Traumatic brain injury is a heterogeneous disorder resulting from an external force applied to the head. The development of cerebral edema plays a central role in the evolution of injury following brain trauma and is closely associated with neurologic outcomes. Recent advances in the understanding of the molecular and cellular pathways contributing to the posttraumatic development of cerebral edema have led to the identification of multiple prospective therapeutic targets. The authors summarize the pathogenic mechanisms underlying cerebral edema and highlight the molecular pathways that may be therapeutically targeted to mitigate cerebral edema and associated sequelae following traumatic brain injury.
Key points
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The development of cerebral edema following TBI is an important factor which contributes to evolution of brain injury following initial trauma.
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Post-traumatic cerebral edema arises from disruption of the blood-brain barrier or dysfunction of cellular ionic pumps and may be classified as vasogenic or cytotoxic edema, respectively.
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Vasogenic and cytotoxic edema arise from unique molecular pathways which which may be targeted therapeutically in pre-clinical models.
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Future studies are needed to determine which therapies targeting cerebral edema may have clinical efficacy in human TBI.
Introduction
Traumatic brain injury (TBI) is a complex and heterogeneous disorder with a tremendous public health burden. It is grossly defined as an alteration in brain function or other evidence of brain pathology caused by an external force which may occur in a multitude of settings – including the highway, at home, at work, during sports activities, and on the battlefield. Despite extensive measures taken to prevent these injuries, TBI continues to have an unacceptably high morbidity and mortality. As of 2010, the Centers for Disease Control and Prevention estimated that 2.5 million emergency department visits, hospitalizations, or deaths were associated with TBI, either alone or in combination with other injuries. TBI was a diagnosis in more than 280,000 admissions. A total of 1.7 million people each year sustain a TBI in the United States. Of those, 275,000 are hospitalized and 52,000 die. It is thought that TBI is a contributing factor in nearly a third of all injury-related deaths in the United States. Furthermore, many of those individuals who survive their initial injury will have clinically evident disability later in life; it has been estimated that up to 5.3 million people are currently living with TBI-related disability in the United States alone.
The deleterious effects of a TBI are not confined to the initial traumatic event. Initial brain trauma initiates a complex cascade of pathophysiologic pathways that lead to evolution of brain injury. These effects have led to the adoption of the crude temporal categories of injury known as primary and secondary injury. Primary injuries are brain injuries that occur at the time of the initial trauma, including cerebral contusions, diffuse axonal injury, penetrating or tissue crushing wounds, extra-axial hematomas, and damage to the cerebral vasculature. Short of preventive measures to mitigate and/or avoid initial injury, in the absence of regenerative therapy very few, if any, therapeutic options are available to reverse injury. Secondary injuries are those that occur in the hours to days following the initial insult and are composed of a diverse array of pathophysiologic phenomena, including hypoperfusion, mitochondrial dysfunction, oxidative injury, as well as disruptions to the blood-brain barrier (BBB).
One process central to the pathogenesis of secondary injury is the development of cerebral edema. In accordance with the Monro-Kellie Doctrine, an increase in brain volume as a result of cerebral edema rapidly leads to an increase in intracranial pressure (ICP). As brain volume begins to increase, cerebrospinal fluid (CSF) is displaced into the spinal thecal sac and blood is compressed from the distensible cerebral veins with little increase in ICP. Once these compensatory mechanisms are exceeded, ICP increases exponentially—a common deleterious cascade observed in severe TBI—and has been shown to correlate with increased mortality and poor functional outcomes. Increases in ICP, in turn, lead to the compression of brain vasculature and decrease the cerebral perfusion pressure, defined as mean arterial blood pressure subtracted by ICP. Mechanical compression of the vasculature and/or reductions in cerebral perfusion pressure may give rise to either focal or global ischemia, which may lead to further edema and ultimately irreversible brain injury.
Evolving brain edema also leads to the genesis of pressure gradients across different intracranial compartments and mechanical displacement of brain structures across compartments, a phenomenon known as herniation . This herniation leads to further neurologic injury through axonal stretch, vascular disruption or compression, and/or a combination thereof and often represents a penultimate event to significant neurologic injury, coma, or death. Despite its importance in neurologic decline, treatment of traumatic intracranial hypertension predominately consists of hyperosmolar therapy, for example, hypertonic saline and mannitol, which lead to the efflux of water from the brain into the systemic circulation and/or surgical decompression. Efforts to expand the treatment armamentarium for intracranial hypertension, including hypothermia and barbiturate induced coma, have largely been unsuccessful. In the present article, the authors focus on the cellular and molecular mechanisms underlying cerebral edema following TBI derived from review of both clinical and preclinical animal models. Special attention is devoted to the cellular and molecular mechanisms underlying its pathogenesis as well as future therapeutic targets.
Introduction
Traumatic brain injury (TBI) is a complex and heterogeneous disorder with a tremendous public health burden. It is grossly defined as an alteration in brain function or other evidence of brain pathology caused by an external force which may occur in a multitude of settings – including the highway, at home, at work, during sports activities, and on the battlefield. Despite extensive measures taken to prevent these injuries, TBI continues to have an unacceptably high morbidity and mortality. As of 2010, the Centers for Disease Control and Prevention estimated that 2.5 million emergency department visits, hospitalizations, or deaths were associated with TBI, either alone or in combination with other injuries. TBI was a diagnosis in more than 280,000 admissions. A total of 1.7 million people each year sustain a TBI in the United States. Of those, 275,000 are hospitalized and 52,000 die. It is thought that TBI is a contributing factor in nearly a third of all injury-related deaths in the United States. Furthermore, many of those individuals who survive their initial injury will have clinically evident disability later in life; it has been estimated that up to 5.3 million people are currently living with TBI-related disability in the United States alone.
The deleterious effects of a TBI are not confined to the initial traumatic event. Initial brain trauma initiates a complex cascade of pathophysiologic pathways that lead to evolution of brain injury. These effects have led to the adoption of the crude temporal categories of injury known as primary and secondary injury. Primary injuries are brain injuries that occur at the time of the initial trauma, including cerebral contusions, diffuse axonal injury, penetrating or tissue crushing wounds, extra-axial hematomas, and damage to the cerebral vasculature. Short of preventive measures to mitigate and/or avoid initial injury, in the absence of regenerative therapy very few, if any, therapeutic options are available to reverse injury. Secondary injuries are those that occur in the hours to days following the initial insult and are composed of a diverse array of pathophysiologic phenomena, including hypoperfusion, mitochondrial dysfunction, oxidative injury, as well as disruptions to the blood-brain barrier (BBB).
One process central to the pathogenesis of secondary injury is the development of cerebral edema. In accordance with the Monro-Kellie Doctrine, an increase in brain volume as a result of cerebral edema rapidly leads to an increase in intracranial pressure (ICP). As brain volume begins to increase, cerebrospinal fluid (CSF) is displaced into the spinal thecal sac and blood is compressed from the distensible cerebral veins with little increase in ICP. Once these compensatory mechanisms are exceeded, ICP increases exponentially—a common deleterious cascade observed in severe TBI—and has been shown to correlate with increased mortality and poor functional outcomes. Increases in ICP, in turn, lead to the compression of brain vasculature and decrease the cerebral perfusion pressure, defined as mean arterial blood pressure subtracted by ICP. Mechanical compression of the vasculature and/or reductions in cerebral perfusion pressure may give rise to either focal or global ischemia, which may lead to further edema and ultimately irreversible brain injury.
Evolving brain edema also leads to the genesis of pressure gradients across different intracranial compartments and mechanical displacement of brain structures across compartments, a phenomenon known as herniation . This herniation leads to further neurologic injury through axonal stretch, vascular disruption or compression, and/or a combination thereof and often represents a penultimate event to significant neurologic injury, coma, or death. Despite its importance in neurologic decline, treatment of traumatic intracranial hypertension predominately consists of hyperosmolar therapy, for example, hypertonic saline and mannitol, which lead to the efflux of water from the brain into the systemic circulation and/or surgical decompression. Efforts to expand the treatment armamentarium for intracranial hypertension, including hypothermia and barbiturate induced coma, have largely been unsuccessful. In the present article, the authors focus on the cellular and molecular mechanisms underlying cerebral edema following TBI derived from review of both clinical and preclinical animal models. Special attention is devoted to the cellular and molecular mechanisms underlying its pathogenesis as well as future therapeutic targets.
Pathogenesis of cerebral edema
Cerebral edema is broadly categorized as being either vasogenic or cytotoxic. Vasogenic edema results from disruption to the BBB formed by cerebrovascular endothelial cells (ECs) and leads to an influx of protein-rich fluid from circulating blood into the brain interstitial fluid. Cytotoxic edema, on the other hand, is a product of failure of homeostatic ion channels and pumps resulting in failure of ionic gradients and an intracellular osmotic shift resulting in cellular swelling, a process more prominent in astrocytes and glial cells.
TBI is a complex and multifaceted injury that leads to dysfunction and/or disruption of multiple cell types. Although TBI-associated brain edema was initially thought to arise predominantly from vasogenic mechanisms, more recent clinical and preclinical studies have demonstrated that cytotoxic edema plays a significant role. For example, MRI studies of patients with closed-head injury revealed a mixed picture of both cytotoxic and vasogenic edema by imaging criteria. However, in the absence of vasogenic edema, a process that depends exclusively on cytotoxic edema would be self-limiting. Similarly, it is likely that the contributions of vasogenic and cytotoxic processes make differential relative contributions through different phases of evolution of edema following brain trauma. In TBI, brain edema is thought to follow a bimodal time course, with one study showing that half of patients had their highest mean ICP recorded during the first 3 days after injury, whereas 25% of patients showed their highest mean ICP after postinjury day 5. In the ensuing subsections, the authors describe in detail the molecular and cellular mechanisms underlying the pathogenesis of posttraumatic vasogenic and cytotoxic edema.
The Blood-Brain Barrier and Neurovascular Unit
The BBB is formed by a continuous lining of brain ECs with specialized properties. Tight junctional complexes composed of transmembrane occludin and claudin proteins are linked to the actin cytoskeleton through adapter proteins, such as zonula occludens-1. Adjacent cells are further connected through adherens junctions composed of cell adhesion molecules, for example, vascular endothelial cadherin molecules. Tight and adherens junctions limit unregulated paracellular flow of circulating proteins and cells from blood into brain. Unlike peripheral vascular beds, brain ECs do not possess fenestrae and display low rates of pinocytosis and nonspecific vesicular transport, so-called bulk flow transcytosis. As a result, molecules that are not sufficiently lipophilic and greater than 40 kDa are excluded from the brain in the absence of highly specialized and regulated transmembrane transport systems in brain ECs.
EC function, however, is further influenced by adjacent cells. Brain endothelium is surrounded by vascular basement membrane, pericytes, and astrocyte end feet. Recent research has demonstrated that pericytes are necessary for establishment and maintenance of endothelial barrier properties. Astrocytes also contribute to barrier functionality and polarity of endothelial transport systems. Their end feet also have a high concentration of aquaporin (AQP) channels that facilitate movement for brain free water in and out of the vascular compartment as well as through the para-vascular compartment. This growing knowledge of the multicellular structure of the cerebrovasculature has led to the coining of the term neurovascular unit , which represents the interconnected relationship of ECs, mural cells (pericytes and vascular smooth muscle cells), glial cells, and neurons. Coordinated bidirectional signaling between cell types is necessary for preservation of vascular structure and function, and injury and/or dysfunction of one cell type influences the structure and function of cells. For example, several signaling molecules, such as cytokines, matrix metalloproteinases (MMPs), and various secondary messengers, may lead to disruption of endothelial barrier properties.
Vasogenic Edema
Vasogenic edema results from disruption of the BBB with extravasation of plasma-derived factors and circulating erythrocytes and immune cells that are normally excluded from the brain parenchyma. Accumulation of protein-rich fluids increases oncotic pressure of the extracellular space and increases total brain volume. The mechanism of vasogenic edema involves (1) mechanical disruption of blood vessels and (2) secretion of propermeability factors from adjacent cells. However, additional factors, for example, detachment of perivascular pericytes, have been described but are poorly understood at the present time. The increase in BBB permeability seen in vasogenic edema follows a bimodal time course with peaks at 1 to 3 hours after injury as demonstrated by immunoglobulin G (IgG) immunoreactivity in the brain followed by an increase in microglia activation seen after 5 days after injury. Therefore, mechanical disruption of brain blood vessels as evidence by contusions, brain petechiae, and increasing CSF concentrations of plasma-derived proteins, for example, IgG and albumin, results in the initial flux of vasogenic edema at the time of the initial injury. Secretion of propermeability factors, on the other hand, likely accounts for vasogenic edema more delayed in onset, especially in the absence of space-occupying mass lesions, which may lead to avulsion of perforating branches of small vessels.
Studies have suggested that the relative contributions of different mechanisms of vasogenic edema depend on injury severity. For example, studies in high-energy blast wave–induced TBI demonstrate an increase in BBB disruption with blast wave overpressure but found no difference in brain IgG immunoreactivity between the immediate postinjury period and 48 hours later. This finding suggests that much of the BBB disruption was mechanical and occurred at the time of injury. In TBI with comparatively milder mechanisms of injury, oxidative stress and inflammatory second messenger cascades make larger contributions to the development of altered BBB permeability, which peaks 6 to 24 hours after injury. Furthermore, mild TBI can induce chronic changes to the brain microvasculature that are evident even months after the initial injury.
Studies in preclinical animal models demonstrate induction of expression of the free-radical–generating enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and inducible nitric oxide synthetase (iNOS) following low-energy brain injury. Generation of free radicals in turn leads to microvascular injury, endothelial apoptosis, and disruption of endothelial tight junctional protein complexes. Oxidative stress also leads to increased expression of the degrading enzymes MMPs in perivascular cells and infiltrating or resident inflammatory cells leading to further degradation of endothelial tight junctions, basement membranes, and cell adhesion molecules.
Simultaneously, posttraumatic neuroinflammation converges with oxidant stress on the vascular interface. Multiple cell types, including microglia, infiltrating inflammatory cells, neurons, and ECs, increase expression and subsequent secretion of proinflammatory cytokines, namely, interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α. These cytokines induce a proinflammatory state and contribute to BBB disruption through loss of tight junction proteins, increased expression of MMPs, and increased levels of other permeability-increasing molecules, for example, bradykinins and the neuropeptide substance P (SP). Disruption in BBB permeability, increased chemokine/cytokine secretion, and increased inflammatory cell adhesion molecules on brain endothelium lead to further recruitment of inflammatory cells from the systemic circulation. Infiltration of peripheral inflammatory cells, such as neutrophils and monocytes, may then lead to further endothelial barrier disruption through generation of reactive oxygen species, proteolytic enzymes, and propagation of inflammatory cascades through secretion of cytokines and chemokines.
Disruption of the BBB leads to nonspecific influx of circulating blood-borne cells, plasma-derived proteins, and other solutes that contribute to neuronal dysfunction and further injury following TBI. Nonspecific protein flux into brain interstitial fluid increases oncotic pressure giving rise to brain edema, which may occlude small vessels and give rise to local hypoperfusion. Many plasma-derived proteins, for example, plasmin, thrombin, fibrin, and erythrocyte-derived free iron, have direct neurotoxic and/or proinflammatory properties. Albumin, in addition to serving as an important determinant of oncotic pressure, increases neuronal irritability and contributes to ictogenesis. Therefore, disruption of the BBB may have several deleterious consequences in addition to the formation of edema following TBI.
Cytotoxic Edema
Cytotoxic edema is defined as intracellular swelling that results from dysfunction of ionic and osmotic flux. An inability to maintain cell membrane electrochemical gradients is one common etiologic factor, most often resulting from ischemia or hypoxia. Impaired oxygen and glucose delivery leads to depletion of cellular adenosine triphosphate (ATP) stores, the source of cellular energy. This depletion in turn leads to impaired function of the sodium ion (Na+)/potassium ion (K+) ATPase. Under physiologic conditions, this active transport system normally pumps 3 Na+ out of the cell and 2 K+ into the cell. In the absence of a functional transporter, Na+ cations begin to flow down their electrochemical gradient and accumulate inside of cells. Several compensatory mechanisms resist the generation of an osmotic gradient, including the passive diffusion of potassium out of the cell. However, this is overcome by increased influx of negatively charged chloride ions (Cl−) via activation of the SLC26A11 channel, which serves as a voltage-gated Cl− channel when the cell is depolarized. In addition, there is dysfunction in several other cellular cation channels, including acid-sensing ion channels (ASICs), the sulfonylurea receptor 1 (SUR1)-regulated NC Ca-ATP channel (SUR1/Transient receptor potential cation channel subfamily M member 4 [TRPM4]), the NKCC1 channel, and the N-methyl-D-aspartate (NMDA) glutamate receptor channel ( Table 1 ). An influx of ions into cells increases cell osmolality, and a compensatory movement of free water down its osmotic gradient through specialized channels closely follows and results in cellular swelling.
| Cation Channel | Permeability | Regulation | Effect in TBI |
|---|---|---|---|
| ASICs | Na+ > Ca2+ | It is activated by H+. | Decreased pH associated with ischemia leads to increased Na+ and Ca2+ influx into cells, contributing to edema and cellular injury. |
| SUR1 regulated NC Ca − ATP channel | All inorganic monovalent cations, impermeable to Mg2+ and Ca2+ | Channel activity is activated by intracellular Ca2+ and inhibited by intracellular ATP; expression in the CNS is induced following hypoxia or injury. | Decreased intracellular ATP stores in hypoxic cells leads to activation of this channel, resulting in membrane depolarization and excessive intracellular Na+ accumulation. |
| NKCC1 channel | 1 Na+: 1 K+: 2 Cl− | Activity is increased by elevated extracellular K+. | Elevated extracellular K+ from ischemia-related depolarization activates channel, leading to dysregulated Na+ and Cl− influx into cells. |
| NMDA glutamate receptor channel | Na+, K+, Ca2+ | The channel pore opens in response to glutamate or glycine binding coincident with membrane depolarization. | Excessive glutamate release by ischemic neurons leads to unregulated influx of Na+ and Ca2+ through NMDA receptor channels, leading to intracellular accumulation of these ions. |
It is important to note, however, that cell swelling itself does not increase brain volume; cytotoxic edema would be a self-limiting process in the absence of ionic and osmotic fluxes across the BBB. Extracellular concentrations of sodium and chloride in the brain interstitial fluid decrease dramatically as these ions move into cells. The result is the generation of an osmotic gradient between the vascular compartment and brain interstitial fluid (ISF). This generation is normally mitigated by an intact BBB. However, in brain trauma, disruption of the BBB and/or heightened endothelial permeability contributes to increased influx of circulating molecules, including both ions and water. The NKCC1 secondary active cotransporter and SUR1/TRPM4 channel are expressed on the luminal EC membrane and mediate ion transport into the ECs. Abluminal non–voltage- or ligand-gated sodium channels may then facilitate diffusion of endothelial sodium into the brain interstitial fluid. This diffusion in turn creates a gradient favoring the shift of water from the vasculature into the brain interstitium.
Although water is able to freely diffuse across most tissues in the body, its movement is highly regulated in the central nervous system (CNS) through a family of specialized transmembrane channels known as AQPs. The predominate AQPs of the CNS are AQP-1 and AQP-4. AQP-1 is expressed at the apical membrane of the choroid plexus epithelium where it aids in CSF secretion into the ventricles. AQP-4, on the other hand, is located at the basolateral membrane of ventricular ependymal cells and astrocyte foot processes where it facilitates CSF-parenchyma and vascular-parenchyma water transport, respectively. In astrocytes, AQP-4 colocalizes with the inwardly rectifying potassium channel Kir4.1, where it is thought to function as a water-potassium transport complex. In normal conditions, this facilitates the clearance of K+ and water from synaptic junctions after the neuronal excitation.
Following brain insults, AQP-4 has been implicated in the osmotic shifts that give rise to cerebral edema. As described earlier, injury leads to excessive cellular osmolality and establishes a strong osmotic gradient. This osmotic gradient leads to the passive diffusion of free water through AQP-4 channels found in the astrocyte foot processes surrounding the cerebral vasculature and subsequent astrocyte swelling, the principal cellular source of this type of cellular edema.
Future treatment targets
As our understanding of the pathogenesis of cerebral edema in TBI has increased, much research has focused on developing novel antiedema treatments. These new therapies can broadly be categorized as affecting differently molecular pathways that give rise to vasogenic and/or cytotoxic edema ( Table 2 ).
| Protein | Function | Cellular Effect |
|---|---|---|
| Vasogenic edema | ||
| MLCK | Modulates cytoskeletal rearrangement | Decreases endothelial tight junction and BBB permeability |
| GR | Multifunctional transcriptional regulator, includes upregulation of tight junction and adherens junction proteins | Increase endothelial tight and adherens junction proteins |
| PPAR | Multifunctional nuclear membrane–associated transcription factors, includes downregulation of proinflammatory molecules | Downregulates proinflammatory molecules that increase endothelial permeability, for example, cytokines or chemokines, or degrade tight junction complexes, for example, MMPs |
| VEGF | Angiogenic factor | Leads to dedifferentiation of brain ECs and loss of barrier properties |
| MMPs | Proteases that degrade components of the extracellular matrix and endothelial junctional complexes | Increases endothelial permeability through degradation of tight and adherens junctions; destabilizes vascular wall through degradation of vascular basement membrane |
| SP | Tachykinin promotes protein extravasation and leukocyte adhesion to ECs | Increases endothelial permeability and adhesion molecule expression |
| Cytotoxic edema targets | ||
| AQP-4 | Transmembrane water channels | Regulate brain water transport, predominately in astrocytes |
| NKCC1 channel | Cotransporter of Na+, K+, and Cl− | Upregulated in brain injury and leads to excessive cellular influx Na+ and Cl− |
| Vasopressin receptors | Initiate downstream signal transduction, including upregulation of AQP-4 | Increase synthesis/release in trauma leads to increased cellular water permeability |
| SUR1-regulated NC Ca-ATP channel (SUR1/TRPM4) | Associates with and regulates the NCCa-ATP channel: a nonselective cation channel regulated by intracellular ATP and calcium | Upregulated in brain injury and leads to nonspecific cation flux into cells |
Therapies Targeting Vasogenic Edema
Myosin light chain kinase
Myosin light chain kinases (MLCK) and its antagonist myosin light chain phosphatase modulate cytoskeletal contraction and/or rearrangement via phosphorylation or dephosphorylation of the regulatory unit of the myosin light chain, which are expressed in brain ECs. In ECs, MLCK plays an important role in regulating barrier permeability.
Endothelial tight junction proteins, for example, claudin or occludin, are linked to the actin cytoskeleton through adaptor proteins, for example, zonula occludins. Thus, contraction or rearrangement of the actin cytoskeleton modulates endothelial permeability. In preclinical models, expression of MLCK peaks 6 hours following TBI and slowly returns to baseline over 48 hours, a period that coincides with the development of cerebral edema. In vitro upregulation of MLCK reduces transendothelial resistance consistent with heightened permeability in response to a myriad of injury factors, including ethanol, methamphetamine, and hypoxia. Treatment with a pharmacologic inhibitor of MLCK either before or following TBI decreased disruption of the BBB and associated cerebral edema. However, the effect of MLCK inhibition on neurologic outcome remains controversial. In a rodent model of closed-head trauma, postinjury treatment with 1-(5-iodonaphthalene-1sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) resulted in improvements in motor and cognitive function as evidenced by improved performance on the wire hang test and 2 object recognition tests, respectively. However, pretreatment of ML-7 before controlled cortical impact did not influence neurologic severity scoring. Given the significant differences in experimental design, including timing of administration, future studies are needed to further confirm a therapeutic role of MLCK inhibition in the treatment of posttraumatic edema.
Degradation of the glucocorticoid receptor
Glucocorticoids, such as dexamethasone and methylprednisolone, are commonly used to treat neurologic conditions with a disrupted BBB, including brain tumors and multiple sclerosis. Glucocorticoids bind to glucocorticoid receptors (GRs) in cerebrovascular ECs leading to the transcriptional upregulation of the tight and adherens junctional proteins, including claudin, occludin, and vascular endothelial cadherin, while inhibiting MMPs and CNS leukocyte infiltration. Upregulation of tight and adherens junction proteins and downregulation of destabilizing enzymes leads to a restoration of endothelial barrier properties in pathologic conditions. However, glucocorticoid treatment is nonspecific and leads to several potentially deleterious side effects in an injured brain, most notably delayed wound healing and hyperglycemia. As a result, glucocorticoids have not been convincingly shown to improve outcomes in patients with ischemic stroke or TBI ; high-dose methylprednisolone treatment is contraindicated in moderate to severe TBI as it is associated with increased mortality. Therefore, the potentially beneficial BBB restorative properties of glucocorticoid therapy are offset by the negative systemic consequences of steroid therapy.
One potential mechanism that may explain this phenomenon is the proteasomal degradation or post-translational modifications of GRs in response to brain injury. For example, in hypoxic conditions, brain ECs lose barrier properties that are unable to be restored with glucocorticoids alone. Treatment with these cells with bortezomib, a pharmacologic proteasome inhibitor, restores the probarrier effects of glucocorticoid therapy. Treatment with bortezomib and dexamethasone similarly led to less edema while restoring endothelial occludin levels and BBB integrity than dexamethasone alone in mice following controlled cortical impact. This treatment was associated with less neuronal damage following brain trauma. Whether these results may be replicated in other experimental TBI models remains to be seen, and whether adjuvant therapy may help augment the response to steroids in the injured brain warrants further investigation.
Peroxisome proliferator-activated receptor
Peroxisome proliferator-activated receptors (PPARs) are nuclear membrane–associated transcription factors that function as nuclear receptors that may be pharmacologically targeted by the fenofibrates and thiazolidinediones, including pioglitazone and rosiglitazone. Three different isotypes (α, β/δ, and γ) are differentially expressed in different organ systems throughout the body, with all 3 being expressed in the CNS. These receptors are activated via a dimerization with retinoid-X receptors, which in turn modulate expression of an array of different proteins with several different phenotypic outcomes, including the downregulation of the proinflammatory transcription factors, such as activator protein 1 (AP-1), signal transducer and activator of transcription protein family (STAT), nuclear factor of activated T-cells (NFAT), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).
The antiinflammatory role of PPAR has led to attention from the TBI community as a possible therapeutic target; pharmacologic agents targeting this pathway, such as fenofibrates or the thiazolidinediones pioglitazone and rosiglitazone, have been applied to several preclinical models. For example, administration of fenofibrate, an agonist of PPARα, reduced brain edema, expression of the inflammatory adhesion molecule intercellular adhesion molecule 1 (ICAM-1), and neurologic deficits in a rat lateral fluid percussion TBI model. At a molecular level, fenofibrate reduced expression of iNOS, cyclooxygenase 2, and MMP-9 as well as markers of oxidant stress. Fenofibrate may, therefore, protect endothelial tight junctions through downregulation of the degradation enzyme MMP-9 and oxidative endothelial injury. In other injury models, such as oxygen glucose deprivation, fenofibrate was shown to have a protective effect on endothelial BBB function. However, the molecular mechanisms of this protection were not described.
Additional studies have begun to explore whether potentiation of PPARγ signaling with treatment of pioglitazone or rosiglitazone may confer benefit in preclinical models of TBI. Collectively, these studies have demonstrated that treatment results in a reduction in lesion size, microglial activation, proinflammatory mediator expression, and neuronal apoptosis while increasing performance on posttraumatic measures of cognition. However, the influence on edema and BBB permeability has yet to be studied in the context of treatment with pioglitazone or rosiglitazone. In vitro studies have demonstrated that rosiglitazone altered endothelial expression of the GTPases Rac1 and RhoA, which play a pivotal role in endothelial cytoskeletal rearrangements and inflammatory cell transmigration across the BBB without influencing tight junction protein level. Whether a similar relationship holds true in TBI is unclear, and future studies are needed to better understand PPAR agonism and how it influences posttraumatic brain edema and BBB function.
In vivo experiments examining the effect of pioglitazone in the setting of TBI demonstrated improved cognitive function after injury, reduced lesion size, and lower levels of microglial activation when compared with controls. Similarly, treatment with rosiglitazone showed similar neuroprotective effects with the addition of reduced proinflammatory gene expression, decreased apoptotic neurons, and increased antioxidant enzymes.
Vascular endothelial growth factor
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor essential to initial brain vasculogenesis as well as sprouting angiogenesis, which is responsible for vascular remodeling in both physiologic conditions and in response to brain injury and pathologic states, such as brain tumors, stroke, and neurodegeneration. VEGF and VEGF receptors are upregulated by the transcription factors hypoxia-inducible factor (HIF)-1 and HIF-2, both of which become highly expressed in the setting of brain ischemia. VEGF leads to dedifferentiation of brain ECs, which assume a leaky and highly proliferative phenotype. The mechanisms through which VEGF induces disruption of the BBB are numerous and affect both paracellular and transcellular pathways. These mechanisms include acquisition of endothelial fenestrae, increased endothelial pinocytosis and bulk flow transcytosis, and increased size of the interendothelial space with downregulation of the tight junction proteins claudin-5, occludin, and zonula occludin-1.
Following TBI, there is an increase in VEGF thought to contribute to reparative mechanisms to restore blood flow to injured brain parenchyma. This increase may come at a cost of leaky immature vasculature, which is prone to rupture and contributes to posttraumatic edema. Experiments using a rat cold-injury model of brain edema showed that antagonism of the VEGF receptor significantly reduced brain water content in a dose-dependent fashion and reduced vascular permeability. Other investigators using in vivo ischemia/reperfusion injury and venous infarction models found that sequestration or antagonism of VEGF reduced radiologically defined brain edema. However, post-TBI treatment with exogenous VEGF may increase neurogenesis, decrease lesion volume, and improve functional outcomes in mice following closed head injury. This information highlights that VEGF influences not only the vasculature but also neuronal health and repair. Thus, targeting VEGF may have both beneficial and deleterious consequences in the posttraumatic setting.
Matrix metalloproteinases
MMPs are a family of neutral proteases that cleave components of the extracellular matrix and are important for wound healing, development, and inflammation. Within the CNS, MMPs are closely associated with the neurovascular unit where they serve to regulate the extracellular matrix that contributes to the BBB. MMPs have been shown to degrade tight junction and basal lamina proteins, thereby contributing to the development of increased BBB permeability and vasogenic edema. In the CNS, ECs express MMP-9; pericytes express MMP-9 and MMP-3; and astrocytes express MMP-2 constitutively. Following TBI, there is an upregulation in MMP expression and activation, which persists from postinjury day 1 to day 7; higher concentrations are found in human CSF. Genetic knockout of MMP-9 reduces lesions sizes, lessens BBB disruption, as well as limits the degradation of zonae occludens-1, the adaptor protein that links tight junctions to the cytoskeleton, when compared with wild-type mice following TBI.
Given this evidence, multiple reports with favorable results have investigated a potential role for pharmacologic inhibition of MMPs immediately following TBI. For example, treatment with a specific inhibitor of gelatinases (MMP-2 and MMP-9) known as SB-3CT reduced lesion volume, dendritic and neuronal degeneration, and microglial activation and astrogliosis while improving sensorimotor and cognitive deficits following controlled cortical impact in mice. In rats, treatment with SB-3CT ameliorated cognitive deficits and hippocampal neuronal loss following fluid percussive injury. A water-soluble derivative of SB-3CT was more recently shown to have a similar effect in reducing lesion volume following TBI. In models of chronic neurodegeneration and subarachnoid hemorrhage, treatment with SB-3CT similarly increased endothelial barrier properties via protection of tight junctional proteins and components of the vascular basement membrane. These changes were associated with and thought to contribute to a protection of neuronal structure and function. Therefore, inhibition of MMPs may have both acute and chronic protective benefits in preserving endothelial BBB properties and may thereby promote neuronal health and repair following TBI.
Substance P
SP is a member of the tachykinin family of kinins along with calcitonin gene–related peptide (CGRP) and neurokinin A. Tachykinins are released by both central and peripheral endings of sensory neurons and function as neurotransmitters. They bind to tachykinin receptors and are key mediators of neurogenic inflammation, with SP primarily binding to the neurokinin 1 (NK1) receptor. Although many members of the kinin family are known to be involved in many aspects of the physiology of neurogenic inflammation, CGRP mediates vasodilation and SP is primarily responsible for enhancing plasma protein extravasation and leukocyte adhesion to ECs. It has recently been recognized that neurogenic inflammation plays a role in the development of vasogenic edema. Further studies found that perivascular SP immunoreactivity increases in the perivascular space following TBI, thereby suggesting that SP, not other neuropeptides, might be responsible for the neurogenic inflammation seen following TBI.
Research examining SP inhibition following TBI has shown promising results. A study using a rat model of TBI found that inhibition of the NK1 receptor with N-acetyl- l -tryptophan 30 minutes after injury attenuated vascular permeability and edema formation as well as improved motor and cognitive outcomes. More recently, it was shown that the addition of a lipophilic trifluoromethyl benzyl ester group to N-acetyl- l -tryptophan to improve membrane permeability of the drug extends efficacy in TBI from 5 to 12 hours after injury. Studies using models of ischemic stroke have similarly shown efficacy of NK1 inhibition on BBB permeability, edema formation, infarct size, and neurologic function. Taken together, these results put forward SP inhibition as a potentially neuroprotective therapeutic for TBI.
Cytotoxic Edema Targets
Aquaporins
AQPs play a key role in maintaining brain-water homeostasis, mediating the flux of water at the interface between the cerebral vasculature and both brain parenchyma and the CSF producing choroid plexus. AQP-4 is localized at the astrocyte foot processes and plays a vital molecular role in the pathogenesis of the glial swelling seen in cytotoxic edema following a myriad of brain injuries, including TBI, brain ischemia, hydrocephalus, water intoxication, and bacterial abscesses. In vitro studies in cultured astrocytes demonstrated a 7.1-fold reduction in osmotic water permeability in AQP-4–deficient cells. Similar findings were demonstrated in organotypic slice preparations in Aqp4 −/− mice confirming that AQP-4 is the primary route for water transport in astrocytes. Experimental models of cryogenic brain injury, an injury associated with predominantly vasogenic edema, have suggested that AQP-4 may a play a role in the resolution of vasogenic edema in addition to its well-established role in cytotoxic edema.
Following TBI, expression of AQPs is increased in both in vitro and in vivo models of experimental brain injury, thus, making it an attractive therapeutic target in TBI. In human subjects, several single nucleotide polymorphisms in AQP-4 are associated with poor functional outcomes following TBI. Preclinical studies in experimental TBI models have shown promising preliminary results. For example, antibody-mediated inhibition of AQP-4 demonstrated a statistically significant reduction in brain water content and neuronal death while improving neurobehavioral outcomes following weight-drop closed-head injury in rats. In juvenile rats, similar protective results were observed following controlled cortical impact with local infusion of small-interfering RNA targeting AQP-4. More recently, controlled cortical impact in Aqp4 −/− mice demonstrated reductions in total brain water and ICP when compared with Aqp4 +/+ mice without differences in BBB permeability. These reductions were associated with milder improvements in neurologic outcomes than have been observed in other injury models with purely cytotoxic injury. This finding led the investigators to conclude that concurrent vasogenic edema may mitigate the potential benefit of AQP inhibition in TBI. Whether treatment with BBB restorative therapy in tandem with AQP inhibition improves outcomes remains to be tested, and future studies are needed to better delineate the role of aquaporin-targeted therapy in TBI.
NKCC1
The NKCC channel is an electroneutral cotransporter of Na+, K+, and Cl−, which is expressed in 2 different isoforms: NKCC1 and NKCC2. NKCC1 is ubiquitously expressed, whereas NKCC2 is found exclusively within the kidney. Within the CNS, the NKCC1 isoform is expressed by glia, neurons, endothelium, and choroid plexus epithelial cells. This cotransporter uses the electrochemical gradient established by the Na+/K+ -ATPase to transport 1 Na+, 1 K+, and 2 Cl− ions across the plasma membrane and into cells. Thus, the NKCC1 is an important regulator of intracellular osmolality and by extension cellular volume.
NKCC1 activity is upregulated following various forms of ischemic and traumatic brain injury. During ischemia or hypoxia, NKCC1 contributes to excessive Na+ and Cl− influx into cells increasing cellular osmolality and leading to the development of cytotoxic edema. In TBI, upregulation and increased phosphorylation contribute to increased activity of the NKCC1 transporter. In vitro treatment of cultured astrocytes with bumetanide, an inhibitor of NKCC1, or small interfering RNA silencing reduced astrocytic swelling following barotrauma. To date, no in vivo studies targeting NKCC1 in TBI have been performed. Other models of brain injury, such as experimental middle cerebral artery occlusion, have demonstrated a 70% reduction in brain water content with bumetanide-mediated NKCC1 inhibition. Whether similar protective effects may be observed following TBI remains to be determined.
Vasopressin receptors
Arginine vasopressin (AVP) is a neuropeptide hormone that regulates plasma osmolality. In addition to peripheral targets, AVP binds to and activates V1 receptors within the CNS, activating downstream signal transduction in several cell types, including decreases in water permeability of CSF-producing ependymal cells, reduced CSF production, changes in water permeability of astrocytes, and changes in AQP-4 expression. Following TBI, AVP synthesis is upregulated both within the hypothalamus, namely, within the paraventricular and supraoptic nuclei, and locally near the traumatic lesion. Selection inhibition of the vasopressin V1a receptor has shown promising results in TBI rodent models. Treatment with a small molecule selective V1a receptor antagonist SR 49059 lessened ICP, brain water content, and gliosis while restoring extracellular sodium concentration of brain ISF following focal cortical contusion induced by controlled cortical impact in rats. These changes were associated with decreased AQP4 expression in perilesional astrocytes offering potential mechanistic insights. More recently, an independent group demonstrated that intracerebroventricular administration, but not systemic administration, of SR49059 decreased brain edema, posttraumatic ICP, and secondary contusion expansion in mice following controlled cortical impact (CCI). Further works are needed to better characterize these effects in other TBI models.
Sulfonylurea receptor 1–regulated NC Ca- –adenosine triphosphate channel
Sulfonylurea receptors (SURs) are integral membrane proteins and belong to the ATP binding cassette family of transmembrane proteins. SURs are not ion transport channels but rather associates with a heterologous pore forming units to form ion channels. In the CNS, SUR1 associates with and regulates the function of the NC Ca-ATP channel, a nonselective cation channel regulated by intracellular ATP and calcium. The SUR1-regulated NC Ca-ATP channel (SUR1/TRPM4) is not constitutively expressed and is upregulated in a variety of brain injury states, including TBI, stroke, and brain tumors. ATP normally inhibits SUR1/TRPM4 flux. Following brain injury, ATP depletion leads to opening of the SUR1/TRPM4 channel, which in turn leads to membrane depolarization, blebbing, and swelling.
Pharmacologic inhibition of the SUR1/TRPM4 with the sulfonylurea glibenclamide, also known as glyburide, reduces the development of cytotoxic brain edema in multiple animal models of brain injury. In vitro studies have shown reduction in membrane blebbing and cellular swelling in isolated astrocytes following ATP depletion. In models of cerebral ischemia, such as middle cerebral occlusion, glibenclamide treatment led to approximately 50% reductions in cerebral edema, infarct volume, and mortality when compared with vehicle-treated rodents. Retrospective cohort analyses have also demonstrated improved outcomes in diabetic patients treated with sulfonylurea medications following ischemic stroke. More recently, these results have been extended to TBI. In rats undergoing CCI, SUR1/TRPM4 was shown to be upregulated within 3 hours following injury, predominantly within brain capillaries. Treatment with glibenclamide or small interfering RNA targeting SUR1 treatment leads to significant reductions in the progressive secondary hemorrhage of the contused brain. Noted reductions in capillary fragmentation and size of the necrotic lesion and improvements on neurobehavioral function, as assayed by vertical exploration or rearing, were observed. An independent group demonstrated reductions in posttraumatic edema and confirmed reductions in contusion size and subsequent evolution but failed to find differences in ICP or biochemical measures, including lactate, pyruvate, and glutamate or changes in motor function, as assessed by beam walking, over the 7 days of study. The source of the discrepancies between these studies is unclear and may reflect differences in experimental design or outcome parameters. Additional studies are needed in other TBI models to either confirm or refute these findings.
These promising results from animal studies prompted the glyburide advantage in malignant edema and stroke-pilot (GAMES-Pilot) trial. A phase I study confirmed the safety of intravenous glyburide administered as a bolus dose followed by 3-day infusion. The investigators have since initiated a multicenter, prospective, open-label phase IIa trial that completed data collection in June 2012. A secondary analysis of GAMES-Pilot subjects compared with controls from the Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET) and Multi Modal Imaging: MRI (MMI-Mri) trial showed improvement in functional measures as well as a trend toward lower mortality among the intervention group, although the study lacked power to demonstrate efficacy. A subsequent case-control study using the GAMES-Pilot subjects demonstrated that intravenous glyburide was associated with reduced surrogate markers of vasogenic edema (namely T2 fluid-attenuated inversion recovery intensity on MRI, apparent diffusion coefficient values, and MMP-9 levels) when compared with historical controls. Given these strong preliminary results, the GAMES-RP trial (phase II) is currently underway to better elucidate the potential efficacy of glyburide pharmacotherapy in ischemic stroke. A similar approach has recently been implemented to investigate the role of glyburide treatment in TBI in a phase II trial with completion of data collection in March 2015. Whether glyburide therapy will show benefit in human patients with TBI remains to be seen.
Related posts:
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Cerebral Metabolism and the Role of Glucose Control in Acute Traumatic Brain Injury
Seizures and the Role of Anticonvulsants After Traumatic Brain Injury
Past, Present, and Future of Traumatic Brain Injury Research
Role of Metabolomics in Traumatic Brain Injury Research
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