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Inflammation andNeuroprotection in Traumatic Brain Injury
Kara N. Corps, DVM, DACVP; TheodoreL. Roth,MS; DorianB. McGavern,PhD
IMPORTANCE Traumatic brain injury (TBI) is a significant publichealth concern that affects
individuals in alldemographics.With increasing interest in themedical and public
communities, understanding the inflammatory mechanisms thatdrive the pathologic and
consequentcognitive outcomes can inform future research and clinical decisions for patients
with TBI.
OBJECTIVES To review known inflammatory mechanisms in TBIand to highlightclinicaltrials
and neuroprotective therapeutic manipulations of pathologic and inflammatory mechanisms
of TBI.
EVIDENCE REVIEW We searched articles in PubMedpublishedbetween 1960 and August1,
2014, using the following keywords: traumatic brain injury, sterile injury, inflammation,
astrocytes, microglia, monocytes, macrophages, neutrophils, T cells, reactive oxygen species,
alarmins, danger-associated molecular patterns, purinergic receptors, neuroprotection, and
clinical trials. Previous clinical trials or therapeutic studies that involved manipulation of the
discussed mechanisms were considered for inclusion. The final list of selected studies was
assembled based on novelty and direct relevance to theprimary focus of this review.
FINDINGS Traumatic brain injury is a diverse group of sterile injuries induced by primary and
secondary mechanismsthat give rise to cell death, inflammation, and neurologic dysfunction
in patients of all demographics. Pathogenesis is driven by complex, interacting mechanisms
thatinclude reactive oxygen species, ion channel and gap junction signaling, purinergic
receptor signaling, excitotoxic neurotransmitter signaling, perturbations in calcium
homeostasis, and damage-associated molecular pattern molecules, among others. Central
nervous systemresident and peripherally derived inflammatory cells respond to TBIand can
provide neuroprotectionor participate in maladaptive secondary injury reactions. The exact
contribution of inflammatory cells to a TBIlesion is dictated by their anatomical positioning as
well as the local cues to which they are exposed.
CONCLUSIONS AND RELEVANCE The mechanisms that drive TBIlesion development as well as
those that promote repair are exceedingly complex and often superimposed. Because
pathogenic mechanisms candiversify over time or even differbased on the injury type, it is
importantthat neuroprotective therapeutics be developed and administered with these
variables in mind. Due to itscomplexity, TBIhas provenparticularly challenging to treat;
however, a number of promising therapeutic approaches are now under pre-clinical
development,and recentclinicaltrials have even yielded a few successes. Given the
worldwide impactof TBIon thehumanpopulation, it is imperative that research remains
active in this area andthat we continue to develop therapeutics to improve outcome in
afflicted patients.
JAMA Neurol. 2015;72(3):355-362. doi:10.1001/jamaneurol.2014.3558
Published onlineJanuary 19,2015.
Video at jamaneurology.com
Author Affiliations: Viral
Immunology and IntravitalImaging
Section, National Institutesof
NeurologicalDisorders and Stroke,National Institutesof Health,
Bethesda, Maryland.
Corresponding Author: DorianB.
McGavern, PhD, Viral Immunology
and IntravitalImaging Section,
National Institutesof Neurological
Disorders and Stroke, National
Institutesof Health, 10 Center Dr,
Bethesda, MD 20892 (mcgavernd
@mail.nih.gov).
Section Editor: HassanM.
Fathallah-Shaykh, MD, PhD.
Clinical Review & Education
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Traumatic brain injury (TBI) is a diverse group of brain inju-
ries thatvary in cause, severity, pathogenesis, and clinical
outcome. As public awareness of TBI and its conse-
quences increases, there is a growing need to understand the un-
derlyingmechanisms anddevelop therapeuticinterventions. Within
theUnited Statesalone, nearly2 million peoplesustaina TBIannu-
ally, contributing to one-third of all injury-related deaths. Individu-
als from all nations and demographics are affected, including ath-letes, military troops, and individuals with unintentional injuries.1-3
Traumatic brain injury is a significant cause of mortality in children
and young adults, and the incidence in older individuals has in-
creased withthe averagelife span.4MildTBI(mTBI)isthemostfre-
quenttype diagnosed, typically resultingin post-TBI survival. Trau-
matic brain injury is suspectedto contribute to a variety of chronic
degenerativeprocesses, including chronic traumaticencephalopa-
thy, Alzheimerdisease,and Parkinsondisease.5 Traumaticbrain in-
jury is initiated by the application of mechanical force to the head,
whichcan occurwith orwithout lossof consciousness.This thentrig-
gers a series of cerebral events that depend in part on the nature
andlocationof theinjury. A major challengeassociated with treat-
ingpatientswithTBIisthediversepathologicandpathogenicmecha-
nisms that become operational after injuries. For example, TBI of-
ten promotes disruptionof blood-brain barrier (BBB)integrity and
theneurovascularunit, which canresultin vascular leakage,edema,
hemorrhage,and hypoxia.Other pathologicmechanisms includecell
death within the meninges and brain parenchyma, stretching and
tearing of axonal fibers, and disruptions at the junctions between
whiteand graymatter, stemmingfrom rotational forces thatcause
shearing injuries.6Allthese primary pathologic mechanisms are ac-
companied by cellularand molecular cascadesleading to inflamma-
tion andadditionalcell death.Thisreviewfocuses onour currentun-
derstanding of the sterile immune reaction to TBI and someclinical
successes in treating patients with TBI.
We searched articles in PubMed published between 1960
and August1, 2014, using the following keywords: traumatic braininjury, sterile injury, inflammation, astrocytes, microglia, mono-
cytes, macrophages, neutrophils, T cells, reactive oxygen species,
alarmins, danger-associated molecular patterns, purinergic recep-
tors, neuroprotection,andclinical trials.Clinical trials or therapeu-
tic studies that involved manipulation of the discussed mecha-
nisms were considered for inclusion. The final reference list was
assembled based on novelty and direct relevance to the primary
focus of this review.
Sterile Immune Reaction to TBI
Centralnervous system (CNS)residentand peripherally derived in-flammatory cellsrespond quickly to brain injuriesand caneven par-
ticipatein the repair process.7,8These responsesare commonlyre-
ferredto as sterile immune reactions. A previous study9 found that
the inflammatory gene expression profile is comparable between
mTBIand severe TBI,suggestinga commonresponse toboth forms
of injury. The acute cellular reaction to TBIincludesastrocytes, mi-
croglia,monocytes or macrophages,neutrophils, andT cells, which
are initiallyactivated in part by purinergicreceptor signaling.10,11 In
the following paragraphs, we describe the inflammatory response
toTBI inmore detail,focusingspecificallyon traditional immunecell
populations.Sterile immune reactionsare at least initiallydesigned
to be beneficial butcan becomedetrimentalin certain situations.
Danger Signals
Pathogens can trigger innate immune activation via pathogen-
associatedmolecular patternmolecules,which are conserved struc-
tureswithina classof microbes recognized by Toll-like receptors or
pathogen-recognition receptors. These innate signaling pathwaysallow plants and animals to respond quickly to invading microbes.
However, it is nowrecognized thattissuedamagein theabsenceof
microbial infection can trigger inflammasome and innate immune
activation throughthe releaseof damage-associatedmolecular pat-
ternmolecules(DAMPs),sometimesreferredto asdangersignals.12
Alarmins are endogenousDAMPs released by cellsundergoing no-
napoptotic death or by cells of the immune system.13 Some ex-
amples of alarmins include HMGB1, S-100 proteins, adenosine tri-
phosphate (ATP),uric acid,DNA or RNA, andinterleukin 1, among
others.AfterTBI, alarminsare undoubtedly released,14andthistrig-
gers a sterile immune reaction designed to restore tissue homeo-
stasis. However, theseverityand duration of injury canfoster mal-
adaptiveimmunereactionsthatbecomeinjurious.Apreviousstudy15
found thatATPrelease anddetectionvia puringericreceptors elicit
an acutely neuroprotective inflammatory response after mild cor-
ticalinjury, butsustainedimmune activationmay notalways beben-
eficial.For example,therapeuticblockade ofinflammasomeactiva-
tionreducedinnateimmuneactivationand severe TBIlesionsize.16
Thus, additional researchis required to better understand therules
that govern pathogenic vs nonpathogenic innate immune reac-
tions after DAMP signaling in theinjured brain.
PurinergicReceptor Signaling
Purinergic receptors are an evolutionarily ancient family of trans-
membranemolecules thatdetect ATP, adenosinediphosphate(ADP),
oradenosine.10,11Thereceptors aredividedinto2 basicclassesbased
onwhethertheyrespondto adenosine (P1receptors)or ATP or ADP(P2receptors).BecauseATP is a cellularsource ofenergy, it is main-
tained ata highintracellular concentration during steady-state con-
ditions. Aftertissue injury, ATPis released fromdamagedcells, which
triggers an immune reaction via purinergic receptor signaling. This
reaction canbe amplified by pannexin and connexinhemichannels
thatpump ATP fromhealthycells intothe extracellularspace. Ster-
ileimmunereactionsgenerallysubsideasATPisconvertedintoaden-
osine through a 2-step reaction that involves ectonucleoside tri-
phosphate diphosphohydrolase1 (CD39) and ecto-5-nucleotidase
(CD73). Astrocytes and microglia each express at least one these
ectoenzymes,17,18 allowing them to dampen ATP-mediated neuro-
inflammation.
Microglia
Microglia are highly dynamic CNS resident innate immune senti-
nels that originate from primitive myeloid progenitor cells during
development.19,20Microgliaparticipate in a variety of homeostatic
CNS functions, including synaptic plasticity and learning,21 andare
often the first responders to any inflammatory event that occurs
within the parenchyma.20 Microglia mediate neuron removal dur-
ingdevelopment via release of reactive oxygen species (ROS) and
can acquire a phagocytic phenotype without an inflammatory
response.20 Microglial expression of genes associated with neuro-
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protection is upregulated with age.22 Microglia express a large
number of surface and cytoplasmic receptors, and cumulative sig-
naling through these receptors determines whether microglia
remain in a ramified, sentinel state or take on various configura-
tions as a result of activation.23 In addition, microglia can sense a
large repertoire of exogenous and endogenous signals, allowing
for dynamic responses to sterile injuries and infectious agents
that can be injurious or neuroprotective, depending on thecontext.22
During CNS autoimmune disease, activated microglia phago-
cytose debrisand downregulate cellular metabolism in contrastto
disease-initiating, peripherally derived macrophages, which ap-
pearto playa moredestructiverole by promotingdemyelination.24
These datasuggestthatmicrogliaare notinherently neurotoxicdur-
ing development of a CNS autoimmune disease. After acute focal
brain injury in rodents, microglia similarly appear to play a neuro-
protective role.15 Using 2-photonmicroscopy, we revealed thatmi-
croglia respond within minutes of brain injury by extending pro-
cesses tothe glial limitansand circumscribingindividualastrocytes,
resemblingahexagonalhoneycombstructure(Figure1A-E,Figure2E
and H,and Videos1 and 2). This reaction was dependenton purin-
ergic receptor signaling (P2X4and P2Y12) and astrocytic ATP-
dependent ATP release via connexinhemichannels. In response to
cell death (eg, astrocytic cell death in the glial limitans), microglia
transformed into phagocytic cells that resembled jellyfish
(Figure 1A-E, Figure 2F andI, andVideos1 and 2). Jellyfish microg-
lia werehighly mobile andoften inserted themselvesinto the dam-
agedglial limitansin place of dead astrocytes, connecting together
to form a phagocytic barrier. This reaction was also dependent on
purinergicreceptorsignaling(P2X4,P2Y6,P2Y12)andconnexinhemi-
channels. When these microglia responses were inhibited locally
throughblockadeof purinergic receptorsignalingor connexin hemi-
channels, the pathologic mechanisms observed after brain injury
were moresevere.One of themost notable changes wasincreased
leakage of materialsthrough the gliallimitans intothe brainparen-chyma.These data suggest that microglianot only clean up debris
fromthe injured brain butalso helpmaintain glial limitans barrier in-
tegrity by sealing thegapsthatresult from dead or damaged astro-
cytes.Moreover, our dataare consistent withpreviousstudies25-28
thatlinkmicrogliainjuryresponses toATPreleaseand purinergic re-
ceptor signaling. Although it is conceivable that microglia re-
sponses become maladaptive overtime or afterexposure to differ-
ent combinations of stimuli,29 we propose that the acute role of
microglia in the focally injured brain is neuroprotective.
Monocytes and Macrophages
Monocytesare a multipotent populationof circulatingbonemarrow
derived leukocytes capable of differentiating into macrophages ordendriticcells afterinvasionof an infectedor injured tissue.30They
are also known to participate in diverse functions, such as phago-
cytosis, cytokine or chemokine release, antigen presentation, im-
munemodulation,and tissue repair. Inthe naivebrain,thereare also
populations of specialized macrophages that reside in the menin-
ges,choroidplexus,andperivascularspaces.31TheirroleinTBIpatho-
genesisis unknown.Anotherstudy15alsofoundthatmeningeal mac-
rophagesareamongthefirstcellstodieafterfocalcorticalinjuryand
may serve as an early source of alarmins and ROS (Figure 1A-C,
Figure2AandB,and Video1).Monocyte-derivedmacrophagescom-
ingfromtheblooddonotreachpeaknumbersinthedamagedbrain
ofanimalsand humansuntil 24to 48 hours after injury.32,33 Mono-
cytes are capable of crossing the bloodcerebrospinal fluid barrier
with neutrophils into the injured brain as a result of CCL2 produc-
tionby choroid plexus epithelium.34 CCL2is significantlyincreased
in the cerebrospinal fluid of patients with TBI.33 Examination of
CCL2/miceafterTBI revealedslight alterations incytokine expres-
sion but no changes in lesion size within the first week of injury.
33
However, when followed for a longer timeframe of 2 to 4 weeks,
CCL2/micehad improved functionalrecovery, suggestinga patho-
genicrole formacrophages during thechronicphase of TBI.Similar
results were obtained in CCR2/ mice after TBI.35 CCR2 is the re-
ceptor for CCL2, and deficiency significantly reduced the number
of lesion macrophages and increased hippocampal neuronal den-
sities, spatial learning, and locomotion when measured several
weeks after brain injury. Collectively, the data obtained in CCL2
and CCR2 knockout mice suggest that monocyte-derived macro-
phages play a pathogenic role in the chronic phase after TBI.
Additional studies are required to determine whether these cells
can participate in brain repair after TBI similar to what has been
described in models of spinal cord injury.36 Whether a macro-
phage is pathogenic or beneficial after tissue injury likely depends
on its state of differentiation.
Neutrophils
Neutrophils arean abundantpopulationof circulating leukocytesthat
are usually among the first responders to tissue injuries in the pe-
ripheryandCNS.37Neutrophilsare oftenviewedas a proinflamma-
tory cell population butareknown toplaya vitalrolein woundheal-
ing through their involvement in phagocytosis, metalloproteinase
release, and growth factor production. After tissue injury, neutro-
phils can help prepare the damaged environment for repair. Neu-
trophilsare rapidly recruitedto theCNS after TBIand enter through
meningealblood vessels andthe choroidplexus.15,32,38,39Theycan
also facilitate the recruitment of monocytes.37
A previous study40
focused on sterile injury of the liver found that ATP released from
the damaged tissue induced inflammasome activation in a P2X7R-
dependent manner. This activation in turn promoted rapid
recruitment of neutrophils through release of chemoattractants
(CXCL1 and CXCL2) and formyl peptides that guided these cells to
thesite of injury. After focal TBI, we observed that neutrophils are
similarly recruited in a P2X7R-dependent manner and arrive
within 1 hour of injury (Figure 2C). 15 Visualization of cellular
dynamics and localization by 2-photon microscopy revealed that
neutrophils localized primarily to the damaged meninges (instead
of theparenchyma), where they swarmed the area andinteracted
with dead cells. Antagonism of this response by blocking P2X7R
signalingincreased theamount of cell death in themeninges, sug-gesting a protective role for neutrophils in the meningeal space
after focal cortical injury.
Neutrophils are not always neuroprotective and have the ca-
pacity tobreak downthe BBBby releasing metalloproteinases,pro-
teases, tumor necrosis factor , and ROS. Inflammatory mediators
released after brain injury can facilitate this process by inducing a
hyperactivated state thatallowsneutrophilsto breach theBBB and
enter the CNS.41 On arrival, neutrophils have the potential to in-
duceneuronalcelldeathusingthesamesolublemediatorsthatbreak
down the BBB.42 A previous study43 revealed that neutrophils are
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themost abundantcell population in circulation after TBIand cause
increasedexpression of oxidative enzymes indicative of activation.
Depletion of neutrophils with antiGr-1 antibodies after controlled
cortical impact in rodents reduced edema, microglia and macro-
phageactivation,and TBIlesion size,but didnot affectvascularleak-
ageat 24 to 48 hoursafter injury.44 These data reveal thatneutro-
phils can be pathogenic after open-skullcortical impact. However,
thecontribution ofneutrophilsto a CNSlesionmay depend ontheir
preciselocalization andstateofactivation. Open-skullcontrolled cor-
tical impact is highly disruptive tomeningeal architecture andlikely
favors neutrophil recruitmentto the heavily damaged brainparen-
chyma. These findings contrast with mild closed-skull cortical in-
jury,which maintainsmeningeal architectureand fostersa more se-
lective patternof neutrophil recruitment.15Todefinitively establish
the contribution of neutrophils to TBI pathogenesis, these cells
shouldbeevaluatedinmanydifferentmodelsofbraininjury.Itiscon-
ceivablethattheir contribution willdifferbasedon thenatureof the
injury.
Figure 1. Pathogenesisof Traumatic Brain Injury (TBI)
ATP
ROS
UDP
Glutamate
Skull
Normal mTBI
Dura mater
Meningealmacrophage
Blood vesselsArachnoid mater
Subarachnoidspace ROS
Fluid leakage,meningealcell death Neutrophils
Jellyfish microgliaMicroglial process
extension to the glial limitans
Glutamate
NMDA
Ca2+ Necroticneuron
UDP
ATPGlial limitans
Pia mater
CSF
Astrocyte
Corticalneuron
Microglia
A
20 m
Parenchymalcell death
250 m
Skull bone
Meningeal macrophages
Parenchyma
Meninges
Microglia
Microglia
B
D
C
E
A, Comparisonof brain anatomy in
the meninges and superficial
neocortexbeforeand after focal mild
TBI(mTBI). Theduramatercontains
numerous small vessels that arelined
by thin,elongatedmeningeal
macrophages.The subarachnoid
spacecontains vessels, fibroblastlike
stromal cells,and cerebrospinal fluid
(CSF).The glial limitans,composedof
astrocytic foot processes, lies
beneath thepia mater andformsa
barrierbetweenthe CSFand
underlyingparenchyma. Mild focal
braininjury mechanically compresses
the meningeal space, compromising
vascular integrityand inducing rapid
necrosis of meningeal macrophages
andstructural cells.Leakageof fluid
from meningeal blood vessels results
in edema,and damaged cells within
the meninges release reactive oxygen
species (ROS) and adenosine
triphosphate(ATP),initiating a sterile
immunereaction. B andC, Maximum
projections (5-m wide) areshownin
thexzplaneof 2-photonz-stacks
captured through thethinned skull of
CX3CR1GFP/+ mice(original
magnification20).
B, A representativeimageof an
uninjured mousereveals the
presence of meningeal macrophages
(green)in theduraand ramified
microglia (green)in thebrain
parenchymabeneaththe glial
limitans (white dotted line).
C, Thirtyminutes after focal mTBI,
meningealmacrophagesdie and
microglia relocate to theinjuredglial
limitans (arrowheads).Skull boneis
shown inblue. D and E,
Histopathologic analysis of the
superficial neocortexby confocal
microscopy8 hours after mTBI
(original magnification20). D, An
uninjuredbrainis shownfor
comparison. Deadcells werelabeled
transcranially with propidium iodide.
Cell nucleiare blue.E, A large lesion
consistingof numerous deadcells
(red)(arrowhead). See Videos1 and
2. UPD indicates uridinediphosphate.
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T Cells
Although T cells play diverse roles in adaptive immune responses
andthe regulation of inflammation, their role (ifany) in TBIpatho-
genesis is not clear. It has been proposed that autoreactive T cells
against CNS antigens, such as myelin basic protein, can be neuro-
protective after spinal cord injury.45 After brain injury, activated T
cellsare recruitedto sites ofdamage,46andROS release may facili-
tate this recruitment by activating endothelial barriers.47 To ad-
dresstheroleof T cells inTBI, a previousstudy48 examined the re-
sponseto closed-skullhead injury in RAG1 knockoutmice that lack
matureT and B cells.No difference in any pathologic or neurologic
parameters was observed between wild-type andRAG1-deficient
mice forup to1 week.Thesedatasuggestthat T cellsplay norolein
early TBI pathogenesis. Additional studies are required to deter-
mine whether T cells actively participate in chronic TBIlesions (be-
yond 1 week) and/or thereparativeprocess.
Therapeutic Modulation of TBI Pathogenesis
The pathogenesis of TBI is complex as reflected by the number of
clinical trials thathave failed to improve outcomes in humans.49,50
The many reasons for these failures have been discussed in other
reviews.49,50Rather thanfocuson thereasonsfor priorfailures, we
instead briefly discuss somesuccessesthat pertain to mechanisms
of pathogenesis and inflammation covered in this review.
Theconceptof freeradicalmediated damage of CNStissue af-
terinjury hasexistedfor several decades.51,52Administration of ef-
fectiveantioxidantshas thepotential to significantlylimit thespread
of damageand inflammationif given soon after brain injury. In ani-
malmodels, a number of previous studies53,54 have yielded prom-
ising results withantioxidants thatneutralizeROS. Forexample,in-
travenousadministrationof thesmall-moleculefree radicalscavenger
edaravone at 2 and 12 hours after weight dropinduced TBI re-
sulted in significantly reduced inflammation, edema, BBB break-
down,lesion size,and neurologicdeficits.53 Inhibitionof NADPHoxi-
dasecomplexassemblywithapocyninalsoreducedROSproduction,
BBB breakdown, and neuronal cell death after weight drop
induced TBI.54 The only caveatof this study was that the apocynin
was injected intraperitoneally 15 minutes before injury. Neverthe-
less,the favorable outcome implicatesNADPH oxidase as a poten-
tial source of ROSafter brain injury.
Using a newmodelof mild cortical injury, we found that trans-
cranial administration of the antioxidant glutathione at 15 minutes
Figure 2. Inflammatory Reaction to Traumatic Brain Injury
A
G
B
E
H
C
F
I
50 m
20 m
50 m
50 m
Normal Ram if ie d Microgl ia Hon eycom b Microgl ia Phagocytic Jel lyfish Microgl ia
Normal mTBI Myelomonocytic Cells
Meningeal Macrophages
D
A-I,The 25-mxymaximum
projections from CX3CR1GFP/+ (A,B,
andD-I)or LysMGFP/+ (C)micewere
captured by 2-photon microscopy
through a thinned skull.A, Meningeal
macrophages (green) are thin,
elongated cellsthat residealong the
dural blood vessels in theuninjured
brain.B, After focal mild traumatic
braininjury (mTBI), meningeal
macrophages undergo necrosis
within30 minutes and disappear
fromthefield of view.
C, Myelomonocytic cells(green)
invadethe damaged meninges within
anhourof braininjury. D and G,In the
uninjured brain,microglia (green)
have small cell bodies andare highly
ramified. Focalbrain injuryinduces
the rapidtransformation of microglia
into at least2 distinct morphologic
patterns.E andH, Honeycomb
microglia extend processes that
circumscribethe bordersbetween
individual astrocytesin the glial
limitans. F andI, Phagocytic jellyfish
microglia are generatedin response
tocelldeathandforma film across
the damaged glial limitans.
High-magnificationviews in panels G
through I aredenoted with white
boxesin panelsD throughF (original
magnification20). See Videos1
and 2.
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or 3 hours after injury significantly reduced inflammation, glial limi-
tansbreakdown,and parenchymal(but notmeningeal)cell deathby
up to approximately 70%.15Pretreatmentwith glutathione reduced
meningealcell deathby approximately50%. Thesedataindicate that
ROS area primary inducer of cell death andinflammationafter focal
brain injury andthatan antioxidant canhavea major effecton lesion
expansion if givenearly.The advantageof passing a neuroprotective
compounddirectlythroughtheskullbone(transcranialdelivery)isthatahighlocaldrugconcentrationcanbeachievedintheCNSwithalim-
itedoff-target effect on theperiphery.
Previous studies55,56 have supported antioxidants as neuro-
protective agents in rats and humans, revealing that administra-
tionofN-acetylcysteine reduces brain damage andimproves recov-
eryafterTBI.N-acetylcysteineisthecellularprecursortoglutathione.
A randomized, double-blind, placebo-controlledclinicaltrial55 was
performed toassess efficacy inmembers ofthe military whoexpe-
rienced a mTBI that resulted from blastexposure. Patients whore-
ceivedN-acetylcysteine within 24 hours had significantly im-
proved recovery during a 7-day period when compared with a
placebo control group. These findings were corroborated in 2 dif-
ferent rodent models of TBI (weight drop and fluid percussion),
whichrevealed thatN-acetylcysteine reversed the behavioral defi-
cits associated with mTBI and moderate TBI.56 Further studies are
needed to determine whether this promising neuroprotective in-
tervention willbe efficaciousin patientswith diverse typesof brain
injury.
Many clinical trials have been completed or are under way to
assess therole of excitotoxicmechanismsin TBIpathogenesis.49,50
With the exception of amantadine, all drugs in this class tested to
datehavenot beeneffective inpromoting recoveryin patientswith
TBI. Amantadine is thought to act as anN-methyl-D-aspartate re-
ceptor antagonist and an indirect dopamine agonist. When pa-
tientswithTBI were treated duringa 4-week periodbeginning4 to
16 weeksafterinjury, amantadineimproved recoveryrelative tothe
placebo control. The mechanismunderlying thispositive effectre-mains unclear. Prevention ofN-methyl-D-aspartate receptor
mediatedexcitatorydamageseemsunlikelygiventhatthe drugwas
administered a monthor more after theinitial injury.57
Manipulationof purinergicreceptorsignaling is another thera-
peutic approach worth considering. Use of specific purinergic re-
ceptor agonists and antagonists should allow therapeutic amelio-
rationof differentTBI lesionparameters.A previous study15 found
that microgliaresponsesafter mTBI weredependent onP2X4,P2Y6,
andP2Y12receptors,whereasP2X7Rsignalingwasnecessaryforneu-
trophil recruitment. It might be possible to promote neuroprotec-
tiveinflammatory responsesthrough therapeutic agonismof these
pathways after brain injury. The challenge, however, with puriner-
gic receptor manipulation is that specific receptors are often ex-pressed on multiple cell populations. A purinergicreceptor agonist
or antagonist will likely affect multiple cell populations simultane-
ously. As an example, a previous58 study found that P2X7R local-
ized to astrocytic end feet and antagonism of this receptor re-
duced astrocyte activation, cerebral edema, and neurobehavioral
abnormalities after controlled cortical impactinduced TBI. A simi-
larprotectiveeffectwasobtainedbyblockingP2X 7Rafterspinalcord
injury, which was linked to receptor expression on spinal cord
neurons.59However, P2X7R isalso expressed oninflammatory cells,
and a previous study15 found that antagonism of this pathway in-
creased meningeal cell death after mTBI, likely due to diminished
neutrophil recruitment. Thus, purinergic receptor modulation can
positively affect one CNS environment and negatively affect an-
other.It willtherefore be important in future studies tomap outthe
exactcontributions of specific purinergicreceptors to differentTBI
lesion parameters beforedeciding which (ifany) arebest to target
therapeutically in patients.
Discussion
The pathogenesis of TBI is initially induced by a mechanical injury
that sets into motion a complex secondary reaction mediated by
ROS, purines, calcium ions, excitatory amino acids, and DAMPs,
amongothers.Thispathogenesis in turntriggers a robuststerileim-
mune reaction that consists of CNS resident and peripherally re-
cruited inflammatory cells. The response is designed to be neuro-
protectiveandpromotewoundhealingbutcanbecomemaladaptive
over time, especially if thelesion remains active for weeks.Among
the earliest soluble mediators are ROS and purines. Both are re-
leased within minutes of brain injury and initiate an inflammatory
cascade. Even after mild focal cortical injury, ROS can damage the
glial limitans that separate themeningesand parenchyma, which re-
sults in lesion expansion within brain tissue. Vascular damage and
leakage represent another early hallmark of TBI pathogenesis that
can foster edema, hypoxia, and tissue destruction. After brain in-
jury,the innateimmune systemquickly mobilizesin responseto pu-
rines andalarmins,and astrocytes helporchestrate this responseby
serving as inflammatory amplifiers. Within minutes, resident mi-
crogliaareamongthefirsttoreactbyfortifyingCNSbarriersandpar-
ticipating in phagocytic cleanup. Neutrophils and monocytes ar-
rive shortly thereafter and preferentially survey injured meningeal
spaces if theCNS architectureremainsintact.Focal brain injury elic-
its an anatomically partitioned immune reaction (at least acutely)
with myelomonocytic cells tending to the damaged meninges andmicroglia responding within the parenchyma. Eventually, myelo-
monocyticcellscanenterthedamagedbrain,andstudies40-42have
found that their presence there is sometimes neurotoxic. How-
ever, sterileimmunereactionsare notinherentlyneurotoxicand are
usuallyelicitedtoprepareadamagedtissueforwoundhealing.Thus,
the entire contribution of immune cellsubsets to TBI lesions needs
to be considered before targeted therapeutic interventions can
be intelligently designed. Another important variable is time. The
exact contribution of immune cells to a TBIlesion mayin fact shift
over time. For example, an initially neuroprotective immune
response may become maladaptive as secondary inducers of tis-
sue destruction diversify.
Although TBI has proven difficult to treat, promisinginterven-tions lieon thehorizon. Given theimportance of ROSin TBIpatho-
genesisandthesuccesswithN-acetylcysteine in patients withmTBI,
clinical pursuit of antioxidant therapy seems warranted. The likely
key to success is early treatment with antioxidants so that TBI le-
sionexpansionand subsequentinflammationcan bestoppedas soon
as they are initiated. Because TBI lesions begin to expand within
hoursof injury, development of strategies to rapidly preserve brain
tissueis paramount. Thekineticsof lesionexpansionmustbe simi-
larlyconsidered whenattemptingto manipulate purinergicand ex-
citatoryneurotransmitterpathways, whichengagerapidlyafter in-
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jury. Therapeutic targeti ng of t hese pathways has the greatest
likelihoodofworkingifadministeredsoonafterinjury.Forthechronic
phase of TBI pathogenesis, more research is required to under-
stand lesion dynamics. Over time, it may become necessary to
dampen maladaptive inflammatory responses andattemptto pro-
mote wound healing reactions, which would be challenging to
achieve without having a better understanding of chronic lesion
dynamics.
Conclusions
Traumatic brain injury encompasses a complex spectrum of inju-
ries that tax the neural-immune interfaceand canresult in perma-
nent neurologic dysfunction. Detailed knowledge of this interface
during the acute and chronic phases of TBI will help us design the
most efficacious interventions.
ARTICLE INFORMATION
Accepted for Publication: October 2, 2014.
Published Online: January19, 2015.
doi:10.1001/jamaneurol.2014.3558.
Author Contributions: DrMcGavernhad full access
to allthe data in thestudy andtakes responsibility
forthe integrityof thedataand theaccuracyof the
data analysis.
Study concept and design: Corps,McGavern.
Acquisition, analysis, or interpretation of data: All
authors.
Draftingof the manuscript: All authors.
Critical revision of the manuscript for important
intellectualcontent: All authors.Obtained funding: McGavern.
Administrative, technical, or material support:
McGavern.
Study supervision: McGavern.
Conflict of Interest Disclosures: Nonereported.
Funding/Support: This work wassupported bythe
National Institutesof Healthintramural program.
Roleof the Funder/Sponsor:Thefunding source
hadno rolein thedesign andconduct of thestudy;
collection, management, analysis, and
interpretation of thedata; preparation,review, or
approval of themanuscript; andthe decision to
submitthe manuscriptfor publication.
Additional Contributions: Ethan Tyler, MA, and
Alan Hoofring, MS, Medical Arts Design Section,
National Institutesof Health, helped withtheillustrationshownin Figure1. MessrsTyler and
Hoofring are salaried employees of the National
Institutes of Healthand were notdirectly
compensatedby ourlaboratoryfor their work.
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