Cooling the Injured Brain: How Does Moderate ...

Current Pharmaceutical Design, 2007, 13, 2310-2322

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Cooling the Injured Brain: How Does Moderate Hypothermia Influence the Pathophysiology of Traumatic Brain Injury Juan Sahuquillo1,* and Anna Vilalta2 1

Department of Neurosurgery Vall d'Hebron University Hospital and 2 Neurotraumatology and Neurosurgery Research Unit, Institut de Recerca Vall d’Hebron, Universidad Autónoma de Barcelona, Barcelona, Spain Abstract: Neither any neuroprotective drug has been shown to be beneficial in improving the outcome of severe traumatic brain injury (TBI) nor has any prophylactically-induced moderate hypothermia shown any beneficial effect on outcome in severe TBI, despite the optimism generated by preclinical studies. This contrasts with the paradox that hypothermia still is the most powerful neuroprotective method in experimental models because of its ability to influence the multiple biochemical cascades that are set in motion after TBI. The aim of this short review is to highlight the most recent developments concerning the pathophysiology of severe TBI, to review new data on thermoregulation and induced hypothermia, the regulation of core and brain temperature in mammals and the multiplicity of effects of hypothermia in the pathophysiology of TBI. Many experimental studies in the last decade have again confirmed that moderate hypothermia confers protection against ischemic and non-ischemic brain hypoxia, traumatic brain injury, anoxic injury following resuscitation after cardiac arrest and other neurological insults.

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Many posttraumatic adverse events that occur in the injured brain at a cellular and molecular level are highly temperature-sensitive and are thus a good target for induced hypothermia. The basic mechanisms through which hypothermia protects the brain are clearly multifactorial and include at least the following: reduction in brain metabolic rate, effects on cerebral blood flow, reduction of the critical threshold for oxygen delivery, blockade of excitotoxic mechanisms, calcium antagonism, preservation of protein synthesis, reduction of brain thermopooling, a decrease in edema formation, modulation of the inflammatory response, neuroprotection of the white matter and modulation of apoptotic cell death. The new developments discussed in this review indicate that, by targeting many of the abnormal neurochemical cascades initiated after TBI, induced hypothermia may modulate neurotoxicity and, consequently, may play a unique role in opening up new therapeutic avenues for treating severe TBI and improving its devastating effects.

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Furthermore, greater understanding of the pathophysiology of TBI, new data from both basic and clinical research, the good clinical results obtained in randomized clinical trials in cardiac arrest and better and more reliable cooling methods have given hypothermia a second chance in treating TBI patients. A critical evaluation of hypothermia is therefore mandatory to elucidate the reasons for previous failures and to design further multicenter randomized clinical trials that would definitively confirm or refute the potential of this therapeutic modality in the management of severe traumatic brain injuries.

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Key Words: Traumatic brain injury, hypothermia, high intracranial pressure, intravascular cooling methods, neuroprotection, thermoregulation. If hypothermia cannot protect, how could any drug be expected to? D. Warner, 2004

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INTRODUCTION Head injury is the leading cause of death in people aged less than forty-four years. The aftermath of such injuries leaves survivors with permanent disabilities resulting in lifelong medical, financial, emotional, familial and social problems.

SCOPE OF THE HEALTH PROBLEM The true incidence of what has been called the silent epidemic of the twentieth century and the persistent epidemic of the third millennium is difficult to ascertain because of the various definitions of TBI in different countries and the variability in the parameters used to calculate death and disability rates. Epidemiological studies of TBI are few, referring mainly to the 1980s and are highly heterogeneous. A comprehensive epidemiological study performed in 1990 established that the incidence of TBI in the USA was 200/100,000 persons/year [1]. Recent data show that 2% of the US population (5.3 million citizens) are disabled as a result of TBI [2]. Surveys in Europe are unfortunately scarce, although nonsignificant differences seem to exist between USA and Europe. In Europe, the magnitude of the problem has only recently been estimated. The World Health Organization (WHO) has studied what is called the global burden of disease (GBD) for different diseases in Europe. Using robust epidemiological parameters, the WHO has *Address correspondence to this author at the Department of Neurosurgery, Vall d’Hebron University Hospital, Paseo Vall d’Hebron 119-129, 08035 Barcelona, Spain; Tel: +34-934893512; Fax: (hospital) +34-934893513, (home) +34-933931930; E-mail: [email protected] 1381-6128/07 $50.00+.00

shown that in Europe, approximately 50% of years lived with disability are caused by brain diseases [3]. Disability adjusted life years (DALY) was one of the main summary measures calculated. Calculation of DALY includes the number of years of life lost because of premature death and the years lived with disability [3]. The main advantage of DALY is that it is independent of the individual disease characteristics, includes direct and indirect health costs and can be used in all societies regardless of their stage of development [3]. In 2000, the leading cause of DALYs in Europe was ischemic heart disease, the second being stroke and the seventh being road traffic accidents. Injury to the brain or spinal cord is the main factor that influences long-term disability due to road traffic accidents [3]. PATHOPHYSIOLOGY OF TRAUMATIC BRAIN INJURY The pathophysiology of brain injured patients and especially of severe TBI (those rendered comatose after impact) is a highly complex issue about which much new information has been added in the last decade. Both primary injury and the added intracranial and extracranial secondary insults stimulate a series of deleterious neurochemical cascades that end in irreversible changes to the cell membranes and cell death. Importantly, most deleterious neurochemical cascades triggered by head injury are temperaturesensitive to even mild changes in core temperature. Although these cascades have been the subject of comprehensive reviews but because being crucial to an understanding of the neuroprotective actions of induced hypothermia, they will be briefly be summarized in this review. Part of this review has been previously published by our group elsewhere [4].

© 2007 Bentham Science Publishers Ltd.

Cooling the Injured Brain

Current Pharmaceutical Design, 2007, Vol. 13, No. 22

THE TRADITIONAL CLASSIFICATION OF HEAD INJURIES The traditional and academic approach to classifying the pathobiology of traumatic brain damage was introduced by Gennarelli and coworkers in the 1980s. Basing their classification on clinicoradiological and neuropathological findings, these authors distinguished between diffuse and focal injuries [5-7]. According to these authors, focal lesions were those large enough to be seen by the naked eye and consequently in the CT scan. These lesions cause neurological dysfunction by direct local damage to the neural tissue but they induce coma only when they are large enough to cause brain shifts, herniations and brainstem compression [5,6]. Contusions, subdural, epidural and intracerebral hematomas are included within this category. In contrast, diffuse injuries are not associated with gross localized and visible lesions. Patients with diffuse brain damage sustain neurological dysfunction that globally affects the brain, usually with no macroscopically significant structural damage detected in the CT scan [5-7]. Diffuse brain swelling, diffuse axonal injury, global ischemic damage and diffuse cytotoxic or vasogenic edema are usually classified within this category [5,6,8]. However, the most devastating diffuse lesion is diffuse primary damage to the white matter or what is now known as diffuse axonal injury (DAI), a lesion that was first described by Strich in 1956 [9]. The interested reader is referred to a recently published review [10]. As stated by Gennarelli, both the cellular and the molecular pathophysiology of both diffuse and focal lesions share many common events and the exact sequence of the damaging cascades set in motion at the moment of impact are difficult, if not impossible, to elucidate [6].

the mechanical impact but are not finished until a variable and sometimes unpredictable time after injury. Consequently, the theoretical therapeutic window for such processes is variable and mostly unknown. A better understanding of glial, neuronal and axonal failure, alterations in the permeability of the blood brain barrier (BBB), as well as evidence of the multiple neurochemical cascades activated by mechanical injury, has blurred the traditional boundaries between primary and secondary injuries. In 1990, Pitts and McIntosh proposed the term tertiary injury to define all anomalous cellular events induced by trauma. These events include alterations in neurotransmitter functions, membrane integrity, ion channel dysfunctions, alterations in ionic homeostasis, induced protein synthesis and abnormalities in different metabolic pathways [14]. To quote these authors “We will not make large advances in brain trauma therapy until we determine the scope of tertiary cell injuries and how to reverse or prevent them” [14]. Although consensus on the term tertiary injury is lacking and the damage included under this heading by various authors is too wide and describes what others would consider to be secondary biochemical events, once again, this concept is a useful framework in which to incorporate new findings that would be difficult to be understood without such a theoretical construct. In this review we will maintain the concept of tertiary injuries because it stresses that after TBI, apart from the obvious morphological lesions visible in the CT scan, there are many phenomena that occur at the cellular and molecular levels. Traumatic brain injuries initiate complex changes that result in glutamate release, increased intracellular calcium, activation of ion channels and triggering of intracellular proteolytic processes. These events are potentially more damaging to the brain than evident macroscopic lesions.

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A NEW CONCEPT: TERTIARY INJURIES The most important new information about the nature of traumatic insults is probably that traumatic brain injuries are highly dynamic and that the various lesions observed after TBI are not single events but processes. These processes are set in motion by

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PRIMARY VERSUS SECONDARY TRAUMATIC LESIONS Without doubt, the most influential classification of TBI was that originally proposed by Adams in the 1970s [11]. According to this classification, traumatic brain lesions are defined as primary or secondary. Primary injuries are those inflicted to the brain immediately on impact, while secondary lesions are defined as those set in motion by the impact but which appear within minutes, hours or days after injury [12]. This model has been and still is a very useful conceptual framework in which new facts about the injured brain can be integrated. Differentiating between primary and secondary injuries emphasizes the difference between unavoidable, immediate-impact lesions and the potentially avoidable, secondary lesions that occur at a variable time after injury. In the 1970s, the most important factor governing the final outcome of a patient with a closed head injury was assumed to be the primary damage sustained by the brain immediately on impact [11]. In the 1980s, despite the undeniable physiopathological importance given to primary injuries, the traditional concept shifted to the pivotal role played by delayed mechanisms of brain damage (secondary injuries) in TBI [11,12]. Research into the influence of secondary injuries, and particularly of ischemic brain damage in TBI did not lose momentum during what was called the “Decade of the Brain” when new information on the pathophysiology of TBI accumulated exponentially as a direct result of increased funding for neurological research. A paradoxical consequence of this information overload is, to quote Gaetz “…that previous outdated knowledge persists, potentially misguiding clinicians and researchers” [13]. The need for clinicians to be aware of, critically evaluate and incorporate the solid evidence already available into the daily clinical practice is therefore mandatory in order to redefine and improve the management of severe TBI.

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A BRIEF GUIDE TO UNDERSTANDING SECONDARY INJURIES Both mechanical injury and secondary lesions, especially secondary intracranial or extracranial induced brain hypoxia, generate multiple cascades that will briefly be summarized. Increased intracranial pressure is the leading cause of death after severe TBI. This increased ICP reduces cerebral perfusion pressure (CPP) and is one of the main causes of brain ischemia. Furthermore, many of the secondary and tertiary injuries progress to dysregulation of intracellular and extracellular water content and consequently to brain edema, which both increases ICP and reduces CPP, thus perpetuating a vicious circle that ends in cell death. ISCHEMIC AND NON-ISCHEMIC BRAIN HYPOXIA One of the most frequent causes of brain tissue hypoxia in head injuries is ischemia. A significant advance in our knowledge of the pathophysiology of traumatic brain injury was the observation made in neuropathological studies that secondary ischemic damage was highly prevalent in the brains of patients who died from their injuries [12,15-17]. Neuropathological findings clearly revealed that ischemia was a key factor in patients who died of a brain injury. Ischemia influences not only mortality but also neurological sequelae after head injury [16]. Some of the causes of ischemia are decreased global cerebral blood flow due to increased ICP, and reduced CPP or regional cerebral blood flow (rCBF) due to vasospasm or brain herniation, with a consequent reduction in the calibre of the compressed arteries by the herniated tissue. An important new piece of evidence is that brain hypoxia follows the same patterns as those described in other organs, the most important difference being that the brain is highly vulnerable when facing this insult. Brain oxygen consumption (VO2) is related to oxygen delivery (DO2) in a biphasic pattern. Above what is known as the critical value of DO2 (DO2crit), VO2 is constant and independent of oxygen delivery [18]. However, when DO2crit is

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reached, VO2 is linearly coupled and dependent on DO2 [18-20]. DO2 is calculated according to the following equation: DO2 = CBF x CaO2 where CBF is cerebral blood flow expressed in ml/100g/min and CaO2 the arterial content of oxygen that is calculated from the following equation: CaO2 = ctHb x 1.34 (SaO2) + 0.0031 PaO2 In this equation, ctHb is hemoglobin content in g/dl, 1.34 is the oxygen binding capacity of hemoglobin, 0.0031 is the oxygen solubility of oxygen in plasma and PaO2 is the arterial partial pressure of oxygen. The solubility of oxygen in plasma increases with hypothermia but at the same time, its affinity to hemoglobin increases. Consequently, the effects on oxygen delivery are difficult to predict and depend on multiple factors. Clinicians usually think about the importance of optimizing oxygen delivery to the brain. However, it is important not to forget that both clinical and experimental evidence supports the concept that under some circumstances, the injured brain is unable to extract enough oxygen from normal or even highly oxygenated blood. This phenomenon has been known for two decades when Schumacker demonstrated this type of dysfunction in different organs in patients with critical illnesses such as adult respiratory distress syndrome and sepsis [21,22]. This alteration in the extraction capacity of oxygen has been named histotoxic hypoxia or cytopathic hypoxia [19,20] and has also been observed in the injured brain. Under these circumstances, increasing oxygen delivery to the brain has no effect on brain metabolism. To detect this complex situation at the bedside, multiparametric neurochemical monitoring is crucial [23].

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cal tissue destruction are usually surrounded by areas of damage but not dead tissue [6]. Furthermore, the high incidence of hypoxic brain damage of ischemic and non-ischemic type found in patients who die after head trauma means that both types of penumbra (ischemic and traumatic) may frequently coexist in the same patient. An interesting and clinically important finding is that both types of penumbra have similar biochemical signatures and share many pathophysiological events. Brain in the traumatic penumbra is especially vulnerable and its fate is highly dependent on its capacity to survive the additional insults that will occur. For a comprehensive review of secondary neurochemical damage, the reader is referred to the review by Siesjö and Siesjö [25,26]. UNDERSTANDING THE UNKNOWN: TERTIARY INJURIES At the cellular level, traumatic events are mediated by four basic mechanisms: excitotoxicity, free radical-induced alterations, inflammatory events and calcium-mediated damage [6,8]. These phenomena are responsible for inducing other lesions such as brain swelling and increased intracranial pressure. Perilesional depolarizations, mitochondrial dysfunction and apoptosis are also common. Because of their importance in an understanding of the neuroprotective capacity of hypothermia, in the following paragraphs we will briefly summarize these anomalous events that could be arbitrarily included among what Pitts and MacIntosh called “tertiary injuries”. All these tertiary injuries can be modulated or partially arrested by induced hypothermia. For the sake of brevity, we will limit our review to the following dysfunctions: excitotoxicity, excitotoxicinduced edema, mitochondrial dysfunction and inflammatory processes after traumatic brain injury.

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THE CONCEPT OF TRAUMATIC PENUMBRA AND ITS CLINICAL SIGNIFICANCE In experimental models of focal infarction induced by reducing rCBF, three different areas have been described. The core of the infarct is the ischemic area in which rCBF is below 20% of the normal values. This area rapidly evolves to necrosis by destructive biochemical phenomena such as lipolysis, proteolysis and disaggregation of the microtubules [24]. However, between this core and the normal brain tissue, there is what is known as ischemic penumbra, defined as the perilesional areas of the core that are functionally altered but still structurally intact. In ischemic penumbra, energy metabolism is still partially preserved but these areas are at high risk of being irreversibly damaged unless prompt reperfusion or neuroprotection are rapidly undertaken. Inflammatory phenomena and apoptosis can lead the cells in the penumbra towards definitive and irreversible damage [24]. Recent reviews on the pathobiology of brain trauma have stressed that brain injury has to be viewed as a clinical syndrome resulting from a complex combination of neural and vascular events that occur after the head has been mechanically distorted [6,8]. An important concept introduced in brain injury has been that of traumatic penumbra to parallel the well accepted term ischemic penumbra. After mechanical load to the head, primary lesions such as diffuse axonal injury or contusions are produced in the brain and, as previously mentioned, multiple neurochemical damaging cascades are rapidly activated. Recent evidence suggests that primary destructive anatomical lesions are frequent in patients who die shortly after impact but that they are less prevalent than previously thought in patients who survive long enough to be admitted in hospitals [6,8]. Mechanical forces acting on the brain result in immediate tissue destruction that can affect the neurons, glial cells and axons as well as the vascular elements [6,8]. As in stroke, traumatic penumbra can be defined as areas of injured but still viable brain tissue. As has been described in severe ischemia, areas undergoing mechani-

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EXCITOTOXICITY AND BRAIN INJURY The neuroprotective effect of hypothermia has been considered to be partly mediated through its capacity to modulate excitotoxic mechanisms. Consequently, a brief review of this challenging phenomenon is warranted. The term excitotoxicity was coined by Olney in 1981 to describe the neuronal death observed when neurons get exposed to very high concentrations of exogenous glutamate or glutamate-agonist compounds [27]. This term has survived because of the robust association between glutamate and neuronal death in experimental stroke models. Glutamate is by far the dominant excitatory amino-acid in the mammalian brain and is released by approximately 90% of neurons when excited [28]. In the presynaptic terminals of neurons, glutamate is normally released from vesicles by exocytosis and diffuses across the synaptic cleft to excite the postsynaptic neurons [29]. The concentrations of glutamate in the brain are compartmentalized in three different locations: 1) intracellular glutamate, 2) synaptic cleft compartment and 3) extracellular space. The intracellular glutamate concentration is in the millimolar scale (around 10 mmol/L) while the extracellular concentration of glutamate is in the micromolar scale and has been estimated to be around 1 mol/L [29]. The glutamate concentration is temporarily very high in the synaptic compartment where it shows a transitory increase to 1 - 2mM/L. However, this concentration is not reflected in the extracellular space (ECS) [30]. Under normal circumstances, glutamate is quickly taken up by astrocytes surrounding the synaptic cleft, thus avoiding excessive and toxic levels in the ECS. The normal concentration of spinal CSF glutamate is on average approximately around 3mol/L [31]. The ECS forms the microenvironment of the neurons and the glial cells and represents approximately 15 to 25% of the total adult brain volume [32]. The ionic composition of the ECS is very close to that of the CSF. Homeostasis of the ECS is maintained by the glial cells. Unlike neurons, which communicate through synaptic



transmission, glial cells communicate amongst one another and with the neurons through the ECS in a way that has been called extrasynaptic transmission [32]. Excitatory amino acids stimulate the postsynaptic neuron through two different types of receptors: ionotropic and metabotropic. Ionotropic receptors can be subdivided in three different types depending on the agonist that stimulates them: NMDA (Nmethyl-d-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate) and kainate [29]. When activated, ionotropic receptors increase permeability to cations, mainly Na+ and Ca++. There is strong evidence that overstimulation of the postsynaptic receptors for glutamate and aspartate might cause injury to neurons in several diseases such as stroke, TBI and epilepsy [29]. Furthermore, various studies have shown that macroglial cells (astrocytes and oligodendrocytes) express glutamate receptors in their membranes. Therefore, glutamate glial receptors are also activated when glutamate is liberated in the synaptic cleft and thus they may be implicated in pathological processes [33]. An intriguing paradox is that the concentrations of glutamate in the ECS necessary to induce endogenous cell death in vivo are very high and far above those measured in models of neurological disorders (e.g. stroke and traumatic brain injury) [30]. This has been considered as indirect evidence that the concentration of glutamate is tightly regulated [30]. Glutamate uptake and all transmembrane ionic shifts (Na+, K+, Ca++ and H+) are followed by water movement into the glial cells, which increases cell volume [32]. For an up-to-date overview of this topic, the reader is referred to Sykova and Chvatal’s review [32]. Changes in glial cell volume modify the geometry of the ECS, increasing its tortuosity and reducing the capacity of molecules to move freely [32]. Overstimulation of glutamate receptors leads to an increase in intracellular calcium. There is strong evidence to support the hypothesis that excitotoxic cell death is mediated by the failure of cytoplasm to maintain Ca++ homeostasis. This process is usually delayed in time, occurring minutes after initial exposure to toxic levels of glutamate and has been called delayed calcium deregulation (DCD). Once DCD is underway, the process of cell death is almost irreversible [34]. The exact mechanism by which cytosolic calcium is increased is unclear, however, three possibilities have been suggested: 1) enhanced Ca++ entrance through the cytoplasmic membrane, 2) massive release of calcium from the mitochondrial matrix to the cytoplasm and 3) failure of the cell to extrude the excessive amounts of Ca++ [34]. As has been explained, NMDA receptors control ionic channels that are permeable to Ca++, Na+ and K+[24]. Experimental models of focal ischemia have shown neuroprotection achieved by blocking such receptors within a short therapeutic window after ischemic insult. Overstimulated AMPA receptors also increase Na+ and K + conductance and facilitate their influx into the cytosol. An interesting observation is that neuroprotection achieved by AMPA receptor blocking has a longer time window than that obtained by blocking NMDA receptors [24].

massive release of excitotoxic amino acids into the extracellular space [24]. Abnormal glutamate accumulation can be identified in both TBI and stroke. According to Lipton et al., several mechanisms are involved: 1) the normal function of the glutamate transporter in astrocytes may be impaired and even the transporter may actually operate in reverse, becoming a source of extracellular glutamate rather than a sink for it. 2) The release of glutamate in the synaptic cleft can normally be potentiated by glutamate itself and by added extracellular glutamate from other sources. One of the features of excitatory neurotransmission is a positive feedback system, with glutamate release causing further glutamate release. 3) Both neurons and astrocytes contain large quantities of glutamate that would be expected to leak out of the injured cells [29]. Probably, the most frequent cause of excess extracellular glutamate is injury to cells and its massive release into the ECS. As described by Lipton “…since each cell contains 10 mmol of glutamate per liter, the potential for disaster is obviously great” [29]. An interesting hypothesis recently introduced by Obrenovitch has challenged the excitotoxic mechanism of neuronal death [30]. However, if glutamate is not a major factor in the pathophysiology of TBI and stroke, as this author suggests, it would be difficult to explain the reason why glutamate antagonists are neuroprotective both in vivo and in vitro. Obrenovitch’s answer to this question merits some thought. According to this author, blocking NMDA receptors in a situation of energy failure is neuroprotective per se because it decreases the metabolic demands [30].

GLUTAMATE IN TBI In TBI, glutamate is not only liberated at the moment of injury, but also increases when brain tissue hypoxia occurs. Consequently, excitotoxic mechanisms in TBI are in part related to the mechanical injury and are in part a consequence of the secondary insults suffered by the brain after injury. Whatever the reason, when oxygen delivery to the brain is significantly reduced, the first phenomenon that is observed is an impairment of the capacity of neurons and glial cells to maintain normal ionic gradients through the cell membrane. Consequently, depolarization occurs in neurons, leading to activation of presynaptic voltage-dependent Ca++ channels and to

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EXCITOTOXIC-INDUCED EDEMA The first visible sign of glutamate overload is cell swelling. This has been called by Marmarou neurotoxic edema to differentiate it from ischemic cytotoxic edema, in which the pathophysiology is energy failure and not overexcitation of the glutamate receptors [35]. Edema is dependent on the entry of excessive amounts of sodium and chloride ions together with water into the cell. But edema by itself does not necessarily lead to cell death. A very appealing finding is that immediate toxicity associated with exposure to glutamate can be blocked by the removal of sodium or chloride from the extracellular medium, but that their absence does not protect neurons from the delayed effects of glutamate toxicity [29].

MITOCHONDRIAL DYSFUNCTION Energy metabolism is systematically altered after severe TBI but some specific alterations are not still fully understood. A traditional and confirmed finding is that in severe TBI, the cerebral metabolic rate for oxygen (CMRO2) is systematically reduced proportionally to the reduction in the Glasgow coma scale score [36,37]. Recent findings support the hypothesis that mitochondrial dysfunction of what has been called the mitochondrial permeability transition (MPT) pore, is of major importance during severe TBI and may explain some paradoxical findings that have been difficult to interpret without significant alteration of the brain’s metabolic “furnaces”. The molecular composition and regulation of MPT pores remain poorly understood, although it is accepted that Ca2+ induces pore opening, whereas Mg2+ and cyclosporin A close the pore [38]. The MPT pore is dysregulated after experimental TBI [39]. Many posttraumatic conditions such as an increase in cytosolic calcium, free radical generation and caspase-3 activation are some of the conditions that could activate the MPT pore [39]. Activation of the pore is characterized by mitochondrial swelling and an increase in the permeability of the inner membrane to solutes of up to 1500 Da [38]. When exposed to exogenous calcium, isolated mitochondrial populations exhibit a dose-dependent increase in swelling rates, indirectly reflecting a greater number of open MPT pores [39]. Some authors have introduced the hypothesis that functional or


structural damage to mitochondrial populations may underlie the sustained hypometabolic state reported after TBI [37,39]. The structural changes observed in mitochondria after experimental TBI such as mitochondrial swelling and membrane destruction are very similar to what is observed when isolated central nervous system (CNS) mitochondria are exposed to calcium [39-41]. Calcium-induced mitochondrial swelling is basically regulated by an opening of the MPT pore [39]. In a very elaborated experiment in which mitochondria were isolated of the rat brain three and 24 hours after lateral fluid– percussion injury, Liftshitz et al. found ultrastructural damage in mitochondria [39]. Morphologically damaged mitochondria show disrupted cristae, thinned outer membranes, and a ballooned appearance [39]. The release of apoptogenic proteins is known to depend on the rupture of the outer mitochondrial membrane [38]. Mitochondrial damage also plays a very important role in primary diffuse axonal injury (DAI). The most recent studies from Povlishokc’s laboratory have shown that the altered ionic permeability of the axolemma is constantly found in DAI and that this increased permeability is a pivotal step in its pathogenesis and in the progression of the dysfunctional injured axon toward secondary disconnection [42,43]. Experimental studies from the same laboratory have suggested that the influx of calcium could be the cause of the mitochondrial swelling observed in many experimental models of DAI [44]. Calcium-induced opening of the permeability transition pore induces mitochondrial failure and consequently a variable amount of disruption in the energy supply to the axolemma [42]. The lack of energy disturbs the axolemma’s ionic homeostasis and provokes further increases in calcium influx, which in turn initiates the degradation process that ends in secondary axotomy [42]. Some studies suggest that, at least in experimental models, there is a short therapeutic window in which this process could be inhibited by certain drugs such as cyclosporin-A [42-45].


et al., cytokines are a “double-edged sword” [46]. Under normal conditions, cytokines are expressed in the normal brain and are neuroprotective. Knockout mice have greater brain damage after TBI than wild-type mice [46]. After TBI, cytokines and especially TNF, play a deleterious role by altering the vascular permeability of the microcirculation and by exacerbating brain edema formation. This increase in brain edema leads to the influx of more inflammatory cells with the resultant production of harmful reactive oxygen species [50]. The inflammatory reaction induced by experimental contusions was studied by Holmin using immunohistochemistry. An inflammatory mononuclear cell response began to be evident on day 2 after injury and reached a peak on days 5 to 6 [52]. The cellular infiltrate found in the injured rats comprised of natural-killer cells, T-helper cells and T-cytotoxic suppressor cells, as well as monocytes and macrophages [52]. In another study by the same group, inflammatory components were studied in contused human brain tissue obtained from the biopsies of brain contusions of patients undergoing surgery 3 hours to 5 days after trauma [53]. In patients studied within 24 hours after trauma, the inflammatory response was limited to vascular margination of polymorphonuclear cells [53]. However, in patients undergoing surgery 3 to 5 days after trauma, a massive inflammatory response consisting of monocytes and macrophages, reactive microglia, polymorphonuclear cells and CD4 and CD8-positive T lymphocytes was detected [53]. These studies corroborated experimental data indicating that in the early phase after contusional trauma, inflammation is mainly intravascular and is dominated by polymorphonuclear cells. However, the inflammation is subsequently localized in the brain tissue [53]. Inflammatory cells may produce several potentially damaging effects in the neurons and glial cells [53]. Both macrophages and microglial cells have been reported to be involved in inflammatory damage by the release of cytotoxic molecules such as oxygen-free radicals and cytokines [48].

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POST-TRAUMATIC INFLAMMATORY PROCESSES Among the known critical factors that are involved in the spread of secondary damage after TBI, there is the acute inflammatory response involving the infiltration, accumulation and activation of polymorphonuclear leukocytes (PL) in the injured brain [46]. Inflammation in the brain can be divided into a local intrinsic response of the brain cells and that recruited from the blood [47]. In the former, microglial cells are activated while in the latter, mononuclear cells and neutrophils are recruited from the blood intervene [47]. In addition, alterations in immunocompetent cells have been observed in head-injured patients [48]. Since the blood-brain barrier can be opened in the acute phase after traumatic brain injury, the entry of circulating cells into the brain contributes to the pathobiology of posttraumatic brain swelling, to the increase in the size of brain contusions and to the perilesional abnormalities found in these lesions. In various experimental models of TBI (weight drop, cortical impact, fluid percussion etc.), accumulation of neutrophils in the injured tissue has been consistently shown early after trauma with peak values occurring at 48 hours [49]. In other studies that used the fluid percussion model, temporal profile of neutrophil accumulation was fairly predictable: it was initiated as early as three hours after injury, peaked at three days and resolved a week after trauma [50]. The consequences of this activation and accumulation include increased microvascular permeability, edema formation, intracranial hypertension and secondary neuronal injury [50]. Activated neutrophils, macrophages and disrupted endothelial cells produce a variety of proinflammatory cytokines that contribute to the ongoing pathological processes [51]. Cytokines are the major effectors of the inflammatory cascade and can be released minutes after trauma, suggesting synthesis by either neuronal or glial cells rather than by leukocytes [46]. Among the cytokines produced are tumor necrosis factor- (TNF-), IL-1, IL-6, leukotrienes, complement, integrin, and platelet activating factor. As described by Morganti-Kossman

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MATRIX METALLOPROTEINASES: NEW PLAYERS IN THE INFLAMMATORY GAME In the last few years, a specific group of neutral proteases, matrix metalloproteinases (MMPs), has been revealed to act as highly important mediators in many neurological diseases, especially in stroke and TBI [47]. MMPs are secreted as latent enzymes (proform) and when activated by multiple mechanisms, could have very deleterious effects on the injured brain. Activated MMPs attack the basal lamina of the cerebral vessels, thus facilitating brain edema and hemorrhage [47]. There are four different families of MMPs: interstitial collagenases, stromelysins, gelatinases and membrane type metalloproteinases [47]. In stroke and TBI, the most important MMPs involved are the gelatinases A (MMP-2) and B (MMP-9) [47]. MMP-2 has a molecular weight of 72 kDa and is normally present in the brain tissue and in the cerebrospinal fluid (CSF). MMP-9 has a molecular weight of 92 kDa and attacks similar substrates. MMP-9 is very scarce under normal conditions but is upregulated in neuroinflammation [47]. The interested reader is referred to Rosenberg’s extensive review [47]. Astrocytes, neurons, oligodendroglia, endothelial cells, pericytes and even microglia produce MMPs, but the types of MMPs and the stimuli that induce them are different from the various cell types [47]. Endothelial cells in culture stimulated with lipopolysaccharide show MMP-9 induction while astrocyte cultures secrete MMP-2 and produce the inactive proform MMP-9 when stimulated with LPS, IL-1ß, or TNF- [54,55]. MMP-2 is observed by immunohistochemistry in astrocytic processes surrounding blood vessels in ependymal and piamater cells of the normal brain [47]. The migration of white blood cells though the BBB is facilitated by MMPs secreted by leukocytes [47]. When an inflammatory stimulus is induced in the brain, MMPs are activated. Experimental studies



have shown that when TNF- is injected directly into the rat brain, MMP-9 is produced after 24 hours and this production is associated with BBB disruption [47]. Acute stroke also induces a significant increase in plasma levels of MMP-9, but the source of this MMP-9 is still uncertain [56,57]. Several experimental studies in the field of neuroinflammation show a common pattern involving both MMPs and the plasminogen/plasmin system in the BBB disruption associated with any inflammatory phenomena originated in the brain. All components of the BBB (capillary tight junctions, astrocytic feet, basal membrane and pericytes) play a role in this drama. When any inflammatory stimulus originates inside the brain, the first line of defense against proteases is tight junctions [47]. The second line of defense is the basal membrane that blocks the diffusion of large molecules and the third line is the astrocytic feet barrier that surrounds the basal membrane. MMPs selectively degrade all components of the basal membrane, allowing this protease digestion to rapidly spread to the extracellular matrix [47]. A pattern of neuroinflammation is emerging from recent clinical and experimental studies in stroke that can be easily extrapolated to TBI. A possible hypothesis on the damaging effect of MMPs in both TBI and stroke is that the reaction begins in the brain cells. Both insults stimulate the activation of the latent MMP-2 present in perivascular astrocytic feet. MMP-2 induces an early opening of the BBB. After 24- 48 hours, a second BBB opening occurs that lasts several days and is associated with vasogenic edema and hemorrhagic conversion [47]. During this delayed opening, MMP-9 has been shown to increase in plasma and this increase is mediated by cytokines, immediate-early genes and the MMP-3 that is contained in pericytes [47]. Neurons, astrocytes and microglial cells release MMPs, while cytokines amplify the response. White blood cells adhere to the endothelial surface of the CNS blood vessels and eventually enter the brain [47]. Greater knowledge of the role played by gelatinases in the pathophysiology of brain edema opens new therapeutic avenues for managing the swollen brain and for modulating neuroinflammation related to stroke and TBI. Many MMPs inhibitors have been described and used experimentally [47] and more recently, the beneficial effects of hypothermia on MMPs have been demonstrated in clinical studies [58,59]. In a non-controlled clinical study of patients with stroke, Horstmann et al., showed that MMP-9 was elevated in patients treated with thrombolysis and was decreased in patients receiving moderate hypothermia [58].

progression from animal to human studies has been criticized as over-hurried and possibly over-ambitious” [60]. Despite these failures, hypothermia is still the most powerful neuroprotective method in animal models of TBI and stroke because it affects a wide range of the pathophysiological processes involved. Instead of focusing on blocking a specific cascade, hypothermia has the advantage of acting at several points of the deleterious pathophysiological events triggered by TBI and stroke [61]. However, enthusiasm aroused by experimental studies and supported by encouraging results from early clinical trials in TBI reported in the 1990s faded when the final report of a multicenter phase III American trial, known as NABISH-I, showed no significant effect of hypothermia on outcome in severe TBI [62]. Greater understanding of the pathophysiology of TBI, new data from both basic and clinical research, the good clinical results obtained in randomized clinical trials in cardiac arrest and better and more reliable cooling methods have given hypothermia a second chance in treating TBI and stroke patients. Furthermore, the discouraging results of previous neuroprotective studies in TBI have made investigators think twice before embarking on clinical trials and many important lessons have been learned from previous failures. An additional but highly important factor is that clinicians have acquired greater knowledge and expertise in the management of patients with hypothermic hypometabolism, thus avoiding iatrogenic problems. An ongoing trial (NABISH-II) is currently in progress in patients with severe TBI and hypothermia on admission. In this specific subgroup, there was some indication of potential benefit in the NABISH-I trial [63]. The present article focuses on the recent past, present and future of both immediate and deferred hypothermia in the management of severe TBI. We will discuss new data on the physiology of the thermoregulation obtained from mammalian hibernators, new experimental data on the neuroprotective effects of hypothermia, and the results of new clinical trials, as well as the preliminary results of clinical studies that have used more effective techniques for intravascular cooling that have recently become available.

MANAGEMENT OF SEVERE HEAD INJURIES The modern paradigm, which is widely accepted in the treatment of head injury, is one that attempts to avoid both intracranial and extracranial secondary lesions and to treat them early, thus providing the injured brain with the optimal conditions in which to recover spontaneously from primary damage. Increased ICP affects more than 50% of patients with severe TBI while ischemia is the most frequent finding in postmortem studies; consequently, both ischemia and high ICP have been the main therapeutic targets in clinical trials conducted in the last decade. At least 20 neuroprotective compounds and therapeutic interventions have been subjected to more than 50 trials in the last three decades [60]. Many competitive and non-competitive glutamate receptor antagonists, free radical scavengers, lipid peroxidation inhibitors and Ca2+ channel inhibitors have been developed and tested in experimental models of TBI [60]. Unfortunately, no neuroprotective drug that has entered phase III randomized clinical trials has been shown to be beneficial in severe TBI. For a recent discussion of this topic, the reader is referred to the paper by Tolias and Bullock [60]. Although the reasons for failure are multiple and have been analyzed in depth in Tolias and Bullock’s review, the main reason for negative results is, to quote these authors, that “The

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INDUCED HYPOTHERMIA: BASIC CONCEPTS There is much confusion regarding the use of proper terminology in thermoregulation and hypothermia clinical research. A good resource for the clinician is the glossary of terms for thermal physiology periodically revised by the Commission for Thermal Physiology of the International Union of Physiological Sciences (IUPS) [64]. According to the IUPS Thermal Commission, Hypothermia is defined as “the condition of a temperature regulator when core temperature is below its range specified for the normal active state of the species” [64]. Induced or deliberate hypothermia is defined by the same group as “The state of hypothermia produced purposefully by increasing heat loss from the body and/or inactivation of heat conservation and heat production by physical and/or pharmacological means” [64]. Clinically induced or regulated hypothermia is defined by Bernard as the controlled lowering of core temperature for therapeutic reasons [65]. Because deep hypothermia causes life-threatening arrhythmias, ventricular fibrillation and cardiac arrest, core temperatures below 28°C can only be safely achieved by using cardiopulmonary bypass (CBP) [66]. Mild-to-moderate induced hypothermia (32-34°C) has been used to achieve neuroprotection in many neurological insults such as anoxic neurological injury after cardiac arrest, severe TBI, major stroke and hepatic encephalopathy, among others. For a comprehensive review of the uses of hypothermia in the ICU, the reader is referred to Bernard and Buist’s review [65]. The common use of the same terminology to describe different types of hypothermia has misled clinicians. The terms mild and


moderate hypothermia have been used interchangeably in many papers to refer to different target core temperatures. Although agreement on terminology is lacking, it is generally accepted that, depending on the core temperature (Tc), hypothermia is classified as mild (33-36°C), moderate (28-32°C), deep (10-28°C), profound (510°C), and ultraprofound (0-5°C) [66]. Despite this widely used classification, the use of the term “moderate hypothermia” varies widely in neurocritical care [67] and, surprisingly, in this classification, the 32-33°C range, which is the most widely used range in the neuro ICU, has simply been forgotten. For the sake of consistency, we will consider the usual lower TC used in the management of stroke and head injury (32-33°C) as moderate hypothermia. A new term, suspended animation, was introduced in 1984 by Safar and Bellamy in the field of resuscitation [68]. Suspended animation is defined by these authors as “…the protection and preservation of the whole organism during prolonged clinical death, for transport and repair (resuscitative surgery) without pulse, followed by delayed resuscitation to complete recovery” [68]. These authors began to investigate this new concept in animal models in 1988 at the International Resuscitation Research Center of the University of Pittsburgh [68]. In experimental models, dogs survived normothermic hemorrhagic shock followed by up to 60 minutes of suspended animation with ultraprofound hypothermic circulatory arrest (at 5°C) and recovered completely without any neuropathological evidence of brain injury [69]. Ironically, research into this technique has mainly been funded by the US Navy. Because epidemiological data from the Vietnam war showed that exsanguinating hemorrhage was the major cause of death in nearly half of all casualties, military interest in this type of research is motivated by the potential to prevent combat casualties due to hypovolemic shock [68]. Suspended animation involves total cardiocirculatory arrest with neuroprotective measures (hypothermia and pharmacological). In a theoretical scenario, circulatory arrest would be induced by protective therapy (drugs and/or hypothermia) and the period of cardiac arrest would be followed by resuscitation, with the aim of achieving complete recovery without brain damage [68]. In the near future, research into this potential area might contribute to the development of new techniques for neuroprotection in neurocritical patients.


Tc. This gradient or error signal activates the appropriate thermoeffector pathways that control heat production and heat loss [70]. The temperature range that does not trigger a thermoregulatory response is known as the interthreshold range and is approximately 0.2°C in awake humans [71]. Fever is a typical example of a change in the setpoint, probably induced by cytokines. In fever, regulated hyperthermia is induced, shivering and non-shivering thermogeneses increase heat production, while cutaneous vasoconstriction reduces heat loss [70]. Traditional induced hypothermia is not regulated but forced. In this situation, TC is forced below Tset and consequently the organism responds with an activation of thermoeffectors to reduce heat loss and increase heat gain [70]. The ideal way to induce therapeutic hypothermia seems to lie in regulating hypothermia or in reducing the Tset to the desired temperature by drugs or internal or external means [70]. When the Tset is reduced, the organism responds by activating thermoeffectors to increase heat loss and to reduce heat production until Tset is reached [70]. While waiting for the ideal technique, intravascular cooling methods enhanced by drugs that can lower the thermoeffector response and/or the setpoint seem a promising avenue of research for the near future.

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REGULATION OF CORE AND BRAIN TEMPERATURE Core temperature in homeotherms is regulated by the central nervous system within an extremely narrow range of setpoints. The setpoint is defined according to the Commission of Thermal Physiology as “The value of a regulated variable which a healthy organism tends to stabilize by the processes of regulation” [64]. When core temperature deviates more than 0.2°C from the setpoint, thermoregulatory mechanisms are triggered in homeotherms, which react quickly with vigorous heat gain and conserving responses to avoid loss of body heat [70].

CYBERBIOLOGY OF THE REGULATION OF CORE AND SHELL TEMPERATURE The main goal of the thermoregulatory system is to maintain a stable core temperature between extremely narrow limits [70]. Thermal physiologists use a simple negative feedback loop model to explain all the phenomena found in homeotherms. What is known as the controller for the setpoint is probably located in the preoptic area and anterior hypothalamus [70]. Under normal conditions, the thermoregulatory system of homeotherms works continuously to maintain the core temperature (Tc) at the setpoint (Tset) [70]. According to this model, the setpoint in the CNS receives input from temperature receptors located in both the shell and the core. The comparator in the CNS compares the Tset with the information received by the various effectors and generates a physiological signal proportional to the difference between the Tset and the

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BRAIN TEMPERATURE The unique characteristics of the human brain compared with all other mammals, including primates, are, according to Mariak: 1) its large weight ratio with regard to the rest of the body and 2) its intensive and continuous metabolism that produces a large amount of heat that needs to be removed and dissipated to avoid an excessive increase in brain temperature [72]. Under normal conditions, heat washout is performed by the circulating blood, which is 0.2°C below brain temperature [72]. The offset between brain and core temperature differs in awake, neurologically intact persons and in patients with a brain insult. In a study of patients who were fully conscious and free of neurological symptoms, Mariak et al., showed that the offset between arterial blood and the average brain temperature in normothermia can be estimated as 0.35–0.51°C [72]. Mariak concludes that “…both brain and rectal temperature just happen to exceed the arterial blood temperature by a similar value; the former because brain temperature is continuously increased by intense metabolism, the latter because of metabolism of the intestinal flora” [72]. In general, core temperature measured at any site (rectum, esophagus, bladder etc.) tends to underestimate the true brain temperature in patients presenting with traumatic brain injury or any neurological insult that impairs consciousness. In a study of 30 neurosurgical patients in whom a thermocouple was inserted through a ventriculostomy, Mellegard et al., showed that intraventricular temperature was at least 1°C higher than central core temperature in one-third of the patients[73]. These authors also found that there was also a temperature gradient within the CNS, with the central parts of the brain being warmer than the surface [73]. Rumana et al., performed a similar study in severe TBI comparing brain temperature in the brain parenchyma with rectal and jugular bulb temperatures [74]. Brain-to-rectal gradients were above 1°C in 60% of the patients and were more than 2°C in 10% [74]. An important conclusion of this study is that temperature measured in the jugular bulb reflects body temperature but not brain temperature [74]. In another relevant study, Mariak et al., retrospectively analyzed the results of direct recordings of cerebral temperature within the subdural space and brain parenchyma in 63 unanesthetized patients following neurosurgical procedures, including 23 with fever >38°C [75]. In all patients, the temperature measured subdurally (brain surface) was higher than the esophageal temperature [75]. A recent review by McIlvoy discussed 15 studies that compared Tbrain with Tc [76] and clearly states that in most studies, brain temperature increased from the brain surface to the ventricles



and that ventricular temperature was always greater than rectal temperature [76]. The gold standard for core temperature is blood temperature measured by a pulmonary artery catheter. However, in clinical practice, either rectal, bladder, or esophageal temperature is usually regarded as a valid estimate of Tc [76]. Although most studies use rectal temperature, ventricular temperature is usually higher than rectal temperature with a mean offset of 0.2 - 2.0°C [76]. Tbrain is usually higher than bladder temperature with an offset of 0.5 2.5°C but with a better correlation with Tbrain than T rectum [76]. There is convincing evidence that any Tc underestimates brain temperature. Thus, in future studies of hypothermia in severe TBI, Tbrain and not Tc should be the target. MECHANISMS OF NEUROPROTECTION BY HYPOTHERMIA Hypothermia is the oldest form of brain protection. Although the neuroprotective effect of deep hypothermia has been known since the early 1950s, the fact that neuroprotection can be achieved by small changes in temperature has only been known since 1987, when Busto et al., reported this finding in experimental models of brain ischemia [77]. Since then, many experimental studies have confirmed that moderate hypothermia confers protection against ischemic and non-ischemic brain hypoxia, traumatic brain injury, anoxic injury following resuscitation from cardiac arrest and other neurological insults. However, the mechanisms that cause the strong neuroprotection provided by deliberate hypothermia in animals is still a matter of debate and are probably multiple. In the words of Corbett and Thornhill “… it is not possible to point to a single mechanism that underlies the robust neuroprotection provided by long duration postischemic hypothermia. Indeed, the remarkable benefit provided by mild hypothermia is likely due to a multitude of actions which make it the ultimate neuroprotective cocktail. This contrasts with neuroprotective drug therapies that typically target a single mechanism and that so far have proven ineffective in clinical trials” [78]. Many posttraumatic adverse events that occur in the injured brain at a cellular and molecular level are highly temperaturesensitive and are thus a good target for induced hypothermia [79]. The basic mechanisms through which hypothermia protects the brain have previously been reviewed by our group [80]. These neuroprotective effects are clearly multifactorial and include at least the following [79,81]: 1) reduction in brain metabolic rate, 2) effects on cerebral blood flow, 3) reduction of the critical threshold for oxygen delivery, 4) blockade of excitotoxic mechanisms, 5) calcium antagonism, 6) preservation of protein synthesis, 7) reduction of brain thermopooling, 8) decrease in edema formation, 9) modulation of the inflammatory response, 10) neuroprotection of the white matter, and 11) modulation of apoptotic cell death. These mechanisms will briefly be reviewed.

It has been shown that all the energy-producing pathways in the brain including the cerebral metabolic rate for glucose (CMRglu), oxygen (CMRO2) and lactate are reduced 2- to 4-fold by a 10°C decrease in temperature. Like other enzymatic catalyzed biological reactions, CMRO2 declines monoexponentially with temperature according to the following formula [82]: CMRO2T1=CMRO2T2 (Q10) - 0.1 (T1-T2), where Q10 is the temperature coefficient and CMRO2T1 and CMRO2T2 are the values of CMRO2 at T1 and T2, respectively [82;83]. Thus, in a non-injured brain and assuming a Q10 of 2, a reduction of 5°C from 37° to 32°C would lead to a reduction of about 33% of the CMRO2. If the normal CMRO2 is on average 3.10 ± 0.56 ml/100g/min [37], a reduction of 5°C would decrease CMRO2 to approximately 1.02 ml/100g/min. Controversies about the effect of hypothermia on brain metabolism surround the true Q10 value for the human brain during moderate hypothermia and the type of metabolism that is modified when temperature is lowered. Several studies in animals (monkeys and dogs) and in human volunteers in whom shivering was suppressed have found a Q10 ranging from 3.8 to 5 [84-87]. This is consistent with the findings of Shiozaki et al., that in severe TBI, Q10 is around 5 [88]. As Hartung and Cottrell commented, it seems that the real Q10 in humans cannot be extrapolated from other animal species, as a consistent finding is that hypothermia induces a more profound CMRO2 depression in primates than in rodents or dogs [87]. Thus, Brain’s Q10 values in humans are much higher than those expected in other species, potentially conferring an additional reason for neuroprotection from hypothermia. However, strong arguments have been posed for and against accepting a Q10 greater than 2 in the brain [89,90] and this highly important issue is still far from being resolved. The second most controversial issue surrounds the type of metabolism that is modified when temperature is lowered. There is convincing evidence to support the concept that brain metabolism is compartmentalized. The classic studies by Michenfelder et al., showed that the reduction of CMRO2 induced by barbiturates was directly related to the inhibition of electrical activity in the brain [91-93]. Depression of all electrical activity (flat EEG) was associated with a 50% depression in CMRO2. Doses higher than necessary to abolish EEG activity did not add additional metabolic suppression. From these studies, brain metabolism was divided into “active or functional” and “basal”. Basal metabolism was related to the oxygen necessary to maintain cell viability and functional metabolism to that necessary for electrical activity [89,94]. Although this concept probably oversimplifies brain physiology, it is useful for understanding brain metabolism. A further subdivision was subsequently introduced by Astrup et al., who showed that in dogs, approximately 40% of basal metabolism could be identified with the Na+-K+ pump and the remaining 60% was associated with synthetic processes and other unknown cell functions [95]. There is convincing evidence that hypothermia lowers mainly the basal metabolic rate and consequently that it acts at a completely different level than barbiturates, which only lower functional metabolism [89]. Therefore, as stated by Nemoto et al. “…the protective effect of mild hypothermia is a function not only of CMRO2 reduction but also of which CMRO2-related processes are inhibited” [89]. Additional studies are still required to clarify this highly important issue.

Reduction in Brain Metabolic Rate Hypothermia reduces whole body metabolism. The traditional concept used to justify hypothermic neuroprotection is that hypothermia confers brain protection because it induces a significant fall in brain metabolic rate. Changes in temperature have been proved to induce significant changes in the rate of chemical reactions in all organs. The reduction of metabolism in any organ can be approximated by what is known as the Van’t Hoff temperature coefficient (Q10), which is on average 2.2 - 2.3 in the human brain [48][82]. This index was formulated by Van’t Hoff, who was awarded the Nobel Prize for Chemistry in 1901 and has been widely used in bioenergetics. The Q10 is obtained as the ratio between the reaction rate at a given temperature (T) and the reaction rate at 10°C above T (T + 10).

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Effects on Cerebral Blood Flow The effects of hypothermia on CBF are a consequence of the well-accepted phenomenon that under normal conditions CMRO2 is tightly coupled to CBF and therefore, a reduction in the former is coupled to a significant reduction in the latter. However, evidence is emerging that suggests that coupling between CMRO2 and CBF is non-linear. Sakoh and Gjedde have recently shown that in experimental pig models of hypothermia, this non-lineal model can


fairly accurately predict the changes in CBF and CMRO2 [82]. Accepting that CMRO2 is tightly coupled to CBF in the normal brain and that hypothermia does not alter this coupling, both CMRO2 and metabolism should be equally reduced. Recent experimental studies by Sakoh and Gjedde confirm that deliberate hypothermia in pigs (from 38°C to 32°C) reduced both CBF and CMRO2 by 50% [82]. One of the paradoxes of the neuroprotection conferred by hypothermia is that brain metabolism is constantly reduced in most patients with a severe TBI. The pivotal paper by Obrist et al., showed that CMRO2 was significantly reduced after severe head trauma and that the reduction was inversely proportional to the Glasgow coma scale score [36]. Recently, new studies have supported this finding. In a cohort of 49 patients with a moderate or severe TBI, Glenn et al., studied brain metabolism [37]. In this series, simultaneous measurements of AVDO2 and CBF were made for the first 10 days after injury and were compared with those in 31 awake healthy volunteers. The results of this study confirmed that in severe TBI, the mean CMRO2 is reduced on average from 3.10 ± 0.56 ml/100g/min in the control group to a mean CMRO2 of 1.4 ± 0.43 ml/100g/min in TBI patients [37]. Consequently, it is difficult to accept that the neuroprotective effect of hypothermia is due to suppression of brain metabolism alone in this particular group of patients in whom depressed CMRO2 is almost a constant finding. A possible working hypothesis is that hypothermia lowers basal metabolism and that coma per se reduces only functional metabolism. The finding that hypothermia can reduce basal metabolism in preference to functional metabolism merits further discussion and research.


creases in glycine were significantly attenuated in the hypothermic group compared with normothermic controls [97]. Another important neurotransmitter, the amino acid D-serine has also been implicated in excitotoxic brain damage. The discovery of the role of D-serine has challenged the traditional concepts of neurotransmitters. D-serine is stored in glial cells but not in neurons and is an endogenous ligand for NMDA receptors[102]. D-serine has been found in the forebrain in very close relationship with the MMDA receptors and specifically in astrocytes that ensheath synapses [102]. It has been well established that glutamate is not the sole agonist of the NMDA receptor and that D-serine is released from astrocytes by glutamate. For an extensive discussion on this issue, the reader is referred to the review by Boehning and Snyder [102]. This study points out that even though glycine potentiates excitotoxicity, glycine is often not co-localized in the vulnerable anatomical regions whereas D-serine is [102]. When D-serine is released, it binds to the glycine site of the NMDA receptor. In summary, although the exact mechanism by which hypothermia modulates excitotoxic damage is still not clear, the glycine and D-serine hypothesis might be an important step toward its elucidation. Further studies on neuroprotective mechanisms of hypothermia should take into consideration both D-serine and glycine.

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Reduction of the Critical Value of Oxygen Delivery Experimental models that allow an strict control of oxygen delivery under highly controlled conditions have shown that moderate hypothermia (33°C) significantly lowers critical oxygen delivery (DO2crit) [18]. Consequently, under hypothermia, both the normal and injured brain seems to be more tolerant to reduced levels of DO2 without inducing a shift toward anaerobic metabolism.

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Blockade of Excitatory Neurotransmitters The injured tissue releases massive amounts of glutamate, inducing what is known as excitotoxic brain damage. The modulation of excitotoxic damage is the second most popular theory to explain the neuroprotection conferred by hypothermia after the hypothesis of reduction of CMRO2. However, some authors have questioned the theory of hypothermia-induced neuroprotection by the modulation of excitotoxic damage. An in-depth discussion of these studies is beyond the scope of this review. Authors such as Kvrivishvili have put forward the alternative hypothesis of glycine to explain the contradictions found in some experimental models of excitotoxic damage [96]. Baker et al., showed that induced hypothermia (29°C) in rabbits exposed to 10 minutes of global ischemia significantly reduced extracellular levels of both glutamate and glycine [97]. These results suggest that the neuroprotective properties of hypothermia may be partly due to its ability to prevent increases in the extracellular concentrations of amino acids that enhance the activity of the NMDA receptor [97]. Glycine is an inhibitory neurotransmitter in the brain stem and spinal cord but causes excitatory effects in rostral brain regions [98]. Glycine binds to a specific site on the NMDA receptor complex and low concentrations of glycine are required for receptor activation [96,98]. The NMDA receptor channel cannot be activated in the absence of glycine, which is abundant in the synaptic cleft [99]. Glycine potentiates excitotoxicity in vivo [100] and glycine antagonists reduce ischemic neuronal damage [101]. Hypothermic neuroprotection might be mediated in part by the ability of hypothermia to block the sustained elevation of the extracellular glycine concentration in response to brain ischemia [97]. In an experimental model of temporary global ischemia, in-

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Hypothermia as a Calcium Antagonist Intracellular calcium concentration is increased when energy fails [99]. Calcium is involved in the regulation of many enzyme systems inside the cell such as calcium/calmodulin-dependent protein kinase II, a multifunctional enzyme complex that mediates many of calcium’s effects. Ca2+ also regulates protein kinase C, an enzyme that plays a pivotal role in neurotransmitter release and gene expression[81]. As already mentioned, excess calcium influx into the cell is partly mediated by excitotoxic mechanisms. Calcium flow into the cell leads to a series of dynamic cascades. Calcium overload in the cell has been implicated both in ischemia and TBIinduced neuron death. Reduced calcium influx might be one of the key mechanisms underlying hypothermia-induced neuroprotection. Calpains are calcium activated cytoplasmic proteases that disrupt the cytoskeleton and nucleus [68]. Calpain I is activated by micromolar calcium, while calpain II is activated by millimolar calcium concentration. In a recent study, Liebetrau et al., have shown that delayed hypothermia beginning 3 hours after initiation of focal ischemia effectively attenuates secondary neuronal damage [103]. Calpain expression in the hypothermia-treated group was significantly reduced, as was the breakdown of its substrate the anti-microtubule-associated protein2, compared with normothermic animals [103]. The same effect has been shown by Buki et al., in traumatic axonal injury [104]. Animal models have demonstrated that the vulnerability of cells to calcium entry is highly individualized. Blocking calcium entry with the compound MK-801 has a neuroprotective effect on the hippocampal neurons, while paradoxically it worsens the outcome of neurons in the cortical layers. As McCarthy suggests, it is clear that “…one neuron’s poison is another neuron’s elixir of life” [105]. Hypothermia significantly reduces calcium influx into the cell by partially blocking glutamate receptor-mediated calcium entrance. Protein Synthesis Preservation The ubiquitin/proteasome proteolytic system is present in all eukaryotic cells and is responsible for the selective degradation of misfolded or damaged proteins. Consequently, ubiquitin plays an essential role in adequate cellular function. Ischemia and other brain insults provoke ubiquitin depletion. One consequence of ubiquitin deficit is excessive accumulation of abnormal proteins induced by brain injury or secondary ischemic events. Hypothermia has been shown to promote ubiquitin recovery in experimental



models of ischemia and could be one of the protective mechanisms through which mild hypothermia protects against delayed neuron death [106]. Reduction in Brain Thermopooling As previously mentioned, under normal conditions brain tissue temperature is usually 0.2 to 0.6 °C above the core temperature [107,108]. The concept of “brain thermopooling” was first described as a secondary insult in severe TBI by Hayashi et al., in 1994 [109]. Thermopooling is characterized by a significant increase in the normal gradient between the core and brain temperature induced by an insuficient CBF to the brain [107,108]. The reduction in CBF decreases the washout effect on brain temperature. Low mean arterial blood pressure may also induce brain thermopooling. The immediate consequence is a high brain temperature with all the deleterious effects already mentioned. According to Hayashi, hypothermia can modulate the increase in brain temperature by reducing the thermopooling effect [107]. However, this hypothesis has not been verified in patients but only introduced in the rationale of a very comprehensive management of TBI with moderate hypothermia followed by a few centers. Further studies are necessary for a better understanding of the thermopooling effect, its potential control by moderate hypothermia and the final impact of this control on clinical outcome. Decrease in Edema Formation Although the characterization and pathobiology of brain edema after head injury is still a matter of debate, a posttraumatic increase in brain water content clearly plays an important role in human TBI [110]. Hypothermia modulates the detrimental effects of injury on the blood brain barrier and consequently decreases the formation of edema [79]. The first experimental evidence that hypothermia reduces vascular permeability was described by the Richmond group in 1992 [111]. In this study it was shown that for rats in which hypothermia was induced prior to injury, there was a greatly reduced protein passage into the brain [111]. Jiang et al., attributed this effect to the hypothermia-induced modulation of the systemic arterial hypertensive response found in normothermic rats [111]. In another experimental model of cortical impact injury in rats, Smith et al., showed that hypothermia significantly attenuated the increased permeability of the BBB after injury [112]. These authors hypothesized that hypothermia might attenuate the free radicalinduced increases in BBB permeability, thus reducing brain edema.

by zymography [116]. This new information opens new horizons in the field of hypothermia in TBI. Neuroprotection of the White Matter In terms of neuroprotection, the white matter can be considered the “forgotten brain”. Most animal models have been focussed on the neuroprotection of neurons and consequently of the gray matter. An appealing concept introduced by Feigin in stroke is that hypothermia causes a more profound neuroprotective effect than any known neuroprotective drug by protecting not only neuronal cell bodies but axons as well (‘brain protection' as opposed to ‘neuron protection' only) [117]. This hypothesis also has some experimental backup in TBI. By using the impact acceleration model, Koizumi and Povlishock demonstrated the protective effects of hypothermia on axonal injury in the absence of any secondary insult [118]. Maxwell and coworkers tested the hypothesis that mild hypothermia may improve injury to the axonal cytoskeleton in experimental models of DAI reproduced by stretching the optic nerve of guineapigs [119,120]. These authors clearly observed that mild hypothermia (32-32.5°C) induced immediately after stretching the optic nerve significantly reduced the number of axons labeled with antibodies against -APP [119]. The data from this study provides quantitative evidence that loss of axonal microtubules and compaction of neurofilaments are significantly reduced in mildly hypothermic animals, at least during the first 4 hours after injury [119]. However, as stated by Maxwell and coworkers, these findings cannot be easily extrapolated to the animals injured by the impact acceleration model. Furthermore, the mechanisms by which moderate hypothermia prevents disturbance of the axons are still highly speculative and outside the scope of the present review.

Modulation of the Inflammatory Response Hypothermia has been shown to reduce the accumulation of PL in the injured brain and to induce a significant reduction in the mediators of inflammatory cascades such as the cytokines IL-6, IL-10 and TNF. Whalen et al., showed that mild hypothermia reduces PL accumulation during the first 4 h of TBI [113]. Additional studies have demonstrated that PL migration both in vivo and in vitro is markedly reduced by profound hypothermia (29°C) [114]. In an experimental model of hemorrhagic shock, Gundersen et al., demonstrated that moderate hypothermia in hypovolemic animals significantly reduced plasmatic levels of IL-6 without significantly modifying TNF [115]. The physiopathological and clinical relevance of this effect on the brain is unclear but the effect of posttraumatic temperature manipulations on the inflammatory response to brain injury deserves further investigation. The effects of moderate hypothermia on MMPs have recently been shown. Like any other enzyme, MMPs are temperaturesensitive and consequently inhibited by hypothermia [116]. In an animal model of MCA occlusion, Wagner et al., showed that hypothermia (33°C) reduced the size of the lesion on diffusion weighted images and decreased the area of BBB breakdown and infarct volume [116]. This study was the first to show that hypothermia led to a significant decrease in MMP-2 and MMP-9 activity as measured

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Modulation of Apoptotic Cell Death Brain cell death occurs by necrosis or apoptosis. Programmed cell death (apoptosis) has been detected both in ischemia and TBI. One problem in the physiopathology of brain injuries is that the same cellular substrates can be damaged by apoptotic caspases or by necrotic proteases [121]. One hypothesis suggests that when an energy supply is present, the form of cell death will be apoptosis, but when an energy supply is lacking, necrotic death will follow [121]. Apoptosis is an active process that requires gene expression and the synthesis of new proteins together with selective downregulation of other proteins [105]. To quote Roy and Sapolsky, “The modes of cell death are distinct but combine to create a picture of cytological chaos; this is an inevitable outcome of a cell’s attempt to superimpose a highly structured process (apoptosis) onto a more chaotic, non-linear process (necrosis)” [121]. For some, apoptotic cell death is the most important mechanism for cell loss following stroke or head injury [105].The important fact regarding apoptotic cell death is that apoptosis requires energy to be initiated and sustained. CONCLUSIONS The new developments discussed in this review indicate that, by targeting many of the abnormal neurochemical cascades initiated after TBI, induced hypothermia may modulate neurotoxicity and consequently, may play a unique role in opening up new therapeutic avenues for treating severe TBI and improving its devastating effects. Many posttraumatic adverse events that occur in the injured brain at a cellular and molecular level are highly temperaturesensitive and are thus a good target for induced hypothermia. The basic mechanisms through which hypothermia protects the brain are clearly multifactorial and include at least the following: reduction in brain metabolic rate, effects on cerebral blood flow, reduction of the critical threshold for oxygen delivery, blockade of excitotoxic mechanisms, calcium antagonism, preservation of protein synthesis, reduction of brain thermopooling, a decrease in edema formation,


modulation of the inflammatory response, neuroprotection of the white matter, and modulation of apoptotic cell death. The multiple effects of hypothermia have prompted clinical researchers to critically review the negative results of clinical trials reported in traumatic brain injury. This re-evaluation, may help to elucidate the reasons for previous a failure and to design further multicenter randomized clinical trials that would definitively confirm or refute the potential of this therapeutic modality in the management of severe traumatic brain injuries. ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Gail Craigie and Mary Jane Smyth in helping us to correct the manuscript. This study was supported in part by by grants PI030153 and PI051092 from Fondo de Investigaciones Sanitarias (FIS) received by Dr. J. Sahuquillo and by the Fifth European Framework of the European Commission (SMILE project, QLK1-CT-2002-02583).

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