Brain, Behavior, and Immunity 26 (2012) 1191–1201

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Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Invited Review

Neuroinflammation after traumatic brain injury: Opportunities

for therapeutic intervention
Alok Kumar, David J. Loane ⇑
Department of Anesthesiology & Center for Shock, Trauma and Anesthesiology Research (STAR), National Study Center for Trauma and EMS,
University of Maryland School of Medicine, Baltimore, MD, United States

a r t i c l e i n f o a b s t r a c t

Article history: Traumatic brain injury (TBI) remains one of the leading causes of mortality and morbidity worldwide, yet
Received 5 April 2012 despite extensive efforts to develop neuroprotective therapies for this devastating disorder there have
Received in revised form 27 May 2012 been no successful outcomes in human clinical trials to date. Following the primary mechanical insult
Accepted 14 June 2012
TBI results in delayed secondary injury events due to neurochemical, metabolic and cellular changes that
Available online 21 June 2012
account for many of the neurological deficits observed after TBI. The development of secondary injury
represents a window of opportunity for therapeutic intervention to prevent progressive tissue damage
Keywords:
and loss of function after injury. To establish effective neuroprotective treatments for TBI it is essential
Traumatic brain injury
Neuroinflammation
to fully understand the complex cellular and molecular events that contribute to secondary injury. Neur-
Neuroprotection oinflammation is well established as a key secondary injury mechanism after TBI, and it has been long
Microglia considered to contribute to the damage sustained following brain injury. However, experimental and
Chronic neurodegeneration clinical research indicates that neuroinflammation after TBI can have both detrimental and beneficial
effects, and these likely differ in the acute and delayed phases after injury. The key to developing future
anti-inflammatory based neuroprotective treatments for TBI is to minimize the detrimental and neuro-
toxic effects of neuroinflammation while promoting the beneficial and neurotrophic effects, thereby cre-
ating optimal conditions for regeneration and repair after injury. This review outlines how post-traumatic
neuroinflammation contributes to secondary injury after TBI, and discusses the complex and varied
responses of the primary innate immune cells of the brain, microglia, to injury. In addition, emerging
experimental anti-inflammatory and multipotential drug treatment strategies for TBI are discussed, as
well as some of the challenges faced by the research community to translate promising neuroprotective
drug treatments to the clinic.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction increase as a result of widespread motor vehicle use (Maas et al.,

Traumatic brain injury (TBI) is associated with significant mor-
bidity and mortality; this devastating disorder has substantial di-
rect, and indirect, costs to society. The Center for Disease Control
and Prevention (CDC) estimate that more than 1.7 million individ-
uals in the United States suffer a TBI annually (Faul et al., 2010).
These numbers, however, greatly underestimate the real incidence,
and costs, of TBI as the CDC data does not include reports of sports-
related concussions or repeated mild TBI from military conflict
zones. Globally, the incidence of TBI is also increasing, particularly
in developing countries where road traffic accidents are on the
⇑ Corresponding author. Address: Department of Anesthesiology and Center for
Shock, Trauma & Anesthesiology Research (STAR), University of Maryland School of
Medicine, 655, West Baltimore Street, Suite #6-011, Baltimore, MD 21201, United
States. Tel.: +1 410 706 5188.
E-mail address: dloane@anes.umm.edu (D.J. Loane).
2008).
TBI is a highly complex disorder that is caused by both primary
and secondary injury mechanisms (Loane and Faden, 2010;
McIntosh et al., 1996). Primary injury mechanisms result from
the mechanical damage that occurs at the time of trauma to neu-
rons, axons, glia and blood vessels as a result of shearing, tearing
or stretching. Collectively, these effects induce secondary injury
mechanisms that evolve over minutes to days and even months
after the initial traumatic insult and result from delayed neuro-
chemical, metabolic and cellular changes. These secondary injury
events are thought to account for the development of many of
the neurological deficits observed after TBI (McIntosh et al.,
1996), and their delayed nature suggests that there is a window
for therapeutic intervention (pharmacological or other) to prevent
progressive tissue damage and improve functional recovery after
injury.
Secondary injury mechanisms include a wide variety of
processes such as depolarizations and disturbances of ionic

0889-1591/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbi.2012.06.008
1192 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201
homeostasis (Gentile and McIntosh, 1993), release of neurotrans-
mitters (e.g. glutamate excitotoxicity) (Faden et al., 1989), mito-
chondrial dysfunction (Xiong et al., 1997), neuronal apoptosis
(Yakovlev et al., 1997), lipid degradation (Hall et al., 2004), and ini-
tiation of inflammatory and immune responses (Morganti-Koss-
mann et al., 2007), among others. These neurochemical events
generate a host of toxic and pro-inflammatory molecules such as
prostaglandins, oxidative metabolites, chemokines and pro-inflam-
matory cytokines, which lead to lipid peroxidation, blood–brain
barrier (BBB) disruption and the development of cerebral edema.
The associated increase in intracranial pressure can contribute to
local hypoxia and ischemia, secondary hemorrhage and herniation
and additional neuronal cell death via necrotic and apoptotic
mechanisms (McIntosh et al., 1996). Although each secondary in-
jury mechanism is often considered to be a distinct event, many
are highly interactive and may occur in parallel.
Considerable research efforts have sought to elucidate second-
ary injury mechanisms in order to develop neuroprotective treat-
ments. Although preclinical studies have suggested many
promising pharmacological treatments, more than 30 phase III pro-
spective clinical trials have failed to show significance for their pri-
mary endpoint (Maas et al., 2010). Most of these trials targeted
single secondary injury mechanisms, but given the multifactorial
nature of the secondary injury process targeting a single factor will
unlikely result in significant improvements in outcome. The com-
plexity and diversity of secondary injury mechanisms have led to
calls to target multiple delayed secondary injury mechanisms,
either by combining agents that have complementary effects or
by using multipotential drugs that modulate multiple injury mech-
anisms (Loane and Faden, 2010; Margulies and Hicks, 2009; Vink
and Nimmo, 2009). This recognition has led to the recent emphasis
on multipotential drug treatments, several of which are now in
clinical trials for human head injury (Vink and Nimmo, 2009). His-
torically neuroprotection treatments for TBI have been dominated
by a neuronocentric view, in which modification of neuronal based
injury mechanisms is the primary or even exclusive focus of the
neuroprotective strategy. However, it is well established that neur-
oinflammation represents a key pathological response to brain in-
jury, and the important role that non-neuronal cells, such as
endothelial cells, astrocytes, microglia, oligodendrocytes, play in
secondary injury-mediated responses is becoming increasingly
recognized (Floyd and Lyeth, 2007; Loane and Byrnes, 2010;
Simard et al., 2010; Ziebell and Morganti-Kossmann, 2010).
In this review we will discuss neuroinflammation as a key sec-
ondary injury mechanism in TBI before focusing on the complex
and varied responses of microglia in terms of their detrimental
and beneficial effects after injury. In addition, we will describe
emerging experimental anti-inflammatory and multipotential drug
treatment strategies that show considerable promise for the treat-
ment of human TBI, and we will discuss some of the challenges fac-
ing basic and clinical researchers in translating novel drug
treatment strategies from the bench to the bedside.
2. The neuroinflammatory response to traumatic brain injury
Neuroinflammation is an important secondary injury mecha-
nism that contributes to on-going neurodegeneration and neuro-
logical impairments associated with TBI. Post-traumatic
neuroinflammation is characterized by glial cell activation, leuko-
cyte recruitment, and upregulation of inflammatory mediators
(Morganti-Kossmann et al., 2007). Although much research has
focused on the detrimental effects of neuroinflammation on the
injured brain, clear beneficial effects can be achieved if neuroin-
flammation is controlled in a regulated manner and for defined
periods of time.
Trauma to the brain results in rupture of the BBB, enabling
recruitment of circulating neutrophils, macrophages and lympho-
cytes to the injured site. The accumulation of blood-born immune
cells within the brain parenchyma has been reported in human TBI
as well as animal models of brain trauma (Morganti-Kossmann
et al., 2001). These cells release inflammatory mediators that mobi-
lize glia and immune cells to the site of injury. In addition to the
infiltration of immune cells, the activation of resident microglia
play a major role in the response to brain injury (Loane and Byrnes,
2010). Elegant in vivo two-photon microscopy imaging studies of
fluorescently labeled microglia following a laser-induced injury
demonstrated rapid proliferation and movement of ramified
microglial cells to the site of injury in response to extracellular
ATP released by the injured tissue (Davalos et al., 2005; Haynes
et al., 2006). The microglial processes then fused to form an area
of containment between healthy and injured tissues, suggesting
that microglia may represent the first line of defense following
traumatic injury (Davalos et al., 2005). However, when microglia
become over-activated or reactive they can induce detrimental
neurotoxic effects by releasing multiple cytotoxic substances,
including pro-inflammatory cytokines (e.g. interleukin (IL)-1b, tu-
mor necrosis factor-a (TNFa), and interferon-c (IFNc)) and oxida-
tive metabolites (e.g. nitric oxide, reactive oxygen and nitrogen
species) (Block and Hong, 2005). Further, the release of pro-inflam-
matory cytokines and other soluble factors by activated microglia
can significantly influence the subsequent activation of astrocytes
and glial scar formation under pathological conditions including
central nervous system (CNS) injury (Zhang et al., 2010).
Astrocyte activation (astrogliosis) is characterized by the in-
crease of intermediate filaments (vimentin and GFAP), increased
cell proliferation and an accompanying cellular hypertrophy
(Herrmann et al., 2008). Similar to microglia, reactive astrocytes
can have detrimental and/or beneficial roles following CNS injury.
Upon activation, astrocytes upregulate a number of neurotrophic
factors [e.g. brain-derived neurotrophic factor (BDNF)] that sup-
port and protect against injury-induced cell death (Zhao et al.,
2004). In addition, astrocytes play a crucial role in regulating extra-
cellular glutamate levels, which can reduce glutamate excitotoxic-
ity to neurons and other cells (Schousboe and Waagepetersen,
2005). Notably, impaired astrocyte performance exacerbates neu-
ronal dysfunction following brain injury and transgenic ablation
of reactive astrocytes increases neuronal cell death and promotes
worse outcome after TBI (Myer et al., 2006); this may in part reflect
the loss of ability to limit the influx of inflammatory cells. Follow-
ing injury, hypertrophic astrocytes surround the lesion site and de-
posit an inhibitory extracellular matrix including chondroitin
sulfate proteoglycans that contributes to the glial scar. This dense
physical and chemical barrier inhibits axonal regeneration and pre-
vents functional connections required for axonal growth and repair
(Cafferty et al., 2007). On the one hand, astrocytes provide neuro-
trophic support and guidance for axonal growth following CNS in-
jury, while on the other, prolonged astrogliosis inhibits axon
regeneration and hinders functional recovery. In fact, improved ax-
onal growth and repair following experimental brain and spinal
cord injury has been demonstrated in transgenic mice deficient
in both vimentin and GFAP (Menet et al., 2003; Wilhelmsson
et al., 2004). Therefore, limiting prolonged astrogliosis may be
important for axon regeneration after traumatic CNS injury.
3. Microglia – mediators of the innate immune response to CNS
injury
Microglia are the primary innate immune cells in the CNS. Un-
der normal physiological conditions these highly dynamic and mo-
tile cells are spread throughout the brain parenchyma and
 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201
constantly survey their microenvironment for noxious agents and
injurious processes (Nimmerjahn et al., 2005). They respond to
extracellular signals and are responsible for clearing cellular debris
and toxic substances by phagocytosis, thereby maintaining normal
cellular homeostasis in the CNS (Hanisch and Kettenmann, 2007).
Therefore, under non-pathological conditions there is continuing
low-level microglial activity in the CNS which is primarily involved
in activity-dependent synaptic pruning and in repair processes
(Tremblay et al., 2011). However, in response to insult, infection
or injury, microglia become dysregulated and highly activated
which results in dramatic changes in cell morphology and behav-
ior. Similar to peripheral innate immune cells microglia express
pathogen recognition receptors (PRRs) such as toll-like receptors
(TLRs) and NOD-like receptors (NLRs), and therefore respond to
pathogen-associated molecular patterns (PAMPs) and endoge-
nously produced danger-associated molecular patterns (DAMPs),
which are secreted by damaged neurons and others cells of the
CNS (Hanisch and Kettenmann, 2007). They also express receptors
for a number of other factors that are released by damaged neu-
rons, including ATP, glutamate, growth factors and cytokines.
Microglia respond to an array of molecules by secreting chemo-
kines, pro- and anti-inflammatory cytokines, neurotrophins and
oxidative metabolites. Furthermore, microglia are antigen present-
ing cells, and upon activation they up-regulate the expression of
cell surface markers such as MHC class II and CD86 among others,
as well as adhesion molecules and complement receptors (Lynch,
2009). A key to maintaining microglia in a quiescent state is the
immunosuppressive potential of the brain microenvironment. In
the healthy brain neurons express a number of immunosuppres-
sive proteins such as CD200, CD47 and fractalkine, which interact
with their receptors on microglia to maintain the microglia in a
quiescent/non-activated state (Harrison et al., 1998; Hoek et al.,
2000; Lynch, 2009). In addition, soluble factors such as neurotro-
phins, anti-inflammatory cytokines and prostaglandins released lo-
cally in the brain by neurons, astrocytes and microglia
downregulate the immune response thereby maintaining microg-
lia in a quiescent form (Lynch, 2009).
Microglia, like macrophages, have multiple activation pheno-
types (Gordon, 2003; Mantovani et al., 2004). Dependent on the
stimuli in their local microenvironment microglia can be polarized
to have distinct molecular phenotypes and effector functions
(Colton, 2009). For example, lipopolysaccharide (LPS) and the
pro-inflammatory cytokine IFNc promote a ‘classically activated’
M1 phenotype, which produces high levels of pro-inflammatory
cytokines and oxidative metabolites that are essential for host de-
fense and phagocytic activity, but that also cause damage to
healthy cells and tissue. Conversely, activating microglia in the
presence of cytokines such as IL-4 or IL-13 promote an ‘alterna-
tively activated’ M2 phenotype (Colton et al., 2006; Ponomarev
et al., 2007). Although there is limited data on resident M2 microg-
lia in the brain, it is thought that much like the M2 macrophages,
these cells can promote angiogenesis, wound healing and tissue re-
pair, extracellular matrix remodeling and suppress destructive im-
mune responses (Colton, 2009). M2 microglia express specific
antigens such as arginase 1 (Arg1), mannose receptor (MRC), found
in inflammatory zone 1 (FIZZ1) and chitinase 3-like 3 (Ym1) (Col-
ton et al., 2006), among others, and cultured microglia exposed to
IL-4 or IL-13 develop the M2 phenotype, which can result in exten-
sive neurite elongation and outgrowth across inhibitory surfaces in
in vitro co-culture systems (Butovsky et al., 2005; Kigerl et al.,
2009). Following an insult or injury to the brain it is likely that
M1 and M2 microglial exist in a state of dynamic equilibrium with-
in the lesion microenvironment. Whether these cells differentiate
into an M1 phenotype that exacerbates tissue injury or into an
M2 phenotype that promotes CNS repair likely depends on the
local signals in the lesion microenvironment. This is further
1193
complicated by the influence of infiltrating blood-borne macro-
phages of M1 or M2 phenotype and other infiltrating cells follow-
ing CNS injury (Kigerl et al., 2009). However, understanding the
molecular mechanisms that polarize and differentiate resident
microglia or infiltrating macrophages towards an M2 phenotype,
thereby promoting CNS repair while limiting inflammatory-medi-
ated secondary injury cascades, may provide an opportunity for
therapeutic intervention after CNS injury.
4. Involvement of pro- and anti-inflammatory cytokines and
chemokines in traumatic brain injury
The neuroinflammatory cascade activated in response to TBI is
mediated by the release of pro- and anti-inflammatory cytokines
and chemokines, and microglia are the primary source of these
inflammatory mediators in the brain. Gene profiling studies in
experimental models of TBI have shown that genes related to neur-
oinflammation are strongly up-regulated in the acute phase after
injury (Kobori et al., 2002; Natale et al., 2003; Raghavendra Rao
et al., 2003). Additional studies focused on microglial related genes
and their temporospatial localization after TBI demonstrated that
markers of activation (e.g. CD68, MHCII), stress responses (e.g.
p22phox, heme oxygenase 1) and chemokine expression (e.g.
CXCL10, CXCL6) were markedly increased after TBI (Israelsson
et al., 2008).
Consistent with the early activation profile of microglia follow-
ing injury, there is rapid elevation of pro-inflammatory IL-1b with-
in hours of TBI in both humans and rodents (Fan et al., 1995;
Winter et al., 2002; Woodroofe et al., 1991). The damaging effects
of IL-1b are mediated through interleukin 1 receptor type 1 (IL-
1RI), which is expressed on microglia and neurons (Lu et al.,
2005b; Pinteaux et al., 2002). This damage is not as a result of
the cytokine itself, but rather its affect on activating other pro-
inflammatory pathways such as TNFa (Rothwell, 2003). Inhibiting
IL-1b in experimental models of TBI has been shown to be neuro-
protective and improve functional recovery (Basu et al., 2002; Lu
et al., 2005a,b; Tehranian et al., 2002; Toulmond and Rothwell,
1995). The potent neurotoxic effects of IL-1b have been shown to
be synergistically enhanced in the presence of TNFa, suggesting
that these important cytokines mediate post-traumatic neuroin-
flammation and brain damage (Chao et al., 1995). Although IL-1b
and TNFa interact with distinct receptors that are structurally
un-related, both cytokines share significant down-stream signaling
pathways that may promote synergy of action. TNFa levels rise
within 1 h of TBI, peak between 3 and 8 h, and return to normal
by 24 h (Fan et al., 1996; Shohami et al., 1994; Stover et al.,
2000). Consistently, TNFa levels are elevated early after injury in
the serum and cerebrospinal fluid (CSF) of severely injured TBI pa-
tients (Goodman et al., 1990; Ross et al., 1994). However, the role
of TNFa in the pathogenesis of TBI is somewhat controversial and
complex in nature with different functional outcomes in the acute
and delayed phases after TBI (Scherbel et al., 1999; Sullivan et al.,
1999). Initial pre-clinical neuroprotection studies targeting TNFa
showed considerable promise, with three different compounds
(dexanabinol {HU-211}, TNF-binding protein, pentoxifylline
{PTX}) showing significant improvements in neurological out-
comes and reducing post-traumatic BBB disruption and brain ede-
ma (Shohami et al., 1997). However, deletion of TNFa in knockout
mice attenuated deficits in the acute phase following TBI, but lim-
ited neurological improvements in the long term (Scherbel et al.,
1999). Another study demonstrated that knockout mice for the
TNFa receptor had exacerbated tissue and BBB damage and im-
paired neurological recovery after TBI (Sullivan et al., 1999), sug-
gesting that endogenous TNFa may be neuroprotective. These
genetic studies indicate that the function of TNFa differs in the
1194 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201
acute and delayed phase after TBI; immediately after injury TNFa
seems to act as a potent inflammatory mediator, but later it is a
neurotrophic factor that is required for neuroprotection and repair.
Interestingly, anti-inflammatory cytokine levels are also chan-
ged after TBI. In humans, IL-10 and transforming growth factor-
b1 (TGFb1) levels are elevated acutely after injury (Csuka et al.,
1999; Morganti-Kossmann et al., 1999), and experimental studies
have shown that IL-10 has beneficial effects following trauma
(Knoblach and Faden, 1998). For example, intravenous administra-
tion of IL-10 after experimental TBI in rats improved neurological
recovery and significantly reduced TNFa and IL-1b expression in
the traumatized cortex and hippocampus. These neuroprotective
effects may be as a result of suppressed microglial activation, as
IL-10 treatment has been shown to decrease production of pro-
inflammatory cytokines (Kremlev and Palmer, 2005). Furthermore,
treatment with the anti-inflammatory cytokine TGFb1 reduced le-
sion size and improved neurological function after injury in rodent
models (Tyor et al., 2002). Notably, the levels of IL-1 receptor
antagonist (IL-1ra) are also elevated following TBI, and IL-1ra has
significant anti-inflammatory properties. IL-1ra plays a major role
in counteracting the biological effects of IL-1b, owing to its ability
to bind to IL-1RI without initiating signal transduction (Allan et al.,
2005). In experimental models of TBI, IL-1ra or IL-1b neutralization
resulted in attenuated pro-inflammatory cytokine and chemokine
production, reduced hippocampal damage and improved neurolog-
ical behavior after injury (Basu et al., 2002; Lu et al., 2005a,b;
Tehranian et al., 2002; Toulmond and Rothwell, 1995).
5. Chronic microglial activation and neurodegeneration after
traumatic brain injury
Chronic microglial activation is considered to be the most dam-
aging response of microglia to injury (Block et al., 2007). DAMPs re-
leased by injured neurons after TBI interact with TLRs and other
PRRs on activated microglia and trigger a vicious self-perpetuating
cycle of damaging events that lead to prolonged and dysregulated
microglial activation that drives pathogenic processes and neuro-
degeneration (Block et al., 2007; Loane and Byrnes, 2010). Human
and animal studies indicate that microglia are chronically activated
for weeks, months and even years after the initial brain trauma,
and may contribute to chronic neurodegeneration and related neu-
rological deficits following injury (Bendlin et al., 2008; Bramlett
and Dietrich, 2002; Maxwell et al., 2006; Smith et al., 1997). Persis-
tent long-term microglial activation has been demonstrated in ani-
mal models of TBI and is associated with increased expression of
pro-inflammatory cytokines (e.g. IL-1b, TNFa) (Holmin and
Mathiesen, 1999). Notably, a recent clinical study utilizing the pos-
itron emission tomography ligand [11C] (R)PK11195 to assess
chronic microglial activation in 10 patients studied more than
11 months after moderate to severe TBI reported significantly in-
creased binding bilaterally at sites distant from areas of focal in-
jury, such as thalamus, and correlated these changes with several
measures of cognitive dysfunction (Ramlackhansingh et al.,
2011). Furthermore, post-mortem studies have also demonstrated
increased microglial activation in the white matter of head-injury
survivors up to 16 years after TBI (Gentleman et al., 2004). The
experimental and clinical evidence now suggest that TBI should
not be viewed as a static, acute neurodegenerative disorder. In-
stead, TBI initiates chronic biochemical processes leading to pro-
longed neuroinflammation and microglial activation, and as such
there may be a longer therapeutic window for the treatment of
head injury than traditionally accepted. In fact, a recent experi-
mental study which used a pharmacological treatment (mGluR5
agonist) to inhibit chronic microglial activation demonstrated that
delayed treatment at 1 month post-injury significantly reduced
both functional deficits and histological outcomes through
4 months post-injury (Byrnes et al., 2012).
Aging also influences microglial activation (Conde and Streit,
2006b; Lucin and Wyss-Coray, 2009), and exacerbated microglial
activation and astroglial responses to injury likely contribute to
the enhanced susceptibility to and poor recovery from TBI in el-
derly patients (Galbraith, 1987; Pennings et al., 1993). In fact,
experimental studies demonstrated that microglial activation
was exaggerated and prolonged in aged mice when compared to
adult mice (Sandhir et al., 2008), and these observations are consis-
tent with reports of elevated microglial activation in the aged brain
following injuries such as facial nerve axotomy (Conde and Streit,
2006a) and cerebral ischemia (Popa-Wagner et al., 2007). It has
been proposed that microglia in the aged brain are ‘primed’ to re-
spond more rapidly, produce more pronounced inflammatory re-
sponses and proliferate more vigorously than microglia in the
young brain following TBI (Conde and Streit, 2006b; Godbout
et al., 2005). Therefore, a hyperactivated and dysfunctional microg-
lial response in the aged hippocampus may contribute to enhanced
neuronal loss and worse cognitive outcomes in the elderly follow-
ing brain trauma.
There is growing awareness of the epidemiological association
between a history of TBI and the development of Alzheimer’s dis-
ease (AD) later in life (Graves et al., 1990; Mortimer et al., 1985;
van Duijn et al., 1992). This link is supported by the identification
of acute and chronic AD-like pathologies in the brains of TBI pa-
tients and in animal models of TBI. Amyloid-b (Ab) plaques, a hall-
mark of AD, may be found in TBI patients within hours of injury
(Ikonomovic et al., 2004; Roberts et al., 1991, 1994), and there
are several pathophysiological mechanisms linking TBI and AD,
such as the accumulation and clearance of Ab peptides after TBI
(Johnson et al., 2010). However, chronic neuroinflammation is a
common neuropathological feature of TBI and AD, and chronic
microglial activation may be a key causative factor. Ab is impli-
cated in the pathology of AD, both through direct toxicity to neu-
rons (Yankner et al., 1989), and by potentiating neuronal damage
by microglial activation (Combs et al., 2000). In AD patients acti-
vated microglia cluster at sites of aggregated Ab and penetrate
the neuritic plaques (Cagnin et al., 2001; McGeer et al., 1987). Fur-
thermore, Ab is a potent pro-inflammatory stimulator of microglia
(Ii et al., 1996; Qin et al., 2002). Interestingly, proinflammatory
cytokines expressed by microglia, such as IL-1b, TNFa and IFNc,
can specifically stimulate c-secretase activity resulting in in-
creased production of Ab and the intracellular domain of APP
(AICD) (Liao et al., 2004). Moreover, TBI induces the expression of
the c-secretase complex proteins in microglia and astrocytes
(Nadler et al., 2008), thereby suggesting a role for glial cells in post-
traumatic accumulation of Ab. It is evident that the interactions
between Ab accumulation/clearance and chronic microglial activa-
tion after TBI are complex and poorly understood. Further detailed
mechanistic studies on these molecular interactions after brain in-
jury are needed because targeting these pathways may provide no-
vel therapeutic opportunities for the treatment of TBI.
6. Challenges translating promising experimental
neuroprotection strategies to the clinic
Neuroprotective treatments that limit secondary injury mecha-
nisms and/or improve behavioral outcome have been well estab-
lished in multiple animal models of TBI. However, translation of
promising experimental neuroprotective treatments to human in-
jury have been very disappointing, with none of the pharmacolog-
ical treatments resulting in any consistent improvements in
outcome in the clinic (Maas et al., 2010). Both conceptual issues
and methodological differences between preclinical research and
 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201 1195

clinical injury have undoubtedly contributed to these translational
difficulties. Apart from the potential shortcomings of the drugs
themselves, a number of flaws in preclinical research and clinical
translation have been recognized. These include (1) inadequate
understanding of secondary injury mechanisms; (2) insufficient
preclinical testing in multiple TBI models (e.g. lateral fluid percus-
sion, controlled cortical impact, closed head injury models), strains,
species (including gyrencephalic), genders and ages; (3) lack of
thorough investigation of pharmacokinetics and therapeutic brain
concentrations of drugs under investigation; (4) failure to ade-
quately examine the therapeutic window for the drug and failure
to use clinically relevant behavioral outcomes; (5) lack of animal
models that include secondary insults such as CNS hypoxia and
systemic injuries despite these insults being commonplace in clin-
ical TBI (6) use of heterogeneous patient populations in clinical tri-
als for TBI; (7) inadequate sample size (lack of power); and (8)
inadequate functional outcome measurements and biomarkers
(molecular and neuroimaging) for clinical TBI. Each of these factors
has contributed to the failure to translate a successful pharmaco-
logical intervention to date. Nonetheless, many of these shortcom-
ings can be addressed with improved preclinical screening of
drugs. Recommendations regarding preclinical experimental de-
sign for the evaluation of neuroprotective agents for TBI have been
made (Loane and Faden, 2010), and they build on the STAIR criteria
for development of neuroprotective treatments for stroke/ischemia
(Fisher et al., 2009; STAIR, 1999), and are in line with NIH guide-
lines for effective therapies for TBI (Margulies and Hicks, 2009;
Saatman et al., 2008). However, the heterogeneity of clinical TBI
and the complex nature of the secondary injury process represent
the most significant hurdles to future clinical trial successes. It is
unlikely that targeting any single secondary injury factor will re-
sult in significant improvement in outcome after TBI. Therefore,
simultaneous targeting of several injury factors using multipoten-
tial drugs may maximize the likelihood of developing a successful
therapeutic intervention to improve outcome in TBI patients.
A number of anti-inflammatory and multipotential drug treat-
ment strategies inhibit post-traumatic neuroinflammation and
microglial activation, and have shown considerable promise in pre-
clinical studies (Fig. 1). In the following section we will review
these neuroprotective drug treatment strategies, focusing on drugs
that are currently in randomized controlled clinical trials for head
injury, or emerging pharmacological treatments that show promise

Fig. 1. Anti-inflammatory and multipotential drug treatment strategies for TBI. Neuroinflammation and microglial activation are key secondary injury mechanisms that
contribute to chronic neurodegeneration and loss of neurological function following TBI. Preclinical studies have identified a number of anti-inflammatory and multipotential
drug treatment strategies that inhibit post-traumatic neuroinflammation and microglial activation in addition to reducing other secondary injury mechanisms such as edema
formation and neuronal apoptosis.
1196 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201
in experimental TBI studies and warrant further investigation.
Unfortunately due to space limitations only a small number of
promising anti-inflammatory drug treatments can be detailed in
this article, and readers are directed to excellent review articles
on other preclinical drug treatment strategies for TBI (Shohami
et al., 2011; Simard et al., 2010; Ziebell and Morganti-Kossmann,
2010).
7. Anti-inflammatory treatments for traumatic brain injury
7.1. Minocycline
Minocycline is a second generation tetracycline that is known to
have neuroprotective properties that are independent of its anti-
microbial activity (Yrjanheikki et al., 1998). Minocycline is a potent
anti-inflammatory drug that suppresses the production of several
pro-inflammatory cytokines (Choi et al., 2005; Seabrook et al.,
2006), and inhibits microglial-mediated neurotoxicity (Tikka
et al., 2001). It has been shown to have significant neuroprotective
effects in spinal cord injury (SCI) models by reducing both inflam-
matory and apoptotic pathways (Festoff et al., 2006; Teng et al.,
2004; Wells et al., 2003). Minocycline has been used in experimen-
tal TBI models and it was demonstrated to improve neurological
recovery after injury due to its ability to inhibit post-traumatic
microglial activation and neuronal apoptosis (Sanchez Mejia
et al., 2001). However, another report demonstrated that minocy-
cline treatment resulted in only a transient improvement in neuro-
logical recovery after TBI, and that there was no difference in
outcomes by day 4 post-injury when compared to vehicle-treated
controls (Bye et al., 2007). Apoptotic mechanisms were not af-
fected by minocycline treatment in this study, but IL-1b and IL-6
levels were decreased in the acute phase after injury and microglial
activation was significantly reduced, which may account for the
transient neuroprotective effects of minocycline. In contrast, recent
studies suggest that blockade of acute microglial activation by
minocycline is neuroprotective and promotes functional recovery
up to 3 months post-injury (Homsi et al., 2010; Ng et al., 2012;
Siopi et al., 2011, 2012). However, the short treatment protocol
used in the latter studies requires that further investigation into
the therapeutic window for minocycline treatment needs to be
performed before it can be considered for clinical translation for
human head injury.
7.2. Peroxisome proliferator-activated receptor (PPAR) agonists
Another class of drugs that display strong anti-inflammatory
properties are the synthetic agonists for PPARs. PPARs are li-
gand-activated transcription factors of the nuclear hormone recep-
tor family (Berger and Moller, 2002). Upon activation by specific
agonists, PPARs bind as heterodimers with a retinoid X receptor
and translocate to the nucleus, where they act as agonist-depen-
dent transcription factors that regulate gene expression by binding
to specific promoter regions of target genes. The PPARs play a crit-
ical physiological role as lipid sensors and regulators of lipid
metabolism, and they are also involved in reproductive, develop-
mental and immune responses. Activation of the PPAR a and c iso-
forms are known to have potent anti-inflammatory effects,
including attenuation of pro-inflammatory mediators, such as
iNOS and COX2 (Bernardo and Minghetti, 2006; Chawla et al.,
2001). PPAR agonists are neuroprotective in acute brain injury
models such as transient focal ischemia (Bernardo and Minghetti,
2006; Sundararajan et al., 2005), and similar anti-inflammatory
and neuroprotective properties have been demonstrated in exper-
imental TBI studies. For example, the PPARa receptor agonist,
fenofibrate, reduced post-traumatic neuroinflammation, oxidative
stress and cerebral edema, and improved neurological function
when administered after TBI (Besson et al., 2005; Chen et al.,
2007b). Similarly, the PPARc receptor agonists, pioglitazone and
rosiglitazone, reduced post-traumatic microglial activation and
promoted anti-oxidant (e.g. Mn-SOD) and neuroprotective chaper-
one protein (e.g. HSP27/70) expression that resulted in improved
functional and histological outcomes after TBI (Sauerbeck et al.,
2011; Thal et al., 2011; Yi et al., 2008). The anti-inflammatory
and neuroprotective effects of pioglitazone are likely to be
PPARc-independent as the beneficial effects of treatment were
not reversed by the PPARc antagonist T0070907 (Thal et al.,
2011). Notably, PPARc receptor agonist are used clinically for the
treatment of type 2 diabetes, and PPARc modulates macrophage
M2 alternative activation states thereby improving resistance to
insulin (Odegaard et al., 2007). Furthermore, rosiglitazone also in-
duces IL-4 expression in the brain and reduces microglial activa-
tion in an IL-4 dependent manner (Loane et al., 2009). Therefore,
PPARc agonists could be used to promote an M2 microglial activa-
tion phenotype through IL-4 dependent mechanisms, which could
potentially modulate the local microenvironment surrounding a
lesion, thereby promoting tissue repair and reducing the damaging
effects of neuroinflammation after TBI.
8. Multipotential drug treatments for traumatic brain injury
8.1. Cell cycle inhibitors
The cell cycle is upregulated in both mitotic (astrocytes and
microglia) and post-mitotic (neurons, oligodendrocytes) cells of
the brain after CNS injury, and post-traumatic cell cycle activation
is associated with caspase-mediated neuronal cell death and glial
cell proliferation (Di Giovanni et al., 2005). Cell cycle inhibitors
have been extensively studied for their role in cancer treatment,
and inhibitors such as flavopiridol, roscovitine and olomoucine,
have been shown to exert powerful neuroprotective effects in
in vitro model systems for neuronal apoptosis (Cernak et al.,
2005; Padmanabhan et al., 1999; Verdaguer et al., 2004), and they
also produce potent inhibitory effects on the proliferation and acti-
vation of astrocytes and microglia (Cernak et al., 2005; Di Giovanni
et al., 2005; Hilton et al., 2008). Given the multipotential properties
of cell cycle inhibitors they are considered to be promising candi-
date drugs for the treatment of TBI.
In experimental studies treatment with flavopiridol, an inhibi-
tor of all major cyclic-dependent kinases (CDKs), after lateral fluid
percussion TBI in rats significantly reduced lesion volume, and im-
proved long-term cognitive and sensorimotor recovery (Di Giovan-
ni et al., 2005). In addition, flavopiridol treatment blocked caspase-
mediated neuronal cell death and significantly reduced glial cell
activation, and these changes were associated with the suppres-
sion of the cell cycle in neurons, astrocytes, and microglia within
the injured cortex. Follow up studies demonstrated that delayed
treatment with flavopiridol as late as 24 h post-injury resulted in
significantly reduced TBI lesion volumes (Cernak et al., 2005). Ros-
covitine is a more selective cell cycle inhibitor, which acts specifi-
cally on CDKs 1, 2, and 5 (Meijer et al., 1997), and it too had
significant neuroprotective actions in rat and mouse TBI models.
In addition to improving functional recovery and reducing the
TBI lesion, central and systemic administration of roscovitine
markedly reduced microglial-mediated neuroinflammation and
associated neurodegeneration up to 28 days post-injury (Hilton
et al., 2008; Kabadi et al., 2012a). Further, genetic deletion of cyclin
d1, a key modulator of the cell cycle, resulted in significantly re-
duced microglial activation and improved histological and func-
tional outcomes after TBI (Kabadi et al., 2012b), thereby
underpinning the key role that cell cycle activation plays in the
 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201
neuroinflammatory response to TBI. Several cell cycle inhibitors
have been studied in randomized clinical trials for cancer (Fischer
and Gianella-Borradori, 2005). Although they are toxic when
administered chronically, cell cycle inhibitors have required only
single dose administration to exert powerful therapeutic effects
in experimental TBI models. Therefore, given their clinically rele-
vant therapeutic window and their multipotential neuroprotective
effects, cell cycle inhibitors such as flavopiridol or roscovitine are
attractive candidates for future clinical translation.
8.2. Statins
The 3-hydroxy-3-methyglutaryl coenzyme A (HMGCoA) reduc-
tase inhibitors (also known as statins) are inhibitors of cholesterol
biosynthesis, and they have additional pleiotropic properties that
make them attractive multipotential neuroprotective drugs (Wible
and Laskowitz, 2010). Statins increase endothelium-derived nitric
oxide production (Eto et al., 2002) and reduce vascular inflamma-
tion (Maeda et al., 2003), thereby improving the microvasculature
after traumatic insult. In in vitro models statins protect cortical
neurons from NMDA-induced excitotoxic death (Zacco et al.,
2003), and statin treatment significantly improves neuronal sur-
vival following TBI (Lu et al., 2004, 2007; Qu et al., 2005; Wang
et al., 2007). Statins may also promote the growth and differentia-
tion of new neurons after brain injury (Lu et al., 2007; Wu et al.,
2008b), and their ability to increase neurogenesis may be in part
due to upregulation of neurotrophic factors (e.g. BDNF) (Chen
et al., 2005; Wu et al., 2008b). Notably, statins exert powerful
anti-inflammatory effects, in part by decreasing the formation of
isoprenoids (Bi et al., 2004; Cordle and Landreth, 2005). In TBI
models, statins have been shown to significantly reduce pro-
inflammatory cytokine production and attenuate microglial activa-
tion and cerebral edema formation, while increasing BBB integrity
(Chen et al., 2007a, 2009; Wang et al., 2007). Moreover, inhibition
of the TLR4 and NFjB signaling pathways are potential mecha-
nisms through which statins modulate the post-traumatic neuro-
inflammatory response (Chen et al., 2009).
Experimental studies have shown that statins target multiple
secondary injury pathways and significantly improve functional
recovery after TBI (Chen et al., 2007a, 2009; Lu et al., 2004, 2007;
Qu et al., 2005; Wang et al., 2007; Wu et al., 2008a). Furthermore,
the therapeutic window for this class of drugs is relatively large,
with treatment 24 h after TBI resulting in long-term functional
improvements and reduced neuronal cell loss (Lu et al., 2004,
2007). As such, statins possess key preclinical neuroprotective
characteristics that make them suitable candidates for clinical
translation. Importantly, statins have a long clinical track record
in critically ill patients; they are easy to administer, are well toler-
ated and have well-defined side effects (Tseng et al., 2005). A small
prospective, randomized, double-blind clinical trial in TBI has been
performed using the statin drug rosuvastatin, and treatment
showed modest improvements in TBI associated amnesia and dis-
orientation time outcomes (Tapia-Perez et al., 2008). Other phase II
clinical trials to evaluate rosuvastatin and atorvastatin for the
treatment of head injury are planned.
8.3. Progesterone
Another neuroprotective drug undergoing clinical trials for head
injury is progesterone; a neurosteroid whose receptors are ex-
pressed in the CNS of both males and females (Camacho-Arroyo
et al., 1994). Progesterone is also thought to have pleiotropic neu-
roprotective properties, and has been reported to improve out-
comes in experimental models of SCI (Gonzalez Deniselle et al.,
2002), stroke (Jiang et al., 1996) and TBI (Roof and Hall, 2000).
Intriguingly, it was first discovered that female rats had better cog-
1197
nitive performance in the Morris water maze than males after
experimental TBI (Roof et al., 1993), and that progesterone-treated
male rats were less impaired in the task than vehicle-treated ani-
mals (Roof et al., 1994). Later studies demonstrated that progester-
one inhibited multiple secondary injury pathways in TBI. For
example, progesterone attenuates glutamate excitotoxicity (Smith,
1991), modulates apoptotic pathways (Djebaili et al., 2005; Yao
et al., 2005), and decreases diffuse axonal injury (O’Connor et al.,
2007). It also reduces lipid peroxidation (Roof et al., 1997), possibly
by upregulating superoxide dismutase (Moorthy et al., 2005), and
reduces cerebral edema formation (O’Connor et al., 2007; Roof
et al., 1996). Notably, progesterone treatment reduces neuroin-
flammation after TBI, by attenuating NFjB signaling, IL-1b, IL-6
and TNFa production, as well as the inflammatory C3 complement
protein (Grossman et al., 2004; Pettus et al., 2005).
Although progesterone has been shown to confer neuroprotec-
tion in models of experimental stroke, SCI and TBI, a recent system-
atic review questions its effectiveness in trauma models (Gibson
et al., 2008). Despite the perceived limitations of the preclinical re-
search, two randomized, double-blind, placebo-controlled phase II
clinical trials for progesterone have been conducted (Wright et al.,
2007; Xiao et al., 2008). Although these trials used different doses
and treatment regimens, both indicated trends toward improved
outcome in progesterone treated patients. The results are promis-
ing since both clinical studies not only showed reduced mortality
but also reduced morbidity as indicated by improved functional
outcomes. In 2010, two major Phase III clinical trials for progester-
one (ProTECT III and SyNAPSe trials) were set up to provide a more
robust answer on the effectiveness of progesterone treatment in
TBI (Stein and Wright, 2010), and the results of these studies are
eagerly anticipated.
9. Conclusions
Neuroinflammation and microglial activation are key secondary
injury mechanisms that contribute to chronic neurodegeneration
and loss of neurological function after TBI. However, the neuroin-
flammatory response to TBI possesses both beneficial and detri-
mental effects, and these likely differ in the acute and delayed
phases after injury. The key to developing future neuroprotective
treatments that target post-traumatic neuroinflammation and
microglial activation is to minimize the detrimental and neuro-
toxic effects of neuroinflammation while promoting the beneficial
and neurotrophic effects, thereby creating optimal conditions for
regeneration and repair after injury. The complex and multifaceted
responses of microglia to injury are essential to these processes,
and research must focus on identifying the signals and mecha-
nisms by which microglia can be guided to promote optimal repair.
Further detailed understanding of the role of classical/M1 and
alternatively/M2 activated microglial phenotypes, particularly in
relation to the chronic neuroinflammatory responses to injury is
required, so that therapeutic approaches that promote the alterna-
tive/M2 phenotype can be developed to support regeneration and
repair after injury.
So complex and intertwined are the secondary injury responses
to TBI that the concept of a single magic bullet strategy for neuro-
protection is no longer accepted. Focus has now turned to multipo-
tential therapeutic strategies that modulate independent
secondary injury mechanisms simultaneously, and several treat-
ments have been identified experimentally and are currently in
or are awaiting human clinical trials for head injury. Notably, many
of these multipotential drug treatments target post-traumatic
microglial activation and neuroinflammatory mechanisms, thereby
demonstrating the major role that these pathways play in the
pathogenesis of this disorder.
1198 A. Kumar, D.J. Loane / Brain, Behavior, and Immunity 26 (2012) 1191–1201

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