Repeated Mild Closed Head Injury Impairs Short-Term

Visuospatial Memory and Complex Learning

Michael J. Hylin,
1
Sara A. Orsi,
1
Natalia S. Rozas,
1
Julia L. Hill,
1
Jing Zhao,
1
John B. Redell,
1
Anthony N. Moore,
1
and Pramod K. Dash
1,2
Abstract
Concussive force can cause neurocognitive and neurobehavioral dysfunction by inducing functional, electrophysiological,
and/or ultrastructural changes within the brain. Although concussion-triggered symptoms typically subside within days to
weeks in most people, in 15%20% of the cases, symptomology can continue beyond this time point. Problems with
memory, attention, processing speed, and cognitive exibility (e.g., problem solving, conict resolution) are some of the
prominent post-concussive cognitive symptoms. Repeated concussions (with loss or altered consciousness), which are
common to many contact sports, can exacerbate these symptoms. The pathophysiology of repeated concussions is not well
understood, nor is an effective treatment available. In order to facilitate drug discovery to treat post-concussive symptoms
(PCSs), there is a need to determine if animal models of repeated mild closed head injury (mCHI) can mimic the
neurocognitive and histopathological consequences of repeated concussions. To this end, we employed a controlled
cortical impact (CCI) device to deliver a mCHI directly to the skull of mice daily for 4 days, and examined the ensuing
neurological and neurocognitive functions using beam balance, foot-fault, an abbreviated Morris water maze test, context
discrimination, and active place avoidance tasks. Repeated mCHI exacerbated vestibulomotor, motor, short-term memory
and conict learning impairments as compared to a single mCHI. Learning and memory impairments were still observed
in repeated mCHI mice when tested 3 months post-injury. Repeated mCHI also reduced cerebral perfusion, prolonged the
inammatory response, and in some animals, caused hippocampal neuronal loss. Our results show that repeated mCHI can
reproduce some of the decits seen after repeated concussions in humans and may be suitable for drug discovery studies
and translational research.
Key words: active place avoidance, axonal injury, context discrimination, neuroinammation
Introduction
A
concussion can cause neurocognitive and neurobeha-
vioral dysfunctions by inducing functional, electrophysio-
logical, and/or ultrastructural changes within the brain. Although
the terms concussion and mild traumatic brain injury (mTBI) are
often used interchangeably, concussion is the term typically used to
describe mTBI in humans. According to the CDC, an estimated 1.7
million people a year sustain a TBI in the United States, with
approximately 75% of these cases considered to be mild TBI or
concussions.
1
This likely underestimates the number of cases, as it
is estimated that close to 40% of people who have experienced a
concussion do not seek medical attention.
2
The incidence of con-
cussion is high in military populations, with studies reporting that
approximately 20% of Operation Enduring Freedom/Operation
Iraqi Freedom (OEF/OIF) veterans have had a clinical diagnosis of
concussion.
3
Prominent neurological symptoms of concussion
include problems with balance, headache, vomiting, nausea, and
sleep disturbances. The most frequently reported cognitive symp-
toms include problems with memory, attention, processing speed,
and complex learning (e.g., problem solving/conict resolution).
Existing literature indicates that in the majority of patients with
concussions, symptoms typically resolve within days to weeks
following the injury.
4
However, in 15%20% of the cases, symp-
tomology can continue beyond the acute time point.
57
While the
neural mechanism(s) underlying the neurological, cognitive, and
behavioral dysfunctions resulting from a single concussion are not
well understood, there is less understanding of the pathobiology of
repeated concussions.
Repeated concussions (which includes loss or altered con-
sciousness) are common to many contact sports. For example,
studies examining the frequency of head injuries in American
football players have reported that sustaining a concussion triples
the risk of sustaining a second concussion.
8,9
Athletes who have
1
Department of Neurobiology and Anatomy, and
2
The Vivian L. Smith Department of Neurosurgery, The University of Texas Medical School at
Houston, Houston, Texas.
JOURNAL OF NEUROTRAUMA 30:716726 (May 1, 2013)
Mary Ann Liebert, Inc.
DOI: 10.1089/neu.2012.2717
716
sustained repeated concussions have been shown to have impaired
visual-spatial perception and complex learning.
10
Postmortem
gross anatomical analyses of the brains from athletes with a history
of repeated concussions indicate enlarged lateral and third ventri-
cles, atrophy of the temporal and frontal cortices, and sclerosis of
the hippocampus.
11
These anatomical changes are consistent with
the observed impairments of episodic memory and high level
cognitive functions in persons sustaining repeated concussions. In
addition, recent studies have indicated that repeated concussions or
subconcussive injuries can lead to the development of chronic
traumatic encephalopathy (CTE), a neurodegenerative disease
characterized by tauopathy.
11,12
As repeated concussions can cause lasting and potentially dev-
astating impairments, there is a need to determine if animal models
of repeated mTBI can mimic the observed neurocognitive and
histopathological consequences of repeated concussions in humans
in order to facilitate translational research. A few experimental
studies in rodents have employed closed head injury (CHI) models
delivering a range of 210 impacts in order to examine the ensuing
histopathological changes and learning decits.
13,14
While these
studies have shown that repeated mCHI can cause spatial learning
and memory decits, inammation, decreased glucose metabolism,
and axonal injury, it has not been examined if repeated mCHI to
rodents can cause learning impairments in cognitive tasks that re-
quire greater exibility. To address this knowledge gap, we de-
livered four mCHI, each 24 h apart, to mice using a controlled
cortical impact (CCI) device as described previously.
1517
Con-
sistent with previous reports, we observed that repeated mCHI
produced no visible brain contusion, and that inammation and
axonal damage were more prominent after repeated mCHI than
after a single injury. Furthermore, repeated mCHI reduced cerebral
perfusion, and exacerbated decits in vestibulomotor and motor
function, short-term visuospatial memory, and conict learning.
Methods
Mild closed head injury
Mild closed head injury (mCHI) was delivered to mice using a
pneumatically driven CCI device, essentially as described previ-
ously.
16,17
Male C57BL/6 mice (2025 g) were anesthetized ini-
tially with 5% isourane in a 1:1 O
2
/air mixture, mounted on a
stereotaxic frame, then maintained with a 2.5% isourane and 1:1
O
2
/air mixture via a face mask. Amidline incision was made and the
soft tissue reected to expose the skull. A stereotaxic frame was
used to immobilize the animal during the surgical incision and en-
sure that a smooth cut was made. The mouse was then transferred to
a foampad designed to make the head level with the body. At 40 sec
after discontinuation of anesthesia, a single impact was applied to
the skull. For this model, our objective was to deliver a mCHI to a
partially awake animal as assessed by the recovery of the tail-pinch
response. Because the response to a tail pinch is an abrupt move-
ment, this could not be tested in the animals prior to the injury as the
resultant movement would upset the positioning of the skull beneath
the impactor. The 40 sec delay was based upon our prior data, which
showed that uninjured mice regain their tail-pinch reex on average
40 sec after discontinuation of anesthesia. A metal impactor tip
(5 mm in diameter) was driven at a velocity of 5.0m/sec to a depth
of 1.0 mm. The center of the tip was located midway between
lambda and bregma, over the sagittal suture. Immediately following
the injury, the animals were monitored for apnea, and when normal
breathing was observed, the scalp was closed using sterile surgical
staples. Duration of apnea, tail pinch, and righting responses were
assessed as described previously.
18,19
Animals with obvious skull
fractures (approximately 25%) were excluded from this study.
Groups of animals received either a single mCHI, or repeated
mCHIs (once per day for 4 days). Single mCHI animals were in-
jured on the day of the last injury for the repeated mCHI. In order to
assess the potential consequences of repeated exposure to anes-
thesia, an additional group of mice received daily anesthesia ex-
posure for 3 days prior to a single mCHI on the fourth day. The
rationale for using four impacts, separated by 24 h each, is based on
previous studies.
13,20
In both these studies, a single mild injury was
insufcient to produce reliable decits in their behavioral tasks.
However, DeFord et al. observed that four injuries at 24 h intervals
resulted in signicant cognitive impairments.
13
Likewise, Meehan
et al. found that between 3 to 5 repeated injuries spaced every 24 h
produced measurable decits in spatial memory.
20
Collectively,
these studies indicated that a minimum of three to four repeated
injuries spaced 24 h apart would be necessary to cause measurable
cognitive decits. Sham animals went through all of the anesthesia
and surgical procedures for 4 days but did not receive a mCHI.
Cerebral blood perfusion imaging
Cerebral perfusion was measured using a PIM3 scanning laser
Doppler device (Perimed, Ardmore, PA). Mice were initially an-
esthetized using 5% isourane with a 1:1 O
2
/air mixture, mounted
on a stereotaxic frame, then maintained with a 2.5% isourane and
1:1 O
2
/air mixture via a nose cone. After the skull was exposed, the
scan eld was set so that the center of the scan was on the midline.
The scanhead was aligned via the targeting laser and a built-in CCD
camera. Distance to the subject was calculated prior to every scan to
ensure consistency, and respiration was monitored throughout the
scanning procedure. Ten consecutive scans were performed, and
the resultant perfusion units averaged to give a perfusion value for
each animal. Pre-injury measurements were obtained as a baseline
for each animal, and used for normalization across animals. Prior to
each days injury, animals were anesthetized, and once breathing
rate had stabilized, they were scanned. After completion of the
scanning, the animals were removed from the stereotaxic device
and injured as described above. Sham and single mCHI groups
were scanned prior to injury (baseline), 2 min, 2 h, and 24 h post-
injury or post-sham surgery. Repeated mCHI subjects were scan-
ned prior to their rst injury (baseline) and then scanned prior to
each subsequent injury. A nal scan was performed 24 h after the
fourth mCHI.
Vestibulomotor and motor functions
Beam balance and foot-fault tasks were used to determine vesti-
bulomotor and motor performances following injury, respectively.
21,22
For beam balance, animals were placed on a narrow wooden beam
(0.5 cm wide) and the duration they remained on the beam was
measured (for a maximum of 60 sec). Animals were given three
daily trials on the beam balance task. Foot-fault was evaluated by
placing the animal on a wire grid (1 1 cm) and the number of foot
misplacements out of a total of 50 steps were counted.
22
A foot-
fault was dened as when a front paw missed and appeared below
the plane of the wire grid. The foot-fault test was repeated three
times to give an average daily score.
Abbreviated Morris water maze
In order to assess short-term memory, we used a modied ver-
sion of the Morris water maze task in which mice are trained to nd
the location of a hidden platform within a single training session
followed by a short-term probe trial.
23,24
Mice were given 10
consecutive training trials with an inter-trial interval (iti) of 15 min.
Each trial was initiated by placing the animal into the water maze at
one of four randomly chosen starting positions. The animal was
allowed to search for the hidden platform for a period of 90 sec, and
the time to nd the platformrecorded. If the animal failed to nd the
REPEATED MILD CLOSED HEAD INJURY 717
hidden platform on any given trial, it was led there by the experi-
menter. Thirty minutes following the last training trial, animals were
tested in a probe trial in which the platformwas removed fromthe tank
and allowed to search for a period of 60 sec. Movement within the
maze was monitored using a video camera linked to tracking software
(Ethovision, Noldus Information Technology, Leesbury, VA, USA).
Measures of memory, including latency to rst platform crossing,
number of crossings, and quadrant preference, were recorded.
Context discrimination
This task assesses an animals ability to distinguish between two
similar contexts, and has been shown to be dependent on hippo-
campal function. Contextual discrimination was assessed essen-
tially as described previously.
25
Animals were pre-exposed
(without shock) to two contexts sharing certain features (horizontal
grid oor, background noise, animal handling to and from the
room) while differing in others (differently spaced grids, scent,
distal cues and oor shape). Animals were given two trials, one in
each chamber, each day. Animals were placed in the shock chamber
and 178 sec later, a 2 sec, 0.75 mA shock was given. In the safe
chamber, animals were free to roam for 3 min and no shock was
given. Animals were exposed to the shock and safe chambers once
a day for 3 days. Discrimination of the two contexts was assessed by
comparing the time spent freezing in each chamber during training.
Active place avoidance and conict learning
Different congurations of this task can be used to assess
learning ranging from simple (passive avoidance) to complex
(conict) learning.
26,27
In the active avoidance conguration, the
animal learns to avoid a region (wedge formed form a 60 arc
measured from the center) where they receive a shock. After the
animal has learned to avoid this shock zone, the shock zone is
moved to a new location, thereby creating a conict. An abbrevi-
ated three-phase protocol, modeled after Burghardt and colleagues,
was used to assess place avoidance and conict learning.
28
Each
phase lasted 20 min and was separated by a 2 h period in which the
animal was allowed to remain in their home cage. In the rst phase
(pre-training), animals were allowed to move around the rotating
platform (moving clockwise at a speed of 1 rpm) with the shock
turned off. This allowed the animals to become familiar with the
environment. This also served as a pre-training baseline to identify
any exploratory differences between the groups. During the second
phase (active avoidance), the shock zone was activated and the
animal had to learn to avoid the area delineated by a 60 arc from
the center of the platform, which was located in a consistent region
of the rotating arena. An animals entrance into the zone resulted in
a brief constant current footshock (500 ms, 60 Hz, 0.3 mA) that was
scrambled across the rods lining the arena. The third phase of the
task consisted of moving the shock zone 180 in order to test
conict learning. The arena was located in a room that had nu-
merous visual cues on the walls. Analysis of the number of times
the animal entered the shock zone was computed using Track
Analysis software (Tracker, Bro-Signal Group Corp., SUNY
Technology Center, Brooklyn, NY).
Immunohistochemistry
Animals were deeply anesthetized using sodium pentobarbital.
Once the animal failed to respond to foot and tail pinch, the chest
cavity was opened and the animal transcardially perfused with
phosphate-buffered saline (PBS) followed by 4% paraformalde-
hyde in PBS. Brains were removed and post-xed overnight. Re-
presentative brains were cryoprotected in a 30% sucrose solution in
PBS. Coronal brain sections (40 lm) were prepared using a cryo-
stat. Immunohistochemistry was performed by incubating brain
sections with primary antibodies (1 lg/mL) overnight at 4C in a
solution consisting of PBS with 0.25% Triton X-100 (PBST), 2.0%
BSA, and 2.5% normal goat or horse serum. After extensive
washing in PBST, tissue sections were incubated for 1 h in species-
specic secondary antibodies conjugated to Alexa-uors. Sections
were washed in PBST, mounted on microscope slides, and visu-
alized using epiuorescence detection.
Silver impregnation
Silver staining was carried out on free-oating sections using a
kit from FD Neurotechnologies (Columbia, MD), essentially as
described by the vendor with one exception: the impregnation time
was extended from 4 min to 6 min in order to maximize the signal-
to-noise ratio.
Statistical analysis
Statistical comparisons were carried out using SigmaStat (Systat
Software, San Jose, CA). Across group comparisons of data col-
lected over time (e.g., behavioral training and blood owmeasures)
were evaluated using a repeated measures two-way ANOVA, fol-
lowed by post-hoc analysis. Group main, or interactions of group
and time, differences were used to compare the groups. Single
measure data (e.g., probe trial data) was statistically compared
using an one-way ANOVA, whereas within group comparisons
(e.g., quadrant preference) were tested using a repeated measures
one-way ANOVA. Data were considered signicant at p < 0.05.
Results
The timeline of the experimental design to assess the conse-
quences of repeated mCHI is shown in Figure 1A.
Acute neurological changes resulting from a single and
repeated mCHI
Duration of apnea, and suppression of tail pinch reex and
righting response were measured for sham, single, and repeated
mCHI groups. A single mCHI caused apnea for 31.8 3.5sec. This
duration of apnea was not signicantly altered by repeated mCHI
(F
(3,11)
=2.277, p =0.099) (Fig. 1B). When compared to sham ani-
mals, single and repeated mCHI animals demonstrated a signicant
increase in the duration of suppression of the tail pinch reex (Fig.
1C) (F
(2,31)
=19.71, p <0.001). However, the duration of this reex
did not change within the repeated mCHI group across days
(F
(3,11)
=2.646, p =0.066). When compared to the shamanimals, the
suppression of the righting responses were signicantly longer in
both single and repeated mCHI animals (Fig. 1D) (F
(2,31)
=30.679,
p <0.001), but did not differ between the two groups. Furthermore,
animals that received repeated anesthesia and a single mCHI (n =9)
were found to have comparable acute neurological responses to
single anesthesia, single mCHI animals (n =9) with the exception of
a modest decrease in suppression of the righting response in the
multiple anesthesia group.
Cerebral perfusion is decreased acutely after mCHI
Sham and single mCHI groups (n =6/group) were scanned using
a PIM3 Imager prior to injury (baseline), 2 min, 2 h, and 24 h post-
injury or sham surgery. Figure 2A shows a representative picture of
a mouse mounted in the stereotaxic frame with the position of the
scan eld (white box) indicated. Color-coded images in Figure 2B
indicate the relative cerebral perfusion of a mouse subjected to a
single mCHI. Black boxes indicate the areas of the cortex used for
quantication. Perfusion in the right and left hemispheres were
averaged. The summary data in Figure 2C shows that cerebral
718 HYLIN ET AL.
perfusion is signicantly decreased by 2min post-injury (F
(3,21)
=4.53,
p =0.013), an effect that normalized by 24 h post-injury. Sham
animals did not show any signicant change in cerebral perfusion
over the monitoring period (data not shown). To determine if the
resolution of cerebral perfusion is inuenced by repeated mCHI,
mice (n = 12) were given four daily injuries, with cerebral perfu-
sion monitored 24 h after each injury. This time point was chosen
based upon the data (Fig. 2C), which indicated that cerebral per-
fusion returned to baseline by 24 h after a single injury. Figure 2D
shows that cerebral perfusion had normalized by 24 h after the
rst, second, and third mCHI. However, after the fourth mCHI,
cerebral perfusion remained signicantly decreased (F
(4,44)
= 3.91,
p = 0.008). Respiratory rate was not signicantly different at any of
the time points examined, nor was the cerebral perfusion signi-
cantly altered in uninjured animals exposed to multiple anesthesia
(data not shown).
Repeated mCHI exacerbates vestibulomotor and
motor decits
Vestibulomotor response was measured daily using a beam
balance task. Testing began the day after the last mCHI and con-
tinued until the injured animals could perform the tasks similar to
uninjured controls. Shamanimals (n =4) were able to remain on the
balance beam for the entire 60 sec duration (Fig. 3A). In contrast,
both single (n = 9) and repeated (n = 7) mCHI animals displayed
vestibulomotor dysfunction and were incapable of balancing on the
beam for the full 60 sec testing period (F
(4,68)
= 38.313, p < 0.001).
Post-hoc analysis revealed that the repeated mCHI animals per-
formed signicantly worse as compared to single mCHI animals
(t
(78)
= 4.884, p < 0.001). The performance of both injury groups
were similar those of shams by day 6.
The results for the foot fault task are shown in Figure 3B (right
forelimb) and Figure 3C (left forelimb). Testing was conducted on
the days animals were tested in the beam balance. For 4 days fol-
lowing injury, animals that received repeated mCHI performed
poorly in this task, making signicantly more foot faults than sham
animals (right forelimb: F
(2,17)
= 46.046, p <0.001 and left fore-
limb: F
(2,17)
= 16.453, p <0.001). It is worth noting that the injury
used in the current study did not result in any signicant motor
asymmetry in the number of foot-faults made by the right or left
forelimb (single mCHI, F
(1,16)
=1.208, p = 0.288; repeated mCHI,
F
(1,12)
=3.55, p = 0.084). Further, by day 6, performance was sim-
ilar among the three groups.
Repeated mCHI impairs short-term visuospatial
memory
Because short-termmemory is often impaired in humans who have
sustained a concussion, visuospatial learning and short-term memory
were assessed using an abbreviated Morris water maze task on day 10,
as described in the Methods section. Figure 4Aillustrates the latencies
to locate the hidden platform for each group. The repeated mCHI
group (n =7) performed poorly as compared to the single (n =9) or
sham (n =8) groups (F
(2,21)
=8.770, p =0.002). When the perfor-
mance of the single mCHI animals was directly compared to the sham
group only (i.e., the repeated mCHI group was omitted), there was a
statistically signicant interaction between the single mCHI and sham
groups (F
(4,60)
=2.877, p =0.03). Post-hoc analysis revealed that these
two groups differed on trials 36. Additional analysis of the training
revealed that when compared to single mCHI and sham animals, the
repeated injury group required more training before they began
searching the center of the maze in order to locate the hidden platform
(Figure 4B). As indicated in Figure 4C, when short-term memory
was tested 30 minutes later by a probe trial, the repeated mCHI
group demonstrated an impaired ability to nd the previous location
of the platform. The swimming path to rst platform crossing
was more circuitous in repeated mCHI animals as compared to the
path taken by sham or single mCHI animals. During the probe,
both the sham and single mCHI groups demonstrated a preference
for the quadrant that previously contained the platform (sham:
F
(3,21)
=11.307, p <0.001; single mCHI: F
(3,24)
=5.395, p =0.006),
whereas the repeated mCHI group randomly searched in all four
quadrants (F
(3,18)
=1.920, p =0.163) (Figure 4D).
FIG. 1. Single and repeated mCHI worsen acute neurological functions. (A) Timeline for the experimental design. Separate groups of
animals were used to assess conict learning and abbreviated Morris water maze. (B) A single mCHI (n = 10) increases the duration of
apnea. For the repeated mCHI group (n =12), apnea was assessed after each injury and did not decrease signicantly across days. (C)
Both the single and repeated mCHI groups demonstrated a suppression of duration of their response to a tail pinch relative to sham
animals (n = 12), but did not differ from each other. (D) Both injury groups showed suppression in their righting response.
REPEATED MILD CLOSED HEAD INJURY 719
FIG. 3. Single and repeated mCHI impairs vestibulomotor and motor functions. (A) Both single (n = 9) and repeated (n = 7) mCHI
animals were impaired in their ability to balance on a narrow beam compared to sham animals (n = 4). Furthermore, repeated mCHI
signicantly worsened performance compared to single mCHI animals. Repeated mCHI mice demonstrated signicantly more foot
faults with both their (B) right forelimb and (C) left forelimb. Both motor and vestibulomotor function were normalized by Day 6 post-
injury. Data is represented as mean S.E.M., *p < 0.05.
FIG. 2. mCHI causes a transient decrease in cerebral perfusion that fails to recover after repeated mCHI. Cerebral perfusion was
measured using a PIM3 scanning laser Doppler device. (A) Picture of a mouse head, as seen from the PIM3 imager, with the scan eld
indicated by white outline. (B) Representative PIM3 scans at the indicated time points after a single mCHI (n = 8) (boxes: areas used for
quantication). Quantication of cerebral perfusion (C) at acute time points following a single mCHI and (D) 24 h after each of the four
repeated mCHIs (n = 12). Data is represented as mean S.E.M., *p < 0.05. Color image is available online at www.liebertpub.com/neu
720
Probe trials were further analyzed for measures of platform lo-
calization. Concentric rings of increasing diameter (2X, 3X, and 4X
platform diameters) centered on the platform were used to assess
the latency and number of entries into the target areas. Repeated
mCHI animals demonstrated higher latencies for all concentric
circles (F
(2,21)
=4.616, p =0.022) (Fig. 5A) and made fewer circle
crossings (F
(2,21)
= 15.852, p < 0.001) (Fig. 5B) when compared to
single mCHI and shams. Analysis of the path length to the rst
platform crossing revealed that repeated mCHI animals travel
signicantly further prior to crossing (F
(2,21)
=8.312 , p =0.002)
(Fig. 5C), taking a more circuitous path in nding the platform.
Additionally, repeated mCHI mice spent more time away from the
target as indicated by a higher mean proximity to platform
(F
(2,21)
=9.569, p = 0.001) (Fig. 5D). However, no differences were
seen in swim speed (F
(2,21)
= 1.554, p =0.235) or latency to nd a
visible platform (F
(2,21)
=0.646, p =0.534). In order to examine if
repeated exposure to anesthesia contributed to the observed cog-
nitive decits, a group of animals were exposed daily to anesthesia
for three days and received a mCHI on Day 4. A second group of
animals received only a single mCHI on Day 4. No difference was
observed between these two groups in either learning or short-term
memory ( p > 0.05).
Mild CHI impairs context discrimination
The ability to discriminate between two similar contexts has
been shown to require hippocampal function. As repeated mCHI
mice were impaired in visuospatial learning and memory, we tested
their ability to discriminate between two contexts that contained
similar visual and auditory characteristics but differed in other
sensory characteristics. Sham animals (n = 8) were able to distin-
guish between two similar contexts after one day of training, as
indicated by signicantly more freezing in the context in which the
mild foot shock was delivered (F
(2,14)
=16.756, p = 0.001) (Fig.
6A). In contrast, both the single mCHI (n = 10) (Fig. 6B) and re-
peated mCHI animals (n =7) (Fig. 6C) required an additional day of
training to distinguish between the shock and safe contexts (single
mCHI: F
(2,18)
=11.878, p < 0.001; repeated mCHI: F
(2,12)
= 6.343,
p = 0.007). No difference was detected between performances of
the single and repeated mCHI groups ( p > 0.05). However, by the
FIG. 4. Repeated mCHI results in learning and short-term memory decits in a visuospatial task. Visuospatial learning and memory
was assessed using an abbreviated Morris water maze task. (A) The repeated mCHI group (n =7) required signicantly longer time to
nd the hidden platform across trials, indicating a learning impairment. (B) The poor acquisition of the repeated mCHI group is in part
due to the animals spending more time in the periphery of the maze, dened as two body widths away from the wall (indicated by the
gray annulus on inset). (C) Representative traces from an animal in each group during a probe trial given 30 min after the completion of
training. (D) During the probe trial, sham (n =8) and single mCHI (n = 9) animals demonstrated a preference for the target quadrant that
contained the platform (black circle in inset), whereas the repeated mCHI injury group did not have a preference for the target quadrant.
Data is represented as mean S.E.M., *p <0.05.
REPEATED MILD CLOSED HEAD INJURY 721
third day of training, all groups demonstrated the ability to dis-
criminate between the two contexts.
Repeated mCHI causes decits in active avoidance
and conict learning
We next examined the performance of a separate group of in-
jured animals using a cognitively demanding task to assess com-
plex learning. Active avoidance is a task that has been previously
shown to rely upon the integrity of the hippocampus.
2830
Mice
(n = 6/group) were initially tested on Day 10 post-injury in an ab-
breviated three-part protocol consisting of a pretraining phase, an
active avoidance phase, and a conict phase. Figure 7A shows
representative traces of animal movements during the pre-training,
active avoidance, and conict learning phases. During the pre-
training phase, all groups explored the rotating platform and fre-
quently entered the inactivated shock zone. This indicated that
there was no inherent bias or tendency to avoid this part of the
platform prior to turning on the shock. During active avoidance
training, sham and single mCHI groups demonstrated a signi-
cantly lower number of entries into the shock zone. In contrast, the
repeated mCHI group entered the shock zone signicantly more
times than either the sham or single mCHI groups (Fig. 7B)
(F
(2,15)
= 39.873, p < 0.001). Two hours after the completion of
active avoidance training, the animals were given a conict training
with the shock zone rotated 180. Repeated mCHI animals were
impaired in their ability to avoid the location of the newshock zone,
making more entries into it than either sham or single mCHI
FIG. 5. Repeated mCHI results in short-term memory decits in a visuospatial task. The repeated mCHI group (n = 7) demonstrated an
inability to learn the general location of the platform as indicated by (A) an increase in the latency to nd the platform, (B) a decrease in
the number of platform crossings during a probe trial, (C) longer swimming paths for the initial platform crossing, and (D) higher mean
proximity to the platform than the sham (n = 8) and single mCHI (n =9) animals. Data is represented as mean S.E.M., *p < 0.05.
FIG. 6. mCHI results in an attenuated ability to discriminate between two similar contexts. Percent time spent freezing in the shock
cage or safe cage after 0, 1, and 2 days of training in a context discrimination protocol. (A) Sham mice (n =8) learn to discriminate
between two similar contexts by test Day 1, as indicated by enhanced fear to the shock chamber relative to the safe chamber. Both (B)
single mCHI (n =10) and (C) repeated mCHI (n =7) mice require an additional day of training in order to differentiate between the two
contexts. Data is represented as mean S.E.M., *p < 0.05.
722 HYLIN ET AL.
animals (Fig. 7A and 7B) (F
(2,15)
= 3.964, p = 0.041). To examine if
these decits observed in the repeated mCHI group persisted, a
second group of repeated mCHI animals was generated and tested
at both 10 days and 3 months post-injury. This cohort exhibited
similar pretraining exploration (40.4 1.3 versus 37.0 3.2 entries,
p =0.314) as well as decits in active avoidance (31.7 6.3 versus
36.3 3.6 entries, p = 0.555) and conict learning (36.6 5.2 versus
39.7 6.1, p = 0.705) at 10 days, as the repeated mCHI cohort
shown in Figures 7A and 7B. At 3 months post-injury (Fig. 7C), the
repeated mCHI animals were still unable to learning the shock zone
as evidenced by no signicant difference in the number of shock
zone entries between the testing phases (F
(2,12)
=1.229, p =0.327)
(Fig. 7D). Figure 7E shows representative heat maps for each of the
tested groups obtained during the conict training phase. The
amount of time an animal spent at any particular location in the
arena is indicated by color coding, with black indicating no time
and red indicating a large amount of time. While both sham and
single mCHI groups spent the majority of their time avoiding the
shock zone learned during the active avoidance phase (indicated by
red wedge), the repeated mCHI groups tended to spend a relatively
larger amount of time in that location.
Repeated mCHI exacerbates neuroinammation and
axonal damage
Following the completion of cognitive testing (on Day 14 post-
mCHI), animals were euthanized, and brains were extracted to
examine cortical contusion, neuronal loss, dendritic damage,
axonal injury, and inammation. No visible contusion was de-
tected in either the single or repeated mCHI animals. Tissue
sections were prepared from representative animals in each group
for immunohistochemical evaluation. No overt neuronal loss was
observed in the majority of animals examined (data not shown).
However, in some repeated mCHI animals (*20%), cell loss in
either the CA1 or CA3 subelds was detected. Silver staining
showed argyrophilic staining in the corpus callosum of mice that
sustained repeated mCHI, suggestive of axonal damage (Fig. 8A).
Figure 8B shows that both single and repeated mCHI increased
GFAP immunoreactivity in the corpus callosum.
17
A similar in-
crease in Iba1 immunoreactivity (a marker for activated micro-
glia) was also seen in the corpus callosum (data not shown). Both
axonal damage and neuroinammation occurred in all the mCHI
animals examined.
Discussion
In the present study, we have examined the neurological, neu-
rocognitive, and histopathological changes resulting from single
and repeated mild closed head injury. Our results revealed ve key
ndings: repeated mild closed head injury 1) reduces cerebral
perfusion, 2) results in a lingering neuroinammation, 3) causes
axonal damage, 4) exacerbates vestibulomotor and motor dys-
function, and 5) impairs short-term visuospatial memory and con-
ict learning.
FIG. 7. Repeated mCHI impairs cognitive exibility. Active avoidance and conict learning were tested using a place avoidance task.
(A) Representative path tracings for an animal in each group (n =6/group) during the pre-training, active avoidance, and conict
learning phases. The shock zone is dened by the red pie wedge. Small red circles represent where the animal was shocked (or would
have been shocked in the pre-training phase). (B) Summary results indicating the number of entries into the shock zone during the
pretraining (shock off), active avoidance, and conict learning phases. (C) Representative path tracings for repeated mCHI animals that
were tested 3 months following injury. (D) Group summary data showing the number of shock zone entries repeated mCHI animals
made following injury when tested at 3 months. The number of entries did not signicantly differ from the number of entries made by
repeated mCHI animals at 10 days. (E) Representative heat maps for an animal in each group during the conict training phase. The color
represents the amount of time an animal spent in a particular location (blue indicates the least amount of time, red indicates the most
amount of time). The red pie wedge represents the location of the shock zone that was used during active avoidance training. The sham
and single mCHI groups avoided this location during the conict training, whereas the repeated mCHI group did not, indicating impaired
short-term memory. Data is represented as mean S.E.M., *p <0.05. Color image is available online at www.liebertpub.com/neu
REPEATED MILD CLOSED HEAD INJURY 723
Both uid percussion injury (FPI) and CCI devices have been
used to cause mTBI in which the force is delivered directly to the
exposed brain.
14,31
While mild FPI and CCI have been shown not to
cause overt brain damage, these injuries do result in impaired
plasticity, axonal damage, neuroinammation, and spatial learning
and memory decits.
3234
For example, Spain et al. have observed
that mild FPI in mice causes evolving axonal damage in addition to
a spatial learning impairment.
35
Recently, it has been shown that
mild FPI in rats alters hippocampal place cell ring; this may be an
underlying mechanism for the spatial learning impairment.
34
Mild
CCI injury in rats also triggers an inammatory response and ax-
onal damage.
32,36
Although these models have yielded interesting
results on the pathophysiology of mild injury, they are not typically
used to model repeated mTBI.
In order to cause closed head injury in rats and mice, investi-
gators have employed the weight-drop method, CCI device, or
projectiles, and have examined the ensuing pathophysiology.
13,37,39
For example, Creed and associates employed the CCI device to
cause closed head injury by delivering the impact directly to the
skull.
38
This injury increased cerebral edema and neurodegenera-
tion in the cortex and dentate gyrus, and caused axonal damage in
the corpus callosum. Daily testing of animals in the Morris water
maze showed impaired learning on Days 13 post-injury but not
when tested on Days 46 post-injury. Chen and colleagues em-
ployed a projectile-based approach in which a sealed microfuge
tube containing dry ice was used to propel the cap into a rats
head.
39
The use of a helmet and adjustment of the distance between
the sealed tube and rat head resulted in a mild TBI with no gross
brain pathology or hemorrhage. In these animals, sensory motor
abnormalities were detected 14 hours post-injury using a Catwalk
test, but no cognitive impairments were observed using the standard
Morris water maze (MWM). We employed an abbreviated MWM
task to examine short-term visuospatial learning and memory.
Using this task, we found that a single mCHI impairs spatial
learning as compared to sham animals when tested on Day 10 post-
injury. Furthermore, when short-term memory for platform local-
ization was assessed using concentric circle analysis (Fig. 5B),
single mCHI animals showed poor recall. These results suggest that
the abbreviated Morris water maze task may be a more sensitive
measure for learning and short-term memory than the standard
spaced version of this task.
It remains unresolved if there is an upper limit (or threshold) for
the number of concussions a person can sustain and still make a full
recovery. A limited number of clinical and experimental studies
have attempted to examine this issue.
15,20,40
It has been reported
that neurocognitive and neurobehavioral decits do not differ
among athletes who sustained 1 or 2 concussions.
41
However, it
appears that more than three mild head injuries may cause irre-
versible decits.
42
Based on these and other ndings, the American
Academy of Neurology recommends termination of the season for
an athlete after the third concussion within that same season. Using
the weight drop model, DeFord and associates examined cognitive
impairments resulting from four concussive injuries to mice de-
livered 24 hours apart.
13
Four repeated weight drops of 100 g or
150 g masses from a height of 40 cm did not cause overt brain
damage, but mice had impaired acquisition in the MWM task.
Whalen and colleagues, using a weight drop model, reported that
ve closed head injuries, each separated by a day, impaired learning
and memory. However, a single closed head injury did not cause
any signicant cognitive decit.
20
Recently, Crawford and col-
leagues used the CCI device to deliver ve closed head injuries to
mice and reported motor decits in the rotarod task and cognitive
decits in the Barnes maze that occurred in the absence of overt
hippocampal cell loss.
17
Consistent with these studies, we observed
that four repeated mCHIs exacerbated performance in the beam
balance and foot-fault tasks. Furthermore, short-term learning and
memory were also impaired in mice sustaining four repeated
mCHIs. At the beginning of MWM training, all animals tend to
swim adjacent to the perimeter of the tank looking for an escape
route. Sham and single mCHI animals quickly learned to abandon
this strategy in favor of an open water search strategy in order to
nd the hidden platform (Fig. 4B). In contrast, the repeated mCHI
mice required more training trials before they switched to this open
water search strategy, though once they had switched to this
strategy they used it for the remainder of the training trials. While
we cannot rule out that enhanced thigmotaxic-like behaviors
FIG. 8. mCHI results in axonal disruption and inammation. Representative photomicrographs of (A) silver staining revealed an
increase in silver impregnated bers in the corpus callosum of the single mCHI group and to a greater degree in the repeated mCHI
group (n = 6/group). (B) Images of the corpus callosum demonstrating that repeated mCHI increases inammation as indicated by an
increase in GFAP (astrocytes) immunoreactivity. Color image is available online at www.liebertpub.com/neu
724 HYLIN ET AL.
contribute to the decits we observed, it appears that the decit is
more related to both an inability to switch the search strategy and to
localize the position of the hidden platform. These decits were
detected in the absence of demonstrable hippocampal pathologies
in the majority of animals, with only 20% of repeated injury mice
having overt hippocampal neuron loss at the magnitude of injury
employed in this study. Although we cannot exclude the possibility
that some animals had cell loss that was more distributed in nature
and not visibly obvious, it appears that the percentage of animals
with cell loss is likely to depend on the magnitude of the impact
force delivered to the skull. Consistent with this, we have observed
that impact forces that cause skull fractures reproducibly cause
visible cortical and hippocampal cell loss (data not shown).
Pattern separation is a neural process by which similar but not
identical experiences, events, or spatial maps are transformed into
discrete non-overlapping representations, allowing for cognitive
exibility.
28,43,44
A conict-based place avoidance task can assess
cognitive exibility. Bergold and colleagues were the rst inves-
tigators to use the active place avoidance task to showthat mild CCI
injury in rats caused decits in the ability to perform this task.
26
Similar to that seen previously, we observed that repeated mCHI
mice performed poorly in the active avoidance version of the task.
Likewise, when the location of the shock zone was moved in order
to increase the need for cognitive exibility, the performance of
repeated mCHI animals was impaired as compared to sham ani-
mals. These decits appear to persist as we have seen decits at
least 3 months after repeated mCHI.
Histopathological assessment showed no visible brain contu-
sion, consistent with a classication of mild injury. However,
recent imaging studies have begun to indicate that even mild brain
injuries can give rise to axonal damage that can be detected using
diffusion tensor imaging (DTI). For example, decreased frac-
tional anisotropy (FA) is often observed in the genu of corpus
callosum and in the internal and external capsules.
45
While de-
creased FA is commonly seen in the chronic stages of mTBI,
increased FA (possibly resulting from axonal edema) has been
observed in the fornix 16 days after injury that correlated with
decreased ANAM score.
46
To assess axonal injury in single and
repeated mCHI animals, we performed silver staining, which
revealed degenerating processes in the corpus callosum that ap-
peared to worsen in animals subjected to repeated mCHI. Inter-
estingly, the immunoreactivities of GFAP (and Iba-1, not shown)
were also found to be increased in the corpus callosum, suggesting
that ongoing neuroinammation may be associated with the ob-
served axonal damage. These changes are similar to those re-
ported by Mouzon et al., who examined the histopathological
changes resulting from ve repeated mCHI, each separated by 2
days.
17
While these studies highlight some of the pathological
changes associated with repeated mCHI, a spectrum of patho-
logical changes ranging from tissue loss to subtle neuronal dys-
function are likely to be observed, depending on injury severity,
injury location, age and gender. Future studies will be required to
examine the inuence of these variables.
In addition to the abovementioned variables, injury interval is
thought to be a dening factor in the degree and types of decits
observed following repeated mild injuries. In a recent study,
Meehan et al. used the weight drop model to examine the effect of
time interval between repeated mCHI in mice on neurocognitive
function.
20
Mice that received ve mCHI injures delivered at one
day or one week intervals had impaired spatial memory. In contrast,
when the interval was increased to one month, no difference was
detected between injured and sham animals, suggesting a window
of vulnerability of at least one week. Using a similar weight drop
model, Vagnozzi et al. examined the window of metabolic vul-
nerability to a second mTBI in rats.
40
This study showed that
maximum reduction of brain levels of ATP, NAA, NAAG, NAD,
and acetyl CoAresulted when the interval for the second impact was
separated by 3 days as compared to 1, 2, 4, or 5 days.
40,47
In addition,
a secondary injury occurring shortly after the rst mild injury has
been shown to prolong this period of metabolic suppression, and
correlates with a signicant increase in hippocampal cell death.
48
A
recent study by Prins et al. shows that when a second mCHI is
delivered 24 hours, but not 3 days, after the rst injury, a larger and
more persistent suppression of cerebral glucose metabolism is ob-
served.
14
Clinical studies have employed neuropsychological test-
ing to assess the duration of vulnerability. A few clinical studies
have reported that, if a concussion is sustained prior to resolution of
the symptoms of the rst concussion have resolved, this can prolong
brain metabolic changes and cause fatality.
49
This condition is re-
ferred to as second impact syndrome (SIS) and has only been re-
ported in young adults.
50
While the occurrence and the mechanism
underlying SIS are controversial, it has been hypothesized that
impaired cerebral autoregulation and brain edema may be under-
lying mechanisms. Our cerebral perfusion results showed that a
single mCHI transiently decreased cerebral blood ow which re-
covered by 24 h post-injury. However, following the fourth closed
head injury, cerebral perfusion remained signicantly suppressed
24 h later. At this time, we are uncertain if this reduction of cerebral
perfusion may be due to impaired autoregulation. Future studies will
address the impact of repeated mCHI on vascular autoregulation,
and on metabolic disturbances in order to help dene the period of
vulnerability.
Acknowledgment
This work was supported by The Institute for Rehabilitation and
Research/Mission Connect; The Vivian L. Smith Foundation and
the Gilson-Longenbaugh Foundation.
Author Disclosure Statement
No competing nancial interests exist for any author.
References
1. Faul M, Xu L, and Wald MM. (2010). Traumatic brain injury in the
United States: Emergency department visits, hospitalizations, and
deaths 2002-2006. Atlanta (GA): Centers for Disease Control and
Prevention, National Center for Injury Prevention and Control.
2. Sosin DM, Sniezek JE, and Thurman DJ. (1996). Incidence of mild
and moderate brain injury in the United States, 1991. Brain Inj 10:
4754.
3. Warden D. (2006). Military TBI during the Iraq and Afghanistan wars.
J Head Trauma Rehabil 21:398402.
4. dHemecourt P. (2011). Subacute symptoms of sports-related con-
cussion: Outpatient management and return to play. Clin Sports Med
30:6372, viii.
5. Bigler ED. (2008). Neuropsychology and clinical neuroscience of
persistent post-concussive syndrome. J Int Neuropsychol Soc 14:122.
6. Dikmen S, Machamer J, Fann JR, and Temkin NR. (2010). Rates of
symptom reporting following traumatic brain injury. J Int Neu-
ropsychol Soc 16:401411.
7. Middleboe T, Andersen HS, Birket-Smith M, and Friis ML. (1992).
Minor head injury: Impact on general health after 1 year. A pro-
spective follow-up study. Acta Neurol Scand 85:59.
8. Guskiewicz KM, Weaver NL, Padua DA, and Garrett WE, Jr. (2000).
Epidemiology of concussion in collegiate and high school football
players. Am J Sports Med 28:643650.
9. Guskiewicz KM. (2003). Assessment of postural stability following
sport-related concussion. Curr Sports Med Rep 2:2430.
REPEATED MILD CLOSED HEAD INJURY 725
10. Matser JT, Kessels AG, Jordan BD, Lezak MD, and Troost J. (1998).
Chronic traumatic brain injury in professional soccer players. Neu-
rology 51:791796.
11. McKee AC, Cantu RC, Nowinski CJ, et al. (2009). Chronic traumatic
encephalopathy in athletes: Progressive tauopathy after repetitive head
injury. J Neuropathol Exp Neurol 68:709735.
12. Stern RA, Riley DO, Daneshvar DH, Nowinski CJ, Cantu RC, and
McKee AC. (2011). Long-term consequences of repetitive brain
trauma: Chronic traumatic encephalopathy. PMR 3:S460S467.
13. DeFord SM, Wilson MS, Rice AC, et al. (2002). Repeated mild brain
injuries result in cognitive impairment in B6C3F1 mice. J Neuro-
trauma 19:427438.
14. Prins ML, Alexander D, Giza CC, and Hovda DA. (2013). Repeated
mild traumatic brain injury: Mechanisms of cerebral vulnerability. J
Neurotrauma 30:3038.
15. Prins ML, Hales A, Reger M, Giza CC, and Hovda DA. (2010). Re-
peat traumatic brain injury in the juvenile rat is associated with in-
creased axonal injury and cognitive impairments. Dev Neurosci
32:510518.
16. Uryu K, Laurer H, McIntosh T, et al. (2002). Repetitive mild brain
trauma accelerates Abeta deposition, lipid peroxidation, and cognitive
impairment in a transgenic mouse model of Alzheimer amyloidosis. J
Neurosci 22:446454.
17. Mouzon BC, Chaytow H, Crynen G, et al. (2012). Repetitive mild
traumatic brain injury in a mouse model produces learning and
memory decits accompanied by histological changes. J Neurotrauma
29:27612773.
18. Dixon CE, Clifton GL, Lighthall JW, Yaghmai AA, and Hayes RL.
(1991). A controlled cortical impact model of traumatic brain injury in
the rat. J Neurosci Methods 39:253262.
19. Hovda DA, Yoshino A, Kawamata T, Katayama Y, and Becker DP.
(1991). Diffuse prolonged depression of cerebral oxidative metabo-
lism following concussive brain injury in the rat: A cytochrome oxi-
dase histochemistry study. Brain Res 567:110.
20. Meehan WP, III, Zhang J, Mannix R, and Whalen MJ. (2012). In-
creasing recovery time between injuries improves cognitive outcome
after repetitive mild concussive brain injuries in mice. Neurosurgery
71: 885892.
21. Zhao J, Pati S, Redell JB, Zhang M, Moore AN, and Dash PK. (2012).
Caffeic acid phenethyl ester protects blood-brain barrier integrity and
reduces contusion volume in rodent models of traumatic brain injury. J
Neurotrauma 29:12091218.
22. Dash PK, Orsi SA, Zhang M, et al. (2010). Valproate administered
after traumatic brain injury provides neuroprotection and improves
cognitive function in rats. PLoS One 5:e11383.
23. Blum S, Moore AN, Adams F, and Dash PK. (1999). A mitogen-
activated protein kinase cascade in the CA1/CA2 subeld of the dorsal
hippocampus is essential for long-term spatial memory. J Neurosci
19:35353544.
24. Guzowski JF, and McGaugh JL. (1997). Antisense oligodeox-
ynucleotide-mediated disruption of hippocampal cAMP response el-
ement binding protein levels impairs consolidation of memory for
water maze training. Proc Natl Acad Sci USA 94:26932698.
25. Frankland PW, Cestari V, Filipkowski RK, McDonald RJ, and Silva
AJ. (1998). The dorsal hippocampus is essential for context discrim-
ination but not for contextual conditioning. Behav Neurosci 112:
863874.
26. Abdel Baki SG, Kao HY, Kelemen E, Fenton AA, and Bergold PJ.
(2009). A hierarchy of neurobehavioral tasks discriminates between
mild and moderate brain injury in rats. Brain Res 1280:98106.
27. Cimadevilla JM, Fenton AA, and Bures J. (2001). New spatial cog-
nition tests for mice: passive place avoidance on stable and active
place avoidance on rotating arenas. Brain Res Bull 54:559563.
28. Burghardt NS, Park EH, Hen R, and Fenton AA. (2012). Adult-born
hippocampal neurons promote cognitive exibility in mice. Hippo-
campus 22:17951808.
29. Cimadevilla JM, Fenton AA, and Bures J. (2000). Functional inacti-
vation of dorsal hippocampus impairs active place avoidance in rats.
Neurosci Lett 285:5356.
30. Cimadevilla JM, Wesierska M, Fenton AA, and Bures J. (2001). In-
activating one hippocampus impairs avoidance of a stable room-
dened place during dissociation of arena cues from room cues by
rotation of the arena. Proc Natl Acad Sci USA 98:35313536.
31. Aiguo W, Zhe Y, and Gomez-Pinilla F. (2010). Vitamin E protects
against oxidative damage and learning disability after mild traumatic
brain injury in rats. Neurorehabil Neural Repair 24:290298.
32. Redell JB, Moore AN, Grill RJ, Jr, et al. (2012). Analysis of functional
pathways altered following mild traumatic brain injury. J. Neuro-
trauma 30:752764.
33. Zhao Z, Loane DJ, Murray MG, Stoica BA, and Faden AI. (2012).
Comparing the predictive value of multiple cognitive, affective, and
motor tasks after rodent traumatic brain injury. J Neurotrauma 29:
24752489.
34. Eakin K, and Miller JP. (2012). Mild traumatic brain injury is asso-
ciated with impaired hippocampal spatiotemporal representation in the
absence of histological changes. J Neurotrauma 29:11801187.
35. Spain A, Daumas S, Lifshitz J, et al. (2010). Mild uid percussion
injury in mice produces evolving selective axonal pathology and
cognitive decits relevant to human brain injury. J Neurotrauma
27:14291438.
36. Levin H, and Robertson CS. (2013). Mild TBI in translation. J Neu-
rotrauma 30:610617.
37. Yuen TJ, Browne KD, Iwata A, and Smith DH. (2009). Sodium
channelopathy induced by mild axonal trauma worsens outcome after
a repeat injury. J Neurosci Res 87:36203625.
38. Creed JA, DiLeonardi AM, Fox DP, Tessler AR, and Raghupathi R.
(2011). Concussive brain trauma in the mouse results in acute cog-
nitive decits and sustained impairment of axonal function. J Neuro-
trauma 28:547563.
39. Chen Z, Leung LY, Mountney A, et al. (2012). A novel animal model
of closed-head concussive-induced mild traumatic brain injury: De-
velopment, implementation, and characterization. J Neurotrauma
29:268280.
40. Vagnozzi R, Tavazzi B, Signoretti S, et al. (2007). Temporal window
of metabolic brain vulnerability to concussions: Mitochondrial-related
impairmentPart I. Neurosurgery 61:379388.
41. Macciocchi SN, Barth JT, Littleeld L, and Cantu RC. (2001). Mul-
tiple concussions and neuropsychological functioning in collegiate
football players. J Athl Train 36:303306.
42. Macciocchi SN, Barth JT, and Littleeld LM. (1998). Outcome after
mild head injury. Clin Sports Med 17:2736.
43. Sahay A, Scobie KN, Hill AS, et al. (2011). Increasing adult hippo-
campal neurogenesis is sufcient to improve pattern separation. Nat-
ure 472:466470.
44. Leutgeb JK, Leutgeb S, Moser MB, and Moser EI. (2007). Pattern
separation in the dentate gyrus and CA3 of the hippocampus. Science
315:961966.
45. Singh M, Jeong J, Hwang D, Sungkarat W, and Gruen P. (2010).
Novel diffusion tensor imaging methodology to detect and quantify
injured regions and affected brain pathways in traumatic brain injury.
Magn Reson Imag 28:2240.
46. Yallampalli R, Wilde EA, Bigler ED., et al. (2013). Acute white
matter differences in the fornix following mild traumatic brain injury
using diffusion tensor imaging. J Neuroimag 23:224227.
47. Giza CC, and Hovda DA. (2001). The neurometabolic cascade of
concussion. J Athl Train 36:228235.
48. Aoyama N, Lee SM, Moro N, Hovda DA, and Sutton RL. (2008).
Duration of ATP reduction affects extent of CA1 cell death in rat
models of uid percussion injury combined with secondary ischemia.
Brain Res 1230:310319.
49. Barkhoudarian G, Hovda DA, and Giza CC. (2011). The molecular
pathophysiology of concussive brain injury. Clin Sports Med 30:33.
50. Doolan AW, Day DD, Maerlender AC, Goforth M, and Gunnar BP.
(2012). A review of return to play issues and sports-related concus-
sion. Ann Biomed Eng 40:106113.
Address correspondence to:
Pramod K. Dash, Ph.D.
Department of Neurobiology and Anatomy
The University of Texas Medical School at Houston
PO Box 20708
Houston, TX 77225
E-mail: p.dash@uth.tmc.edu
726 HYLIN ET AL.