Traumatic brain injury (TBI) is characterized by physical trauma to the brain caused by biomechanical forces in the form of violent impact, blow, or jolt, causing the brain to strike against the inside of the skull, or when an object is thrust through the skull and subsequently damages brain tissue (Blennow, Hardy, & Zetterberg, 2012; Hernandez-Ontiveros et al., 2013). Cases of TBI can be characterized by severity, anatomical areas affected, and the forces that caused them, with symptoms varying from mild to moderate to severe (Hernandez-Ontiveros et al., 2013). Mild symptoms include dizziness, nausea, confusion, headache, blurred vision, ringing of the ears, fatigue, change in sleep patterns, behavioral or mood changes, and reduced memory, concentration, attention, or thinking (Blennow et al., 2012; Hernandez-Ontiveros et al., 2013). Loss of consciousness may also occur. As the name suggests, the symptoms of moderate to severe TBI are even more serious, including intermittent headaches, repeated vomiting or nausea, seizures, inability to awaken, dilation of the pupils, slurred speech, weakness or numbness in the extremities, loss of coordination, increased confusion, restlessness, agitation, and attention and memory deficits (Hernandez-Ontiveros et al., 2013). The most serious of symptoms include intracranial bleeding from torn tissue, cerebral contusions, massive edema, alterations of endogenous neurotransmitter mechanisms, and possible death (Blennow et al., 2012; Hernandez-Ontiveros et al., 2013).
The general physiological response to TBI following the cascade of biochemical reactions caused by mechanical impact involves an immense immune response, plus neurodegeneration with secondary repair. The innate immune response is activated by damage to the system that transports oxygen, nutrients, and other important substances to the brain to ensure its proper functioning, as well as damage to neural cell bodies. Massive influx of leukocytes capable of producing antibodies or responding to a cell-mediated immune reaction occurs simultaneously with this innate immune response. Following this is the production of proinflammatory cytokines, which promote an inflammatory response around the site of injury, and prostaglandin, a lipid compound which regulates these inflammatory effects. The adaptive immune response then begins, promoting the production of antibodies and proliferation of a subtype of white blood cells involved in a diverse array of processes during the immune response, called lymphocytes. As both the innate and adaptive immune responses greatly influence the speed of tissue repair, cytokines and other immune cells secreted during these responses have served as a significant target for pharmaceuticals to favor tissue restoration (Kelso & Gendelman, 2014).TBI affects up to 1.7 million Americans every year, with 235,000 hospitalized for non-fatal TBI, 1.1 million treated in emergency rooms, and 50,000 cases resulting in death, on average (Hernandez-Ontiveros et al., 2013; Kelso & Gendelman, 2014). Approximately 43.3% of Americans treated for TBI have consequential disability one year following injury, adding up to as many as 5.3 million living with disability after hospitalization with TBI in the United States (Hernandez-Ontiveros et al., 2013). TBI has recently come to the forefront of public consciousness due to increasing numbers of wounded soldiers returning from wars in the Middle East, as well as increasing numbers of lawsuits surrounding TBI caused by traumatic impact in sports like boxing and hockey, especially in the National Football League (NFL) (Blennow et al., 2012; Kelso & Gendelman, 2014). Because of this, it is imperative to understand the pathophysiology of acute neurological injury as a result of TBI.
The diagnostic measures for TBI are still quite primitive, with only gross, very broad criteria to categorize the stage and progression of the condition. Further, once the condition has been characterized, there still exists a large dearth of prognostic methods through which to predict patient outcomes. However, cytokines show great promise in improving these gaps in medical knowledge, both as diagnostic and prognostic tools. Not only can this understanding lead to revolutionary methods in axonal repair and regrowth as a result of physical brain trauma; there are also a significant number of crossovers with degenerative neurological diseases such as Alzheimer’s and Parkinson’s diseases (AD and PD, respectively), amyotrophic lateral sclerosis (ALS), stroke, and Huntington’s disease (Kelso & Gendelman, 2014).
Types of TBI
Among medical professionals, there is little agreement as to how to define or diagnose concussion. One internal consensus statement describes concussion as “a complex pathophysiological process affecting the brain, induced by traumatic biomechanical forces” (Blennow et al., 2012). In general, no abnormalities can be seen on structural brain imaging in the case of a concussion. No loss of consciousness occurs from mild concussion; however, more severe cases may involve prolonged periods of unconsciousness. This type of brain injury resulting from physical trauma is common in sports like football, particularly in lineman and linebackers who may be exposed to upwards of 1,000 impacts per season. Transient symptoms of concussion manifest in what is clinically recognized as postconcussion syndrome (PCS) in 40-80% of all cases (Blennow et al., 2012).
Catastrophic Brain Injury
Catastrophic brain injury refers to acute severe brain injury, with intracranial bleeding or cerebral contusions which may result in death. The most common cause of death as a result of catastrophic brain injury is subdural hematoma, during which blood gathers between the dura mater and the brain, increasing intracranial pressure (ICP) and damaging delicate brain tissue to a fatal degree. During the second half of the 20th century, over 400 football players died from catastrophic brain injury in the United States (Blennow et al., 2012).
Chronic Traumatic Encephalopathy
Chronic Traumatic Encephalopathy, commonly known as CTE, was first described by H.A.S. Martland in 1928 as “punch drunk syndrome” in boxers who had experienced chronic brain damage. J.A. Millspaugh later described the condition as “demential pugilistica” in 1937, which is more commonly used. This condition is characterized by neurofibrillary tangles in cortical areas, atrophy in the cerebellum, gliosis (nonspecific reactive change of glial cells in response to CNS damage), hypopigmentation of the substantia nigra, and cavum septum pellucidum. CTE is officially described as a chronic brain syndrome/neurodegenerative disorder due to effects of repetitive brain trauma that typically occurs during midlife. However, there are no generally accepted guidelines for clinical diagnosis to differentiate CTE’s neuropathological effects from those of aging or AD. One-third of CTE cases are progressive in nature. Symptoms vary extensively, most likely due to the wide variety of areas affected during injury, and generally involve neurological and cognitive complaints paired with psychiatric and behavioral disturbances. CTE has recently gained attention in the NFL as retired players have come forth with stories and lawsuits regarding their experiences with CTE as a result of repeated brain trauma from physical impact (Blennow et al., 2012).
The Innate Immune Response
In general, two subtypes of the immune system exist: the innate immune system, and the adaptive immune system. Innate immunity provides the body’s first-line defense against both invading pathogens as well as injuries that occur as a result of events such as traumatic brain injury. The immune system does this by recognizing and responding to pathogens in a nonspecific way, marked by significant upregulation of proinflammatory cytokines. However, unlike the adaptive immune response, innate immunity provides only a temporary defense against pathogens and trauma.
Immediately after a pathogen enters the body or trauma occurs, damage-associated molecules are recruited, activating microglia and astrocytes, which trigger an immune response cascade. The innate immune system is generally responsible for recruiting these types of immune cells to sites of infection and inflammation, accomplished through the synthesis and secretion of cytokines. Following this, factors which amplify the inflammatory response are activated to identify bacteria, activate further immune cells, and promote clearance of cell debris and antibody complexes. Other actions of the innate immune response include the identification and removal of foreign bodies by leukocytes; providing a barrier, both physical and chemical, to infectious agents; and eventually activating the adaptive immune system to provide a more long-term, specific response to the original source of inflammation (Kelso & Gendelman, 2014)..
The nonspecific inflammation characteristic of the innate immune response is stimulated by way of a collective group of immune cells called mononuclear phagocytes. These cells include blood-borne monocyte-derived macrophages and neutrophils, which generally promote inflammation during the period of time in which the innate immune system is active. Monocyte-derived macrophages are specialized cells which clear cellular debris following inflammation, and as the term implies, are derived from monocytes, an important type of white blood cell. Overall, over 100 genes related to inflammation are significantly upregulated immediately following brain trauma, making evident the complexity of this initial immune response (Kelso & Gendelman, 2014).
Similar to monocyte-derived macrophages, neutrophils are also a type of white blood cell, and are the most prevalent type of leukocyte in the blood. Generally, neutrophils are the first cells that enter the site of neuronal injury after TBI, appearing within only five minutes of initial impact. After only two hours, they peak in numbers in both the subarachnoid and subdural spaces of the brain. These components of the innate immune response following TBI are highly mobile, allowing them to quickly enter tissue to destroy foreign material. Infiltration of tissue by neutrophils is enabled by a complex set of mechanisms beginning with the release of proinflammatory cytokines mentioned above. These chemical messengers promote the binding of integrins to adhesion molecules on the surface of neutrophils in order to form initial contact, and then to intracellular adhesion molecules to securely form adhesion. Once established, neutrophils migrate across the endothelium to inflammatory sites in damaged tissue and proceed with their phagocytic and clearance duties (Kelso & Gendelman, 2014).
As soon as they are present in brain tissue, neutrophils may continue to promote infiltration of damaged cells by secreting vascular endothelial growth factor (VEGF). VEGF is a mediator of the production and secretion of chemokines, as well as the blood-brain barrier (BBB) disruption and worsening edema that occurs following physical trauma. Neutrophil recruitment is overall a significant component early in the TBI pathogenesis (Kelso & Gendelman, 2014). Though neutrophils themselves are likely not a useful biomarker of TBI, as they disappear quickly after the initial innate immune response, their interactions with certain pro- and anti-inflammatory cytokines show promise in the future characterization and diagnosis of traumatic brain injury.
Microglia are an equally significant component of the innate immune response. After neuronal injury and disruption of the BBB following impact, microglia are activated within minutes; in fact, they are one of the first immune cells recruited during this proinflammatory phase, along with neutrophils and astrocytes. Microglia generally display three different phenotypes: (1) a resting phenotype, (2) an activated phenotype that is found in areas involved in CNS inflammation, and (3) a reactive phenotype which is present in areas of trauma or infection in general. Additionally, microglia polarize into two major subtypes: M1, the “classically activated” subtype, and M2, the “alternately activated” subtype (Hernandez-Ontiveros et al., 2013). Depending on the subtype activated, microglia can have either neuroprotective or cytotoxic effects (Tang & Le, 2015). In addition to the “Yin and Yang” paradigm of these microglial subtype functions, these two subtypes also differ by the pro- and/or anti-inflammatory cytokines that they secrete.
M1 microglia are the archetypal cell of innate immunity in response to injury and invasion of pathogens. As such, they tend to promote inflammation with the excessive secretion of proinflammatory cytokines and predominate in the location of injury (Tang & Le, 2015). They are marked by high production of cytokines IFNγ (interferon-gamma), TNFα (tumor necrosis factor-alpha), IL-1 (interleukin-1), IL-8, and IL-12; superoxide; nitric oxide (NO); and reactive oxygen species (Hernandez-Ontiveros et al., 2013; Tang & Le, 2015). Though these chemical signals aid in the rapid recruiting of further immune cells to respond to injuries like TBI, they have neurotoxic effects and promote a malicious cycle between dying neurons and acute inflammation as a result. This classically activated phenotype is associated with conditions thought to be chronic neuroinflammatory diseases as well, such as type 2 diabetes (Tang & Le, 2015).
In contrast with pro-inflammatory and neurotoxic M1 microglia, M2 microglia are immunosuppressive and reparative in nature, tending to ameliorate inflammation and clear cellular debris. This anti-inflammatory subtype is rapidly activated following the onset of classically-activated M1 microglia, leading to healing at the site of injury, return to tissue homeostasis, and reparation of gene expression. These remedial properties are caused by the secretion of anti-inflammatory cytokines, including IL-4, IL-13, IL-10, and TGFβ (transforming growth factor-beta) (Tang & Le, 2015; Hernandez-Ontiveros et al., 2013). These cytokines antagonize those secreted by M1 microglia, thus suppressing inflammatory cytokine signaling: for example, IL-4 and IL-13 secreted by M2 microglia likely counteract the effects induced by pro-inflammatory cytokines such as IL-8, IL-6, and TNFα produced by M1 microglia. M2 microglia are also capable of reducing NO release, preventing neuronal injury. In addition to the secretion of anti-inflammatory cytokines, M2 microglia are also able to enhance the release of neurotrophic factors, a family of proteins responsible for the growth and survival of neurons. As M2 microglia have the great potential to repair and possibly reverse neuronal damage, much research has been invested in ways to harness the capabilities of these cells in order to repair damage caused by blunt force trauma as a result of TBI, as well as neurodegenerative diseases such as multiple sclerosis and AD.
Inflammatory Cytokines as Biomarkers for TBI
Overall, activation of the immune system in the CNS is being increasingly recognized as a key part of the onset and progression of numerous neurological disorders in addition to TBI, such as AD and PD. Though there are many commonly used biomarkers to characterize and diagnose TBI, such as neuron-specific enolase (NSE), myelin basic protein (MBP), and S-100 protein B (S100B), S100B is the most studied biomarker for TBI pathogenesis. It is true that there exists a strong correlation between S100B levels and severity of TBI, as well as a link between elevated S100B expression and unfavorable outcomes; however, there are factors which still limit the ability of this biomarker to be used as a diagnostic and prognostic tool. For one, S100B does not easily cross the BBB. In addition, serum levels of S100B increase after peripheral trauma, even in the absence of brain injury, and it is has been shown to not always be a reliable predictor of outcome (Woodcock & Morganti-Kossmann, 2013). As such, a large part of TBI pathology currently being investigated is the collective group of proteins produced by blood leukocytes and glial cells in response to TBI, known as cytokines, which are secreted in response to neuronal injury. Inflammatory cytokines hold the potential to provide information about the extent of tissue damage, and are easily and readily measured with immunological assays (Woodcock & Morganti-Kossmann, 2013). However, the use of these mediators as diagnostic factors is in need of further substantiation, as they are not widely accepted as a method by which to anticipate and predict symptoms indicative of poor outcome of TBI, such as elevated ICP (Woodcock & Morganti-Kossmann, 2013). The evidence favoring the use of a few of these cytokines for the characterization and diagnosis of TBI is reviewed below.
TNFα is a potent pro-inflammatory cytokine produced by microglia and astrocytes, though it can be an anti-inflammatory cytokine as well. In general, both laboratory and clinical experiments show that increased expression of TNF is detrimental. However, one study has shown that mortality rates increased and long-term recovery was impaired in TNF receptor knock-out mice, a conflicting result which reveals the dual role of TNF as a pro- and anti-inflammatory cytokine (Woodcock & Morganti-Kossmann, 2013).
In laboratory studies modeling TBI in rats, TNF mRNA has been detected prior to the cytokine protein itself in the injured rat brain, showing that upregulation of TNF precedes infiltration of leukocytes at the location of injury. This could prove useful in a clinical setting both as a diagnostic and prognostic factor, especially as TNF has been shown to reflect injury severity. For example, for severe injury, significant increases in TNF are observed, but for mild injury, no change in TNF is seen. There is conflicting evidence as to whether there is increased expression of TNF in brain homogenates over the course of 24 hours in the closed brain injury model of focal TBI, however, as well as observed differences in the timing of TNF peaks depending on brain region. Additionally, no correlation has been found between TNF and the Glasgow Coma Scale (GCS), ICP, or neurological outcome in humans, once again revealing conflicting results that need further data for verification (Woodcock & Morganti-Kossmann, 2013).
Like TNFα, IL-10 can act as both a pro- and anti-inflammatory cytokine; however, its properties are primarily anti-inflammatory. It has potent inhibitory effects on the production of several pro-inflammatory cytokines and mediators, such as IL-1β and TNF. This is the most important function of IL-10, as these cytokines are known to play a key role in the launch and proliferation of inflammation in response to brain trauma (Woodcock & Morganti-Kossmann, 2013).
In laboratory investigations, rats with brain injury who were treated with IL-10 showed improved outcomes, as well as reduced levels of IL-1β and TNF in brain tissues. Similarly, clinical studies have shown elevated levels of IL-10 in the CSF and serum of patients with isolated, severe TBI, possibly suggesting that as injury becomes more severe, levels of IL-10 increase in an effort to ameliorate inflammation. However, other studies have found no correlation between BBB function and IL-10 levels.
In general, IL-10 expression increases early in the pathogenesis of brain trauma, reaching peak levels within 2-8 hours of injury. Higher levels have been linked to better outcomes in some studies, proposing possible prognostic utility; however, not all studies have shown this link. Additionally, some studies found no correlation between IL-10 and outcome, emphasizing the need for more research to better characterize the injury-specific mechanisms by which IL-10 acts in response to trauma (Woodcock & Morganti-Kossmann, 2013).
IL-8 is a pro-inflammatory cytokine part of the CXC chemokine family, and is secreted by glial cells, macrophages, and endothelial cells. It is an important mediator in the activation and movement of neutrophils in response to chemical signals. In addition, IL-8 is released from astrocytes in response to other cytokines like IL-1β and TNF, as both are expressed early after initial injury. Expression of IL-8 has been shown in many other conditions besides TBI, including some cancers, bacterial infections, and cardiovascular disease. This suggests further research of IL-8 may provide a biomarker that is useful for many clinical conditions besides TBI (Woodcock & Morganti-Kossmann, 2013).
In healthy humans, IL-8 is detected at very low levels. Following TBI, however, there is a significant increase in IL-8 concentration in the CSF, with levels appearing to peak early in the TBI pathogenesis. In plasma, increased levels of IL-8 following TBI have been reported as well; however, these increases are more variable and are lower in magnitude compared to increases seen in the CSF (Woodcock & Morganti-Kossmann, 2013).
There appears to be prognostic value for IL-8 in the context of the GCS, patient age, and the Acute Physiological and Chronic Health Evaluation (APACHE II). Serum biomarker levels are associated with unfavorable outcomes 6 months later, and serum concentrations of IL-8 72 hours after injury are significantly higher in those who do not survive brain trauma compared to those who do. In general, studies have shown that increased IL-8 in the CSF is associated with mortality, further suggesting IL-8 as a useful prognostic biomarker (Woodcock & Morganti-Kossmann, 2013).
Traumatic brain injury is a serious public health issue, affecting up to 1.7 million Americans every year (Hernandez-Ontiveros et al., 2013). These numbers are steadily increasing worldwide due to the return of wounded soldiers from war and increasing numbers of sports-related head injuries. However, there is still a severe lack of effective ways to accurately characterize and diagnose TBI and to predict patient outcomes.
Cytokines and other immune mediators secreted by fast-acting immune cells such as microglia and astrocytes have shown promise as both diagnostic and prognostic biomarkers for TBI. In order to identify potential biomarkers, researchers have been increasingly studying pro-inflammatory cytokines, such as IFNγ, TNFα, IL-1, and IL-12, as well as anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, and TGFβ, secreted immediately following initial injury. TNFα, IL-10, and IL-8 currently show the most promise as biomarkers for TBI pathogenesis, though there are often conflicting results between laboratory and clinical evidence for the utility of these as well as other cytokines released during the immune response. Because of this, further research is needed to better identify the role of these immune mediators so as to better determine their utility as diagnostic and prognostic methods through which to characterize TBI. Not only will this benefit victims of traumatic brain injury, but also those of many other neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s Disease, and multiple sclerosis.