Concussive brain trauma in the mouse results in acute cognitive deficits and sustained impairment of axonal function.
ABSTRACT Concussive brain injury (CBI) accounts for approximately 75% of all brain-injured people in the United States each year and is particularly prevalent in contact sports. Concussion is the mildest form of diffuse traumatic brain injury (TBI) and results in transient cognitive dysfunction, the neuropathologic basis for which is traumatic axonal injury (TAI). To evaluate the structural and functional changes associated with concussion-induced cognitive deficits, adult mice were subjected to an impact on the intact skull over the midline suture that resulted in a brief apneic period and loss of the righting reflex. Closed head injury also resulted in an increase in the wet weight:dry weight ratio in the cortex suggestive of edema in the first 24 h, and the appearance of Fluoro-Jade-B-labeled degenerating neurons in the cortex and dentate gyrus of the hippocampus within the first 3 days post-injury. Compared to sham-injured mice, brain-injured mice exhibited significant deficits in spatial acquisition and working memory as measured using the Morris water maze over the first 3 days (p<0.001), but not after the fourth day post-injury. At 1 and 3 days post-injury, intra-axonal accumulation of amyloid precursor protein in the corpus callosum and cingulum was accompanied by neurofilament dephosphorylation, impaired transport of Fluoro-Gold and synaptophysin, and deficits in axonal conductance. Importantly, deficits in retrograde transport and in action potential of myelinated axons continued to be observed until 14 days post-injury, at which time axonal degeneration was apparent. These data suggest that despite recovery from acute cognitive deficits, concussive brain trauma leads to axonal degeneration and a sustained perturbation of axonal function.
- SourceAvailable from: Matthew T. K. Kirkcaldie[Show abstract] [Hide abstract]
ABSTRACT: Traumatic brain injury (TBI) from penetrating or closed forces to the cranium can result in a range of forms of neural damage, which culminate in mortality or impart mild to significant neurological disability. In this regard, diffuse axonal injury (DAI) is a major neuronal pathophenotype of TBI and is associated with a complex set of cytoskeletal changes. The neurofilament triplet proteins are key structural cytoskeletal elements, which may also be important contributors to the tensile strength of axons. This has significant implications with respect to how axons may respond to TBI. It is not known, however, whether neurofilament compaction and the cytoskeletal changes that evolve following axonal injury represent a component of a protective mechanism following damage, or whether they serve to augment degeneration and progression to secondary axotomy. Here we review the structure and role of neurofilament proteins in normal neuronal function. We also discuss the processes that characterize DAI and the resultant alterations in neurofilaments, highlighting potential clues to a possible protective or degenerative influence of specific neurofilament alterations within injured neurons. The potential utility of neurofilament assays as biomarkers for axonal injury is also discussed. Insights into the complex alterations in neurofilaments will contribute to future efforts in developing therapeutic strategies to prevent, ameliorate or reverse neuronal degeneration in the central nervous system (CNS) following traumatic injury.Frontiers in Cellular Neuroscience 12/2014; 8:429. · 4.18 Impact Factor
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ABSTRACT: Although the mechanisms that contribute to the development of TBI-related deficits are not fully understood, it has been proposed that altered energy utilization may be a contributing factor. The tuberous sclerosis complex, a heterodimer composed of hamartin/Tsc-1 and tuberin/Tsc-2, is a critical regulatory node that integrates nutritional and growth signals to govern energy utilizing processes by regulating the activity of mechanistic Target of Rapamycin complex 1 (mTORC1). mTORC1 activation results in enhanced protein synthesis, an energy consuming process. We show that mice that have a heterozygous deletion of Tsc2 exhibit elevated basal mTORC1 activity in the cortex and the hippocampus while still exhibiting normal motor and neurocognitive functions. Additionally, a mild closed head injury (mCHI) that did not activate mTORC1 in wild-type mice resulted in a further increase in mTORC1 activity in Tsc2+/KO mice above the level of activity observed in uninjured Tsc2+/KO mice. This enhanced level of increased mTORC1 activity was associated with worsened cognitive function as assessed using the Morris water maze and context discrimination tasks. These results suggest that there is a threshold of increased mTORC1 activity after a TBI that is detrimental to neurobehavioral performance, and interventions to inhibit excessive mTORC1 activation may be beneficial to neurocognitive outcome.Journal of Neurotrauma 07/2014; · 3.97 Impact Factor
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ABSTRACT: For the past 25 years, controlled cortical impact (CCI) has been a useful tool in traumatic brain injury (TBI) research, creating injury patterns that includes primary contusion, neuronal loss, and traumatic axonal damage. However, when CCI was first developed, very little was known on the underlying biomechanics of mild TBI. This paper uses information generated from recent computational models of mild TBI in humans to alter CCI and better reflect the biomechanical conditions of mild TBI. Using a finite element model of CCI in the mouse, we adjusted three primary features of CCI: the speed of the impact to achieve strain rates within the range associated with mild TBI, the shape, and material of the impounder to minimize strain concentrations in the brain, and the impact depth to control the peak deformation that occurred in the cortex and hippocampus. For these modified cortical impact conditions, we observed peak strains and strain rates throughout the brain were significantly reduced and consistent with estimated strains and strain rates observed in human mild TBI. We saw breakdown of the blood-brain barrier but no primary hemorrhage. Moreover, neuronal degeneration, axonal injury, and both astrocytic and microglia reactivity were observed up to 8 days after injury. Significant deficits in rotarod performance appeared early after injury, but we observed no impairment in spatial object recognition or contextual fear conditioning response 5 and 8 days after injury, respectively. Together, these data show that simulating the biomechanical conditions of mild TBI with a modified cortical impact technique produces regions of cellular reactivity and neuronal loss that coincide with only a transient behavioral impairment.Frontiers in Neurology 06/2014; 5:100.
Concussive Brain Trauma in the Mouse Results
in Acute Cognitive Deficits and Sustained
Impairment of Axonal Function
Jennifer A. Creed,1Ann Mae DiLeonardi,1Douglas P. Fox,2
Alan R. Tessler,2,3and Ramesh Raghupathi1,2
Concussive brain injury (CBI) accounts for approximately 75% of all brain-injured people in the United States
each year and is particularly prevalent in contact sports. Concussion is the mildest form of diffuse traumatic
brain injury (TBI) and results in transient cognitive dysfunction, the neuropathologic basis for which is traumatic
axonal injury (TAI). To evaluate the structural and functional changes associated with concussion-induced
cognitive deficits, adult mice were subjected to an impact on the intact skull over the midline suture that resulted
in a brief apneic period and loss of the righting reflex. Closed head injury also resulted in an increase in the wet
weight:dry weight ratio in the cortex suggestive of edema in the first 24h, and the appearance of Fluoro-Jade-B-
labeled degenerating neurons in the cortex and dentate gyrus of the hippocampus within the first 3 days post-
injury. Compared to sham-injured mice, brain-injured mice exhibited significant deficits in spatial acquisition
and working memory as measured using the Morris water maze over the first 3 days (p<0.001), but not after the
fourth day post-injury. At 1 and 3 days post-injury, intra-axonal accumulation of amyloid precursor protein in
the corpus callosum and cingulum was accompanied by neurofilament dephosphorylation, impaired transport
of Fluoro-Gold and synaptophysin, and deficits in axonal conductance. Importantly, deficits in retrograde
transport and in action potential of myelinated axons continued to be observed until 14 days post-injury, at
which time axonal degeneration was apparent. These data suggest that despite recovery from acute cognitive
deficits, concussive brain trauma leads to axonal degeneration and a sustained perturbation of axonal function.
Key words: axonal injury; axonal transport; compound action potential; concussion; spatial learning; traumatic
brain injury; working memory
each year, and is particularly prevalent in contact sports
(Bazarian et al., 2005; Ropper and Gorson, 2007). The diag-
nosis of concussion is based on a rapid onset of impaired
neurologic function such as loss of balance and amnesia
(McCrory et al., 2009). Although 80–90% of concussions re-
solve spontaneously within 10 days, some functional deficits
persist for weeks to years (Geurts et al., 1999; McCrea et al.,
2003). Impaired cognitive functions include loss of concen-
tration, deficits in working memory, and reduced speed of
processing (Levin et al., 1987; McCrory et al., 2009). In this
oncussive brain injury (CBI) accounts for approxima-
tely 75% of all brain-injured people in the United States
and visual memory tasks (Lovell et al., 2003, 2004; Maddocks
and Saling, 1996), and information processing (Hinton-Bayre
et al., 1999; Niogi et al., 2008), and impaired attention (Kraus
et al., 2007). Although standard neuroimaging fails to identify
structural abnormalities, diffusion tensor imaging can detect
axonal injury (Arfanakis et al., 2002; Niogi et al., 2008; Smits
et al., 2010; Wilde et al., 2008). These imaging findings have
been supported by post-mortem examination of several cases
of concussed brains demonstrating abnormal axonal mor-
phology and disrupted axonal transport (Blumbergs et al.,
Experimental models of CBI, using either fluid-percussion
or weight-drop injury in rats and cats, traditionally have been
defined by brief periods of apnea, suppression of electrical
brain activity, and loss of pressure autoregulation (Dixon
1Program in Neuroscience,
Administration Medical Center, Philadelphia, Pennsylvania.
2Department of Neurobiology and Anatomy, Drexel University College of Medicine, and
JOURNAL OF NEUROTRAUMA 28:547–563 (April 2011)
ª Mary Ann Liebert, Inc.
et al., 1987; Foda and Marmarou, 1994; Hamm, 2001; Yamaki
et al., 1994), ionic dyshomeostasis such as increased extra-
cellular potassium (Katayama et al., 1990), and prolonged
calcium accumulation (Fineman et al., 1993), as well as
changes in patterns of regional glucose utilization (Hovda
et al., 1990; Yoshino et al., 1991). Accompanying these acute
alterations were intra-axonal swellings characterized by
the presence of compacted neurofilaments and disrupted
microtubules, leading to accumulation of organelles and
vesicles containing b-amyloid precursor protein (b-APP)
(Buki and Povlishock, 2006; Foda and Marmarou, 1994;
Lewen et al., 1995; Povlishock et al., 1983; Stone et al., 2000).
Moreover, both myelinated and unmyelinated axons within
the corpus callosum exhibited reduced compound action
potentials (CAPs) up to a week after injury (Baker et al., 2002;
Reeves et al., 2005) indicative of functional deficits. However,
behavioral analysis in brain-injured animals revealed acute
(within 3 days) and chronic (up to 1 month) deficits in vesti-
bulomotor and cognitive function (Hamm, 2001; Tang et al.,
1997a; Yamaki et al., 1997; Zohar et al., 2003), along with post-
concussive syndrome (Ryan and Warden, 2003; Williams
et al., 2010).
Diffuse traumatic brain injury (TBI), including CBI, has
been modeled in mice (Laurer et al., 2001; Longhi et al., 2005;
Spain et al., 2010; Tang et al., 1997a, 1997b; Zohar et al., 2003).
Injury induced by a weight-drop, fluid-percussion, or a
modified cortical impact device resulted in diffuse neurode-
generation in the cortex and hippocampus, and b-APP-
positive intra-axonal swellings in the thalamus, corpus
2010; Tang et al., 1997b; Tashlykov et al., 2007). As was ob-
served in rats, closed head injury in mice resulted in long-term
behavioral dysfunction characterized by learning deficits, de-
pressive behavior, and increased passive avoidance (Milman
et al., 2005; Spain et al., 2010; Tang et al., 1997a; Zohar et al.,
2003). In contrast, impact to the intact skull using a silicone-
tipped indenter only resulted in a transient deficit in motor
function with no effect on spatiallearning ability(Laureret al.,
acute neurochemical, microscopic, and anatomical patho-
physiology of concussive brain trauma, they do not appear to
model the hallmark of concussion: transient neurologic (cog-
nitive) dysfunction. With this in mind, we developed a model
manner similar to previously published studies in immature
rats (DiLeonardi et al., 2009; Huh et al., 2008).
over the midline suture between the lambda and bregma
sutures resulted in spatial learning and working memory
deficits only in the first 3 days after trauma, which were re-
solved by day 4 post-injury. Axonal swellings in the corpus
callosum containing APP, dephosphorylated neurofilament,
or synaptophysin, were observed up to 3 days post-injury, as
well as degenerating axons at 14 days post-injury. These
structural alterations in injured axons were accompanied by
functional deficits that manifested as reductions in CAPs and
decreased retrograde transport which were present up to 2
weeks post-injury. In addition to alterations in the white
matter, the cortex underlying the impact site was edematous
over the first 24h post-injury and contained Fluoro-Jade-B-
positive neurons. Neuronal degeneration was also observed
within the hilus of the dentate gyrus up to 3 days post-injury.
Collectively, these observations are indicative of the ongoing
degeneration of the concussed brain despite resolution of
Adult male C57BL/6J mice (18–35g; Jackson Laboratories,
Bar Harbor, ME)were used for this study (Table 1). Mice were
in a controlled temperature environment with a 12-h light-
dark cycle. All procedures used followed the guidelines set by
the U.S. Public Health Service Policy on Humane Care and
Use of Laboratory Animals, and the National Institutes of
Health (NIH) Guide for the Care and Use of Laboratory An-
imals, and were approved by the Drexel University Institu-
tional Animal Care and Use Committee.
Concussive brain injury
Mice were subjected to closed head injury in a manner
similar to that used in immature rats (Huh et al., 2008). An-
esthesia was induced with isoflurane (1.5%; Webster Veter-
inary, Sterling, MA) inhalation via a nose cone, and the
corneas were kept moist during surgery by applying Lu-
britears ointment (Bausch and Lomb, Tampa, FL). Body
temperature was maintained throughout theentire procedure
by using a heating pad at 37?C. The scalp was swabbed with
povidone-iodine and a subcutaneous injection of lidocaine
was administered prior to making a 1.0-cm midline rostral-to-
caudal incision to expose the skull. The periosteum was re-
flected (Fig. 1A), and the animals were placed in a standard
mouse restrainer (Braintree Scientific, Braintree, MA) with the
head supported by a soft foam pad to make it level with the
body. The restrainer was positioned under the cortical impact
device (Custom Design & Fabrication, Richmond, VA), and
the 5-mm-diameter hemispheric metal impactor tip was zer-
oed by touching it to the sagittal suture midway between the
bregma and the lambda (Fig. 1B). At 30sec after removal of
anesthesia, the impactor was electronically driven perpen-
dicularly onto the exposed sagittal suture at a velocity of
5.0m/sec to a depth of 1.5mm farther than the zero point.
Immediately after the impact, the righting reflex was evalu-
ated by measuring the time required for the mice to regain
their normal posture over three consecutive attempts when
placed in the supine position. The animals were re-anesthe-
tized so that the scalp incision could be closed with 4-0 silk
suture. Sham-injured mice were subjected to the same pro-
cedures without receiving an impact. All animals were al-
lowed to recover on a heating pad set at 37?C, and upon
becoming ambulatory were returned to their home cages.
Transection of the corpus callosum was performed as pre-
viously described (Schalomon and Wahlsten, 1995). Mice
(n=7) were anesthetized with sodium pentobarbital (65mg/
kg IP) and immobilized in a stereotaxic frame. The scalp was
swabbed with povidone-iodine and a subcutaneous injection
of lidocaine was administered prior to making a 1.0-cm
midline rostral-to-caudal incision to expose the skull. A
5-mm-diameter craniectomy was made over the sagittal su-
ture midway between the bregma and the lambda. The dura
548CREED ET AL.
was removed and an ultra-fine micro-knife (15? cutting angle,
0.15-mm thickness, carbon steel tip; Fine Science Tools, Foster
City, CA) secured to a stereotaxic arm was lowered slowly
between the two cortical hemispheres at the bregma suture,
taking care not to disrupt the sagittal sinus. When the blade
reached a depth of 2mm, it was moved 3mm in the posterior
direction along the sagittal groove. No bleeding was ob-
served, indicating that the vein was not breeched. Following
the transection, a cranioplasty was glued to the skull using
Vetbond, and the scalp incision was closed with 4-0 silk su-
ture. All animals were allowed to recover on a heating pad set
at 37?C, and upon becoming ambulatory were returned to
their home cages.
Tissue water content
An increase in tissue water content, anindication of edema,
was determined using the wet weight:dry weight method
(Dempsey et al., 2000). Briefly, at either 6 or 24h following
surgery or injury, the animals were euthanized using Eu-
thasol?(Virbac AH Inc., Fort Worth, TX) and decapitated.
The brain was removed and a 3-mm-thick coronal section
Table 1. Acute Neurological Status Responses following Closed Head Injury
StudyExperimental groupn Apnea (sec) Righting reflex (sec)Skull fracture (%)
Tissue water content
Retrograde axonal transport
Spatial learning (days 1–3) and working
memory (days 7–9)
Spatial learning (days 4–6)
Working memory (days 1–3)
Compound action potentials
*p<0.05 compared to study-matched sham animals.
Immediately following impact, mice were evaluated for time of loss of breathing (apnea), righting reflex, and the presence of skull
fractures. Separation either by recovery time within a study or across the multiple experiments showed no significant differences in the
injured animals for both latency to the righting reflex and the duration of apnea. Apnea, righting reflex, and skull fracture values are
presented as the mean–standard deviation.
NA, not applicable.
metal indentor tip was zeroed onto the surface of the skull, centered between the lambda and bregma sutures and over the
sagittal suture. (C). Note the location of the minor fracture perpendicular to the sagittal suture typically observed immedi-
ately following impact.
Model of concussive brain trauma. (A) The skull was exposed by reflecting the periosteum. (B) The hemispheric
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT 549
representing the area directly below the impact site was iso-
lated and placed on an ice-cold glass plate. The cortex, hip-
pocampus, and thalamus from injured and sham (uninjured)
animals were dissected and immediately weighed to obtain
the wet weight (WW). The samples were then dried in a
convection oven at 70?C for 48h and weighed to determine
the dry weight (DW). Water content was then calculated us-
ing the equation: [(WW – DW)/WW] * 100.
Retrograde axonal transport
The neuronal tracer Fluoro-Gold (FG; FluoroChrome Inc.,
Englewood, CO) was used to assess retrograde axonal
transport (Schmued and Fallon, 1986). Mice were anesthe-
tized with sodium pentobarbital (65mg/kg IP) and im-
mobilized in a stereotaxic frame. The scalp was swabbed with
povidone-iodine and a subcutaneous injection of lidocaine
was administered prior to making a 1.0-cm midline rostral-to-
caudal incision to expose the skull. A hand drill was used to
expose the dura above the target site (2.0mm anteroposterior
from the bregma suture, and 2.0mm mediolateral from the
sagittal suture), and FG (1lL of a 2% solution in de-ionized
water) was manually injected into the cortex (0.5mm deep
from the dura) over 3min using a 10-lL gas-tight Hamilton
syringe with a beveled tip (0.47mm outer diameter, 26 gauge;
SGE Analytical Science, Austin, TX). To mitigate backflow
the injection. Sham- and brain-injured mice received FG in-
jections at 1, 5, or 12 days following surgery and/or injury,
while callosotomy mice were injected with FG immediately
after the transection; all mice were euthanized 48h later. FG-
positive cells were counted in three coronal sections (1.46,
1.94, and 2.42mm posterior to the bregma) in 4 adjacent non-
overlapping fields (10· magnification) covering the dorsal-
ventral cortex in the hemisphere contralateral to the injection
site. Values represent the mean number of FG-positive cells
across each of the four high-powered fields (HPF) in each of
the 3 sections.
following CBI or sham-injury, the brains were processed for
immunohistochemical and histologic analysis as previously
described (Huh et al., 2008; Saatman et al., 2006). One set of
40-lm-thick coronal sections taken between +1.1mm and
-3.8mm relative to bregma (10–11 sections/set) was moun-
ted onto gelatin-coated slides and stained with 2% cresyl vi-
olet and 0.2% cyanine R (Nissl-myelin). A second set of
sections was mounted and stained with Fluoro-Jade-B (Che-
micon, Temecula, CA) as previously described (Huh et al.,
2008; Tong et al., 2001). All Fluoro-Jade-B-labeled cells in each
section were manually counted in the following manner: the
total number of Fluoro-Jade-B-positive cells was counted in
the cortex (both hemispheres) in each of 8 sections (approxi-
mately 480lm apart) between 0.86mm anterior to bregma
and 3.4mm posterior to bregma, and are presented as an
arithmetic mean per section. Five coronal sections (480lm
apart) between 1.34mm and 3.18mm posterior to bregma
were used to count all Fluoro-Jade-B-positive cells in the
hippocampus. Sets of adjacent sections were analyzed
for the presence of b-APP, polyclonal antibody to the
C-terminus of the protein (1:2000; Zymed, Carlsbad, CA),
dephosphorylated 200-kDa neurofilament protein (1:2500,
clone SMI32; Covance, Princeton, NJ), and synaptophysin
(1:1000, clone SVP-38; Sigma-Aldrich, St. Louis, MO), using
standard procedures (DiLeonardi et al., 2009; Huh et al., 2008;
Saatman et al., 2006). As a negative control, one or two
sections from each animal were incubated with all reagents
except the primary antibodies. APP-immunoreactive profiles
were quantified in three coronal sections (1.46, 1.94, and
2.42mm posterior to bregma) from each brain as previously
described (DiLeonardi et al., 2009).
At 24h, and 3 and 7 days following CBI or sham-injury, the
corpus callosum was dissected on an ice-cold glass plate and
placed in buffer (50mM Tris HCl [pH 8.0], 150mM NaCl,
2mM EDTA, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet
P-40, and 0.5% sodium deoxycholate) containing the compo-
nents of the Complete Roche system and pepstatin A (20lg/
mL) to inhibit proteases, and 50mM sodium vanadate to in-
hibit phosphatases. Tissue samples were sonicated and then
centrifuged at 14,000g for 10min to separate the supernatant
fraction (s1) from the pellet. Samples of the s1 fraction (25lg
total protein/well) were subjected to immunoblot analyses
using a monoclonal antibody to myelin basic protein (MBP,
1:1000, clone SMI-99; Covance). Membranes were then re-
probed with actin (1:1000, clone AC-40, Sigma-Aldrich),
which served as a loading control. Protein expression was
quantified using densitometry with the GeneSnap imaging
system and software (SynGene, Frederick, MD). The inte-
grated density values (IDV) of the 21.5-kDa and 18.5-kDa
MBP bands were normalized to the IDV of the actin band.
Spatial learning and working memory assessment
Spatial learning in sham- and brain-injured mice was as-
sessed using the Morris water maze as previously described
(Huh et al., 2008). Each mouse was trained over 3 consecutive
days (days 1–3 ordays 4–6 post-injury), with 4 trials each day,
and allotted a maximum of 60sec to locate the eccentrically-
placed submerged platform in a 1-m-diameter water maze.
was computed as the average of 4 trials. At 24h following the
platform and the latency to reach the platform was recorded
in 2 trials (visible platform trial); the swim speed of the animal
was also calculated. Working memory was assessed in the
Morris water maze as previously described (Hoane et al.,
2003), with the minor modification that the mice were re-
leased from a fixed starting point located on the periphery of
the maze for all trials. The animals were required to locate the
hidden platform using visual cues placed outside and around
the pool. The platform was positioned in a different location
on each of the 3 testing days. Each mouse was trained over 3
consecutive days(days 1–3or days7–9) with4 trialseach day,
and allotted a maximum of 60sec to locate the platform. The
first trial of every day was deemed the ‘‘learning trial,’’ and
the average of trials 2–4 was computed (Hoane et al., 2003).
Compound action potential measurements
CAPs in the corpus callosum were evaluated as previously
described (Reeves et al., 2005). The mice were anesthetized
550CREED ET AL.
with isoflurane (5%), and decapitated, and the brains were
quickly removed. Coronal slices (450lm) were cut on a Vi-
bratome and placed in artificial cerebrospinal fluid (aCSF; pH
7.4), composed of (in mM): NaCl 130,KCl 3.5, Na2H2PO41.25,
and gassed with 95% O2/5% CO2. The sections were incu-
bated for 1h at room temperature and then transported to the
recording chamber where they were submerged andperfused
with aCSF (22?C) at a constant rate (3–4mL/min) for the re-
mainder of the experiment. Recordings were made at room
temperature (22?C) in order to differentiate the myelinated
et al., 2005). A bipolar, tungsten stimulating electrode (inter-
tip distance *0.5mm) was lowered into the corpus callosum
about 0.5mm lateral to the midline. A glass extracellular re-
1mm from the stimulating electrode across the midline.
Evoked CAPs were recorded using an Axoclamp 2b amplifier
and digitized at 100kHz. The amplitude of the CAPs was
measured from standardized input–output curves from each
slice, which were generated by increasing the intensity of
stimulus pulses in 10 equal current steps (200lsec duration,
0.1Hz), ranging from the threshold (the current where CAPs
were first observed) to the maximum. To examine refractori-
ness, pairs of pulses were presented for which the inter-pulse
interval increased in 0.5-msec steps from 3msec to 12msec
using the maximum current level determined from the input–
output curve for that slice.
Quantitative analysis was performed on waveforms that
represented the average of 4 successive sweeps in each of 2
slices per animal. The difference from thefirst positive peak to
the first trough was determined as the amplitude of the my-
elinated fibers (N1), while the difference between the second
peak and the second trough was determined as the amplitude
of the slower conducting unmyelinated fibers (N2). To ana-
lyze refractoriness of the N1 and N2 components, the CAP
amplitude elicited by the second pulse (C2) in each paired
stimulation was divided by the CAP amplitude elicited by the
first pulse stimulation (C1), and this ratio was plotted as a
function of the inter-pulse interval. The inter-pulse interval
corresponding to the point at which the amplitude of the
second pulse achieved 50% of that of the first pulse was
All quantification was performed by an evaluator who was
blinded to the injury status and survival time of the sample.
All data are expressed as the mean–standard deviation.
Comparisons of tissue water content, histologic data, immu-
noblot data, and acute neurologic parameters were deter-
mined using factorialone-way
(ANOVA), followed by Newman-Keuls post-hoc testing if
ANOVA rejected the null hypothesis. Spatial learning,
working memory, and CAPs were analyzed using repeated-
measures ANOVA, followed by Newman-Keuls post-hoc
tests. Probe trial scores, swim speeds, and latencies to the
time point. Refractoriness was compared using the Mann-
Whitney U test. Only p values <0.05 were considered to be
Acute neurological responses
following closed head injury
Closed head injury in adult mice did not result in acute or
delayed mortality. However, impact with the metal-tipped
indenter resulted in a minor fracture perpendicular to the
sagittal suture (arrow in Fig. 1C). Sham-injured mice righted
themselves spontaneously within 15–20sec after removal of
anesthesia. In contrast, the return of the righting reflex (in-
dicative of the duration of unconsciousness) was significantly
longer after closed head injury (140–170sec, p<0.05, Table 1).
Brain-injured mice experienced a brief period of apnea rang-
ing from about 20–40sec, which was not observed in sham-
injured mice (p<0.05, Table 1). Neither latency to the righting
reflex nor the duration of apnea in the injured animals sepa-
rated either by survival time post-injury within a study or
across the multiple experiments was significantly different
from each other, indicative of the consistency of the injury
across the different groups.
Transient cognitive deficits following closed head injury
Closed head injury in the adult mouse resulted in spatial
acquisition deficits in the Morris water maze when tested on
days 1–3 post-injury (Fig. 2A). Although both sham- and
brain-injured mice were able to locate the hidden platform
over the 3-day testing period [day effect, (F(2,26)=4.1,
p<0.04)], the time taken by brain-injured mice to reach the
submerged platform was significantly greater compared to
their sham-injured counterparts [injury effect, (F(1,13)=7.1,
p<0.025)]. When a separate group of sham- and brain-injured
mice was tested for spatial learning on days 4–6 post-injury,
no deficit was observed [Fig. 2B, injury effect (F(1,16)=0.03,
p=0.86)], and all animals learned the location of the platform
over the3daysoftraining [dayeffect,(F(2,32)=26.6, p<0.001)].
Closed head injury did not appear to cause motor impair-
ment, based on the observation of similar swim speeds be-
tween sham-injured (21.0–3.1cm/sec) and brain-injured
to be an issue in the brain-injured mice because the latency to
the visible platform was not significantly different between
(11.2–2.4sec). In addition to spatial learning deficits, brain-
injured mice exhibited deficits in working memory in the
acute (days 1–3) post-traumatic period, with an injury effect
[Fig. 2C, (F(1,13)=6.6, p<0.025)], but not at a later time point
(days 7–9) post-injury [Fig. 2D, (F(1,13)=2.5, p=0.13)].
Traumatic axonal injury following closed head injury
In sham-injured animals b-APP immunoreactivity was not
observed in the corpus callosum (Fig. 3A). Closed head injury
resulted in intra-axonal b-APP immunoreactivity that was
observed as swellings in otherwise contiguous axons at 24h
bulbs at 3 days post-injury (arrowhead in Fig. 3C). Periso-
matic axons containing b-APP were occasionally observed
within the cortex below the impact site, and in the dorsome-
dial and dorsolateral thalamic nuclei (data not shown). In
addition to the ventral hippocampal commissure and fimbria,
swollen axonal profiles containing b-APP were observed
along the central portion of the corpus callosum, extending
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT 551
away from the midline to white matter regions under the
cingulum and in the lateral aspect of the white matter tract
(top panel in Fig. 3J). By 3 days post-injury, the pattern of
axonal b-APP staining was restricted to the fimbria and cor-
pus callosum (middle panel in Fig. 3J). By 7 days, b-APP im-
munoreactivity was scarce (bottom panel in Fig.3J), and by14
days staining was absent (data not shown). Quantification of
theextent ofb-APP-reactiveaxonal profilesrevealed aninjury
effect (F(3,19)=63.4, p<0.0001, Fig. 3K). Post-hoc analysis in-
dicated that there were significantly more injured axonal
profiles at 24h (p<0.001) and 3 days post-injury (p<0.01)
compared to sham-injured animals, greater axonal injury at
24h than at 3 (p<0.001) or 7 days (p<0.001), and at 3 days
compared to 7 days (p<0.001). At 7 days post-injury, the
extent of axonal b-APP reactivity was not significantly dif-
ferent from that in sham-injured animals (Fig. 3K).
Axonal synaptophysin (SYP) immunoreactivity was used
to specifically evaluate transport impairment in the ante-
rograde direction. In brains from sham-injured mice, immu-
noreactivity for SYP demonstrated a diffuse granular pattern
in the cortex (data not shown), and was absent in underlying
white matter (Fig. 3D). Following closed head injury, SYP
staining was evident in axons and appeared as swellings in a
linear pattern along the length of the axon at 24h (arrows in
Fig. 3E), and as larger diameter swellings at 3 days following
injury (arrowhead in Fig. 3F). Few if any SYP-positive axonal
profiles were present at 7 days following injury (data not
shown). Axonal immunoreactivity for SYP wasonly observed
in the corpus callosum below the site of impact (Fig. 3J).
The presence of dephosphorylated neurofilaments within
axons (Christman et al., 1994), detected using the SMI-32 an-
tibody, has been used as an indicator of TAI. Compared to
sham-injured brains (Fig. 3G), intra-axonal SMI-32 immuno-
reactivity was observed at 24h post-injury (Fig. 3H and I),
either as increased varicosity (arrows in Fig. 3H), or as ter-
minal bulbs (arrowhead in Fig. 3H). At 3 (arrowhead in Fig.
3I) and 7 days post-injury, the extent of SMI-32-positive axo-
nal swellings was reduced.
Axonal degeneration following closed head injury
Fluoro-Jade-B histochemistry was used to determine if TAI
was associated with axonal degeneration (Schmued et al.,
1997). In sham-injured animals (Fig. 4A), and at 24h following
closed head injury (Fig. 4B), there was no evidence of Fluoro-
Jade-B-positive axonal profiles within any white matter tract
analyzed. At 7 (data not shown) and 14 days post-injury (Fig.
4C), Fluoro-Jade-B staining was evidentinthe corpuscallosum
as swollen axonal segments (arrow in Fig. 4C), and terminal
bulbs (arrowhead in Fig. 4C), and in the cingulum and lateral
white matter tracts. These observations suggest that axons
continue to degenerate after accumulation of APP, synapto-
physin or dephosphorylated neurofilament are no longer
visible. Despite the presence of TAI and axonal degeneration,
Nissl-cyanineR histochemistry did not revealovert differences
in the corpus callosum (Fig. 4D and E), cingulum, and lateral
injured animals. Immunoblot analysis did not show any dif-
ferences in the expression of MBP in white matter tracts
between sham-injured and brain-injured animals at any time
point post-injury, for either the 21.5-kDa (F(3,12)=2.8, p=0.09)
or 18.5-kDa band (F(3,12)=1.3, p=0.32, Fig. 4F).
Retrograde axonal transport deficits
following closed head injury
The presence of acute TAI and chronic axonal degeneration
led us evaluate the integrity of retrograde axonal transport
using Fluoro-Gold (FG). Confirmation that FG transport oc-
Latency to platform (sec)
Spatial learningWorking memory
memory (C and D) abilities as described in the methods section. Repeated-measures analysis of analysis of variance (AN-
OVA) revealed an injury effect in both the spatial acquisition (A; p<0.025) and the working memory (C; p<0.025) paradigms
on days 1–3 post-injury. No deficits were observed in spatial learning on days 4–6 post-injury (B), or in working memory on
days 7–9 post-injury (D). All values are presented as mean–standard deviation.
Cognitive deficits following concussive brain trauma. Mice were tested for spatial learning (A and B), and working
552 CREED ET AL.
b b-APP IR (%)
axonal immunoreactivity for b-amyloid precursor protein (b-APP, A–C), synaptophysin (SYP, D–F), and dephosphorylated
neurofilament (SMI-32, G–I), in the corpus callosum at 24h (B, E, and H), and 3 days (C, F, and I) following impact. Note the
absence of immunoreactivity for all three proteins in sham-injured brains (A, D, and G). b-APP-positive profiles were
identified as swellings along contiguous axons (arrows in B), and terminal bulbs (arrowhead in C). SYP immunoreactivity
was present as punctate staining (arrows in E), and larger swellings (arrowhead in F). SMI-32-positive profiles were apparent
as swellings (arrows in H), and retraction balls (arrowheads in H and I). (J) Schematic diagram showing the spatial distri-
bution of b-APP (gray circles), SYP (white circles), and SMI-32 (black circles), at 24h (top panel), 3 days (middle panel), and 7
days (bottom panel) post-injury. (K) Quantification of the extent of intra-axonal b-APP immunoreactivity in white matter
tracts below the site of impact. All values are presented as mean–standard deviation (*p<0.01, **p<0.001 compared to sham-
injured brains; #p<0.001 compared to 3 or 7 days post-injury; scale bar=100lm for panels A, D, and G, and 20lm for all
Traumatic axonal injury following concussive brain trauma. Representative photomicrographs demonstrating intra-
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT553
curred via callosal fibers was based on the observation that
transection of the corpus callosum led to a complete absence
of FG-positive cells in the homotypic cortex contralateral to
the injection site (Fig. 5B). Furthermore, intense FG staining at
the site of injection (Fig. 5A) to similar extents in sham-injured
and brain-injured animals suggested that closed head injury
did not affect uptake of FG at axon terminals. At 2 days after
the injection of FG into the cortex of sham-injured animals,
FG-positive cell bodies were detected in the homotypic cortex
contralateral to the injection site (Fig. 5C). Higher magnifica-
bodies (inset in Fig. 5C). In contrast, when FG was injected at
12 days following closed head injury and the animals were
sacrificed 48h later, there was a noticeable reduction in the
extent of FG-labeled cells in the cortex contralateral to the
injection site (Fig.5D). Quantitative analysisrevealed thatthis
reduction was present as early as 24h post-injury, and was
sustained until 12 days [Fig. 5E, (F(3,24)=3.1, p<0.05 by one-
way ANOVA)]. Post-hoc analysisindicated that the number of
FG-positive cells in the sham-injured brains was significantly
greater than at 1 (p<0.04), 5 (p<0.025), and 14 days
(p<0.025, Fig. 5E) post-injury.
Compound action potentials
following closed head injury
Evoked CAPs were used to determine whether structural
and functional alterations in the white matter were associated
Jade B–reactive axons in the corpus callosum at 14 days post-injury (C), but not in either sham-injured animals (A), or at 24 hs
following injury (B). Note the appearance of swollen axonal segments (arrow in C), and terminal bulbs (arrowhead in C).
Panels D (sham-injured) and E (7 days post-injury) are photomicrographs of Nissl-cyanine R staining in the corpus callosum.
(F) Representative immunoblots of myelin basic protein from lysates of corpus callosum of sham-injured animals, and at 24
hours (Inj 24h), 3 days (Inj 3 days), and 7 days (Inj 7 days) post-injury, demonstrating the characteristic 21.5- and 18.5-kDa
bands. Quantification of optical density as a function of actin (loading control) is presented in the graph. All values are
presented as mean–standard deviation (scale bar=20lm for panels A–C, and 50lm for panels D and E).
Axonal degeneration following concussive brain trauma. Representative photomicrographs demonstrating Fluoro-
554 CREED ET AL.
with axonal conduction deficits. As observed in adult rats
(Reeves et al., 2005), evoked CAPs in adult sham-injured
and brain-injured mice resulted in a biphasic waveform
comprised of an initial segment (N1), representing the
fast-conducting myelinated axons, followed by a second
component of the waveform (N2), which characterized the
slower-conducting unmyelinated axons (Fig. 6A). Quantita-
tive analysis of CAPs recorded from injured slices revealed a
decrease in amplitude for the N1 component [injury effect,
(F(1,18)=16.6, p<0.001 Fig. 6B)], which was independent of
survival time [injury·time effect, (F(1,18)=0.02, p=0.90, Fig.
6B)]. The amplitude for the N2 component did not show a
difference between the sham-injured and brain-injured
p=0.56, Fig. 6C)]. Refractoriness, indicative of the time re-
quired to depolarize the membrane from a hyperpolarized
state into a range in which a second action potential can be
initiated, for the myelinated (N1) fibers did not differ between
the sham and injured groups at both 24h (p=0.70), and 14
days (p=0.12, Fig. 6D). For the unmyelinated (N2) fibers,
the refractoriness was no different between slices from sham-
injured and brain-injured mice at 24h (p=0.70); however, at
14 days post-injury, the amount of time required for refrac-
toriness to reach 50% was significantly longer in slices from
Fluoro-Gold (FG)-labeled cell bodies in the cortex. (A) FG-positive cell bodies at the site of injection in sham-injured mice. (B)
Note the absence of FG-labeled cell bodies in the cortex contralateral to the site of injection following transection of callosal
fibers. (C) FG-positive cell bodies in the homotypic cortex contralateral to the site of injection in sham-injured mice. (D) At 14
days post-injury; note the particulate nature of FG (see also inset in C). (E) Quantification of FG-positive cells was performed
as described in the methods section. All values are presented as mean–standard deviation (HPF, high-powered field;
*p<0.05 compared to sham-injured animals; scale bar=100lm for panels A–D, and 10lm for inset in panel C).
Impaired retrograde transport following concussive brain trauma. Representative photomicrographs demonstrating
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT 555
injured brains than in those from sham-injured brains
(p<0.04, Fig. 6E). The conduction velocity of either the N1 or
the N2 component was not affected by injury at either sur-
vival time point (data not shown).
Cortical damage following closed head injury
In sham-injured animals, there was no evidence of Fluoro-
Jade-B reactivity in any region of the cortex (Fig. 7A). At 24h
granular and agranular retrosplenial cortex in both hemi-
spheres (Fig. 7B). The pattern of Fluoro-Jade-B-positive cells
often extended laterally along the cortex corresponding to the
primary and supplementary motor cortex, the lateral parietal
cortex, and the trunk region of the primary sensory cortex.
Quantitative analysis of Fluoro-Jade-B-positive cells in the
cortex revealed an injury effect (F(3,20)=26.8, p<0.0001);
post-hoc analysis demonstrated significantly more Fluoro-
Jade-B-positive cells at 24h compared to sham-injured
animals (p<0.001), and at 3 (p<0.001) and 7 days (p<0.001)
following injury (Fig. 7C). In addition, there was a greater
extent of cortical Fluoro-Jade-B staining at 3 days post injury
compared tosham-injured animals(p<0.03).Nisslstainingof
adjacent sections revealed shrunken and intensely-stained
cells at 24h (Fig. 7E) and 7 days post-injury (Fig. 7F), com-
pared tosimilarregionsinthesham-injured animals(Fig.7D).
Although impactwiththemetal-tipped indenterfractured the
of the majority of injured animals; however, hemorrhage in
the cortex immediately below the impact site was present in
2/6 animals from at 24-h survival time point (data not
shown). Despite the lack of intracerebral hemorrhage, closed
head injury in the mouse resulted in a significant increase in
the tissue water content, reflective of edema, in the cortex
Representative traces of evoked CAPs in sham- and brain-injured mice at 24h post-injury. Evoked CAPs of (B) myelinated
(N1) and (C) unmyelinated (N2) axons in the corpus callosum were measured as described in the methods section. Repeated-
measures analysis of variance of CAP amplitude revealed an injury effect for the myelinated (p<0.001), but not the un-
myelinated, component. Refractoriness for the (D) myelinated and (E) unmyelinated fibers at 24h and 14 days post-injury
(*p<0.04). All values are presented as mean–standard deviation.
Inhibition of compound action potentials (CAPs) of myelinated fibers following concussive brain trauma. (A)
556CREED ET AL.
compared to sham-injured animals over the first 24h post-
surgery/injury [injury effect, (F(1,25)=4.6, p<0.04; no time
effect, Fig. 8)]. Similar analysis in the hippocampus and
thalamus did not reveal increases in tissue water content at
any time post-injury (Fig. 8).
Neurodegeneration in the hippocampus
following closed head injury
shown), or the granule layer of the dentate gyrus (Fig. 9A and
B), was not apparent up to 7 days following CBI. At 24h,
patches of intensely-stained, non-neuronal cells were ob-
served in the granule cell layer (Fig. 9A). Fluoro-Jade-B-
24h (Fig. 9C), and were additionally observed in the granule
cell layer of the dentate gyrus at 3 days (data not shown); little
to no evidence of neurodegeneration was present at 7 days
post-injury (Fig. 9D). Quantitative analysis of Fluoro-Jade-B-
positive cells in the dentate gyrus revealed an injury effect
(F(3,20)=10.5, p<0.001). Post-hoc analysis demonstrated that
there were significantly more Fluoro-Jade-B-positive cells at
24h compared to sham-injured animals (p<0.001), at 3
(p<0.01) and 7 days (p<0.001) following injury (Fig. 9E).
Immunoreactivity for synaptophysin was assessed between
24h and 14 days post-injury as a measure of synaptic alter-
ations in the hippocampus (Fig. 9F–H). The classic trilaminar
pattern of synaptophysin immunoreactivity in the molecular
layer of the dentate gyrus was present in both sham-injured
(Fig. 9F) and brain-injured animals at 24h (Fig. 9G), 3 days
(data not shown), 7 days (data not shown), and 14 days (Fig.
9H) post-injury, indicating that no overt loss of synapses ac-
companied CBI in the mouse.
This report describes a model of concussion that is char-
3 days 7 days
Fluoro-Jade-B reactivity in the retrosplenial cortex in (A) sham-injured animals, and (B) at 24h post-injury. (C) Quantification
of the number of Fluoro-Jade-B-positive cells in the cortex below the site of impact. All values are presented as
mean–standard deviation (*p<0.05, **p<0.001 compared to sham-injured brains; #p<0.001 compared to 3 or 7 days post-
injury). Panels D–F are photomicrographs of Nissl staining in the retrosplenial cortex of (D) sham-injured brains, and at (E)
24h, and (F) 7 days post-injury. Note the increased cellularity in the brain-injured animals (scale bar=100lm for all panels).
Neurodegeneration in the cortex following concussive brain trauma. Representative photomicrographs illustrating
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT557
learning and working memory over the first 3 days post-
injury. The cognitive deficits were accompanied by increased
tissue water content in the cortex, diffuse neurodegeneration
in the retrosplenial cortex and the hilus of the dentate gyrus,
and TAI in the corpus callosum and cingulum, leading to
axonal degeneration. Direct evidence of functional damage to
axons was provided by decreased retrograde transport and
lowered amplitude of CAPs of myelinated axons. Im-
portantly, edema, neurodegeneration, TAI, and cognitive
deficits were only observed in the acute (1–3 days) post-
traumaticperiod,whereas axonaldegeneration anddeficits in
to 2 weeks post-injury, suggesting that the concussed brain
continues to demonstrate evidence of cellular dysfunction
despite resolution of behavioral deficits.
Cognitive deficits have been observed in existing models of
concussive brain trauma in both rats and mice (Hamm et al.,
1996; Lyeth et al., 1990; Spain et al., 2010; Tang et al., 1997a;
Zohar et al., 2003). Brain injury as a result of impact to the
intact skull of mice resulted in spatial learning deficits (Zohar
et al., 2003), and in latent learning deficits at 1 and 2 weeks
trauma resulted in spatial learning deficits from 1 week up to
3 months following injury in mice (Spain et al., 2010) and rats
post-injury in rats (Hamm et al., 1996). In the present study,
impairments in spatial learning and working memory were
observed only in the first 3 days post-injury, validating the
transient nature of post-concussive cognitive deficits; con-
cussed patients suffer acute memory loss and difficulty main-
taining attention, which resolve within 2 weeks following
injury (McCrory et al., 2009). While differences in mouse strain
may explain some of these discrepancies in cognitive findings,
it is unlikely that the transient nature of our deficits can be
righting reflex latency was similar to that reported in previous
Interestingly, Longhi and colleagues reported that unilateral
impact on the intact skull did not produce any spatial learning
deficits over the first 3 days (Longhi et al., 2005), suggestive of
the importance of the location of the impact in producing dif-
fuse brain damage.
Traumatic axonal injury (TAI) is a hallmark of diffuse TBI,
including concussion (Blumbergs et al., 1994; Oppenheimer,
1968). Importantly, a strong correlation between TAI, as in-
dicated by decreased fractional anisotropy in white matter
tracts, and cognitive dysfunction has been demonstrated in
humans (Kraus et al., 2007; Kumar et al., 2009; Wozniak et al.,
2007). Intra-axonal APP accumulation is recognized as a
marker of TAI and indicates impaired axonal transport (IAT;
Buki and Povlishock, 2006), although the link between APP
accumulation and behavioral deficits is not clearly under-
stood. Spain and colleagues suggest that intra-axonal APP
accumulation may represent the cellular basis for impaired
spatial learning following TBI (Spain et al., 2010). However,
spatial learning deficits were not observed following con-
cussive TBI, despite the presence of APP accumulations
within axons (Longhi et al., 2005), while Tweedie and col-
leagues failed to observe intra-axonal APP accumulation in
animals that exhibited depressive behavior following TBI
(Tweedie et al., 2007). Because APP can be transported in both
anterograde (Buxbaum et al., 1998; Koo et al., 1990) and ret-
rograde (Papp et al., 2002) directions, we provide additional
evidence that transport in either direction is impaired. Thus
the presence of SYP, a component of synaptic vesicles (Wie-
denmann and Franke, 1985), in axonal swellings strongly
implies IAT in the anterograde direction, an observation
similar tothatseen following brain injury in therat (Shojo and
Kibayashi, 2006). In addition, we demonstrate for the first
time evidence of retrograde transport impairment in the brain
following a traumatic injury, which validates an earlier report
of reduced Fluoro-Gold transport to the retinal ganglion cell
layer from the superior colliculus following optic nerve
stretch injury (Saatman et al., 2003). The cellular mechanisms
underlying IAT have yet to be described and may be medi-
ated by alterations in axonal structure (Buki and Povlishock,
2006). In this regard, neurofilament compaction, which occurs
as a result of dephosphorylation, may lead to IAT, and has
Tissue water content (%)
6 h 24 h 6 h 24 h6 h 24 h 6 h 24 h6 h 24 h 6 h 24 h
and thalamus as described in the methods section. Factorial analysis of variance revealed that brain trauma significantly
increased edema in the underlying cortex compared to sham-injured animals over the first 24h post-surgery/injury. All
values are presented as mean–standard deviation (*p<0.05 compared to sham-injured brains).
Brain edema following concussive brain trauma. Tissue water content was measured in the cortex, hippocampus,
558 CREED ET AL.
3 days 7 days
Shown are representative photomicrographs of Nissl-stained sections, demonstrating a lack of change in the cellularity in the
granule cell layers of the dentate gyrus at 24h (A) and 7 days (B) post-injury. Fluoro-Jade-B-positive cells were present in the
hilus of the dentate gyrus at 24h (C), but not at 7 days (D) post-injury. (E) Quantification of Fluoro-Jade-B-positive cells was
performed as described in the methods section. All values are presented as mean–standard deviation (*p<0.001 compared to
sham animals; #p<0.01 compared to 3 or 7 days post-injury). Synaptophysin immunoreactivity was visualized as the classic
trilaminar pattern of dark-light-dark staining in the molecular layer of the dentate gyrus in both sham-injured (F) and brain-
injured animals at 24h (G) and 14 days (H) post-injury (scale bar=100lm for all panels).
Neurodegeneration and synaptophysin immunoreactivity in the hippocampus following concussive brain trauma.
CONCUSSION IMPAIRS AXONAL ACTION POTENTIAL AND TRANSPORT559
been recognized as another prominent characteristic of TAI
following TBI (Chen et al., 1999; Christman et al., 1994; Pov-
lishock et al., 1997). Although co-localization experiments
were not performed in the present study, axons containing
dephosphorylated neurofilament were observed in regions
where axons containing accumulated APP or SYP were
present, suggestive of an association between transport im-
pairment and cytoskeletal alterations. It must be noted that in
certain tracts of the traumatically-injured rat brain, co-locali-
zationofaccumulated APP andcompacted neurofilamenthas
not been observed (DiLeonardi et al., 2009; Marmarou et al.,
Impaired axonal transport was accompanied by decreased
axonal conductance within the corpus callosum, confirming
the results of earlier studies of diffuse TBI in adult rats, in
which reductions in the amplitude of compound action po-
tentials in both myelinated (Baker et al., 2002; Reeves et al.,
2005) and unmyelinated axons were observed (Reeves et al.,
2005). However, unlike in previous reports (Baker et al., 2002;
Reeves et al., 2005), we did not observe any recovery of CAP
amplitude up to 14 days post-injury. While it may be possible
that there is no causal link between axonal conduction and
cognitive deficits, the discrepancy between this sustained re-
duction in CAP amplitude and the transient cognitive deficits
may also reflect the differential sensitivity of the two outcome
measures. Thereduction inCAPamplitudein ourstudies was
restricted to myelinated axons, whereas in the brain-injured
adult rat, the unmyelinated axons were more susceptible
(Reeves et al., 2005). In part, the differences between our ob-
servations and those published previously may be attributed
to species and injury model differences, and was less likely a
the rat were greater than those observed in the mouse (Reeves
et al.,2005).We suggestthatthe sustained deficits observed in
the present study may reflect axonal degeneration detected
using Fluoro-Jade-B histochemistry (Goda et al., 2002; Hallam
et al., 2004; Huh et al., 2008; Ohlsson et al., 2004; Schmued
et al., 1997; Shojo and Kibayashi, 2006; Tong et al., 2002). It
must be noted that the immunohistochemical markers of TAI
(APP, SYP, and desphosphorylated neurofilament) were not
as apparent after the first 3 days post-injury, suggesting that
evaluation of damage in the chronic period may require ad-
ditional indicators of axonal injury.
It has been suggested that neurodegeneration in the cortex
and hippocampus may be one mechanism underlying the
post-traumatic behavioral deficits (Longhi et al., 2005; Spain
et al., 2010; Tang et al., 1997b; Tashlykov et al., 2007). The
presence of Fluoro-Jade-B-positive neurons in the hilus may
explain the transient spatial learning deficits (Hicks et al.,
1993; Tang et al., 1997a), although we did not find evidence of
neurodegeneration in areas CA2 and CA3 of the hippocam-
pus, in contrast to a previous study (Tang et al., 1997b). It is
important to note that hippocampal cell death is not always
associated with learning deficits, as suggested by the obser-
vations seen following diffuse TBI in the rat (Miyazaki et al.,
1992; Reeves et al., 1997). While it is not certain if there are
deficits in long-term potentiation in brain-injured mice, we
did not observe overt alterations in SYP immunoreactivity in
the dentate gyrus, as has been reported in the rat (Phillips
et al., 1994). Diffuse neurodegeneration in the retrosplenial
cortex, possibly due to the observed edema (Longhi et al.,
2005; Tang et al., 1997b; Zohar et al., 2003), may explain the
deficits in working memory. Animals with lesions of the ret-
rosplenial cortex make more errors in the eight-arm radial
arm maze task of working memory compared to controls
(Keene and Bucci, 2009; Pothuizen et al., 2010). It must be
noted that neither overt neuronal loss nor caspase activation
(data not shown) was observed, despite the presence of both
Fluoro-Jade-B staining and morphologic changes, suggesting
that neuronal dysfunction, rather than neuronal loss, may
underlie the behavioral deficits seen.
In summary, we have developed a clinically-relevant
model of concussive brain injury in the adult mouse that ex-
hibits acute cognitive deficits accompanied by axonal injury.
reduced transport and conduction in axons, along with evi-
dence of axonal degeneration, suggest that the concussed
brain continues to demonstrate evidence of cellular dysfunc-
tion and damage. This model allows the development of
strategies to evaluate the mechanisms underlying axonal
damage uncomplicated by an accompanying contusion, with
a view toward developing treatment strategies that are tar-
geted to the concussed patient.
We would like acknowledge the efforts of Ms. Rachel Tang
for the analysis of the electrophysiology data. These studies
were supported in part by grants from the NIH (no. HD41699
and NS065017 to R.R.), and a VA Merit Review grant to R.R.
Author Disclosure Statement
No competing financial interests exist.
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