect (17) than humans.

Furthemore. the magnitude of the CO, Bohr effect in sheep is dependent on the SaO2.

Although these data are available in the literature. no equations for the calculation of the

fixed and the COz Bohr effect in sbeep have been developed.

3.1.2 The Ot dissociation cccrve in septic sl~ock

In contrat to other diseases. which also induce a mismatch between O2 delivery and

Or demand. case reports of septic patients descnbe a lefi-shift nther than a right-shifi of

the ODC 102. 10% 108. a situation which would hrther inhibit tissue Or extraction due

to the high aafnity of O2 to hemoglobin. This shifi of the ODC was associated with a

depression of 2.3-diphosphoglycerate levels (2.3-DPG). However. assessrnent of the

ODC in sepsis from these studies is confounded by a number of factors. Patients in these

case reports were in shock. had received massive blood transfusions and had alterations

in the acid-base-status of the blood. Each of these factors by itself c m affect the position

of the ODC (20. 35. 37). Therefore. Watkins et al. argued that there is no direct effect of

sepsis on the ODC and that the lefi-shifi c m be avoided by proper transfusion

management and by maintaining normal inorganic phosphate levels (1 8 1).

There are only a few controlled studies available which address hemoglobin Or

affinity in sepsis. Patients with adult respiratory distress syndrome had a lower p5O than

healthy volunteers (93). This finding was unrelated to semm phosphate levels. Lehot and

coworkers reported that patients rvith septic shock had a low p5O which rose with

recovery (1 10). However. this study does not answer the question whether the shifi in the

ODC's position is due to sepsis or due to shock. It also remains unclear whether a sepsis-

induced lefi-shift in the ODC cûn explain some of the Oz extraction deficit usually seen

in sepsis.

3.1.3 Goals and hypot/teses

The first goal of this analysis was to establish a correction factor for the totai Bohr

effect in sheep by using data available in the literature and to reconstruct individual O2

dissociation curves from blood gas data obtained dunng the hypoxia study described in

chapter 2. The second goal was to compare effects of possible sepsis induced shifts in the

ODC on systemic and myocardial O? extraction.

Based on the clinical literature cited above. we hypothesized that

1. the Oz dissociation curve is Mt-shified in normotensive hyperdynarnic sepsis.

2. the impairment in systemic as well as myocardial O? extraction in sepsis could be

explained by a lefi-shified Oz dissociation curve.

3.2 Methods

3.2.1 Establishing the total Bohr effect for sheep

As demonstrated in Eqn. 4. the COZ and the fixed Bohr effect have to be estirnated to

calculate the total Bohr effect. Magimiss et al. measured the CO1 Bohr effect in sheep

( 1 15). As the CO2 Bohr effect in sheep is less influential on the Or-hemoglobin affinity at

very hi& and very Iow O2 saturations. respectively. the Dill's ratio has to be calculated

dependent on the S 0 2 . A second order polynome (Eqn. 5 ) was applied to fit Maginnis'

data. A least square regression mode1 was used to estimate the factors a b. and c:

A l n ( p ~ z ) l ~ p ~ = c r + b ( ~ ~ ~ / l ~ ~ ) + c ( ~ O ~ / 100)' Eqn. 5

The fixed Bohr effect is estimated by the BE (Eqn. 4). Since sheep have a lower buffer

capacity than humans. Gattinoni and Samaja developed a set of equations (Eqn. 6 to Eqn.

8) to calculate base excess in sheep (70). This modified base excess was used instead of

the BE supplied by the blood gas analyzer used in this study (Radiometer Copenhagen,

Denmark).

BE=.;l+BxpCOi

where A and B are defined as

Eqn. 6

A = -13.09 x ( p ~ - 7 ) ' +11.02 x (pH-7)-16.5 Eqn. 7

B = 1 . 2 4 ~ ( p ~ - 7 ) ' i - 0 . 3 6 ~ (pH- 7)-0.18 Eqn. 8

From H1astalafs data. who measured the COz Bohr effect as well as the fixed Bohr

effect in sheep (86). it was calculated. how much the pOz would increase when the pH

was dropped fiom 7.4 to 7.0 while keeping pCOr constant at 42 mmHg (fixed Bohr

effect). The base excess was calculated by using Eqn. 6. and Eqn. 4 was resolved to

calculate k:

Eqn. 9

where Alog pOzcfixed) describes the change of p02 due to the fixed Bohr effect and

Alog pOzccoz, the change of POr due to the COz Bohr effect. The effect of temperature on

the ODC has not been measured in sheep. Therefore. the human correction factor of

Ap02/ATemp = 0.024 was app t ied ( 160).

To cover the complete range of the ODC. p02 and Oz saturation measurements in each

sheep were grouped from al1 sample sites (carotid artery. pulmonary artery and coronary

sinus). and from al1 hypoxic stages. First. p01 values were corrected (p02corr) for

differences in pH and temperature. As common in sheep physiology. standard ODCs

were drawn at a temperature of X ° C and a pH of 7.40 ( 132).

The O2 dissociation curve was reconstructed with the Hill-equation (85):

Eqn. 10

The p5O and the Hill coefficient n of the Hill equation were estimated with non-linear

regression by using the procedure N L N of SAS 6.07 (SAS Institute Inc.. Cary. NC.

USA). Non-linear regression was applied by using the Gauss method as recomrnended by

OiRiordan et al. ( 1 39).

Variables are shown as mean and standard srror of rnean. Data were analyzed by

analysis of variance by using a two factor design witli repeated measurernents on one

factor. The Tukey test was used as a post-hoc test for pairwise comparisons. The effect of

p j 0 on Oz extraction was detennined by analysis of covariance. A p-value of less than

0.05 was considered to be statisrically significant.

3.3.1 Reconstruction of the O? Dissociation Cirrve

First. the COz Bohr effect was estimated by using Maginniss' data (1 15). The least

square regression technique on Eqn. 5 yielded the results dernonstrated in Table 9. To

what extent different Oz saturations affect the Dill's ratio is shown in Fig. 1 1.

Parameter Value

Table 9: Results of the regression analysis for the CO2 Bolir effect.

CO,-Bohr effect (Dills ratio)

Fig. 1 I : Effect of the hemoçlobin O2 saturation on the COz Bolir effect in sheep. Second order regression on data obtained by Maginniss et al. ( l 15)

Next step was the calculation of the fixed Bohr eEect. According to Eqn. 6, a

metabolic acidosis with a pH of 7.0 and a pC02 of 42 mmHg results in a BE of

-19 mmoWl. From Hlastala's data (86) it was calcuiated. how much the p 0 2 would

increase when the pH was dropped from 7.4 to 7.0 while keeping pCO- 7 constant at

42 mmHg. As Hlastala measured the fixed Bohr effect to be -0.27. Alog pOz(fixed)

calculates to 1.28. If this drop in pH is due to an increase in pCOr, Alog pOtccoz, wodd

calculate to 0.164. When applying Eqn. 9 on these data. k was calculated to be -0.007.

Tlius. the total Bohr effect in sheep c m be calculated by using Eqn. 4 when the CO2 Bohr

effect is replaced by Eqn. 5 and the fixed Bohr effect is estimated by the base excess:

~otalBohreffecr = -0.349 - 0.264 x (SO, / 100) + 0.222 x (SO: / 100)' Eqn. 11

- 0.007 x BE

Al1 pOz values were corrected for the total Bohr effect according to Eqn. I l and for

temperature by using a ratio of Ap0-T=0.024. Table 10 shows the artenal blood gas

analysis at baseline and the four stages of hypoxia. In both groups, pCO- 3 decreased and

pH increased with progressive hypoxia (respiratory alkalosis). There were no differences

in temperature between the groups while both groups demonstrated a slight but

significant increase in temperature at the end of the study.

58

Baseline Hypoxia 1 Hypoxia 2 Hypoxia 3 Hypoxia 4

sharn

CLP

sham

CLP

sharn

CLP

sham

CLP

sharn

CLP

sharn

CLP

Table 10: Arterial blood gas analysis during Iiypoxia Data are given as mean + SEM. Resillts of tlie Tukey-test for painvise comparisons: *: pe0.05 between stages. T: p<0.05 to baseline. #: p<O.OS between groups. pOz: uncorrected pOZ. BE: calculated base excess (see text).

In each individual sheep. the ODC was reconstructed by using the Hill equation (Eqn.

10). Fig. 12 shows a reconstructed ODC for one sheep as an example. The mean ? in

sharn sheep was 0.9910.00 versus 0.9710.0 1 for the CLP group (not significant). CLP

sheep demonstrated significantly lower p50 than the sham sheep while t hex was no

difference in the Hill coeficient n (Fig. 13).

In chapter 2. it was demonstrated that maximum 0 2 extraction was 0.73*0.03 in the

sham group versus 0.54*0.03 in the CLP group (p<O.Oj). Systemic 0 2 E was lower over

the whole experiment in CLP sheep compared to sham animals. as demonstrated by a

significant group effect for this parameter (p<O.Ol). When the p50 was introduced as a

covariate into the analysis of variance. tlie group effect increased from 0.004 to 0.035.

Fig. 14 shows the relationship between p5O and the maximum 02E achieved during

liypoxia. There was a significant linear relationship between those two variables (r2=0.26,

p=0.03).

SO, [%] 100

Fig. 12: Reconstruction of the O? dissociation curve for one sham sheep as an example The pOz wns corrected according to Eqn. I 1 . Tlie parameters of the Hill equation including 95% confidence intervals were estimated as follows: p5O: 46.6 (46.1/47.1) mmHg. n: 2.842 (2.756/2.929). r' = 0.99.

- - sham - 4

- ' CLP

Fig. 13: Tlie p5O and the Hill coefficient n in CLP and sham sheep. Data are expressed as mean and sern. 3: p<O.O5 (iinpaired t-test)

maximum 0, extraction 1.0 -

0 sham a CLP

Fig. 14: Relationsliip benveen maximum sysremic 0: extraction v e n u s p5O. Regression equation: maï.O1E=0.06 + 0.0 I xp5O: r2=0.76

Coronaq circulation also ciemonstrated a difference in myocardial O- 7 extraction

reserve during hypoxia (see chapter 2). Linear regession b e ~ e e n maximum myocardial

0 2 E and p5O yielded no significant correlation (r' = 0.02). There was also no significant

correlation between p5O and myocardial O2 extraction reserve (r' = 0.00).

3.4 Discussion

This retrospective analysis was designed to descnbe the O? dissociation c u v e (ODC)

and its effect on systemic and myocardial Oz extraction in a sheep mode1 of

hyperdynamic sepsis. It was found that the ODC is lefi-shifted in septic sheep

accompanied by a sipificant depression in maximum systernic O7 extraction during

acute hypoxia. That an increase in Oz-hemoglobin affinity rnay limit Oz extraction is in

agreement with previous studies on non-septic animals where p50 was lowered by

carbamylation of the blood. During progressive hypovolemia dogs with an

sxperimentally lowered p5O reached a lower maimurn 0, extraction than the control

group with a normal p50 (258). Another study lowered p5O in dogs and measured

perfiusion of the brain ( 188). [t was found that a lefi-shifi of the ODC Iimited regional O2

consurnption of the brain although cerebral blood flow was increased in the low p50

group. The most likely explanation for the limitation of O2 extraction during an increased

Or-hemoglobin affinity is a reduction of O? unloading on the capiIlary level which would

decrease the capillary-tissue pOz gradient. This gradient is an important parameter of O2

diffusion and. hence. Oz extraction. Functioning of intracellular mitochondria is a

function of O2 partial pressure (187) and the amount of p 0 2 transferred from the

hemoglobin into the ce11 therefore Iimits mitochondrial function.

When increasing Or-hemoglobin affinity. hemoglobin binds Or more easily which

increases O2 saturation as well as the arterial O? content and. hence, O2 delivery. Shifiing

the ODC to the left is an important mechanism to maintain tissue Oz delivery during

hypobaric hypoxia in high altitudes. In humans. the left-shifi in hi& altitudes is initiated

by tachypnea foilowed by respiratory alkalosis (1 55). Therefore. a lefi-shift of the ODC

was considered to be of advantage during nomobaric hypoxia. as well. Or delivery was

significantly greater in hamsters during Iiypoxia when p50 was lowered by sodium

cyanate compared to untreated animals (1 65). Howevrr. in rabbits during severe hypoxia.

a lower p5O depressed systemic O2 extraction. Mathematical modeling of the data

suggested a limitation to O7 diffùsion (78). It was concluded that an increased

hemoglobin affinity is not of advantage durinç nomobaric hypoxia.

A depression in the level to which O? extraction increases when O? delivery is not

meeting tissue O2 demands is characteristic of sepsis and is probably due to dterations in

the distribution of blood flow at both the regional and microcircuIatory leveis of the

circulation (135). The data presented in this thesis suggest another issue contributing to

the O2 extraction defect in sepsis. It was found that 26% of the variation in maximum

systemic O- extraction could be explained by differences in p5O. Similar to maintenance

of O? delivery in high altitudes. it was suggested that a therapeutically initiated drop in

p5O might be an advantageous concept for increasing 0- delivery in septic patients (1 83).

However. maintaining whole body Oz delivery in patients wi th septic shock is usually

sufficiently achieved by fluid- and catechotarnine therapy as well as blood tnnsfusion

and mechanical ventilation. On the other hand. there is no concept available to treat the

depression in O2 extraction during sepsis. As mentioned above. a significant reduction in

p50 is able to depress O2 extraction even in non-septic animals. The combination of

impaired capillary perfusion and an increase in O?-hemoglobin affinity may be therefore

a significant factor in causing tissue injury which underlies multiple organ dysfùnction in

sepsis (3).

Since p02 values were corrected for temperature. pH. and base excess, it seems

unlikely that the left-shifi of the ODC in the septic sheep \vas caused by environmental

factors. but rather by direct effects of sepsis on the erythrocytes. Such effects may include

( i ) change of hemoglobin type (ii) depression of 2.3-DPG levels and (iii) alteration of red

blood ce11 homeostasis.

D u h g stress, i.e. chronic anemia. slieep can produce reticulocytes with a different

hemoglobin type within Four dap . This hemoglobin type C has a lower p5O than any

other hemoglobin type (171). Although the henioglobin type was not determined in our

experiment with only two days difference between the laparotomy and the hypoxia trial,

it seems unlikely that this issue has biased the results. Furthemore. only sheep with

hemoglobin A are known to change to Iiemoglobin C (50). The high p50s obtained in this

study suggests the hemoglobin type B having a p5O of about 42 mmHg (50).

In humans. it has been suggested that sepsis depresses 7.3-DPG levels and. therefore,

lotvers the p50 (1 72). However. sheep red blood cells contain only very low levels of 2,3-

DPG. The importance of 2.3-DPG in mature sheep is not clear. Although it has been

suggested that 2.3-DPG levels in sheep still have some affect on the ODC (6). other

phosphates may play the role that 2.3-DPG has in humans (90).

Among the many types of tissues affected during sepsis. the red blood ce11 is a target

of the septic impact. as well. Endotoxin in the presence of leukocytes increases the

viscosity of red ce11 membranes leading to a decreased deformability of erythrocytes

( 169). Binding of endotoxin to red ce11 membranes may induce a breakdown of red blood

cells (48). Furtherrnore. the red blood ce11 deforrnability correlated with the arterio-

venous oxygen difference in septic patients (149). thus. influencing O2 utilization in this

disease. However. intracelluiar homeostasis of erythrocytes like changes in intracellular

pH have not been studied in sepsis. As ce11 membranes are an important part in

maintenance of the intracellular environment. an alteration of erythrocyte homeostasis

seems likely dunng sepsis. A loss of the regulation of intracellular pH would naturally

affect the position of the ODC. Future studies would be desirable to clarify cellular

pathophysiology of erythrocytes during sepsis.

When differences in systemic Oz extraction capacity between sham and septic animals

can partly be explained by differences in p5O. it seems reasonable to assume that

differences in myocardial O2 extraction reserve can also be attributed to different

positions of the ODC. However. this analysis did not show any correlation between p5O

and maximum myocardial Or extraction or myocardial O2 extraction reserve. In healthy

dogs, a lowered p5O did not alter coronary autoregulation (14). The authors also found

that a left-shified ODC decreased myocardial O2 extraction without inhibiting myocardial

O2 extraction reserve. Even in sepsis. O2 extraction is very high in the coronary

circulation compared to whole body O2 extraction. The hemoglobin of the coronary sinus

blood is saturated with about 70% of oxygen. Therefore. additional O2 unloading during

hypoxia happens at the lower part of the ODC. As differently positioned ODCs converge

at low pOz values (1 58). an increased O? hemoglobin affinity will not affect O2 extraction

in this p 0 2 range.

3.4.1 Meth odolo,9ical considerat ions

The data used to establish a p 0 2 correction for differences in pH were drawn fiom the

literature. These studies mostly described differences of sheep hemoglobin to human

hemoglobin and were not primarily designed to develop an equation for the total Bohr

effect. However. the publications included enough data about the CO2-Bohr effect as well

as the fixed Bohr effect that modifications to the blood gas calculator by Severinghaus

( 160) became possible. In fact. the studies that established the Bohr effect in sheep (87.

115) used methods similar to Severinghaus. When regression analysis was used to

describe the saturation dependency of the CO? Bohr effect- a hi& correlation coefficient

was achieved. No temperature correction for sheep kvas found in the literature. Thus. a

systemic error might be introduced by using the human correction. However. as there was

no difference in temperature between s h m and CLP sheep. this error would be of similar

magnitude in both groups.

Several equations have been developed to describe the O? dissociation curve since the

Hill equation becomes inaccurate at O? saturations below 20% and above 98% (139).

Aberman et al. published a different equation that should better resemble the shape of the

ODC (2). However. this equation was designed for the human standard ODC and

calculation with this equation is not possible with shifted ODCs. Therefore. this equation

was not suitable for reconstructing ODC from sheep. O'Riordan et al. reviewed nine

different equations to fit blood &as data including the Hill equation (139). Several other

models produced lower residual surn of squares in regression analysis than the Hill

equation suggesting a better accuracy. However. those models have other disadvantages.

Since these models are more cornplex. up to seven parameters have to be estimated by

nonlinear regression. Thus. either a large amount of data is necessary for sufficient

accuracy of the model or the error range of one or more parameters will include zero.

These parameters do not have a physiologic meaning like the p50 in the Hill equation. It

was concluded that the Hi11 equation is a sufficient accurate model to descnbe ODC data

and that other models do not add information of physiologic interest (139).

The data analysis in this study was of retrospective nature. Furthermore. the ODC was

not determined by direct measurement on a single blood sample. but by calculation of the

ODC from several blood samples drawn from different sample sites obtained over a four

hour period. However. the results were remarkably consistent: Al1 regession calculations

delivered hi& correlation coefficients that were not different benveen the groups.

Although 0 2 transport in sheep is very similar to hurnans (141), red blood ce11

physiology has remarkable differences. As rnentioned above. sheep erythrocytes do not

contain 2.3-DPG. sheep may switch hemoglobin types. and the p50 is much higher in

sheep than in hurnans. Considering these facts. one should be cautious to draw

conclusions about the effects of sepsis on red blood cells and the position of the O2

dissociation cune in humans. Thus- future studies are necessary to investigate whether

sepsis induces an increase in 02-hemoglobin affinity in patients.

From this analysis it was concluded that the O? dissociation curve is lefi-sbitted in

normotensive, hyperdynamic septic sheep. most likely because of a direct effect of sepsis

on the erythrocytes. The increase in the Or hemoglobin affinity in sepsis is such that it

may depress mâuimum systemic Or extraction. [n the presence of an already altered

microcirculation. this rnay further restrict the 0: supply reserve in sepsis. On the other

band. the lefi shift of the O2 dissociation cuwe does not limit myocardial O? extraction

reserve. This is most likely due to the convergence of ODCs at low pOzs where changes

in the position of the ODC are less influential on Oz unloading.

Chapter 4 - Coronary circulatory reserve during modest anemia

4.1 Introduction

Sepsis disturbs the meiabolic 0: reserve of the circulation since the capacity to

au*gnent cardiac output. to approprîately distribute blood flows between organs and to

extract O? are al1 diminished in this syndrome (see chapter 1). The study described in

chapter 2 found that the sepsis-associated circulatory dysfunction may be generalized to

the lieart. In sheep rendered septic by cecal ligation and perforation. the capacity to

augment both coronary blood tlow and myocardial O2 extraction \.as depressed when the

heart's metabolic O2 reserve was stressed by progressive hypoxia. As the heart's O2

demands are increased by the hyper-rnetabolic milieu imposed by sepsis. this depressed

metabolic O? reserve in combination with changes in myocardial Oz delivery. which are

normally inconsequential. could iead to ischernia (1 78). In this circurnstance. rnyocardial

dysfùnction complicating the consequences of local ischemia would further limit cardiac

output reserve. thereby promoting injury to non-cardiac circulations. As the analysis in

chapter 3 demonstrated that the lefi-shified ODC was associated with a depression in

ma~imum O2 extraction in the systemic but not in the coronary circulation. it would be of

interest whether other parameters of O2 carrying capacity such as the hem~giobin level

would affect the coronary circulatory reserve.

[sovolemic hemodilution is normally accompanied by a generalized circulatory

compensation to maintain tissue oxygenation. To maintain whole body O? delivery.

cardiac output increases by elevatinp stroke volume ( 1 74). Several mechanisms support

this effect: i) increased venous retum due to reduced blood viscosity (106). ii) facilitation

of lefi ventricular ernptying by reduced blood viscosity. (45) and iii) increased

myocardial conuactility due to activation of the sympathetic nerve system (73). This

increase in heart work is supported by a redistribution of organ blood flows to the heart

( 176. 174). This redirected Oz delivery originates from non-vital organs. for example.

from the splanchnic circulation wliere oxygenation is then supported by increasing tissue

O? extraction (120). The increase in O2 extraction is initiated by the drop in blood

v i s c o s i ~ which reduces vascular hindrance and optimizes microcirculatory blood flow

(1 13). Capillaq recruitment does not play a role in increasing Oz extraction during

hemodihtion ( 177).

In sepsis. hemodilution limited appropriate increases in myocardial 0 2 delivery at

hemoglobin levels in sepsis wliich. in contrast. were well tolerated in non-septic animals

(126). These data may have been the consequence of the impact of sepsis on Oz

extraction reserve (134). since the ability to redistribute O? delivery away frorn the gut in

septic animals was also depressed during modest anemia. These results suggest that

hemodilution might be of disadvantage for the coronary circulation during sepsis. This

concept is also of clinical interest since clinical guidelines emphasize a low transfusion

trigger (7). A recent clinical study on intensive care patients - the TNCC - trial

(transfusion in critical care) (82) - even dernonstrated a higher rnortality in critical care

patients that were transfùsed to a higher hemoglobin level. However. this difference was

not demonstrable in more severely injured patients (APACHE II score > 20) which

would include patients with sepsis. as well.

The goal of this study was to describe the coronary circulatory reserve in sepsis at

different hemoglobin levels since inild anemia is a chnically relevant situation in the

clinical setting. I t was liypothesized that isovolemic hemodilution to create modest

anemia in mature sheep rendered septic by cecal ligation and perforation would depress

the heart's ability to support circulatory compensation in this syndrome. The second goal

of this study was to describe the effects of different hemoglobin levels on the systemic Oz

extraction reserve.

4.2 Methods

The animal rnodel used in this study is equivalent to the rnodel in the hypoxia trial

described in chapter 2. Therefore. the procedures will only be repeated briefly. Fifteen

mature. male Suffolk sheep weighing 39-76 kg (average 56.7 kg) undenvent

instrumentation fotlowing one week of acclimatization in the laboratory. On the first day

of study. the study animal was anesthetized with halothane and 100% O-, (5-6 Vmin) via

mask. after which the trachea was intubated and the sheep was ventilated with 100%

oxygen. As described in chapter 2. a saline-filled Sitastic catheter (0.135 inch OD. Dow

Corning. Midland, ME. USA) \vas inserted into the lef atrium and the c o r o n q sinus kvas

retrogradely cannuiated via the hemiazvgous vein. Saline-filled silastic catheters

(0.125 inch OD) were also placed into the lefi femoral and carotid arteries. while the Ieft

extemai jugular vein was cannulated with a No. 8 French introducer (Cordis, Miami. FL,

USA). Afier recovery. the sheep were placed in metabolic cages and were allowed Free

access to food and water. Ringer's lactate. 4 mVk@h. was administered post-operatively

to maintain adequate hydration. Analgesia was provided with meperidine. 100 mg

admixed with each 1000 ml OF Ringer's lactate. Catheter patency was maintained by

intermittent flushes with heparinized saline ( 1 .O00 U heparin/jOO ml saline).

4.2. I Experimeniui Protocol

The study design is s h o w in Fig. 15 and is a modification of the hypoxia trial

described in chapter 2 (see also Fig. 4). Three days after the initial surgery, a 7 French

Swan Ganz catheter (mode1 93- 13 1 : American Edwards Laboratories. Santa Ana, CA,

USA) was tlow-directed into the pulrnonary artery through the jugular vein introducer. A

non-septic study was then performed with the sheep conscious. Systemic arterial and

pulrnonary artery pressures and cardiac output were measured. Blood was simultaneously

obtained from the carotid artery. central vein and the coronary sinus to mesure blood

gases, hemoglobin and lactate. Afier this non-septic study. al1 animals undenvent a cecal

ligation and perforation as described in chapter 2. Then. a sterile tracheostomy was

performed. A 39 Fr. low-pressure cuffed tracheostorny tube (Sliiley. USA) was inserted

into the trachea and connected to a system providing hurnidified and warmed air.

Pte-septic study Cecal ligation and perforation Trac heostomv

Study Manoewer #2 ./ Baseline study

(4Sh rs) 4 hypoxia tria _

I O

I 24 tirs

I -18 hrs

4 C

Study Manoewer # l isovo kmic hernodilution (hb: 70-80 or blood transfusion (hb: (00-120 -1)

Fig. 15: Study design of the hemodilution/blood transfusion trial

Sheep were randomiy allocated to either a red blood ce11 (RBC) transfusion group

(group T) or a hemodilution group (group H). In the hemodilution group. blood was

drawn from the carotid artery and pentastarch was infused isovolemicalIy into the

extemal jugular vein to reach a Iiemoglobin-Ievel of 70 g/L. Sheep in the RBC

transfusion group received fresh packed RBCs taken from a donor sheep on the

transfusion day. titrated to a hemoglobin of 120 g/L. The transfusion and hemodilution

interventions were completed during the first 24 hours after peritoneal contamination to

allow circulatory compensation to be adequately expressed before the hypoxia

intervention. A pilot study demonstrated that the interval between randomization and the

hypoxia study was. however. short enough to maintain the group differentiation

according to different hemoglobin levels. Additionally to the hemodilution and blood

transfusion. respectively. pentastarch was infused in both to maintain left atnal pressures

at non-septic levels throughout the time between laparotomy and study 48 hours later.

Analgesia was continued as previo~isly detailed. and increased if the sheep showed

discornfort.

Forty-eight hours afier the CLP. sheep were connected to a semi-open system to lower

the inspired Oz concentration (FiO2) by mixing room air with nitrogen (Bird Corp. 0 2

blender. Palm Springs. CA. USA). This system was connected to a metabolic monitor

(DeltaTrac II. Datex - Instrumentation Corp.. Helsinki. Finland) to directly rneasure

systemic 0 2 consumption. We repeated al1 rneasurements described for the non-septic

study. A set of radioactive microspheres was then injected to allow the later calculation

of organ blood flows. while coronary sinus btood was obtained to calculate myocardial

O2 consumption.

Similar to the study described in chapter 2. coronary circulatory reserve was

chailenged by a hypoxia trial. L'sing a polarographic 0 2 monitor (mode1 5570.

Ventronics. USA). the Fi02 was subsequently reduced through up to f o u successive

stages. adjusted to achieve a similar decrease in arterial 0 2 content (Caoz) between

stages. Hernoglobin and artenal saturations were measured at and in between each

stage to calculate the Caoz. Twenty minutes of equilibration time was allowed at each

experimental level or until the DeltaTrac showed steady state conditions. Then

rneasurements were repeated. as described in the 48 hour baseline study, including the

infusion of another set of radioactive microspheres. After the final stage of hypoxia. the

study animal was euthanized with pentobarbital and organs were then harvested for

gamma counting to calculate organ blood flows.

Hemodynamic measurements including measurement of systemic and pulmonary

artery pressures as well as cardiac output were done by using the same technique and

equipment as has been described in chapter 2.2. Cardiac outputs were measured in

tripkate by the thennodilution technique using a cardiac output computer (mode1 9570A,

Hewlett Packard. USA). The cardiac output was indexed to the body surfhce area and

heart work was estimated as the product of cardiac index and mean aonic pressure (1).

Blood gas sarnples were stored on ice p io r to analysis with an ABL-3 blood gas analyzer

(Radiometer. Copenhagen. Denmark). During the experiment. the hemoglobin and the O?

saturations were measured by a CO-oximeter (OSîvI-II Hemoximeter, Radiometer,

Copenhaçen, Denmark). Subsequentl y. hemoglo bin levels were confirmed by a Coulter

Ce11 Counter (mode1 5, Burlington. Ont.. Canada). Lactate was rneasured by a Greiner G-

400 Chemistry Analyzer ( S wi tzerhd).

In the initial hypoxia trial described in chapter 2. both 0 2 delivery as well as Oz

consurnption was calculated from cardiac index as well as arterial and mixed venous

blood gases (see appendix for equations). Since variation in cardiac index will affect QOz

as well as Oz consumption. similar changes of these two parameten might be due to

mathematical coupling rather than to a true physiologie variation (8). In this study, O2

consumption was measured with a metabolic monitor (DeltaTrac II. Datex -

Instrumentation Corp., Helsinki. Finland) to obtain an independent mesure of this

parameter. The DeltaTrac calculates O2 consumption from the inspiratory as well as

expiratory O2 concentration and the tidaI volume.

The microsphere technique wvas used to quantitate organ blood flows through the

different stages of the expenmental protocol. The methodology has been outlined in

detail in chapter 2.2. We used latex spheres with a diameter of 15 Fm labeled with either

-%SC. j9Zn. SjSr, '5Nb. or IJCe obtained frorn New England Nuclear (Dupont Canada Inc..

Mississauga. Ont.. Canada). After mixing the spheres for five minutes with a Vortex

Mixer (Mode1 58223. Scientific Producrs. Evanston. IL. USA). an arnount equivalent to

approximately 25 to 30 pCi was injected into the lefi atrium. Using an

infusiodwithdrawal purnp (Harvard Apparatus Co.. USA) sampling of the reference

blood from the carotid artery and the fernoral artery (10 ml/min) was started during

injection of the spheres and was continued for 90 seconds after the injection.

ABer sacrifice. the heart \vas obtained: the lefi and right atrium were removed h m

the heart and discarded. The ventricles were counted together to represent coronary blood

flow. Random sarnples were taken from the liver. diaphragm. small and large gut. while

the brain, kidneys. pallbladder and pancreas were processed as wliole organs. The

gastrocnemius muscle was obtained to represent skeletal muscle blood flow. Al1 tissues

were cut in 1 cm long pieces and placed on a Petri dish. After drying for 72 hours in a

heater (Biological Safety Cabinet. Nuaire. Plymouth. MA. USA). tissue was placed into

plastic tubes. Tissue and the reference biood samples were then counted in a multichannel

Automatic Gamma Counter System. Series 1185 (Scarle Anaiytic Inc.. Des Plaines, IL,

USA). Radioactivity of? each isotope in each organ was determined by the stripping

technique (see chapter 2.23). The counts of the two blood reference sarnples were

averaged and organ blood flow was calculated by Eqn. 3. Al1 organ blood flows are

expressed as rnl/min/lOOg wet weight of tissue. The quality of the microsphere injection

was assessed by linear regression between the blood flow to left and right kidneys. r+vas

0.98 in the hemodilution group versus 0.97 in the transhsion group. The regression Iines

were not different between the groups and the slopes were not statistically different from

1.

A11 data are expressed as mean '. standard error of mem. The data were analyzed by

analysis of variance using a two-factor design as it is supported by SPSS 6.0. Therefore-

eKects are s h o w as group effect (hemodilution vs. transhsion). hypoxia effeci. and

interaction. A p-value of less than 0.05 was considered to be statistically significant.

Dependent physiological parameters were analyzed by regression analysis using the least-

square method. To compare regression lines between the two groups. a variable

containing the values O or 1 for the two groups (so-called durnmy variable) was added to

the regression model. Two regression lines are significantly different if the durnrny

variable is a significant part of the regression model ( 1 O 1 ).

4.3 Resuits

4.3.1 Effects of the Izemodilurion and red blood cell transfusion

Post-mortem examination confirmed purulent ascitic fluid with an exudative reaction

around a necrotic cecum. Forty-eight hours following CLP. the hemoglobin level was 77

+ 3 g/L in the hemodiluted group and 1 17 k 4 =/L in the transfused group. To establish

these endpoints. 745 + 65 ml of whole blood was withdrawn from the hemodiluted sheep

and replaced with pentastarch. and the transfused sheep received 3 14 lr 177 ml of packed

RBCs. Exclusive of the pentastarch infused to isovolemically replace blood withdrawn in

the hemodiluted group. H sheep received 39.8 k 3.9 mVkg24h and the T sheep received

35.0 I 2.1 mI/kg/24h (p: n-S.) during the 48 hours afier CLP. Table 1 1 surnmarizes the

effect of these interventions on circulatory and systemic O2 rnetabolism values in the two

study groups. just before beginning the hypoxia intervention.

The study's primary interventions. hemodilution or transfùsion. achieved the desired

endpoints regarding the hemoglobin concentration 48 hours following CLP (Table 11).

Cornpared to pre-CLP evaluation. hemodynamic consequences of peritoneal

contamination included an unchanged rnean merial blood pressure and an increase in al1

of the cardiac index. h e m rate and left atrial pressure ( L M ) in both study groups (Table

I l ) . Systernic 0 2 delivery increased betrveen the baseline and 48 hour study in the T

moup. but not in the H group. Simultaneously. systemic 0 2 extraction fell during the 18 C

hours following CLP in the T group. wliile calculated systemic Oz consumption and

arterial lactate ievels (H group: 0.3 + 0.05 mmol/l to 0.3 t 0.08 mmollL: T group: 0.5 t

0.14 mmol/L to 0.4 k 0.12 mmollL: p: n.s.) remained unchanged.

- -

Hemodilution Transfusion

General

Temperature ["Cl

Hematocrit

Pa02 [mmHg]

Hetnody namics

BPM [mmHgl

CVP [mrnHLd

LAP [mmHg]

C 1 [r/min/m2/

S V R [dyneis'h2]

Systemic O2 metnbolism

O' delivery [ml/rnin/nz2]

0: consumption

[mliminim2]

0: extraction -

Table 1 I : Effects of different liemoglobin Ievels on systemic hemodynamics before and afier CLP AI1 data are expressed as mean * standard error of mean. Significance is: *: pC0.05, **: p<0.01 between the non-septic baseline measurernent and 48 liours afier CLP: #: p<0.05, ##: pcO.0 1 between groups.

4.3.2 Effects of lrypoxirr and li tmoglobk ïevef on systemic Iirmorlynamics

Table 12 compares the baseline study to the finai hypoxic stage of selected

hemodynamic and O2 metabolism values. The final FiOz approximated O. 1 I in both study

groups by the end of the fourth study stage: thus. the artenal pOz fell significantly in both

groups. By the final hypoxic stage. the overd1 depression in arterial O1 contents was

significantly greater in the T compared to the H çroup (Fip. 16). Neither mean blood

pressure nor mean pulmonary artery pressures werr affected by the hypoxic intervention

in either study group. The cardiac index was greater at baseline in the H cornpared to the

T group. Coincident with an increase in the lieart rate. the cardiac index increased in both

goups between the baseline and final hypoxic study stages. and rernained higher

throughout al1 hypoxic stages in the H versus T group.

BaseIine Last Stage

Pa0:

rlnmHsl

Cardiac index

[/in idrn']

0, delivery

[TI l/m in/m;]

O? consurnption

[ml/inin/m2/

Oî extraction

lactate

hemo

Tram

hemo

Tram

Hemo

lrans

herno

hemo

hemo

hemo

Table 1 2: Effects of anemia and hyposia on systemic O? transport Effects of liypoxia on systernic O? metabolism variables comparing the 48 Iirs baseline study versus the last stage of Iiypoxia by the groups liernodilution (hemo) versus transfusion (tram). Analysis of variance: **: p<O.O I (liyposia effect); #: p<O.OS. ##: p<O.O 1 (group effect); there was no significant interaction.

Table 12 also records the depression in systemic Oz delivery that occurred between

baseline and the final hyposic study stages. During this study penod. the systemic O2

consumption remained unchanged tliroughout al1 four stages of Iiypoxia in the H group

(Fig. 17). Reducing the FiOl was accornpanied by an increase in systemic Oz extraction

in both study groups (H group p < 0.01: T group p < 0.01). A significant group effect

during hypoxia in systemic O? extraction (p < 0.0 1) was likely explained by the lower

baseline value in the T group as the ma.xima1 value \vas similar in both study groups. In

both study groups. arterial lactate rose modestly. but significantly by the final stage of

s tudy .

C hemodilution transfusion

pre 48hrs h4 h2 h3 h4 pre 48hrs h l h2 h3 h4

Fig. 16: Hemoglobin Ievels and arterial O? content before and after liemodilution/transfusion Before CLP (pre). hemoglobin levels were similar between the hvo groups and differed according to tlie hemodilution and transfusion. respectively. 48 Iioun after CLP. No p-values have been calculated since the alterations oftlie parameters in this figure were purpose of the snidy design. Stages II 1 to 114 corresponds to tlie four stages of Iiypoxia.

100 - - -O-... -. - -.

7 5 - - * - 0.4

2 hemodilution - vo, 50 - transfusion ' ' ' O$ .*-. .

Caoz [ml O//]

Fig. 17: Changes of systernic O2 extraction and VO-, over the change of arterial 0: content during the hypoxia trial. Results of the analysis of variance: OzE: Iiyposia effect: p<O.O 1 ; group effect: p<O.O 1. VOz: Iiypoxia effect: n-S.: group effect: n.s. Tliere was no significant interaction.

4.3-3 Effects of liypoxia ami lr ernogiia bin ievel on nzyocnrdial O2 meta bolism variables

Fig. 18 shows the effect of study interventions on both myocardial O? consumption

and heart work, the latter estimated as the mean blood pressure times cardiac index

product. This figure demonstrates that the progressive reduction in FiO2 was

accompanied by significant and similar increases in myocardial work and myocardial Oz

consumption in both study groups. Accordingly. coronary blood flow increased in both

groups with progressive hypoxia. but was significantly greater in the H group throughout

the entire hypoxia intervention (p < 0.01). The lefi ventricular

subendocardiaVsubepicardial flow ratios were not affected by the hypoxic intervention in

either study group. although they were significantly lower in the H g o u p than in the T

group during al1 study stages (Fig. 19).

-7 g rooo - S - s

Fig. 1 8: Changes of Iiean work and myocardial O? consumption over the change of arterial O? content.. Analysis of variance for Iieart work estimated by the product of mean blood pressure and cardiac index: Iiypoxia effect: pcO.0 1: group effecr: n s . Analysis of variance for myocardial VOz: Iiypoxia effect: pcO.05: group effect: n.s.

left ventricular endo:epi flow ratio 2 0 1

Hernodilution Transfusion

Fig. 1 9: Left-ventricular subendocîrdial/su bepicardial blood f!ow ratios. As tliere is no interaction or time effect. the bars i n tliis figure represent the flow-ratios of all stages for eacli group. The flow ratios differ with p=0.076.

Although myocardial Oz delivery increased as the Fi02 kvas reduced in hemodiluted

and transfused sheep (p < 0.01). there were no group differences during any of the

hypoxic study penods (Fig. 10). At baseline. myocardial Or extraction was similar in the

H (0.78 + 0.04) and T (0.79 f 0.02) study groups. but was greater in the T group

throughout the entire hypoxia intervention (p = 0.03). ANOVA did not find an overdl

statistical change over the time course. suggesting a different behavior of myocardial 0 2 E

over time. However. the interaction lacked statistical significance. Myocardial O?

extraction reserve was O.03iO.01 in the H-group and 0.05iO.01 (n.s.) in the T-group. In

both groups. myocardial O2 extraction reserve was different from O (H group: pcO.05: T

group: p 4 . 0 1). Fig. 2 1 demonstrates that coron- sinus p 0 2 was greater in the H

compared to the T group at any ievel of arterial pO? (p < 0.05).

- 9 hemodilution 1 I transfusion

Fig. 20: Changes of myocardial 0. delivéry and inyocardial 0 2 E over the change of arterial O? content Analysis of variance for myocardial Cl2 delivery: Iiyposia effect: p<O.O 1 : group effect: n.s. Aiialysis of variance for myocardial 02E: Iiyposia effect: 1i.s.: groiip effect: p<0.05.

Hernodilution

0 Transfusion - - - -

arterial pO2 [mmHg]

Fig. 2 I : Relationship between arteriaI and coronary sinus p02 . Regression-analysis was done on each sheep separacely and resulted in following average regression equations: hemodilution-group: pa02 = 0. I6(*O.O4) x pcs02 + l4.83(*2.O 1 ), ?= 0.5910.1 1 : transfusion-group: paO2 =0.22(M.02) x pcsOl + S. i 8(*1.80), r2= 0.8W0.05. The slope of the regression lines and r2 were not significantly different. The intercept differed with pcO.05 indicating that at a given arterial p02, hemodiluted slieep Iiad a greater coronary sinus p02 than transfused sheep. Data in the equations are expressed as mean*standard error of mean.

The maximum increase in coronary blood flow during the hypoxic intervention was

significantly greater in the H (766.3 k 87.4 ml/min/lOOg) compared to the T group (422.7

+ 53.7 mVmin/100g; p < 0.01). Fig. 22 compares changes in coronary blood flow and

myocardial O2 consumption as the Fi@ was reduced. The slopes of the two regression

lines differed (p < 0.01), tlius demonstraring that coronary blood flow increased more in

H than in T sheep when myocardial O? demands rose.

1200 Hemodilution

Transfusion

a.'

myocardial 0 2 consumption [ml/min/?OOg]

Fig. 22: Myocardial O2 consumption versus coronary blood flow. Regression-analysis was done on each slieep separately and resulted in following average regression equations: H-group: CBF = 3 l .Oj(*;.j8) x VrnyO? - 228.5(+.90.5). +=0.7910.12; T- Uroup: CBF = 17.12(*1.52) x VmyO? - I 13.1(*3O.J) . r2=088h0.08. The intercept of the 5

regression lines and r' ivere not significantly diflerent. The slope differed with p<O.O 1. Data in the equations are expressed as meanistandard error of mean.

4.3.4 Effects of izypoxiu and ltemoglobin level on regional Or delivery reïationFltips

Fig. 73 demonstrates that the hypoxic intervention was accompanied by an increase in

the regional O? delivery (QOr) to the heart in both goups. Q 0 2 to the brain was not

affected by the hypoxic trial. Both groups reduced Q02 to the gallbladder, kidneys and

spleen. There was a statistically significant reduction in regional Q 0 2 to liver. pancreas.

rumen. small and large gut in the transfusion group only. The hemodilution group oniy

showed a tendency to reduce QOr to these organs without being statisticaily significant.

hemodilution transfusion

Fig. 23 : Percent change of organ QO, during hypoxia at different hemoglobin levels *: p<0.05, **: piO.0 1 change different from G . #: p<O.Oj group difference.

4-4 Discussion

There is a considerable variation in both practice and opinion regarding the

appropriate RBC transfusion trigger in sepsis. Despite the recommendation of large

medical societies (26. 39. 164). intensive care physicians tend to apply a high transfusion

trigger (81). In a recent study-. a multicenter trial demonstrated that mortality in critical

care patients was reduced when a Iow transfusion trigger was applied compared to a more

liberal transfusion regime (81). However. this difference in mortality disappeared in a

subgroup of more severely il1 patients. Since patients with sepsis may be counted to this

a-oup. there is a lack of physiologic data about effects of modest anemia in sepsis to C

rxplain these findings. This experiment \vas designed to mesure and compare the effect

of two clinicaily relevant RBC transfusion strategies on the coronary circulation's

metabolic Oz reserve in septic sheep.

Sepsis is characterized by tissue injury which may lead to the multiple organ

dysfirnction syndrome (MODS). Sepsis is also a hyperrnetaboiic state, where tissue O2

demands are markedly increased. Where research has linked outcornes from sepsis with

an inability to match O? delives to these elevated tissue O? demands (190), treatment

strategies have emphasized optimizing increasing systemic Or delivery by (i) increasing

the cardiac output with intravascular volume expansion and/or inotropic therapy. and/or

(ii) increasing arterial O2 content with RBC transfusion and rneasures which improve

artenal oxygenation (i.e.. supplemental Oz and positive end-expiratory pressure). As it is

not uncornmon to find modest anemia in septic patients. the appropriateness of

transfùsing RBCs to increase tissue O? availability has to be considered (145. 152).

A finely regulated control system distributes O2 delivery to match the tissues'

metabolic O2 demand. Because the mocardium has a limited ability to increase Oz

extraction. isovolemic hemodilution in heaith is accompanied by an increase in

convective O2 delivery to the heart (126. 174). Such compensation is facilitated by a

redistribution of O2 delivery away from organs with a greater O2 extraction reserve, such

as the splanchnic circulation (100. 120). The effectiveness of this compensation is evident

in guidelines which propose that anemia in previously healtliy patients can be tolerated to

hemoglobin levels as low as 60-70 g/I (7).

In conuast to health. an argument rnay be made that the RBC transfusion trigger

should be higher in sepsis. For exarnple. sepsis is a hypermetabolic process. and

increasing the metabolic rate in healthy animal models elevates the gut's optimal

liematocrit (1 00). Second. sepsis is characterized by circulatory abnormalities that impact

on tissue Oz delivery. including myocardial depression (46). an impaired redistribution

capacity of Oz delivery from the splanchnic organs to heart and brain (24) and

microcirculatory dysfunction whicli limits Or extraction capacity (1 07). When reviewing

the Literature about the effects of modest anemia on organ blood flow. the foHowing

results were demonstrated. (i) Hernoglobin concentration exerted independent and

negative effects on regional 0: delivery in septic sheep (68): (ii) sepsis in sheep

depressed the capacity to increase both coronary blood flow and myocardial Or extraction

during acute hypoxia when compared to control sheep (see chapter 2); and (iii) the

ability to appropriately increase myocardial Oz delivery during acute hypoxia in septic

rats occurred only in animals transfused to maintain hematocrit levels greater than 45%

( 126). In contrast. and confirming previous work (36. 96). nonseptic rats maintained

myocardial Oz delivery reserve to hypoxia with hematocrits less than 30%: and (iv) the

hemoglobin dissociation cuve was left-shified in septic sheep. cornpared to nonseptic

study conditions (see chapter 3). Taken together. these data are indirect evidence that the

coronary and the systemic circulation rnay have a benefit from elevated hemoglobin

levels. This wouid be clinicaIly acknowiedged as a need to transfuse RBCs earlier in

sepsis than in health. However. the benefits of blood transfüsion in septic patients have to

be investigated by a clinical study addressing the effects of different hemoglobin levels

on morbidity and rnortality in patients with sepsis.

4.4.1 Hemoglobin Ievels ami the systemic circulation 's mrtabofic O7 reserve

Before the hypoxic intervention. both study groups demonstrated a circulatory profile

which is typical of sepsis. Demonstrating that anemia imposed an added stress on the

central circulation, H sheep had a higher cardiac index than T sheep, and this difference

was maintained across al1 subsequent study stages. As O? delivery was sequentially

reduced during acute hypoxia. an expected increase in systemic Oz extraction was similar

in both study groups. In the first hypoxia trial presented in chapter 2. it was confirmed

that sepsis in this animal mode1 depressed the capacity to extract Oz. Data from the

current expenment therefore demonstrate that the animal's hemoglobin status does not

influence the progression of this lesion. As rneasurement of systemic Oz extraction

reflects the sum of changes occumng within individual organ circulations. it is possible

that changes according to hemoglobin status might have occurred in individual organ

circulations. While hemodilution is known not to reduce regional O2 delivery by increase

of organ blood flows (174). severe hypoxia reduced regional 0 2 transport to al1 non-

cardiac organs observed during this study regardless of the hemoglobin Levet. In the

previous hypoxia-study it was found that sham animals redistributed Q02 h m the gut to

the heart while septic sheep did not (see chapter 2). It was suggested that septic sheep

were unable to increase 0 2 E suficiently to give up blood flow. In this snidy, a significant

reduction in regional Q02 to the gut was found in the transfusion group only. This might

reflect a normalization of the gut's 0 2 E reserve in the transfusion group. However. this

remains speculation since the regional OzE of the gut was not measured .

4.4.2 Hemogiobin lr vels and t[z e coronary circrdutiort % metabolic 0- reserve

Reducinp Oz content was accornpanied by an increase in the cardiac index times blood

pressure product. a surrogate endpoint for heart work (1). A parallel increase in

rnyocardial 0 2 consumption was the consequence of an increase in flow work and

confirms that the metabolic coupling of O? availability to changing Or demands remains

intact in hyperdynamic sepsis. However. the rnechanism by which myocardial 0 2

consurnption was supponed differed according to the study subject's hemoglobin status.

Thus. (ij c o r o n q blood flow was greater at al1 levels of rnyocardial Oz consumption in

H compared to T animals. and (ii) the rna~irnurn rnyocardial 01 extraction achieved was

greater in the T compared to H animals.

In health. hemodilution is accompanied by an increase in coronary blood flow which

may be greater than necessary to satisfy myocardial 01 demands. Such relative

overperfùsion has been attributed to the drop of btood viscosity with hemodilution ( 1 79,

and is accornpanied by a maldistribution of coronary blood flows from subendocardial to

subepicardial layers after severe hemodilution (43). In this study. the subepicardial

redistribution of coronary blood flows occurred already afier rnild hemodilution.

Therefore. the greater dependence on using coronary blood flow reserve to support

myocardial oxygenation in hemodiluted animals is a pattern consistent with the effects of

hemodilution alone, albeit occurring at an earlier stage.

This enhanced dependence on coronary flow reserve in hemodiluted animals could

also be explained by an effect of anemia to impair the heart's Or extraction reserve.

Hemodilution is normally accompanied by a modest increase in myocardial O2 extraction

(43). whiIe the study in chapter 2 found that sepsis b h t s this compensation. The current

study is consistent with this previous experiment. narnely. an acute reduction in

myocardid O7 availability was not accompanied by a significant increase in this organ's

O2 extraction. However. it was found that the capacity to augment myocardial Or

extraction was greater in sheep transfused to normal hemoçlobin levels. when normalized

to arterial O2 contents (Fig. 20).

Tt is conceivable that the increase in blood viscosity that would have accompanied

transfusion to normal hemoglobin Leveis in the septic sheep explain the ability of this

a o u p to extract O2 in the heart's circulation at greater levels than in the anemic group. Ci

Sepsis is characterized by elevated blood flows (34. 143). impaired arteriolar reactivity

( 1 18) and a loss of MC-perfused capillaries ( 107). The usual microvascular response to

normal isovolemic anemia includes changes in both microcirculatory hematoctit and

RBC flow distribution, because of a dedine in both vascular hindrance and blood

viscosity (1 50). Routing an elevated blood flow through circulatory networks with fewer

perfused capillaries would boost RBC flow rates in remaining, perfused capillaries and,

by shortening transit time through the capillaries. potentially impede Oz extraction. An

increased blood viscosity in the transfùsed group could therefore have led to greater RBC

transit times compared to the H group. and thereby provided more time for capillary O2

exchange to occur. We did not measure rnyocardial transit times or directly assess the

heart's microcirculation. but the higher coronary sinus pOz at any level of arterial p02 in

the hemodilution group might confimi such microcirculatory differences between the two

snidy groups. Crystal also proposed that excessively elevated RBC flow rates could

explain an inability of the right ventrïcle to maximally extract Or during hemodilution

(44). It is also possible that greater nitric oxide release in the peripheral microcirculation

in the T versus H group could have provided fùrther cytoprotective fiinction in this model

(41). Some experirnents support the possibility that increasing RBC levels minimizes

progression of the septic microcirculatory injury. in a study demonstrating that significant

increases in the number of stopped flow capillaries was time dependent in septic rats

(107). post-hoc analysis demonstrateci a negative relationship between the number of

stopped-flow capillaries and the systemic hemoglobin concentration (148).

To obviate potential confounding effects of anaesthetic agents on both regional and

microregional circulations. this study was carried out with the experimental animal awake

(JO). Afier baseline studies. the animals were then exposed to acute hypoxia to determine

whether the circulation's usual metabolic Or reserve was altered by hemoglobin status

(22. 178). As the hypoxic intemention was accompanied by a modest increase in the

arterial lactate concentration. it is probably reasonable to conclude that acute

compensation to maintain Oz availability in this animai model was exceeded during the

final hypoxic study stage ( 128).

Al1 animals demonstrated arterial lactate leveis below 0.5 mmol/l 48 hrs afier CLP.

These values are lower than usually seen in tliis mode1 (24); CLP sheep from the hypoxia

trial presented in chapter 2 had lactate leveis above 1 mmol/l. However, a necrotic cecurn

with panperitonitis was observed in al1 CLP sheep during the post-mortem examination.

Furthemore. the maximum Oz extraction during severe hypoxia was similar in al1 CLP

sheep and significantly lower than in the sham sheep presented in chapter 2. This can be

seen as evidence that the impact due to sepsis did not change between the two studies of

this thesis.

Animals allocated to the T group had a mean study hemoglobin which approximated

normal hemoglobin levels in sheep. while the mean study hemoglobin in the H animals

was simihr to values proposed as target values above wliich no transfüsion is necessary

in clinical guidelines (7. 39. 8 1). As sepsis lengthens the time required to ensure steady

state conditions in the central and regional circulations (67), interventions to distinguish

anemic versus nonanemic sheep were completed 24 hours before the hypoxic

intervention. Further. the multitude of different relevant blood groups in sheep (18)

makes it almost impossible to find a compatible donor sheep. Therefore it is

recommended to give RBCs only once to avoid hemolytic side effects (18). Et was

detemined in a pilot study that different hemoglobin levels can be maintained for 24 hrs.

As reduced RBC deformability complicating storage may Iimit tissue 0- availability in

sepsis (1 16). RBCs stored in CPDA-1 was taken from a donor sheep on the sarne day as

RBC transfiision.

In sumrnary. this is the first study to examine the effects of two clinically relevant

hernoglobin levels on systemic and regional Oz delivery in an animal mode1 of

normotensive hyperdynamic sepsis. Trmsfusing to a normal hemoglobin level does not

change the systemic O? extraction reserve that is depressed by sepsis. The metabolic

coupling between rnyocardial O? demand and coronary blood flow remained intact in

both groups. However. mild hemodi~ution inflicted changes on the regional and

microregional blood flow of the heart which are considered udavorable and are usually

only seen afier severe hemodilution. In hemodiluted septic animals. these changes

include a maldistribution of coronary blood flow to the subepicardial layer and a lower

myocardial O2 extraction.

Chapter 5 - Discussion

The studies in this thesis were designed to investifate the coronary circulatory reserve

in a sheep mode1 of normotensive sepsis. This topic is of interest since circulatory

dysfunction is a characteristic feature of sepsis (143). On the other hand. it is discussed

whether the coronary circulation sliares in this dyshnction (5. 47. 52). Coronary

circulatory reserve to maintain myocardial 0 2 demand consists of a coronary flow reserve

and a myocardial O2 extraction reserve (see chapter 1.3). It was found that coronary flow

reserve was intact but significantly reduced in septic sheep. In other organs. loss of

arteriolar reactivity is mediated by the massive release of nitric oxide in sepsis (94). This

loss of vascular reactivity induces the inability of appropriate vasoconstriction (3 1) and

causes the drop in systemic vascular resistance typically seen in septic patients (143).

Vasodilation in the coronary circulation also is depends on nitnc oxide (80). and it is

known that the myocardium releases NO during sepsis (69). As regulation of c o r o n q

blood flow relies on vasodilation and vasoconstriction. it seems likely that NO release

during sepsis mediates die loss of coronary flow reserve (Fig. 24). Although there was

still some correlation between increase in heart work and increase in coronary blood

flow. the data presented in chapter 2 indicate a more passive circulation in septic sheep

resulting in an exhaustion of coronary circulatory reserve nt a greater arterial 0- 7 content

in septic than in sham sheep.

When septic sheep are isovolemically hernodiluted. the animals achieved greater

coronary blood flows than sheep with a normal hemoglobin level (see chapter4).

However. distribution of intramyocardial blood flow was markcdly disturbed in

liemodiluted sheep. This behavior in coronary blood tlow was only seen in animals that

have been hemodiluted to a mucli lower hemoglobin than in the study demonstrated in

chapter 4(43). Therefore. the very high coronary blood flow during mild hemodilution in

septic animals is probably not due to the restoration of coronary flow reserve but the

result of the sum of two effects: coronary vasodilation due to sepsis and an increased

blood flow due to a drop in blood viscosity. This conclusion is supported by the finding

that hemodiluted sheep generated a greater coronary blood flow for a given myocardial

O2 consumption (Fig. 22). On the other hand. transfusing sheep to a higher hemoglobin

level did not improve coronary flow reserve but normal subendocardiaVsubepicardial

flow ratios were maintained. Thus. rnild hemodilution amplifies the impact of sepsis on

the coronary circulation which might be prevented by maintaining a normal hemoglobin

level.

- - - - - - nitric oxide

c o r o n q vasodilation

microcirculatory dysfunction

I C

0 2 extraction deficite

Fig. 24: Proposed pathophysiolozy describing the loss of coronary circulatory reserve in sepsis

The second mechanism of coronary circulatory reserve is the ability to fùrther increase

myocardial Oz extraction. Cliapter 7 demonstrated that myocardial Oz extraction reserve

was abolished in septic sheep. The O2 extraction deticit is also a typical feature of sepsis

and is annbuted to microcirculatory dysfunction ( 1 07). Chapter 3 demonstrated that a

Ieft-shifi in the ODC might be partially responsible for an O2 extraction deficit in sepsis.

While there \vas a significant correlation between the p50 and the maximum Oz

extraction. this relationship was not found in the coronary circulation. It was aiready

pointed out that this difference may be due to the convergence of differently positioned

ODCs at very low pOz values as they are found in the coronary sinus. Thus. the failure to

increase myocardial Oz extraction dunng Iiyposia cannot be attributed to a lefi-shifted

ODC. In agreement with the morphologic data available (83) it is proposed that the loss

of myocardial 0 2 extraction reserve in sepsis is due to a microcirculatory failure (Fig.

24). Messmer hypothesized that hemodilution might be beneficial in States of an altered

microcirculation since rheologic properties improve at a lower hematocrit (121). In the

hemodilution experiment demonstrated in chapter 4. there was no difference in systemic

maximum extraction in hemodiluted sheep compared to transfùsed slieep. Similarly to

the CLP sheep in chapter 2. hemodiluted septic sheep did not show a reserve in

myocardial OzE. As discussed in chapter 4. this finding might be explained by the very

hi& coronary blood flows dunng hypoxia preventing a complete O2 unloading in the

myocardial microcirculation. In transfused sheep. where coronary blood flows are lower

than in hemodiluted sheep. some increase in myocardial 02E was observed. However,

this increase in rnyocardial O? extraction reserve did not result in a restoration of O2

extraction reserve as demonstrated in sham sheep (see chapter 2).

Adequacy of myocardial 0 2 delivery is essential for the maintenance of cardiac

function. Since the septic coronaq circulation is characterized as being hyporesponsive

to an increase in myocardial O-, demand. it seems likely that rnyocardial ischemia occurs

when the septic hem is stressed. Two clinical studies independently found that the

coronary circulation behaves like the systemic circulation in sepsis demonstrating a high

blood flow and a low Oz extraction (47. 52). Dhainaut concluded that the low myocardial

O2 extraction might support the hypothesis of cardiac ischemia although he did not find

myocardia1 lactate production (52). This contradicts the conclusions from other authors

which excluded myocardial ischemia as cause of cardiac failure in sepsis due to the

elevated coronary blood florv (5. 47). However. as it was already-discussed above, this

elevated bhod flow may be due to inadequate NO mediated coronary vasodilation rather

than to an increased metabolic need alone. Although this might ensure adequacy of

coronary blood flow at rest. this plienomenon might be at the expense of flow reserve.

5.1 Tissue hypoxia in sepsis

One reason for the ongoing discussion about the association of myocardial ischemia

and cardiac dysfunction in sepsis is the scientific proof of circulatory dysfunction on one

hand, and the failure to End direct evidence of tissue ischemia on the other hand. It is

interesting that some authors questioned the hypothesis that tissue hypoxia in sepsis is

responsible for the development of the multiple organ dysfunction syndrome (MODS)

frequently cornplicating sepsis (166). It is argued that skeletal muscle oxygen tensions

were not necessarily low in sepsis (28). Furthemore. gastric mucosal acidosis was not

associated with rnucosal ischemia in a porcine mode1 of sepsis. Finally. elevated lactate

levels in sepsis mighr not indicate tissue hypoxia in sepsis since lactate production rnay

occur despite an adequate organ O2 delivery due to an altered activity of the pyruvate

dehydrogenase in this disease (95). Suter et al. argued that MODS might be caused by

reperfusion injury. mediator release. and leukocyte-endothelium interactions rather than

by tissue hypoxia (1 66).

However. the lack of data confirrning tissue hypoxia in sepsis might be due to a lack

of methodologies detecting the ischemic injury in scpsis. Morphologic data from septic

sheep support the hypothesis of lesions on a microscopie level where necrotic cells are

observed next to uninjured cells (83). Currently available techniques rnay be unable to

detect such a hypoxic injury. Even on the regional level. minor disturbances in organ

blood flow distribution are not detecred by systernic parameters like Oz delivery and Or

consumption. respectively ( 1 36). it seeins reasonable that tissue hypoxia in sepsis would

only be recognized by tissue oximetry. As reviewed by Vallet and Lund. one of the

necessary characteristics of such a technique has to be a jood spatial resolution (173).

None of the techniques currently available (polarographic electrodes. gastric tonometry,

nuclear magnetic resonance etc.) lias the spatial resolution to detect cellular hypoxia.

Positron emission tornograpliy may be a promising technique to Fulfill this task (92).

However. the positron-emitting hypoxic market ["FI tluorornisonidazole. which binds to

Iiypoxic cells in inverse proportion to cellular oxygen tension. does not show affinity to

necrotic cells.

As reviewed by Suter et al. (166). the development of MODS is most likely due to

multiple factors like reperfusion injury. rnediator release. and Ieukocyte activation.

However. there is also enough evidence that tissue hypoxia complicates sepsis. As

already demonstrated in chapter 1. reguiation of tissue 0: delivery is depressed on the

systemic. regional. and microregional level (1 07. 1 18). The prognosis of septic patients is

also determined by the adequacy of 0: delivery. Several studies demonstrated a higher

survival rate in septic patients when systemic 0, delivery was maintained at greater than

5.2 Limitations to the animal model

Cecal ligation and perforation causes an injury which may be comparable to patients

with sepsis. However. some restrictions should be taken in considerations when

transferring resdts and conclusions from this experirnents to the clinical situation. The

blood flow response to a challenge to the circulation (Le. hypoxia) may differ depending

on the focus of infection. The increase of coronary blood flow during severe hypoxia was

supported by a redistribution of blood flow away from the gut in non-septic sheep but not

in CLP sheep (chapter 2). This observation might be due to the direct impact of cecal

ligation and perforation to the zut rather than an effect of sepsis. Further research is

necessary to cl&@ whether such an effect on the distribution of cardiac output is also be

seen in other rnodels of sepsis with a different focus of infection such as pulmonary

infection. Blood flow to the skeletal muscle increased during hypoxia while the opposite

reaction would be expected to fürther support blood flow to vital organs. Sheep were

awake and standing in the metabolic cage during the whole hypoxia trial. hcreases in

skeletal muscle blood flow may be therefore an artifact of the animal's movements and

are rnost likely not be observed in a septic patient.

The CLP mode1 used in the experiments of this thesis simulates early sepsis as CLP

sheep since the animals do not show a sign of organ dysfünction. Furthemore. fluid

resuscitation without vasopressor support is sufficient to maintain a normal blood

pressure. Patients with sepsis are fiequently sedated. mechanically ventilated. and receive

some kind of catecholamine treatment each of which would affect the circulatory

response to a stress such as hypoxia. However. the goal of this thesis was to demonstrate

that there is already a considerable alteration of c o r o n q blood flow regulation during

earl y stages of sepsis.

It might be argued that severe hypoxia is not a relevant model to descnbe alterations

in blood flow regulation since severe hypoxia is not a frequent complication of sepsis or

septic shock. However, hypoxia has not been chosen as a stress to simuIate a clinical

condition but as a tool that allows a caretiil titration of challenge to the coronary

circulation.

5.3 Future Work

One drawback of this thesis is the lack of parameters of cardiac ischemia. Although it

was mentioned in section 5.1 that techniques detecting ischemia in sepsis might be

difficult to apply. some difference should be found during severe hypoxia if coronary

circulatory reserve fails to maintain myocardial Oz demand. Additionally to the

difficulties detecting the septic injury. measuring the degree of ischemia in the

rnyocardium is even more difficult. Lactate is a commonly used parameter to assess

adequacy of systemic O? delivery and Huckabee introduced measurements of

transmyocardial lactate metaboIism as a measure of cardiac ischemia (94). Several other

parameters such as excess lactate and anaero bic metabolic rate. which took pyruvate and

myocardial O1 consumption into account. were introduced to increase the accuracy to

detect myocardial ischemia (94. 138). However. doubt about the quality of those

parameters were risen when none of these parameters could predict ischemia in patients

with coronary heart disease although they cornplaint about chest pain (133). The main

problem is that the myocardium extracts lactate from the blood as a nutrient but at the

same time has a baseline lactate release even if no ischemia is present (71). Furthemore,

the heart changes its lactate metabolism dependent on arterial lactate levels as it occurs

dunng severe hypoxia (72). This becomes more important in sepsis as the septic heart

prefers lactate rather than glucose as a nutrient (52). Especially when applying

transrnyocardial lactate metabolism in the studies presented in this thesis. the complexity

of myocardial lactate metabolism makes it difficult to assess ischemia by just observing

the net lactate metabolism from arterial and coronary sinus lactate measurements.

By infusing "c-labeled lactate. the amount of myocardial lactate production and

consumption c m be quantified (71. 77). When myocardial net lactate production occurs.

coronary sinus blood is diluted by iinlabeled lactate produced by cardiac cells. We were

trying to apply this technique to the sheep mode1 to identify the onset of cardiac ischemia

during hypoxia. "c-labeled lactate was administered as continuous infusion during the

whole hypoxia trial. At eacli stage. arterial and coronary sinus blood was taken,

centrifuged and the serum deproteinized with acetonitrile. Since myocardium and liver

will produce L'~-glucose from the infüsed '"c-lactate. lactate has to be separated tiom

glucose. The purification was done by solid phase extraction (Sep Pak Vacuum Manifold,

Millpore, Milford. Massachusetts). The negatively charged lactate is trapped in the

cartridges filled wiîh resin. which expresses NH2'-groups. while the neutral glucose 14 molecule passes. Then, the lactate was eluted with 1M phosphate (pH8), and the C-

lactate was measured in a scintillation counter.

However. the methodology did iiot produce reliable results. Even during severe

hypoxia and the onset of cardiac failure. no lactate production was determined. A

possible problem was the administration of '"c-labeled lactate for several hours while it

was infused at one time point only in the original paper. The myocardial tissue may 14 become saturated with "c-glucose produced in the liver and starts to produce C-lactate

when ischemia occurs. Nevertheless. application of this technique in the CLP model

would be a very valuable source of information about the adequacy of myocardial QOz.

Iiifusing ' ' ~ - ~ l u c o s e rnight be an option to receive information how much 'k-lactate was

metabolized during the experiment. More work is necessary to establish this technique in

the septic sheep model.

A main conclusion in this thesis is that myocardial O2 extraction reserve was

depressed in septic sheep. This phenornenon was attributed to microcirculatory failure.

Although this is in agreement with the literature (52). there is currentiy no direct evidence

for microcirculatory dysfunction in the septic myocardium. There are two possible

techniques to study the microcirculation: The intravital microscopy and intravital staining

techniques. Intravital microscopy is difficult to apply in the beating heart due to its

movement. Nevertheless. a tloating objective was designed which autornatically keeps

moving tissue in focus (1 1). Naturally. this technique only allows observation o f the

subepicardial layer only. The microcirculation of the heart kvas mostiy examined by

staining techniques where a dye such as fluorescein is injected (75. 176). Afier the animal

is sacrificed. the coronary microcirculation may be studied by fluorescent microscopy.

The accuracy of this methodology relies very much on the speed of preparation since

fluorescein quickly starts to extravasate which causes the tluorescein to stain tissue

instead of capillaries. A dye staining endothelial cells would allow for more preparation

tirne. We tried to apply Ulex europaeus 1 lectin in a rat model. Ulex europaeus I lectin

binds to surface proteins of endothelial cells (91) and had already been used in isoiated

perfiised organs (16). ïhere are different fluorescent markers available for lectins. This

would allow us to intravitally stain the microcirculation at two different experimentai

stages. i.e. normoxia and hypoxia. However. we did not achieve sufficient staining when

the lectin was injected into a living animal. Potential reasons may include the presence of

binding sites for Ulex europaeus I lectin in the blood and the problem that the lectin may

not be given enough time 10 interact with the endothelium in an environrnent with

flowing blood. Other dyes. i.e. monoclonal antibodies against proteins on the endothelial

ce11 membrane. should be invesrigated for their potentiai use as intravital dyes.

A novel finding was the possible interaction between the position of the ODC and 0 2

extraction in sepsis. Although this phenornenon does not seem to play a role in the

coronary circulation. there was a significant contribution of the Ot hemoglobin affinity to

maximum OrE in other organs. However. the study presented in chapter 3 was of

retrospective nature and erythrocyte physiology of sheep is markedly different to human

red blood cells. More work about the O?-hemoglobin affinity in septic patients is

therefore needed. This is especially important as the red blood ce11 seems to be a target

ce11 during sepsis (48. 149) and alteration of the Or-hernoglobin affinity may be one

symptom.

5.3 Conclusion

The purpose of this thesis was to describe the coronary circulatory reserve consisting

of a blood flow and an 0 2 extraction reserve in a normotensive hyperdynamic sepsis

mode1 in sheep. The studies in this thesis confirmed previous work that sepsis induces the

coronary blood flow to increase and the myocardial O2 extraction to drop. Metabolic

control of coronary blood flow rernained intact durinç sepsis. However. it was found that

sepsis siçnificantly reduces coronary blood flow reserve and abolishes rnyocardial Oz

extraction reserve resulting in lower tolermcc to hypoxia in septic sheep compared to

sham sheep. Loss of coronary blood flow reserve may be attnbuted to the massive release

of nitric oxide characteristic of sepsis while loss of myocardial O? extraction reserve is

most Iikely due to microcirculatory failure. Therefore. the depression of coronary

circulatory reserve in sepsis is evidence that the coronary circulation shares in the

circulatory defect seen in other organs during sepsis. Since coronary circulatory reserve is

a necessary mechanism to maintain myocardial 0: demimd, ischemia may occur when

the septic heart is stressed.

Further. it is concluded that sepsis induces a lefi-shift of the O2 dissociation curve and.

thereby, restricting O? unloading at the capi l lq levd by an increased Oz-hemoglobin

affinity. However. this phenornenon does not play a role in the coronary circulation due

to the special quality of the coronary microcirculation where a very high 01 extraction

exists already at rest. A further increase in myocardial O2 extraction is not limited by a

lefi-shified O? dissociation curve since different ODCs converge at low pOz values.

Coronary blood flow reserve in sepsis was not restored by blood transfusion but

maintaining a normal hemoglobin level in septic sheep allowed for some increase in

myocardial O2 extraction. It is concluded that mild hemodilution is of disadvantage for

the coronary circulation due to the generation of very high coronary blood tlows. The

increase in coronary blood flow during hemodilution is such that a maldistribution of

coronary blood flow occurs that is norrnally seen afier severe hemodilution only.

Appendix

Appendix 1 Ab breviations

ARDS

BE

Cao-,

CBF

ccsoz

CI

CLP

cvoz CVR

EDRF

IL

N O S

L A P

iMDS

MODS

N

NO

0 2 E

ODC

pso

PAF

PEEP

QO,

RBC

Sa02

Adult Respiratory Distress S_vndrome

Base excess

Arterial O- 7 content

Coronary blood flow

Coronary sinus O- 7 content

Cardiac index

Cecal ligation and perforation

Mixed venous content

Coronary vascular resistance

Endothelium derived relayant tàctor

InterIeukin

Inducible nitric oxide synthase

Lefi atrial pressure

Myocardium depressant substances

Multiple Organ Dysfunction Syndrome

Hill coefficient

Nitric oxide

O-, extraction

O7 dissociation curve

PO2 at an 0 2 saturation of 50%

Platelet activating factor

Positive endexpiratory pressure

O2 delivery

Red blood cells

Arterial O2 saturation

SIRS

svo2 SVR

TNF

VOt

S ystemic Inilammatory Response Syndrome

Mixed venous O2 saturation

S ystemic vascular resistance

Tumor necrosis factor

O2 consumption

Appendix TI Calculated parameters

Cao?

cvoz CcsO.,

CVR

MyocardiaI lactate

consurnption

Myocardial 0 2 E

iMyocardial Q02

Myocardial VOz

02E

QOz

SVR

1.36 x SaO2 x hernoglobin +- (arterial p 0 2 x 0.003)

1.36 x SvOl x hemoglobin + (mixed venous pOz x 0.003)

1-36 x Scs02 x hemoglobin i- (coronary sinus p 0 2 x 0.003)

mean urterkl hlaoti presszwe - central venous pressure CBF

CBF x (arterial lactate - coronary sinus lactate)

C d z -CcsO,

cno, CBF x Ca@

CBF x (Cao2-CcsOÎ)

Cclo? -CvO,

79.9 x (mean crrrerid presszire - central venozrs pressure)

CI

All parameters are calculated by using standard equations published in Snyder et al.:

Oxygen transport in the critically ill. 1987. (1 63)

Appendix III Ethics Approvaf

The study protocols of both animal studies descnbed in chapter 2 and chapter 4

were approved by the University Council on Animal Care in accordance with the

guidelines set down by the Canadian Council on Animal Care. During the experiment. al1

animal care was provided by a physician and qualified animal health technicians.

nie approvals of the ethics cornmittee for the following studies are attached to this

appendtx:

mEthics approval for the study descnbed in chapter 2 and chapter 3.

~Ethics approval for the study descnbed in chapter 4.

Oear Dr. Sibbald:

Your "Apptication To Usa Animafs for Research ar Teaching" entltted:

"Causes and Consequeoces of Abnarmalities of Myocardial O2 Delivery in Sepsis in a Larçe Animal Madel"

has been apprdved by the University CounciI an Animal Care. This approval expires in one year on the lasf day of the manth. The number for this praject iS 3 930t6-1

1. This nurnber musc ba indicated when ordering animais for this pruject. 2, Anirnals for other projects may not be ordered under this number. 3 - if no nurnber auDears un thia amoroval please contact this office wherl qqn t amroval is

received, If the a~~l lcat ion for fundinu is not successful and it vau wish to pra=eed with the oraiect, request mat an iniernal sclentific Deer review he ~erforrned bv vour animal a r e cornmittee.

4. Purcriases of animais other than tnrough this systern mus: be clcarcd through the ACVS office. HeaftR certificates wiii be requit&-

ANMALS APPROVED

Sheq - 40

ETHICAL ACCEPTANCE OF ANIMAL RESEARCH CONFORM~TÉ A L'É~H~OUE EN MATIÈRE DE REPORT OF THE ANtMAL CAR€ COIVIMITTEE RECHERCHE SUR CES ANIMAUX

RAPPORT DU c 0 M n - É DE PROTECTION DES ANIMA

Rsquired for all ApplIc8llmc Froposing Rssearch lnuohlng Anima& Obligatoirr pour 10ums las 8amsndes conesmant dms rocharches dw anlmaux

FunOs from the Mediml Raseerch Councll of Ciinoda may not be uscd fer Les fanas que !e Conseil de recherches m+iicailss Cu Canada a accoi reaearch invdving animais wles UIW rooearcn propose& has b a n founa ne ?ourront servir i d e s recnecches sur des rnimdux h moins qu mcceoteble by m Animat Care Corninittee oppdntmd and opemirhg In Camitè de pmtrctlon dss onimmux &aW et air196 confomr6rnenl ecead wlth the Gutde to th. Cam and U s u of Expmrlmonwl himals or Manuel sur la -in et I'uliiisoUoii des animaux d'rxpérimenmtlon 1 tbe Crnidian Cauncil on Anlmal Csre (CCAC) (Vol, 1 (1980). Vol. 2 ttDûd)l. 1 (1986). ml. 1 (1 9B4)1 bu Cmtrrlt eanidien do pmtoctian das anim

(CCPA) n'ait convenu qua le rcrohercho pmpoaie répand aux non eîablies par le CCPA-

(The cornpierad Ionn mucd be iacàvad by Councll not Iatar t h m aixty (Le farmulntro dament rempli M t Blre reçu par k Conseil au plus i (601 dsys eftrr iha dacidlime date for the recuipt of ths spptleatlan, If thm soixante (BO) jour6 rprds Le date [imite Qe réception de la demanda form is not provldod within th18 perlod, the rppiiutlon wilI not be le f-ulAre n'est pl6faumi au but de ce delai. la csndldalurm nm e con&iderecî-) pas iludiee,)

STATEMENT FROM THE lNSTITUTIQNf IN WHlCH THE DECLARATION DE L'INSnTUflONr OÙ SE D ~ R O O C E I RESEARCH WlLL BE PERFORMEO LA RECHERCHE

The Anlmsl Cure Cornmittes established by Le Comlté de protscilan des anlmaux établi par

T h e Jn ivers i ty of Western O n t a z i g , London, O ~ t a r i o

has exarnined the protocot for the research funds entitîed: a étudie le protomle de la recherche intituihe: Does Seps i s M o d i f y the Circulatory Response to V a r i a b l e B e m a t o c r i t s ?

(Use tlie same Me as on Uie mpplicallbn subrnitted to thta Council) (Utlllscr ie meme trlre que celui indiqub Sur IR demande présernëe au Conseil}

William J. Sibbeld MD F R c B O ~ ~ ~ ~ ~ Knre l Tyml BASc TkD Christapher G, ~ ï l i s PN) BEN?

iName(s> 01 aapiicaritls] ss appeanng on lhs aodication submitted ta me CouncJj tNom(s) du(des1 canaidattsj tels qu'ils a~Daraissent sur la demande suurne au Canseill

and found the proposed protoc01 involving animais to meet the standards of the Canadian Cauncil cm Animal Care. m d ïhat the facilities in which the animals will be hcrused and used cornply with the CCAC requirernents.

Signatures: n

' lnstithtion includes universities. hospitals, rwearch instihrtes br companies

et a convenu que 1s protocole propos6 sur des animaux rc pond aux nomes Btablies par le Conseii Canadien de grc tectlon des animaux, et que les installations qui abriteront le animaux qui serviront B I'expërimentation sont conformes au exigences du CCPA. .,

MIrlrele %. Bailey, ~ . V . R L IHL OHIYBSin OF V;E$TEZ GfiTAfljn

- - - - - - - - - r Rosserch lnrolvlng A

Animais) I (Noms en lcnrss moutees du ad1ëgue d e l'institution' an metiére de mchmlre sur des enimaux)

14 , 1991 d

Date

'Par institution, ori entend tes universités. les hbpitaux, le: instituts de recherche ou les çompagqies

105

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