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Taccone, F. (2014). Brain and sepsis, from macro- to microcirculation (Unpublished doctoral dissertation). Université libre de Bruxelles, Faculté de Médecine – Médecine, Bruxelles.

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UNIVERSITÉ LIBRE DE BRUXELLES

FACULTÉ DE MEDECINE

BRAIN AND SEPSIS:

FROM MACRO- TO MICROCIRCULATION

Dr. Fabio Silvio TACCONE

Department of Intensive Care

Hôpital Erasme

Advisor: Prof. Jean-Louis VINCENT Co-Advisor: Prof. Daniel DE BACKER

This Work bas been presented to obtain the doctoral degree

iphylosophiae doctor, PhD) in Medicine

Acknowledgements

First of ail, 1 would like to thank Prof. Jean-Louis Vincent, who inspired me with his unbelievable strength and passion in the daily work and research. He supported since my first arrivai in the Erasme and was présent during ail the steps of my scientific activity. He invited me to start a PhD project few years ago and significantly contributed to ail my results. The best “Chef’ and mentor I may wish to anyone.

Then, l’d like to thank ail the people who helped me during this long and hard work; Prof Daniel De Backer, who was essential for the high expertise in the

microcirculatory field and who gave me ail the necessary suggestions and support to develop this project and make it published; Dr. Fuhong Su, who showed me how difficult can be the experimental modelling and who guided ail my experiments. 1 would also like to thank Drs. Xinrong He, Ali Abdelhadii, Charalampos Pierrakos, Katia Donadello, Laura Penaccini, Alessandro Devigili and ail those who spent some time with me in the laboratory, discussing about research, medicine and the ... meanings of life.

A spécial thank goes to my friend Dr. Sabino Scolletta, who was more than just a colleague during his two years in Erasme and who supported me during the whole work. Also, l’d like to thank Prof. Cathy De Deyne and Prof. Olivier Dewitte, because they instructed me on how to perform neurosurgery in animais and how to aneilyse brain metabolism with very advanced techniques.

I would like to thank Mr. Hassane Njimi for his time (and skills! ! !) in the statistical analysis of the data; Dr. Karen Pickett, who had the hard task to correct my

“Mediterranean English”; Mrs. Marie-Rose André and ail the ICU staff (Véronique, Colette, Alain, Aurelie, Ina, Dominique) for the precious help and presence.

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in the laboratory and supported me when I finally came back to clinics, first as a Clinical Fellow, then as Senior ICU staff physician.

Also, this Work could not hâve been realized without the help of the Fondation Erasme, which provided economical and technical assistance during three years for my research, and the European Society of Intensive Care Medicine, which gave me the Basic Science Research Award in 2009 and contributed with this grant to complété my Project.

Last but not the least, I would like to thank my family in Italy (my parents, Walter and Virginia, and my brother, Massimiliano), who I had to leave more than 10 years ago for this “new life” in the cold Belgium and who supported me with their love during ail this time.

Jury Members Prof. Jean-Paul SCULIER — President

Department of Intensive Care and Oncologie Emergencies Institut Jules Bordet - Université Libre de Bruxelles Bruxelles (Belgium)

Prof. Kathleen MCENTEE - Secretary Laboratory of Physiology, Faculty of Medicine Université Libre de Bruxelles

Bruxelles (Belgium)

Prof Anne-Pascale MEERT

Department of Intensive Care and Oncologie Emergencies Institut Jules Bordet - Université Libre de Bruxelles Bruxelles (Belgium)

Prof Hazim KADHIM

Department of Pathology -Neuropathology

Centre Hospitalier Universitaire Brugmann - Université Libre de Bruxelles Bruxelles (Belgium)

Prof. Jean-François PA YEN

Department of Anesthesiology and Critical Care Hôpital Albert Michallon

Grenoble (France) Prof Bernard VIGUÉ

Department of Anesthesiology and Intensive Care Hôpital Kremlin-Bicetre - Université Paris-Sud Le Kremlin-Bicêtre (France)

Prof. Jean-Louis VINCENT - Advisor Department of Intensive Care

Hôpital Erasme - Université Libre de Bruxelles Bruxelles (Belgium)

Prof. Daniel DE BACKER - Co-Advisor Department of Intensive Care

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TABLE OF CONTENTS

Summary (in English and French) 13

INTRODUCTION

Brain dysfunction during sepsis 20

Pathogenesis of SAE: a short overview 23

Alterations in brain perfusion during sepsis 29

Cérébral Blood Flow; normal ranges, physiology and régulation

How to monitor cérébral perfusion and autoregulation in septic patients Brain Perfusion in Experimental Sepsis

Brain Perfusion in Human Sepsis

What is microcirculation?

How is microcirculation regulated? How to monitor microcirculation?

Mechanisms of microcirculatory alterations during sepsis Microvascular abnormalities during sepsis

Microcirculation 45

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RESULTS

1. Cérébral autoregulation is influenced by carbon dioxide levels in patients with septic shock.

2. Cérébral microcirculation is impaired during sepsis: an experimental study. 3. Sepsis is associated with altered cérébral microcirculation and tissue hypoxia in

experimental peritonitis.

4. Effects of reversai of hypotension on cérébral microcirculation and metabolism in experimental sepsis.

DISCUSSION AND PERSPECTIVES

CONCLUSIONS

List of Abbreviations

ALI: Acute Lung Injiiry

ANOVA: Analysis Of Variance

APACHE II: Acute Physiology and Chronic Health Evaluation

ARDS: Acute Respiratory Distress Syndrome

BBB: Blood-Brain Barrier

BOLD: Blood Oxygénation Level Dépendent Contrast

CA: Cérébral Autoregulation

CAI: Cérébral Autoregulation Index

CBF: Cérébral Blood Flow

CI: Cardiac Index

CMRO2: Cérébral Metabolism Rate Of Oxygen

CNS: Central Nervous System

COR: Cerebrovascular-C02 Reactivity

CPP: Cérébral Perfusion Pressme

CRP: C-Reactive Protein

CT: Computed Tomography

CVOs: Circumventricular Organs

CVP: Central Venons Pressure

CVR: Cerebrovascular Résistance

DO2: Oxygen Delivery

EEG: Electroencephalography

eNOS: Endothélial Nitric Oxide Synthase

ETCO2; End-Tidal Expired Carbon Dioxide Concentrations

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Fi02: Inspired Fraction Of Oxygen

GCS: Glasgow Coma Score

HbT: Total Tissue Hemoglobin

HES; Hydroxyethyl Starch

ICAM-1; Inter-Cellular Adhesion Molécule 1

ICU: Intensive Care Unit

ICP: Intracranial Pressure

IFN-y: Interferon Gamma

iNOS: Inducible Nitric Oxide Synthase

L-NAME: N-Nitro-L-Arginine Methylester

L-NMMA: N-Monomethyl-L-Arginine

LPR: Lactate/Pyruvate Ratio

LVSWI: Left Ventricular Stroke Work Index

MAP: Mean Arterial Pressure

MCA: Middle Cérébral Artery

MD: Microdialysis

MPI: Mean Flow Index

MOF: Multiple Organ Failure

MRI: Magnetic Résonance Imaging

NIRS: Near-Infrared Spectroscopy

NO: Nitric Oxide

NPC: Number of Perfiised Capillaries

NSE: Neuron-Specific Enolase

OPS: Orthogonal Polarization Spectral

Pa02: PAOP: Pb02: PET: PSPV: PVD: RL: ROC: ROS: SAE: SAH: SDF: SOFA: SPECT: SSEP: St02: SVO2: SVR: SVRI: TBI: TCD: TNF-a: TPC: TVD: VIP:

Arterial Oxygen Tension

Pulmonary Artery Occlusion Pressure Brain Oxygen Tension

Positron Emission Tomography Proportion Of Small Perfused Vessels Perfused Vessels Density

Ringer’s Lactate

■•S.

Receiver Operating Characteristic Reactive Oxygen Species

Sepsis-Associated Encephalopathy Subarachnoid Hemorrhage

Sidestream Dark Field

Sequential Organ Failure Assessment

Single Photon Emission Computed Tomography Somato-Sensory Evoked Potential

Tissue Oxygen Saturation

Mixed Venons Oxygen Saturation of Hemoglobin Systemic Vascular Résistances

Systemic Vascular Résistances Index Traumatic Brain Injury

Trans-Cranial Doppler

Tumor Necrosis Factor Alpha Thoraco-Pulmonary Compliance Total Vessel Density

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Summary

Brain dysfimction is a frequent complication of sepsis and is usually defmed as “sepsis- associated encephalopathy” (SAE). Its pathophysiology is complex and related to a number of processes and pathways, while the exact mechanisms producing neurological impairment in septic patients hâve not been completely elucidated. Alterations in cérébral blood flow (CBF) bave been suggested as a key component for the development of SAE. Réduction of CBF may be caused by cérébral vasoconstriction, induced either by inflammation or hypocapnia. More importantly, the nahoral mechanisms that protect the brain from reduced/inadequate CBF can be impaired in septic patients, especially in those with shock, and this further contributes to cérébral ischemia if blood pressure drops below a critical threshold. Hypercapnia is associated with a narrower autoregulatory plateau, which may potentially results in large CBF variations when mean arterial pressure (MAP) varies within usual targets. However, few data are available on the rôle of PaC02 on cérébral autoregulation (CA). Finally, as SAE occurs also in patients without hémodynamie instability, alterations in brain tissue perfusion could occur independently from hypotension; thus, alterations in cérébral microcirculation, which largely régulâtes régional flow and blood-cellular nutrients exchanges, could contribute to SAE. In septic animais, these microcirculatory abnormalities could be implicated in the development of electrophysiological abnormalities observed during sepsis and contribute to neurological alterations. However, these fmdings were limited by several factors, including the technique used to assess the microcirculation, the short time of observation and the limited amount of fluid resuscitation used in those models.

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patients with PaC02 < 35 mmHg, 7/9 (77%) with PaC02 between 35 and 42 mmHg, and 3/3 (100%) with PaC02 > 42 mmHg had impaired CA. The Receiver Operating Characteristic (ROC) analysis showed that a PaCÛ2 threshold of 38 mmHg had a sensitivity of 50% and a specificity of 100% for the prédiction of impaired CA, with an area imder the ROC curve of 0.76 (95% confidence interval: 0.52-0.91).

Résumé

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Utilisée pour évaluer la microcirculation, le temps d’observation très court et la quantité limitée de fluides administrés au cours de la réanimation liquidienne dans ces modelés.

Dans la première partie de ce travail, j’ai décrit les anomalies de la CA et l’impact de la PaC02 sur la CA chez des patients en choc septique. En étudiant 21 patients en ventilation mécanique, j’ai pu observer que 14 d’entre eux avaient une CA altérée, y compris 7/14 avec une PaC02 < 40 mmHg et 7/7 avec ime PaC02 > 40 mmHg (p = 0.046). De plus, 4/9 (44%) avec PaC02 < 35 mmHg, 7/9 (77%) avec PaCOa between 35 and 42 mmHg, and 3/3 (100%) avec PaC02 > 42 mmHg avaient une CA altérée. L’analyse selon la « Receiver Operating Characteristic » (ROC) montrait une sensibilité de 50% et une spécificité de 100% pour prédire une CA altérée, avec un seuil de PaC02 de 38 mmHg (l’aire sous la courbe de l’analyse ROC était à 0.76 [95% ICs: 0.52-0.91]).

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Brain dysfunction during sepsis

Septic shock and related multi-organ failure (MOF) remain a major cause of morbidity and mortality in intensive care Lmits (ICUs) worldwide [1]. The infectious stimuli, associated with a widespread reaction characterized by the release of numerous circulating pro- inflammatory molécules, can potentially impair the fimction of several organs [2]. Brain dysfunction occurs early during sepsis and is commonly characterized by the development of an altered mental State; however, cérébral abnormalities hâve been also described in late course of sepsis, often accompanied by MOF, hypotension and other systemic events [3, 4]. Hippocrates first reported the association between infections and cérébral dysfunction more than 2500 years ago [5], and sir William Osler also described, later on, the occurrence of “delirium” in patients with ongoing sepsis [6]. Nevertheless, concomitant hepatic or rénal failure, electrolyte and metabolic disturbances, altered glucose homeostasis, hypotension, hypoxemia, hypothermia or neurological side-effects of different pharmacological agents may concomitantly occur in septic patients, complicating the différentiation between sepsis-related brain dysfunction and encephalopathy from other causes [7].

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of brain dysfünction [25]. Finally, sérum levels of NSE and S-lOOB protein hâve been correlated with poor outcome in septic shock patients [26]. Magnetic résonance imaging showed variable degrees of vasogenic edema, related to blood-brain barrier (BBB) breakdown, or ischémie lésions surrounding the Virchow-Robin spaces in septic brain [27].

Pathogenesis of SAE: a short overview

SAE is still poorly imderstood. The pathophysiology is likely to be multifactorial (Figure 1) [35]. The concept of altered brain fimction related to the presence in the blood, and possibly in the brain, of micro-organisms and/or their toxins is considered obsolète as altered CNS fimction is observed also in patients without bacterial bloodstream invasion [36], Disseminated cérébral micro-abscesses hâve been suggested as a cause of SAE [37], although they hâve not been reported in recent post-mortem studies [38]. The spécifie rôle of spécifie bacterial products like endotoxin is unlikely, as the incidence of SAE is similar in Gram- positive and Gram-negative bacteremia, as well as fimgemia and even sepsis with imidentified pathogens [13]. Encephalopathy occurs also in non-infectious conditions, such as pancreatitis or trauma, suggesting that it would be mostly related to the systemic inflammatory response [39,40].

The rôle of inflammation on brain dysfunction has been widely investigated in several in

vitro or experimental models of sepsis; although some of the reported fmdings hâve also been

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astrocytosis through activation of its receptor [44]. The intra-cerebral administration of interleukin-1 and IFN-y in animais induced the same slow-waves EEG patterns than those observed in patients with SAE [45, 46],

More importantly, sepsis-induced systemic inflammation can directly affect brain homeostasis by triggering the two important pathways that are implicated in the response to stress and in the immime System modulation: the circumventricular organs (CVOs), which lack a BBB and hâve a direct communication with circulating mediators of sepsis and the vagus nerve, which is triggered by viscéral inflammation [7]. Once systemic and/or viscéral inflammation are detected by these pathways, an abnormal activation of brain signaling spread to behavioral, neuroendocrine and neurovegetative structures, affecting directly microglial and neuronal cells fimctions and modulating neurosecretion and neurotrasmission [47, 48]. Inflammation rapidly alters the fimction of hypothalamus, which régulâtes the endogenous production of corticosteroids and ceux modulate the inflammatory response [49] and may significantly impair the reticular activating System fimctions, which control consciousness and attention [50]. The cholinergic and serotoninergic releases are altered, while an increase in the y-aminobutyric acid receptor density is observed in the forebrain of septic rats [51, 52]. Brain tissular norepinephrine and epinephrine concentrations are decreased in the forebrain and brain stem of septic animais together with the down-regulation of the cérébral D-adrenergic System [53]. Also, a significant and early increase of plasma aromatic aminoacids during abdominal murine sepsis was foimd to be associated with the development of encephalopathy [54]. The activation of CVO also promotes the entry of leucocytes into the CNS, with further increase of local inflammation [55].

peroxidation in the cérébral vessels Eind the snrrounding parenchyma, which eventually cause structural membrane damage and promote inflammation [56]. Cérébral oxidative stress can also be triggered by the decrease of heat-shock factors or by hyperglycemia [57, 58]. Bacterial endotoxin and inflammatory cytokines increase cérébral tissue levels of glutamate via the up- regulation of iNOS; glutamate can trigger brain cells damage by allowing high intracellular levels of calcium and the activation of several en2ymes that alter cell structures, such as components of the cytoskeleton, membrane, and DNA [59]. Within the brain parenchyma, the activation of astrocytes via the toll-like receptors by the mediators of inflammation [60] can further increase the vulnerability of neurons to glutamate and free radical-mediated injuries [56, 61, 62]. Moreover, sepsis also alters the synthesis of ascorbate, which may provide antioxidant and protective effects on neurons in response to glutamate and ROS release [63]. The complément System, which normally contributes to eliminate bacteria, may be

excessively activated during sepsis and hâve deleterious effect through the activation of glial cells, sécrétion of pro-inflammatory cytokines and génération of other toxic products on brain fimction [64]. Edema formation could be further promoted by the activation of aquaporin channels, which regulate the presence of water in the brain tissue [65]. Alterations in the glucose uptake and metabolism or régional deregulation of intracellular calcium homeostasis hâve also been suggested to contribute to the pathogenesis of SAE [50, 66]. Ail these

pathways cause mitochondrial dysfimction by inhibiting the mitochondrial électron transport Chain and uncoupling oxidative phosphorylation, which ultimately leads to bioenergetic failure and apoptosis of brain cells [38, 58].

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Circadian rhythm Autonomk dysfunction

T Ach and 5 MT fGABA

Figure 1: Schematic représentation of meehanisms involved in the pathogenesis of sepsis- associated encephalopathy. Pro-inflammatory cytokines are released during sepsis. They can either activate vagal fibers or enter the brain causing neurologie dysfunction (see text for details).

5-HT, serotonin (5-hydroxytryptamine); Ach, acétylcholine; BBB, blood-brain barrier; CNS, central

nervous System; CO, carbon monoxide; CVOs = circumventricular organs; ECs, endothélial cells;

GABA, gamma-aminobutyric acid; GLT, glutamate; HPA, hypothalamic-pituitary-adrenocortical;

HSF, heat-shock factors; IL, interleukin; LPS, lipopolysaccharide; NF-k|3, nuclear factor kappa B; NO,

nitric oxide; NTS, nucléus tractus solitarius; PAF, platelet activating factor; PG, prostaglandin; PVN,

paraventricular nuclei; RAS, reticular activation System; ROS, reactive oxygen species; TNF-a, tumor

Alterations in brain perfusion during sepsis

A decrease in brain perfusion is a major déterminant of SAE [74], Alterations of systemic blood flow, associated with tissue hypo-perfiision and poor oxygen distribution, are a key feature of sepsis [2]; indeed, several studies bave evaluated the link between cérébral perfusion abnormalities and brain dysfunction in sepsis. As brain perfusion is primarily dépendent on mean arterial pressure (MAP), which is generally reduced in severe sepsis and septic shock, it would be important to monitor the adequacy of cérébral blood flow (CBF) and oxygénation during sepsis as well as to evaluate the integrity of flow régulation, in order to adapt blood pressure levels and avoid the development of secondary brain ischémie events in such patients [38].

Cérébral blood flow: normal ranges, physiology and régulation.

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and potentially damage brain tissue. On the other hand, low CBF (<18-20 mL/lOOg/min) induces brain ischemia and tissue death when CBF falls below 8-10 mL/lOOg/min. Systemic administration of vasopressors would only minimally affect CBF, provided the BBB is not altered [78]. Pressure autoregulation is thought to be controlled by the baroceptor reflexes and both the upper and the lower limits of CA can be affected by many factors, including

sympathetic nerve activity, arterial carbon dioxide tension (PaC02) and pharmacologie agents [79-82],

The control of CBF is dépendent not only on MAP but also on four other major déterminants, including Chemical, metabolic, neural and myogénie factors (Figure 3) [77]. Although this division may be somewhat artifîcial and these control mechanisms probably operate in concert, it is useful to consider each separately. CBF is extremely sensitive to changes in arterial PaC02 (Chemical), displaying marked increases during moderate hypercapnia and réduction during hypocapnia. Changes in CBF in response to changes in PaC02 are referred to as cérébral C02-reactivity (COR).

The magnitude of cérébral circulatory response is approximately 5% change in CBF for each 1 mmHg change in PaC02, within a range of 25 to 60 mmHg [83]. In contrast, acidosis and alkalosis, for blood pH values ranging from 6.7 to 7.6, hâve little effect upon the CBF [84]. Changes in PaC02 are detected by carotid artery chemoreceptors and this regulatory mechanism can be altered by a lésion of the tegmental reticular formation [77]. The effect of PaC02 seems to be related to changes in local brain perivascular pH rather than direct carbon dioxide action on smooth vascular cells, but could also be mediated by K'^-dependent

channels or adenosine, while vasodilatation induced by hypercapnia can be abolished by prostaglandin synthesis inhibition [85-89]. Astrocytes contribute in maintaining the

induced by hypercapnia is markedly attenuated by moderate hypotension, which contributes to exhaust the capacity of cérébral vessels to fiirther dilate [83]. On the opposite, with marked hypercapnia, the capacity to maintain a constant CBF during hypotension is lost, and CBF will décliné as CPP déclinés [91]. Finally, CBF is less sensitive to changes in arterial oxygen tension (Pa02) over the normal physiological ranges. If Pa02 falls below 50 mmHg, CBF increases markedly (at 30 mmHg CBF is almost twice as high as at 100 mmHg Pa02). Conflicting results hâve been published on the effects of hyperoxia on cérébral perfusion; moderate hyperoxia induced a mild decrease in CBF in healthy volimteers [92], while ventilation with 100% of oxygen has only minimal effects upon CBF in patients with traumatic brain injury [93].

Local CBF is also tightly coupled to neuronal activity (metabolic) so that CBF may adjust to the level of energy génération in the brain (“neurometabolic coupling”). The régional cérébral metabolic activity is represented by the metabolism of oxygen (CMRO2) and glucose

[94]. The précisé mechanisms responsible for this coupling remain elusive and alterations in the concentrations of local métabolites or the génération of several vasoactive Chemical substances (i.e., H and K ions, adenosine, vasoactive intestinal peptide-VIP or products of arachidonic acid) hâve been proposed as pivotai mediators [95, 96]. Astrocytic processes extensively unsheathe cérébral artérioles and, through the link between neuronal synapses and the cérébral vasculature, are in a strategie position to convey cellular signais to the blood vessels [97]. The brain metabolism can also significantly affect the magnitude of CBF

modification to changes of PaC02; indeed, factors that reduce neurons activity (e.g., sédation reduces oxygen and glucose consumption) reduce the cérébral circulation response to

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is that part of cérébral metabolism would use lactate generated by the metabolism of glucose in the astrocytes, through the activation of glycolytic pathways [100]; lactate itself has some vasodilating effect on cérébral artérioles and could contribute to modulated brain perfusion in response to cérébral cells demand [101, 102].

The third factor implicated in CBF régulation is the perivascular innervation (neuro- vascular reactivity) (Figure 3), which dépends not only on the extrinsic nerve supply from the cranial ganglia to the cérébral vasculature, but also on the intra-cerebral neurons linked to these vessels. Most of the extrinsic neuron fibers are sympathetic and can be summarized in three different types, each with distinct origins and neurotransmitters. The first consists of sympathetic neurons arising principally from the superior cervical ganglion (producing norepinephrine and neuropeptide-Y, thus vasoconstriction); the second consists of

parasympathetic neurons in the spheno-palatine and otic ganglia (producing acétylcholine and VIP); the third consists of sensory fibers originating in the trigeminal ganglion (producing substance P and calcitonin gene-related peptide, thus vasodilation) [103]. Nevertheless, attempts to manipulate CBF by either cervical sympathectomy or symphathetic stimulation resulted in variables CBF changes in experimental models [104,105]. Also, the

parasympathetic nerves may hâve some effect only in pain-mediated cérébral vasodilatation while trigeminal fibers can substantially affect CBF only in particular conditions, such as hypertension and seizures [103]. Thus, CBF appears to be primarily regulated by local metabolism with only minor modulation by extrinsic nerves. Among local neuromodulators, dopaminergic axons innervate the intra-parenchymal microvessels and dopamine may directly regulate cortical blood flow [106].

muscle of the vascular wall, stimulating the reflex contraction of radial fibers, which eventually resuit in vasoconstriction [108]. A growing body of evidence suggests that endothelium-dependent pathways are the primary mediators of myogénie control of CA [109]. The endothélium appears to act as the transducer of transmural forces that would stimulate the release of vasodilating substances, such as NO and L-arginine [110, 111]. The rôle ofNO has been confirmed by the vasodilatation of large cérébral arteries and pial artérioles in response to the application of acétylcholine in vivo [112]; this process was dépendent on the NOS activity and could be blocked by NOS antagonist, such as N-monomethyl-L-arginine (L- NMMA) [113].

mmHg

Metabolic Régulation Adenosine PaCO Perivascular pH Adenosine MAP-based Régulation r ^ Myogénie Régulation ^ ,t 1 Extrinsic 1 L... N6urai Régulation 1 Intrinsk |

r.

Carotid sinus, aortic arch (Baroreceptors) Endothe ium Brain acttvity Chemical Régulation Brian Stem Chemoreceptors Sc-o/iy

Figure 3: Schematic représentation of multiple mechanisms of cerebrovascular control. The control of cérébral blood flow is dépendent on five major déterminants: mean arterial

pressure, and Chemical, metabolic, neural and myogénie factors (see text for details). Adapted from [73].

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How to monitor cérébral perfusion and autoregulation in septic patients

Cérébral perfusion bas been initially evaluated using the Kety-Schmidt technique, which applies the Fick principle (arterial and bulb jugular venons content at different time-points of a tracer is proportional to the global blood flow) to calculate CBF; different tracers, such as N2O, xénon or argon hâve been used [118]. Using the same Fick principle, the CMRO2 and the cérébral metabolic rate for glucose could be also calculated [119]. Similarly, the indicator- dilution technique can also estimate CBF, through the injection of a dye solution (i.e.

indocyanine green) and arterial and bulb jugular dye concentrations, which are used to build dilution curves and calculate the mean transit time for the indicator [120]. These methods are radier invasive and time-consuming and hâve also a low temporal resolution.

Neuroimaging can be used to measure global and régional CBF. The CT-perfiision is nowadays routinely used in clinical practice and consists of the sequential scanning of selected brain areas during the injection of a bolus of contrast medium, when it passes

through the cérébral vasculature [121]. Additional imaging techniques include fimctional MRI and positron émission tomography (PET) Scan that can both be used to measure global and régional CBF [122, 123]. Other techniques include single photon émission computed tomography (SPECT), which uses a gamma-emitting tracer (the 99-Technetium), and the Xenon-enhanced CT scan, which évaluâtes the brain distribution of Xénon, after the

There is no general consensus on which is the best method to monitor CA. Testing CA requires to apply a hémodynamie stimulation, such as an increase of MAP through the administration of vasoactive agents, manipulating the ventilator to induce PaCOi changes, the modification of venons retum (i.e., through thigh euff release, application of négative body pressure, tilting test) or the compression of the carotid artery [127]. Thereafter, dynamic changes of CBF are recorded to quantify the reactivity of autoregulatory forces. This approach is limited because it only allows assessment of CA to précisé time-points, i.e. during the different manipulations. Another option to assess CA, without the potentially harmful effects of hémodynamie manipulations, is to analyze the continuons dynamic trends of MAP and CBF over time. In this setting, the most commonly used tool to estimate CA is TCD, although some data suggest that cérébral NIRS could also be a valuable method [126]. Brain autoregulation could be continuously assessed by calculating the moving corrélation coefficient between MAP and middle cérébral artery velocities (VMCA) (so called, Mx index) [128], or between MAP and cérébral oxygénation estimated by NIRS (Tox index) [126]. Briefly, values of MAP and VMCA are calculated every 10 seconds by bedside softwares and Mx/Tox indexes are obtained as the moving linear corrélation coefficient over the last 30 consecutive values. A positive corrélation coefficient indicates a close linear relationship between pressure (MAP) and flow (VMCA), thereby suggesting pressure dependency of CBF and impaired CA, while a coefficient close to zéro or négative (<0.3) indicates intact CA.

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vasodilatation of cérébral artérioles and allows testing cerebro-vascular reactivity to CO2 also in septic patients [129].

Brain Perfusion in Experimental Sepsis

Several experimental papers bave investigated cérébral perfusion during sepsis. In one study on dogs, CBF showed a 30% decrease within 15 min after endotoxin administration, while the arterial blood pressure was still not markedly changed [130]. In a second canine study, CBF decreased immediately after the administration of endotoxin and consistently remained below control values [131]. Cerebrovascular résistances (CVR) initially decreased, then progressively increased to levels significantly higher than normeil and were associated with the lowest CBF levels in the later stages of shock. Nevertheless, other studies also demonstrated that CBF was unchanged during sepsis induced in rats and sheep, while others reported an increase in CBF [132-135]. These seemingly controversial findings may be related to different models (endotoxin vs. bacteria) and species used.

Cerebrovascular reactivity to pressure changes was well maintained in several experimental model of sepsis [139, 136, 137], but in others reactivity to PaCOi changes was altered [138, 139]. In one study, Hinkelbein et al. [140] foimd no significant différence in the global CBF between non-septic and septic animais, despite the presence of significant

hypocapnia in the sepsis group. Although one possible explanation to these findings is that the cerebrovascular tone becomes unresponsive to carbon dioxide stimuli, authors also suggested that, during the hyperdynamie phase of sepsis, brain hyperemia might develop, which

Local inflammation is another important factor influencing brain autoregulation in experimental models. In one study, CBF was increased with preserved autoregulation in rats with pneumococcal sepsis, even if there was a right shift of the lower threshold of MAP at which CBF was kept constant [141]. Importantly, if these animais had also a direct brain injury, represented by concomitant bacterial meningitis, CA was completely impaired, suggesting that pneumococcal bacteremia itself can trigger only cérébral vasodilatation but does not affect CA in the absence of direct brain inflammation. The importance of

inflammation on brain autoregulation was also underlined in a paper by Rosengarten et al., in which cérébral hyperemia induced by transient carotid compression in septic rats was

significantly impaired in those animais receiving high dose endotoxin and having lower MAP [142].

Although some controversy still exists, some studies suggest that sepsis can

significantly impair brain perfusion and CBF régulation. Whether modulating brain perfusion with hémodynamie augmentation and the use of vasopressors may improve brain perfusion after sepsis is still imclear. In one study on a model of sepsis induced by continuous infusion of Pseudomonas aeruginosa [136], CBF remained stable during the hypotension phase even if a redistribution of cardiac output in other organs than the brain was observed. Interestingly, when norepinephrine was used to restore normal MAP, cerebrEd perfusion was unaffected, and the same was observed after the administration of L-NMMA, which inhibits the NO production ffom iNOS. As Meyer and Lingnau [132, 133] observed a significant decrease in CBF after NOS inhibition usingN-nitro-L-Arginine-methylester (L-NAME), it is still

possible that more sélective iNOS inhibitors like L-NMMA, as compared to L-NAME, would leave the constitutive NOS imtouched and thus prevent excessive vasoconstriction.

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in parallel with decreased EEG activity and was associated with reduced glucose uptake, measured by PET-scan, and increased inflammation [143]. These data suggested that an early drop of CBF could be related to régional changes in neuronal activity and energy demand and may be independent from MAP changes in septic animais. Although different areas of the brain showed significant différences in cérébral metabolic changes during sepsis (i.e. an increase of 27 to 33 % in the septal nucléus and raphe nucléus and a decrease of 14 to 27 % in the auditory cortex, latéral geniculate, superior colliculus, hippocampus, pariétal cortex, and locus coeruleus) [50], the influence of cérébral metabolic changes on brain perfusion during a septic process may add a new key of interprétation.

Brain Perfusion in Human Sepsis

The concept of reduced cérébral perfusion as a major déterminant in SAE development was supported by a rétrospective analysis of patients developing sepsis after surgery, which showed that hypotension was the only predictor of delirium in [144]. Also, in an autopsy study analyzing the brain of patients who died ffom sepsis, multiple ischémie lésions could be identified in different areas of the brain and were attributed to hypotensive events, which may occur in the presence of preexisting cerebrovascular disease as well as in case of impairment ofCBF autoregulation [38].

Several studies on healthy volimteers or septic patients hâve been conducted to imderstand the changes of CBF during a severe infections process, as well as brain autoregulatory

experiment on healthy volnnteers, when sepsis was induced using an intravenous bolus of

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Table 1. Clinical studies on cérébral hemodynamics in sepsis.

* Only in patients with septic shock; * ** In case of lower MAP and higher values of PaC02; *** Associated with reduced PaC02

In mechanically ventilated septic patients [148], authors showed that there was only a moderate réduction of cerebrovascular reactivity to PaC02 when compared to values in awake Controls, but consistent with values obtained during sédation and anaesthesia. Bowton et al.

[74] also reported a normal response to CO2 changes in septic patients. More recently, in ongoing sepsis for more than 48 hours, Thees et al. showed CO2 reactivity was to he intact in

10 septic patients treated by mechanical ventilation [120]. Hypocapnia was associated with a réduction of CBF without affecting CMRO2, which was already within low ranges at baseline. None of the characteristics of patient population, including Acute Physiology and Chronic Health Evaluation (APACHE) II score, température, MAP, CI had any significant association with CO2 reactivity. In contrast with these fmdings, Terborg et al. [149] observed, in a small cohort of brain injured patients developing severe sepsis and septic shock, that

cerebrovascular reactivity to PaCÛ2 was severely impaired, independently of changes in MAP. Nevertheless, the pre-existing neurological illness of these patients may hâve affected the results. In another study by Bowie et al., on 12 patients, 3 of them had normal

cerebrovascular reactivity, 7 had impaired vasomotor tone and two had a greater response to CO2 changes [150]. Recently, Szatmâri et al. found septic patients with SAE had altered cerebro-vascular reactivity as assessed by an acetazolamide injection test [129]. Finally, the cerebrovascular reactivity to PaC02 in patients with septic shock was lower than in control patients, with a significantly lower reactivity in patients receiving dexmedetomidine than propofol [151].

Conceming CA, in a First study on 10 patients with sepsis and altered mental status, Matta et al. [148] showed that CA was intact within the First 24 hours after ICU admission, when phenylephrine infusion was used to increase MAP within the normal pleateau of

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Microcirculation

Interestingly, as SAE occurs also in patients without hémodynamie instability, one may argue that alterations in oxygen supply to the cérébral tissue during life-threatening infections could occur independently from low cardiac output and hypotension. Thus, it would be the microcirculation, which largely régulâtes tissue perfusion and blood-cellular nutrients exchanges, the key component in the development of tissue hypoxia in septic patients.

What is microcirculation?

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Microcirculation has then three major sectors: pre-capillEiry (résistive), capillary (swap) and post-capillary (capacitive). In the pre-capillary sector, artérioles and pre-capillary sphincters participate. Their funetion is to regulate blood flow and tissue perfusion before blood enters the capillaries and venules by the contraction and relaxation of the smooth muscle found on their walls. As a conséquence, microcirculation blood flow remains constant despite of changes in systemic blood pressure. This mechanism is présent in ail tissues and organs of the hiunan body. The second sector is the capillary sector, which is represented by the capillaries, where substance and gas exchange between blood and interstitial fluid takes place. Finally, the post-capillary sector is represented by the post-capillary venules, which also allows free movement of some substances [160]. Microcirculation also serves to regulate température, to activate the coagulation cascade and it is involved in the response to inflammation as well as in the angiogenesis process [158]. Most vessels of the microcirculation are lined by flattened cells, the endothélium, which acts as a continuous, sélective, semi-permeable séparation between the vessel lumen and the surrounding tissue, controlling the passage of water, ions and small molécules and the transit of white blood cells into and out of the bloodstream. Through the presence of several intercellular tight junctions and desmosomes, it maintains its integrity and limits the pénétration of circulating pathogens into peripheral tissues [161].

How is microcirculation regulated?

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vasodilation. Importantly, it should be remembered that capillary hematocrit is less than systemic hematocrit, due to the necessary presence of a plasma layer at the endothélial surface. Accordingly, hematocrit is proportional to capillary radius, so vasodilation will markedly increase local oxygen delivery as the resuit of a combined increase in flow and in oxygen content. Finally, it should be noted that adaptation of capillary perfusion at the organ level does not dépend on systemic arterial pressure and cardiac output but, of cotirse, will resuit in increased cardiac output if venous retum increases as a resuit of a major increase in capillary flow, as during exercise or feeding.

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100

Vein

Venule

Pre-capill.

sphincter

Arteiy

Ca^illaries

Tissue cells

POISEUILLE'S LAW

AP r4 7T

8

How to monitor?

The évaluation of the microcirculation can include assessment of its transport and exchange functions, permeability, and régulation of inflammation and coagulation. In our Work, we hâve predominantly focused on the transport and exchange functions. By définition, any device looking at the microcirculation can only evaluate the microcirculation in the microvascular bed in which it is implemented. The ability of that spécifie window to represent other beds dépends on the mechanisms implicated in microvascular disease (generalized, diffuse but somewhat heterogeneous or localized), on organ microvascular architecture, and on local factors (local vasoconstriction/pressure). Some areas may be more relevant than others, as illustrated by a relationship with microcirculatory alterations in that area with outcome. Nevertheless one should, at best, consider that the area being investigated is a window that reflects the minimal alterations that are likely to be observed in other areas, provided that local factors do not exacerbate the lésion in the investigated area [172].

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Laser Doppler techniques are frequently used to measure microvascular blood flow. They can be applied on various tissues and probes can even be inserted in the upper digestive tract through a nasogastric tube [178]. As laser Doppler techniques provide measurements of blood flow in relative units (mV), one can only assess relative changes ffom baseline. The main limitation of this technique is that it measures flow in a variable voliune of tissue and it is imable to detect it in individual vessels. Moreover, the sampling volume of current laser Doppler devices is between 0.5 and 1 mm^, so that the flow that is measured represents the average flow in at least 50 vessels, including artérioles, capillaries, and venules of variable size, direction, and perfusion. Reflected-mode confocal laser scaiming microscopy is an attractive development as it can both allow measurements of vascular density, diameters, and blood flow, with a semi-quantitative évaluation of heterogeneity of perfusion, especially on human skin [179,180].

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and gut [189-192]. In intact humans, this technique can be applied to the skin, conjunctiva, gingiva, sublingual area, iléostomies or colostomies, and rectal mucosa [193-198]. In the sublingual area, which is the area that has been investigated most, capillaries and venules of variable size (resolution is 2-3 Dm) can be visualized; artérioles are usually not visualized because they are located in deeper layers. Red blood cells are identifîed as black bodies and tissue perfusion can be characterized in individual vessels.

Measurements of O2 tension or satiuation in a piece of tissue reflect the balance between O2 transport and O2 consumption in that tissue. These measurements are, therefore, influenced by flow but also by hemoglobin content, arterial PO2, and O2 consumption. Venons O2 saturation is often considered as a gauge for the circulation [199], but this measurement can be misleading. As illustrated in Figure 6, venons O2 saturation is a poor indicator of microvascular dysfunction: venons O2 saturation can be high or low for the same degree of microvascular shimting. Several studies hâve shown that measuring SVO2 does not provide much information about microvascular alterations [200, 201].

PO2 can be measured in tissues with Clark-type électrodes, which are made of multiple platinum wires that measure PO2 in the surroimding tissue. These électrodes accurately

measure tissue PO2 when oxygen is homogenously decreased but they are not suitable in

Réflectance spectroscopy measures tissue oxygen saturation (SO2). Light generated with a rapidly rotating filter disk at 64 different wavelengths of 2-nm incréments in the range 502-628 nm is directed through a microlight guide to the tissues. The use of different wavelengths allows SO2 measurement due to light absorption by oxy- and deoxy-hemoglobin. The resolution of the probe is very sharp (1 nm) allowing SO2 measurements in a very small area. Nevertheless, the depth of the tissue sampled is quite large [204], so that the sampling volume is not so small and reporting only the mean value of tissue SO2 is misleading [205, 206], and no conclusions can be drawn on the presence or absence of hypoxie areas. Near- infrared spectroscopy (NIRS) is a technique that utilizes near-infrared light to measure chromophores (oxy- and deoxyhemoglobin, myoglobin, and cytochrome 3) in tissues [207]. The fractions of oxy- and deoxyhemoglobin are used to calculate tissue O2 saturation (St02). In addition, total light absorption is used to compute total tissue hemoglobin (HbT) and the absolute tissue hemoglobin index (THI), two indicators of blood volume in the région of microvasculature sensed by the probe [208]. The NIRS signal is limited to vessels that hâve a diameter less than 1 mm (artérioles, capillaries, and venules), but, as 75% of the blood in a

skeletal muscle is venous, NIRS StÛ2 measurements mostly represent local venons

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hence, in the majority of studies, the thenar eminence has been used because the thickness of skin and adipose tissue covering this muscle is less influenced by any increase in fluid content or body mass index.

Tissue CO2 represents the balance between CO2 production and flow to the tissue. It is influenced by arterial CO2, so that the tissue to arterial gradient, or PCO2 gap, is usually calculated. The PCO2 gap reflects more the adequacy of flow than the presence of tissue hypoxia, imless very high PCO2 gap values are reached [214, 215]. Tissue PCO2 can be measured by électrodes inserted in tissues, probes in contact with the tissue, or tonometry. In experimental conditions, there was a close relationship between mucosal PCO2 and mucosal perfusion [216]. In patients with sepsis, there was no corrélation between the gastric PCO2 gap and total splanchnic perfusion [217], although changes in mucosal PCO2 correlated with

changes in mucosal perfusion [218]. Sublingual and buccal PCO2 monitoring hâve been

developed [219, 220]; although attractive, it is unfortunately not easily available at the présent time and available for research purposes only.

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z:i

X

Magnifying Multiple light sources

lens _(latéral) Reflected light |(deeper layers) Screen Reflected light (superflclal)

Figure 5: Orthogonal polarization spectral (OPS) imaging technique (Upper Panel). Polarized light is directed to the tissue. Light reflected by the superficial layers is still polarized and discarded by the orthogonal filter. Light reflected ffom the depth of the tissues has

encountered many scattering events and has lost its polarized characteristics so is not

discarded by the orthogonal filter; this light is absorbed by hemoglobin contained in red blood cells so that these will be seen as gray/black bodies on the screen. Sidestream dark field

Normal flow

I>ow but homo{;enous flow

miymin 24 (\ 02 = !H») ml/min Sv02 60% ml/min 24 (\ 02 = 96) ml/min 0, ml/min i 0 (\'02 = 48) 0 * ml/min S\02 80%

Figure 6: Impact of heterogeneous perfusion on tissue metabolism and venous oxygen saturation. Normal situation (top panel): 02 is delivered at 240 ml/min in four perfused capillaries. The tissues extract oxygen to meet cellular oxygen consiunption (VO2). Low flow but homogeneous perfusion (middle panel): half the oxygen is delivered to the tissue but ail the capillaries are perfused. The amount of oxygen is sufficient to meet oxygen requirement of the cells. Hence, VO2 is preserved even though venous oxygen saturation is severely decreased. Heterogeneous flow (bottom panel): even though total oxygen delivery is

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Mechanisms of microcirculatory alterations during sepsis

Several mechanisms, including endothélial dysfimction, altered balance between levels of vasoconstrictive and vasodilating substances, glycocalyx alterations, and interactions with circulating cells (Figure 7), are implicated in microcirculatory alterations during sepsis. The crucial issue is to imderstand which are the major mechanisms that contribute to the microvascular alterations présent in septic conditions and, more importantly, whether these could be improved with therapy.

Multiple studies hâve shown that endothélial dysfimction occurs in sepsis, as evidenced by a decreased sensitivity to vasoconstricting but also vasodilating agents. However, most of these trials used large arteries, up to first-order artérioles, and it is not known to what extent the findings may apply to more distal artérioles and capillaries. In addition, communication between endothélial cells may be altered. Experimentally, Tyml et al. showed that the communication rate between microvessels 500 pm apart was markedly impaired [223]. The study of post-ischemic hyperemia provides some indirect evidence that endothélial dysfimction may play a rôle. Using laser Doppler and NIRS techniques, several authors hâve reported that the post-ischemic hyperemic response is blunted in patients with sepsis and that these alterations are related to the severity of organ dysfimction and outcome [209, 224].

Activation of coagulation may play a key rôle in the pathogenesis of microcirculatory alterations. In mice challenged with endotoxin, fibrin déposition occurred in a significant proportion of capillaries; the addition of anticoagulant factor decreased the nmnber of non- perfused capillaries, whereas the number was increased after the addition of procoagulant factors [226]. However, microthrombi formation is infrequently observed in experimental sepsis [227].

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Endothélial dysfunction

(Impaired sensitivity of

vasoconstrictive/vasodllating

adhesion of RBC

and WBC to

endothélium

Red blood cell

White blood celi

Microvascular abnormalities during sepsis

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The first hypothesis of this work was that sepsis could directly cause brain damage by altering brain global perfusion. In clinical practice, several therapeutic protocols exist to guide the management of patients suffering from severe sepsis or septic shock, including early-goal directed therapy, prompt administration of antimicrobiais or protective limg ventilation; however, we still lack a spécifie neuro-protective approach to prevent or minimize secondary ischémie brain injuries occurring during sepsis. This is probably due to the limited data available on the mechanisms inducing brain hypoperfusion in septic patients. However, most studies evaluating CBF in this setting showed a reduced CBF but did not provide a relevant link between these observations and the development of SAE. More than the absolute measure of CBF, the évaluation of CA appears to be fimdamental for the hémodynamie management of septic patients; as such, impaired CA may leave brain tissue unprotected against possibly harmfiil effects of blood pressure changes during sepsis, leading to cérébral ischemia. As PaCOa is the main déterminant of cérébral vaso-reactivity, we evaluated whether normal or high PaC02 levels (i.e. excessive vasodilation) could further alter CA in patients with septic shock; PaCÛ2 is frequently manipulated in clinical practice and a potential association of PaCÛ2 and CA abnormalities should alert ICU physicians on the potential deleterious effects of carbon dioxide changes on CBF variations during septic shock.

As SAE occurs also in patients without hémodynamie instability, the second hypothesis of this work was that alterations in cérébral microcirculation, which régulâtes cérébral tissue perfusion and oxygénation, would be one of the key components in the development of brain hypoxia during sepsis. As cérébral microcirculation assessment is not feasible in clinical practice yet, we explored this hypothesis in an experimental model of peritonitis-induced septic shock, using a multimodal monitoring of brain microvascular flow, oxygénation and metabolism. In particular, we evaluated: a) whether microvascular

between changes in microvascular flow, cérébral oxygénation and metabolism; c) the effects of changes in systemic hemodynamics on cérébral microcirculation and oxygénation. These fmdings may identify some new mechanisms associated with SAE development and

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Cérébral Autoregulation is Influenced by Carbon Dioxide Levels

in Patients with Septic Shock

Fabio SUvio Taccone * Diego Castanares-Zapatero * Daliana Peres-Bota * Jean-Louis Vincent *

Jacques Berre’ * Christian Melot

Introduction

Septic shock and related multiple organ failure (MOF) remain major causes of morbidity and mortality in intensive care miits [235]. Acute détérioration in mental status during severe infections, the so-called “sepsis-associated encephalopathy” (SAE), can develop in up to 70% of these patients [9,12]. Clinical signs of SAE range from mild disorientation to lethargy and coma and there may be associated neurophysiological

disturbances [15, 25]. Importantly, SAE can be an early sign of sepsis and may contribute to long-term cognitive complications and increased mortality [28, 29].

The pathogenesis of SAE remains unclear [17]. Varions factors hâve been suggested to contribute to the pathogenesis, including cérébral effects of circulating inflammatory mediators, disruption of the blood-brain barrier, impaired astrocytic fiinction, altered neurotransmission, and induced neuronal apoptosis [7, 8]. A rétrospective study identified severe hypotension as an important risk factor for SAE and the authors therefore suggested that SAE may be primarily related to ischémie damage rather than to other causes [144]. This concept was supported by an autopsy study in which ischémie lésions were the major

pathologie findings in the brain of patients who died in septic shock [38]. Although

impairment of tissue perfusion and blood flow redistribution are typically considered as being involved in the pathogenesis of organ failure, it remains unclear whether sepsis can induce profound and sustained brain damage by reducing global or régional cérébral blood flow

In healthy individuals, CBF is kept constant over a wide range of mean arterial pressmes (MAP). This phenomenon, called cérébral autoregulation, is accomplished by changes in cérébral vascular tone and is generally observed for MAP values between 50 and 150 mmHg [73]. There are conflicting reports regarding disturbances in cérébral

autoregulation during sepsis. In a recent study, Pfister et al. [125] reported impaired cérébral autoregulation in 12/16 patients with severe sepsis and septic shock, which was associated with clinical delirium, higher levels of slOOp, and worse outcome. However, Matta and Stow

[148] reported intact cérébral autoregulation in 10 patients with early sepsis. Impaired

cérébral autoregulation may leave brain tissue unprotected against possibly harmful effects of blood pressure changes during sepsis, potentially leading to cérébral ischemia. With its potent vasodilating properties, PaC02 is an important regulator of the cérébral circulation and

hypercapnia can directly increase CBF [236]. Although the vascular response to PaC02 in the brain has been shown to be markedly attenuated by moderate hypotension in experimental studies [83], Thees at al. recently reported normal reactivity to CO2 in patients with sepsis [120]. Interestingly, changes in CBF induced by changes in PaC02 can influence the pressure limits of arterial blood pressure, referred to as the autoregulatory plateau, within which cérébral autoregulation opérâtes to keep CBF constant [237]. Thus, during hypercapnia, this autoregulatory plateau is narrowed to between a lower upper limit and a higher lower limit of MAP [91].

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However, the rôle of PaC02 on cérébral autoregulation dnring sepsis bas not been investigated. The purpose of this study was, therefore, to evaluate cérébral autoregulation in patients with septic shock and to assess the influence of PaC02 concentrations on the autoregulatory cérébral capacity in these patients.

Methods

This prospective study was conducted in the 35-bed Department of Intensive Care of a University hospital. The hospital Ethics Committee approved the study protocol, and

informed consent was obtained from patient’s next of kin. We enrolled consecutive adult patients with septic shock, as defined by standard criteria [238], for less than 72 h who were being treated with mechanical ventilation. Exclusion criteria were âge <18 years, intracranial infection, a history or clinical evidence of a neurologie disease, or significant arrhythmias. Patients in whom cérébral vessel waveforms were hardly obtained using TCD or who had significant sténoses of the extracranial and intracranial cérébral arteries on echo-Doppler examination were also excluded.

In ail patients, démographie data, pre-existing chronic diseases, and admission diagnosis were collected. The GCS was used to assess the neurologie status on admission. The source of sepsis and relevant microbiological results were recorded. Acute lung

injury/acute respiratory distress syndrome (ALI/ARDS) were diagnosed according to standard criteria [239]. Acute rénal failure was defmed by a sequential organ failure assessment

total protein, albumin, and CRP concentrations were recorded on the day of the study. ICU and hospital length of stay, overall mortality and cause of death were noted.

Each patient had continuons monitoring of heart rate, CVP, MAP (using a radial or fémoral arterial cathéter); pulmonary artery pressures and cardiac output were continuously measured using the thermal dilution technique via a triple-lumen pulmonary artery cathéter (Vigilance; Baxter Edwards Critical-Care, Irvine, CA), previously inserted for diagnostic or therapeutic purposes. CI and systemic vascular résistance index (SVRI) were calculated using standard formulas. Arterial and venons blood gases, oxygen saturation, and hemoglobin were determined at baseline (ABL 700; Radiometer, Copenhagen, Denmark). End-tidal expired carbon dioxide (ETCO2) was continuously measured (SC9000; Drager Medical, Germany). Volume-control ventilation was provided with a tidal volume of 6-8 ml/kg and a plateau pressure not exceeding 30 cmH20. The inspiratory oxygen fraction was set to maintain a Pa02 above 70 mmHg. Morphine was used for analgesia. Sédation was provided by a

continuons infusion of midazolam, titrated as clinically required to target a Brussels sédation score of 4 [243]. No other agents were used for analgesia or sédation during the study period. Fluid and drug administration remained unchanged throughout the study.

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were kept in the 30-degree head up position throughout the procedure. PaC02 was measnred immediately before starting each test and at the end of the procedure to confirm stable levels. Cérébral vascular résistance (CVR) was calculated as CVR = MAPA'^MCA. Changes in cerebrovascular résistance (ACVR) were estimated from the changes in (AVMCA) in response to changes in MAP (AMAP). The cérébral autoregulation index (CAI) was

calculated as the ratio of the relative changes in CVR and MAP (CAI = AMAP%/ACVR%); the normal limits are between 0 and 2 [243].

Statistical analyses were performed using a SPSS 13.0 program for Windows NT software package (2004). Descriptive statistics were computed for ail study variables. A Kolmogorov-Smimov test was used, and histograms and normal-quantile plots were examined to verily the normality of distribution of continuons variables. Discrète variables were expressed as coimts (percentage) and continuons variables as means ± SD or médian with interquartile ranges (IQR: 25th-75th percentiles), depending on the data distribution. Categorical variables were compared by chi-square or Fisher’s exact test, as appropriate. Continuons variables were compared using a Student’s t test. Non-parametric tests were used if the data were not normally distributed. We conducted a receiver operating characteristic (ROC) analysis to détermine the best PaCOi cut-off of, in terms of sensitivity and specificity, for the prédiction of impaired CA. Différences at a level of p < 0.05 were considered

statistically significant.

The study included 21 patients with septic shock (2 women, 19 men); their clinical characteristics are summarized in Table 2. Ten patients (47%) had a history of arterial hypertension and 2 (9%) of insulin-requiring diabètes; GCS at ICU admission, before mechanical ventilation and sédation, ranged ffom 5 to 15 (médian: 13). The duration of septic

shock was 2 days for 14 patients and 3 days for 7; 17 patients (80%) had acute rénal failure (médian créatinine 1.9 [ranges; 1.3-4.1] mg/dl and médian urea 84 mg/dl [ranges: 45-125] mg/dl) and 14 (66%) had ALI/ARDS on the study day. Twelve patients (57%) were treated with hydrocortisone, 12 with dobutamine, and 8 (39%) with activated protein C. Eight patients (38%) eventually died because of complications related to sepsis. The remaining patients were discharged without gross neurologie sequelae.

At study inclusion, MAP was 65 ± 6 mmHg, VMCA 60 ± 20 cm/s, and médian PaCOi 35 [28^9] mmHg. Norepinephrine infusion was increased from 7 [2-70] to 20 [8-110] meg/min to raise MAP from 65 ± 6 to 96 ± 13 mmHg. Ail other major variables, including CI, remained constant throughout the study (Table 3), except for a significant increase in SVRI with the higher doses of norepinephrine. No relevant changes in PaC02 were observed between the start and the end of the procedure.

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Discussion

The major finding of this study is that CA is often impaired in the early phase of septic shock. Impaired CA seemed to be more frequent in patients with hypercapnia, suggesting that PaC02 may alter the responsiveness of cérébral vasculature to arterial pressure stimuli. Steady State conditions were maintained diuing the study, and we avoided hyperglycemia, extreme acidosis, and uremia, and changes in température and hemoglobin concentration, ail factors that are known to affect cérébral autoregulation [148]. The drugs used for sédation or analgesia and norepinephrine do not influence cérébral autoregulation [244-246], so that one can assume that modifications in VMCA were due only to changes in MAP.

Hyperventilatory hypocapnia probably reduces CBF in septic patients without compromising cérébral metabolism, as indicated by an unchanged cérébral metabolic rate of oxygen (CMRO2) [120]. However, in a canine model of sepsis, only normocapnie animais showed an increase in CMRO2 of over 40% [247], suggesting that, in the presence of normal or high

PaCÛ2 levels, CBF and cérébral metabolism could be uncoupled during sepsis, probably

because of loss of CA. Autoregulation of CBF is a sensitive mechanism, which can be impaired by varions pathological conditions, and has a direct impact on delayed ischémie events and poor outcome [248, 249]. Even though the précisé mechanisms of CA remain controversial, vascular caliber changes are mediated by a complex interplay of myogénie and

metabolic mechanisms. In a study describing the effects of CO2 on CA [91], the