ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1267

Minimal volume ventilation in lunginjury

With special reference to apnea and buffertreatment

STAFFAN HÖSTMAN

ISSN 1651-6206ISBN 978-91-554-9727-9urn:nbn:se:uu:diva-305369

Dissertation presented at Uppsala University to be publicly examined in Grönwallsalen, Ing70, Akademiska Sjukhuset, Uppsala, Friday, 2 December 2016 at 09:00 for the degree ofDoctor of Philosophy (Faculty of Medicine). The examination will be conducted in Swedish.Faculty examiner: Professor Hans Hjelmqvist (Medicinsk vetenskap, Örebro Universitet).

AbstractHöstman, S. 2016. Minimal volume ventilation in lung injury. With special reference toapnea and buffer treatment. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1267. 71 pp. Uppsala: Acta Universitatis Upsaliensis.ISBN 978-91-554-9727-9.

A fairly large portion of patients receiving surgical or intensive care will need mechanicalventilation at some point. The potential ventilator-induced lung injury (VILI) is thus of interest.One of the main causal factors in VILI is the cyclic energy shifts, i.e. tidal volumes, in thelung during mechanical ventilation. The problem can be approached in two ways. Firstly, onecan utilize apneic oxygenation and thus not cause any tidal injuries at all. Secondly, and moretraditionally, one can simply lower the tidal volumes and respiratory rates used. The followingdescribes a series of animal experiments exploring these options.

In the first two papers, I explored and improved upon the methodology of apneic oxygenation.There is a generally held belief that it is only possible to perform apneic oxygenation by priordenitrogenation and by using 100% oxygen during the apnea. As 100% oxygen is toxic, thishas prevented apneic oxygenation from more widespread use. The first paper proves that it isindeed possible to perform apneic oxygenation with less than 100% oxygen. I also calculated thealveolar nitrogen concentration which would conversely give the alveolar oxygen concentration.The second paper addresses the second large limitation of apneic oxygenation, i.e. hypercapnia.Using a high dose infusion of tris(hydroxymethyl)aminomethane (THAM) buffer, a pH > 7.2could be maintained during apneic oxygenation for more than 4.5 hours.

In the last two papers, THAM’s properties as a proton acceptor are explored during respiratoryacidosis caused by very low volume ventilation. In paper III, I found that THAM does not,in the long term, affect pH in respiratory acidosis after stopping the THAM infusion. It does,however, lower PVR, even though the PaCO2 of THAM-treated animals had rebounded to levelshigher than that of the controls. In the last experiment, I used volumetric capnography to confirmour hypothesis that carbon dioxide elimination through the lungs was lower during the THAMinfusion. Again, the PaCO2 rebounded after the THAM infusion had stopped and I concludedthat renal elimination of protonated THAM was not sufficient.

Keywords: VILI, THAM, buffers, apneic oxygenation, respiratory acidosis, hypercapnia, lowvolume ventilation, mechanical ventilation, ultra-protective ventilation

Staffan Höstman, Department of Surgical Sciences, Anaesthesiology and Intensive Care,Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.

© Staffan Höstman 2016

ISSN 1651-6206ISBN 978-91-554-9727-9urn:nbn:se:uu:diva-305369 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-305369)

Dedicated to Ellen, Lisa, Ida and Eva-Marie

List of papers

This thesis is based on the following papers, which are referred to in the textby their Roman numerals.

I Höstman S, Engström J, Sellgren F, Hedenstierna G, Larsson A.Non-toxic alveolar oxygen concentration without hypoxemia duringapnoeic oxygenation: an experimental study. Acta anaesthesiologicaScandinavica. 2011; 55(9):1078–84

II Höstman S, Engström J, Hedenstierna G, Larsson A. Intensivebuffering can keep pH above 7.2 for over 4 h during apnea: anexperimental porcine study. Acta anaesthesiologica Scandinavica.2013; 57(1):63–70

III Höstman S, Borges J, Suarez-Sipmann F, Ahlgren K M, Engström J,Hedenstierna G, Larsson A. THAM reduces CO2-associated increasein pulmonary vascular resistance: An experimental study inlung-injured piglets. Critical Care. 2015; 19(1):331

IV Höstman S, Kawati R, Perchiazzi G, Larsson A. THAM administrationreduces pulmonary carbon dioxide elimination, causing rebound inarterial carbon dioxide tension. An experimental study inhypoventilated pigs Manuscript

Reprints were made with permission from the publishers.

Contents

Part I: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Ventilator Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Ventilator-induced lung injury - VILI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Stress and Strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Atelectasis during anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Lung protective ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Low volume ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Positive end expiratory pressure - PEEP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Ultraprotective ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Apneic Oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Carbon dioxide and buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20THAM - tris(hydroxymethyl)aminomethane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

THAM vs. NaHCO3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Carbon dioxide and inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Part II: Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Part III: Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Experimental protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Apneic oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27THAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Details of the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Main experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Alveolar nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Estimation of THAM dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Main experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Lung injury model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Estimation of THAM dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Dead space reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Main experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Cytokine analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Outline of the main experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Statistical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Part IV: Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Part V: Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Advances in protective ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57The oxygen and nitrogen problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57The CO2 problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

With regard to apneic oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58With regard to minimal VT ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

The THAM problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59The volume problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

List of Tables

Table 1: Acid-base balance and oxygenation in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Table 2: Hemodynamics in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Table 3: Hemoglobin, glucose, electrolytes and diuresis in Paper II . . . . . . . . 46

Table 4: Acid-base, oxygenation, venous admixture and oxygenconsumption in Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Table 5: Acid-base status, oxygenation and shunt fraction in Paper IV . . . 53

Table 6: Urine pH and output in Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Table 7: Hemodynamics in Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

List of Figures

Figure 1: Outline of the experiment in Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Figure 2: Linear regressions in Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Figure 3: Arterial pH progression in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Figure 4: Base excess progression in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Figure 5: Arterial PaO2 progression in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Figure 6: Arterial PaCO2 progression in Paper II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Figure 7: Acid-base and arteriovenous ∆PaCO2 progression in Paper III . . 50Figure 8: Arterial PaCO2 at the different stages in Paper IV . . . . . . . . . . . . . . . . . . . . . . 51Figure 9: VCO2 at the different stages in Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

AbbreviationsADS – Anatomical dead spaceALI – Acute lung injuryANOVA – Analysis of varianceAO – Apneic oxygenationARDS – Acute respiratory distress syndromeARF – Acute respiratory failureBE – Base excessCO – Cardiac outputCO2 – Carbon dioxideECCO2R – Extra corporeal carbon dioxide removalEELV – End-expiratory lung volumeFiO2 – Fraction of inspired oxygen.FRC – Functional residual capacityg – Gram, unit of mass.HA – Hypercapnic acidosisHC – HypercapniaHCO−3 – Carbonate ionLPV – Lung protective ventilation. In reality LVV with PEEP.LVV – Low volume ventilationm – Meter, unit of lengthMAP – Mean arterial pressureMPAP – Mean pulmonary arterial pressureMOF – Multi-organ failureN – Newton, unit of force or weightNaHCO3 – Sodium bicarbonatePAO2 – Alveolar partial pressure of O2Pa – Pascal, unit of pressurePaCO2 – Arterial partial pressure of CO2PaO2 – Arterial partial pressure of O2PEEP – Positive end-expiratory pressurepKa – The acid dissociation constantpp – Percentage point(s)PPLAT – Plateau pressurePVR – Pulmonary vascular resistanceRR – Respiratory rates – Second, unit of timeSD – Standard deviationSVR – Systemic vascular resistanceTHAM – Tris(hydroxymethyl)aminomethaneVILI – Ventilator-induced lung injuryVT – Tidal volume

11

Part I:IntroductionThe matter of ventilators, their use and that of carbon dioxide and buffers.

This body of text is mainly concerned with apneic oxygenation, low volumeventilation (LVV), buffering of respiratory acidosis and the recent small ad-vances made at the Hedenstierna Laboratory by my co-researchers and myself.To the reader, apneic oxygenation might seem of little consequence in the realworld of medicine if we do not first establish some facts regarding low volumeventilation and other related subjects which will be handled accordingly laterin the text.

Ventilator Therapy

Depending on how one defines acute respiratory failure (ARF), a country likeSweden will have a total incidence of 7500 to 14500 cases of ARF per year.1, 2

In this report ARF is defined as acute respiratory failure needing ventilatortherapy. Thus, at the start of the 2000s, it was also estimated that somewherebetween 40%-65% of patients receiving ICU care, would also need mechani-cal ventilation.3–6

Ventilator-induced lung injury - VILIUntil the late 1990s it was considered good care to ventilate the critical carepatient, needing ventilator therapy, with tidal volumes (VT) as high as 10 to 15ml/kg and often without positive end-expiratory pressure (PEEP).7, 8 It was,however, found that, at least in animals, excessive airway pressures can causepulmonary edema9–12 and damage to the lung endothelium and epithelium.This in turn, or in parallel, would cause release of inflammatory mediatorsthat might affect the other internal organs.13–15 Prompted by this discovery,and smaller preliminary clinical trials,16–20 the ARDS Network investigatorsperformed a larger clinical trial proving that low VT with PEEP did indeed im-prove survival and lessen the number of days on ventilator treatment, and thatinterleukin-6 concentrations fell more rapidly and to a lower level in the in-tervention group.21 Belief in the beneficial effects of low volume ventilation,or more specifically lung protective ventilation (LPV), i.e. low VT with suf-ficient PEEP, has since been strengthened in a number of meta-analyses,22, 23

the latest by Serpa Neto et al.24 The detrimental effects on lung function ofthe ventilator and particularly sub-optimal ventilator settings has come to beknown as ventilator-induced lung injury (VILI).

Stress and StrainIn physics, stress is defined with a unit of kg/(m*s2) or Pascal (Pa) which is thesame unit as that of pressure.∗ It describes the internal tension in a material,owing to an external force applied to that material. Strain is viewed as theamount of deformation of a material, compared to the resting state. Strainis dimensionless.25 In practical pulmonary pathophysiology, stress is viewed

14

as more or less the equivalent of pressure. The relationship between end-expiratory lung volume (EELV) and VT is viewed as strain26, 27 although therelationship is not perfect.28

It is also useful to remember Hooke´s Law – "ut tensio, sic vis" or "as theextension, so the force", i.e there is a linear relationship between stress andstrain.25 This is, however, only true when not approaching the boundaries ofthe material’s stress tolerance. In reality the stress-strain plots of lungs arenot linear but very nearly so, even in ARDS patients.29 The lungs connectivetissue consists of elastin and collagen fibers where the elastin fibers providethe elastic recoil properties and the collagen provide a boundary for maximalstretch. In other words, elastin is the rubber band and collagen is the string inthe system.26–28 Stretching the lung close to or beyond its strain boundarieshas been shown to produce lung-damage10–13, 30 and nowadays is a way ofproducing an ARDS model in pigs.31, 32

In the 1970s Mead et al. published a somewhat involved theoretical modelof the internal stresses in the lung.33 They postulated that it is at least theoret-ically plausible that the internal stresses in lung parenchyma neighboring thatof atelectatic regions would be much higher than what one would normallyintuit. As an example, stresses of 140 kPa can develop at reasonably nor-mal airway pressures. Although this theoretical model has never been proven,there is evidence that even in undamaged rat lung, the local strains are hetero-geneous and, in hotspots, four times higher than the global strain.34

Atelectasis during anesthesiaPulmonary atelectasis, as it is thought of today (i.e. in relation to mechanicalventilation), was first rudimentarily characterized by Bendixen et al.35 afterit had been noted that general anesthesia often causes hypoxemia in the pa-tient.36 This phenomenon has subsequently been further elucidated by Heden-stierna and coworkers.37–39 Sometimes it is viewed as simply a complicationof sub-optimal ventilator care during anesthesia40 but, this notwithstanding,atelectasis is certainly detrimental to the patient and closely related to the con-cept of VILI. Atelectasis is formally defined as areas of lung tissue that arecollapsed and not participating in the tidal exchange of gases.38–41 Argumentshave also been made that atelectasis is not only, or not at all, a problem ofalveolar collapse, but rather a state where the lungs are filled with fluid.42

There are 3 main mechanisms explaining the formation of atelectasis.43, 44

∗As a side note, it is important to realize that stress and pressure are not necessarily thesame. A 4 meter high slab of granite (stress) will exert about 106*103 kg/(m*s2) or 106 kPa onthe ground beneath it. This is equivalent to a diving depth of 10.6 meters (pressure), which iscommonplace but no one would survive beneath 4 meters of solid rock. Thus, it is conceptuallyuseful to view stress as a vectored quantity and pressure as a scalar, at least within a reasonablysmall frame of reference.

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Atelectasis formation through compressionCompression atelectasis occurs when the alveoli are permitted to collapse dueto a lowered transmural pressure (i.e. the pressure between the inside of thealveoli and the pleura). The term compression thus does not refer to any exter-nal forces but rather to the net effect of lowering the intra-alveolar pressurerelated to the pleura. There are several reasons why the transmural pres-sure might fall. In the awake subject, the diaphragm allows for differentialpressures between the thoracic and abdominal cavities. When the patient isintubated, the diaphragm shifts cephalad,37 causing the pleural pressures torise. Relaxation does not seem to affect the magnitude of diaphragmatic dis-placement but the most affected parts of the diaphragm vary depending onwhether the patient is paralyzed or not.37, 45 There are, however, conflictingstudies46, 47 but the FRC is, nevertheless, lower during anesthesia and intuba-tion and it seems reasonable to conclude that this gives rise to compressionatelectasis as the thoracic volume is actually compressed. A second factor thatmight contribute to compression atelectasis is the shift of blood volume fromthe thoracic cavity (thus likely pooling in the abdominal cavity and elicitingpressure on the diaphragm) that occurs during anesthesia.37

Atelectasis formation through gas resorptionGas resorption atelectasis (sometimes called re absorption atelectasis) formseither through one or through a combination of both mechanisms.48 In the firstmodel there is occlusion of part of the airways and thus no ventilation distal tothe occlusion. As O2 is normally extracted at a rate of about 250 ml/min andCO2 at a rate of 200 ml/min, there is a net loss of volume and thus the totalpressure, keeping the alveolus open, falls and atelectasis is formed. Because ofthe high uptake of oxygen the potential for re absorption atelectasis formationis higher when higher FiO2 is used.49–52

In the second mechanism the VA/Q ratio is posited to fall below a criticalvalue where the in-flow of gas to the lung unit is balanced with the diffusionof gas (O2) to the blood. When the VA/Q ratio falls below this value, the lungunit will collapse.48 With higher FiO2 and lower VA/Q, this becomes morelikely.53, 54

Atelectasis formation because of surfactant impairmentThe pulmonary surfactant is a mixture of phospholipids, neutral-lipids andapoproteins that cover the alveolar area and reduce the surface tension of thealveoli, stabilizing them and ensuring alveolar patency.40, 41 In the ICU setting,this mechanism of atelectasis formation might not be of the same magnitudeas the other two as is seems that surfactant is negatively affected mainly byfluorinated hydrocarbons (anesthetic gases)55, 56 and its release is increased bylarge or very large VTs.57–61 However, surfactant is inactivated by inflam-matory mediators, as well as plasmaproteins, that leak into the alveoli duringARDS.

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VILI and multi-organ failureAs ventilator therapy can elicit an inflammatory response one must ask one-self: Does VILI by itself cause or augment multi-organ failure (MOF)? MOFis associated with high mortality and of the patients that suffer from MOF,those with ARF do worse.5, 62 In 1999 Ranieri et al. published a study of37 patients where they found that protective ventilation elicited a lower cy-tokine response.63 The same group has since re-examined the data post hocand also found a higher MOF scoring (mostly due to kidney failure) amongthe patients who did not receive protective ventilation.63 In addition to this,there are experimental data from animals suggesting that protective ventilationlowers the cytokine response compared to that of septic controls, in animalswith sepsis-induced ALI and protective ventilation.15 In 2006, Villar et al.published a study (the ARIES study) more or less re-iterating the 1998 Amatostudy, where they confirmed the effectiveness of lung protective ventilation onmortality but importantly, they also included the change in number of failingorgans as a secondary end point. In this study there was a clear difference (0.3vs 1.2, p<0.001) in the number of acquired organ failures pre randomizationversus post study.

Lung protective ventilationLow volume ventilationSince the ARDSNet study21 LVV is commonplace and considered the stan-dard of care when ventilating any patient with ARF.8 The standard of 6 ml/kgpredicted body weight and a good PEEP setting is far from perfect however, asthere are still areas of hyperinflation.30 It is also disconcerting that the scien-tific community remains divided as to whether LVV (and PEEP) has actuallyhad any effect on mortality from ARDS.24, 64

Recently, Futier et al showed that adhering to the LVV paradigm is bene-ficial during anesthesia for major abdominal surgery. By limiting the VT to6-8ml/kg predicted body weight, mandating peep levels to 6-8 cmH2O andperforming recruitment maneuvers, the number of patients needing ventila-tory support within 7 days after surgery dropped by 69% and the incidence ofmajor pulmonary and extra-pulmonary complications after surgery droppedfrom 27.5% to 10.5%.65∗

In 2009, Terragni et al. published a study with even smaller VTs and con-comitant extra corporeal CO2 removal, and found a decrease in inflammatorymarkers.66 However, others have done similar experiments without the same

∗In this study they included nearly all patients with any risk of pulmonary complications(i.e. high risk patients were included in both groups) and thus it should come as no surprisethat treating high risk patients with 0 PEEP and high VT will produce a significant amount ofpulmonary complications.

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beneficial effects but they did not use the higher PEEP levels used in Terragni’sstudy.67 Hager et al. indirectly question whether low VT or low plateau pres-sure (PPLAT) is the more important parameter.68 To further complicate thepicture, Amato et al.69 have recently published data arguing that the drivingpressure might be the most important parameter and the recent paper by Chi-umello et al.70 lends further strength to this idea.

Positive end expiratory pressure - PEEPIn the 1960s to early 1990s, PEEP seems to have been viewed primarily as anoxygenation tool17–20, 71, 72 but today the subject of PEEP is, in many ways,closely related to the recruitment-derecruitment concept. Ventilating a patientwith a too low PEEP-setting will cause parts of the lung to collapse with everyexpiration and re-open at inspiration.73, 74 If not re-opened either an atelec-tasis will form, or the continual recruitment-derecruitment will damage thelung.75, 76 In the ALVEOLI study, the ARDSNet investigators tried to furtherelucidate the posited positive effects of PEEP on mortality from ARDS but hadto stop mid-study due to futility criteria. Grasso et al later studied the effectson recruited lung volume by raising PEEP, using a FiO2-to-PEEP table andfound that roughly half of the patients received negligible benefit (measuredas gained alveolar volume) from the higher PEEP setting. This showed thatblindly raising PEEP by using a "one size fits all" table, is probably of no useunless the patient actually recruits lung volume.77 In the elegant 2006 Villar etal. ARIES study, only the sickest of ARDS patients where included. They setthe PEEP at the lower inflection point + 2 cmH2O (mean=14.1 cmH2O, on day1) and VT at about 7ml/kg, whereas the controls had PEEP at 9.0 cmH2O (onday 1) and VT at about 10ml/kg. They found a rather large effect on mortality,lung mechanics and oxygenation and, in parity with the 2005 Grasso study,suggested that PEEP should be individualized for each patient.78

Ultraprotective ventilationAs discussed above, mechanical ventilation has evolved, from a simple methodto deliver oxygen and remove carbon dioxide - to something much more com-plex where the physician has to be continously vigilant so as not to cause harmwhen ventilating the patient. Any exchange of energy (tidal volume) in thelung has the potential to cause damage to the lung and under that assumption,no tidal volume at all would be the ultimate in protective ventilation.

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Apneic Oxygenation

Apneic oxygenation is oxygenation without tidal movement of gas. The mass-transfer of oxygen during apneic oxygenation is most easily explained by firstlooking at the expanded alveolar gas equation.

PAO2 = FiO2 · (PAT M−PH2O)−PaCO2(1−FiO2[1−RQ])

RQ(1)

Specifically the subtrahend:

−PaCO2(1−FiO2[1−RQ])

RQ

Which expands to:

−PaCO2−PaCO2 ·FiO2[1−RQ]

RQ⇒−PaCO2

RQ+

PaCO2 ·FiO2[1−RQ]

RQ

In clinical practice, this is usually simplified to an approximation:

−PaCO2

RQ

The correcting factors explain the mass-transfer of oxygen (or inspired gasmixture) that is due to the difference (RQ) between CO2 and O2 movement inthe alveoli.79

The reason the subject becomes hypoxic when using FiO2 less than 1.0,is because the mass-transfer explained above, also moves the nitrogen in theinspired gas mixture, to the alveoli. This produces a constant rise in alveolarpartial pressure of N2 when the nearly insoluble N2 remains locked on thealveolar side. In fact, Holmdahl showed that when ventilating dogs with 50%O2 and then performing apneic oxygenation, also with 50% O2, the oxygenuptake ceased after 3 minutes. When air was used, oxygen uptake ceasedalmost immediately.80 Fear of the likely O2 toxicity81–83 that would result hasso far been one of the limitations to using apneic oxygenation to any greaterextent in clinical practice.

Another and perhaps the greatest limitation to a more extensive use of ap-neic oxygenation is that of hypercapnia. As no tidal transfer of gas occurs, noCO2 is removed and the subject rapidly becomes hypercapnic with respiratoryacidosis.

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Carbon dioxide and buffers

THAM - tris(hydroxymethyl)aminomethaneTHAM or Tris, as it is commonly referred to by chemists, is an inert bufferwith a pKa of 7.82 at 37◦C. As buffers are more effective the closer the pH ofthe system is to the buffer’s pKa, THAM is a much more potent buffer than anybuffer involving carbonate ions, as the apparent pKa of these is 6.1 at 37◦C.∗

THAM acts as a proton acceptor and generates HCO−3 through the reactiondescribed in equation 2 where R−NH2 represents unprotonated THAM.

R−NH2 +H2O+CO2⇔ R−NH+3 +HCO−3 (2)

Note, that this reaction consumes CO2 and by adding THAM to a solution onecan lower the PaCO2 of said solution by driving the reaction in eq 2 to theright side.

THAM vs. NaHCO3

NaHCO3 owes its buffering potential to the open-endedness of the contextin which it is considered in medicine. The ion as such, has limited buffercapacity in physiological pH ranges but through continuous removal of CO2it forms a sufficient buffer for the human body under normal circumstances.The drawback (vs THAM) of this mechanism is that the resultant CO2 has tobe removed by raising the minute ventilation. This is pictured in the formulasbelow.

NaHCO−3 +H+→ Na++H2CO3

H2CO3→ H2O+CO2

Carbon dioxide and inflammationDuring the last 15-20 years, there has been a considerable amount of researchdone regarding the potential anti-inflammatory and thus potentially beneficialeffects of high CO2 i.e. hypercapnia (HC), which usually leads to respiratory

∗The true pKa of carbonic acid is 3.6 but in biological systems the apparent pKa (i.e. in-cluding the effects of CO2 in aqueous solution) is commonly used.

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or hypercapnic acidosis (HA). What seems clear is the apparent dampeningof the innate immune response by HC, providing anti-inflammatory and pro-tective effects in ex-vivo and in-vitro experiments.84–90 There are, however,concerns regarding the concomitant attenuation of epithelial wound closure.91

Going forward into more clinically relevant in-vivo models, one needs to real-ize the distinction between aseptic models where inflammation and injury wasmainly induced by injurious ventilation with or without bacterial lipopolysac-charides or other toxins, and the septic models where bacteria were aerosolizedinto the trachea. In the aseptic in-vivo models, nearly all published researchhints at or shows overt protective effects of HC on lung function and inflam-mation.90, 92–97 However, there are a few published results98, 99 where HC didnot provide protection or even worsened lung-injury. With these results onhand it certainly seems clear that there is a dampening effect of HC on theinnate immune system, even in-vivo, but the specific effects and modes of ac-tion are perhaps not fully elucidated yet. The question of; whether there is anybenefit from HC becomes much less clear when septic models are examinedand it seems time is a factor. In shorter studies100–102 where the aerosolizedbacteria are given less time to produce lung damage there may be some bene-fit from HC but when the time-periods are extended,103, 104 HC is of no use oreven deleterious. This is probably due to impaired bacterial killing.103 Thereis also the question of if or in what way buffering HA changes the possiblebeneficial effects of HC. In fact, there is some evidence85 that buffering HAwith sodium bicarbonate is deleterious in an ex-vivo model.

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Part II:AimsDue to the increasing evidence that low - or very low - volume ventilation hasprotective effects in very diverse groups of patients we wanted to explore thepossibilities of further lowering the VTs needed.

The specific aims of the studies were:

I Firstly, to explore whether it is possible to perform apneic oxy-genation, using less than 100% oxygen, which had not been donepreviously. Secondly, to determine the actual alveolar nitrogen(and indirectly oxygen) concentration during apneic oxygenationfrom the pre-apneic values

II To prove our hypothesis that by using large amounts of THAMbuffer, one can keep an animal in an apneic state for several hourswithout serious adverse effects on pH or oxygenation

III To explore whether THAM buffer can be used to attenuate thenegative effects of hypercapnia associated with very low volumeventilation

IV To further quantify and survey the CO2 rebound that was ob-served in Paper III

The results from these studies are meant to give more information on possi-ble methods to further lower the VT needed to properly ventilate any patientneeding ventilator therapy.

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Part III:Materials and Methods

Animals

All experimental protocols were approved by the Uppsala Animal Ethics Com-mitte. Current Swedish regulations and legislation were followed in the designand conduct of the experiments. All personnel involved in the experimentshold the pertinent qualifications for animal experiment work.

A total of 38 healthy Swedish country breed piglets where used. They werebought from a for-purpose breeder. The animals had free access to feed andwater until being transported to the Hedenstierna laboratory.

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Anesthesia

All animals received identical anesthesiaOn arrival at the experimental facility, the animals where pre-medicated

with an intra-muscular injection of xylazine 2.2 mg/kg and tiletamine/zola-zepam 6 mg/kg.

After waiting for at least 5 minutes and always until the animal was sedated,it was placed upon a table in supine position and a venous cannula was intro-duced into an auricular vein. Fluids and anesthetic drugs were infused andthe depth of anesthesia was checked by absence of response to painful stimulibetween the hind or front toes on either side.

Anesthesia was maintained with a mixed infusion of ketamine 30 mg/kg/h,midazolam 0.1 mg/kg/h and fentanyl 4 µg/kg/h, and after checking that anes-thesia was sufficient to prevent responses to painful stimulation, relaxationwas achieved by rocuronium 0.3 mg/kg/h.∗Depth of anesthesia was checkedintermittently by following the hemodynamic parameters and checking for re-sponse to painful stimulus as stated above. If it was deemed to be insufficient,additional 4–10 µg/kg doses of fentanyl were given. If several doses of fen-tanyl were needed, the infusion rate of the anesthetic mixture was raised by10%. If the animal was insufficiently paralyzed (i.e. fighting the ventilator),an additional dose of rocuronium (0.1 - 0.3 mg/kg) was given after first con-firming the depth of anesthesia.

∗In papers I – III pancuronium is stated as the relaxant used. This is an error. Rocuroniumwas used in all experiments.

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Experimental protocols

Apneic oxygenationApneic oxygenation (AO) was performed with different levels of PEEP. InPaper I the animals were randomized to receive either 5 or 10 cmH2O of PEEPand in paper II all pigs received AO with a PEEP level of 20 cmH2O as thisapnea was of much longer duration and atelectasis had to be prevented. Apneawas performed by ventilating the animal at the planned PEEP level, clampingthe endotracheal tube on end expiration and then connecting the tube to aPEEP valve set at the desired PEEP level. Pressure was held by infusing 1-3liters/min of O2 into the breathing circuit. Overflow O2 was allowed to escape.

THAMIn the Swedish pharmacopeia, THAM dosage is calculated according to equa-tion 3

Dose(mmol) = weight(kg)∗∆Base Excess(mmol/l)∗0.3(l/kg) (3)

Paper ISeven pigs (22-27 kg) where anesthetized according to the above description.

InstrumentationA tracheostomy was performed using an 7 or 8 mm internal diameter endo-tracheal tube , and the lungs were ventilated in a volume-control mode by aServo-I with VT 8 ml/kg, inspiratory : expiratory ratio (I : E) 1:2, FiO2 0.7,and PEEP 5 cmH2O. The respiratory rate (RR) was adjusted to keep end-tidalCO2 at 5-6 kPa . Just before the tracheostomy, a bolus of fentanyl 10–20 µg/kgwas given intravenously (i.v.). During the first hour, 10 ml/kg/h Ringer’s ac-etate was infused i.v., and then the infusion rate was lowered to 5 ml/kg/hi.v. After open dissection of the neck vessels, an arterial catheter was insertedinto the right carotid artery for blood sampling and blood pressure monitoring,and a central venous catheter was inserted via the right external jugular vein.

27

In addition, a pulmonary arterial catheter for measurement of cardiac out-put (CO) and pulmonary artery pressure was introduced via the right externaljugular vein and the position verified by pressure monitoring. CO was ob-tained as the mean of three values measured by thermodilution after injectionof 10 ml ice-cold saline into the central venous catheter . A bladder catheterwas inserted suprapubically to measure hourly urine production. Electrocar-diographic monitoring was started, and oxygen saturation by pulse oximetrywas measured at the base of the tail.

Details of the experimentThe outline of the experiment is shown in Figure 1. To achieve four differentlevels of alveolar N2 concentrations, the animal was ventilated in randomizedorder with two different FiO2 levels (0.6 and 0.8) at two different PEEP levels(5 and 10 cmH2O). The high FiO2 was used in order to maintain adequatealveolar PaO2 when alveolar CO2 increases during the apnea. The AO wasthen performed with 100% O2 for 10 minutes with CPAP equal to the previ-ous PEEP. The alveolar N2 concentration at the end of the experimental periodwas measured after a passive release of the CPAP and was compared with thepredicted N2 concentration. It was assumed that during the first minutes of ap-nea, the N2 in the ADS, by convection and molecular diffusion, mixed with thegas in the alveolar compartment. The predicted alveolar N2 concentration wascalculated using equation 4. Before the apneic challenge, the alveolar oxygentension (PAO2 before)was calculated according to the alveolar gas equation(Eq. 1). PAO2 at the end of apnea (PAO2apnea) was calculated according to thefollowing assumptions. The change in PaO2 during apnea was considered tobe caused by the change in alveolar N2 tension (∆ PAN2) and the change inalveolar CO2 tension (∆ PACO2). The alveolar CO2 tension was assumed tobe equal to PaCO2. Thus, PaO2apnea was calculated as PAO2before - ∆ PAN2 -∆ PACO2.

Main experimentA lung recruitment maneuver was performed before each test and then thelungs were ventilated with the randomized FiO2 and PEEP level with VT 8ml/kg and a rate of 20 - 25 min−1 for 20 minutes in order to allow for N2 equi-libration. At the end of this period, blood samples were drawn for blood gasmeasurements; hemodynamic parameters were registered as well as VT andpeak and end-inspiratory plateau pressures, and PEEP. EELV and ADS weremeasured with a washin/washout technique using sulfur hexafluoride,105 andend-tidal N2 concentration was obtained from a small catheter with its tip 2cm below the tracheal tube connected to a calibrated N2 analyzer. Thereafter,the endotracheal tube was connected to an oxygen source with a PEEP valve

28

set at the same pressure as the PEEP during the preceding ventilation. Theflow from the oxygen source was adjusted to 2 l/min. After 10 min, new bloodsamples were obtained and the hemodynamic parameters were registered onceagain. Thereafter, airway N2 concentration was measured during an expirationcaused by the release of the PEEP valve. It was assumed that this N2 valuerepresented the N2 concentration in the alveolar compartment. The lungs wereagain ventilated by the ventilator set at FiO2 and PEEP in accordance with therandomization procedure, but ventilated at 1.5 times the previous minute ven-tilation for about 10 min in order to normalize PaCO2 before VT and ventilatorrate were adjusted back to the settings used before the apnea. After a further 10min, the procedure was performed again with the new PEEP and FiO2 settings.Thereafter, the experiment ended, and the animal was killed by an overdose ofpotassium chloride given intravenously.

29

Figure 1. Outline of the experiment in Paper I

Alveolar nitrogenAs N2 has a very low solubility coefficient in blood, we hypothesized that anyN2 in the airways and in the lung would be trapped in the alveolar compart-ment, diffusing neither from nor to the blood stream significantly. Thus, the

30

change in alveolar concentration of N2, due to diffusion, would be negligi-ble during a period of AO with 100% O2. The total amount of N2 (Eq. 4)would then be determined by the end-tidal N2 concentration during conven-tional ventilation before the start of AO multiplied by the alveolar volume,the latter calculated as end-expiratory lung volume (EELV) minus anatomicaldead space (ADS).

N2% = 100 · End tidal N2 · (EELV +apparatus dead space)EELV −anatomical dead space

(4)

Paper IISix pigs (23.5-30 kg) where anesthetized according to the description above.

InstrumentationA tracheostomy was performed in the manner described in Paper I and theanimal was mechanically ventilated with VT 10 ml/kg, inspiratory:expiratoryratio 1:2, FiO2 1.0, and PEEP 5 cmH2O. The respiratory rate was adjusted tokeep end-tidal CO2 below 5 kPa. During the first hour, 20 ml/kg/h Ringer’sacetate was infused i.v., and then the infusion rate was lowered to 5 ml/kg/hi.v. and increased, if needed, to counter urinary losses. Catheters were intro-duced and monitoring was started in the manner described in Paper I with theaddition of a second large bore central venous catheter that was introduced inthe left external jugular vein to facilitate the THAM infusion.

PreparationsAfter setting up the instrumentation, blood was sampled for measurement ofacid-base status, blood gases, oxygenation, hemoglobin, lactate, electrolytesand glucose content. Also, samples of urine were taken for measurements ofNa2+, K+ , and Cl− content as well as pH. Baseline hemodynamic parameterswere recorded as well. After baseline sampling and parameter retrieval, a lungrecruitment maneuver was performed. If the animal was considered hemody-namically unstable at this point, 50 ml boluses of hexaethyl starch were givenuntil a MAP of at least 50 mmHg was achieved.

Estimation of THAM doseAssuming that a pig exhales about 7ml CO2/kg/min,106 one can calculate theamount of buffer needed to keep the pH within reasonable levels by neutral-izing the proton load using THAM buffer.We estimated the needed bufferingcapacity as 20 mmol THAM i.e. 6 ml THAM (3.3 mmol/ml)/kg/h.

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Main experimentAfter the recruitment maneuver, the THAM infusion was started at 6 ml/kg/hin the central vein, as well as the AO with a PEEP of 20 cmH2O. The ex-cess oxygen escaped through the PEEP valve to the atmosphere. No breathingefforts were seen or recorded as assessed by capnography. During the AO,hemodynamic parameters were recorded at 5, 10, 15, and 30 min and then atevery 30 min during the experiment. In addition, at every 30-min interval, ar-terial and venous blood gases, as well as urine were sampled, and blood gases,blood glucose concentration, blood and urine electrolytes were analyzed (asabove). If plasma K+ was > 6 mM, 5-10 ml/h of a 30% glucose solution con-taining insulin 0.5 IU/ml was administered and discontinued if K+ decreased< 5.5 mM. The experiment was continued until 4.5 h from the start of AO hadpassed or until the animal perished or was deemed unfit to continue the exper-iment. In one animal, we continued the experiment for 6 h. At the end of theexperiment, the animal was killed by an i.v. overdose of potassium chloride.

Paper IIIEighteen pigs (23.0-30.5 kg) where randomized into three groups. Those thatwould receive hypoventilation only (NT), those that would also receive a 1hour infusion of THAM (1T) and those that would receive 3 hours of THAMinfusion. Anesthesia was performed as described above.

Lung injury modelAs a lung injury model, we lavaged the lung 8 times with 30ml/kg 0.9% NaClsolution heated to a temperature between 37◦ and 39◦ Celsius. The animalwas given, at most, a few minutes to recover to an oxygen saturation of atleast 95%, measured by pulse oximetry, if the oxygenation dropped during thelavage.

Estimation of THAM doseWe targeted a pH of 7.35 because, as stated in the study by Weber et al.,107

there was a rise in mean pulmonary arterial pressure (MPAP) at a pH of ap-proximately 7.2. Inserting these values into the Henderson-Hasselbalch equa-tion and solving for HCO−3 = 10(7.2−6.1) ∗0.23∗15 = 43mEq/L, we could findthe mEq/L amount of buffer needed as the difference between this value andnormal bicarbonate (HCO−3 ), which was assumed to be 20 mEq/L. The buffer-ing capacity needed for 1h of infusion was then estimated as weight in kilo-grams * 23 * 0.4 = 9.2 * weight. The recommended dosing for THAM isnormally calculated with a coefficient of 0.3, not 0.4, but in a pilot study a 1h

32

infusion with coefficient 0.3 was found to be an inadequate dose. Therefore,the coefficient was raised.

InstrumentationA tracheostomy was performed in the manner described in Paper I and theanimal was mechanically ventilated with VT 8 ml/kg, inspiratory:expiratoryratio 1:1, FiO2 0.5, PEEP 5 cmH2O and RR 25/min. The respiratory rate wasadjusted to keep end-tidal CO2 below 5 kPa. During the first hour, 10 ml/kg/hRinger’s acetate was infused i.v., and then the infusion rate was lowered to5 ml/kg/h i.v. Catheters were introduced and monitoring was started in themanner described in Paper I.

Dead space reductionIn a typical ventilator-patient circuit, the air that is re-breathed usually startsfrom the endotracheal tube or the Y-piece in the ventilator, depending onwhether the ventilator utilizes bias-flow. By using a double lumen tube thatwas cut just distal of the proximal cuff, we could remove the Y-piece from theventilator and use the two lumina to separate inspiration from expiration, thuseliminating dead space down to the tip of the tube.108

PreparationsAfter the instrumentation, FiO2 was set to 1.0 and a lung recruitment maneu-ver was performed to homogenize lung volume history. When the animal hadstabilized, baseline blood gas, oxygenation, hemoglobin, lactate, electrolytes,glucose and acid-base status values were recorded as well as baseline hemo-dynamic parameters. PEEP was set to 0 cmH2O; an airway pressure-volume(PV) loop was obtained,109 and functional residual capacity (FRC) was ob-tained using a sulfur hexafluoride washin/washout method.105 After baselinemeasurements, lung injury was induced. Blood gases and hemodynamics weresampled again as well as FRC and compliance of the respiratory system asobtained from the maximum slope of the expiratory PV loop.109 Next, the tra-cheal tube was replaced with a double lumen endotracheal tube to reduce deadspace according to the above description. A second lung recruitment maneu-ver was performed, and the ventilator was set to PEEP 10 cmH2O, VT 6 ml/kg,and I:E 1:2, and the RR was adjusted to target a pH between 7.38 and 7.42 intwo successive blood samples obtained within a 15-minute time window.

33

Main experimentWhen the pH had stabilized, time point 0 measurements were registered. Hy-poventilation was initiated by reducing VT to 3 ml/kg, and the THAM infusionwas started through a central venous line, in the two buffering groups. Arte-rial and mixed venous blood gases were obtained at 5, 10, 15, 30, 60, 90, and120 minutes and then every hour up to 6 hours from initiation of hypoventila-tion. In addition, the hemodynamic variables were registered at similar timepoints, except at 5 and 10 minutes. Further data were collected by drawingvenous samples for cytokine analysis, measuring FRC and compliance andperforming a lavage of the basal right lung with normal saline. Thereafter theleft lung was removed and dissected to obtain tissue samples from the ventral,medial, and dorsal parts at the hilar level. The tissue and lavage samples werefrozen in liquid nitrogen and stored at -80◦ C. The animals were killed with anintravenous dose of potassium chloride under deep anesthesia.

Cytokine analysisThe tissue samples were homogenized in lysis buffer, with the addition ofprotease inhibitor. Cytokine content was assessed using an enzymelinkedimmunosorbent assay (ELISA) system, for porcine tumor necrosis factor α

(TNF-α), interleukin (IL) IL-1β /IL-1F2, and IL-6. The detection limits of theassays were 125 pg/ml for TNF-α , 62.5 pg/ml for IL-1β /IL- 1F2, and 125pg/ml for IL-6. Total protein content of the supernatant was measured usinga Coomassie Plus Assay. Cytokine content of tissue lysates were normalizedagainst total protein content of the homogenate.

Paper IVSeven pigs (22- 27 kg body weight) where anesthetized according to the abovedescription.

InstrumentationA tracheostomy was performed in the manner described in Paper I and theanimal was mechanically ventilated with VT 6 ml/kg, inspiratory : expiratoryratio 1:2, FiO2 1.0, PEEP 10 cmH2O and RR 36/min. During the first hour, 10ml/kg/h Ringer’s acetate was infused i.v., and then the infusion rate changedevery hour to counter urinary losses. Catheters were introduced and moni-toring was started in the manner described in Paper I. An additional centralvenous catheter was inserted, distal from the heart in the cranial caval vein toenable sampling of blood without contamination of THAM. Expired CO2 wasmeasured using a NICO.

34

PreparationsAfter instrumentation, a lung recruitment maneuver was performed to homog-enize lung volume history. Before the animal was allowed to enter the mainexperiment, a stable pH of 7.38 - 7.42 was achieved by changing the tidalvolume in small increments.

Outline of the main experimentThe main experiment consisted of three stages; 1) Two hours of respiratoryacidosis (RA), 2) three hours of THAM infusion (THAM), and 3) two hoursof observation (OB).

Respiratory acidosisRespiratory acidosis was induced by lowering the RR by 50% and the VT by33%. The I:E ratio was also changed to 1:4. This was done because it hadbeen noted in pilot experiments that the NICO machine had difficulties recog-nizing the cut-offs between expiration and inspiration if the RR and VT weresmall and the I:E was set to 1:1. This new setting would lower the alveolarventilation by approximately 33%. During the next 5 minutes small changesin VT were allowed in order to achieve an alveolar minute volume as close to33% as possible.

Before the RA stage was started, time point 0 measurements were obtained.These included arterial and mixed venous blood gases, acid-base status andelectrolytes and hemodynamic measurements. In addition, central venousblood gases and acid base status were measured. These measurements wererepeated at 1, 5, 10, 15, 30, 60, 90 and 120 minutes.

THAM infusionWe estimated the needed amount of THAM by using the arterial PaCO2, pHand BE values from the last arterial blood sample in the RA stage. Targetinga pH of 7.35, the Henderson-Hasselbalch and Siggaard-Andersen equationswere used to calculate the target BE and thus the needed buffer amount asthe difference between actual BE and target BE. This amount of buffer wasinfused every hour over 3 hours. Values were recorded in the same mannerand at the same time points (and once more at 180 minutes) as described forthe RA stage. However, during this stage, mixed venous blood was sampledat every measurement time point. When all measurements had been made attime point 180, the OB stage commenced. The 180 min THAM values servedas 0 min OB values as well.

ObservationThe THAM infusion was stopped and observation commenced with measure-ments and sampling in the manner described for the RA stage.

35

Statistical methods

In all experiments a p-value of 0.05, or lower, was considered statisticallysignificant. The statistical procedures were performed using Sigmastat orSigmaplot (Systat Software, San Jose, CA, USA) and using the R110 softwarepackage

In Paper I, we were mainly interested in the relationship between the pre-dicted and measured N2 concentrations after AO. We decided that a N2 con-centration difference of > 5 percentage points (pp) to be unacceptable andestimated the standard deviation (SD) to be around 3pp. With these bound-aries, the above stated alpha level and a desired power of 0.8, we calculatedthat at least 5 animals would be needed. A Shapiro-Wilks test was used toconfirm normality. Parametric data were compared using a Student’s t-testand correlations between variables were expressed using linear expressions.

In Paper II, we wanted to detect a pH lower than 7.1 and based on earlierresults and a cursory power calculus with the same boundaries as in Paper I,we found that 6 animals would be adequate. The Friedman repeated measuresanalysis of variance(ANOVA) on ranks tests, along with the Tukey HonestSignificant Difference test as a post hoc test, were used for the inferentialstatistics. Data were presented as median with inter-quartile range.

In Paper III, a power analysis indicated that we would need 6 animals ineach group to detect a pH difference of 0.1 which was our main parameter. Inthe cases where the data resembled a exponential distribution, a logarithmictransformation was performed to improve on normality. In the cases wherenormality was still not achieved, a Kruskal-Wallis one way ANOVA was per-formed. For the data that was normal, a one-way ANOVA was performed.Tukey’s Honest Significant Difference test was used as a post hoc test. Resultswere presented as mean and standard deviation.

In Paper IV, it was estimated that we would need at least 6 animals forinferential tests on exhaled CO2. Repeated measures ANOVAs with a Holm-Sídak corrected, paired pairwise t-test as a post hoc analysis were used forinferential statistics. Data were tested for normality and homoscedasticity.Results were presented as mean with standard deviation.

36

Part IV:Results

Paper IThe measured and predicted N2 concentrations where 46.9±4.3% and 48.2±2.5% at an FiO2 of 0.6 and a PEEP of 5 cmH2O. Similar values at FiO2 0.8 andPEEP of 5 cmH2O were 23.3± 3.5% and 25.2± 2.2%, respectively, at FiO20.6 and PEEP of 10 cmH2O 47.0± 2.6% and 46.8± 2.8%, respectively, andat FiO2 0.8 and PEEP of 10 cmH2O 24.7± 3.23% and 23.9± 3.1%, respec-tively. The difference between measured and predicted alveolar N2 concentra-tion when using all data points was: −0.5± 3%, P = 0.369. The differencebetween predicted and measured alveolar N2 concentration when using thedata from each individual pig was −0.5± 2%, P = 0.587. The linear regres-sion equation between the measured and predicted N2 concentration using alldata points was: measured [N2] = 0.12+ 0,984 ∗ [N2predicted ], R2 = 0.95, P <0.001 (Left panel in figure 2). The difference between the N2 concentrationmeasured before and the N2 concentration measured at the end of the apneicperiod using all data points was 4±2%, P < 0.001. The linear regression equa-tion between the N2 measured before and the N2 measured at the end of apneausing all data points was: N2 end apnea = −0.05+ 1.11 ∗N2 be f ore apnea, R2 =0.95, P < 0.001 (right panel in Figure 2)

Paper IITwo animals died before the end of the experiment; one after 210 min becauseof cardiac arrest caused by untreated hyperkalemia and the other one after 240min because of fluid-unresponsive hemodynamic instability. The data are pre-

sented as median and interquartile range (IQR).

Acid base balance and oxygenationpH decreased from 7.5 (7.5, 7.5) to 7.3 (7.2, 7.3) (P < 0.001) at 270 min (Fig-ure 3, Table 1), with no change between 5 min 7.4 (7.4, 7.5) and 270 min.PaCO2 (Figure 6) increased from 4.5 (4.3, 4.7) to 25 (22, 28) kPa at 270 min(P < 0.001), and base excess (BE) (Figure 4) increased from 5 (2, 6) to 54 (51,57) mEq/l at 270 min (P < 0.001). HCO−3 followed the same pattern as baseexcess (BE) (P < 0.001). Arterial oxygen tension (Figure 5) decreased from70 (67, 75) to 41 (37, 45) kPa at 270 min (P < 0.001).

HemodynamicsCO was stable at approximately 2 l/min during the experiment (P = 0.178)(Table 2). MAP decreased by approximately 10 mmHg (P = 0.039), and heartrate increased during the first hours but returned to the base-line value at theend of the experiment (P < 0.001). No changes in systemic vascular resistance(SVR) were found. MPAP increased from 20 (19, 22) to 28 (28, 30) mmHg (P< 0.001). Pulmonary vascular resistance tended to follow the same pattern asMPAP (P = 0.251).

Hb concentration, electrolytes, and fluid balanceHb increased by approximately 20 g/l (P < 0.001) (Table 3). Na+ decreasedby approximately 15mM (P = 0.015), with the major changes during the first90 min. K+ increased from 3.8 (3.6, 3.9) to 6.7 (5.9, 6.8)mM(P = 0.027), andCl− increased by approximately 5mM (P = 0.012).

UrineNa+ concentration was unchanged during the experiment (P = 0.801), but K+

decreased from 36 (23, 100) to 6 (5, 8)mM (P = 0.037). Cl− was approxi-mately 90mM (P = 0.034) during the experiment. Urinary pH increased from6 (5, 6.5) to 8.5 (8.3, 8.5) at 270 min (P = 0.007). Diuresis increased to amaximum of 780 (690, 870) ml/h between 90 and 150 min, and decreased to310 (210, 360) ml/h at 270 min (P = 0.006). Fluid balance at 270 min was+940 (740, 1240) ml. One animal was given 0.5 mg furosemide just after 3 hand 40 min.

38

Figure 2. Linear regressions in Paper I, Left panel: Measured vs. predicted alve-olar nitrogen concentration. Right panel: Measured alveolar N2 at the end of apneavs. measured end-tidal N2 before apnea. The lines indicate the regression and 95%confidence interval of the regression curve.

39

Figure 3. The progression of pH in Paper II

40

Figure 4. The progression of base excess in Paper II

41

Figure 5. The progression of arterial PaO2 in Paper II

42

Figure 6. The progression of arterial PaCO2 in Paper II

43

Tabl

e1.

Aci

d-ba

seba

lanc

ean

dox

ygen

atio

nat

the

diffe

rent

mea

sure

men

tpoi

nts

inPa

per

II

Tim

e(m

in)

030

6090

210

270

p

pH7.

53(7

.5, 7

.54)

7.37

(7.3

4,7.

4)7.

35(7

.33,

7.39

)7.

33(7

.31,

7.38

)7.

29(7

.25,

7.34

)7.

27(7

.24,

7.31

)<0

.001

P aC

O2

(kPa

)4.

5(4

.3,4

.7)

10(9

.0,1

2)13

(11,

15)

16(1

3,17

)24

(19,

27)

25(2

2,28

)<0

.001

BE

(mm

ol/L

)5.

6(2

.2,6

.0)

17(1

5,19

)24

(23,

27)

31(3

0,33

)51

(47,

53)

54(5

1,57

)<.

0001

HC

O− 3

(mm

ol/L

)28

(25,

28)

43(4

1,45

)51

(50,

54)

59(5

7,62

)82

(76,

85)

84(8

0,88

)<0

.011

P aO

2(k

Pa)

70(6

7,75

)64

(62,

66)

61(5

9,64

)57

(54,

58)

46(4

5,51

)41

(37,

45)

<0.0

01S v

O2

(%)

51(4

4,61

)53

(52,

53)

66(6

2,74

)71

(61,

76)

68,(

61,7

6)0.

053

Lac

tate

(mm

ol/L

)1.

5(1

.3,1

.8)

2.2

(2.1

,2.9

)2.

4(1

.8,2

.8)

1.9

(1.3

,2.2

)1.

6(1

.3,2

.3)

3.1

(2.2

,3.9

)0.

041

N6

66

66

4

44

Tabl

e2.

Hem

odyn

amic

sat

the

diffe

rent

mea

sure

men

tpoi

nts

inPa

per

II

Tim

e(m

in)

030

9015

021

027

0p

CO

(L/m

in)

1.8

(1.5

,2.1

)1.

7(1

.6,1

.7)

2.2

(1.9

,3.0

)2.

5(1

.9,3

.5)

2,8

(1,8

,3,5

)2,

0(1

,7,2

,5)

0.17

8M

AP(

mm

Hg)

65(6

2,74

)57

(55,

66)

62(5

9,71

)58

(54,

62)

53(4

6,56

)55

(53,

57)

0.03

9C

VP(

mm

Hg)

13(1

1,14

)17

(16,

18)

13(1

2,14

)13

(10,

14)

13(1

1,15

)15

(13,

16)

<0.0

01PC

WP(

mm

Hg)

14(1

2,15

)19

(17,

21)

17(1

6,17

)16

(14,

17)

17(1

5,18

)19

(18,

19)

<0.0

01H

R(m

in−

1 )90

(80,

100)

76(7

2,79

)10

6(9

2,11

6)12

0(1

13,1

24)

115

(114

,115

)92

(89,

97)

<0.0

01

SVR

(dyn·s/c

m5 )

2300

(209

0,26

90)

1950

(175

0,24

00)

1740

(131

0,20

20)

1520

(116

0,18

90)

1240

(114

0,20

00)

1630

(120

0,20

30)

0.25

1

MPA

P(m

mH

g)20

(19,

22)

25(2

3,25

)26

(23,

26)

26(2

3,26

)26

(26,

27)

28(2

8,30

)<0

.001

PVR

(dyn·s/c

m5 )

300

(270

,320

)25

8(2

30,2

82)

275

(238

,340

)28

5(2

08,3

37)

257

(232

,533

)47

9(2

82,6

82)

0.07

9N

66

66

54

45

Tabl

e3.

Blo

odhe

mog

lobi

n,gl

ucos

e,pl

asm

aan

dur

ine

elec

trol

ytes

,and

diur

esis

atth

edi

ffere

ntm

easu

rem

entp

oint

sin

Pape

rII

Tim

e(m

in)

030

9015

021

027

0P

Hb-

bloo

d(g

/L)

82(8

0,85

)72

(68,

75)

77(7

4,79

)88

(82,

90)

96(7

9,10

2)10

0(9

4,10

5)6

<0.0

01G

luco

se-p

lasm

a(m

mol

/L)

8.4

(7.3

,10.

2)7.

3(7

.0,8

.0)

6 (5.5

,6.3

)5.

7(5

.0,8

.3)

4.2

(3.7

,4.8

)8.

3(8

.1,9

.0)4

0.00

2

Na+

-pla

sma

(mm

ol/L

)13

5(1

34,1

36)

126

(126

,128

)12

1(1

21,1

22)

120

(118

,121

)12

0(1

18,1

21)

120

(118

,121

)30.

015

K+

-pla

sma

(mm

ol/L

)3.

8(3

.6,3

.9)

4.5

(4.3

,4.8

)5.

1(5.

0,5.

3)6.

1(5

.6,6

.5)

6.4

(6.0

,6.9

)6.

7(5

.9,6

.8)3

0.02

7

Cl−

-pla

sma

(mm

ol/L

)10

2(1

00,1

04)

100

(99,

101)

97 (95,

100)

98 (95

,101

)10

2(9

9,1

06)

107

(104

,110

)30.

012

Diu

resi

s(m

l/h)

95 (43,

130)

110

(81,

110)

530

(490

,570

)78

0(6

30,8

70)

460

(430

,620

)31

0(2

10,3

60)4

0.00

6

Cl−

-uri

ne(m

mol

/L)

92 (77,

138)

138

(107

,144

)93 (8

8,10

2)80 (7

5,89

)84 (7

4,95

)92

(79,

106)

40.

034

K+

-uri

ne(m

mol

/L)

36 (26,

100)

14 (7.2

,40)

10 (9,1

6)9 (7

,11)

8 (5,1

0)6

(5,8

)40.

037

Na+

-uri

ne(m

mol

/L)

31 (21,

43)

20 (20,

20)

30 (21,

44)

30 (22,

44)

26 (20,

32)

26(2

2,34

)40.

801

n6

66

66

The

supe

rscr

iptn

umbe

rsin

the

270

min

colu

mn

indi

cate

nva

lues

fort

hest

atis

tic

46

Paper IIIAll values reported in groups of three are in the order controls (NT), 1-h in-fusion (1T), 3-h infusion (3T) of THAM. P-values reported are with regard toNT unless otherwise specified.

Blood gas and acid-base status (Table 4, Figure 7)Arterial pH was largely different between the groups during THAM infusionbut there were no differences between groups at 360 minutes. PaCO2 rosein all groups. At the end of infusions, both the 1T and 3T groups showed asecond phase of PaCO2 increase, whereas NT changed very little after 120minutes. There was a large difference in PaCO2 at 360 minutes (13.8± 1.5kPa vs. 15.2± 3.3 kPa, p = 0.55; 22.6± 1.7 kPa, p < 0.001). The differencebetween mixed venous and arterial carbon dioxide tension was lower in both1T and 3T groups starting from the 15-minute time point (1.8± 0.5 kPa vs.0.8±0.4 kPa, p = 0.001; 0.6±0.2 kPa, p < 0.001). After the infusion, the 1Tgroup rebounded to levels similar to that of NT. At 360 minutes, the valueswere 2.1±0.8 kPa vs. 2.2±0.6 kPa (p = 0.96) and 1.0±0.7 kPa (p = 0.03).At 360 minutes, the BE values were 3.4±3.2 mEq/L vs. 10.2±2.1 mEq/L (p= 0.002) and 27.8±3.1 mEq/L (p < 0.001). HCO−3 showed an increase similarto that of BE. Arterial oxygen tension (PaO2) trended toward decrease and waslower at 360 minutes in the 3T group (p < 0.001) compared with controls andthe 1T group. The 3T group had a higher shunt fraction than the 1T group at360 minutes (p = 0.03).

HemodynamicsThe MPAP was lower in the 3T and 1T groups from the 30- and 60-minutetime points, respectively. At 360 minutes, however, only the 3T group wasdifferent from the control animals (25± 5 mmHg vs. 21± 2 mmHg, p =0.17; 18± 2 mmHg, p = 0.008). PVR was similar to MPAP, and the 3Tgroup was still different at 360 minutes (450± 141 dyn · s/cm5 vs. 329± 77dyn · s/cm5, p = 0.11; 255± 43 dyn · s/cm5, p = 0.0081). CI exhibited a ris-ing trend over time in all the groups, with 3T separating from the rest at 360minutes (3.5± 1.0 L/min/m2 vs. 3.8± 0.75 L/min/m2, p = 0.42; 5.0± 0.57L/min/m2, p < 0.001). MAP and SVR did not differ between the groups atthe time points examined, but SVR showed a decreasing trend over time in allthree groups.

Inflammatory markers - ELISAThere were a few missing (i.e. below the detection limit) values in the 1Tand 3T groups, indicating that they might be generally lower than in the con-trols. In the tissue samples, there was an effect on the IL-6 concentration(detected by two-way ANOVA) in the THAM strata, and post hoc analysisshowed a p-value of 0.014 compared with controls, with the 3T group being

47

higher (11.8±11.7 pg/mgprotein) than the 1T group (4.5±2.0 pg/mgprotein) andnon-significant values being found for the rest (p =0.086 for 3T vs NT and p =0.423 for 1T vs NT). Two-way ANOVA of the TNF-α and IL-1β concentra-tions was not done, owing to many missing values. A Kruskal-Wallis one-wayANOVA on ranks was performed for every tissue strata, with the missing val-ues set below the minimum measured values. No differences were detected.

Lung mechanicsFRC and compliance of the respiratory system decreased with the lavages (p<0.001) but did not differ between the groups.

ElectrolytesArterial sodium concentration fell during the THAM infusion in both the 1Tand 3T groups, and arterial potassium concentration increased.

Paper IVPaCO2 and exhaled carbon dioxidePaCO2 increased during RA from 6.2± 0.4 to 15.2± 1.4 kPa, decreased byTHAM to 12.2± 1.1 kPa and rebounded during OB to 16.6± 1.2 kPa. Allvalues are significantly different from each other (Table 5, Figure 8).

VCO2 (ml/min) was at normoventilation 132± 15 and fell to 109± 12 atsteady state of acidosis. During the THAM it fell further to 74± 12 but thenrose again to 111±15 at the end of OB (Table 5, Figure 9). A repeated mea-sures ANOVA showed that all strata were different to each other except RAvs. OB (p = 0.39).

By calculating the expected VCO2 and comparing that to the total cumula-tive VCO2, we estimated the effect of THAM on the CO2 elimination throughthe lungs during RA. Three hours of acidosis would have produced a meanof 19.6± 2.2 liters of CO2 but the actual cumulative exhaled CO2 during theTHAM stage was 13.8±1.8 liters of CO2 (p < 0.001, paired t-test).

Acid-base status, oxygenation and shunt fraction (Table 5)As expected, the arterial pH exhibited marked swings during the experiment.Starting at 7.40± 0.02 (which was one of the criteria for starting the experi-ment), it fell during RA to 7.07±0.04 and due to the effects of THAM buffer,rose again to 7.41± 0.04 and finally settled at 7.24± 0.03 at the end of the2-hour OB stage. The BE values showed a progression reflecting the changein pH. Starting at 3.7±2.5, they fell to 1.6±3 at the end of RA. After 3 hoursof THAM, they had risen to 29.5±3.2 and then fell to 23.4±3.3 after 2 hoursof OB. The arterial HCO−3 values showed similar changes. Refer to Table 5for information about which strata achieved significance.

48

Table 4. Arterial acid base values, arterial and venous oxygenation, oxygen consump-tion and venous admixture in Paper III

Parameter Group Start 60min 180min 360min

pH NT 7.40(0.01) 7.14(0.03) 7.11(0.05) 7.12(0.06)1T 7.41(0.02) 7.34(0.05)* 7.18(0.05)* 7.16(0.07)3T 7.40(0.01) 7.35(0.02)* 7.39(0.01)*§ 7.16(0.03)

PaCO2 NT 6.0(0.5) 12.3(1.2) 13.5(1.4) 13.8(1.5)(kPa) 1T 5.8(0.3) 10.2(0.9)* 14.6(2.4) 15.2(3.3)

3T 5.9(0.7) 10.6(0.7)* 13.1(0.5) 22.6(1.7)*§PaO2 NT 63.7(7.4) 48.1(12.0) 45.2(12.3) 46.4(11.8)(kPa) 1T 65.0(8.6) 59.8(6.3) 49.4(9.0) 53.3(5.0)

3T 56.0(4.6) 49.7(4.1) 32.6(7.6)§ 30.0(7.7)*§BE NT 2.7(2.3) 1.3(2.2) 1.8(2.8) 3.4(3.2)(mEq/l) 1T 2.4(1.5) 13.8(3.5)* 10.9(3.1)* 10.2(2.1)*

3T 2.4(3.0) 16.0(0.6)* 31.2(2.2)*§ 27.8(3.1)*§VO2 NT 124(22) 118(23) 120(9) 121(21)(ml/min) 1T 142(18) 131(20) 139(14)1 140(26)

3T 123(18)1 117(7) 127(7) 134(13)QS/QT NT 0.11(0.04) 0.17(0.04) 0.20(0.07) 0.20(0.08)(fraction) 1T 0.09(0.02) 0.11(0.03)* 0.17(0.05)1 0.15(0.04)

3T 0.12(0.02)1 0.15(0.03)§ 0.24(0.04) 0.26(0.06)§Lactate NT 1.3(0.23) 0.7(0.08) 0.6(0.14) 0.6(0.14)(mmol/l) 1T 1.4(0.14) 1.1(0.19)* 0.6(0.14) 0.7(0.19)

3T 1.2(0.15) 0.9(0.31) 0.9(0.23)* 1.3(0.59)*§∆PaCO2 NT 2.2(0.6) 1.9(0.5) 2.2(0.7) 2.1(0.8)(kPa) 1T 2.6(0.3) 0.7(1.1)* 2.1(0.3) 2.2(0.6)

3T 2.7(0.3) 0.6(0.4)* 0.2(0.6)*§ 1.0(0.7)*§QS/QT; venous admixture obtained at inspired oxygen fraction 1.0, ∆PaCO2; Arteriovenous difference incarbon dioxide tension. NT; controls which did not receive THAM, 1T; group with THAM infusion over1 hour, 3T group with THAM infusion over 3 hours. The time points refer to time after start of THAMinfusion or corresponding time points in the controls (NT). ∗ = Value is different from NT group. § = Valueis different from 1T group. 1 = only 5 subjects in statistic due to technical error.

49

Figure 7. Acid-base and arteriovenous ΔPaCO2 progression in Paper III

50

Figure 8. Arterial CO2 tension at different time points in Paper IV. Start RA = nor-moventilation/start of respiratory acidosis. End RA = end of respiratory acidosis. EndTHAM = end of THAM infusion, and End OB = end of observation. Values are meanswith standard deviations presented as error bars.

51

Figure 9. VCO2 at different time points in Paper IV. Start RA = normoventilation/startof respiratory acidosis. End RA = end of respiratory acidosis. End THAM = end ofTHAM infusion, and End OB = end of observation. Values are means with standarddeviations presented as error bars.

52

Tabl

e5.

Aci

d-ba

sest

atus

,oxy

gena

tion

and

shun

tfra

ctio

nin

Pape

rIV St

artR

AE

ndR

AE

ndT

HA

ME

ndO

B

Art

eria

lpH

7.40±

0.02

ap7.

07±

0.04∗

7.41±

0.04

ap7.

24±

0.03∗

Art

eria

lcar

bon

diox

ide

tens

ion

(kPa

)6.

2±

0.4∗

15.2±

1.4∗

12.2±

1.1∗

16.6±

1.2∗

Art

eria

loxy

gen

tens

ion

(kPa

)75

.9±

6.9∗

64.7±

4.6s

65.9±

4.7s

63.2±

3.8s

Art

eria

lhem

oglo

bin

satu

ratio

n(%

)98±

0ap97±

0∗98±

0a98±

0sa

Art

eria

lbas

eex

cess

(mE

q/l)

3.7±

2.5tp

1.6±

3.0tp

29.5±

3.2∗

23.4±

3.3∗

Art

eria

lbic

arbo

nate

ion

conc

entr

atio

n(m

mol

/l)27

.6±

2.3∗

23.5±

2.2∗

53.7±

3.7∗

47.2±

4.8∗

Art

eria

lhem

oglo

bin

conc

entr

atio

n(g

/l)84±

784±

1086±

885±

8A

rter

iall

acta

teco

ncen

trat

ion

(mm

ol/l)

1.4±

0.4ap

0.6±

0.1st

1.1±

0.4a

0.8±

0.2s

Ven

ous

pH7.

30±

0.03∗

7.03±

0.04∗

7.40±

0.04∗

7.22±

0.03∗

Ven

ous

carb

ondi

oxid

ete

nsio

n(k

Pa)

8.6±

0.5∗

17.2±

1.6st

12.8±

1.2∗

17.9±

1.2st

Ven

ous

oxyg

ente

nsio

n(k

Pa)

5.3±

0.6∗

9.3±

1.0∗

6.8±

0.5∗

8.5±

0.5∗

Ven

ous

hem

oglo

bin

satu

ratio

n(%

)51±

11∗

74±

5s76±

4s72±

20s

Ven

ous

base

exce

ss(m

Eq/

l)4.

8±

2.5tp

2.3±

2.5tp

30±

3.0∗

24.0±

3.6∗

Ven

ous

bica

rbon

ate

ion

conc

entr

atio

n(m

mol

/l)27

.5±

2.3∗

24.1±

2.3∗

53.7±

3.4∗

46.1±

3.8∗

Art

erio

veno

usca

rbon

diox

ide

tens

ion

diff

eren

ce(k

Pa)

2.4±

0.4tp

2.1±

0.6tp

0.6±

0.2∗

1.3±

0.4∗

End

tidal

CO

2(k

Pa)

6.3±

0.4∗

15.9±

1.4st

12.6±

1.4∗

16.9±

1.5st

Shun

tfra

ctio

n(%

)7±

1∗13±

2s13±

3s13±

4s

s,a,

t,p

indi

cate

diff

eren

cefr

omSt

artR

A(s

tart

ofre

spir

ator

yac

idos

is),

End

RA

(end

of12

0m

inut

esre

spir

ator

yac

idos

is)

End

TH

AM

(end

of18

0m

inut

esof

TH

AM

)and

End

OB

(end

of12

0m

inut

esob

serv

atio

n)re

spec

tivel

y.*

indi

cate

sth

atth

est

ratu

mw

asdi

ffer

entf

rom

allt

heot

hers

trat

a.

53

Table 6. Urine pH and output in Paper IV

Start RA End RA End THAM End OB

Urine output (ml/hour) - 104±46t 797±420ap 353±208t

Urine pH 4.8±0.6t p 4.8±0.2t p 7.6±0.3sa 7.1±0.3sa

s, a, t, p indicate difference from Start RA (start of respiratory acidosis), End RA (end of 120 minutes respi-ratory acidosis) End THAM (end of 180 minutes of THAM) and End OB (end of 120 minutes observation)respectively. * indicates that the stratum was different from all the other strata.

Urine pH increased significantly when THAM was started and continued tobe high during the THAM and OB stages (Table 6).

PaO2 decreased when end-tidal CO2 increased at RA (indicating an increasein alveolar PaCO2 competing with alveolar PaO2). However, the calculatedshunt fraction increased from 7% to 13% (p < 0.001), but then remained con-stant during the experiment.

Hemodynamics (Table 7)There were no changes in MAP, CVP or PCWP. CO and heart rate increasedduring RA (p< 0.001). MPAP rose from 18± 2 at normocapnia to 26± 2mmHg at the end of RA but fell to 20± 2 mmHg at the end of THAM andstayed at a similar level of 21± 2 mmHg at the end of the OB stage (p <0.001).

54

Tabl

e7.

Hem

odyn

amic

sin

Pape

rIV

Star

tRA

End

RA

End

TH

AM

End

OB

Hea

rtR

ate

(1/m

in)

85±

10∗

99±

11∗

128±

8∗11

5±

4∗

Mea

nar

teri

alpr

essu

re(m

mH

g)74±

1474±

578±

1873±

15M

ean

pulm

onar

yar

teri

alpr

essu

re(m

mH

g)18±

2ap26±

2∗20±

2a21±

2sa

Cen

tral

veno

uspr

essu

re(m

mH

g)41

0±

110±

18±

19±

14Pu

lmon

ary

capi

llary

wed

gepr

essu

re(m

mH

g)11±

112±

110±

112±

2C

ardi

acou

tput

(l/m

in)

1.8±

0.5∗

2.9±

1.0s

3.6±

0.5s

3.7±

0.7s

s,a,

t,p

indi

cate

diff

eren

cefr

omSt

art

RA

(sta

rtof

resp

irat

ory

acid

osis

),E

ndR

A(e

ndof

120

min

utes

resp

irat

ory

acid

osis

)E

ndT

HA

M(e

ndof

180

min

utes

ofT

HA

M)

and

End

OB

(end

of12

0m

inut

esob

serv

atio

n)re

spec

tivel

y.*

indi

cate

sth

atth

est

ratu

mw

asdi

ffer

entf

rom

allt

heot

hers

trat

a.

55

Part V:Discussion

Advances in protective ventilation

As mentioned in the introduction, ventilation with no tidal volumes at all or atleast the minimal possible volume, will also mimize the damage done by dy-namic energy shifts in the lung. In light of this, we need new methods that canminimize or even abolish the energy shifts but still satisfy the patient’s needswith regards to oxygenation and carbon dioxide removal. In a perfect worldone would perhaps ventilate the patient by sucking, not pushing gas into thelungs. Ironically, this is how the iron-lung worked. Formerly it was thoughtthat the main limitations of apneic oxygenation were the resultant (1) hyper-capnia and (2) hypoxia that would occur when using less than 100% O2 duringthe apnea. Due to the risks of oxygen toxicity, 100% O2 (and thus AO) hasnever been considered a viable option other than when diagnosing brain death.If one were to forgo the rather extreme method of AO and instead strive forconventional mechanical ventilation with the minimum possible VT, hyper-capnia with its resultant hemodynamic side-effects again, becomes a limitingfactor.

The oxygen and nitrogen problemsIt has been shown that AO with a PEEP of 20 cmH2O and FiO2 1.0 in conjunc-tion with extra corporeal carbon dioxide removal (ECCO2R) can keep PaO2and PaCO2 at reasonable levels during 7h of AO.111 However, another grouphas also performed similar experiments with only 5 cmH2O of PEEP and wasnot able to maintain adequate oxygenation.∗ The reason 100% O2 is neededis the fact that if any amount of nitrogen is allowed to enter the airway it willtravel to the alveoli and as it is not very soluble, will remain in the alveoli.This in turn lowers the partial pressure of oxygen.80, 112 However, one needsto realize the dichotomy between FiO2 and PAO2. The alveolar damage seenwhen using high FiO2 is, of course, due to the alveolar concentration of O2and not the FiO2 per se. In Paper I, we proved that AO is indeed possible withan alveolar O2 concentration less than 100% and thus the - perhaps - smallerof the two limitations is more or less abolished.

During AO one would not only want to have less than 100% O2 in thealveoli but also to know or at least estimate the concentration of N2 in thelungs. This, in turn, would give a reasonable estimate of the actual alveolar O2

∗As stated in Paper I, this is likely due to the lower PEEP level as the experiments wereotherwise very similar.

57

concentration, which would provide the safety needed to perform AO duringlonger periods of time, with or without ECCO2R. In Paper I, we also triedto estimate the alveolar N2 concentration and this was successful within areasonable degree of estimation error.

The CO2 problemWith regard to apneic oxygenationAs already stated, the other limitation of AO, and probably the main one, ishypercapnia due to the fact that the subject receives no tidal exchange of gas.In a study by Holmdahl in nine apneic dogs receiving 100% oxygen, threedied within 1 hour. The survivors had a pH of about 6.5 after 1 h and PaCO2had increased to about 47 kPa.80 Furthermore, Draper and Whitehead showedin a similar animal model that the animals died within 2 h.113 One can onlyspeculate as to why the animals died but two reasons come to mind. Firstlythe acidosis per se was deep and probably lethal in and of itself. Secondly, thevery high PaCO2 will compete with PaO2 and at the PaCO2 levels reported,one can easily see how the PAO2 would fall low enough to prohibit adequateoxygenation. This has so far been treated mainly with ECCO2R but there isalso published research using buffers114 and dialysis.115 Previous work onTHAM-buffered AO has been done on dogs114 and humans.80 However, theexperimental setups differ somewhat from ours as Nahas´s dog experimentswere done with de-nitrogenation (ventilating with FiO2 1.0 for 1 hour) andonly one hour of apnea. Our experiments also included much more extensiveblood-gas and hemodynamic measurements. There is also the fact that dogshave collateral ventilation116 which pigs do not. It is unlikely, though, thatthis would have made any difference as the oxygenation was performed withan endo-tracheal tube in both experiments. The human research on AO andTHAM was done with tracheal insufflation of O2 through a broncoscope whichmight have caused some CO2 to escape through the endo-tracheal tube.112 TheAO in Holmdahl’s experiments was also of very short duration. In Paper II,we managed to keep all animals except one alive for the whole duration of theexperiment, i.e. 4.5 hours. At this end-point they were well-oxygenated andthe pH was > 7.2.

With regard to minimal VT ventilationIn Paper III, we found an unexpected effect in the rebound of PaCO2 after theTHAM infusion had been stopped. We then confirmed in Paper IV that, asmight be expected, there was less CO2 exhaled when giving THAM, whichhints at an accumulation of CO2. From a purely chemical stand point, this isperhaps not surprising, as the THAM infusion (raising the pH) will push the

58

equilibrium towards more HCO−3 and less CO2, which is also confirmed bythe experiments in Papers III and IV.

In Paper III, there was no protocol to counter the urinary losses that occurwhen giving the highly osmotic THAM. As this might have affected the elim-ination of THAM via the kidneys, this might explain why the rebound effectis more pronounced in Paper III than in Paper IV.

The THAM problemAs mentioned above, one of the animals in Paper II expired due to hyper-kalemia which is a known side effect of THAM. The doses used were verylarge and in Paper II the mean base excess was 54 mEq/l at the end of theexperiment. All pigs in Paper II also elicited hyperemic skintone and in themiddle of the experiment their hemodynamics were hyperdynamic. This ispartly due to the osmotic effects of THAM and partly due to a perhaps, asof yet, unreported toxic effect of THAM, when given in large doses. Thispresents another set of problems if the method in Paper II were ever to be usedclinically.

The volume problemIn Papers II, III and IV we used THAM to normalize the pH of the animalsduring no ventilation or very low volume ventilation. As mentioned above,the high doses and prolonged infusion of THAM used in Paper II did seemto produce detrimental effects with regards to hemodynamics and perhaps aninflammatory response. However, the ELISAs in Paper III seem to refute thatargument, at least at that particular THAM dose. Using the methodology inPaper III, one could theoretically abolish the concomitant rise in PVR oftenseen in patients that are hypercapnic. It is notable that even after the THAMinfusion had been stopped and the animals had returned to a similar pH asthat of the controls, the PVR was still lower than in the controls. It is notclear whether high CO2 (hypercapnia) is beneficial but if one were to acceptthat notion, an infusion of THAM during low or very low volume ventilationmight even be beneficial as it lowers PVR∗ but does not seem to have anysignificant lowering effect on the PaCO2 in the long run.

∗Even in Paper II, there was only a slight increase in PVR even though the PaCO2 hadincreased to 25 kPa.

59

Conclusions

1. In light of the results in Paper I one might go as far as to claim that ap-neic oxygenation with an fraction of inspired oxygen of less than 1.0 isclearly possible. We also managed to estimate the actual alveolar N2 toa reasonable degree of accuracy. With that knowledge and ignoring anytrace gases, one can conversely also estimate the alveolar O2 concentra-tion.

2. In Paper II, we set out to prove that by using a THAM infusion as the soleproton scavenging technique, one could prolong the duration of apneicoxygenation significantly. At the end of this experiment the animals hada pH > 7.2 and a normal P/F ratio after 4.5h of apneic oxygenation, albeitwith an FiO2 of 1.0.

3. In Paper III, we were surprised to find that the PaCO2 of the animals roseto even higher levels after the THAM infusion and deemed this worthyof further investigation. We also found that despite these high PaCO2levels, the PVR was still at levels similar to those of the baseline, thusmitigating the load on the right heart.

4. In Paper IV, we confirmed our suspicions that, due to the buffering effectof THAM, less CO2 was exhaled and as the PaCO2 again rose above theexpected levels, we also concluded that the renal elimination of CO2 wasinsufficient.

60

Acknowledgements

This thesis has been made possible by the support of the Department of Surgi-cal Sciences, Faculty of Medicine, Uppsala University and the Department ofAnesthesiology and Intensive Care, Akademiska Sjukhuset, Uppsala, Sweden.

All animal experiments were done at the Hedenstierna Laboratory.

My superiors, colleagues and all the personnel of the Central ICU at AkademiskaSjukhuset. We have a great ambiance. Let’s keep it that way.

Anders Larsson, for showing surprising amounts of patience and knowingwhen to use the carrot, and when to use the stick.

Rafael Kawati, for teaching me more every day and for being a paragon ofprofessionalism.

Göran Hedenstierna, bright, kind and humble.

Gaetano Perchiazzi, for inspiring me to think further.

Fernando Suarez-Sipmann, also for being inspiring, helpful and sticking toscientific principles.

João Batista Borges, for our many interesting discussions.

Agneta Roneus, Karin Fagerbrink, Anders Nordgren, Kerstin Ahlgren,Monika Hall, Maria Swälas, Maria Berquist, Bo Sandhagen, for muchhelp with and for teaching me the practicalities of animal experimentation.

Sten Rubertsson, for supporting me in this endeavor.

Joakim Engström, well-mannered and well-tempered, always helpful. A fel-low nerd. This is our millenium.

Henrik Reinius, for your sense of duty and responsibility, and the way youmake people feel good about themselves.

61

Viveka Lindén, for being one of the strongest people I know. Yours is a rarebreed we need more of.

Filip Fredén, for showing us what the right amounts of intellect and humilitylook like. You possess both in abundance.

Ewa Wallin, Ing-Marie Larsson, Anna Aronsson, for sharing your experi-ence in the daily life of a researcher.

Erik Lindgren, David Smekal, Jyrki Tenhunen, for providing positive dis-traction in times of need.

Eva-Marie, Ellen, Lisa, Ida. Because you are. My love for you lights thepath I walk on.

62

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