Interactions of Oxidative Stress and Neurovascular Inflammation in the Pathogenesis of Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a major cause of death in the young age group and leads to persisting neurological impairment in many of its victims. It may result in permanent functional deficits because of both primary and secondary damages. This review addresses the role of oxidative stress in TBI-mediated secondary damages by affecting the function of the vascular unit, changes in blood-brain barrier (BBB) permeability, posttraumatic edema formation, and modulation of various pathophysiological factors such as inflammatory factors and enzymes associated with trauma. Oxidative stress plays a major role in many pathophysiologic changes that occur after TBI. In fact, oxidative stress occurs when there is an impairment or inability to balance antioxidant production with reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels. ROS directly downregulate proteins of tight junctions and indirectly activate matrix metalloproteinases (MMPs) that contribute to open the BBB. Loosening of the vasculature and perivascular unit by oxidative stress-induced activation of MMPs and fluid channel aquaporins promotes vascular or cellular fluid edema, enhances leakiness of the BBB, and leads to progression of neuroinflammation. Likewise, oxidative stress activates directly the inflammatory cytokines and growth factors such as IL-1β, tumor necrosis factor-α (TNF-α), and transforming growth factor-beta (TGF-β) or indirectly by activating MMPs. In another pathway, oxidative stress-induced degradation of endothelial vascular endothelial growth factor receptor-2 (VEGFR-2) by MMPs leads to a subsequent elevation of cellular/serum VEGF level. The decrease in VEGFR-2 with a subsequent increase in VEGF-A level leads to apoptosis and neuroinflammation via the activation of caspase-1/3 and IL-1β release.

Introduction

Traumatic brain injury (TBI) is characterized by physical brain injury that temporarily or permanently impairs cognitive function and is a major cause of death and disability worldwide, particularly among the young population. TBI causes approximately 1.5 million deaths and hospitalizations annually in the USA. TBI is classified based on the origin of mechanical forces as blast, blunt, and ballistic. In ballistic injury, the penetrating object pierces the skull and enters to the brain. Then the penetrating object may be expelled out, depending on the kinetic energy at the time of contact, from the opposite side along with some attached brain tissues or stuck inside the brain [1]. In blunt injury, the head of the victim collides with stationary or moving object. During the process of collision, the head encounters a directional force in a local region. Furthermore, the head and the brain translate or rotate depending on the magnitude and direction of the impacting force. The blast injury is complex and the facets of the injuries include components of both blunt and ballistic injuries. Based on the severity of TBI, patients are typically categorized into mild, moderate, and severe by using the Glasgow Coma Scale, a system used to assess coma and impaired consciousness [2]. The Glasgow Coma Scale is divided into three components—eye opening, verbal response, and motor responses. These are usually added together to produce a total score. A Glasgow Coma Scale score of 13–15 is defined as mild, 9–12 as moderate, and 3–8 as severe [2]. The mechanisms of brain tissue injury associated with TBI have been classified as primary and secondary. Primary injury is the result of mechanical forces applied to the skull and brain at the time of impact, which is believed to be irreversible [3]. The primary injury leads to skull fractures, brain contusions, axonal injuries, rupturing of blood vessels, and intracranial hemorrhages [4]. Secondary injury, on the other hand, evolves over time [5]. These are characterized by a complex cascade of biochemical events that lead to elevated intracranial pressure, blood-brain barrier (BBB) disruption, neuroinflammation, brain edema, cerebral hypoxia, ischemia, and delayed neurodegeneration [6-8]. Furthermore, a series of molecular, neurochemical, cellular, and pathophysiological mechanisms contribute to secondary injury. Secondary brain injury may be reversible; therefore, therapeutic intervention can be targeted. We and some other investigators showed that oxidative stress plays a pivotal role in secondary brain injury [9-11].

Oxidative stress is the biochemical and physiological stress or damage caused by the free radicals and can be neutralized by antioxidants. Free radicals contain unpaired electrons that are formed when oxygen is partially reduced and these free radicals attack cell components. Free radicals can then form long-lasting toxic materials that can make effects even beyond its site of production [12]. These toxic species and free radicals are collectively called reactive oxygen species (ROS) and reactive nitrogen species (RNS). The excitotoxicity and enervation of the endogenous antioxidant system (e.g., superoxide dismutase, glutathione peroxidase, and catalase) favors the production of a high level of ROS/RNS that induces peroxidation of cellular and vascular structures, protein oxidation, cleavage of DNA, and impairment of the mitochondrial electron transport chain [13]. These mechanisms are adequate to contribute to neuroinflammation, early or late apoptotic programs, and immediate cell death. In this review, we discuss about oxidative stress in TBI and its effect on BBB dysfunction, neuroinflammation, neurodegeneration, and impairment of cognitive function. In addition to these, a prior description on the major biomarkers of TBI is essential as they are the primary measures of brain damages.

Biomarkers of Traumatic Brain Injury

The use of markers for tissue damage in the central nervous system (CNS), such as S100 calcium-binding protein β (S100β) and neuron-specific enolase (NSE), has been proposed as potentially useful in order to quantify the severity of the TBI early in the process [14]. S100β is a calcium-binding protein having a molecular mass of 21 kDa. It is mainly released by glial cells, but it is also released by some other cells such as adipocytes, bone marrow, skeletal muscle, and melanocytes [15, 16]. NSE is a glycolytic protein having a molecular mass of 78 kDa. It is mainly released by neurons and some other cells like smooth muscle, adipose tissue, platelets, and red blood cells. NSE is widely used to assess neuronal damage [17]. The release of S100β and NSE from the CNS into peripheral circulation is believed due to disturbed membrane integrity of brain cells and increased permeability of the BBB after a traumatic event [15, 18]. However, the exact mechanism of cellular release is not yet fully studied. The serum measurements for S100β and NSE within 6 h of TBI would give a clear picture of the severity of brain injury and can be correlated with clinical outcome [16]. In our analysis, significantly higher levels of S100β and NSE were found in the blood samples of mild traumatic brain injury (mTBI) shock wave-exposed animals when compared with controls. The maximum level of S100β was found at 6 h, whereas leaking of NSE across the damaged BBB into the bloodstream continued to increase even at 24 h after the primary blasts [9].

Cleaved-tau (C-tau) protein is another important biomarker for TBI. Tau is a microtubule-associated phosphoprotein predominantly expressed in axons of neurons within the CNS. TBI results in the proteolysis of tau, producing a cleaved product called C-tau [19]. Serum C-tau levels are dependent on both compromised BBB as well as the degree of neuronal damage in the brain. A number of investigators reported the elevated levels of tau protein in CSF and serum following TBI [20-22]. A significant increase in the level of C-tau at 6 h of posttrauma in the serum of cortical contusion injury rats has been reported [19]. However, some studies suggested that tau protein is a poor biomarker for mild TBI [23]. Recently, Goldstein et al. [24] reported the high level of phosphorylated tau protein in TBI patients. Phosphorylation of tau protein is developmentally regulated such that fetal tau is more phosphorylated than adult brain tau [25]. Phosphorylation inhibits the ability of tau to bind to microtubules making them less stable. A few other biomarkers have also been reported in TBI, viz. glial fibrillary acidic protein (GFAP), isozyme of creatine kinase, and myelin basic protein (MBP) (see review [26]).

Oxidative Stress in Traumatic Brain Injury

The secondary injury of TBI is primarily due to oxidative stress. It has a significant role in the etiology of progressive neuropathology in TBI. ROS and RNS are the main sources of oxidative stress in brain injury. ROS includes superoxide (O_2^•−), hydroxyl radical (HO•), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl). RNS refer to various nitric oxide (NO)-derived compounds, such as peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂). RNS have different reactivities and the half-lives of RNS are generally longer than O_2^•−and HO• [27]. There are a number of enzymes involved in free radical generation, viz. the NADPH oxidase family, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), cytochrome P450 (CYP450), cyclooxygenase (COX), lipoxygenase (LOX), and xanthine oxidase (XO). The role of these enzymes has been reviewed extensively [28-31], and the mechanisms of their activation and inhibition are beyond the scope of this review.

The most common free radical in TBI is superoxide (O_2^•−). It is produced when oxygen molecules gain an electron from other molecules. This superoxide causes tissue damage by promoting hydroxyl radicals from hydrogen peroxide (H₂O₂) and peroxynitrite when combined with NO. The power house organelle, mitochondria, is the major source of O_2^•−in brain injury [32]. In addition, the enzyme NADPH oxidase is a major contributor to posttraumatic cellular ROS production [11, 33]. In the cell, NADPH oxidase produces superoxide by transferring electrons from NADPH across the membrane and combining these to molecular oxygen to produce superoxide anion [34]. Another prooxidant enzyme, iNOS, has a significant role in the production of RNS by catalyzing the generation of NO from l-arginine. Several authors extensively studied the role of NO in generating oxidative stress in TBI [35-37]. NO has both neurodestructive and neuroprotective roles [36, 38]. In TBI, the neurodestructive role of NO is its involvement in the generation of toxic free radicals by lipid peroxidation and protein nitration. RNS are produced by coupling this NO with superoxide to form peroxynitrite (ONOO⁻). Recently, we have reported that the biochemical damage of the CNS is induced by the upregulation of these free radical-generating enzymes such as NADPH oxidase 1 (NOX1) and iNOS in TBI rats [9]. Induction of these enzymes by shock wave exposure paralleled the signatures of oxidative and nitrosative damage (4-hydroxynonenal (4-HNE)/3-nitrotyrosine (3NT)) [9]. The increased level of NOX activity in the cerebral cortex and hippocampal CA1 regions with an early peak at 1 h has been reported [11]. The significantly high levels of 4-HNE and 3NT at 3 h post-exposure and returned to control levels at 24 h post-exposure have also been reported [10]. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, reveals that the O_2^•−induction occurs in neurons at 1 h after TBI. By using the NOX inhibitor, apocyanin, oxidative stress damage can be minimized [11]. Oxidative/nitrosative stresses modify proteins via carbonylation, nitration, and peroxidation. Synaptic proteins also may be affected through these modifications [39].

Hydrogen peroxide (H₂O₂) is a relatively stable molecule and it is formed by dismutation of superoxide radicals. It is generated from nearly all sources of oxidative stress and can diffuse freely in and out of cells and tissues [40]. It aggravates cell proliferation and induces cell death either via apoptosis or necrosis. Monoamine oxidase is another important source of H₂O₂ [41]. H₂O₂ can be activated by transition metals in their reduced states to hydroxyl radical (HO•), which is a potent oxidant and aids to excess tissue damage [40].

Mitochondria are well involved in the generation of ROS. They themselves are targets of oxidative stress and also important in redox signaling from the organelle to the rest of the cell [42]. In 1966, Jensen first reported that ROS produced through mitochondrial respiratory chain [43]. Later, Chance and colleagues showed that the isolated mitochondria produce H₂O₂ [44-46]. During the Q cycle, it has been shown that electron “leakage” to O₂ yields superoxide anion radical (O_2^•−) and hydrogen peroxide (H₂O₂). Thus, it appears that the cytochrome bc1 complex may be a significant source of ROS during TBI wave induction. An additional source of mitochondrial ROS generation may be the NADH dehydrogenase complex, in which O_2^•−is produced during autooxidation of the flavin mononucleotide (FMN) [47]. Therefore, mitochondrial electron transport constitutes the major intracellular source of ROS. Formation of mitochondrial ROS promotes inappropriate activation of the mitochondrial permeability transition, leading the cells to proapoptotic pathways [48].

Biomarkers of Oxidative Stress

ROS can be measured either directly or indirectly following the formation of oxidative by-products of lipids, proteins, or nucleic acids. Different types of oxidative stress biomarkers have been extensively reviewed recently [49, 50]. However, here we discuss about the major biomarkers of oxidative/nitrosative stress briefly.

4-HNE, isoprostanes (IsoPs), and malondialdehyde (MDA) are the major by-products of lipid peroxidation. 4-HNE is one of the most abundant and active lipid peroxides and is derived from the peroxidation of n-6 polyunsaturated fatty acids such as arachidonic and linoleic acids. IsoPs are a family of stable, prostaglandin-like compounds generated from the peroxidation of arachidonic acid [51]. MDA is potentially atherogenic lipid peroxide and generated in vivo via peroxidation of polyunsaturated fatty acids [52]. Another lipid biomarker of oxidative stress is 8-epi-PGF2a. This derivative is produced in vivo by free radical peroxidation of arachidonyl-containing lipids [51].

Nitrosative stress, mainly detected by the presence of 3NT, has been demonstrated in TBI [9]. Protein tyrosine nitration is mediated by RNS such as peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂) and results in a nitro group adduct on susceptible tyrosine residues [53]. S-glutathionylation is another protein modification marker of oxidative stress. S-glutathionylation is the formation of a disulfide bridge between a reactive cysteine residue and the abundant cellular tripeptide glutathione [54]. Bursell and King have been reported S-glutathionylation of hemoglobin as a potential marker of oxidative stress [55].

Oxidative stress damages nucleic acids either by DNA fragmentation (single- and double-stranded DNA breaks) or modification and loss of bases due to oxidative stress [56]. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is widely used as an index of DNA oxidative damage.

ROS can be detected directly by electron paramagnetic resonance (EPR; or electron spin resonance). Using EPR, the superoxide can be detected with superoxide-specific spin probes [57]. In our previous study, we used 4-HNE and 3NT (Fig. 1a) as major biomarkers of oxidative/nitrosative stress in TBI rats besides the direct measurement of ROS (cumulative detection of both hydroxyl and superoxide free radicals) using EPR [9]. In the next section, we discuss BBB permeability and neuroinflammation in TBI.

Fig. 1.

Induction of oxidative/nitrosative stress and activation of MMP-2 and AQP-4 in primary blast (123-kPa peak overpressure) induced mTBI rat brain microvessels. Immunofluorescent staining of nitrosative stress marker, 3-nitrotyrosine (3NT) in mTBI rat brain cortex (a); matrix metalloproteinases-2 (MMP-2) in mTBI rat in intact brain microvessels (b); aquaporin-4 (AQP-4) (red) and endothelial marker, GLUT1 (green) in brain microvessels of rats exposed to primary blast (c). Cell nuclei were counterstained with DAPI (blue) in c. Scale bar (yellow bar in last panel)=5 μm in all panels. For details, see Abdul-Muneer et al. [9]

Blood-Brain Barrier Impairment in TBI

The blood-brain barrier (BBB) is a regulating interface between the peripheral blood circulation and the CNS. In 1885, Paul Ehrlich first studied the existence of the BBB. The BBB is composed of brain microvascular endothelial cells (BMVEC), astrocytes, basement membrane, and pericytes and neurons and are constituting a “neurovascular unit” [58, 59]. Pericytes and endothelial cells are encircled by the basal lamina; a thin basement membrane supports the abluminal surface of the endothelium and is composed of collagen type IV, heparin sulfate proteoglycans, laminin, fibronectin, and other extracellular matrix proteins [60]. Astrocytes are adjacent to the endothelial cell with astrocytic end feet sharing the basal lamina. In addition to astrocytes, the microglia are also closely associated with the brain endothelium and are involved in the integrity of the BBB. These glial and endothelial cells functionally interact with each other in a paracrine manner.

Under physiological conditions, the BBB ensures constant supply of nutrients (oxygen, glucose, and other substances) for brain cells and guides the inflammatory cells to respond to the changes of the local environment. BMVEC possess unique barrier functional properties and are connected by tight junctions (TJ), which are composed of transmembrane proteins occludin/claudin-5 and intracellular zonula occludens (ZO-1, ZO-2, and ZO-3) [59]. TJ provides structural integrity and low permeability of the monolayer. Several intrinsic signaling pathways are involved in the modulation of expression and subcellular localization of TJ proteins [61].

The BBB compromise or damage leads to several neurodegenerative diseases and other neurological complications. Signs of BBB compromise are seen in neurological disorders including stroke [62], Alzheimer’s disease [63], HIV-1 encephalitis [64], multiple sclerosis [65], and TBI [66]. Besides the pathological conditions or diseases, drug or alcohol abuse compromises the integrity of the BBB [67-69]. This review addresses the neurological complications induced by oxidative stress and other neuroinflammatory agents associated with TBI.

In our recent study, we have shown that oxidative stress induces cerebral vascular injury (BBB damage) and neuroinflammation via activation of matrix metalloproteinases in single (one time only shock wave pressure exposure) or repeated (more than one shock wave pressure exposure on the same animal) exposures to low-intensity blast over pressure [9]. In 123-kPa shock wave pressure-induced mTBI in rats, the immunofluorescence and Western blotting methods showed the diminished expression of tight junction proteins in the brain microvessels of treated rats when compared with controls [9]. The peroxidation of membrane lipids is one of the consequences of oxidative stress in TBI, which would affect the permeability of the BBB [70]. Hydroxyl radicals (•OH) may play an important role in peroxidation of membrane lipids in the formation of 4-HNE that increases the permeability of the BBB [71]. The role of lipid peroxidation has been proven by administrating an inhibitor of lipid peroxidation that blocks posttraumatic damage of the BBB [72]. These pathological processes can contribute to long-term neurological disorders.

Infiltration and accumulation of immune cells into brain parenchyma in acute posttraumatic injury is another consequence of BBB damage [73]. Intercellular adhesion molecule (ICAM-1) helps the migration of immune cells into the damaged tissue by mediating the adhesion of these cells to the endothelium. Upregulation of ICAM-1 has been reported in several experimental TBI models [74, 75]. A number of studies have been addressed to study the BBB disruption by analyzing the level of S100β in CSF [76-78]. The BBB permeability has been proven by injecting intravenously the visible tracers biotin-dextrin-amine 3000 (BDA-3 K, 3 kDa) and horseradish peroxidase (HRP, 44 kDa) at 4 h or 3 or 7 days post-TBI, in which both small and large molecular weight tracers were detected in the contusion area and even in remote regions of the injury. However, the larger tracer molecule (HRP) was not detected at later posttraumatic time periods [6]. In another study, the passive BBB dysfunction has been proven by analyzing the CSF-plasma albumin quotient in TBI patients [79].

Several authors showed evidence of BBB disruption following TBI by using pharmacological inhibitors and some other agents, in which they could attenuate TBI-mediated BBB breakdown, upregulation of matrix metalloproteinases (MMPs), aquaporins (AQPs), and other inflammatory agents. Tail intravenous injection ofpoloxamer 188 (anti-inflammatory drug) in TBI mice could reduce TBI-induced brain edema and restored BBB integrity, suppressed TBI-induced neural cell death, and improved neurological function [80]. Neutrophil depletion with an antipolymorphonuclear leukocyte antibody (anti-PMN) before inducing intracerebral hemorrhage in the rat striatum reduces neutrophil infiltration, BBB breakdown, and expression of MMP-9 [81]. In another study, Lopez et al. [82] showed that treatment with orexigenic hormone ghrelin decreases BBB permeability and the level of perivascular AQP-4 and S100β following TBI in mice.

Role of Matrix Metalloproteinases in BBB Permeability

MMPs are zinc-dependent endopeptidases, collectively called matrixins, that participate mainly in extracellular matrix (ECM) degradation. Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous inhibitors [83, 84]. The regulation of MMP expression and activation is complex and tightly controlled, and loss of this control may result in diseases such as arthritis, cancer, atherosclerosis, aneurysms, nephritis, tissue ulcers, and fibrosis [85, 86]. MMPs are broadly divided into two general classes: the secreted MMPs and membrane-type MMPs [87]. To date, 24 different vertebrate MMPs have been identified, of which 23 are found in humans. MMPs can be grouped according to their domain structure into collagenases, gelatinases, stromelysins, and matrilysin. The actions of MMPs are strictly controlled by endogenous MMP inhibitors (MMPIs) and tissue inhibitors of MMPs (TIMPs). TIMPs are specific inhibitors of matrixins that participate in controlling the local activities of MMPs in tissues [88]. Overexpression of MMPs results in an imbalance between the activity of MMPs and TIMPs that can lead to a variety of pathological disorders [85]. There are four types of TIMPs: TIMP-1, TIMP-2, TIMP-3, and TIMP-4.

Activation of MMPs due to oxidative stress degrades basement membrane proteins resulting in loss of brain endothelium stability and increases BBB permeability in vivo [89-91] and in vitro [92]. Studies have revealed that activation of MMPs and degradation of microvascular basement membrane play an important role in stroke [93-95]. An increase in the activity of MMPs also degrades basement membrane proteins and disrupt TJ assembly, thus enhancing BBB permeability [96, 97]. Hanumegowda et al. [98] have reported that the loss of TJ function in brain endothelium is associated with the increase in activity of MMP-2 or MMP-9. Moreover, activation of MMPs leads to brain-blood microvessel disruption, featuring leukocyte infiltration and activation of resident brain macrophages (microglia) [94]. Recently, Reijerkerk et al. [99] reported that diapedesis and transendothelial migration of monocytes due to MMP mediated occludin protein degradation in rat brain endothelial cell line (GP8/3.9).

In TBI, several investigators have reported the involvement of MMPs in degrading various types of ECM proteins and TJ proteins of the BBB [100, 101]. MMP has a critical role in the pathophysiology of synaptic loss and BBB breakdown in TBI, alcohol abuse, stroke, and neurodegeneration [68, 102, 103]. We also demonstrated that the induction of free radical-generating enzymes causes oxidative damage, impairment of the BBB, and neuroinflammation via activation of matrix metalloproteinases and fluid channel activator aquaporin-4 in mTBI [9]. As depicted in Fig. 1b for MMP-2, in our study, the level of expression of the three types of MMPs (MMP-2, MMP-3, and MMP-9) was increased in mTBI rats [9]. There are several reports on the upregulation of MMPs, particularly MMP-2, MMP-3, and MMP-9 and their role in causing acute disruption of the BBB, leading to vasogenic edema and following cell death in TBI [104, 105]. Recently, Ranaivo et al. [106] showed the high level of MMP-9 in combination with mild stretch followed by IL-1β treatment in in vitro model of cell stretch to investigate the effects of mild mechanical insult on astrocyte injury. Higashida et al. [107] reported that when inhibiting MMP-9, the permeability of the BBB significantly ameliorated in TBI rats.

Role of Vascular Endothelial Growth Factor in BBB Disruption

Vascular endothelial growth factor (VEGF) is a heparin-binding protein that is considered the most potent proangiogenic growth factor involved in vascular permeability, vascular dilation, endothelial proliferation, and angiogenesis [108, 109]. Three high-affinity tyrosine kinase receptors exist for VEGF, viz. VEGFR-1 (fms-like tyrosine kinase-1), VEGFR-2 (fetal liver kinase-1/kinase domain region), and VEGFR-3 (fms-like tyrosine kinase-4), all of which are expressed almost exclusively on endothelial cells [110]. The vascular endothelial growth factor receptor-2 (VEGFR-2) plays a central role in the proliferation and apoptosis of vascular endothelium [111, 112]. VEGFR-2 (also known as KDR or Flk-1), is a 200–230-kDa high-affinity receptor for VEGF-A, the processed forms of VEGF-C and VEGF-D, and VEGF-E. It is expressed in both vascular endothelial and lymphatic endothelial cells; its expression has also been demonstrated in several other cell types such as megakaryocytes and hematopoietic stem cells [113]. Vegfr-2^−/−embryos die by embryonic days 8.5–9.5, exhibiting defects in the development of endothelial and hematopoietic precursors, indicating that the receptor is crucial for vascular development [114].

Previous studies suggested that BBB disruption is due to the proteolytic degradation of the vascular membrane. Among proteases, MMPs, in particular MMP-2 and MMP-9, are able to digest the endothelial basal lamina and, therefore, may play a major role in promoting BBB permeability [115-117]. Valable et al. [118] reported that in ischemic animals, administration of VEGF leads to BBB permeability as to an induction of MMP-9 activity. The elevation of MMP-9 levels in stroke is also reported [119].

There are reports on the cleavage of VEGFR-2 by MMP-7 and MMP-9, where it cleaves VEGFR-2 at multiple positions (e.g., Leu-Ser/Met-Leu, Leu-Ser/Ile-Arg) [120]. Tran et al. [121] reported that the plasma MMP activity causes cleavage of extracellular, but not intracellular, domain of VEGFR-2 on the endothelium. This cleavage reduces the ability of the cell to bind VEGF agonists and may be one of the reasons for the enhanced apoptosis [121]. The inhibition of MMP with doxycycline attenuates VEGFR-2 cleavage as well as endothelial apoptosis [121]. Lee and Agoston [122] reported that a blockade of VEGFR-2 signaling with a selective inhibitor, SU5416, abrogates prosurvival response and induced high activation of caspase-3/7 and leads to cell death in TBI. In our recent study, we found that alcohol-mediated upregulation of MMPs leads to VEGFR-2 degradation in rats. In correlation with this observation, we found profound elevation of VEGF-A in the bloodstream and that in turn activates caspase-1 and causes cell apoptosis. This was evidenced from the treatment of VEGFR-2 kinase inhibitor, ki8751, which induces cell apoptosis [68]. The increase in the level of VEGF after TBI causes edema formation and neutrophilic invasion to the brain [123]. The enhancement of the level of VEGF-A further increases the level of ROS [124]. However, there are reports on the neuroprotective role of VEGF, where VEGF increases neurogenesis and angiogenesis and reduces lesion volume after TBI [125]. To resolve this controversy of whether VEGF damages or protects the brain tissue after TBI will be a field for future study.

Neuroinflammation in TBI

Several studies have shown that cytokines, chemokines, and growth factors have significant roles in the pathophysiology of TBI. Shortly after brain injury, there is mass production of proinflammatory cytokines, such as IL-1β and tumor necrosis factor-α (TNF-α) as well as transforming growth factor-beta (TGF-β), which further exacerbates the trauma condition of the brain with oxidative stress and MMPs to cause delayed recovery [126, 127]. The mRNA and protein concentrations of these cytokines have been shown to increase markedly in the acute posttraumatic period following experimental brain trauma in rats [128-130]. These posttraumatic inflammatory cascades also contribute to BBB dysfunction, which eventually leads to the influx of inflammatory cells from the blood to the brain [73].

In the event of TBI, IL-1β is the most studied cytokine. IL-1β is produced by glial cells and can act on neurons and other brain cells. IL-1β triggers inflammatory reactions and leads to recruitment of immune cells, disruption of the BBB and formation of edema, and loss of neurons [131-134]. The high level of IL-1β has been detected in CSF and brain tissue within early hours of brain injury in humans as well as in experimental animals [135, 136]. The role of IL-1β in the formation of brain edema, neuroinflammation, and neurodegeneration has been evidenced by using IL-1β inhibitor in experimental rats [137, 138]. Intracerebroventricular administration of an IL-1β-neutralizing antibody reduces cerebral edema and tissue loss and improves late cognitive outcome following TBI in mice [137]. Active caspase-1 is essential for the cleavage of pro-IL-1β into its mature, biologically active forms [139-141]. There are several reports on the activation of caspase-1 in TBI [142, 143]. Similarly, there are few reports on the upregulation of other types of interleukins such as IL-6, IL-8, IL-10, IL-12, and IL-18 in post-TBI [128, 144, 145]. Some of these interleukins have neuroprotective actions against injury [146-148].

TNF-α has important roles in neuronal development, cell survival, synaptic plasticity, and ionic homeostasis in the CNS. The dysregulation of TNF-α signaling has been implicated in the pathophysiology of several CNS conditions, specifically relating to immune cells infiltration [149] and increased BBB permeability [150]. TNF-α has been detected in the CSF and serum of patients with TBI [144, 151]. Several authors proved the increased activity of TNF-α in association with the loss of sensorimotor and cognitive functions in TBI models by pharmacologically or genetically inhibiting its activity [129, 152, 153].

Several in vivo studies showed that TGF-β is a major regulator of injury response [154]. This growth factor can be synthesized by nearly all cells of the CNS [155, 156] and its increased level has been detected in several CNS disorders including TBI [157, 158]. The high level of TGF-β in CSF has been detected within initial days of head-injured patients [158]. This high level of TGF-β in CSF than in serum may be as a result of damaged BBB. In 2009, Cacheaux et al. [159] made a striking investigation wherein rats treated with a drug that blocks TGF-β, it prevents epilepsy after brain damage.

Traumatic Brain Injury and Edema Formation

The formation of cerebral edema is one of the major factors leading to high mortality and morbidity in TBI. Cerebral edema may account for up to half of the mortality in all victims of TBI [160]. There are two types of edema: cellular and vasogenic [161]. Cellular edema is characterized by an increase in water content in the intracellular compartment. Vasogenic edema results from the breakdown of the BBB and this allows intravascular proteins and fluid to penetrate into the parenchymal extracellular space [162]. Usually, vasogenic edema forms in the first few hours after TBI, followed by cellular edema that develops more slowly over the next few days and prolongs up to 2 weeks [163]. TBI may lead to swelling of the brain tissue and elevate intracranial pressure (ICP), which may also lead to edema formation and BBB disruption [164].

Several mediators are involved in the formation of edema after TBI. Among them, AQPs are the key player in the development and resolution of cerebral edema [165]. AQPs are integral membrane proteins belonging to a larger family of major intrinsic proteins that form pores in the membranes of mammalian cells [166]. There are 13 known AQPs, and among these, AQP-1, AQP-4, and AQP-9 are highly expressed in the brain [167]. AQP-4 is predominantly expressed in astrocytic end feet. In our study, we reported a high level of AQP-4 in 123 kPa wave pressure-induced mTBI (Fig. 1c) and these AQP-4 were seen to be co-localized with GFAP (astrocytic marker) in close proximity to intracerebral vessels in mTBI brain tissue [9]. Several studies have shown that AQP-1, AQP-4, and AQP-9 expression is clearly altered in both experimental and clinical brain injury [168-170]. Several investigators reported that upregulation of AQPs after brain injury promotes edema formation [171-173] and it is evidenced that pharmacological inhibition of AQP-4 can control the formation of edema in TBI [172, 174, 175]. Blocking of hypoxia-inducible factor-1 alpha (HIF-1α), a transcription factor known to activate the expression of AQP-4 and AQP-9, by 2-methoxyestradiol (2ME2) reduces water content or edema formation in injured animals [172]. Higashida et al. [107] reported that the formation of edema can be reduced after inhibition of AQP-4, MMP-9, or HIF-1α.

Another important mediator of edema formation is MMPs. We have discussed the role of MMPs in the pathogenesis of TBI in the previous section. The role of MMPs in edema formation has been proven by using a pharmacological inhibitor of MMPs such as minocycline or TIMP-1, in which reduced BBB disruption and edema, reduced inflammatory response, and improved cognitive function have been observed [176-178]. Some other vasoactive agents also cause BBB disruption and formation of cerebral edema. These vasoactive agents include kinins, such as bradykinins and tachykinins [162, 179]. Donkin and Vink extensively reviewed about these vasoactive agents that cause edema formation in TBI (reviewed in [162]).

In our recent study, we established that the biochemical cerebral vascular/brain injury occurs within a window of 6–24 h after exposure to low-frequency shock wave pressure. Oxidative/nitrosative stress initiates neurovascular injury via the induction of NOX1 and iNOS by blast wave. The signatures of oxidative/nitrosative damage (4-HNE/3NT) in the microvessels paralleled an induction of NOX1/iNOS. The association of cerebral vascular oxidative injury with the downregulation of TJ proteins and the perivascular units with the subsequently enhanced immune cell infiltration justified the BBB dysfunction. Loosening of this BBB by shock wave exposure is exacerbated by oxidative stress-induced MMPs and fluid channel aquaporin-4. Our findings suggest that impairment of the BBB and perivascular units by MMPs promotes BBB leakiness, vascular fluid cavitation, edema formation, neuroinflammation, and subsequently neurotrauma [9].

Antioxidant Defenses and Clinical Strategies in TBI

The living cell has several defense mechanisms to compete with constant exposure to oxidative stress. The antioxidant defense system, which is one of the major defense mechanisms, assists the living cell to cope directly with oxidative stress and to prevent oxidative damage. The brain cells, which are particularly susceptible to oxidative damage, contain various types of antioxidants in which some of them are exclusive to the brain. There are two major groups of antioxidant system, viz. enzymes and low molecular weight antioxidants [180]. Superoxide dismutase (SOD), catalase, peroxidases, peroxyredoxins, and thioredoxins are several of the major enzymes directly regulating the levels of ROS and RNS. Ascorbic acid, glutathione (GSH), vitamin E, lipoic acid, and coenzyme Q are the low molecular weight antioxidants in the brain. Though the application of antioxidants is promising as therapeutic agents, there are limitations in the use of exogenous antioxidants. They are less amenable to the trafficking across the BBB; moreover, their instability and the fast rate of metabolism and toxicity at a higher dose are the major challenges for their therapeutic use [181]. Despite these limitations, targeted applications of natural and modified antioxidants are promising in developing therapeutic interventions.

Several researchers have been concentrating on neuroprotection in TBI patients or models by inhibiting or reducing oxidative/nitrosative stress and neuroinflammation. The ability of several agents or pharmacological inhibitors to attenuate oxidative stress and secondary cell death has been established in different models of TBI. The superoxide scavengers such as SOD [182], OPC-14117 [183], tempol (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) [184], α-phenyl-N-tert-butyl nitrone (PBN) [185], etc. showed to protect the brain from the pathophysiology of TBI. The antioxidant α-tocopherol is hydrophobic and is able to prevent lipid peroxidation [186] and edema [187]. Melatonin is another hydroxyl and superoxide radical scavenger, which reduces brain edema, neuronal death, and memory deficits [188]. Resveratrol, an antioxidant found to be rich in grapes, has been shown to be a promising neuroprotectant in TBI models, possibly by inhibiting lipid peroxidation and minimizing other oxidative damages [189]. Kline et al. examined the neuroprotective effects of bromocriptine (BRO), a dopamine D2 receptor agonist with significant antioxidant properties, with results of reduced lipid peroxidation in a rodent model of focal brain trauma [190]. Tirilazad, a steroid, attenuates cerebral and vasogenic edema by inhibiting lipid peroxidation after TBI [191]. Cannabinoids, a natural psychoactive drug, prevent lipopolysaccharide-induced neurodegeneration in the rat substantia nigra in vivo through inhibition of microglial activation and NOX1 [192]. Likewise, there are myriads of reports on the use of an antioxidant drug against TBI in animals and humans, and several of them give better improvement physically and cognitively.

Similarly, several clinical and basic science trials have been conducted by using different novel treatment strategies to improve the consequences of TBI. The most recent clinical trials have investigated drugs such as steroids (e.g., progesterone) [193], calcium channel inhibitors (e.g., dantrolene) [194], vitamin E [195], amantadine [196], citicholine [197], dexanabinol [198], minocycline [199, 200], and magnesium sulfate [201]. Unfortunately, no interventions have been successful enough in practice to be implemented as standard care [202]. Development of standard care practice by integrating various therapeutic approaches has great significance especially in the scenario of increasing TBI incidents.

Conclusions

This review has highlighted the role of oxidative stress in the intricacy of BBB pathogenesis and cellular and molecular inflammatory cascades elicited after TBI. Figure 2 depicts the schematic representation of oxidative and inflammatory pathways in TBI. The physiological and biochemical responses to TBI, including MMP activation, VEGF accumulation, and neuroinflammation, have been discussed in this review. Furthermore, the development of cerebral edema and its possible reasons and its contribution to the high mortality and morbidity associated with TBI have been addressed. However, while writing this review, we understood that the exact role of VEGF in trauma formation after BBB impairment and apoptosis is not yet fully elucidated. The therapeutic aspects by attenuating BBB impairment, blocking of MMP activation, and reducing neuroinflammation are other scopes of future study. Resolving the issue of whether VEGF damages or protects the cells or tissues after TBI will also be an area of focus for the field.

Fig. 2.

Schematic representation of oxidative stress-induced BBB disruption and neuroinflammation in traumatic brain injury. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the main sources of oxidative stress in brain injury. ROS include superoxide (O_2^•−), hydroxyl radical (HO•), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl). RNS refer to various nitric oxide (NO)-derived compounds, such as peroxynitrite (⁻OONO) and nitrogen dioxide (NO₂). Superoxide (O_2^•−) causes tissue damage by promoting hydroxyl radicals from hydrogen peroxide (H₂O₂) and peroxynitrite (−ONOO) when combined with nitric oxide (NO). ROS activate matrix metalloproteinases (MMPs) that further exacerbate the condition and lead to BBB disruption via degradation of the extracellular matrix and tight junction proteins. Further, MMPs are involved in degradation of vascular endothelial growth factor (VEGFR) and lead to an increase in the level of VEGF that in turn causes ROS and activates caspase-1/3, which leads to cell death. At the same time, ROS or RNS also activate different inflammatory cytokines and growth factors such as IL-1β, TNF-α, and TGF-β, which cause BBB disruption and neuroinflammation

Normal functioning of the BBB is the key factor for brain repair and homeostasis. Attenuation of BBB permeability is a promising approach to control brain edema and associated neuroinflammation. Targeting on restoring BBB function after injury is the main objective for neuroprotection in TBI. It will allow a more reliable delivery of brain-targeted therapeutic agents/drugs. In terms of potential therapeutic strategies and interventions, future treatment of TBI will require a detailed understanding of cerebral physiology and function as well as the pathological and restorative responses toward therapeutic agents. The development of innovative research designs with antioxidants and other anti-inflammatory agents would provide a better therapeutic strategy to treat the pathophysiology of mTBI before advancing toward posttraumatic stress disorder (PTSD).