PREDICTORS OF BRAIN INJURY AFTER EXPERIMENTAL HYPOTHERMIC CIRCULATORY ARRESTAn experimental study using a chronic porcine model

MATTIPOKELA

Department of Surgery,University of Oulu

OULU 2003

MATTI POKELA

PREDICTORS OF BRAIN INJURY AFTER EXPERIMENTAL HYPOTHERMIC CIRCULATORY ARRESTAn experimental study using a chronic porcine model

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium 1 of the University Hospitalof Oulu, on October 10th, 2003, at 12 noon.

OULUN YLIOPISTO, OULU 2003

Copyright © 2003University of Oulu, 2003

Supervised byProfessor Tatu JuvonenDocent Kai Kiviluoma

Reviewed byDocent Mikko HippeläinenDocent Jorma Sipponen

ISBN 951-42-7105-X (URL: http://herkules.oulu.fi/isbn951427105X/)

ALSO AVAILABLE IN PRINTED FORMATActa Univ. Oul. D 745, 2003ISBN 951-42-7104-1ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2003

Pokela, Matti, Predictors of brain injury after experimental hypothermic circulatoryarrest An experimental study using a chronic porcine modelDepartment of Surgery, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland Oulu, Finland2003

Abstract

There is a lack of reliable methods of evaluation of brain ischemic injury in patients undergoingcardiac surgery. The present study was, therefore, planned to evaluate whether serum S100β protein(I), brain cortical microdialysis (II), intracranial pressure (III) and electroencephalography (EEG)(IV) are predictive of postoperative death and brain ischemic injury in an experimental survivingporcine model of hypothermic circulatory arrest (HCA).

One hundred and twenty eight (128) female, juvenile (8 to 10 weeks of age) pigs of native stock,weighing 21.0 to 38.2 kg, underwent cardio-pulmonary bypass prior to, and following, a 75-minuteperiod of HCA at a brain temperature of 18°C. During the operation, hemodynamic,electrocardiograph and temperature monitoring was performed continuously. Furthermore, metabolicparameters were monitored at baseline, end of cooling, at intervals of two, four and eight hours afterHCA and before extubation. Electroencephalographic recording was performed in all animals, serumS100β protein measurement in 18 animals, cortical microdialysis in 109 animals, and intracranialpressure monitoring in 58 animals. After the operation, assessment of behavior was made on a dailybasis until death or elective sacrifice on the seventh postoperative day.

All four studies showed that these parameters were predictive of postoperative outcome. Animalswith severe histopathological injury had higher serum S100β protein levels at every time intervalafter HCA. Analysis of cortical brain microdialysis showed that the lactate/glucose ratio wassignificantly lower and the brain glucose concentration significantly higher among survivors duringthe early postoperative hours. Intracranial pressure increased significantly after 75 minutes of HCA,and this was associated with a significantly increased risk of postoperative death and brain infarction.A slower recovery of EEG burst percentage after HCA was significantly associated with thedevelopment of severe cerebral cortex, brain stem and cerebellum ischemic injury.

In conclusion, serum S100β protein proved to be a reliable marker of brain ischemic injury asassessed on histopathological examination. Cerebral microdialysis is a useful method of cerebralmonitoring during experimental HCA. Low brain glucose concentrations and high brain lactate/glucose ratios after HCA are strong predictors of postoperative death. Increased intracranial pressureseverely affected the postoperative outcome and may be a potential target for treatment. EEG burstpercentage as a sum effect of anesthetic agent and ischemic brain damage is a useful tool for earlyprediction of severe brain damage after HCA. Among these monitoring methods, brain corticalmicrodialysis seems to be the most powerful one in predicting brain injury after experimentalhypothermic circulatory arrest.

Keywords: aortic arch surgery, brain infarction, cerebral ischemia, cerebral microdialysis,electroencephalography, Hypothermia, hypothermic circulatory arrest, intracranialpressure, S100b protein

To Saara, and my mother and father

AcknowledgementsThis work was carried out at the Cardio-thoracic Research Laboratory of the Departmentof Surgery, Oulu University Hospital, during the years 1998–2003.

I owe my deepest gratitude to my supervisor, Professor Tatu Juvonen, Head of theDepartment of Surgery, who introduced and developed in this University a chronicporcine model of hypothermic circulatory arrest for studying cerebral protection methodsduring complex cardiac and aortic arch surgery. I am indebted to him for havingintroduced me to this research issue and for providing me with continuous support duringthese studies.

I wish to thank my friends and co-workers Jussi Rimpiläinen M.D., Ph.D. and PekkaRomsi M.D., Ph.D. for having taught me surgical skills, and with whom I also sharedunforgettable, alpine skiing in Chamonix (2000–2002). I also thank my friend and co-worker Vesa Anttila M.D., Ph.D. for having taught me surgical skills. I am grateful toDocent Kai Kiviluoma, Docent Vilho Vainionpää, Timo Salomäki M.D., Ph.D. andKauko Korpi R.N for taking care of cardio-pulmonary bypass in the laboratory. I alsothank my younger research colleagues, Janne Heikkinen M.S., Timo Kaakinen M.S.,Erkka Rönkä M.S. and Sebastian Dalhbacka M.S. You were irreplaceable in this work. Inaddition, I am especially grateful to Docent Fausto Biancari, who helped me, forfinalizing the last three studies with great skill and giving irreplaceable help with myEnglish for presentations and for this thesis manuscript.

I also thank my co-authors: Professor Jorma Hirvonen, who has examined thehistology of the brain in these studies, also after his retirement; Ari Mennander, M.D.,Ph.D., for his contribution in brain histological analysis, Docent Ville Jäntti, Pasi LepolaM.Sc., Minna Mäkiranta, M.Sc., Elina Remes M.S., for EEG monitoring and analysis;and Pasi Ohtonen M.Sc. for statistical analysis and support.

I thank our research laboratory staff, and especially Seija Seljänperä, Tanja and VeikkoLähteenmäki and its director Hanna-Marja Voipio, D.V.M., Ph.D., for providing facilitiesfor the present work.

I am grateful to Docent Mikko Hippeläinen, and Docent Jorma Sipponen, forreviewing the present manuscript.

I would like to thank the previous Chief of Cardio-thoracic and Vascular Surgery,Professor Pentti Kärkölä, and the current Chief of Cardio-thoracic Surgery, Docent MarttiLepojärvi, and the surgeons in the Division of Cardio-thoracic and Vascular Surgery.

My special thanks to Martti Lepojärvi, Esa Salmela and Vilho Vainionpää for havinghelped my family.

My deepest thanks to my friends Jussi Koivunen, M.D., Jussi Piuva, M.D., JyrkiVainionpää M.Sc. (eco.), Antti Kummu M.Sc (eco.), Jouni Ruokonen, M.D., Anna Terho,M.D., Hanna Rahtu, M.D., Petri Kynsilehto M.Sc. (eng.), Markus Kulmunki M.Sc. (eco.),Sami Karppi M.Sc. (eco.), Sami Niska M.Sc. (eng.), Tero Pinola, M.D., Mikko Gärding,M.D. and Tarmo Heinonen, M.D. I am privileged to have friends like you.

I am deeply grateful to my parents Marja-Leena and Risto Pokela for their love andsupport.

Finally, I wish to express my loving thanks to my girlfriend Saara Ojala.This work was financially supported by my parents and by grants from the Oulu

University Hospital, Oulu University, The Finnish Foundation for CardiovascularResearch, The Finnish Medical Foundation, Aarne and Aili Turunen Foundation, the IdaMontin Foundation and the Sigrid Juselius Foundation.

Oulu, May, 2003 Matti Pokela

AbbreviationsADP Adenosine diphosphateATP Adenosine triphosphateCK-BB Creatine kinase brain-brain isoenzymeCMRO2 Cerebral metabolic rate of oxygenCSF Cerebrospinal fluidCPB Cardiopulmonary bypassECF Extracellular fluidEEG ElectroencephalographyGFAP Glial fibrillary acidic proteinHCA Hypothermic circulatory arrestICP Intracranial pressureIQR Interquartile rangeMRI Magnetic resonance imagingNIRS Near-infrared spectroscopyNMDA N-methyl-d-aspartateNSE Neuron-specific enolasePCr PhosphocreatineRCP Retrograde cerebral perfusionSCP Selective antegrade cerebral perfusionSEP Somatosensory evoked potentials

List of original publicationsThis thesis is based on the following articles, which are referred to in the text by theirRoman numerals:

I Pokela M, Anttila V, Hirvonen J, Rimpiläinen J, Vainionpää V, Kiviluoma K, Men-nander A & Juvonen T (2001) Serum S100ß protein predicts brain injury after hypo-thermic circulatory arrest in pig. Scand Cardiovasc J 34: 570–574.

II Pokela M, Biancari F, Rimpiläinen J, Romsi P, Hirvonen J, Vainionpää V, KiviluomaK, Anttila V & , Juvonen T (2001) The role of cerebral microdialysis in predictingthe outcome after experimental hypothermic circulatory arrest. Scand Cardiovasc J35: 395–402.

III Pokela M, Romsi P, Biancari F, Rimpiläinen J, Vainionpää V, Kiviluoma K, Hirvo-nen J, Rönkä E, Heikkinen J, Kaakinen T, Salomäki T & Juvonen T (2002) Increaseof intracranial pressure after hypothermic circulatory arrest in a chronic porcinemodel. Scand Cardiovasc J 36: 302–307.

IV Pokela M, Jäntti V, Lepola P, Romsi P, Rimpiläinen J, Salomäki T, Vainionpää V,Biancari F, Hirvonen J, Juvonen T (2003) EEG burst recovery is predictive of braininjury after experimental hypothermic circulatory arrest. Scand Cardiovasc J 37:154–157.

Contents

Abstract Acknowledgements Abbreviations List of original publications 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.1 Hypothermic circulatory arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172.1.1 Brain metabolism during hypothermic circulatory arrest . . . . . . . . . . . . 18

2.1.1.1 Depletion of brain energy sources . . . . . . . . . . . . . . . . . . . . . . . 182.1.1.2 Depolarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.1.1.3 Biochemical cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.1.1.4 Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.1.1.5 Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.1.2 Recovery of brain metabolism after circulatory arrest . . . . . . . . . . . . . . . 222.1.3 Reperfusion injury after hypothermic circulatory arrest . . . . . . . . . . . . . 22

2.2 Predictors of brain injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2.1 Blood and cerebrospinal fluid markers of brain injury . . . . . . . . . . . . . . 23

2.2.1.1 Creatine phosphokinase isoenzyme BB . . . . . . . . . . . . . . . . . . . 242.2.1.2 S100β protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.2.1.3 Neuron-specific enolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272.2.1.4 Glial Tissue-Specific Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . 282.2.1.5 Other markers of brain injury . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2.2.2 Electrophysiological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.2.2.1 Electroencephalography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.2.2.2 Somatosensory evoked potentials . . . . . . . . . . . . . . . . . . . . . . . . 31

2.2.3 Brain tissue analyzing methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.2.3.1 Cerebral microdialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.2.3.2 Tissue monitoring probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.2.3.3 Near-infrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.2.3.4 Intracranial pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

3 Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.1 The chronic porcine model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.2 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.3 Preoperative management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.4 Anesthesia protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.5 Anesthesia methods and monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.6 Experimental protocol of hypothermic circulatory arrest . . . . . . . . . . . . . . . . . 434.7 Intracranial parameters monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.7.1 Cortical microdialysis (studies II–IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.7.2 Intracranial pressure (study III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454.7.3 Brain tissue oxygenation and temperature monitoring

(study III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.8 Postoperative management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.9 Histopathologic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474.10 Electroencephalography monitoring (study IV) . . . . . . . . . . . . . . . . . . . . . . . . 484.11 Measurement of serum S100β protein (study I) . . . . . . . . . . . . . . . . . . . . . . . . 494.12 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

5 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515.1 Serum S100β protein (study I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515.2 Cerebral microdialysis (study II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525.3 Intracranial pressure (study III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555.4 Electroencephalography (study IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596.1 General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596.2 Experimental model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606.3 Serum S100β protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616.4 Brain microdialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616.5 Intracranial pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636.6 Electroencephalography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

1 IntroductionDespite advances in anesthesia, cardiopulmonary bypass (CPB) and surgical techniques,brain ischemic injury remains a major source of morbidity and mortality after cardiacsurgery (Roach et al. 1996). In particular, cerebral protection is of major concern duringaortic arch surgery (Ergin et al. 1994, Ohmi et al. 1998). The diagnosis of cerebral injurycurrently relies on clinical neurological examination, computed tomography (CT) andmagnetic resonance imaging (MRI) (Ali et al. 2000). These methods are usually notalways suitable during or immediately after cardiac surgery. Invasive methods ofmonitoring such as cerebral microdialysis, which requires a drill hole to the skull, cannotbe routinely used in cardiac surgery (Mendelowitsch et al. 1998). Electro-encephalography (EEG) and somatosensory-evoked potentials (SEP) are currentlyavailable non-invasive methods based on brain electrical activity, but both have certainlimitations (Kawada et al. 1996, Stecker et al. 1996). Near-infrared spectroscopy (NIRS)is another non-invasive method for cerebral monitoring, but its specificity and sensitivityare unsatisfactory (Nemoto et al. 2000).

Because of such limitations in invasive and non-invasive cerebral monitoring, severalproteins synthesized in astroglial cells or neurons have been proposed as markers ofcentral nervous system cell injury as detected in the serum or in the cerebrospinal fluid(CSF) (Maas 1977a, Maas 1977b). Bakay and Ward (Bakay et al. 1986) suggested that anideal serum or CSF marker should have high specificity and sensitivity for brain injury,be released only after irreversible damage of brain tissue, and have a rapid appearance inthe serum or CSF. In clinical practice biochemical markers should be interpretable in theserum, since the sampling of CSF is impractical (Ingebrigtsen and Romner 2002).

In the '70s, levels of lactate dehydrogenase, creatine kinase and glutamic oxaloacetictransaminase were shown to be markers of brain injury (Maas 1977a). Nowadays, S100βprotein is the only biochemical marker showing some potential in predicting brainischemic injury after cardiac surgery (Johnsson 1996). New markers of brain injury arecurrently under evaluation and, among them, glial fibrillary acidic protein (GFAP) is oneof the most interesting (Herrmann et al. 2000a).

Experimental surgery provides the opportunity for testing invasive and non-invasivemethods of brain monitoring with a throughout histopathological evaluation of brainischemic injury, which is not possible in the clinical field. Indeed, the gold standard ofevaluation of cerebral injury is histopathological analysis of the brain specimens, and

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according to our findings, these should preferably be from a chronic animal model (Kurthet al. 1999, Mennander et al. 2002). Furthermore, postoperative death is without doubtone of the main outcome measures, and this further calls for the need of a survivinganimal model for a better evaluation of brain protection methods. Even ifsemiquantitative methods of evaluation of behavioral outcome have been developed(Redmond et al. 1993, Midulla et al. 1994, Miura et al. 1996), neurological evaluation inanimal models of brain ischemic injury is somewhat more difficult.

In the present series of experimental studies, methods for predicting brain injury wereevaluated in a chronic porcine model of 75 minutes of hypothermic circulatory arrest(HCA) at 18°C of brain temperature. During these studies, several methods of cerebralmonitoring were employed. In the present four studies, the value of serum S100β protein,cortical microdialysis, intracranial pressure and quantitative electroencephalography inpredicting brain ischemic injury and outcome after HCA have been evaluated.

2 Review of the literature

2.1 Hypothermic circulatory arrest

The clinical indications for the use of HCA during cardiac surgery have increased withtime (McCullough et al. 1999). HCA was introduced by Niazi and Lewis as a method ofprotecting the brain during circulatory arrest (Niazi & Lewis 1957), but Charles Drewwas probably the first surgeon to use HCA (Dobell & Bailey 1997). Griepp, in the '70s,popularized this technique (Griepp et al. 1975), and nowadays it is employed both incomplex congenital heart diseases and in aortic arch surgery, when interruption of bloodflow to the brain is required (Griepp et al. 1997b). A bloodless operative field free ofclamps and cannulas is the major advantage of HCA. However, such a technique has as amajor limitation the time constraint, 40 minutes having been recognized as the safe limitof HCA, Table 1 (Griepp et al. 1991, Bellinger et al. 1995, McCullough et al. 1999).

Selective antegrade cerebral perfusion (SCP) and retrograde cerebral perfusion (RCP)are alternative methods for brain protection during complex congenital heart diseases andaortic arch surgery (Juvonen et al. 2000). Although technically more demanding, SCP isnot associated with such a strict time constraint, as it provides continuous blood flow tothe brain and has been used in aortic arch surgery with good results (Veeragandham et al.1998). The disadvantages of SCP are a perceived higher risk of embolic stroke in patientswith supra-aortic arterial disease (Frist et al. 1986) and the presence of cannulas in theoperative field. Studies on RCP during HCA showed controversial results of outcome,and it remains partly unclear whether RCP provides brain protection (Reich et al. 2001).A major problem is the lack of strong evidence of a clear metabolic support provided byRCP (Ono et al. 2000). Anyway, aortic surgery using RCP in association with HCA canbe performed with acceptable results (Deeb et al. 1999).

The lack of homogenous, prospective large clinical studies prevents any conclusion onthe superiority of SCP and RCP over isolated HCA. Despite more than four decades ofexperience with the use of HCA, this method is associated with a risk of immediatepostoperative mortality ranging from 6.1% to 15.0% and of stroke from 0 to 9.9% (Erginet al. 1994, Coselli et al. 1995, Liddicoat et al. 2000). Indeed, much worse results havebeen reported, especially in high-risk patients (Hagl et al. 2002).

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2.1.1 Brain metabolism during hypothermic circulatory arrest

Interruption of blood flow to the brain for a few minutes is associated with severe andoften irreversible brain ischemic injury (Fessatidis et al. 1993a). Ischemic brain injurydevelops through three different sequential phases: depolarization, biochemical cascadeand reperfusion injury in Fig. 1.

Fig. 1. Pathogenesis of ischemic brain injury.

2.1.1.1 Depletion of brain energy sources

During complete cessation of blood flow, the brain tissue is converted into a closedsystem. The brain energy sources are limited to the pre-existing levels of high-energycompounds such as phosphocreatine (PCr), adenosine triphosphate (ATP) and adenosine

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diphosphate (ADP) (Ljunggren et al. 1974b), and to the ATP produced when glucose andglycogen are anaerobically degraded to lactate (Siesjö 1978). Sources of cellular energyare degraded in this order: PCr, glucose, ATP and glycogen in Table 1 (Ljunggren et al.1974b). During normothermia, useful energy sources are used within one to three minutesof ischemia, whereas deoxygenation of brain tissue occurs in only a few seconds (Siesjö1978). During hypothermia, the metabolic rate of the brain is significantly slower and theenergy sources last longer (Shin'oka et al. 2000) Table 2. In fact, in the case of HCA at18 °C, the concentrations of PCr and ATP fell to zero in 18 ± 4 and 29 ± 5 minutes(Sutton et al. 1991). The deoxygenation time during hypothermia is also much longerthan in normothermia (Kurth et al. 1992). During brain ischemia glucose, pyruvate andglycogen are metabolized anaerobically, thus quickly leading to accumulation of lactateand CO2 (Ljunggren et al. 1974a), which is associated with a decrease in pH Table 1.

Table 1. High-energy phosphates and glycolysis metabolites during total ischemia in therat. Values are means ± standard error of mean (Ljunggren et al. 1974a).

Table 2. Safe duration of HCA and cerebral metabolic rate at different temperatures.Calculation based on assumption that there is a 5-min tolerance for circulatory arrest at37°C (McCullough et al. 1999).

Parameter Control 1 minute 3 minutes 10 minutesDuration of Ischemia

Glucose (mmol/kg) 4.35 ± 0.22 0.15 ± 0.21 0.33 ± 0.21 0.24 ± 0.16Lactate (mmol/kg) 1.60 ± 0.01 12.01 ± 0.80 13.71 ± 1.05 14.03 ± 1.05Pyryvate (µmol/kg) 105 ± 5 102 ± 10 30 ± 17 13 ± 5Lactate/Pyryvate ratio 15.2 ± 0.7 123 ± 9 1045 ± 265 3896 ± 2291PCr (mmol/kg) 5.04 ± 0.04 0.22 ± 0.10 0.10 ± 0.03 0.09 ± 0.03ATP (mmol/kg) 3.06 ± 0.02 1.33 ± 0.16 0.32 ± 0.06 0.09 ± 0.02ADP (mmol/kg) 0.27 ± 0.00 1.13 ± 0.07 0.78 ± 0.05 0.51 ± 0.03CO2 (kPa) 6.05 ± 0.09 13.2 ± 0.4 – 15.1 ± 0.4

pH 7.04 ± 0.01 6.60 ± 0.01 – 6.48 ± 0.01

Temperature (°C) Cerebral metabolic rate(% of 37°C)

Safe duration of HCA (min)

37 100 530 56 (52–60) 9 (8–10)25 37 (33–42) 14 (12–15)20 24 (21–29) 21 (17–24)15 16 (13–20) 31 (25–38)10 11 (8–14) 45 (36–62)

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2.1.1.2 Depolarization

Complete arrest of the cerebral circulation leads to cessation of neuronal electricalactivity within 11 seconds and depolarization of cell membranes within 70–100 seconds.These can be delayed by moderate (28°C) hypothermia to 18 seconds and 194–310seconds (Nakashima et al. 1995, Bart et al. 1998). The effect of hypothermia on brainmetabolic rate has not been extensively studied, but during deep hypothermia (15°C),depolarization would occur 10 to 20 minutes after the start of HCA according toMcCullough and colleagues´ calculations (McCullough et al. 1999). After effectiveenergy sources during circulatory arrest have been depleted, the energy-demanding cellmembrane potentials and ion gradients for Na+, K+ and Ca2+ across the cell membranescan no longer be regulated since the Na+/K+-pump stops through lack of energy (Hansen1985). After depolarization, the loss of ion homeostasis is rapid (Hansen 1985). Depletionof high-energy phosphates leads to degradation of macromolecules of key importance forthe cell membrane and cytoskeletal integrity, to failure of the membrane ion pump and,thus, to the influx and accumulation of calcium ions, sodium, chloride with osmoticallyobligated water and efflux of cellular potassium (Siesjö 1992a). If the interruption ofcerebral blood flow persists for more than 5–10 minutes in normothermia, irreversiblecell damage is likely (Ljunggren et al. 1974b, Astrup et al. 1981).

2.1.1.3 Biochemical cascade

Normally, there is a large electrochemical potential in the cell, including a 10000-foldhigher extracellular than intracellular concentration of Ca2+, which is associated with a60 to 90 mV negative electrical potential (Kristian & Siesjö 1998). As a result of thedepolarization, there is a massive influx of Ca2+ via voltage-sensitive channels, leading toa 500-fold increase in the intracellular calcium and a concomitant secretion of glutamate(Dunlap et al. 1995, Kristian & Siesjö 1998). The main route of entry of Ca2+ is throughchannels gated by glutamate receptors (Li et al. 1995) when the cells of the presynapticmembrane depolarize and release neurotransmitters, i.e. glutamate (Santos et al. 1996).The ischemia-induced release of monoamines is completely Ca2+-independent, but acomponent of glutamate release is Ca2+-dependent (Fig. 1) (Santos et al. 1996). Inhypothermic conditions the release of extracellular amino acids, including glutamate, isconsiderably decreased (Nakashima & Todd 1996, Li et al. 1999).

2.1.1.4 Glutamate

The concept of excessive glutamate accumulation in the extracellular space leading toneuronal injury is widely accepted (Lipton & Rosenberg 1994). An association betweenaccumulation of glutamate and neurologic injury has been observed after HCA (Redmondet al. 1994). However, the relationship between accumulation of extracellular glutamateand subsequent neuronal cell death is not direct (Siesjö et al. 1995), but the final common

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pathway of ischemic neuronal injury involves excessive stimulation of glutamatereceptors and accumulation of glutamate (Fig.1)(Lipton & Rosenberg 1994). In corticalor hippocampal neurons, substantial excitotoxic damage to the brain tissue is expected tooccur when the extracellular glutamate concentration reaches 2 to 5 µmol/L (Lipton &Rosenberg 1994).

Because glutamate is highly neurotoxic, it is continually cleared from the extracellularspace in normal conditions (Santos et al. 1996). Neuronal uptake recycles glutamate byrestorage in vesicles for re-release. Glial cells contain a similar, high-affinity activetransport that ensures efficient removal of glutamate from the synapse. The decrease inNa+ and K+ gradients resulting from the energy depletion of the synaptosomes underischemic conditions promotes the reversal of the neurotransmitter transporters. Thedecrease of neurotransmitter uptake may also contribute to the rise in the extracellularconcentration of different transmitters observed during brain ischemia. (Santos et al.1996)

2.1.1.5 Calcium

Increasing free calcium is lethal when it is sustained above a certain level, because Ca2+

damages the mitochondrial respiration mechanism and induces uncontrolledCa2+-dependent cascade reactions (Siesjö & Bengtsson 1989). It is likely that initialdepolarization and calcium influx, but not glutamate, during ischemia represents atriggering event for irreversible cell damage (Siesjö et al. 1995). A component ofglutamate release is also Ca2+-dependent (Santos et al. 1996). On the other hand, theresulting over-stimulation of neuronal glutamate receptors, particularlyN-methyl-d-aspartate (NMDA) receptors, leads to excessive influx of calcium throughreceptor-gated ion channels (Fig.1)(Zipfel et al. 1999). It has been shown that even mildhypothermia (34°C) during ischemia is able to decrease the calcium influx after ischemicdepolarization (Wang et al. 2000).

An increased intracellular Ca2+ concentration starts numerous secondary processeswhich accelerate ischemic neuronal damage (Siesjö 1992b). These mechanisms includeactivation of phospholipases, phospholipase A2, endonucleases, nitric oxide synthesis,reactive oxygen species, caspains, and proteases, since they affect proteinphosphorylation by altering the activity of protein kinases and phosphatases (Siesjö1992b, Zipfel et al. 1999). It is also clear that a coupling exists between influx of calciuminto cells and the production of reactive oxygen species such as [O2

–], H2O2, and [OH–](Kristian & Siesjö 1998). Phospholipase A2 catalyzes the breakdown of membrane lipidsforming fatty acids and arachidonic acid, which can later undergo further metabolism bycyclo-oxygenase, thus producing oxygen radicals; and there is a correlation between therelease of arachidonic acid and neuronal damage (Dumuis et al. 1990).

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2.1.2 Recovery of brain metabolism after circulatory arrest

Following normothermic ischemia of up to 15 minutes duration, the recovery of oxidativeenergy metabolism occurs in 1 to 3 minutes (Ljunggren et al. 1974b). After an ischemicinsult during mild hypothermia, the recovery of energy metabolism is even faster(Kimura et al. 2002). However, after ischemia, capillary perfusion is disturbed, and afternormothermic ischemia of 10 to 20 minutes duration, it was only 30% of normal withouta change in ICP (Krause et al. 1988).

The recovery of cerebral metabolism after HCA is different compared to thatfollowing normothermic ischemia. Cerebral blood flow and the cerebral metabolic rate ofoxygen (CMRO2) are both impaired during reperfusion after HCA (Jonassen et al. 1995,Rodriguez et al. 1995). These changes are associated with impairment in intracellularbrain oxygenation (Greeley et al. 1991). Furthermore, it has been shown that thereductions in CMRO2 are directly proportional to the duration of HCA (Mault et al.1993). The recovery of brain-tissue high-energy phosphates after HCA is also remarkablyslow, taking over 40 minutes (Filgueiras et al. 1996, Shin'oka et al. 2000). A similardelayed recovery is seen in brain-tissue oxygenation and carbon dioxide partial pressure(Hoffman et al. 1996b). The recovery of acidosis after HCA is also delayed, taking evenlonger than the normalization of carbon dioxide levels (Hoffman et al. 1998). Recovery tonormal values in glucose, lactate and pyruvate was found to take over 4 hours after HCA(Rimpiläinen et al. 2001, Rimpiläinen et al. 2002, Romsi et al. 2002b).

The reason for such a delayed recovery of brain metabolism after HCA is notcompletely known. HCA and CPB cause endothelial dysfunction in cerebral microvessels(Sellke et al. 1996, Cooper et al. 2000). Abnormal cerebral vasoconstriction mediated byvasoconstrictors such as endothelins, oxygen free radicals, and thromboxane A2 may befactors leading to impairment of CMRO2.

Perfusion strategies after HCA could have an important effect on CMRO2, and apH-stat strategy has been shown to be associated with higher cerebral blood flow andbetter recovery of energy metabolism (Hiramatsu et al. 1995, Kurth et al. 1998).Hyperventilation could also cause impairment of cerebral blood flow and is associatedwith increased glutamate, lactate and lactate/pyruvate ratio in the brain tissue (Marion etal. 2002). An increase in ICP may lead to vessel compression and decrease of brain tissueperfusion (Hekmatpanah 1970, Taylor 1998a).

2.1.3 Reperfusion injury after hypothermic circulatory arrest

The hallmark of reperfusion injury after brain ischemia is an inflammatory reactioncharacterized by leukocyte infiltration, primarily neutrophils (Feuerstein et al. 1994).Brain-inflammatory cells, such as microglia, also play a role in the development ofischemic brain injury (Feuerstein et al. 1994).

Leukocyte migration into brain tissue is critically dependent on the expression ofspecific adhesion proteins located on the surface of endothelial cells and on the activationof leukocytes (Arvin et al. 1996). Leukocyte activation during CPB is the result ofoperative trauma and, most important, contact between the artificial surface of the bypass

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circuit and leukocytes (Asimakopoulos & Taylor 1998). A strong inflammatory responsehas also been shown to be related to HCA operations (Asimakopoulos & Taylor 1998,Taylor 1998b, Rimpiläinen et al. 2000, Tassani et al. 2002).

Cytokines are a group of polypeptides acting as mediators in immuno-endocrineinteractions, and are necessary for the optimal functioning of leukocytes (Rothwell et al.1996). Tumour necrosis factor α and interleukin 1-β seem to play a significant role inbrain immune and inflammatory activities and in ischemic brain injury (Tonnesen et al.1996, Feuerstein et al. 1998). Brain tissue is capable of producing cytokines, whenproduction is induced by endotoxins and neurotoxins such as kainite (Jean et al. 1998).Induced cytokine production, including tumour necrosis factor α and interleukin 1-β, hasalso been observed after transient cerebral ischemia (Liu et al. 1993, Liu et al. 1994). Inoperations using HCA, the production of cytokines is also induced by CPB alone (Hattleret al. 1995, Wan et al. 1997) , and HCA causes a further increase in their concentration inthe blood (Rimpiläinen et al. 2000, Hovels-Gurich et al. 2002). Activated neutrophilsmay induce cerebral edema via regional cytokines (Dewanjee et al. 1998).

Since tumour necrosis factor α, interleukin 1-β and endotoxins can up-regulateendothelial cell-derived adhesion molecules, there is a clear connection between cytokineproduction, surface expression of adhesion molecules and the resultant inflammatoryreaction (Arvin et al. 1996, Hill 1998). Furthermore, CPB increases the generation ofsuperoxides and the production of adhesion molecules in endothelial cells duringreperfusion (Jean et al. 1998).

The adhesion molecules initiate adhesion of leukocytes to the walls of cerebral bloodvessels, leading to their transendothelial migration into the interstial fluid phase andrelease of their lysosomal contents (proteolytic enzymes, leukotrienes and oxygen freeradicals) (Paparella et al. 2002). These agents are stimulators of lipid peroxidation in theendothelial cells, leading to cellular dysfunction, edema and even cell death (Jordan et al.1999). Leukocyte activation, adhesion to the walls of the vessels and infiltration intotissue also interfere with normal microvascular perfusion (Feuerstein et al. 1998), andcould also cause microvascular thrombosis (Boyle et al. 1999).

2.2 Predictors of brain injury

2.2.1 Blood and cerebrospinal fluid markers of brain injury

Identification of biochemical markers of brain injury would represent a major stepforward in the noninvasive assessment of the efficacy of neuroprotective methods. Undernormal circumstances, the marker should not be available in measurable amounts in theserum or CSF; after ischemic brain injury it is released from the brain into the serum orCSF in measurable amounts. Several proteins have been suggested as markers of braininjury, and the most important ones evaluated are listed in Table 3.

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Table 3. Most important biochemical markers of brain injury.

2.2.1.1 Creatine phosphokinase isoenzyme BB

Creatine phosphokinase has three isoenzymes: the muscle type (CK-MM), the heart type(CK-MB) and the brain type (CK-BB). The function of creatine phosphokinase is totransfer an energy-bond between ATP, PCr and ADP (Bakay et al. 1986). The molecularmass of CK-BB is 40 to 53 kDa, and it is normally found in the serum in very low

Marker Studies CommentCerebrospinal fluid markers of brain injury

Creatine-Kinase BB (CK-BB)

Cardiac surgery (Lundar & Stokke 1983).Experimental HCA (Fessatidis et al. 1993b).

In clinical use, not very good marker of brain injury.

S100β protein Cardiac surgery (Sindic et al. 1982). Not very sensitive in cardiac surgery (Sell-man et al. 1992)

Cleaved tau protein (c-tau)

Severe head-injury patients (Zemlan et al. 2002)

Not evaluated in cardiac surgery.

Neuron-specific enolase (NSE)

Cardiac surgery (Sellman et al. 1992). Pediatric cardiac surgery (Schmitt et al. 1998).

Sensitive for blood contamination (Schmitt et al. 1998).The predictive value for brain injury is not clear yet (Schmitt et al. 1998)

Glial fibrillary acidic protein (GFAP)

Normal-pressure hydrocephalus in patients (Albrechtsen et al. 1985).

No commercially available measurement ready yet. Serum analysis could be good enough. Coming in the future?

Serum markers of brain injury

Creatine-Kinase BB (CK-BB)

Cardiac surgery with HCA (Lundar et al. 1983a)

Hypothermia increases the values. (Johnsson 1996). CK-BB is not a reliable marker of brain damage in cardiac surgery (Johnsson 1996). In clinical use.

S100β protein Cardiac surgery (Westaby et al. 1996, Blomquist et al. 1997, Kilminster et al. 1999, Georgiadis et al. 2000, Herrmann et al. 2000b)

Contamination from adipose tissue (Jönsson et al. 1999). Best available marker of brain injury in cardiac surgery (Johnsson 1996).

Myelin basic pro-tein (MTB)

Head injury (Thomas et al. 1978) No commercially available measurement (Ingebrigtsen & Romner 2002). Myelin basic protein is not sufficiently examined in car-diac surgery (Johnsson 1996)

Cleaved tau protein (c-tau)

Head injury (Chatfield et al. 2002)Bacterial meningitis (Irazuzta et al. 2001)

Not evaluated in cardiac surgery. Future?

Neuron-specific enolase (NSE)

Stroke patients (Missler et al. 1997) (Wunderlich et al. 1999).Cardiac surgery (Rasmussen et al. 2002).

Hemolysis increases the values (Johnsson et al. 2000). Not valuable in cardiac surgery (Johnsson et al. 2000).

Glial fibrillary acidic protein (GFAP)

Stroke patients (Herrmann et al. 2000a) No commercially available measurement ready yet. Not evaluated in cardiac surgery yet. Very specific for brain tissue, future?

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concentrations (the upper normal serum concentration is 3µg/L) (Phillips et al. 1980).CK-BB is not normally found in CSF, and it does not cross the intact blood-brain barrier(Maas 1977a, Maas 1977b). Somer introduced CK-BB as a potential marker of braininjury (Somer et al. 1975). In brain injury, an elevated concentration of CK-BB wasmeasured from CSF and serum (Kaste et al. 1977). The CSF concentration of CK-BB inpiglets correlated with increasing periods of HCA (Fessatidis et al. 1993b) and has beenshown to be a valuable marker of neurological outcome after cardiac arrest (Roine et al.1989). Lundar observed an association between high CK-BB activity in the CSF andadverse neurologic outcome after HCA (Lundar et al. 1983a).

However, CK-BB as a marker of cerebral injury has several weaknesses. A few yearsago the radioimmunoassay method had a cross-reaction between CK-BB and CK-MB,but nowadays this problem has been solved (Bell et al. 1978). Another methodologicalweakness is the high extracerebral concentrations of CK-BB as compared, for example,with S100β protein, NSE or especially GFAP (Table 4). Hypothermia also increases theCK-BB concentration in serum, both with and without CPB (Vaagenes 1986, Vaagenes etal. 1987).

In summary, CK-BB is not a very specific marker of brain injury after cardiac surgery,new markers having been shown to be superior.

Table 4. Relative concentrations of CK-BB, NSE, and S100β protein in tissues ascompared with brain tissue concentration in humans. (Johnsson 1996).

Human tissues CK-BB NSE S100β protein Brain cortex 100 % 100 % 100 %Adipose tissue – – 2.8–5.6 % *Rectum 49.1 % 1.9 % 2.5 %Stomach 35.3 % 2.6 % 0.7 %Urinary bladder 35.3 % 2.6 % 2.0 %Prostate gland 31.9 % 2.0 % 0.1 %Small intestine 19.2 % 1.9 % 2.1 %Uterus 22.1 % 2.6 % 0.2 %Vein 12.1 % 1.9 % 0.2 %Thyroid gland 11.3 % 1.1 % 0.2 %Gall bladder 5.4 % 1.4 % 1.7 %Kidney 5.7 % 2.6 % 0.3 %Lung 3.5 % 0.9 % 0.2 %Mammary gland 0.5 % 0.1 % 1.8 %Spleen 0.7 % 1.5 % 1.8 %Aorta 0.8 % 2.5 % 0.1 %Liver 0.3 % 0.5 % 0.1 %Skeletal muscle 0.3 % 0.2 % 0.7 %Heart – – 0.2 %– = No data. *(Haimoto et al. 1987)

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2.2.1.2 S100β protein

The S100 protein is a small dimeric cytosolic calcium-binding protein with a molecularweight of 22 kDa (Zimmer et al. 1995). It exists in various forms depending on its chain(α or β) structure (Zimmer et al. 1995). The αα-form is found in striated muscles, heartand kidney (Haimoto & Kato 1988, Hasegawa et al. 1993). The isoforms αβ and ββ arepredominantly present in astroglial and Schwann cells, and are commonly referred to asthe brain-specific S100β protein (Ingebrigtsen & Romner 2002); they are also found inthe adipose tissue at lower concentrations and in other tissues (Table 4) (Haimoto et al.1987, Johnsson 1996).

An increased serum and CSF concentration of S100β protein indicates both neuronalinjury and increased permeability of the blood-brain barrier (Persson et al. 1987).Elevated serum S100β protein levels are found in the blood and CSF after cerebral stroke,subarachnoid hemorrhage, cranial trauma, coma after cardiac arrest and many otherneurological disorders (Sindic et al. 1982, Persson et al. 1987, Missler et al. 1997,Grocott et al. 1998, Rosen et al. 1998, Raabe et al. 1999). Because of this, serum S100βprotein has been suggested as a promising marker of brain injury in cardiac surgery(Johnsson 1996, Westaby et al. 1996). S100β protein is eliminated from the serum by thekidney and excreted in the urine. Its exact biologic half-life is not known, but recentstudies suggest that it is well below 60 minutes (Jönsson et al. 2000).

The first study on S100β protein measurement after cardiac surgery was published in1992 (Sellman et al. 1992), and showed no measurable S100β protein concentration inthe CSF after CPB. A certain association between serum S100β protein levels andpostoperative cerebral complication was published in 1995 (Johnsson et al. 1995). Since apositive correlation between CPB perfusion duration and S100β protein was observed,serum S100β protein level was considered to be indirect proof of brain injury (Westaby etal. 1996). Increased serum levels of S100β protein were demonstrated to be significantlyassociated with cerebral embolic event (Grocott et al. 1998) and adverse earlypostoperative neuropsychological outcome by numerous studies (Westaby et al. 1996,Blomquist et al. 1997, Kilminster et al. 1999, Georgiadis et al. 2000, Herrmann et al.2000b). The association between increased serum S100β protein concentrations andlong-term neuropsychological outcome is, on the other hand, less clear (Herrmann et al.2000b, Westaby et al. 2000).

During operations under HCA, a positive correlation has been demonstrated betweenS100β protein serum levels and the duration of HCA and CPB respectively (Lindberg etal. 1998, Bhattacharya et al. 1999, Wong et al. 1999).

However, an increased serum level of S100β protein is observed in almost all patientsundergoing CPB, and there was a variation in S100β protein levels at different samplingtimes (Jönsson et al. 1998). Serum levels and the degree of variation decreased graduallywith time after termination of extracorporeal circulation, and an increase in S100β proteinduring CPB as a marker of brain injury was questioned (Jönsson et al. 1998). In fact, ithas been shown that S100β protein is released from mediastinal adipose tissue duringcardiac operations (Jönsson et al. 1999), and an increased concentration of S100β proteinwas detected in cardiotomy suction blood.

The conclusion based on increased serum S100β protein levels after CPB as a markerof disturbance of the blood-brain barrier during CPB could also be erroneous (Bokesch

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1999, Lloyd et al. 2000), because CPB did not actually increase the serum S100β protein(Anderson et al. 2001).

The timing of increased S100β protein in the serum has been pointed out as being ofkey prognostic importance: when the serum level of S100β protein was elevated 48 hoursafter coronary artery bypass surgery, it had a negative predictive value for medium-termsurvival (Jönsson et al. 2001) and long-term survival (Johnsson et al. 2003) andcorrelated positively with the size of the infarcted brain tissue (Jönsson et al. 2001). Infact, it has been shown that seven hours after the end of CPB, the S100β protein releasedfrom the fat cells is not detected in the blood, and an increased S100β protein levelcorrelates with decreased memory function (Svenmarker et al. 2002).

In summary, the S100β protein is a potential marker of brain injury. Serum analysis ofS100β protein is flamed by contamination during CPB, which causes a certain limitationin its accuracy. However, at 7, and surely 48, hours after surgery it is a very good markerof brain injury and a predictor of outcome.

2.2.1.3 Neuron-specific enolase

Enolases are a family of ubiquitous glycolytic enzymes occurring as series of dimericisoenzymes including three subunits, the α, β and γ chains (Cooper 1994). The isoformsγγ and αγ are restricted to neurons, where they act as glycolytic enzymes in cytoplasm,and are named neuron-specific enolase (NSE). NSE is located in the cytoplasm ofneurons, and has a molecular weight of 78 kDa (Ingebrigtsen & Romner 2002). Thebiologic half-life of NSE in serum is 24 hours (Ishiguro et al. 1983). It was first used as atumor marker for small-cell lung cancer, neuroblastoma, and other malignancies ofneuroendocrine origin (Cooper 1994). Later, it was introduced as a marker of braindamage (Steinberg et al. 1984, Sellman et al. 1992).

Serum NSE levels have been shown to correlate with infarct volume (Missler et al.1997) and functional impairment after stroke (Wunderlich et al. 1999). In cardiac surgery,postoperative serum concentrations of NSE have a predictive value with respect to earlyneuropsychological and neuropsychiatric outcome after cardiac surgery (Herrmann et al.2000b). In patients with traumatic head injuries, a serum NSE concentration of more than10µg/L is considered pathologic (Raabe et al. 1998). A recent study suggested that NSEis the most useful marker of brain injury, and the most appropriate timing for NSE bloodsampling is 36 hours after coronary artery bypass grafting (Rasmussen et al. 2002).However, this study included only 15 patients, and a significant correlation with theoutcome was observed only in one of the several sampling points after surgery. Thisfinding is thus far from being conclusive.

The measurement of NSE in the serum is also associated with a number ofmethodological weaknesses. NSE protein is present at relatively high levels inerythrocytes, thus even mild haemolysis is enough to increase significantly the serumlevels of NSE (Brown et al. 1980). Furthermore, high extracerebral concentrations ofNSE can also be detected in other tissues (Table 4). Certainly, this is a major problemwhen enclosing this marker in a situation likely to be associated with hemolysis, such asCPB (Pierangeli et al. 2001). Because of these problems, serum NSE has failed to be a

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specific and efficient marker of brain injury during and shortly after CPB (Georgiadis etal. 2000, Johnsson et al. 2000) and during HCA (Hovels-Gurich et al. 2001).

Certain problems with serum analysis have lead to attempts to use CSF NSE as amarker of brain injury. A significant increase in CSF NSE levels has also beendemonstrated in patients suffering traumatic brain injury (Ross et al. 1996) and after CPB(Sellman et al. 1992). A correlation between CSF NSE levels and the Glasgow comascale score has been demonstrated after traumatic brain injury (Ross et al. 1996). Themethodological weakness of CSF NSE is that the measurement shows high sensitivity toblood contamination during sampling, because NSE is present in blood cells in significantconcentrations (Schmitt et al. 1998). The predictive value of CSF NSE levels in thedetection of brain injury after CPB is estimated to be limited, and even elevated CSF NSElevels might be related to blood-brain barrier disturbances (Schmitt et al. 1998).

In summary, the value of the NSE as a marker of brain injury after cardiac surgery withCPB is highly questionable.

2.2.1.4 Glial Tissue-Specific Protein

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressedalmost exclusively in the astrocytes, where it represents the major part of thecytoskeleton. GFAP is a monomeric molecule with a molecular mass ranging from 40 to53 kDa (Missler et al. 1999). Increased concentrations GFAP in the CSF have beenshown in normal-pressure hydrocephalus (Albrechtsen et al. 1985), dementia (Teunissenet al. 2002) and stroke (Aurell et al. 1991). GFAP is a very sensitive and specific markerof rapid astrocytic response to injury and diseases (Eng & Ghirnikar 1994). Themeasurement is not sensitive to the effect of haemolysis, the concentration of GFAP isstable for at least three freezing and thawing cycles and normal freezer storage (van Geelet al. 2002). Hermann and colleagues provided the first systematic clinical evaluation of astrong association between serum levels of GFAP and severity of stroke (Herrmann et al.2000a). Serum GFAP protein seems to be a promising marker for brain injury, and itseems to fulfill the demand for a highly specific marker for brain injury (Herrmann et al.2000a). However, further investigations are required to evaluate its prognostic accuracyin patients undergoing cardiac surgery.

2.2.1.5 Other markers of brain injury

A number of molecules and proteins have been suggested as markers of neuronaldamage. Adenylate kinase is an intracellular cytoplasmic enzyme that is present inneurons as well as in other cells such the erythrocytes. In the case of ischemic braindamage, the CSF concentration of adenylate kinase is increased significantly (Ronquist &Frithz 1982), and its increase has also been observed in the case of neurologicaldysfunction after CPB (Åberg et al. 1984). However, its association with neurologicaloutcome is rather weak (Johnsson 1996). The problem is the high concentration of

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adenylate kinase in the blood, and contamination from blood is possible unless specificprecautions are taken (Bakay et al. 1986).

ICAM-5 (telencephalin) is only expressed in the somatodendritic membranes oftelencephalic neurons. 48 hours after hypoxic-ischemic injury, the level of ICAM-5 waselevated in serum, and it was suggested as a potential marker for somatodendriticneuronal damage (Guo et al. 2000).

CSF cleaved tau proteins are structural microtubule binding proteins primarilylocalized in the axonal compartment of neurons. Elevated CSF cleaved tau proteins havebeen demonstrated in patients suffering from traumatic head injury and inmultiple-sclerosis patients (Zemlan et al. 1999). CSF tau protein levels have been shownto predict the increase of ICP and the clinical outcome after traumatic head injury(Zemlan et al. 2002). Cleaved tau proteins can also be measured from blood, and apreliminary study showed increased C-tau levels in patients with unfavorable outcomeafter severe head injury (Chatfield et al. 2002). Further studies are required to explorewhether C-tau could be used as a marker for brain ischemic injury (Chatfield et al. 2002).

Myelin basic protein (MBP) is detectable in developing oligodendroglia, and it isbound to the extracellular membranes of central, and to a lesser extent peripheral, myelin(de Vries et al. 2001). However, as a marker of brain injury, NSE is superior compared tomyelin basic protein (Garcia-Alix et al. 1994).

Endothelin-1 is identified as a most potent vasoconstrictor peptide with 21-aminoacids. It is present in endothelial cells, were it also exists in two different isopeptides,endothelin-2 and endothelin-3 (Lampl et al. 1997). Endothelin-1 has been shown toparticipate in astrocyte activation and oxidative stress after trauma (Beuth et al. 2001). Itis elevated in the CSF after stroke (Lampl et al. 1997), and after traumatic head injuryboth in the CSF and blood (Beuth et al. 2001). A correlation between the CSFconcentrations of endothelin-1, the volume of the lesion, and the Matthew Scale score hasbeen shown after stroke (Lampl et al. 1997). After traumatic head injury, a correlationbetween the concentration of CSF endothelin and the Glasgow coma scale has beenobserved (Beuth et al. 2001). Studies have shown that an increase in endothelin-1 mayexacerbate brain injury associated with head injury or stroke (Sato & Noble 1998, Park &Thornhill 2000). Further studies are required to evaluate the role of CSF endothelin as apredictor of outcome in ischemic brain injury.

2.2.2 Electrophysiological methods

2.2.2.1 Electroencephalography

The electroencephalogram (EEG) is a well-established method for monitoring brainelectrical function. EEG is a method for recording cerebral electrical potentials, includingaction potentials and postsynaptic potentials (Binnie & Prior 1994). The EEG is verysensitive in detecting regional synaptic depression accompanying cerebral ischemia andeven hypotension without hypoxia (Gavilanes et al. 2001). An EEG recorded aftercerebral ischemia has been shown to predict the extent of cerebral damage (Binnie &

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Prior 1994, Stecker et al. 2001). Many abnormalities in EEG are known to be associatedwith brain ischemic injury. The following have been shown to be associated withischemic brain injury after cardiac surgery: increased slow activity (theta and delta); adecrease in fast activity (alpha); a decrease in EEG frequency in several channels,indicating general slowing of the EEG (Sainio 1974, Arroyo et al. 1993, Vanninen et al.1998); EEG seizures after pediatric cardiac surgery using HCA; (Helmers et al. 1997,Rappaport et al. 1998) slow recovery of EEG power after experimental HCA (Mezrow etal. 1995).

Both after cardiac arrest and HCA, the EEG recovers from electrical silence throughburst suppression to continuous EEG even when no cerebral injury occurs. A delay insuch an EEG burst-suppression recovery is an indicator of brain damage (Binnie & Prior1994, Stecker et al. 2001), and when EEG remained in burst suppression after recoveringto normothermia, patients suffered severe postoperative neurological complications(Stecker et al. 2001). The EEG amplitude also rises after ischemia, but because of thelarge variation in amplitude, it is not a good marker of injury (Sainio 1974). A similarEEG frequency after ischemia as detected preoperatively has been shown to predict goodneurological outcome (Arroyo et al. 1993), but analysis is quite sensible to artefacts. Inpediatric cardiac surgery, postoperative clinical and EEG-detected seizures wereassociated with a consistent pattern of worse developmental and long-term neurologicalfunction and with cerebral damage as detected by MRI (Rappaport et al. 1998). Whetherclinical or EEG-detected seizures are markers of brain injury or themselves contributorsto development of injury remains unclear (Helmers et al. 1997). An EEG poweramplitude of less than 500 µV2 two hours after HCA strongly predicts clinically evidentneurologic impairment (Mezrow et al. 1995). The impact of injured brain areas on EEGabnormalities such as recovery is not well known.

Neuromonitoring during cardiac or carotid surgery has been considered a sensitivemethod for identifying cerebral ischemia (Edmonds et al. 1996, Sebel 1998).Unfortunately, many non-injurious processes may produce the same EEG changes asoccur during hypoperfusion or hypoxia (Austin et al. 1997). Because of the complexity ofconventional EEG analysis and its susceptibility to electrical and mechanical artifact, itsprognostic value has been questioned (Witoszka et al. 1973). However, after HCA, EEGseems to be of prognostic importance (Stecker et al. 2001). Quantitative EEG is asensitive method in the detection of slight cerebral functional changes, and useful inidentifying patients with suspected cerebrovascular problems before surgery (Toner et al.1998). EEG monitoring during CPB has a positive effect on outcome when aninteroperative approach to increase brain blood flow was adopted in the case of EEGabnormalities (Hansotia et al. 1975). On the other hand, after cardiac surgery, subclinicalbrain injury was observed in quantitative EEG in one fifth of the patients three monthsafter surgery (Vanninen et al. 1998).

In summary, quantitative and classic EEG analysis is a sensitive marker of brain injury.Even partial ischemia is seen on EEG, and subclinical injury could be seen in EEG.Specificity of the EEG analysis could be variable, depending on the abnormal finding andthe method of analysis. Furthermore, interpretation of quantitative EEG is complex, andmay require special expertise (Toner et al. 1998).

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2.2.2.2 Somatosensory evoked potentials

Somatosensory evoked potentials (SEP) are the electrophysiologic responses of thenervous system to sensory stimulation (Chiappa & Ropper 1982a,b). The evaluation ofSEP is an established form of neuromonitoring. SEP reflects the functional integrity ofspecific sensory pathways. A loss of cortical SEP is a good indicator of cerebralhypoperfusion, and is able to identify patients at risk of stroke during carotid surgery(Schwartz et al. 1996, Beese et al. 1998). Loss of SEP response is an indicator of cerebralischemia during hypothermic low-flow CPB (Wilson et al. 1988).

In patients who have undergone HCA, delayed recovery of SEP has been shown to bea good marker of cerebral injury (Taylor et al. 1985), and intraoperative SEP alteration isassociated with transient or permanent neurological sequences with high specificity(Ghariani et al. 1999). Acute, unilateral decreases in amplitude of the cortical potentialare more useful than changes in latency in detecting intraoperative stroke (Stecker et al.1996).

There is a problem inherent in the use of SEP amplitude asymmetry as a criterion forcerebral injury, since it is strongly biased towards detecting unilateral or asymmetriccentral nervous system lesions (Stecker et al. 1996). When SEP is used during CPB withmild hypothermia, the commonly used rule that a 50% decrease in evoked potentialamplitude suggests a neurologic injury is too conservative, and it is not uncommon to see90% decreases in amplitude in patients without strokes (Stecker et al. 1996).

In summary, SEP is a sensitive marker of stroke during CPB, but its specificity is notsatisfactory.

2.2.3 Brain tissue analyzing methods

2.2.3.1 Cerebral microdialysis

Evaluation of changes in the chemical microenvironment is important in order tounderstand the mechanisms underlying the development of brain ischemic injury. Thesechanges of extracellular biochemical microenvironment in tissues can be monitored by amicrodialysis tool in vivo (Benveniste et al. 1984, Tossman & Ungerstedt 1986). Themethod of microdialysis was introduced in the 60s, when push-pull cannulas, dialysissacs, and dialyrodes were inserted into animal tissues to study biochemistry directly(Ungerstedt 1991). The first report on microdialysis technique dates back to 1966, whenBito described a fluid-filled semipermeable membrane implanted in dogs (Bito et al.1966). Tossman and Ungerstedt simplified the technique, introducing the currentmicrodialysis method (Tossman & Ungerstedt 1986).

Technique of microdialysis. In vivo microdialysis measures the chemical compositionof the extracellular fluid (ECF). Microdialysis, as the name suggests, functions on theprinciple of the diffusion of water-soluble substances through the semipermeable dialysismembrane until equilibrium is attained (Fig. 2) (Benveniste 1989, Ungerstedt 1991).

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Depending on the permeability of the membrane, the molecular weight of thesesubstances can be up to 100 kDA (Benveniste & Huttemeier 1990).

Fig. 2. Principles of microdialysis. The semipermeable membrane at the probe tip allowsexchange of soluble molecules between the probe and surrounding tissue.

Nowadays there are a variety of different probes. The basic probes consist of a smallpolycarbonate tube or probe with a diameter of 0.2–0.6 mm and a length of dialysis areaof 10 to 30 mm at the tip of the probe (Fig. 3). Substances from the interstitial fluid candiffuse through the membrane into the perfusion fluid (Ungerstedt 1991). Furthermore,the probe is connected to a microperfusion pump and constantly perfused with aphysiological solution at a constant flow rate of 0.1 to 5 µL/min, which is collected forlater analysis (Di Chiara 1990). Depending on the availability of analysis methods, everysoluble molecule in the interstitial space can, theoretically, be measured by microdialysis.

Fig. 3. Microdialysis probe. The perfusion fluid enters through the inlet cannula and passesthrough an inner cannula to the tip of the probe, as shown in enlargement of the tip, then backthrough the dialysis area between inlet cannula and the membrane. The fluid leaves the probethrough the outlet cannula to a changeable sample vial, modified from Ungerstedt (1991).

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The recovery of a particular substance is defined as the ratio, expressed as a percentage(%), of the concentration in the dialysate to the concentration in the interstitial fluid. Theperfusion flow rate is inversely related to the relative recovery (Fig. 4), restricting the sizeof the samples and the minimum time between samples. When recovery is less than100%, the concentration in the dialysate depends both upon the supply of substances fromthe blood capillaries and how much the cells take up from the interstitial fluid. Forexample, a glucose supply to the microdialysis catheter can decrease due to a decrease inthe capillary blood flow and/or to an increase in the cell uptake of glucose. (Ungerstedt1991)

Fig. 4. Principles of recovery. Recovery depends upon the flow of the perfusion fluid in thecatheter and the length of the dialysis membrane. The diagram shows an example of recoverywith three different lengths of the dialysis membrane with different dialysate flow rate, datafrom Ungerstedt (1991).

Monitoring brain metabolism and ischemia. Cerebral microdialysis has been used tostudy chemical changes in several cerebral pathological processes, for exampleParkinson, epilepsy, malignant neoplasia, brain ischemia and stroke, traumatic braininjury and subarachnoid haemorrhage in an intensive care setting. During the last fewyears, this method has been developed into a promising tool for monitoring and targetingtherapy of brain injury (Persson & Hillered 1992, Muller 2002). A methodological studyof microdialysis showed the high quality of one of the most-used brain catheters,CMA70, and clinical analyzer CMA 600 (Hutchinson et al. 2000).

Normal and ischemic brain tissue concentrations of glucose, lactate and pyruvate, andthe lactate/pyruvate and lactate/glucose ratios, are shown in Tables 5 and 6.

0,0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 3,0 3,3 3,6 3,9 40

20

40

60

80

100

Longer membrane Shorter membrane

Rec

over

y (%

)

Flow rate (uL/min)

34

Table 5. Interstitial concentrations of glucose, lactate, pyruvate, glycerol, urea, andglutamate and the lactate/pyruvate ratio in normal human and piglet brain tissue(Reinstrup et al. 2000).

The brain depends almost exclusively on the aerobic consumption of glucose forenergy production (Goodman et al. 1999). However, information on brain ECF glucoselevels under normal conditions in response to brain ischemia/hypoxia is scant (Valtyssonet al. 1998). The brain glucose concentration reflects the balance of the supply from theblood and utilization by cells (Fellows et al. 1992). The results of the rat ischemia modelsupport the contention that brain glucose may be a valuable marker of severe ischemiaand may help to differentiate between partial and complete ischemia (Valtysson et al.1998). In the case of complete ischemia, glucose is depleted almost totally from the braintissue (Ljunggren et al. 1974a, Rimpiläinen et al. 2001), whereas with a lesser degree ofischemia it is decreased, but still found in the ECF (Tables 1 and 6) (Schulz et al. 2000).

Fig. 5. Ischemic glycolysis.

Several studies have shown that high-energy metabolites ATP and PCr, along withglucose and glycogen stores, are consumed during the first few minutes duringnormothermic ischemia, and at this time lactate has reached its maximal concentrationand glucose has disappeared from the brain tissue (Ljunggren et al. 1974a). Lactateincreases during total or, in particular, partial ischemia (Schulz et al. 2000). However, theabsence of a rapid increase of ECF lactate represents an important finding fordistinguishing between intracellular lactate and ECF lactate (Persson et al. 1996). Studies

Brain tissue microdialysis Glucose Lactate Pyryvate Glycerol Gluta-mate

Lactate/pyryvate ratio

Urea

Human brain

Anesthetized (1.0 µl/min) 1.2 ± 0.6 1.2 ± 0.6 70 ± 24 28 ± 16 17 ± 12 22 ± 6 2.4 ± 1.3

Awake (1.0 µl/min) 0.9 ± 0.6 1.4 ± 0.9 103 ± 50 42 ± 29 7 ± 5 21 ± 6 2.5 ± 1.3

Awake (0.3 µl//min) 1.7 ± 0.9 2.9 ± 0.9 166 ± 24 82 ± 44 16 ± 16 23 ± 4 4.4 ± 1.7

Piglet brain

Anesthetized (0.3 µl/min) 1.8 ± 0.7 2.0 ± 0.8 124 ± 93 35 ± 11 7 ± 6 18 ± 5 7 ± 6

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have shown that lactate increased mainly during recirculation rather than during completeischemia, and during repolarisation rather than depolarization (Persson et al. 1996). Itshould be recalled that the lactate production depends on substrate availability, and in thecase of a fast lack of glucose and pyruvate, lactate production is consequently limited(Fig. 5) (Persson et al. 1996).

Pyruvate metabolism resembles glucose metabolism. During severe ischemia its brainconcentration decreases, and in the case of complete ischemia, pyruvate is used toproduce lactate (Table 6) (Schulz et al. 2000). After an ischemic insult, an increase ofpyruvate levels is a better marker of reperfusion than glucose levels (Persson & Hillered1992).

The lactate/pyruvate ratio is a well-known marker of cell ischemia (Hillered et al.1990), and a much more reliable marker of cerebral ischemia compared to lactate orpyruvate alone (Valtysson et al. 1998). The use of a ratio has the further advantage ofabolishing the influence of changes in recovery over the dialysis membrane (Persson &Hillered 1992). When mitochondrial function is impaired, as during anoxia or severeischemia, the intracellular NADH/NAD+ ratio increases together with accumulated [H+]and drives the lactate dehydrogenase reaction towards lactate (Valtysson et al. 1998).Pyruvate may also be consumed when α-ketoglutarate and alanine is formed fromglutamate and pyruvate by alanine aminotransferase (Siesjö 1978). Changes in the brainlactate/pyruvate ratio appear to closely reflect the intracellular redox state (Siesjö 1978).

Table 6. Brain concentrations of markers of energy metabolism and neuronal injury inpatients with or without symptoms of ischemia (Schulz et al. 2000).

The lactate/glucose ratio is also a reliable marker of ischemia (Zauner et al. 1997,Goodman et al. 1999). When cerebral oxygenation is partially reduced, lactateaccumulates in the extracellular space. When severe enough, such episodes are associatedwith depletion of glucose from the extracellular space. This state may lead touncompensated anaerobic glycolysis in which neurons and astrocytes compete for theextracellular glucose in a desperate bid for a trickle of adenosine triphosphate production.In such a severe metabolic state, an increase in lactate/glucose ratio is observed, and isassociated with a poor clinical outcome Table 6 (Zauner et al. 1997, Goodman et al.1999).

Other markers of ischemia and cell damage. Glutamate is released from neuronsduring ischemia and accumulates in the interstial space. It is responsible for initiation of a

Marker Normal value SevereIschemia

Glucose (mmol/L) 2.12 ± 0.15 0.54 ± 0.15

Lactate (mmol/L) 3.05 ± 0.32 6.73 ± 1.09

Pyruvate (µmol/L) 151 ± 11.5 84.2 ± 35.8

Lactate/glucose ratio ratio 1.62 ± 0.18 16.7 ± 4.7

Lactate/pyruvate ratio ratio 19.3 ± 1.7 97.8 ± 32.2

Glutamate (µmol/L) 14.0 ± 3.33 119 ± 58.4

Glycerol (µmol/L) 81.5± 12.4 354 ± 88.5

36

pathological influx of calcium leading to cell damage. Glutamate concentrations havebeen shown to predict the postoperative outcome of patients with subarachnoidalhemorrhage (Persson et al. 1996). Glutamate has been shown to increase in ECF duringischemia and also in secondary ischemia (Hillered et al. 1990, Boris-Moller & Wieloch1998). It is an indirect marker of cell damage, but it is sometimes difficult to interpret itschanges due to the fact that the neuronally released glutamate is mixed with the largemetabolic pool of glutamate (Lipton & Rosenberg 1994). In experimental models ofHCA, brain glutamate levels increased after prolonged HCA, but without a clearassociation to postoperative outcome (Rimpiläinen et al. 2001, Rimpiläinen et al. 2002).Difficulties in detecting a narrow peak level of glutamate, as well as the commonlyobserved variability in individual increase of glutamate, may explain the lack ofcorrelation with the postoperative outcome.

Degradation of membrane phospholipids is a well-known phenomenon in acute braininjury (Hillered et al. 1998). Glycerol is an integral component of the cell membrane.Loss of energy during brain ischemia leads to an influx of calcium, which triggers eventsfor membrane phospholipid degradation to glycerol (Fig. 6) (Kristian & Siesjö 1998).During and after the ischemic condition, the release and production of glycerol isunbalanced. Glycerol concentrations rise during and after cerebral ischemia, and glycerolhas been shown to be a sensitive and reliable marker of cell damage in experimentalcerebral ischemia (Hillered et al. 1998, Frykholm et al. 2001). When compared withglutamate, brain glycerol has a wider peak after HCA, and its decrease takes severalhours, making the measurement much easier.

Fig. 6. Biochemical pathways of membrane phospholipids degradation to glycerol modifiedfrom (Frykholm et al. 2001). Phospholipids are liberated from the cell membrane through theaction of phospholipases. Further degradation occurs in the cytosol. In ischemia, theseprocesses are augmented by Ca2+ overload or induction of phospholipases. In the diagram,enzymes are shown in italics.

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2.2.3.2 Tissue monitoring probes

Direct brain tissue oxygen monitoring in patients with traumatic head injury was firstreported by Maas (Maas et al. 1993). The placement of intracranial chemistry probes canbe accomplished with techniques similar to those used for standard ICP probes(McKinley et al. 1996). The microsensor probe is 0.8 mm in diameter, and the sensingarea is 18 mm2 (Fig. 7).

Fig. 7. Tissue oxygenation monitoring probe with a diameter of 0.8mm.

Later, a probe by which it is possible to monitor cerebral tissue oxygen, carbondioxide, pH and temperature was introduced (Zauner et al. 1995). A fiber-optic probewith sensors for monitoring the partial pressure of tissue oxygen (Pti02), carbon dioxide(PtiCO2) and pH has been proved to provide results comparable to those achieved byother tissue monitoring methods (McKinley et al. 1996).

Brain tissue oxygenation. The normal level of brain PtiO2 is 33 to 36 mmHg (Hoffmanet al. 1996a). Monitoring of PtiO2 by tissue probe is safe, reliable and sensitive indetecting changes in brain tissue oxygenation with results comparable with jugular veinoxygen saturation measurement (Kiening et al. 1996). A brain PtiO2 below 8 mmHg forlonger than 30 minutes is associated with increased extracellular glutamate and cerebralinfarction (Kett-White et al. 2002). The quality of the oxygenation measurement isexcellent, provided the first hour after insertion (adaptation time) is excluded (Dings et al.1998).

Brain tissue carbon dioxide pressure (PtiCO2). The normal level of brain PtiCO2 isabout 49 mmHg (Hoffman et al. 1996a). There are conditions associated with anincreased or decreased PtiCO2 such as increased cerebral vascular constriction (CarmonaSuazo et al. 2000). The increased brain tissue PtiCO2 observed in patients with acompromised cerebrovasculature is consistent with ischemic tissue PtiCO2 (Hoffman etal. 1996a). However, as a marker of ischemia, PtiO2 is faster and more reliable thanPtiCO2 (Hoffman et al. 1996b). The decrease of PtiCO2 after hypothermic ornormothermic ischemia is a marker of reperfusion, and faster than PtiO2 or pH (Hoffmanet al. 1996b).

Brain tissue pH. Continuous monitoring of pH and PtiCO2 allows the precisemonitoring of the acid-base status of the brain tissue. The normal level of brain pH isabout 7.25 in humans (Hoffman et al. 1999). It has been shown by this method that a pHof less than 7.0 for more than 20 minutes is associated with severe brain injury (Hoffmanet al. 1996a). Interestingly, monitoring of brain tissue pH provides indirect evidence ofICP (Hoffman et al. 1996a).

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In summary, a combination of brain tissue pH, PtiCO2 and PtiO2 measurement bymeans of a minimally invasive tissue monitoring probe gives valuable and reliableinformation on tissue oxygenation, carbon dioxide pressure and acid-base balance.

2.2.3.3 Near-infrared spectroscopy

Near-infrared spectroscopy (NIRS) is a non-invasive optical monitoring techniqueproviding information on vascular oxygenation by showing changes in tissueoxyhemoglobin, deoxyhemoglobin and total hemoglobin. It also provides information oncellular oxygenation by detecting changes in oxidized cytochrome a, a3 (CytOx), the lastenzyme of the respiratory chain (Jöbsis 1977, Wray et al. 1988). The basis of thismonitoring method is near-infrared light, which, in the spectral range of 700 to 1000 nm,is absorbed less than visible light and can penetrate much further, up to a depth of 6 cminto the tissues (Wray et al. 1988). These optical methods allow transmissionspectroscopy to be performed in vivo.

Fallon et al first reported on its use in patients undergoing cardiac surgery forinvestigation of cerebral hemodynamics during CPB (Fallon et al. 1993). NIRSmeasurements with magnetic resonance imaging (MRI) during HCA showed highcorrelation between the CytOx value and PCr levels and histological brain injury afterHCA (Shin'oka et al. 2000). The oxygenated hemoglobin signal nadir time in NIRS is auseful predictor of safe duration of circulatory arrest. Interestingly, when the nadir timewas below 25 minutes during HCA of 15 or 25 °C, there was no behavioral orhistological evidence of brain injury (Sakamoto et al. 2001b). NIRS has also been used toverify the safe level of brain oxygenation during total arch replacement employingselective brain perfusion (Yamashita et al. 2001).

The usefulness of these measurements has been questioned because the measurementsare relative to baseline, and CytOx provides a small signal and therefore is vulnerable toartifact (Matsumoto et al. 1996, Nollert et al. 1998). Another pitfall of this method is thefact that hematocrit interferes with the cytochrome signal (Kurth & Uher 1997, Sakamotoet al. 2001a).

Clinical experience with cerebral oximetry after stroke and cardiac arrest have shownsome methodological characteristics. Oximetry by NIRS reflects the balance betweenregional oxygen supply and demand. In dead or infarcted nonmetabolizing brain,saturation may be near normal because of sequestered cerebral venous blood in capillariesand venous capacitance vessels, and because of contribution from overlying tissue(Nemoto et al. 2000). Indeed, NIRS has failed to be an indicator of cerebral ischemiaduring carotid surgery (Beese et al. 1998).

In summary, NIRS is a non-invasive, feasible and safe method for measuring cerebraloxygenation independent of brain function, cerebral blood flow and metabolism. It has allthe advantages to make it the gold standard for real-time brain monitoring during cardiacsurgery (Nollert et al. 2000). NIRS measurements, especially the CytOx signal, correlatewell with high-energy phosphates and have a high sensitivity for predicting histologicalbrain damage after HCA. However, the CytOx signal also has several limitations, such as

39

a small signal and high artifact effect, depending on the hematocrit value (Nollert et al.2000).

2.2.3.4 Intracranial pressure

An increase in ICP occurs after brain infections, head injury, ischemic injury andintracranial bleeding. It has been shown to be a major factor contributing to the severityof brain ischemic injury in neurosurgical patients (Juul et al. 2000, Schneweis et al.2001), as it is associated with derangements in blood flow supply to the brain(Hekmatpanah 1970, Nagai et al. 1997). Such disturbances in cerebral blood flow beginat the microcirculatory level with the collapse of capillaries, which is associated withsloughing of red blood cells and formation of microemboli. A further increase in ICP mayalso involve larger intracranial arteries and veins, thus worsening the blood supply to thebrain (Hekmatpanah 1970). Nakai employed a microvascular laser-Doppler flow-meter tomeasure the blood flow into the middle cerebral artery, and showed that with the increasein ICP, the flow patterns appeared in the following order: normal flow, sharp wave,systolic flow, systolic spike and no flow (Nagai et al. 1997). ICP threshold levels andcerebral perfusion pressure (CPP) predicting adverse outcome have been studied inpatients with severe head injury (Chambers et al. 2001). CPP threshold levels for adverseoutcome were 45mmHg in children and 55 mmHg in adults, and for ICP, 35 mmHg inboth adults and children (Chambers et al. 2001). A reduction of ICP to less than 20mmHg is considered a main therapeutic target in patients with severe head injury (Juul etal. 2000).

There is evidence that a significant increase in ICP also occurs during and after CPB(Lundar et al. 1983b, Lundar et al. 1985, Johnston et al. 1991, McDaniel et al. 1994). Theetiology behind such an ICP increase during CPB remains unknown. Interestingly, a studyemploying MRI has shown an increased content of cerebral water after CPB, which wasnot found in patients undergoing off-pump coronary artery bypass surgery (Anderson etal. 1999). Obstruction of CSF venous flow has been suggested as a contributor toincreased ICP (Philpott et al. 1998). Increased postoperative ICP has been observed to beassociated with lower recovery of EEG and adverse neurological outcome afterexperimental HCA (Hagl et al. 2002).

Only a few studies have been performed to investigate the role of ICP in predicting theoutcome after cardiac surgery. Anyway, there is evidence that a modest increase of ICPmay also put the patient at high risk of cerebral hypoperfusion, especially in the case ofdecreased mean arterial pressure (Philpott et al. 1998). Thus, the quality of perioperativeand postoperative monitoring of cerebral function would be greatly improved ifintracranial compliance and pressure (ICP) could be continuously monitored. In cardiacsurgery, a new non-invasive method for monitoring intracranial compliance allowson-line ICP monitoring and could be performed with reliable results (Michaeli &Rappaport 2002, Paulat et al. 2002).

3 Aims of the studyThe aims of the present research were:

1. To investigate the relationship between serum S100β protein concentration andhistopathological brain injury after prolonged HCA (I).

2. To evaluate which of the cerebral microdialysis parameters are predictors ofpostoperative survival and histopathological brain injury after HCA (II).

3. To investigate whether intracranial pressure is predictive of postoperative outcomeafter HCA (III).

4. To evaluate whether derangements in EEG burst percentage recovery is associatedwith the development of ischemic brain injury after HCA (IV).

4 Methods

4.1 The chronic porcine model

This chronic porcine model was adopted from the Mount Sinai School of Medicine inNew York by professor Juvonen (Juvonen et al. 1998a, Juvonen et al. 1998b). The modelhas undergone significant methodological improvement at the Cardio-thoracic ResearchLaboratory of Oulu University during the years 1998–2003 (Anttila et al. 1999, Anttila etal. 2000a, Anttila et al. 2000b, Rimpiläinen et al. 2000, Rimpiläinen et al. 2001,Rimpiläinen et al. 2002, Romsi et al. 2002b, Romsi et al. 2002c, Romsi et al. 2002a,Pokela et al. 2003).

4.2 Animals

128 female juvenile (8 to 10 weeks) pigs of native stock, weighing 21.0 to 38.2 kg,underwent CPB preceding and following a 75 minutes period of HCA at 20°C. Most ofthese animals (112/128) also belong to our previous experimental studies (Anttila et al.2000b, Rimpiläinen et al. 2000, Rimpiläinen et al. 2001, Rimpiläinen et al. 2002, Romsiet al. 2002b, Romsi et al. 2002c).

4.3 Preoperative management

All animals received humane care in accordance with the “Principles of LaboratoryAnimal Care” formulated by the National Society for Medical Research and the “Guidefor the Care and Use of Laboratory Animals” prepared by the Institute of LaboratoryAnimal Resources and published by the National Institutes of Health (NIH publicationNo. 85–23 revised 1985). The study was approved by the Research Animal Care and UseCommittee of the University of Oulu.

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4.4 Anesthesia protocols

Anesthesia was induced with ketamine hydrochloride (10 mg/kg intramuscularly) andmidazolam (1 mg/kg intramuscularly) in all animals of study I, in 45 out of 56 animals instudy III and in 20 out of 44 animals in study IV. In the remaining animals of studies II–IV, anesthesia was induced by midazolam (1 mg/kg administered intramuscularly) andmedetomidine hydrochloride (0.4 mg/kg intramuscularly). Before endotrachealintubation, anesthesia was deepened with thiopental sodium (125–250 mg administeredintravenously), and after intubation the animals were maintained on positive pressureventilation. Muscular paralysis was maintained with pancuronium bromide (0.1 mg/kgintravenously) and anesthesia with isoflurane (1.2%). Cefuroxime, 1.5 g, was given asantibiotic prophylaxis at anesthesia induction, 8 hours after the start of rewarming, andbefore extubation.

4.5 Anesthesia methods and monitoring

A peripheral venous catheter was inserted into the right ear for administration of drugsand to maintain fluid balance with Ringer acetate. An arterial catheter was positioned intothe left femoral artery for arterial pressure monitoring and blood sampling. Athermodilution catheter (CritiCath®, 7-F, Ohmeda GmbH & Co, Erlangen, Germany) wasplaced through the left femoral vein to allow blood sampling, pressure monitoring in thepulmonary artery and for recording the blood temperature and cardiac output. A 10-Fcatheter was placed into the urinary bladder for urine output monitoring. Coretemperatures were monitored continuously from the blood, rectum, esophagus, andepidural space.

During the experiment, hemodynamic and metabolic measurements: electro-cardiography (ECG), heart rate, systemic and pulmonary arterial pressures, central venouspressure, pulmonary capillary wedge pressure, cardiac output, temperatures from theblood, rectum, esophagus, epidural spaces, respiratory gases (O2, CO2, isoflurane)) weremonitored by the Datex AS/3 anesthesia monitor (Fig. 8) (Datex Inc., Espoo, Finland).Samples for arterial and venous pH, oxygen and carbon dioxide partial pressures, oxygensaturation, oxygen concentration, hematocrit, hemoglobin, sodium, potassium andglucose [Ciba-Corning 288 Blood Gas System; Ciba-Corning Diagnostic Corp, Medfield,Mass]; lactate [YSI 1500 analyzer; Yellow Springs Instrument Co, Yellow Springs, Ohio];leukocyte differential count (Studies II, III, IV) [Cell-Dyn analyzer; Abbot, Santa Clara,CA, U.S.A.]; and creatine kinase [CK] and its isoenzymes (Studies II, III, IV) [CK-MM,CK-MB, CK-BB, Hydrasys LC-electrophoresis, Hyrys-densitometry; Sebia, France])were taken and analyzed at baseline, at the end of cooling (immediately before institutionof HCA), 30 minutes, 2 hours, 4 hours, and 8 hours after the start of rewarming, andbefore extubation.

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Fig. 8. Anesthesia monitoring during cooling perfusion. (Datex AS/3)

4.6 Experimental protocol of hypothermic circulatory arrest

Through a right thoracotomy on the fourth intercostal space, the right mammary vesselswere ligated, the pericardium was opened and the heart and great vessels were exposed.A membrane oxygenator (Midiflow D 705, Dideco, Mirandola, Italy) was primed with 1liter of Ringer acetate and heparin (5000 IU). After systemic heparinization (500 IU/kg),the ascending aorta was cannulated with a 16-F arterial cannula and the right atrialappendage was cannulated with a single 24-F atrial cannula. Non-pulsatile CPB wasstarted at a flow rate of 100 ml/kg/min and the flow was adjusted to maintain a perfusionpressure of 50 mmHg. A 12-F intracardiac sump cannula was positioned into the leftventricle through the apex of the heart for decompression of the left side of the heartduring CPB. A heat exchanger was used for core cooling.

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Fig. 9. Experimental protocol of 75 minutes of hypothermic circulatory arrest is summarized inpicture.

Protocol is summarized in Fig. 9. A cooling period of 60 minutes was carried out toattain a brain temperature of 18°C. Then, a 75-minute period of HCA was started. Duringperfusion, pH was maintained using alpha-stat principles at 7.40 ± 0.05 with an arterialCO2 tension of 5.0 to 5.4 kPa, uncorrected for temperature. The ascending aorta was thencross-clamped just distal to the aortic cannula and cardiac arrest was induced by injectingpotassium chloride (3 g) via the aortic cannula. Topical cardiac cooling with ice slush wasbegun and maintained throughout the HCA period. During HCA, the epidural andintracerebral temperatures were maintained at a level of 18°C with ice packs over thehead.

After a 75-minute HCA, rewarming was started and furosemide (40 mg), mannitol(15 g), methylprednisolone (80 mg), lidocaine (40–150 mg) and calcium chloride (1375mg) were administered. Weaning from CPB occurred approximately 60 minutes after thestart of rewarming, cardiac support after CPB was provided by dopamine (0–30mg/h).Urine output was supported by furosemide (20–80mg) to attain adequate water balance.The animals were kept in isoflurane anesthesia until the following morning in study I. Inthe studies II–IV, animals were extubated, and moved into a recovery room 8 hours afterHCA.

4.7 Intracranial parameters monitoring

4.7.1 Cortical microdialysis (studies II–IV)

A microdialysis catheter (CMA 70; CMA/Microdialysis, Stockholm, Sweden) was placedinto the brain cortex to a depth of 15 mm below the dura mater. The catheter was

1720232629323538

Bra

in te

mpe

ratu

re (C

°)

Protocol

HCA75 min CPBCPB

0h 1h 2h 4h 8h-75min

Respirator

Extu

batio

n

-135min Time after HCAAne

sthe

sia

indu

ctio

n

45

connected to a 2.5 ml syringe placed into a microinfusion pump (CMA 106; CMA/Microdialysis, Stockholm, Sweden) and perfused with a ringer solution at a flow rate of0.3µL/min (Perfusion Fluid CNS; CMA/Microdialysis, Stockholm, Sweden). Sampleswere collected at baseline, 30 minutes of CPB cooling, start of HCA, 30 minutes of HCA,end of HCA and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7 and 8 hours after HCA. Theconcentrations of cerebral extracellular glucose, lactate, glutamate and glycerol weremeasured immediately after collection using ordinary enzymatic methods with amicrodialysis analyzer (Fig. 10) (CMA 600; CMA/Microdialysis, Stockholm, Sweden).

Fig. 10. Microdialysis analyzator (CMA 600).

4.7.2 Intracranial pressure (study III)

An intracranial pressure-monitoring catheter (Codman Micro-Sensor ICP Transducer,Codman ICP Express Monitor, Codman & Shurtleff, Inc., Raynham, MA, USA) wasplaced through a hole located at the left side 0.5 cm posterior to the coronal suture. ICPwas continuously recorded. The ICP monitor is shown in Fig. 11.

46

Fig. 11. Intracranial pressure monitor (Codman).

4.7.3 Brain tissue oxygenation and temperature monitoring(study III, IV)

Two catheters for the measurement of intracerebral tissue oxygen partial pressure(Revodoxe Brain Oxygen Catheter-Micro-Probe, REF CC1.SB, GMS, Mielkendorf,Germany) and temperature (Thermocouple Temperature Catheter-Micro-Probe REF.C8.B, GMS, Mielkendorf, Germany) were inserted through a hole located at the right sideanteriorly to the coronal suture. These parameters were monitored continuously by aLicox CMP Monitor (Fig. 12) (GMS, Mielkendorf, Germany).

Fig. 12. Brain tissue oxygen and temperature monitor (Licox CMP Monitor).

4.8 Postoperative management

Postoperatively, all animals were evaluated at least once a day to maintain sufficient painmedication with buprenorfine (0.3–0.9 mg/day intramuscularly). Intravenous fluid andmask oxygen were administered during the first postoperative day. During the followingday, the animals were helped to drink if they were not able to.

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4.9 Histopathologic analysis

Perfusion fixation. Each surviving animal was electively sacrificed on the seventhpostoperative day. Immediately after intravenous injection of pentobarbital (60 mg/kg)and heparin (500 IU/kg), the thoracic cavity was opened, and the descending thoracicaorta was clamped. Ringer solution (1 L) was perfused through the ascending thoracicaorta through the upper body, and blood was suctioned from the superior vena cava untilthe perfusate was clear of blood. Then a 10% formalin solution (1 L/15 min) was infusedthrough the brain in the same manner to accomplish perfusion fixation (Studies III andIV). Immediately thereafter, the entire brain was harvested, weighed, and immersed in10% neutral formalin.

Fixation protocol and histological analysis. The brain was allowed to fix further for 1week en bloc. Thereafter, 3-mm thick coronal samples were sliced from the left frontallobe, thalamus (including the adjacent cortex) and hippocampus (including the adjacentbrainstem and temporal cortex), and sagittal samples from the posterior brainstem(medulla oblongata and pons) and cerebellum were obtained. The specimens were fixedin fresh formalin for another week. After fixation, the samples were processed as follows:rinsing in water for 20 minutes and immersion in 70% ethanol for 2 hours, 94% ethanolfor 4 hours, and absolute ethanol for 9 hours. Then, the specimens were kept for 1 hour inan absolute ethanol-xylene mixture, 4 hours in xylene and embedded in warm paraffin for6 hours. The specimens were sectioned at 6 mm and stained with hematoxylin and eosin.The sections of the brain specimens of each animal were screened by an experiencedsenior pathologist (J.H.) unaware of the experimental design or the identity and fate ofeach animal.

Quantitative scoring of histological findings in study I. Visual estimation and gradingof brain ischemic injury in the sampled regions was carried out at histologicalexamination as follows: 0 = no morphological signs of damage; 1 = edema oreosinophilic or dark neurons or dark/shrunk cerebellar Purkinje cells; 2 = at least twosmall hemorrhages and 3 = clearly infarctive foci. The total score was the sum of scoresin each specific brain area (cortex, thalamus, hippocampus, posterior brainstem and brainstem). To allow semiquantitative comparison between animals, a total histological scorewas calculated by adding all the regional scores.

Quantitative scoring of histological findings in studies II–IV. The signs of injury werescored as follows: 1 (slight edema, dark or eosinophilic neurons or cerebellar Purkinjecells); 2 (moderate edema, at least 2 hemorrhagic foci in the section); 3 (severe edema,several hemorrhagic foci, infarct foci [local necrosis]). The total regional score was thesum of the scores in each specific brain area (cortex, thalamus, hippocampus, posteriorbrainstem, and cerebellum). A total histopathologic score was calculated by summing allthe regional scores to allow semiquantitative comparison between the animals.

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4.10 Electroencephalography monitoring (study IV)

Cortical electrical activity was registered by 4 stainless-steel screw electrodes of 5 mm indiameter implanted in the skull over the parietal and frontal areas of the cortex using adigital electroencephalography (EEG) recorder (Nervus, Reykjavik, Iceland) and anamplifier (Magnus EEG 32/8, Reykjavik, Iceland) (Fig. 13). The sampling frequency was256 Hz and bandwidth 0.03 to 100 Hz. All EEG recordings were referenced to a frontalscrew electrode, which, together with a ground screw electrode, was implanted over thefrontal sinuses.

Fig. 13. Electroencephalography monitor (Nervus).

Isoflurane was administered at a steady level of 1.2%. EEG was recorded for 10minutes to get a baseline recording of steady burst-suppression activity before the coolingperiod. After HCA, EEG recording was restarted and continued until extubation. Theduration of bursts was measured from 5-minute EEG samples at 1-hour intervals. Artifactperiods were excluded from each 5-minute sample and, after that, the sum of bursts wascalculated as a percentage of the sum of artifact-free bursts and suppressions. This burstpercentage was used as a measure of EEG activity in the analysis (Fig. 14).

49

Fig. 14. An example of EEG burst percentage analysis in a pig after 75 minutes of HCA.Characters a–f shows EEG sample of different time points after HCA and left side samecharacters show the exact time intervals and percentage of EEG recovery. Made by PasiLepola.

4.11 Measurement of serum S100β protein (study I)

Central venous blood samples were taken for S100β protein analysis at specific timeintervals: before the operation and at 2, 4, 7 and 20 hours after the end of HCA. After fastcooling, all samples were centrifuged and the serum frozen. S100β concentrations were

50

analyzed using a luminescence immunoassay kit (Sangtec-100®, LIA-mat) (SangtecMedical AB, Bromma, Sweden).

4.12 Statistical analysis

Statistical analysis was performed using the statistical software SPSS (Statistical Packagefor Social Sciences 10.0.7, SPSS Inc., 444 North Michigan Avenue, Chicago, IL 60611,USA). Data on continuous variables are expressed as the median with interquartile range(25th and 75th percentiles). The Wilcoxon’s test was used to evaluate any difference incontinuous variables between the baseline measurement and the other intervalmeasurements. The Student’s t-test or the Mann-Whitney test was used to evaluate anydifference between continuous variables at different time intervals. The Pearson’scorrelation coefficients (r) with their significance levels were calculated in order toestimate the correlation between continuous variables in study III. Kendall`s rankcorrelation was used to estimate the correlation coefficient (τ) to parametrical data.Spearman’s correlation coefficients with their significance levels were calculated in orderto estimate the correlation between continuous variables in study IV. The analysis ofvariance for repeated measures was performed. Logistic regression was used to evaluatethe impact of ICP on the outcome, and the ICP values at each study interval were used asindependent variables in study III. The mean EEG recovery zero to seven hours afterHCA was calculated for EEG measurements in study IV. A probability value of <0.05was considered significant.

5 ResultsAll animals that were included in the present studies were stable during the surgicalprocedures and survived at least until the extubation. The major findings of these series ofstudies are summarized in Table 7 and (Fig. 15–18).

Table 7. Predictors of postoperative death, brain infarction and severe brain ischemicinjury as detected at histological examination.

5.1 Serum S100β protein (study I)

Serum S100β protein concentrations are presented in Fig. 15. The median baseline S100βprotein concentration was 0.4 mg/L (IQR, 0.2–0.7 mg/L). Two hours after HCA, S100βprotein levels were higher than the baseline levels, the median being 0.7 mg/L (IQR,0.4–1.0 mg/L) (p=0.005).

Ten out of eighteen animals (56%) survived 7 days after surgery and were electivelysacrificed. Animals that died had higher serum S100β levels 20 hours after HCA, but thedifference was not statistically significant.

The median total perfusion time was 136 minutes (IQR, 129–147 minutes). Themedian cooling CPB time was 61 minutes (IQR, 60–68 minutes) and warming CPB timewas 75 minutes (IQR, 67–78 minutes). The total period of CPB tended to correlate withthe S100β protein levels (τ=0.322, p=0.075).

A distinct brain injury was observed in all animals but one. Brain infarction occured inseven animals. The correlation between histopathological injury and S100β protein

Parameter Mortality Brain infarction Brain histology

Serum S100β protein level – + + + +

Brain microdialysis + + + + + +

Intracranial pressure + + + + –

EEG burst percentage recovery – + + + + +

– = not predictive; + = tended to be predictive; ++ = predictive; +++ = very predictive

52

concentrations at each recording point after HCA are shown in Figure 15. The strongestcorrelation was observed 7 hours after HCA. Animals with brain infarction hadsignificantly higher S100β protein concentrations seven hours after HCA than thosewithout brain infarction (median 0.28 mg/L vs. 0.18 mg/L, p=0.048) (Fig. 15). The sametrend was found at all recording points.

Fig. 15. Serum S100β protein concentration in 18 pigs that underwent a 75-minute period ofHCA. a. overall serum concentrations of serum S100β protein. c. Serum S100β protein concent-ration in animals that had or not brain infarction. b,d. Scatter plots showing the correlation bet-ween serum S100β protein and the severity of histological brain injury at 7- and 20-hour inter-vals after HCA. In figures a and c, values are reported as the median plus 25th and 75th IQR.

5.2 Cerebral microdialysis (study II)

The overall postoperative mortality rate was 48.6%, and the mortality rate on the day ofthe operation was 39.2%. Among 38 animals that survived until the 7th postoperative day,histological examination revealed brain infarction in 50% of them.

The Mann-Whitney test showed that animals that survived until the 7th postoperativeday had constantly higher brain glucose levels during all postoperative intervals exceptthe 75-minute interval of HCA and 30-minute interval after the start of rewarming, whenglucose levels were nil (tests of between-subjects effects, p=0.017). Such differenceswere statistically significant from the 90-minute to the 7-hour intervals after the start ofrewarming and tended to be significant even at the 8-hour interval (p=0.072) (Fig. 16). It

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is worth noting that a difference in brain glucose level was present even during thepre-HCA period. The blood venous concentrations of glucose were also higher amongsurvivors throughout all the intervals, but a significant correlation with the brain glucoselevels was observed only at 2-hour and 4-hour intervals after the start of rewarming.

The other microdialysis parameters did not show such large differences betweensurviving and dead animals. However, apart from brain lactate during the first twopostoperative hours, higher levels of brain lactate, glycerol and glutamate were observedthroughout the study intervals among animals that died postoperatively, and suchdifferences became statistically significant from the 4-hour point after the start ofrewarming (Fig. 16). Interestingly, brain glutamate and, in particular, glycerol levels,were significantly negatively correlated with the brain glucose levels.

Among animals that survived, brain lactate levels were slightly, but not significantly,higher during the rewarming phase until the 2-hour interval. However, after HCA, thelactate/glucose ratio was significantly lower among survivors during the wholepostoperative period (tests of between-subjects effects, p=0.014) (Fig. 16).

The analysis of the data in the subgroup of animals that survived until the 7th

postoperative day revealed that brain glucose, again, was higher among the animals thatdid not develop brain infarction. However, such a difference was statistically significantonly at the 30-minutes of cooling and 30-minutes of HCA intervals. Brain lactateconcentrations were higher, but without statistical significance, from the start ofrewarming to the 2-hour interval, and this finding was coupled with anot-statistically-significant lower brain lactate/glucose ratio during the same intervals.Postoperatively, brain glycerol levels were higher, but not reaching statistical significancein the surviving animals that developed brain infarction.

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Fig. 16. Microdialysis data in animals that died or survived. Values are reported as medians, * = p <0.05 between the groups, # = p < 0.01 between the groups of each interval.

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5.3 Intracranial pressure (study III)

The 7-day survival rate was 60.7%. Among the animals that survived until the 7thpostoperative day, 20 of them (58.8%) had brain infarction.

A significant increase in ICP as compared with the baseline level was observed fromthe end of cooling (p=0.047), and the difference became greater during all thepostoperative intervals (p<0.0001) (Fig. 17). CPP at the 8-hour postoperative intervaltended to decrease because of a continuous increase in ICP, which was significantlyhigher at the 8-hour postoperative interval (median: 14 mmHg, IQR, 12–17) as comparedwith the 4-hour interval value (median: 11.5 mmHg, IQR 10–14) (p<0.0001). It ispossible that the rise of central venous pressure could have had a minimal effect on theincrease of ICP. In fact, central venous pressure, despite having seen significantly higherthan the baseline level, tended to decrease from the peak value reached at the 2-hourpostoperative interval, and at the 8-hour postoperative interval the median value was 4.5mmHg. Furthermore, there was no statistically significant correlation between the ICPvalues and the central venous pressure values, cardiac index values and otherhemodynamic parameters at any study interval.

According to the Pearson correlation test, ICP was significantly correlated with brainconcentrations of lactate at the 2-hour interval after the start of rewarming (r=0.302, p=0.024) and with the brain lactate/glucose ratio at the end of the cooling interval (r=0.347,p=0.011) and at the 2-hour interval after the start of rewarming (r=0.396, p=0.003).

Animals that died postoperatively tended to have higher ICP levels during all thepostoperative intervals (tests of between-subjects effects: p=ns) (Fig. 17). According tothe Student’s t-test, the ICP was significantly higher at the 4-hour postoperative intervalin the animals that died postoperatively (p=0.040). The logistic regression analysisshowed that the ICP at the 4-hour postoperative interval was predictive of postoperativedeath (p=0.034; OR 1.221). The mean peak ICP was 16.6 mmHg (IQR, 13.6–21.0) inanimals that died and 15.2 mmHg (IQR, 12.8–19.6) in those that survived 7 days (p=ns).

The same tendency was observed among animals that survived until the 7thpostoperative day and that developed brain infarction or not (tests of between-subjectseffects: p=ns). However, the logistic regression analysis showed that ICP at the 2-hourpostoperative interval was predictive of postoperative brain infarction (p=0.032; OR1.685). The animals that developed brain infarction had higher mean arterial pressure andcerebral perfusion pressure at the 40-min and 2-hour postoperative intervals, in thepresence of a slightly higher ICP. The mean peak ICP was 17.9 mmHg (IQR, 13.3–19.9)in the animals that developed brain infarction and 14.1 mmHg (IQR, 11.8–16.4) in thosethat did not develop brain infarction (p=ns).

The animals that died or developed brain infarction postoperatively had higher ICPvalues postoperatively as compared with those that survived without developing braininfarction (tests of between-subjects effects: p=ns) (Fig. 17), and, according to theStudent’s t-test, such a difference reached significance at the 2-hour (p=0.015) and 4-hourpostoperative intervals (p=0.035). The logistic regression analysis showed that the ICP atthe 2-hour postoperative interval was predictive of postoperative death or brain infarction(p=0.018; OR 1.625). The peak ICP was 17.2 mmHg (IQR, 13.7–20.8) in animals thatdied or developed brain infarction and 14.1 mmHg (IQR, 11.8–16.4) in those thatsurvived 7 days without developing brain infarction (p=ns).

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Fig. 17. a. Intracranial pressure significantly increased from the end of cooling as comparedwith the baseline level; b,c,d: Intracranial pressure values in different outcome groups,*: p<0.05 according to the Student’s t-test. Values are shown at the median plus the 25th and75th IQR.

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5.4 Electroencephalography (study IV)

Brain infarction developed in 13 animals (48.1%); in 12 cases (44.4%) it involved thecortex, in one the thalamus (3.7%) and in another the hippocampus (3.7%). The mediantotal histopathological score was 13 (IQR: 8–16). The cortex had a medianhistopathological score of 4, the hippocampus, cerebellum and brain stem of 2, and thethalamus of 1.

All animals were in electrocerebral silence immediately after HCA, and at the 1h–20min interval after the start of rewarming 15 animals (55.5%) were still in electrocerebralsilence.

The median of mean EEG burst percentage from the start of rewarming to the 7h–20min interval was 34.0% (15.2–46.7%) (Fig. 18a). At the 7 h-20min interval themedian EEG burst percentage was 88.9% (49.3–98.9%). The mean EEG burst percentagesignificantly correlated with the total brain histopathological score (ρ=–0.588, p=0.001,Fig. 19a). EEG burst percentage from the 2h-20min to 7h-20min intervals correlated withthe total brain histopathological score and with the cortex, brain stem and cerebellumscores, the strongest correlation being at the 6h 20min interval (Fig. 19b).

The EEG burst percentage at each study interval and the mean EEG burst percentagewere not significantly associated with brain infarction. The mean EEG percentage ratewas higher, but not significantly so, among the animals without brain infarction (38.5%vs. 32.4%). The EEG burst percentage at the 3h-20min interval was significantlyassociated with the development of brain infarction in the brain cortex (p = 0.02) (Fig.18b).

The mean EEG burst percentage significantly correlated with brain glucoseconcentration at 1h (ρ= 0.387; P= 0.046), brain lactate concentration at 2h (ρ= –0.431; p=0.025), and the brain lactate/glucose ratio at 1h-30min from the start of rewarming (ρ=–0.433; p= 0.024). Correlation was also observed at the 7h-20min interval between brain

microdialysis glycerol and EEG recovery (ρ= –.403; p= 0.037).

Fig. 18. a-b. a. EEG percentage in pigs after 75 minutes of HCA with blood temperature curveand b. with or without brain infarction. EEG burst percentage: [EEG burst time/(EEG bursttime + EEG suppression time)] x 100. Dot line, median temperature; Black line, EEG median;error bars, IQR.

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6 Discussion

6.1 General discussion

The aim of the present studies was to evaluate the role of different monitoring methods inpredicting brain injury after experimental HCA. Two major outcome end-points weretaken into account in this analysis, i.e. postoperative mortality and stroke, the later beingconsidered the most relevant cause of morbidity and death after HCA. The lack ofadequate parameters for intraoperative monitoring of the brain which also work aspredictors of postoperative outcome forms the basis of the present research. Modernneuroimaging techniques, such as computed tomography and MRI are sensitive andspecific methods for evaluation of brain injury, but these tools are not always suitable orwidely available on a routine basis for neuromonitoring during or immediately aftercardiac surgery (Ali et al. 2000).

Measurement of biochemical markers from peripheral blood would certainly be morefeasible in the clinical field than the above-mentioned imaging methods, as bloodsampling is relatively noninvasive and easily repeatable. Although such markers are notlikely to provide the surgeons and anesthesiologists with prompt information on theoccurrence of brain ischemic injury during the operation, they would be of great help inthe identification of adverse cerebral events even if subclinical. Indeed, these noninvasivemethods would render possible a biochemical grading of the severity of brain ischemicinjury which, nowadays, mostly relies on neurological evaluation. Thus, even if not allcerebrovascular events occurring after cardiac surgery are of clinical importance in theimmediate and long-term, methods for an adequate identification and estimation of theirseverity would represent a major step forward in the experimental and clinical researchsetting. Several proteins measurable from peripheral blood have been introduced asbiochemical markers of brain injury. Unfortunately, most of them have a low accuracy,and to date only S100β protein seems to have some potential as a biochemical marker ofbrain ischemic injury in cardiac surgery (Johnsson 1996). Studies of potential newmarkers are ongoing, and preliminary reports on GFAP showed promising results(Herrmann et al. 2000a).

EEG is a non-invasive method of neuromonitoring which has been extensivelyemployed during cardiac surgery (Edmonds Jr 2002). However, there is a lack of data

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regarding its role in the case of HCA and, in particular, there is a lack of a simple,standardized EEG method.

Among the invasive methods, cerebral monitoring by microdialysis and measurementof ICP have found a place during neurosurgical operations and in neurointensive careunits, as these parameters have been shown to be strong predictors of outcome (Persson etal. 1996, Zauner et al. 1997, Goodman et al. 1999). However, in cardiac surgery, theinvasive nature of these methods prevents the monitoring of ICP and microdialysisparameters in the clinical field, and their use is limited to the experimental setting.

This research focused on the evaluation of the latter methods of neuromonitoring in aparticularly severe experimental setting of brain ischemic injury such as prolonged HCA,a condition that has profound adverse effects on cerebral metabolism. The relatively largenumber of animals collected from our previous studies and included in the presentresearch provided good support for an adequate statistical analysis of the data. In fact, it isusually the size of the study population which represents a major limitation inexperimental studies.

6.2 Experimental model

A chronic porcine model was used in these studies. The pig represents the animal ofchoice for experimental cardiac surgery, because porcine anatomy and physiology arevery close to those in humans (Rawlings et al. 1973, Griepp et al. 1997a). Non-humanprimates are likely to be better for experimental issues, but their use is not ethically andeconomically feasible. Dogs and sheep have been used in cardiac surgery research withgood results (Baumgartner et al. 1998, Nagashima et al. 1999), but their use is moreexpensive as compared with pigs, they are not readily available in this area and, in thecase of dogs, public opinion is strongly against their use in experimental surgery.

Although the use of a chronic animal model is much more complex to develop andmaintain, it is logically more close to clinical conditions, and permits retrieval ofinformation on two major research end-points, i.e., mortality and development of braininfarction. The cause of postoperative death after both experimental and clinical HCA iscertainly multifactorial (Juvonen et al. 2001), however, cerebral complications areconsidered a major determinant of postoperative mortality. In fact, the severity of brainischemic injury caused by a 75-minute period of HCA, as not infrequently occurs in theclinical field, is such as to clearly surpass the degree of ischemic injury to any otherorgan. Anyway, mortality can be viewed as a non-specific end-point, hence the need for apost-mortem histopathologic evaluation of the brain for a better definition of the extent ofbrain injury. On the other hand, histopathological grading of brain ischemic injury maysuffer the lack of clear-cut parameters, as the only way to compare histological findings isto use semiquantitative methods. The grading system used herein is a modification, byProf. Hirvonen, of the Mt. Sinai Hospital’s system (Juvonen et al. 1998a), but a largenumber of other methods are currently in use by other groups.

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6.3 Serum S100β protein

The presence in the blood of proteins normally confined to the cytoplasm of brain cells isconsidered indirect evidence of brain damage. Several proteins have been suggested asmarkers of brain damage, S-100β protein being the most promising.

In the present study, S-100β protein failed to show any significant association withpostoperative mortality, but a certain higher serum concentration at the 20-hour intervalafter HCA in animals that died has been observed. This finding can be explained by themultiple determinants of death in these animals and by the lack of a large population. Theimportant finding of study I was the positive correlation between histological brain injuryand serum S100β protein levels. The best correlation between the serum levels of S100βprotein and the brain histological score was observed at 7 and 20 hours after HCA,respectively. Other authors have also suggested that the optimal timing seems to bebetween 7 to 48 hours after the operation (Jönsson et al. 2001, Svenmarker et al. 2002,Johnsson et al. 2003). Serum levels of S100β protein tended to be higher in animals thatdeveloped brain infarction during all the postoperative period intervals. However, in theanalysis, the animals that died immediately after the experiment were also included, thusevidently not having yet developed brain infarction detectable at histopathologicalevaluation. Anyway, the results are in line with those of clinical studies showing anassociation between stroke and postoperative serum levels of S100β protein (Jönsson etal. 2001).

Interestingly, the baseline S100β protein values were unexpectedly high, with a largestandard deviation. This might have been caused by brain injury during induction ofanesthesia or, more likely, by extracerebral contamination from adipose tissue as a resultof subtle trauma during animal capture. Indeed, serum S100β protein is not accurate whenmeasured before the 8th postoperative hour after cardiac surgery because ofcontamination from mediastinal adipose tissue, especially when cardiotomy suction isused (Jönsson et al. 2001, Svenmarker et al. 2002). According to the present findings,serum concentration of S100β protein is a good marker of brain injury after HCA whenmeasured at least seven hours after surgery.

6.4 Brain microdialysis

Cerebral microdialysis allowed direct, reliable on-line biochemical neuromonitoring atmultiple intervals during the ischemic and reperfusion phases of the present experimentalmodel of HCA. This tool provided useful information for a better understanding of thepathophysiologic mechanisms underlying the development of brain ischemic injury in amodel of HCA that closely resembles clinical conditions. In this study, cortical brainglucose, lactate, glycerol and glutamate levels were measured by microdialysis before,during and after HCA. Cortical microdialysis parameters were, herein, showed to bepredictors of postoperative death. This striking finding was observed despite the fact thatthe microdialysis probe monitors just a small cortical area of the brain, possible differentmetabolic changes in different brain regions thus possibly remaining undetected.

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However, according to the findings of study III, cortical areas were the most vulnerable tothe development of brain infarction, and this may explain the high predictive value ofcerebral microdialysis limited to the cortical area.

Interestingly, changes in brain glucose metabolism were the most important predictorsof postoperative death. Higher brain glucose concentrations were observed in animals thatsurvived during the pre-ischemic as well as during the post-ischemic period, and higher,but not significant concentrations of glucose were observed among those animals that didnot develop brain infarction. This finding somewhat contradicts the results of studieswhich suggested the detrimental effect of hyperglycemia after cerebral ischemic events(Lin et al. 1998). Indeed, other studies have provided evidence of the neuroprotectiveeffect of glucose (Vannucci et al. 1996, Schurr et al. 1997, Schurr 2001). The animals thatdied had higher lactate/glucose ratios and a lower brain glucose concentration ascompared with animals that survived (Fig. 16). The predictive importance of brain lactate/glucose ratio in this context is suggested also by the correlation with the postoperativeincrease of ICP (study III) and with the recovery of EEG burst percentage (study IV). Thelactate/glucose ratio has also been demonstrated to be a predictor of adverse outcome inpatients with traumatic brain injury (Zauner et al. 1997, Goodman et al. 1999).

Another important finding of the present study is the temporary increase in brainlactate concentration during the early phases of reperfusion. A few studies demonstratedthat aerobic utilization of lactate, and not of glucose, fuels the recovery of synapticfunction during reoxygenation (Schurr et al. 1997, Bliss & Sapolsky 2001, Schurr 2001).Astrocytes continue to produce lactate until their own glycogen store or delivery ofglucose is insufficient. Thus, physiologic compensatory glycolysis is not associated withdepletion of glucose from extracellular space. In this context, the brain lactate/glucoseratio is a good marker of preservation of brain glucose metabolism.

Glutamate has been suggested as a key factor for the development of ischemic braininjury, and it has been regarded as an important marker of irreversible brain cell injury(Redmond et al. 1994). In this study, brain glutamate concentrations were significantlyhigher in animals that died than in those that survived only during the last study intervals,the peak values not having been different between survivors and deaths. However,cerebral microdialysis with a low flow rate is not very sensitive to the short-lived peakvalue of the glutamate, and this could explain the weak association between brainconcentrations of glutamate and postoperative outcome.

Ischemic injury of the brain cell membranes has been shown to lead to thedevelopment of brain edema (Ayata & Ropper 2002), and it has been shown that glycerolis a marker of cell membrane injury (Hillered et al. 1998, Frykholm et al. 2001). Herein,brain glycerol concentrations after HCA were shown to be significantly increased inanimals that died at several postoperative time intervals. Brain concentrations of glycerolwere also constantly higher among animals that developed brain infarction as comparedwith those that did not, but the differences did not reach statistical significance.

In summary, cortical microdialysis is a valuable method for evaluating cerebralmetabolism and predicting outcome after HCA.

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6.5 Intracranial pressure

ICP is a main determinant of outcome in neurosurgical patients. Indeed, ischemic braindamage is associated with a certain increase in ICP, which eventually may have animportant impact on the cerebrovascular circulation. Interestingly, an increase in ICP canbe detected in cardiac patients undergoing CPB and, to a major extent, after HCA(Lundar et al. 1983c, McDaniel et al. 1994, Hagl et al. 2002). A significant increase inICP after experimental HCA has been observed herein, although to a much lesser extentthan that observed in neurosurgical patients (Marik et al. 1999, Juul et al. 2000). Anyway,such a relatively small increase of ICP was associated with a certain increased risk ofdeath and the development of brain infarction in animals which underwent HCA (Fig.17). However, a slight increase in ICP after global brain ischemia, as frequently occursduring and after complex cardiac procedures, could lead to a significant worsening of theischemic injury. In fact, it may result in severe disturbances of cerebral blood flow at themicrocirculatory level with collapse of capillaries, which is associated with sloughing ofred blood cells and formation of microemboli (Hekmatpanah 1970). Thus, a relativelyslight increase in ICP may easily result in compression of peripheral small vessels. Thismay explain the present findings of development of brain infarction mostly in the cortex,where the peripheral vessels are much smaller and, thus, more easily compressible than inother parts of the brain. In this sense, it is interesting to note that the bottom of the cortexsulcus was the most frequent site of brain infarction, probably because of adversehydrodynamic conditions occurring at this site.

Interestingly, in this study, ICP was significantly correlated with cortical brainconcentrations of lactate and the lactate/glucose ratio two hours after HCA. Thesefindings support the hypothesis of a relevant impact of ICP on the risk of postoperativebrain infarction and mortality by affecting brain glucose metabolism.

ICP was not significantly associated with any extracranial hemodynamic derangement,specifically, a rise of central venous pressure. Thus, ICP can be viewed in this setting asan independent risk factor probably secondary to severe global ischemic brain injury.

6.6 Electroencephalography

The evaluation of EEG findings after experimental HCA was done in order to establishwhether a slow EEG burst percentage recovery could be considered a reliable marker ofHCA-related brain injury, and whether brain microdialysis parameters provideinformation on metabolic derangements possibly underlying abnormal EEG recovery(IV).

The major finding of this study was a strong correlation between quantifiedhistological brain injury and the recovery of EEG burst percentage (Fig 19a). EEG burstpercentage recovery predicted the severity of ischemic injury of the cortex, cerebellumand brain stem, but not of the thalamus and hippocampus, probably because ischemicinjury of these latter areas is not easily detected by EEG. The present findings are of greatclinical interest, since myoclonic status epilepticus, either violent as myoclonic jerking or

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mild as loss of motor activity, occurring during the EEG burst suppression pattern is aclear sign of severe cerebral injury after cardiac arrest (Young et al. 1988). Such eventshave been shown to be associated with histologically evident injury of the brainstem andcerebellum.

A certain constant, but not statistically significant, difference in EEG burst percentagerecovery was detected between animals with and without brain infarction through the firstpostoperative hours after the start of rewarming, thus suggesting that a significantdifference would probably have been detected in a larger study series.

Importantly, the mean EEG burst percentage after rewarming significantly correlatedwith the brain concentrations of lactate and glucose and the lactate/glucose ratio duringthe early postoperative hours (IV). In fact, as demonstrated in study II, low brain glucoseconcentrations and high lactate/glucose ratios immediately after rewarming from HCAare important predictors of poor outcome. This observation thus provides evidence ofmetabolic derangements possibly underlying such a reduced EEG burst percentage andthe associated increased risk of brain ischemic injury.

This study demonstrated a better correlation between ischemic brain injury and EEGfindings than earlier studies (Fessatidis et al. 1993b, Mezrow et al. 1995), probablybecause in our study variations in EEG burst percentages were quite small and all theanimals underwent the same experimental protocol. Furthermore, EEG burst percentagewas chosen as a monitoring method in this study because of its distinctive advantages. Infact, it is not as sensitive for artifacts as the amplitude- (Sainio 1974) and frequency-based EEG methods (Sainio 1974, Mezrow et al. 1995). In addition, the EEG burstpercentage method is easy to perform, not requiring a neurophysiologist, and analysis ofthe data can also be performed reliably by special software (Särkelä et al. 2002).

Based on our findings, the EEG burst percentage after HCA is a strong predictor ofseverity of histologically evident brain ischemic injury of the cortex, brainstem andcerebellum. It is not yet clear whether EEG burst percentage would remain just as a usefulmarker of significant brain dysfunction in experimental and clinical studies or whether itwould become a target for postoperative therapeutic intervention in daily clinical practice.

7 ConclusionsRecalling the purpose of the present investigation, the results can be summed up asfollows:

1. Increased serum concentrations of S100β protein correlated with histopathologicallyevident brain ischemic injury after HCA in pigs.

2. Cerebral microdialysis is a useful method for cerebral monitoring duringexperimental HCA.

3. High brain lactate/glucose ratios measured by means of cortical brain microdialysisafter HCA is strong predictor of postoperative death.

4. Intracranial pressure increases significantly after 75 minutes of experimentalhypothermic circulatory arrest, and such an increase is associated with an increasedrisk of postoperative death and brain infarction.

5. EEG burst percentage as a sum effect of the anesthetic agent and ischemic braindamage gives an early prediction of the severity of brain ischemic injury after HCA.

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