Novel therapeutic strategies for traumatic brain injury: acute antioxidant reinforcement.

CNS Drugs (2014) 28:229–248 DOI 10.1007/s40263-013-0138-y

REVIEW ARTICLE

Novel Therapeutic Strategies for Traumatic Brain Injury: Acute Antioxidant Reinforcement Rodrigo Fernańdez-Gajardo • Jose´ Manuel Matamala Rodrigo Carrasco • Rodrigo Gutie´rrez • Ro´mulo Melo • Ramoń Rodrigo

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Published online: 15 February 2014 Ó Springer International Publishing Switzerland 2014

Abstract Traumatic brain injury (TBI) is the most important cause of disability in individuals under the age of 45 years and thus represents a significant social and economic burden. Evidence strongly suggests that oxidative stress is a cornerstone event leading to and propagating secondary injury mechanisms such as excitotoxicity, mitochondrial dysfunction, apoptosis, autophagy, brain edema, and inflammation. TBI has defied conventional approaches to diagnosis and therapy development because of its heterogeneity and complexity. Therefore, it is necessary to explore alternative approaches to therapy development for TBI. The aim of this review is to present a therapeutic approach for TBI, taking into account the evidence supporting the role for oxidative stress in the pathophysiological processes of secondary brain injury. The role of agents such as mitochondria-targeted antioxidants (melatonin and new mitochondria-targeted antioxidants), nicotinamide adenine dinucleotide phosphate (NADPH) inhibitors

R. Fernańdez-Gajardo R. Carrasco R. Gutie´rrez R. Rodrigo (&) Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Independencia 1027, Casilla, 70058 Santiago 7, Chile e-mail: [email protected] R. Fernańdez-Gajardo J. M. Matamala R. Melo (&) Department of Neurological Sciences, Faculty of Medicine, University of Chile, Jose Manuel Infante 553, Providencia, Santiago, Chile e-mail: [email protected] R. Fernańdez-Gajardo J. M. Matamala R. Carrasco Laboratory of Biomedical Investigation, Faculty of Medicine, University of Chile, Jose Manuel Infante 553, Providencia, Santiago, Chile

(antioxidant vitamins and apocynin), and other compounds having mainly antioxidant properties (hydrogenrich saline, sulforaphane, U-83836E, omega-3, and polyphenols) is covered. The rationale for innovative antioxidant therapies based on current knowledge and particularly the most recent studies regarding this field is discussed. Particular considerations and translational potential of new TBI treatments are examined and a novel therapeutic proposal for TBI is presented.

1 Introduction Traumatic brain injury (TBI) is the most important cause of disability in individuals under the age of 45 years. TBI is a growing public health problem, and the World Health Organization (WHO) has predicted that deaths from road traffic incidents, primarily due to TBI, will double between 2000 and 2020, placing TBI as the third leading cause of global mortality and disability by 2020 [1]. Patients surviving severe TBI suffer permanent neurological and psychological disabilities that represent a significant socio-economic burden. Wittchen et al. [2] estimated that TBI costs extend to €33 billion per year in Europe alone, becoming one of the priorities in the national research agendas of many countries. The overall neurological recovery and long-term morbidity continues to be a significant healthcare challenge, despite progress in prevention measures and early resuscitation [3]. There is no effective therapy for TBI that has been approved by any regulatory agency. TBI has defied conventional approaches to diagnosis and therapy development because of its heterogeneity and complexity. Therefore, it is necessary to explore alternative approaches to TBI therapy development.

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The aim of this review is to present an update of the evidence supporting the role of oxidative stress in the pathophysiological processes of secondary brain injury after TBI. Even though oxidative stress has a demonstrated pathogenic role in TBI, the therapeutic effects of antioxidants have shown disappointing results, likely due to pharmacological problems. Furthermore, this review will cover the role of agents such as mitochondria-targeted antioxidants, nicotinamide adenine dinucleotide phosphate (NADPH) inhibitors and other compounds having mainly antioxidant properties. In addition, it discusses the rationale for innovative antioxidant therapies based on the most recent studies focused on neuroprotective agents and what has been learned from the deficiencies in the design of previous studies. Finally, a novel therapeutic proposal for TBI is presented.

2 Pathophysiology of Traumatic Brain Injury (TBI) TBI results in various types of brain damage. The primary injury comprises the mechanical forces at the time of the injury, which result in direct mechanical damage to neurons, axons, glia, and blood vessels as a result of shearing, tearing, or stretching [4, 5]. The resultant injury includes hemorrhage, contusions, and laceration, with immediate clinical effects [6]. In treatment terms, this type of injury is exclusively sensitive to preventive but not therapeutic measures. The secondary type of injury is non-mechanical damage, which involves consecutive pathological processes initiated at the moment of trauma, including excitotoxicity, mitochondrial dysfunction, apoptosis, autophagy, and inflammation, among others. Secondary injury cascades are thought to account for the development of many of the neurological deficits observed after TBI [7], and their delayed nature offers a therapeutic window for treatment to prevent, attenuate, or at least delay the resultant neurological deficits [8]. Although each secondary injury mechanism is often considered to be independent, most are highly interactive and may occur concomitantly. An increasing body of evidence suggests that reactive oxygen species (ROS)-induced oxidative stress may contribute to the pathophysiology of secondary damage after TBI [9, 10]. ROS initiate and perpetuate tissue damage through complex mechanisms, including excitotoxicity, metabolic failure, and disturbance of intracellular Ca2? homeostasis [11]. This damage could occur localized either in a focal area or diffusely, depending on the nature of the injury. The form whereby this damage could take place is through phenomena such as necrosis, apoptosis, and autophagy.

3 Oxidative Stress ROS and reactive nitrogen species (RNS) are families of highly reactive species formed either enzymatically or nonenzymatically in mammalian cells. Oxidative stress arises from an imbalance between the generation of these species due to increased pro-oxidant activity over the antioxidant defense system such that the latter becomes overwhelmed [12]. Some of the most important reactive species are superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH), nitric oxide (NO), and peroxynitrite. These are produced in physiological conditions, mainly as a byproduct of mitochondrial respiratory chain, NADPH oxidase, xanthine oxidase (XO), nitric oxide synthase (NOS), cyclooxygenase, lipoxygenase, and cytochrome p450, among others. On the other hand, antioxidant mechanisms are present in cells to remove ROS or even prevent their generation. The antioxidant potential of cells is determined by substances such as reduced glutathione (GSH), vitamin C, and vitamin E. In addition, antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), GSH peroxidases (GSH-Pxs), heme oxygenase (HO), and glutathione reductase (GR) are important in achieving ROS depuration. ROS exert cell damage either directly or through behaving as intermediates in diverse signaling pathways, including deoxyribonucleic acid (DNA) damage, protein carbonylation, and lipid peroxidation, which is the most studied mechanism of oxidative damage [13]. Lipid peroxidation refers to the oxidative degradation of lipids, and causes alterations in cell membrane fluidity and permeability, and a decrease in membrane ATPase activity, leading to cell damage [14]. It is a self-propagating chain reaction characterized by three distinct steps: (i) initiation, occurring when a free radical extracts a hydrogen atom from a polyunsaturated fatty acid (PUFA), converting it into a lipid radical; (ii) propagation, arising when the unstable lipid radical is oxidized to form a lipid peroxyl radical, which in turn is capable of reacting with another PUFA to produce a second lipid radical and a lipid hydroperoxide; and (iii) termination, when the lipid radical reacts with another radical or radical scavenger, giving rise to a stable non-radical end product. ROS involvement in both structural and functional cell damage leading to cell death is depicted in Fig. 1. Because of the short half-life of ROS, their direct measurement is difficult to perform in human subjects. The measurement of products formed by ROS attack to biomolecules, such as F2-isoprostanes, F4-neuroprostanes, isofuranes, malondialdehyde (MDA), 4-hydroxynonenal, 8-hydroxy-20 -deoxyguanosine, and protein carbonyl, are

Antioxidants in Traumatic Brain Injury Treatment

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Table 1 Pharmacological features of main antioxidant drugs tested to prevent traumatic brain injury-induced brain damage Compound

Mechanism of action

Brain penetration

Route/vehicle

Longest known therapeutic window

References

Melatonin

Free radical scavenging, antioxidant enzyme modulator. Improves efficiency of the mitochondrial electron transport chain

Fully crosses BBB. Transcellular lipophilic pathway

Intraperitoneal/0.5 % ethanol

1h

[127–136]

Mitochondriatargeted antioxidants (XJB-5-131)

Free radical scavenging, recycling and SOD-mimicking capacities

Fully crosses BBB

Intravenous/50:50, vol/ vol, cremophor EL:ethanol dissolved in saline (1:3)

5 min

[147, 148, 150]

Vitamin C

Water-soluble antioxidant, enzyme modulator

Ascorbic acid: SVCT2 transporter

Intravenous/0.9 % saline

Up to 8 h

[165, 166, 171, 172, 177]

Dehydroascorbate: GLUT family transporter Vitamin E

Lipid-soluble antioxidant, enzyme modulator

No studies available

Oral/vegetable oil

Up to 8 h

[177, 180, 181]

Apocynin

NADPH oxidase inhibitor

Fully crosses BBB

30 min

[184–189]

Hydrogen-rich saline

OH scavenging, antioxidant enzyme modulator

Fully crosses BBB. Paracellular aqueous pathway

Subcutaneous injection/ 5 % ethanol in saline solution Intraperitoneal/0.9 % saline

5 min

[192, 193, 202]

Sulforaphane

Nrf2 activator


Intraperitoneal/1 % dimethyl sulfoxide in 0.9 % saline

1h

[203–205]

U-83836E

ROS scavenging, lipid peroxidation inhibitor


Intravenousintraperitoneal/0.9 % saline

15 min

[206–208]

Resveratrol

HO-1 induction, ROS scavenging, PPARa activator, SIRT1 activator

Partially crosses BBB

Intraperitoneal/0.9 % saline

5 min

[212–216]

PycnogenolÒ

Free radical scavenging, binding of iron and copper, direct inhibition of prooxidative enzymes


Intravenous/6 % dimethyl sulfoxide in 0.9 % saline

4h

[222, 223, 226]

Epigallocatechin3-gallate

Free radical scavenging, binding of iron.

Fully crosses BBB

Oral/drinking water

Immediately after TBI

[226–229]

Wogonin

Free radical scavenging. NADPH, XO, and iNOS inhibitor


Intraperitoneal/30 % dimethyl sulfoxide

10 min

[230–236]

Omega-3 PUFAs

Ion channels modulation, PPAR activation, Nrf2-mediated survival response

Fully crosses BBB

Oral/fish oil

Up to 24 h

[239–241, 248– 256]

BBB blood–brain barrier, GLUT glucose transporters, HO heme oxygenase, iNOS inducible, NOS nitric oxide synthase, NADPH nicotinamide adenine dinucleotide phosphate, Nrf2 nuclear factor erythroid 2-related factor 2, OH hydroxyl radical, PPAR peroxisome proliferator-activated receptor, PUFA polyunsaturated fatty acid, ROS reactive oxygen species, SIRT sirtuin, SOD superoxide dismutase, SVCT sodium-dependent vitamin C transporter, TBI traumatic brain injury, XO xanthine oxidase

useful tools for oxidative stress assessment [15]. The more sensitive and early biomarkers of oxidative stress are lipid peroxidation products, such as F2-isoprostanes derived from non-enzymatic peroxidation of arachidonic acid. Because of their formation mechanism, specific structural features that distinguish them from other free radical-generated products, and their chemical stability, F2-

isoprostanes can provide a reliable index of lipid peroxidation in vivo in a variety of clinical settings associated with oxidative stress [16]. However, since docosahexaenoic acid (DHA) is the major PUFA in the brain, F4-neuroprostane levels in cerebrospinal fluid (CSF) are likely a more specific indicator of possible neurological dysfunction than F2-isoprostanes [17].

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Fig. 1 Involvement of ROS in damage to biomolecules. (i) Lipid peroxidation to membrane phospholipid polyunsaturated fatty acids: (a) initiation (b) propagation (c) termination. Products of lipid peroxidation: 4-HNE, F2-isoprostanes, MDA. Structural damage by lipid peroxidation products. (ii) Structural changes of proteins (e.g. carbonylation) leading to functional impairment and cell death. ROS damage to mitochondrial (iii) and nuclear DNA (iv), leading to mitochondrial dysfunction and cell death, respectively. ETC electron transport chain, Fe?2 ferrous iron, Fe?3 ferric iron, L1 first

polyunsaturated fatty acid, L2 second polyunsaturated fatty acid, L1 first lipid radical, L2 second lipid radical, LO lipid alkoxyl radical, LOO lipid peroxyl radical, LOOH lipid hydroperoxide, MDA malondialdehyde, mt mitochondrial, NAD? nicotinamide adenine dinucleotide, NO nitric oxide, OH hydroxyl radical, ONOO– peroxynitrite, O2- superoxide, PARP poly(ADP-ribose) polymerase, ROS reactive oxygen species, 4-HNE 4-hydroxynonenal, 8-OHdG 8-hydroxy-20 -deoxyguanosine

Recent studies have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor, plays an indispensable role in the induction of endogenous antioxidant enzymes. Upon exposure to ROS, Nrf2 translocates from the cytoplasm to the nucleus, where it sequentially binds to the antioxidant response element (ARE), inducing the production of many phase II detoxifying and antioxidant enzyme genes such as HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) [18, 19]. The Nrf2-ARE pathway is considered to be a multiorgan protector and is reported to play an important role in several central nervous system (CNS) diseases, including TBI [20– 23]. The CNS has some distinctive characteristics that increase therapeutic opportunities for antioxidant agents to reduce oxidative damage. In contrast to other cells, neurons are especially vulnerable to increases of both ROS and

RNS, due to low antioxidant enzyme activity, high O2 consumption, and high levels of iron all acting as prooxidants under pathological conditions [24, 25]. Furthermore, their high levels of PUFAs are a rich source for lipid peroxidation reactions, due to both the myelin content and the high membrane-to-cytoplasm ratio intrinsic to brain cells [26]. It is of interest to note that the optimal antioxidant effect is expected to be achieved by crossing the blood–brain barrier (BBB), giving rise to a direct cellular effect of antioxidants [27]. Nevertheless, BBB integrity is altered in patients with TBI, possibly allowing for higher drug concentrations compared with physiological conditions. In addition, effective antioxidation can be expected at the level of endothelial interface, which otherwise could be activated by ROS towards the production of chemokines, decreased availability of NO, down-regulation of PGI2 and endothelium-derived hyperpolarizing factor.


Evidence indicates that oxidative stress can cause a reduction in brain-derived neurotrophic factor (BDNF) and a subsequent decline in cognition and neuroplasticity [28]. BDNF modulation has been suggested as a common step in the effects of antioxidants on synaptic plasticity by conferring resistance to cognitive dysfunction after TBI [29]. 3.1 Oxidative Stress in TBI Interestingly, TBI is a significant factor in the overall antioxidant potential in humans. Several studies have reported increased levels of lipid peroxidation biomarkers in TBI patients, such as thiobarbituric acid reactive substances (TBARS) [30–32], protein carbonyls [30], and 8-iso-PGF2a [10, 33]. It is noteworthy that 8-iso-PGF2a has been demonstrated to be strongly correlated with oxidative stress and excitotoxicity [10], and that its plasma levels are highly associated with the 1-year clinical outcome [33]. Additionally, a recent case-controlled study revealed significantly increased CSF isofurans and F4-neuroprostanes in TBI patients, confirming that TBI results in increased oxidative stress [17]. Regarding ROS sources, mitochondrion is a major intracellular producer of ROS [34, 35] and is considered to be the major source following TBI [36]. However, recent studies in mice demonstrated a significant contribution of NADPH oxidase activity and superoxide production to pathology of TBI [37]. NADPH oxidase accounts for most of the non-mitochondrial ROS production as its inhibition almost completely prevents the oxidative damage after TBI. This enzyme activity increases in a biphasic manner, with observed peaks at 1 h and 24–96 h, which parallels elevations of O2-, allowing for a therapeutic window to intervene. A recent report suggested that NADPH oxidase also plays a critical role in microglia activation in the cerebral cortex at 24–48 h following TBI [38].

4 Secondary Injury Mechanisms and Oxidative Stress 4.1 Excitotoxicity Excitotoxicity is widely recognized as an important process in secondary damage and cell death following acute brain injury and has been associated with increased levels of oxidative stress-related biomarkers [10]. The high glutamate quantities binding to the N-methyl D-aspartate (NMDA) receptor promote substantial calcium (Ca2?) influx. In turn, Ca2? overload can trigger many downstream neurotoxic cascades, including the uncoupling mitochondrial electron transfer from ATP synthesis, and the activation and overstimulation of enzymes, such as calpains, protein kinases, endonucleases, NOS and

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NADPH oxidase [39, 40]. Neuronal production of NO and O2- is recognized as a crucial step linking NMDA receptor overstimulation to neuronal cell death, even in neighboring neurons and astrocytes [41–45]. Despite NMDA-receptor antagonists protecting neurons in experimental settings, these agents have not demonstrated therapeutic efficacy in in vivo models [46–48]. Therefore, understanding the mechanisms of glutamate toxicity beyond the initial stimulation of Ca2? influx, such as increased ROS production, is important for developing efficient neuroprotection strategies. 4.2 Mitochondrial Dysfunction Mitochondrial dysfunction is a leading event occurring in the cascade of apoptosis and necrosis pathways, leading to cell death in TBI. Patients with TBI and profound mitochondrial impairment have a poor prognosis [49], and the magnitude of the deficit in cerebral energy metabolism after TBI has been shown to be the best predictor of outcome [50, 51]. In response to several ROS-producing stimuli, such as Ca2?, H2O2, peroxynitrite generation, NAD/NADH ratio or pH, mitochondrion assumes a high-conductance state that allows the deregulated entry of small solutes into the mitochondrial matrix [52], even several days after the TBI event. Consequently, the mitochondrial permeability transition generates disruption of its electrical membrane potential [53]. This causes disturbance of the electron transport chain, leading to further ROS increase, osmotic swelling, and rupture of the outer membrane, triggering caspase cascade activation and ultimately cell death [54]. Oxidative mitochondrial DNA (mtDNA) damage also plays an important role in mitochondrial dysfunction. mtDNA lacks introns and, being close to an ROS source, is prone to oxidative damage. Because mtDNA encodes 13 polypeptides of the respiratory chain, its damage decreases respiratory function, thus enhancing ROS generation and causing a vicious cycle of ROS–mtDNA damage [55, 56]. Ultimately, mitochondria seem to provide a common pathway of three major forms of cellular death: apoptosis, necrosis, and autophagy [57–65]. 4.3 Apoptosis TBI triggers a complex cascade of apoptotic events that cause delayed tissue damage. Several apoptotic signaling pathways are modulated by redox status [66]. Indeed, specific ROS such as H2O2 or O2- have been implicated as critical mediators of apoptotic cell death [67, 68]. Apoptotic morphologies have been reported in post mortem analysis of brain tissue of patients surviving up to


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12 months post-TBI [69], indicating a comparatively wide therapeutic window for intervention. Initiation of apoptosis may occur through extrinsic or intrinsic pathways. As a global effect over the extrinsic pathway, ROS mediate death receptor activation and apoptotic induction through ROS-induced receptor clustering and formation of lipid-raft-derived NADPH oxidase platforms [70–73]. In particular, NADPH oxidase-dependent H2O2 and O2- generation has been recently considered a key regulatory mechanism of Fas-induced apoptosis through FLIP down-regulation [74]. Moreover, whether TNFR1 activation induces anti- or proapoptotic effects depends on the level and duration of Jun N-terminal protein kinase (JNK) activation by ROS [75]. Intrinsic apoptosis is caused by the release of mitochondrial intermembrane space proteins, through caspasedependent and independent pathways. Previously discussed ROS-stimulated mitochondrial membrane permeabilization is considered a critical step in the release of pro-apoptotic proteins such as cytochrome c, Smac/Diablo, apoptosisinducing factor (AIF) or endonuclease G [76, 77]. 4.4 Autophagy Autophagy is a caspase-independent process in which cellular proteins and organelles are sequestered in doublemembrane vesicles known as autophagosomes, delivered to lysosomes, and ultimately digested by lysosomal hydrolases [78, 79]. It is controlled by genes from the autophagyrelated protein (ATG) family [80] and occurs after both experimental and clinical TBI [81, 82]. Oxidative stress has been reported to serve as an important autophagy stimuli during periods of nutrient deprivation, ischemia/reperfusion, hypoxia, cell stress, and in TBI [83–86]. ROS are also essential for physiological autophagy to proceed [87]. During cellular starvation and nutrient deprivation, there is an increased generation of mitochondrial-derived H2O2 through a phosphoinositide 3-kinase (PI3K)/beclin-1 pathway [83], leading to oxidation and consequent inhibition of ATG4, ultimately promoting autophagosome maturation. Increased ROS also activate the ubiquitinproteosome system, degrading Bcl-2 [88, 89], thus allowing for beclin-1 promotion of autophagic cell death [90]. Additionally, antioxidant enzymes such as CAT and SOD are targeted by autophagosomes, thereby increasing ROS presence and creating a positive autophagic feedback loop, leading to cell death [83]. 4.5 Edema Cerebral edema often leads to elevations in intracranial pressure, local hypoxia and ischemia, herniation, and

subsequent neuronal cell death via necrosis and apoptosis. Increased intracranial pressure has in fact been singled out as one injury factor that closely predicts outcome following severe brain injury [91, 92]. Vasogenic and cytotoxic edema are the most clinically relevant types of edema in TBI. Vasogenic edema is caused by a weakened BBB mediated by ROS, mostly through the activation of matrix metalloproteinases (MMPs) that degrade collagen and laminin in the basal lamina and disrupt the integrity of the vascular wall, increasing BBB permeability. NO, a seemingly important molecule in MMP activation, has been suggested to regulate MMPs via direct activation or up-regulation of MMP-9 [93, 94]. In contrast, cytotoxic edema is characterized by intracellular swelling of neuronal, glial, and endothelial cells in the absence of any measurable breakdown of the BBB [95]. Mitochondrial dysfunction has been proposed as the main cause of propagation of cytotoxic brain edema [96, 97]. It has been proposed that ion transporters responsible for cellular swelling can be modulated by ROS, through the peroxidation of membrane phospholipids, oxidation of sulfhydryl groups, and protein modification [98]. Excitotoxicity may also contribute, promoting intracellular accumulation of sodium (Na?) [99]. In addition, oxidative stress also supports cytotoxic edema formation through interactions with other mediators of cellular swelling as occurs in lactic acidosis, triggering the release of iron from its binding site. This enhances OH formation by increasing intracellular H2O2 levels and by supplying protons during the chain reaction between O2- and NO [100]. 4.6 Inflammation Inflammation is considered critically important in TBI [101]. Both primary and secondary insults activate the release of cellular mediators, including pro-inflammatory cytokines, prostaglandins, free radicals, and complement. Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b and IL-6 are up-regulated within hours of injury [102, 103]. Moreover, recent data provide evidence for the cerebral production of these cytokines and show a stereotyped temporal pattern after TBI [104]. These processes induce chemokines and adhesion molecules that in turn mobilize immune and glial cells in a concurrent and synergistic way. BBB disruption, partially mediated by ROS, allows systemic immune cells to enter the CNS compartment, supporting the local inflammatory response [105, 106]. Because compromised BBB function may facilitate the acute inflammatory response after TBI, it may well serve as a target for anti-inflammatory drug development [107].


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Fig. 2 Role of oxidative stress in the pathological processes accounting for brain damage in traumatic brain injury. ROS play a key role not solely in the development of neuronal death, but also as mediators of other deleterious pathways (e.g. excitotoxicity, mitochondrial dysfunction, inflammation, autophagy, apoptosis). ATG4 autophagy-related protein 4, BBB blood-brain barrier, CAT catalase,

ETC electron transport chain, FAS fas cell surface death receptor, ICP intracranial pressure, mPTP mitochondrial permeability transition pore, mtDNA mitochondrial DNA, NADPH reduced nicotinamide adenine dinucleotide phosphate, NF-jB nuclear factor-jB, NOS nitric oxide synthase, ROS reactive oxygen species, SOD superoxide dismutase, TNFR1 tumor necrosis factor receptor 1

Infiltrating neutrophils and microglia/macrophages may generate toxic levels of NO mainly via the inducible NOS (iNOS) isoform. However, the activity of neuronal NOS (nNOS) [108] and NADPH oxidase expressed at the plasma membrane of microglia can also contribute to bursts of ROS generation [109]. Local ROS-induced expression of cytokines from astrocytes also mediates the activation of microglia [110]. Furthermore, NO is responsible for cytotoxicity through the inhibition of adenosine triphosphate (ATP)-producing enzymes and stimulation of pro-inflammatory enzymes such as cyclooxygenase-2 [43, 111]. Inflammatory cells generate ROS and RNS, which, in turn, are capable of activating these same inflammatory cells through several mechanisms, including ROS activation of nuclear factor (NF)-jB. NF-jB appears to play a major role in perpetuating the immune response to TBI, and is known to increase genetic transcription of proinflammatory mediators such as TNFa and IL-6 [112]. It is important to remember that the balance of the inflammatory and immune response under sufficient

homeostatic control promotes healing and repair. Taken together, the varied actions of inflammatory cells support the concept that the immune response is a two-edged sword [112, 113].

5 Antioxidant Strategies in TBI TBI has challenged most conventional measures aimed at improving survival, quality of life, and neurological recovery, thus creating a substantial need for further research on agents that are able to significantly reduce brain damage. There is considerable evidence supporting the role of oxidative stress as an initiator and perpetuator of pathological processes in secondary brain injury (Fig. 2). According to this evidence, acute antioxidant enhancement has been successfully used to reduce brain damage, even in the subacute phases of TBI as discussed below. Nevertheless, efforts should focus on therapeutic strategies more likely to succeed, such as those aimed at reducing mitochondrial dysfunction and inhibiting the major

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non-mitochondrial ROS source NADPH oxidase. Consequently, we categorize antioxidant drugs in terms of their cellular target as follows. Drugs having major effects other than that of antioxidants, for instance minocycline and estrogen, were not analyzed.


In summary, melatonin administration in appropriate doses appears to be a safe, physiologic, multifunctional, and potentially highly effective strategy for reducing the deleterious effects of TBI in humans. 5.1.2 Mitochondria-Targeted Antioxidants

5.1 Antioxidants Primarily Protecting Mitochondria 5.1.1 Melatonin Melatonin, a metabolite of the essential amino acid tryptophan, is a pleiotropic compound that exerts multiple physiological actions, including those derived from its antioxidant properties [114]. It is secreted by the pineal gland and fortifies the antioxidant system by scavenging free radicals, stimulating antioxidant enzymes and the synthesis of GSH, protecting antioxidant enzymes from oxidative damage and augmenting the efficiency of the mitochondrial electron transport chain [115–119]. Interestingly, it has a lower side-effect profile and produces fewer pharmacokinetic and pharmacodynamic interactions than xenobiotic antioxidants [114, 120–122]. Due to its amphiphilic structure, melatonin has no barriers to its distribution. Moreover, the highest levels of intracellular melatonin are found within mitochondria, being able to interact with lipid membranes and stabilize the mitochondrial inner membrane [123], making melatonin a highly potent antioxidant specifically in protecting against mitochondrial oxidative damage [122, 124–126]. Melatonin treatment at different doses (10–100 mg/kg) reduces brain lipid peroxidation induced by various free radical-generating agents and processes such as iron, ischemia-reperfusion, radiation, and painful stimulation [127–130]. In experimental TBI models, melatonin increases brain antioxidant levels, decreases NF-jB activation, and improves cognitive function [131–133]. Additionally, it can significantly decrease brain edema, BBB permeability, and intracranial pressure at 72 h after TBI [134]. One combinational treatment of low-dose melatonin (5 mg/kg) and minocycline (40 mg/kg) failed to improve functional and histopathological impairments following TBI in a rat model [135]. However, a recent therapy with melatonin (10 mg/kg) and dexamethasone (0.025 mg/kg) was able to attenuate (i) the amount of lesion volume, (ii) the loss of motor function, (iii) the expression of proapoptotic proteins, (iv) iNOS, and (v) MMP in a rat model [136]. In patients suffering TBI, a study outlining the modifications of melatonin revealed that endogenous CSF melatonin concentrations increased post-TBI [137]. This may be explained by the possibility that deleterious oxidative stress resulting from TBI induces a physiologic endogenous response, stimulating production of this antioxidant.

Since mitochondria are the main source and target of ROS, antioxidants that act in mitochondria might be more effective than other antioxidants [119, 138]. However, the inner membrane is highly impermeable to most molecules. Interestingly, peroxidation of the inner membrane cardiolipin produces a high diversity of oxygenated products that seem to be required to activate the neuronal death program [139–146]. Mitochondria-targeted antioxidants are a family of membrane-penetrating cations that specifically accumulate in the inner mitochondrial membrane [147] due to its negative charge [148]. This family of antioxidants includes stable nitroxide radicals, which have the ability to combine radical scavenging action with recycling and SOD-mimicking capacities. Tempol, a representative of this group, has been shown to improve motor function in a TBI rat model; however, high millimolar concentrations were required to achieve this improvement [149]. It has been found that the new mitochondria-targeted antioxidant XJB-5-131 (a conjugate of 4-amino TEMPO) effectively accumulates in mitochondria and crosses the BBB. This leads to decreased accumulation of cardiolipin oxidation products as well as substantial reduction in neuronal death both in vitro and in vivo, and significantly reduced behavioral deficits and cortical lesion volume [150]. MitoQ, a ubiquinone derivative cation, has also demonstrated promising results in neuroprotection [151, 152]. MitoQ decreased oxidative stress, synaptic loss, and astrogliosis in a transgenic mouse model of Alzheimer’s disease, as well as prevented caspase-3 and -7 activation [153]. Moreover, Skulachev [154] found that mitochondriatargeted plastoquinone-derivatives (SKQ1 and SKQR1) interrupt the cardiolipin peroxidation chain reaction by already formed ROS, and, at higher concentrations, lower the rate of ROS formation in the respiratory chain due to mild uncoupling. The latter can increase proton leakage, thereby reducing oxygen stress by lowering the mitochondrial electrical gradient [155–160]. Furthermore, because the mitochondrial electrical gradient favors cytoplasmic Ca2? entry, a mild mitochondrial uncoupling can attenuate mitochondrial Ca2? cycling [161, 162]. Plastoquinonederivatives are also rechargeable antioxidants, which are reduced in the respiratory chain complex III [163, 164]. Mitochondria-targeted antioxidants are a new strategy to reduce ROS overproduction, and could have significant advantages over more conventional approaches.


5.2 NADPH Oxidase Inhibition 5.2.1 Vitamin C Vitamin C (ascorbic acid, ascorbate) is a potent watersoluble antioxidant in humans. It also behaves as an enzyme modulator, causing the up-regulation of endothelial NOS (eNOS) and down-regulation of NADPH oxidase [165]. Even more, ascorbate in aqueous compartments can recycle vitamin E in membranes by reducing the a-tocopheroxyl radical back to a-tocopherol [166]. Given the lipid-rich environment of the brain, the sparing or recycling of a-tocopherol may be a very important role of ascorbate. Vitamin C plasma concentrations are approximately 80 lmol/L or less following oral administration, with peak values not exceeding 220 lmol/L, even after maximum doses of 3 g six times daily (18 g total) [167]. In contrast, intravenous vitamin C use sustains plasma concentrations at levels as high as 15 mM [168]. As O2- reacts with NO at a rate 105-fold greater than the rate at which O2- reacts with ascorbic acid, a 10 mM concentration of ascorbate is needed to support its competition with NO for O2- [169]. Therefore, vitamin C plasma concentrations high enough to efficiently scavenge ROS can only be achieved by intravenous supplementation. On the other hand, plasmatic concentrations of vitamin C that inhibit NADPH oxidase (10–100 lmol/L) [165] can be achieved both orally and intravenously. Remarkably, single doses of vitamin C as high as 200 g do not produce complications except in cases of severe renal impairment or glucose-6-phosphate dehydrogenase deficiency [170]. Specific ascorbate transporters generate the trans-plasma membrane gradient of ascorbate, which is mainly accumulated in the brain [171] in part by the sodium-dependent vitamin C transporter 2 (SVCT2). Moreover, neurons can also take up dehydroascorbate, the two electron-oxidized form of ascorbate, by facilitated diffusion of the ubiquitous glucose transporters of the GLUT family [172]. Antioxidant and pharmacodynamic properties allow ascorbate to protect neurons from NMDA-induced excitotoxicity [173, 174] and to prevent lipid peroxidation induced by various oxidizing agents [175], especially in combination with a-tocopherol, in cell cultures [176]. However, to our knowledge, there are no TBI rat models testing the effects of vitamin C. Interestingly, there is one clinical trial of vitamin C performed in patients with severe TBI and the radiologic diagnosis of diffuse axonal injury [177]. Patients received a total dose of 32 g of intravenous vitamin C during the first 7 days after TBI, with a maximum single dose of 10 g on the first day, resulting in a significant earlier stabilization of the perilesional edema compared with the placebo group. Although the absence of many parameters of clinical

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importance and monitoring techniques may make the interpretation of these results challenging, they are encouraging results regarding the use of vitamin C in humans. Nevertheless, it is important to mention that given the metabolism of ascorbate, doses administered on different days cannot be considered accumulative [178], and the higher single dose used in this study was not high enough to achieve plasmatic concentrations above 10 mM. It is important to mention that, even when vitamin C transporters may be able to achieve millimolar concentrations in neurons in vivo in physiological conditions, TBI has been demonstrated to decrease the plasmatic levels of vitamin C [179], suggesting a role for vitamin C supplementation in head trauma. Furthermore, because vitamin C transport through the SVCT is supported by the Na? electrical gradient, which is altered in patients with TBI, its ability to maintain high levels of intracellular ascorbate may be reduced. Therefore, vitamin C is a promising and remarkably safe alternative for neuroprotection after TBI, and further studies, in particular the testing of intravenous administration of this vitamin, are strongly encouraged. 5.2.2 Vitamin E Vitamin E, mainly a-tocopherol, is the major peroxyl radical scavenger in biological lipid phases such as cell membranes [180, 181], and its antioxidant mechanism is related to the inhibition of lipid peroxidation and NADPH oxidase [165]. a-tocopherol can be restored by reduction of the a-tocopheroxyl radical with redox-active reagents such as vitamin C or ubiquinol [182]. A recent study in a TBI rat model established that vitamin E dietary supplementation (500 IU/kg during 4 weeks before TBI) protects the brain against the effects of mild TBI on long-term synaptic plasticity and cognition, influencing molecular systems such as BDNF and Sir2 [29]. In rats, a-tocopherol reduced the expression of neurite outgrowth inhibitor-A and its receptor, and improved the recovery of animal nerve function after TBI [183]. One clinical trial of vitamin E has been performed in patients with severe TBI and the radiologic diagnosis of diffuse axonal injury [177]. For 7 days, patients received vitamin E at 400 IU/day intramuscularly, which resulted in improved clinical outcome and reduced mortality at discharge. Despite promising results, clinical trials testing vitamins C and E, isolated or in combination, are needed to change current therapeutic measures in patients suffering TBI. 5.2.3 Apocynin Apocynin is a natural organic compound isolated from the medicinal herb Picrorhiza kurroa. It is the most commonly


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used NADPH oxidase inhibitor and exerts its function in neutrophils [184–186], macrophages, and endothelia [184] through the inhibition of p47phox subunit translocation to the membrane. A recent study revealed that pretreatment with 100 mg/ kg of apocynin prevented ROS production, thus reducing microglial activation, BBB disruption, and neuronal death after weight-drop brain injury in rats [187]. In another study, treatment with apocynin 30 min before TBI reduced brain edema and improved spatial learning function assessed using the Morris water maze [188]. Recently, Ferreira et al. [189] demonstrated that treatment with apocynin (5 mg/kg) 30 min and 24 h after TBI improved cognition impairment, and reduced plasma inflammation biomarkers and lesion volume. There is limited evidence regarding the role of apocynin in neurological consequences of TBI and no clinical trials have been performed in TBI patients. Moreover, apocynin should be used with caution in humans because it may not be specific for NADPH oxidase, rather functioning as a non-specific antioxidant, also inhibiting Rho kinase inhibitor [190, 191]. 5.3 Other Antioxidant Strategies 5.3.1 Hydrogen-Rich Saline Molecular hydrogen [H2], recognized as a novel antioxidant for preventive and therapeutic applications, scavenges OH in living cells [192], decreases lipid peroxidation, and directly induces the expression of CAT and SOD [193]. Scavenging OH serves as a critical detoxification process as there is no physiological system known to accomplish this function [194]. The principal advantages of H2 as a potential antioxidant are (i) its ability to penetrate biomembranes and diffuse into the cytosol, (ii) its penetration through the BBB, (iii) its low cytotoxicity even at high concentrations [195–198], and (iv) its smaller adverse effects than those of other antioxidants [199]. Moreover, the administration of H2 via an injectable hydrogen-rich saline (HS) is accurate and safe [200, 201]. In a TBI rat model, HS significantly reduced oxidative stress, increased levels of Sir2, and counteracted cognitive decay (possibly through BDNF-mediated synaptic plasticity) [202]. HS also dose-dependently attenuated the increase of BBB permeability, brain edema, and lesion volume in TBI-challenged rats [193]. To date, there are no clinical trials testing the effect of HS in TBI. Even though this is a potent strategy to be tested, the relationship between H2 concentration and its beneficial effects has yet to be elucidated in preclinical studies.

5.3.2 Sulforaphane Sulforaphane, a naturally occurring isothiocyanate abundant in cruciferous vegetables, is well known for its chemopreventive and antioxidant properties. Although the molecular targets of this molecule are not completely characterized, the best-known effect of sulforaphane is to induce Nrf2-dependent gene expression [203]. In vitro studies demonstrated that sulforaphane can disrupt the Nrf2/Keap1 interaction, leading to Nrf2 stabilization and nuclear localization and the expression of ARE-containing phase II genes, which play a major role in the detoxification of ROS produced by xenobiotics [19]. It has been demonstrated that sulforaphane attenuates BBB permeability, reduces cerebral edema, and improves cognitive function after TBI [203, 204]. Behavioral improvements were only observed when the treatment was initiated 1 h post-injury but not after 6 h post-injury. Hong et al. [205] conducted a recent study aimed at determining whether a causal relation exists between the Nrf2-ARE pathway activation by sulforaphane and its antioxidant effect in TBI in a rat controlled cortical impact (CCI) model. In Nrf2-knockout mice, oxidative damage was exacerbated after TBI and the protective effects of sulforaphane were non-existent, which is consistent with a specific role for Nrf2. These studies support the use of sulforaphane in the treatment of TBI, and the fact that Nrf2 activation may be a prime candidate for the attenuation of oxidative stress and subsequent neurotoxicity. Nevertheless, considering the narrow time window of sulforaphane use observed in preclinical studies, the success of sulforaphane in clinical settings is uncertain. 5.3.3 U-83836E U-83836E is a synthetic ROS scavenger that allows for further inhibition of lipid peroxidation. In a TBI animal model, U-83836E attenuated cortical oxidative damage and preserved mitochondrial function following TBI, including aerobic respiration and Ca2?buffering capacity [206]. In a recent study, U-83836E produced a dose-related attenuation of post-traumatic calpain-mediated cytoskeletal (a-spectrin) proteolysis [207] by preserving the neuronal intracellular Ca2? homeostatic mechanisms that are compromised after TBI [208]. It is important to note that published clinical studies have largely validated the use of a-spectrin degradation and its proteolytic fragments in CSF as biomarkers, and the levels of these markers appear to correlate with TBI diagnosis, Glasgow Coma Scale score and, most importantly, outcome [209]. Further studies to determine the pharmacokinetics of U-83836E in humans are strongly


recommended, as the compound appears to inhibit a crucial step of the secondary brain injury cascade of events. 5.3.4 Resveratrol Resveratrol, a powerful polyphenol, has been observed to scavenge ROS and prevent lipid peroxidation in several pathologic conditions by its antioxidant activity [210, 211], and peroxisome proliferator-activated receptor (PPAR)a activation, which is recognized to have anti-inflammatory actions. In vitro studies have demonstrated that resveratrol induces HO-1, counteracting the intracellular increase of heme, a pro-oxidant agent [212, 213]. Furthermore, animal studies have revealed that resveratrol may be beneficial due to activation of the SIRT1 pathway [214], which has been observed to proceed in a substrate sequence-selective way [215]. In TBI rat models, resveratrol 100 mg/kg diminished lipid peroxidation and cerebral edema, reduced XO and NO levels, elevated glutathione after TBI [211], and improved motor performance and visuo-spatial memory [216]. A recent study revealed that resveratrol-treated animals present a reduction in microglial activation and brain levels of IL-6 and IL-12. These findings support the strong participation of the anti-inflammatory properties of resveratrol in the reduction of secondary brain injury after experiencing TBI [217]. Given the encouraging results in animal models, further studies testing the effect of resveratrol on TBI in humans are warranted. 5.3.5 Flavonoids Flavonoids are the most common group of polyphenolic compounds in the human diet [218]. They possess neuroprotective properties in part due to their free radicalscavenging properties and their ability to modulate intracellular signals [219–221]. Several flavonoids, generally water-soluble compounds with low affinity for mitochondria, have traversed the BBB in relevant in vitro and in situ models [220]. 5.3.5.1 Pycnogenol Pycnogenol is a patented combinational bioflavonoid [222] containing three main antioxidant mechanisms: (i) free radical scavenging; (ii) binding of iron and copper, and (iii) direct inhibition of pro-oxidative enzymes such as lipoxygenase, NOS, and XO [223]. One in vivo model testing Pycnogenol in moderate TBI reduced the levels of oxidative stress and neuroinflammation and increased the levels of key synaptic proteins [224]. In a more clinically applicable approach, it has also been demonstrated that a single intravenous treatment of

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Pycnogenol (1, 5, or 10 mg/kg) is neuroprotective after TBI, with a therapeutic window of at least 4 h after the trauma [225]. Pycnogenol is a reasonable drug to be considered as a potential therapeutic agent for TBI in humans. 5.3.5.2 Epigallocatechin-3-Gallate EGCG is the main green tea polyphenol, accounting for more than 10 % of the extract dry weight, and is able to prevent and/or reduce the deleterious effects of ROS, such as O2- and OH [226]. Moreover, EGCG immediately crosses the BBB after its administration [227, 228]. In a rat model, different EGCG schemes reduced ROS production, DNA damage, and lipid peroxidation after TBI [229]. EGCG increased bcl-2 and decreased expression of NF-jB in neurons, suggesting an anti-apoptotic, anti-autophagic, and anti-inflammatory effect. These findings support the idea that EGCG could reduce neuronal damage if used after the occurrence of TBI. Hence, EGCC is a new possible therapeutic agent for patients suffering head trauma. 5.3.5.3 Wogonin Wogonin, found in the root of the Chinese herb Scutellaria baicalensis Georgi, is widely used in treating allergic and inflammatory diseases [230]. In addition to the flavonoid’s neuroprotective properties, wogonin is also a potent enzymatic inhibitor of NADPH oxidase [231], XO [232], and iNOS [233–235]. In a TBI rat model, wogonin reduced inflammation and improved long-term sensory-motor recovery after TBI. Wogonin also reduced cortical contusion volume (significant decrease of lesion size of 24.2 %), BBB permeability, and brain edema after TBI [236]. To our knowledge, this promising study is the only one evaluating the effects of wogonin in a TBI model. Wogonin may also be considered among NADPH oxidase inhibitors, being one of the most rational therapeutic approaches for TBI treatment. 5.3.6 Omega-3 Omega-3 PUFAs are essential for brain development [237] and play a crucial role in brain function. Conversely, omega-6 PUFA metabolism leads to the production of proinflammatory products. Since there is competition between omega-3 and omega-6 metabolisms, omega-3 availability in the organism strongly depends on the dietary omega6:omega-3 ratio, which is higher than recommended in many countries around the world [238]. The main omega-3 forms are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). DHA is the major form in the CNS, representing 10–20 % of the total fatty acid composition and has been shown to possess neuroprotective effects, having numerous specific targets, including ion channels, nuclear receptors, and G-protein coupled receptors [239–

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241]. Studies showed that omega-3 fatty acids are neuroprotective against excitotoxic processes [242–244] and also possess well recognized antioxidant and anti-inflammatory properties [245–247]. On the other hand, omega-3 PUFAs are highly prone to lipid peroxidation, increasing ROS in a regulated fashion. Furthermore, chronic exposure to low-to-moderate ROS levels, which is expected after continued omega-3 administration, is thought to induce an Nrf2-mediated survival response [248]. Two recent studies in TBI rat models evaluated acute omega-3 supplementation with 10 and 40 mg/kg/day doses of omega-3 preparation (EPA:DHA = 2:1) during 30 days after head injury, which resulted in reduced axonal damage and caspase-3 levels [249, 250]. In a new combined approach using exercise and DHA supplementation after TBI, this treatment optimally counteracted the effects of TBI on cognitive function and several parameters of synaptic plasticity, as well as plasma membrane homeostasis [251]. Recent studies also demonstrated that omega-3 pretreatment (during 30 days) reduced oxidative stress and energetic impairment after TBI [252, 253], with behavioral improvement and reduced axonal dysfunction, microglial activation, and caspase-3 levels at doses of 40 mg/kg/day [254]. In addition, Pu and colleagues [255] recently demonstrated for the first time that omega-3 supplementation during 60 days prior to TBI protects against white matter injury in vivo and in vitro. It is noteworthy that dietary supplementation with omega-3 PUFAs in humans is extremely safe and can be used in acute phases of TBI as well as a prophylactic measure [256]. Given these favorable results and pharmacological profile of omega-3 PUFAs, additional studies in humans are warranted. Alternatively, other therapies based on the combined use of hyperbaric and normobaric hyperoxia have been used in severe TBI, reporting significantly improved biomarkers of oxidative metabolism in relatively uninjured brain and pericontusional tissue, as well as reduced intracranial hypertension [257]. It is likely that the mechanism whereby this neuroprotection occurs is related to the Nrf2 pathway, as increased ROS are expected following hyperbaric hyperoxia, allowing for cytoprotective activation mediated by this pathway. Therefore, antioxidant therapies should not be administered together with hyperoxia, otherwise blunting the cytoprotective mechanism. 5.4 Translational Limitations of Experimental TBI Treatments Currently, there is no neuroprotective agent that has been demonstrated in a large phase III clinical trial to improve neurological outcome. Despite the encouraging preclinical


results, more than 30 phase III prospective clinical trials have failed to display significance for their primary outcome [258–260]. Most of these studies of putative neuroprotective agents have been focused on a single component of a complex cascade of injury, which is not appropriate in a multifactorial process [261–263]. As previously explored, antioxidant drugs meet the theoretical requirements to be potentially effective therapies in TBI. Despite this promise, in the past decades, three large phase III clinical trials testing the effect of the antioxidants PEG-SOD [264], tirilazad [265], and dexanabinol [266] produced disappointing results. Nevertheless, a post hoc analysis showed that tirilazad reduced mortality in males with subarachnoid hemorrhage from 43 % in placebo-treated to 34 % (p \ 0.026), which is consistent with previously proved efficacy of tirilazad in animal models [267] as well as in aneurysmal subarachnoid hemorrhage trials [268, 269]. However, tirilazad development was discontinued and no additional trials confirmed its neuroprotective effect. Patients often present more than one abnormality or injury type due to neurotrauma, which is difficult to replicate in animal TBI models [270]. A method of precise definition and classification of TBI, incorporating patientspecific biomechanics and neurochemical factors, is necessary to improve further investigations. Moreover, disparities in TBI management may influence trial results. There are many other critical differences between clinical and preclinical studies that have potential relevance for translation, such as (i) the influence of anesthetic drugs (used in TBI models) on the primary outcomes of the study, (ii) potential drug/anesthetic interactions [271], (iii) methodological analysis of results, (iv) the use of genetically identical populations in TBI models, and (v) the time course of pathophysiological and therapeutic effects, among others. However, the latter is one of the most important contributions from experimental approaches, improving the knowledge of molecular processes, therapeutic windows, pharmacological properties, and time suitability of different drugs (Table 1). TBI is commonly associated with alterations in drug pharmacokinetics [272] that are even likely to vary among TBI patients. In addition, alterations in BBB integrity, albumin, and other serum protein concentrations, and the metabolic capacity of the cytochrome P450 and other hepatic enzyme systems and immune response after TBI, may alter the concentration–time profile or tissue concentrations that might be typical of a non-TBI patient [272, 273]. Unfortunately, robust descriptions of therapeutic windows, pharmacokinetics, and pharmacodynamics for new agents are often not available. Consequently, the use of well known antioxidants such as vitamin C, vitamin E,


melatonin, and polyphenols represent a valuable therapeutic alternative.

6 Concluding Remarks and Perspectives TBI is an increasing public health problem, constituting the most important cause of disability in individuals under the age of 45 years. It results in both primary and secondary injuries and is commonly associated with systemic lesions. Secondary injury involves a complex cascade of consecutive pathological processes initiated at the moment of head trauma. As oxidative stress is a central event in the damaging pathways involved in TBI, it should be considered in potential therapeutic implications. Currently, there is no neuroprotective agent that has been demonstrated in a large phase III clinical trial to improve neurological outcomes. Efforts should focus on developing novel strategies, with thorough preclinical studies and clinical trials that consider the translational barriers of a heterogeneous pathology such as TBI. To date, antioxidant vitamins have suggested beneficial effects in patients with severe TBI and the radiologic diagnosis of diffuse axonal injury [177]. In addition, the synthetic lazaroid tirilazad has proved to be effective in severely injured males with subarachnoid hemorrhage [265]. However, as evidence is limited, no recommendations of particular antioxidants can be made regarding characteristics of patients, trauma mechanism, or clinical severity. From a mechanistic viewpoint, the recommendation for further research should be made on the basis of existing results regarding the two main therapeutic targets related to oxidative stress cell damage. First, for NADPH oxidase inhibition: high-dose vitamin C and vitamin E arise as noteworthy alternatives for treatment, since they have demonstrated safety and have produced promising results in clinical studies derived from the knowledge of their pharmacological properties. Second, for functional stabilization of the mitochondrial electron transport chain complexes: (i) melatonin, which is a well known, safe, physiologic, multifunctional, and possibly highly effective strategy for TBI according to preclinical studies, and (ii) the new mitochondria-targeted antioxidants. The latter being emerging compounds that have been successfully tested in other pathological models, with outstanding theoretical properties to reduce secondary brain damage in TBI. Furthermore, we must not forget that many studies of putative neuroprotective agents have failed mostly because they focused on a single component of a complex injury cascade. On the other hand, it is likely that the use of pleiotropic drugs such as antioxidants, and the combination

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of the aforementioned mechanistic recommendations, may improve the neuroprotective effect and increase the therapeutic window of drug therapies. The potential use of unconventional treatments, such as antioxidant defense system reinforcement, could play a key role in the management of these patients. This reinforcement appears as a safe, low-cost and multifunctional novel therapeutic approach in TBI patients. As previously explained, antioxidant therapies are effective over a long period of time, allowing for suitable use in clinical settings. Due to the encouraging preclinical results and the antioxidant drug profiles, acute antioxidant reinforcement is emerging as a highly cost-effective alternative for neuroprotection in TBI patients. However, further studies in humans are needed in order to allow the full establishment of the effectiveness of these therapies. Acknowledgments

No funding declared.

Conflict of interest

Authors declare no conflicts of interest.

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Neuroprotective strategies for traumatic brain injury: improving clinical translation.

Perspectives on molecular biomarkers of oxidative stress and antioxidant strategies in traumatic brain injury.

Compensatory strategies for people with traumatic brain injury.

Endogenous neurogenesis following ischaemic brain injury: insights for therapeutic strategies.

Acute Rehabilitation in Traumatic Brain Injury.

Imaging Evaluation of Acute Traumatic Brain Injury.

Extracellular ezrin: a novel biomarker for traumatic brain injury.

A novel traumatic brain injury model for induction of mild brain injury in rats.

Thaliporphine derivative improves acute lung injury after traumatic brain injury.

Neurostimulation for traumatic brain injury.

Therapeutic benefits of phosphodiesterase 4B inhibition after traumatic brain injury.

Erythropoietin for traumatic brain injury.

Plasma copeptin level predicts acute traumatic coagulopathy and progressive hemorrhagic injury after traumatic brain injury.

Effects of Patient Preinjury and Injury Characteristics on Acute Rehabilitation Outcomes for Traumatic Brain Injury.

Novel therapeutic strategies for ischemic heart disease.

Novel therapeutic strategies for multiple myeloma.

New antioxidant drugs for neonatal brain injury.

Neurosensory Symptom Complexes after Acute Mild Traumatic Brain Injury.

[Acute treatment of patients with severe traumatic brain injury].

Psychiatric Disease and Post-Acute Traumatic Brain Injury.

Acute sports-related traumatic brain injury and repetitive concussion.

Antioxidant Therapeutic Strategies for Cardiovascular Conditions Associated with Oxidative Stress.

Diffuse brain injury induces acute post-traumatic sleep.

Deferoxamine attenuates acute hydrocephalus after traumatic brain injury in rats.