Traumatic brain injury (TBI) is the biggest cause of death and disability in the under 40s in the developed world (Langlois et al., 2006; Hyder et al., 2007), both in the civilian and military context. The overall majority of TBIs is a single event; however, repeated injuries among soldiers and athletes are associated with the development of Chronic Traumatic Encephalopathy (CTE) although the pathology of CTE can also be seen after single TBI and its causation remains uncertain (Omalu et al., 2011; Goldstein et al., 2012). Survivors often suffer from debilitating cognitive, emotional and physical impairments. This is further aggravated by the constant failure of clinical trials (reviewed in Loane and Faden, 2010; Gruenbaum et al., 2016), leaving patients without any treatment.

Brain injury can trigger neurodegeneration, which is a major determinant of long-term outcome. Head injury is a major risk factor for the development of dementia, suggested by the presence of amyloid-β (Aβ) plaques in around 30% of post-mortem brain tissue from TBI patients (Roberts et al., 1994). Additionally, amyloid plaques have been observed in surgically removed tissue surrounding contusions in brains of survivors of TBI (Ikonomovic et al., 2004; DeKosky et al., 2007). Recently, it has been shown by Positron Emission Tomography (PET) imaging that the distribution of amyloid plaque in TBI survivors overlaps with that in patients with Alzheimer's disease (AD) but also involves the cerebellum, an area of the brain not typically involved in AD (Scott et al., 2016). Other pathological features of AD are also present in TBI, including increased phosphorylated tau and acetylcholine deficiency (Jordan, 2000; Tran et al., 2011; Goldstein et al., 2012; McKee and Daneshvar, 2015; Shin and Dixon, 2015). In particular, repetitive mild TBI leading to CTE is characterized by perivascular tau pathology, which is also irregularly distributed in the depths of cortical sulci. Another AD susceptibility factor is the Apolipoprotein E isoform 4 (ApoE4), which has been linked with exiguous neurological outcome after TBI (Verghese et al., 2011). This has been associated with alterations in the integrity of the blood brain barrier (BBB) in individuals with ApoE4 genotype (Methia et al., 2001; Nishitsuji et al., 2011).

Additionally, TBI has been linked with Parkinson's disease (Jafari et al., 2013), various psychiatric disorders leading to increased risk of suicide, an overall increase in mortality (McMillan et al., 2011) and peripheral immune suppression (Hazeldine et al., 2015). There are currently no neuroprotective drug treatments available and a major challenge is to understand how brain trauma causes neurodegeneration and other long-term impairments.

A common feature of the neurological pathologies developing as a consequence of TBI is that they initiate and are potentiated by an inflammatory response. Microglial activation occurs early after experimental (Chiu et al., 2016) and human TBI (Ramlackhansingh et al., 2011; Johnson et al., 2013) and can persist for years, detectable both in vivo and post-mortem. Sites of activation often coincide with neuronal degeneration and axonal abnormality (Maxwell et al., 2010; Giunta et al., 2012). It was hypothesized that the inflammatory response to TBI therefore could be associated with the subsequent development of neurodegenerative disorders. However, new evidence indicates that glial activation may have also reparative/restorative effects. Thus, it is critical to investigate what causes this inflammatory response, what are the consequences for neuronal degeneration and survival and whether this can be modified with anti-inflammatory therapeutic approaches.

In this review, we analyse the role of microglia in TBI patients and animal models, from imaging studies used to visualize changes in microglia activation in vivo and ex vivo to studies in post-mortem brains of TBI patients and the prospects of therapies targeting microglia activation in TBI.

TBI occurs when brain structure and physiology are disrupted due to an extrinsic biomechanical insult to the cranium, resulting in neuronal, axonal and vascular damage. In response to TBI, the brain orchestrates a complex immunological tissue reaction similar to ischemic reperfusion injury (Werner and Engelhard, 2007). It was suggested that, as macrophages, microglia can migrate to the site of the injury, in order to establish a protective environment mitigating deleterious consequences of the injury (Faden et al., 2016). The acute function of microglia in response to TBI is to eradicate cellular and molecular debris. Microglial removal of damaged cells is a very important step in the restoration of the normal brain environment. Damaged cells release Danger-associated molecular patterns (DAMPs), which can become potent inflammatory stimuli, resulting in further tissue damage (Solito and Sastre, 2012; Zhang et al., 2012). Moreover, activated microglia are also capable of releasing noxious substances such as pro-inflammatory cytokines, reactive oxygen species (ROS), nitrogen species and excitatory neurotransmitters i.e., glutamate, which exacerbate damage (Kreutzberg, 1996). While pro-inflammatory cytokines are directly deleterious, they also stimulate the release of glutamate from microglia in an autocrine/paracrine fashion. Therefore, depending on the released amount, this can lead to direct neurotoxic effects on neurons, synapses and dendrites, through ionotropic glutamate receptors and interfere with the glutamate buffering ability of astrocytes by inhibiting astrocytic glutamate transporters (Takaki et al., 2012).

It was initially hypothesized that there is a temporary transition in function of the inflammatory milieu, which in the latter phases favors a protracted inflammatory profile associated with chronic microglial activation, precipitating neurological manifestations, although this view has been found to be far too simplistic.

As in other CNS injuries, it seems that microglia activation in TBI results in different phenotypes, corresponding to neurotoxic or neuroprotective priming states. Depending on the stage of the disease and the chronicity, microglia are stimulated differentially and this leads to particular activation states, which correspond to altered microglia morphology, gene expression and function. Microglia are activated in-situ by pro-inflammatory cytokines such as IFN-γ, IL-1α, IL-6, and TNF-α (Ziebell and Morganti-Kossmann, 2010) and become primed. Microglia morphology can switch from a “normal,” ramified shape to a hypertrophic, “bushy” morphology. In response to extensive tissue damage or pathogen invasion, microglia can change into an amoeboid morphology, primarily acting in a phagocytic/macrophage fashion and being difficult to differentiate from infiltrating macrophages (Figure 1). In general, these activation states are classified along a spectrum with M1 or “classically activated” at one end and M2 or “alternatively activated” and the opposite end, similar to macrophages. The proinflammatory M1 phenotype favors the production and release of cytokines that can exacerbate neural injury (Hu et al., 2015). In contrast, the M2 is associated with the release of neurotrophic factors that promote repair and a phagocytic role. However, in recent years, transcriptomic analysis has revealed that microglia and macrophages display a much broader transcriptional repertoire than M1 and M2, depending on the different environmental signals received (Hickman et al., 2013; Xue et al., 2014). Therefore, it is rare to find clear M1 or M2 microglial phenotypes in chronic diseases, since these states are transitory and eventually include mixed activation states. This seems particularly important in TBI as indicated by the many animal studies showing a mixed expression of different markers associated with both M1 and M2 phenotype (Table 1), also described as transitory state (Mtrans) by some authors (Kumar et al., 2016a). Additionally, the inevitable recruitment of bone marrow-derived monocytes into the injured brain parenchyma following experimental TBI and the subsequent differentiation into macrophages complicates the interpretation of the findings, especially with regard to the ability of these cells to change phenotype.

Additional complications arise from the clinical observation that outcome after TBI can be different between male and female adults and post-pubescent adolescents in some but not all studies (Coimbra et al., 2003), with females having lower mortality and less complications (Phelan et al., 2007; Berry et al., 2009; Ley et al., 2013). This can be attributed, at least partially, to astrocytes and microglia, as recently reviewed (Caplan et al., 2017). Gonadal steroid hormones such as progesterone and estradiol are directly involved in the glial response under injury conditions in and during development (Lenz and McCarthy, 2015). Moreover, as progesterone can shift macrophage polarization from M1 to M2 phenotype (Menzies et al., 2011), is seems possible that similar effects could occur in microglia as well, whether the concept of microglia polarization is accepted or not.

The polarization states of microglia could therefore be relevant in the progression of TBI into other neurological disorders such as AD and could interfere with the recovery of the patients and the effectiveness of particular anti-inflammatory treatment.

In addition, the role of infiltrating macrophages and other immune cells are also relevant in the context of microglia dynamics; injury type and parameters may play an important role, with focal models of TBI generally eliciting a more pronounced and localized inflammatory response due to significant tissue damage and pronounced infiltration of peripheral immune cells through the compromised BBB. In line with this, factors that have been implicated in BBB breakdown after injury, such as ApoE (mentioned above) and the extracellular enzymes matrix metalloproteinases (MMPs) have been found to contribute to contusion expansion and vasogenic edema after TBI (Guilfoyle et al., 2015).

In the past five decades, several animal models have been developed to characterize the biomechanical and pathophysiological patterns of TBI and to provide reliably ways to test treatment strategies. These models almost exclusively employ a single-hit injury and only in the past decade repeated injury models emerged that try to mimic the effect of repetitive injuries. The different animal models address distinct pathophysiological aspects of TBI and have different etiological and construct validity (O'Connor et al., 2011). The most widely applied models of TBI are open-head models, that induce an injury via the intact dura mater upon the underlying cortical tissue (reviewed in Xiong et al., 2013), with focal, diffuse and/or mixed injury patterns, depending on the type of injury. In order to do so, rigid impactors (Controlled Cortical Impact, CCI; weight drop, WD), fluidic pressure (Fluid Percussion Injury, FPI) and blast waves are employed. Additionally, high-velocity probes or projectiles are used to model penetrating brain injury. The CCI, FPI and the various WD models have a long history and were extensively characterized in the past, including assessment of long-term outcome (Kochanek et al., 2002; Immonen et al., 2009).

With the recognition that multiple head injuries, even of sub-threshold severity, are connected to chronic sequel, such as CTE (McKee et al., 2016; Vile and Atkinson, 2017), new models were developed to account for those findings. A growing number of studies in repeated injury rodent models (Shitaka et al., 2011; Klemenhagen et al., 2013; Mannix et al., 2014; Semple et al., 2016; Robinson et al., 2017) indicate that the glial response, in particular increases in microglia numbers and changes in morphology, shares an overall similar pattern with the traditional single-injury models. Interestingly, a few studies showed that repeated injuries can exacerbate the glial response, e.g., microglia or astrocyte cell density, as compared to a single injury (Ojo et al., 2013; Petraglia et al., 2014; Xu et al., 2016; Gao et al., 2017; Tyburski et al., 2017). Recent findings in human post-mortem tissue support the overall notion that repeated injuries result in a stronger glial response by showing that repeated head injury was a predictor of CD68 cell density and phosphorylated tau (Cherry et al., 2016).

The involvement of microglia in the complex pathophysiological cascade following brain injury has long been recognized. Shortly after the description of microglia by Del Rio-Hortega, accumulation of “microglia-like” cells were reported in the tissue around autologous blood injections and a cortical stab wound (Carmichael, 1929; Dunning and Stevenson, 1934) in the rabbit. Similar to reactive astrocytes, microglia are versatile cells, which is exemplified by their heterogeneous morphology (Boche et al., 2013). Perivascular microglia respond to pressure on the thinned mouse skull (Roth et al., 2014) by changing into jellyfish morphology; CCI causes some microglia to align with axon initial segment, the site where action potentials are generated (Baalman et al., 2015) and midline FPI yields several rod-like microglia (Ziebell et al., 2012).

Depending on the employed markers and methods, significantly increasing microglia activation in animal models of TBI is usually apparent from day 1 to 3 post-injury (p.i.) (Bye et al., 2007; Elliott et al., 2011) and can persist chronically beyond 28 days p.i., a usual cut-off time-point for many animal studies. A few studies have elegantly emphasized the long-term nature of microglia activation after experimental TBI, primarily with immunohistochemical analysis showing that major histocompatibility complex (MHC)-II positive cells were still present in the ipsilateral hemisphere at 3 months after WD injury (Holmin and Mathiesen, 1999). Recently, a significant loss of ramified, but increase of hypertrophic microglia in the injured rodent cortex at 1 year after moderate CCI in mice was demonstrated (Loane et al., 2014a). This suggests that, although microglial activation occurs early after TBI, it can change the phenotype and function over time (see Table 1).

Measurement of microglial activation in vivo has become possible using PET and Single Photon Emission Computed Tomography (SPECT) due to the development of radioligands that bind to the 18-kDa translocator protein (TSPO). TSPO is a five-transmembrane domain protein localized in the outer mitochondrial membrane (Jaremko et al., 2014). The best studied putative function of TSPO relates to its role in transporting cholesterol into the mitochondrial inner membrane space, the rate-limiting step of steroid and neurosteroid biosynthesis (Papadopoulos et al., 2006). However, this function has been lately questioned due to the findings from knockout mice that did not show abnormal steroidogenesis (Morohaku et al., 2014; Tu et al., 2014). Despite almost 40 years of study, the precise functional role of TSPO is far from clear (Selvaraj and Stocco, 2015).

The relevance of microglia in the general pathophysiological response to TBI is recognized as a potential therapeutic avenue (reviewed in Chio et al., 2015) and has prompted several studies to investigate the effects of certain drugs on microglial polarization in brain injury models. Although studies in animal models indicate that microglia/macrophages respond to TBI with a transient M2 phenotype, followed by a shift to M1, and that the number of M1 cells is strongly correlated with the severity of white matter injury (Koh and DiPietro, 2011), this may differ in non-human and human primates. Therefore, therapies that prime microglia/macrophages toward the beneficial M2 phenotype after TBI may offer new anti-inflammatory strategies (Table 2). Several of these drugs, including minocycline, minozac, etanercept and the PPAR agonists fenofibrate and pioglitazone are FDA approved (Garrido-Mesa et al., 2013; Kim et al., 2014) confirming their safety and tolerability.

In recent years, there has been a growing interest in investigating the activation of microglia in TBI, because of the potential role in the progression of those patients to neurodegenerative and psychiatric diseases. The studies of microglia in animal models of TBI have allowed the manipulation of microglial numbers (by genetic or pharmacological ablation), indicating their important role in neuroprotection especially at early times post-injury, although this sequence seems to be model and species-specific. In addition, this has provided insights into the time course of the different profile of microglial activation phenotypes in the injury site and the spreading of inflammation to other areas of the brain, such as the thalamus.

In vivo molecular imaging of TSPO potentially provides an extremely useful biomarker of microglial activation and the effect of immunomodulatory drugs. In TBI patients, it has allowed the observation of chronically activated microglia located in areas of persistent white matter damage, even long time post injury. However, it currently only provides a one-dimensional measure, i.e., the amount of microglial activation within a particular brain region. It is clearly too simplistic to describe microglial activation in vivo along a single dimension (i.e., from “low activity” to “high activity”). Crucially, on the basis of current evidence, TSPO molecular imaging cannot discriminate activation phenotype, but probably reflects the (potentially uneven) summation of activity from microglia across all functional states.

In addition, there has been some debate regarding the possibility that certain TSPO ligands also recognize astrocytes or other immune cells in the brains of TBI patients. Other limitations of the application of second-generation ligands include that the binding affinities are influenced by a common polymorphism (rs6971) in the TSPO gene which causes a single amino acid substitution (A147T) in the protein (Owen et al., 2012). Because 147T TSPO binds ligands with lower affinity than 147A, this produces three classes of binding affinity across a population, which studies must therefore control for. Even after accounting for TSPO genotype, however, many second-generation TSPO ligands show high between-subject variability in uptake when using analysis methods which rely on measurement of the ligand in arterial blood (Guo et al., 2012). As for [¹¹C]PK11195, high and variable plasma protein binding may be a factor (Lockhart et al., 2003). Methods of analysis such as the simplified reference tissue model (SRTM) have been developed that do not require arterial blood data, but rather use the PET imaging data from a reference region (or reference tissue) instead (Gunn et al., 1997).

To improve the interpretation of the TSPO signal, a detailed characterization is needed of how TSPO expression in humans varies with the diversity of microglial phenotypes seen in vivo. Future TBI work needs to provide a description of the time-course of microglial phenotype change after TBI and its relationship to TSPO expression. Novel PET ligands showing specificity for distinct functional subtypes of microglial activation would be of great utility, with cannabinoid type 2 and purinergic receptors, such as P2Y12 and P2X7, possibly providing suitable targets in the future. Another option is to combine TSPO PET imaging with different biomarkers that disambiguate the TSPO signal in a particular context, in particular neurofilament light (NFL). NFL levels in CSF have been proposed as a valid biomarker to accurately assess the level of trauma and predict the clinical outcome of the patient (Zhang et al., 2016).

Recent investigations have indicated that simple suppression of microglia activation can exert only limited beneficial effect and the inhibition of M1-like responses might be detrimental similar to a simple stimulation of M2-like phenotypes, as indicated by increased fibrosis which seems modulated by Arginase-1 in peripheral and central infection (Hesse et al., 2001; Aldrich and Kielian, 2011). In addition, pharmacological treatment with the anti-inflammatory drugs tested in several models may not have similar effects when administered in TBI patients, as seen in clinical trials for AD (Lleo et al., 2007), limiting the potential therapeutic impact. It seems therefore imperative for future studies that target microglia polarization as therapeutic strategies, to assess several markers in the target cells within a sufficient temporal window in order to show a long-term positive outcome.

MS wrote most of the introduction, the manipulation of microglia in animal models and the treatments section; CD wrote most of the section on animal models of TBI and preclinical TSPO imaging in animal models, made the tables, the references and wrote part of the conclusions; GS wrote most of the part of the imaging with TSPO, made two figures and part of the conclusions; SG contributed with the image of microglia (Figure 1) and edited the manuscript.