Barriers in the brain: a renaissance?

Barriers in the brain: a renaissance?

Review Barriers in the brain: a renaissance? Norman R. Saunders, C. Joakim Ek, Mark D. Habgood and Katarzyna M. Dziegielewska Department of Pharmacol...

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Review

Barriers in the brain: a renaissance? Norman R. Saunders, C. Joakim Ek, Mark D. Habgood and Katarzyna M. Dziegielewska Department of Pharmacology, University of Melbourne, Parkville, VIC 3010, Australia

Barrier mechanisms regulate the exchange of molecules between the brain’s internal milieu and the rest of the body. Correct functioning of these mechanisms is critical for normal brain activity, maintenance and development. Dysfunctional brain barrier mechanisms contribute to the pathology of neurological conditions, ranging from trauma to neurodegenerative diseases, and provide obstacles for successful delivery of potentially beneficial pharmaceutical agents. Previous decades of research have yielded insufficient understanding for solving brain barrier problems in vivo. However, an awakening of interest and novel approaches are providing insight into these mechanisms in developing and dysfunctional brain, as well as suggesting new approaches to circumventing brain barrier mechanisms to get therapeutic agents into the central nervous system. Introduction The study of blood–brain barriers is perhaps the only field of neuroscience that has declined until recently. The reasons for an awakened interest are due to revived appreciation that brain barrier mechanisms play critical roles in brain development and pathophysiology of several neurodevelopmental (e.g. autism [1,2], schizophrenia [3,4] and cerebral palsy [5]) and neurodegenerative disorders (e.g. multiple sclerosis [6,7] and Alzheimer’s [8] and Parkinson’s [9] diseases). These mechanisms also present formidable obstacles to getting therapeutic agents into the CNS for treating neurological and psychiatric disorders. There is increasing appreciation that neuroactive compounds developed solely through in vitro experimental and screening systems might not be therapeutically useful, as their access to the brain in vivo is often obstructed by the brain’s barriers. Understanding the morphological and physiological nature of these barriers in developing, mature and aging brains is critical for our ability to restore these mechanisms when they are disturbed in pathological conditions. The term ‘blood–brain barrier’ describes a series of mechanisms that control the internal environment of the brain. Stability of this environment and its distinctness from that of the rest of the body are essential for normal brain development and function [10]. Underlying the cellular mechanisms that determine the brain’s internal environment is a fundamental physical barrier at the level of intercellular junctions between cells forming the interface between blood and brain (blood–brain barrier) and in the choroid plexuses (blood–cerebrospinal fluid [CSF] barrier); see Figure 1. These cell junctions (tight junctions) Corresponding author: Saunders, N.R. ([email protected]).

occlude, or at least severely attenuate, movement through intercellular spaces between endothelial cells in the blood– brain barrier and epithelial cells in the choroid plexus blood–CSF barrier. This diffusion restraint allows ion and other concentration gradients to be set up by transfer mechanisms within cells of the barrier interfaces. These mechanisms include blood-to-brain transfer of amino acids and glucose vitamins, two-way exchange of ions that provide the ionic stability necessary for neuronal activity and brain-to-blood transfer mechanisms such as p-glycoprotein that are an important defense for the brain but a great impediment to drug entry. Tight junctions in effect extend the properties of single cells to the entire interfaces between blood, brain and CSF. However, more than just endothelial cells are involved: astrocytes, with foot processes that surround the endothelial cells; pericytes between the endothelial cells and astrocytes and basement membrane (Figure 1). The term ‘neurovascular unit’ is also used to encompass these components of ‘the’ blood–brain barrier, as well as to get away from this rather misleading term. Some methodological limitations To summarize the methods currently used in brain barrier research [11] is outside the scope of this short review. However, a problem, particularly for people new to the field, is that published descriptions of methods often do not adequately address potential limitations and pitfalls. A particular reason why the field of brain barrier biology has been in decline is a lack of innovation and an adherence to methods that have been around for decades. The most commonly used test of barrier integrity is to repeat Ehrlich’s 19th century experiment and inject a dye. This is akin to electronic researchers insisting on the continued validity of cat’s whiskers or crystal diode radios for their experiments. Although dyes (Evans blue in recent years; trypan blue in many earlier studies) appear to be tightly bound to albumin in vitro providing the dye is present in low concentration [12], in vivo there are conflicting reports, probably because of differences in the amount of dye injected. Thus, it is unclear to what extent dyes entering the brain across a disturbed barrier reflect entry of protein or unbound dye. The large molecule horseradish peroxidase (HRP), which can be visualized at the electron microscopical (EM) level, has been widely used. But technical problems with diffusion of the reaction product and toxic effects in some species (see Ref. [13]) often seem to be ignored. Different-sized dextrans or naturally occurring plasma proteins avoid these problems [13–15]. For quantitative studies of any duration it is also essential that blood levels of a marker during the course of an experiment

0166-2236/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2008.03.003 Available online 9 May 2008

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Review are estimated, otherwise brain/plasma concentration ratios (a common way of representing brain barrier permeability data) can reflect changes in blood levels rather than permeability. Estimation of blood contamination of

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brain samples is important, but often overlooked. It is also essential that an appropriate measure of barrier function is used. For example, it is inappropriate to use a measure of passive permeability (e.g. Evans blue or dextran) to

Figure 1. Schematics of the sites of the barrier interfaces (indicated in orange) in the adult and developing brain. (a) The blood–brain barrier is a barrier between the lumen of cerebral blood vessels and brain parenchyma. The endothelial cells (Endo) have luminal tight junctions (arrowhead) forming the physical barrier of the interendothelial cleft. Outside the endothelial cell is a basement membrane (bm) which also surrounds the pericytes (Peri). Around all these structures are the astrocytic endfeet processes from nearby astrocytes (As endfoot). All these structures together are often referred to as the neurovascular unit. (b) The blood–CSF barrier, a barrier between choroid plexus blood vessels and the CSF. The choroid plexus blood vessels are fenestrated and form a nonrestrictive barrier (small arrows); however, the epithelial cells (Ep) have apical tight junctions (arrowheads) that restrict intercellular passage of molecules. (c) The meningeal barrier is the least studied and structurally most complex of all the brain barriers. The blood vessels of the dura are fenestrated and provide little barrier function; however, the outer cells of the arachnoid membrane (Arach) have tight junctions (arrowheads), and this cell layer is believed to form the physical barrier between the CSF-filled subarachnoid space (SAS) and overlaying structures. The blood vessels in the arachnoid and on the pial surface (PIA) have tight junctions with similar barrier characteristics as cerebral blood vessels although lacking the surrounding pericytes and astrocytic endfeet. (d) The fetal CSF–brain barrier, a barrier between the CSF and brain parenchyma, has only been shown to be a functional barrier in the early developing brain (see Ref. [19]). In early development, the neuroependymal cells are connected to each other by strap junctions (open arrowheads) that are believed to form the physical barrier restricting the passage of larger molecules such as proteins but not smaller molecules such as sucrose. (e) The adult ventricular ependyma. During development, the neuroependymal cells flatten and lose their strap junctions. The mature ependyma does not restrict the exchange of molecules at least as large as proteins between CSF and brain.

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Review estimate barrier dysfunction when studying a mechanism involved in cellular transfer. It is sometimes assumed that the concentration of a marker in CSF is an estimate of its blood–brain barrier permeability (e.g. Ref. [15]), whereas it more likely reflects entry across the choroid plexuses, perhaps with a contribution from the cerebrovascular route. Many laboratories have endeavored to mimic in vivo properties of blood–brain and blood–CSF interfaces using invitro models [16]. There were hopes that this would provide a rapid screening tool for evaluating barrier permeability of new drugs. This has so far not proved very successful, and it was acknowledged at the recent inaugural meeting of the International Brain Barrier Society that these models still require considerable improvement (http://www.ibbsoc.org/PDFs/NeuralBarriers.pdf). The recent innovation of incorporating pericytes into these models appears promising [17]. Developmental aspects The nature of the internal environment in which the brain grows is likely to have a fundamental impact on its development. For several decades, the emphasis of developmental neurobiologists has been on cellular and

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molecular mechanisms, with, until recently, little attention paid to other factors [18]. As in the adult, early experiments to test the integrity of the barrier in developing animals also relied on injected dyes (trypan blue or Evans blue). These gave conflicting results, probably because of differences in the amount of solution injected into fragile immature animals (see Ref. [19]). A key point in debates about the maturity of brain barriers in embryos and fetuses turns on determining functional effectiveness of tight junctions in cerebral vessels and choroid plexus epithelium. Tight junctions in developing brain have been studied extensively using transmission EM and freeze fracture [20]. The most recent advance uses small hydrophilic molecules that can be measured quantitatively in plasma and CSF and their route of penetration into brain and CSF visualized at the EM level. These studies showed that tight junctions in the earliest vessels that enter neocortex appear to be impermeable to markers even smaller than sucrose (342 Da) and inulin (5 kDa, Figure 2). It is important to stress that these markers define functional permeability. Tight junctions undergo developmental changes in their ultrastructure and molecular configuration [21–23], although the latter has

Figure 2. Micrographs of blood–brain and blood–CSF barriers at early stages of brain development. (a) Neocortical blood vessels on the brain surface of a newborn opossum (Monodelphis domestica) 30 min after an intraperitoneal injection of a small inert tracer (3 kDa biotin-dextran amine). The tracer, visualized as a dark reaction product, is clearly confined to the lumen of these blood vessels, which are the first to grow into the neocortex of the developing opossum brain (scale bar is 50 mm). (b) Electron micrograph of an intercellular junction between two adjacent endothelial cells in the newborn opossum brain (blood–brain barrier). Note the biotin-dextran amine tracer (dark reaction product) which is present in the lumen of the blood vessel, but does not pass beyond the tight-junction complex (arrow) located at the luminal end of the intercellular cleft (scale bar is 200 nm). (c) Electron micrograph of an intercellular junction between two adjacent choroid plexus epithelial cells (blood–CSF barrier) in a newborn opossum brain 30 min after the tracer was injected into the intraperitoneal cavity. Note that the tight junction (arrow) restricts the passage of this tracer along the intercellular cleft toward the cerebrospinal fluid in the ventricles (scale bar is 100 nm). (d) Electron micrograph of an intercellular junction between two adjacent choroid plexus epithelial cells in an embryonic rat brain at 15 days gestation. In this animal, the tracer was administered into the cerebrospinal fluid on the other side of the blood–CSF barrier rather than into the peritoneal cavity as in (c). Note that by 10 min after administration, the tracer is clearly visible in the intercellular cleft, but is unable to pass beyond the tight-junction complex (arrow) located at the ventricular end of the intercellular cleft (scale bar is 200 nm). Reproduced with permission from Refs [27–29].

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Review mainly been studied in vitro and the functional relevance is unclear. The findings that cerebral blood vessels in immature brain were impermeable to even small molecules [24,25], whereas brain/plasma and CSF/plasma concentration ratios are much higher than in adults, raised questions about the route of entry for these molecules and the reasons for the decline in concentration ratios with age [26]. It appears that both small, hydrophilic molecules and proteins enter the CSF via a transcellular route in the choroid plexuses. Once in CSF these molecules, including proteins, are taken up by some of the cells in brain that have contacts with CSF. The reason for the much higher brain/plasma and CSF/plasma concentration ratios in the immature brain is not that the barrier interfaces are more permeable; initially these markers enter a closed space (cerebral ventricles and brain itself), but as the brain develops, there is a progressive increase in CSF secretion and ventricular spaces open up to provide a drainage pathway that limits the accumulation of markers in CSF. This results in lower brain/plasma and CSF/plasma concentration ratios [26,27]. Astrocyte involvement in early barrier formation remains unclear, as most work has been done in vitro [16] and tight junctions are present before astrocytic endfeet ever come into contact with endothelial cells. However, astrocytes might be important for induction of other barrier mechanisms. Recent work points to the crucial involvement of pericytes in influencing vascularization in the fetal brain [28,29]. An intriguing parallel has been drawn between factors such as netrin that guide neurites during CNS development and their involvement in guidance of blood vessels in immature brain [30], but it needs to be determined whether pericytes and the basement membrane contribute to development of specific features of barrier mechanisms. It is of fundamental interest to understand the development of exchange mechanisms that are superimposed on the tight junction-based diffusion restraint and examine how the nature of the internal environment resulting from these mechanisms contributes to brain development. Earlier attempts to tackle this problem have been hampered by the difficulty of distinguishing between developmental differences in barrier transport and metabolic usage of, for example, amino acids. One way around this difficulty is the application of powerful molecular techniques to define gene expression patterns in endothelial cells in the developing brain, an approach only recently applied. This is yielding some spectacular insights into barrier properties, for example the expression of hemoglobin in endothelial cells [31] and a range of transcriptional changes that occur with age [32]. Little is known about effects of barrier disruption during brain development, but an instance where it might be important is the recent demonstration of barrier breakdown in blood vessels of white matter tracts of developing brain, following systemic inflammation. This is associated with damage to these tracts, but only at a critical period in brain development [5,33,34], and is reminiscent of white matter damage leading to cerebral palsy that occurs in some prematurely born infants of mothers with an intrauterine infection at 22–28 weeks gestation [35,36]. 282

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Neurodegenerative diseases To do these important aspects of brain barrier pathology justice is not possible here, but recent detailed reviews are available. Important findings of contributions of brain barrier functions in diseases such as Alzheimer’s [8], motor neuron disease [37] and multiple sclerosis [6,7] have been described. There is increasing recognition that these disorders involve complex changes in brain barrier functions, including specific transport mechanisms, inflammatory mediators, oxidative stress and abnormal angiogenesis (http://www.ibbsoc.org/PDFs/Neurodegeneration.pdf). However, it is not clear to what extent these changes are a causal contributor to the disease or whether barrier dysfunction is a consequence and reinforces the primary pathological process within the brain. Neurotrauma Neurotrauma produces an instant disruption of blood vessels and thus disruption of brain barrier function in the affected areas. This is followed by inflammatory responses that contribute to further localized changes in barrier function and secondary damage to surrounding tissue. Defining the timeframe and properties of barrier dysfunction following injury is important because it delimits the period when the CNS is at risk of secondary damage due to entry of toxic molecules. However, it also represents a therapeutic window through which drugs have a better chance of reaching damaged areas. The most frequently used method for investigating this disruption has been to inject dyes such as Evans blue or the protein HRP. As indicated above these have serious limitations, some of which might account for the reported variable time courses of barrier opening following a CNS insult. Recently, the use of a range of inert permeability markers of different molecular size confirmed that there is a period of leakage of large molecules (plasma protein-sized) lasting for 4 h post-brain injury [13], but unexpectedly there is a much longer period (at least 4 days) of increased permeability to molecules of <10 kDa in the region of damage (Figure 3). This suggests a useable therapeutic window to treat brain injuries with molecules <10 kDa. Earlier studies showed that attenuating the inflammatory response appeared to have positive neurological outcomes. More recent studies [38,39] showed that an increase in barrier permeability following spinal cord trauma is not just a simple opening of barriers but that there is an upregulation in transport systems for inflammatory cytokines such as tumor necrosis factor a and leukemia inhibitory factor in endothelial cells around the injured area. Thus, brain and spinal cord barriers have an active role in the inflammatory response. These studies highlight the key role of brain barriers in pathophysiology of neurotrauma and show that regulating their function is a powerful way to manipulate the delicate balance between harmful and beneficial events following injury (http://www.ibbsoc.org/PDFs/Injury.pdf). An area of considerable uncertainty in neurotrauma barrier pathology is the role of the cerebrovascular endothelium in generation of life-threatening cerebral edema that accompanies severe head injury. The mechanism of action of the commonly used treatment (intravenous hyperosmolar solutions, e.g. mannitol) for raised intracranial

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Figure 3. Time course of barrier disruption following brain injury in adult mice for different size permeability markers. The permeability markers were injected intravenously 10 min before collection of brain tissue. The large horseradish peroxidase (HRP) marker stops leaking from the injury site by around 4 h postinjury. By contrast, the smaller (10 kDa and 3 kDa) biotin-dextran amines (BDA) and very small biotin-ethylene diamine (BED) continue to leak from the injury site for over 4 days postinjury. By 5 days postinjury, there is no visible leakage of BDA or BED after 10 min circulation time. Data are from Ref. [40].

pressure caused by this edema is also not fully understood [40], nor is there ‘gold standard’ clinical trial evidence for its effectiveness [41]. The treatment is thought to be dependent on blood–brain barrier restriction of small molecules, with significant outward movement of water across the cerebral vasculature. However, small molecules do slowly penetrate into the brain and with repeated treatment can cause ‘rebound’ inward water transfer, thus exacerbating the initial clinical condition [42]. Even the route for water entry or exit across cerebrovascular endothelial cells is unclear. Thus far, no aquaporins have been demonstrated in cerebral endothelial cells and it is suggested that water crosses by diffusion and/or endocytosis [43], as was assumed for other cells before the discovery of aquaporins. Drug targeting to the brain Direct targeting of drugs to the brain requires overcoming five main obstacles: (i) stability and half-life of the drug in blood, (ii) selective accumulation and (iii) enhanced uptake across barrier interfaces, (iv) drug residence time in CNS and (v) specificity of distribution to target sites inside brain. Complex tight junctions between barrier-forming cells prevent paracellular passage of compounds from the lumen of blood vessels into brain interstitial fluid, and thus passive entry into the CNS is effectively restricted to lipid-soluble compounds. Many early pharmacological strategies to enhance entry of drugs into the brain involved chemical modifications of drugs to make them more lipid soluble (lipidization), either by replacing hydrophilic side groups with hydrophobic groups or by masking them with hydrophobic groups. However, these lipidized drugs are not site directed, and when administered in vivo distribute widely and can become substrates for efflux mechanisms at the brain’s barrier interfaces that export a broad range of lipid-soluble drugs back into blood [44]. As understanding of structure and function of the brain’s barrier interfaces expanded, it became apparent that many essential compounds required for normal brain growth and function (e.g. glucose and amino acids) enter in much greater amounts than could be accounted for by their

low lipid solubility. This led to the discovery of numerous inwardly directed uptake mechanisms that selectively transport specific compounds into the brain (carriermediated and receptor-mediated pathways). Also, many cationic (positively charged) compounds also exhibit greater entry into brain than would be expected from their low lipid solubility (absorptive-mediated pathway; e.g. Ref. [45]). Thus, evolution appears to have solved many of these drug delivery obstacles for essential compounds, and considerable attention has focused on the drug delivery potential of these endogenous pathways (see Figure 4). Carrier-mediated uptake has been utilized to deliver compounds that resemble endogenous ligands and to carry attached drug cargos with them. These chemical delivery systems have been extensively reviewed [46,47]. Competition with endogenous substrates and lower affinity for the receptors can be limiting factors, and this has directed attention primarily toward higher-capacity transporters (e.g. GLUT1) and those with broader substrate specificity (e.g. L-system amino acid transporter). Receptor-mediated endocytosis systems (e.g. transferrin and insulin receptors) are attractive uptake pathways because they can accommodate much larger cargos than classical transporter proteins [48]. These systems require binding of ligands to receptors to initiate endocytosis, but can also internalize specific antibodies directed at parts of the receptors exposed at the luminal surface of cerebral blood vessels (e.g. OX26). These antibody binding sites are remote from the ligand binding sites and do not appear to adversely affect normal ligand binding and initiation of endocytosis. Large compounds conjugated onto these antibodies are also carried into the cell ‘Trojan horse style’ hidden inside the endosome. It is not clear how these antibody conjugates exit the endothelial cells, but studies using antibody-targeted siRNA [49] and antibody-targeted BDNF [50] have shown that this approach is effective when applied in vivo. The types of compounds deliverable by these carrier systems is limited by their ability to survive exposed in plasma. For example, compounds potentially useful for genomic and proteomic therapies (e.g. DNA, mRNA and oligopeptides) rapidly degrade in blood or aggregate into 283

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Figure 4. Schematic diagram of a blood–brain barrier interface. The main pathways for drug entry into the brain are (a) passive diffusion if the compound is sufficiently lipid soluble, (b) inwardly directed carrier proteins (e.g. transporters) that bind substrates at the luminal surface and then release them into the endothelial cytoplasm for subsequent export by carriers located at the abluminal membrane, (c) absorptive-mediated endocytosis (AME) if the compound has sufficient cationic (+ve) charge to enable localized electrostatic disruption of the cell membrane phospholipids and (d) receptor-mediated endocytosis (RME), where binding of a substrate to a receptor at the luminal surface results in the formation of an endocytotic vesicle that carries the receptor/substrate complex into the cell. Outwardly directed carrier proteins (e) can significantly reduce rates of entry into brain for many lipid-soluble compounds.

deposits. Accordingly, there has been an expansion of research into enclosed capsular-based delivery systems (liposomes and nanoparticles) because of the protection they offer and reduced renal clearance if larger than 3– 5 nm [51]. Recent research into these nonviral delivery vectors has concentrated on the composition of the capsules and surface coatings to improve plasma solubility and stability, minimize immunogenicity and reduce nonrenal clearance; for example, incorporation of polyethyleneglycol units onto nanoparticles markedly extends plasma residence times [52]. More recently, site-directed targeting of these capsules has been achieved by attachment of antibody fragments that enable uptake through receptor-mediated pathways. This rapidly expanding field of molecular capsule design has been extensively reviewed [53]. For an overview of drug targeting strategies, see http://www. ibbsoc.org/PDFs/Delivery.pdf. Introduction of novel methods In recent years, a sign of revival in the field has been entry of new researchers and introduction of methods not previously used to study brain barrier problems. These have included genomic and proteomic analysis of adult [54] and immature endothelial [32] and choroid plexus epithelial [55] cells. Another promising approach is the use of imaging techniques. Thus, Nedergaard and colleagues have used two-photon microscopy to image microvasculature responses to photolysis of Ca2+ caged in astrocytic endfeet around vessels in neocortex of mice in vivo. This indicated a role for astrocytes in control of cerebral blood flow [56]. In a mouse model of Alzheimer’s disease, vascular instability from abnormal astrocytic activity might contribute to pathology of the condition [57]. The resolution of this technique 284

is good enough to study permeability of single cerebral blood vessels in vivo, under normal and pathological conditions; combined with video recording, it gives the prospects of following the time course of events at the blood–brain interface. A combination of imaging and electrophysiological techniques has been used to study the possible role of pericytes in control of cerebral microvessels [58]. Nanoparticles for improved in vivo imaging resolution [59,60] and drug delivery are being introduced. The higher resolution of synchrotron-generated radiation has only recently begun to be used for brain barrier studies [61]. The application of novel imaging techniques in other fields of neuroscience has been a major component of the spectacular advances in, for example, developmental neurobiology and cognitive neuroscience. Conclusion The ionic stability, neurochemical environment and normal functioning of the brain are crucially dependent on the integrity of its barrier systems. Without this stability, complex functions performed by the brain would be impossible and ‘our sensory experience of the outside world would be confined to a series of flashes and bangs’ (H. Davson, unpublished). The importance of specific barrier mechanisms for particular features of brain development remains largely to be determined. It is becoming more apparent that dysfunction of brain barrier mechanisms in a variety of pathologies is more than disruption of the normal (tightjunction) diffusion restraint and that such dysfunction might be a part of the disease process, rather than a consequence. A wide range of increasingly complex strategies is being tested to overcome barrier mechanisms to get therapeutic agents to their targets in the brain for a variety of nervous system disorders. After a decline in interest and

Review Box 1. Some outstanding questions o What is the relation between different barrier mechanisms and specific features of brain development? o What are the mechanisms of barrier dysfunction and their contribution to different brain pathologies? o Can the period of barrier dysfunction be utilized clinically for positive outcomes in different brain disorders? o What happens to cellular inward and outward transfer mechanisms following trauma and in neurodegenerative diseases? o Can CNS-directed drug delivery systems that deal with all five targeting obstacles be designed? o Can in vitro systems that will be effective substitutes for wholeanimal models be developed?

activity in brain barrier research over several decades, there are now signs of a significant resurgence in this important field [62], (Box 1). Acknowledgements The authors would like to acknowledge support received from the National Institutes of Health (RO1 NS043949–01A1), the National Health and Medical Research Council, Australia, the Victorian Neurotrauma Initiative and in earlier times the Medical Research Council, UK and the Wellcome Trust.

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