Influence of Post-Traumatic Stress Disorder on
Neuroinflammation and Cell Proliferation in a Rat Model
of Traumatic Brain Injury
Sandra A. Acosta1., David M. Diamond2,3, Steven Wolfe2., Naoki Tajiri1., Kazutaka Shinozuka1,
Hiroto Ishikawa1, Diana G. Hernandez1, Paul R. Sanberg1,4, Yuji Kaneko1, Cesar V. Borlongan1*
1Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, Florida, United
States of America, 2James A. Haley Veterans Affairs Medical Center, Tampa, Florida, United States of America, 3Department of Psychology, Center for Preclinical & Clinical
Research on PTSD, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, United States of America, 4Office of Research and
Innovation, University of South Florida, Tampa, Florida, United States of America
Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric
disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI
with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell
proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled
cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based
histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions
examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral
peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and
sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the
hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic
niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating
cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone
and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD
revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive
deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least
within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did
not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may
evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized
Citation: Acosta SA, Diamond DM, Wolfe S, Tajiri N, Shinozuka K, et al. (2013) Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell
Proliferation in a Rat Model of Traumatic Brain Injury. PLoS ONE 8(12): e81585. doi:10.1371/journal.pone.0081585
Editor: Krishnan M. Dhandapani, Georgia Health Sciences University, United States of America
Received October 4, 2013; Accepted October 23, 2013; Published December 9, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Supported by USF Veterans Reintegration Research Funds and Department of Defense W81XWH-11-1-0634. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: This work was supported by a grant from the Department of Defense and intramural support from the USF Veterans Reintegration
Committee and the Neuroscience Collaborative program. The opinions expressed in this publication are those of the authors and not of the Department of
Veterans Affairs or the US government. Senior author Dr. Cesar Borlongan is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all
the PLOS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Approximately 2 million Americans every year suffer traumatic
brain injury (TBI) . Due to medical advances, the mortality rate
associated with TBI has declined from 24.9 per 100,000 US
residents in 1979 to 17.8 per 100,000 US residents in 2007 [2,3].
However, an estimated 90,000 survivors will experience loss of
physical and cognitive functions . As a consequence, there is an
increase of TBI-related chronic illnesses such as memory
impairments and, neuropsychological disabilities including depres-
sion, anxiety, and post-traumatic stress disorder (PTSD), which
impedes quality of life and contributes to a high cost of disability
annually [4,5]. These TBI-induced neuropsychological disabilities
either persist or develop late in life and may precipitate anxiety
disorders and PTSD in veterans and civilians [6,7,8,9]. However,
there is no clear evidence on how these psychiatric morbidities
interact with chronic TBI .
Accumulating evidence indicates TBI closely presents with
neurological impairments, which progressively worsen over time,
and lead to secondary injuries instigating a diffused neuroin-
flammatory response [1,5,10,11,12] and neurogenic alterations
[13,14,15]. Although these early immunological and neural
disturbances are becoming recognized in the laboratory, the
long-term pathological consequences of TBI have remained
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underexplored. In particular, whether traumatic stress at the time
of TBI exacerbates chronic neuroinflammation and suppressed
neurogenesis is not fully understood. To this end, the present in
vivo study recognized the gap in knowledge on the pathological
link between TBI and PTSD, and embarked on characterizing the
neuroinflammatory response, neuronal cell loss, cell proliferation
and neuronal differentiation by integrating an animal model of
chronic TBI with a well-established animal model of PTSD
[16,17,18]. The emergence of PTSD as a major co-morbidity
factor associated with TBI is an urgent clinical unmet need.
Because TBI has become the signature wound of wars in Iraq and
Afghanistan, improving the clinical outcome will most likely
require treating TBI, as well as co-morbid disorders, including
Materials and Methods
Experimental procedures were approved by the University of
South Florida Institutional Animal Care and Use Committee
(IACUC). All animals were housed under ambient conditions
(20uC, 50% relative humidity, and a 12-h light/dark cycle), and
necessary precautions were undertaken throughout the study to
minimize pain and stress associated with the experimental
treatments. All studies were performed by personnel blinded to
the treatment conditions.
TBI surgical procedures
Ten-week old Sprague–Dawley rats (n=24) were subjected to
either moderate TBI using a controlled cortical impactor (CCI)
(n=12, n=6 TBI alone and n=6 TBI-PTSD) or sham treatment
(no TBI) (n=6 sham surgery-no PTSD and n=6 sham surgery-
PTSD). Deep anesthesia was achieved using 1–2% isoflurane, and
it was maintained using a gas mask. All animals were fixed in a
stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA).
After exposing the skull, the CCI rod impacted the brain at the
fronto-parietal cortex (coordinates of 20.2 mm anterior and
+0.2 mm lateral to the midline) with a velocity of 6.0 m/s
reaching a depth of 1.0 mm below the dura and remained in the
brain for 150 milliseconds. The CCI rod was angled 15u degrees
vertically to maintain a perpendicular position in reference to the
tangential plane of the brain curvature at the impact surface. A
linear variable displacement transducer (Macrosensors, Pennsau-
ken, NJ), which was connected to the impactor, measured the
velocity and duration to verify consistency across animals. Sham
control injury surgeries (i.e., uninjured animals) consisted of
animals exposed to anesthesia, scalp incision, craniectomy, and
suturing. An electric drill was used to performed the craniectomy
of about 4 mm radius centered from bregma 20.2 anterior and
+0.2 mm lateral right. A computer operated thermal blanket pad
and a rectal thermometer allowed maintenance of body temper-
ature within normal limits. All animals were closely monitored
post-operatively with weight and health surveillance recording as
per IACUC guidelines. Rats were kept hydrated at all times, and
the analgesic ketoprofen was administered after TBI surgery and
as needed thereafter. Pre and post TBI, rats were fed with regular
rodent diet from Harlan (Harlan 2018).
Post-traumatic stress disorder regimen
Rats were exposed to an adult cat for 1 hr on two occasions,
separated by 10 days (Days 1 and 11). They experienced non-
tactile (visual, olfactory, auditory) cues of the cat in our model of
PTSD, as we described previously [16,17,18,19,20,21]. Social
instability was produced with pseudorandom changes in the pairs
of cage cohorts on a daily basis. Rats experienced social instability
for a total of 31 days (Days 1–31). Therefore, the two cat exposures
overlapped with the period of social instability. The combination
of social instability and cat exposure produces remarkable PTSD-
like behavioral, physiological, endocrine and pharmacological
abnormalities in stressed rats [16,17,18]. The TBI surgical
procedure occurred one day following the second cat exposure.
Thus, TBI was induced 11 days after stress induction, and then
post-TBI recovery took place during a subsequent period of 20
days of stress. This approach was designed to mimic battlefield
conditions in which TBI occurs in already stressed soldiers, and
then their recovery must occur in conjunction with post-TBI stress.
Hematoxylin and eosin analysis
Under deep anesthesia, rats were euthanized at 8 weeks after
TBI surgery, and perfused through the ascending aorta with
200 ml of ice cold phosphate buffer saline (PBS), followed by
200 ml of 4% paraformaldehyde (PFA) in PBS. H&E staining was
performed to confirm the core impact injury of our TBI model. As
shown in our previous studies [22,23,24], we demonstrated
primary damage to the fronto-parietal cortex. Lesion for impacted
area is approximately 29.669.7 mm2. In addition, H&E staining
was analyzed in the hippocampus. Starting at coordinates AP-
2.0 mm and ending AP-3.8 mm from bregma, coronal brain
sections (40 mm) covering the dorsal hippocampus were selected. A
total of 6 sections per rat was used (n=3 randomly selected rats
per group). Cells presenting with nuclear and cytoplasmic staining
(H&E) were manually counted in the CA3 neurons. CA3 cell
counting spanned the whole CA3 area, starting from the end of
hilar neurons to the beginning of curvature of the CA2 region in
both the ipsilateral and contralateral side. Sections were examined
with Nikon Eclipse 600 microscope at 20X.
Under deep anesthesia, rats were sacrificed 8 weeks after TBI
surgery, and perfused through the ascending aorta with 200 ml of
ice cold phosphate buffer saline (PBS), followed by 200 ml of 4%
paraformaldehyde (PFA) in PBS. Brains were removed and post-
fixed in the same fixative for 24 hours followed by 30% sucrose in
phosphate buffer (PB) for 1 week. Coronal sectioning was carried
out at a thickness of 40 mm by cryostat. Staining for the cell cycle–
regulating protein Ki67, migrating neuronal marker DCX, and
activated microglial cell markers OX6 was done on every sixth
coronal section throughout the entire striatum and dorsal
hippocampus. Sixteen free-floating coronal sections (40 mm) were
incubated in 0.3% hydrogen peroxide (H2O2) solution followed by
1-h of incubation in blocking solution (0.1 M phosphate-buffered
saline (PBS) supplemented with 3% normal goat serum and 0.2%
Triton X-100). Sections were then incubated overnight with Ki67
(1:400 Novacastra), DCX (1:150 Santa Cruz), and OX6 (major
histocompatibility complex [MHC] class II; 1:750 BD) antibody
markers in PBS supplemented with 3% normal goat serum and
0.1% Triton X-100. Sections were subsequently washed and
biotinylated secondary antibody (1:200; Vector Laboratories,
Burlingame, CA) in PBS supplemented with 3% normal goat
serum, and 0.1% Triton X-100 was applied for 1 h. Next, the
sections were incubated for 60 minutes in avidin–biotin substrate
(ABC kit, Vector Laboratories, Burlingame, CA). All sections were
then incubated for 1 minute in 3,30-diaminobenzidine (DAB)
solution (Vector Laboratories). Sections were then mounted onto
glass slides, dehydrated in ethanol and xylene, and cover-slipped
using mounting medium.
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Figure 1. OX6 + + expression in ipsilateral gray matter subcortical regions of TBI and TBI-PTSD rats. Figures 1A–C represent quantitative
data of estimated volumes (mm3) of OX6+ in A) cortex, B) striatum, and C) thalamus. Figures 1D–G represent OX6+ immunostaining of the ipsilateral
side of cortex in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures H-K represent OX6+ immunostaining of the ipsilateral side of striatum in
H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures L-O represent OX6+ immunostaining of the ipsilateral side of thalamus in L) sham-no
PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Note that in Figure 1C, N, the upregulation of activated microglia cells reach significance only for the group
of chronic TBI combined with PTSD. Cortex, F3,20=11.90,***p,0.0004; striatum, F3,20=6.629, **p,0.0036; thalamus, F3,20=5.999, ** p,0.0076. Scale
bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 mm.
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Immunohistochemistry techniques were used in order to tag
three different cell markers. Activated microglia cells were
visualized by staining with OX6, an antibody against antigen
presenting cell; major histocompatibility complex class ll or MHC
ll+. In order to determine the cell proliferation after chronic TBI,
an antibody against Ki67 (protein present in all active cell cycle
phases) was used . Doublecortin (DCX), an antibody against
immature migrating neurons, was used to determine neuronal
proliferation. Moreover, positive stainings were analyzed with a
Nikon Eclipse 600 microscope and quantified using Stereo
Investigator software, version 10 (MicroBrightField, Colchester,
VT). The estimated volume of OX6 positive cells was examined
using Cavalieri estimator probe of the unbiased stereological cell
technique  in analyzing the cortex, striatum, thalamus,
cerebral peduncle, corpus callosum, and cerebellum areas such
as white matter (WM), granular cell layer (GCL), and molecular
layer (ML). Ki67 and DCX positive cells were counted within the
subgranular zone (SGZ) in both hemispheres (ipsilateral and
contralateral), using the optical fractionator probe of unbiased
stereological cell counting technique. The sampling was optimized
to count at least 300 cells per animal with error coefficients less
than 0.07. Each counting frame (100 X 100 mm for OX6, Ki67,
and DCX) was placed at an intersection of the lines forming a
virtual grid (125 X 125 mm), which was randomly generated and
placed by the software within the outlined structure.
For data analyses, contralateral and ipsilateral corresponding
brain areas were used as raw data providing 2 sets of data per
treatment condition, therefore one-way analysis of variance
(ANOVA) was used for group comparisons, followed by subse-
quent pairwise comparisons (post hoc tests Bonferonni test). All
data are presented as mean values 6 SEM. Statistical significance
was set at p,0.05 for all analyses.
In the preliminary analyses of the data, comparisons between
sham treatment ipsilateral and sham treatment contralateral side,
across all brain regions studied, did not significantly differ
(p’s.0.05). Thus, the data from both sides of the sham treatment
groups were combined. In addition, the contralateral side across
all treatment groups also did not significantly differ (p’s.0.05),
thus analyses were focused on comparing the ipsilateral sides from
treatment groups. Analyses revealed there were no significant
differences between sham and PTSD alone treatment across all
brain regions examined (p’s.0.05).
Upregulation of MHC ll+ activated microglia cells in
chronic TBI alone and combined TBI-PTSD
To test the hypothesis of whether upregulation of microglia cells
associated with chronic TBI was further exacerbated in a PTSD
model with chronic TBI, different subcortical gray and WM areas
were examined. The estimated volume of activated microglia cells
(MHC ll+) was calculated using an anti-OX6 antibody. ANOVA
revealed significant treatment effects in MHC II+ expression in the
three brain regions examined (cortex, F3,20=11.90,***p,0.0004;
striatum, F3,20=6.629, **p,0.0036; thalamus, F3,20=5.999, **
p,0.0076). Pairwise comparisons revealed TBI alone and
combined TBI-PTSD resulted in a significant upregulation in
the volume of MHC II-labeled activated microglia cells in gray
matter areas ipsilateral to TBI when compared to PTSD alone and
sham treatment (p,0.05) (Figure 1A, B, C). Moreover, TBI alone
and combined TBI-PTSD, did not significantly differ between
each other in the volume of MHC ll+ in ipsilateral cortex
(Figure 1A), striatum (Figure 1B), and thalamus (Figure 1C)
Further analysis showed significant treatment effects in MHC
II+ expression in the white matter areas (corpus callosum,
F3,20=8.611, **p,0.0017, cerebral
**p,0.002, fornix, F3,20=8.368, **p,0.002). Pairwise compari-
sons revealed that TBI alone and combined TBI-PTSD also
instigated an increase of activated microglia cells (MHC ll+)
volume in ipsilateral white matter areas compared with PTSD
alone and sham treatment (p’s,0.05) (Figure 2). Significant
exacerbation of microglia cells in the cerebral peduncle was
evident in the TBI and TBI-PTSD rats compared to PTSD alone
and sham treatment (p,0.05). In addition, a significant increase in
activated microglia cells in the fornix of TBI alone and TBI-PTSD
group was found (p,0.05), relative to PTSD alone and sham
treatment (p,0.05). TBI alone and TBI- PTSD resulted in an
equivalent upregulation of activated microglia cells in corpus
callosum, cerebral peduncle and fornix around the injury side
(p,0.05) (Figure 2A, B, C).
The estimated volume of activated MHC II+ microglia cells was
also calculated in the GCL, WM, and ML of the cerebellum.
ANOVA revealed no detectable treatment effects on upregulation
of activated microglia cells in any of the examined cerebellar
regions known already the four treatment groups (Figure 3)
Chronic TBI impairs hippocampal cell survival and
proliferation, but not neuronal differentiation in
Next, we examined the effects of PTSD on chronic TBI by
evaluating the total number of surviving neurons in the
hippocampal CA3 region, the estimated number of positive
dividing cells within SGZ, and SVZ and the estimated number of
positive neuronal differentiating cells within SGZ, and SVZ where
examined. ANOVA revealed significant treatment effects on
**p,0.0017), with post hoc tests demonstrating that both TBI
alone and combined TBI-PTSD significantly reduced CA3 cell
survival in the ipsilateral hippocampus relative to ipsilateral PTSD
alone and sham treatment (p’s,0.05) (Figure 4A). There was no
significant difference in the number of surviving neurons in the
CA3 between TBI alone and TBI-PTSD animals (p.0.05)
(Figure 4A). Furthermore, analyses of cell proliferation, as
evidenced by number of positive Ki67 cells in the SGZ of the
hippocampus, and SVZ of the lateral ventricle revealed significant
**p,0.0012). Post hoc tests revealed that TBI alone and combined
TBI –PTSD significantly reduced cell proliferation in SGZ and
SVZ in a similar manner when compared to PTSD alone and
sham treatment. Both TBI alone and combined TBI-PTSD
prompted a decline of proliferating cells only in the ipsilateral side
of SGZ and SVZ compared to the corresponding hemispheres of
PTSD alone and sham treatment (Figure 4B, and Figure 5A)
(p,0.05). Finally, ANOVA revealed no significant treatment
effects (F3,20=1.9597 ns p=0.1512, F3,20=0.324 p=0.8076) on
the neuronal differentiation in the SGZ and SVZ (Figure 4C,
Figure 5B). Interestingly, while cell survival and proliferation were
altered by TBI and combined TBI-PTSD, there were no
significant differences produced by these injuries on the neuronal
differentiation across all treatment groups (p.0.05).
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Figure 2. OX6 + + expression in ipsilateral subcortical white matter regions of TBI and TBI-PTSD rats. Figures 2 A–C represent quantitative
data of estimated volumes (mm3) of OX6+ in A) corpus callosum, B) cerebral peduncle, and C) fornix. Figures 2D–G represent OX6+ immunostaining of
the ipsilateral side of corpus callosum in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 2 H–K represent OX6+ immunostaining of the
ipsilateral side of cerebral peduncle in H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures2 L-O represent OX6+ immunostaining of the
ipsilateral side of fornix in L) sham-no PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. Corpus callosum, F3,20=8.611, **p,0.0017, cerebral peduncle
F3,20=8.550, **p,0.002, fornix, F3,20=8.368, **p,0.002. Scale bars for D, E, F, G, H, I, J, K, L, M, N, O are 1 mm.
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In the present in vivo study, we demonstrated that exacerbation
of activated microglia cells was detected at 8 weeks in chronic TBI
and is associated with CA3 cell loss, and dysfunctional cell
proliferation in the hippocampus. In this first assessment of
histological pathology, we have found that PTSD did not
exacerbate TBI-induced neuroinflammation and neurodegenera-
tion. After eight weeks post-TBI, chronic TBI alone, and chronic
TBI combined with PTSD showed a similar significant augmen-
tation of intensified activated microglia cells in cortical and
There was a 20-fold increase of activated microglia cells in
cortex for TBI alone and TBI combined with PTSD when
compared to PTSD alone and sham treatment. There was a 12-
fold increase of activated microglia cells in both TBI and TBI-
PTSD in the striatum. The TBI-PTSD group showed 30-fold
increase relative to sham treatment; however, it failed to reach
statistical significance when compared with TBI alone group. For
the TBI alone and TBI-PTSD, there were 10-fold and 30-fold
increments, respectively, of activated microglia cells present in the
thalamus. The corpus callosum showed 100-fold increases of active
microglia cells compared with PTSD alone and sham treatment.
In the cerebral peduncle area, there were 10-fold and 15-fold
increments in TBI and TBI-PTSD, respectively, relative to PTSD
alone and sham treatment. The area of the hippocampal fornix
showed 10-fold and 8-fold increments in TBI and TBI-PTSD,
respectively, compared with PTSD alone and sham treatment.
Furthermore, cerebellar analysis showed that there were no
significant increments of activated microglia cells in the white
matter (WM), granular cell layer (GCL), and molecular layer (ML)
relative to sham treatment. In parallel, distinct hippocampal areas
were assessed to determine neurodegeneration. Both TBI and
TBI-PTSD showed significant declines of CA3 hippocampal
neurons relative to stress and sham treatment. By comparing our
TBI model with the TBI-PTSD model, we can conclude that
chronic PTSD exposure did not further generate an increase in
neuronal cell death in the CA3 region of the hippocampus. Of
note, analysis of the contralateral side showed no significant
differences in CA3 cell loss across treatment groups.
Further examination of the hippocampus revealed significant
declines in the proliferative capacity of newly born cells within the
SGZ. There was about 40% decrease in cell proliferation within
the SGZ in both TBI and TBI-PTSD when compared with PTSD
alone and sham treatment. The analysis showed that the injury
caused by TBI alone was associated with impaired cell differen-
tiation in the SGZ of the hippocampus since there was no further
impairment on cell proliferation when TBI is combined with
PTSD. Moreover, histological analysis of the SVZ showed that the
proliferative ability of the cells in this neurogenic niche was also
affected by chronic TBI alone which was not further impaired
when TBI was combined with PTSD. Of note, in both neurogenic
niches, SGZ and SVZ, the cell differentiation profile was not
Figure 3. OX6 + + expression in white matter, granular cell layer and molecular layer of the cerebellum of TBI and TBI-PTSD rats.
Figure 3 A represents quantitative data of the estimated volumes (mm3) of OX6+ in three distinct regions of the cerebellum; white matter (WM),
granular cell layer (GCL) and molecular layer (ML). Figures 3 B–F represent OX6+ immunostaining of the cerebellum in B) sham-no PTSD, C) sham-
PTSD, D) TBI-PTSD, E) TBI. As shown in Figure 3 A, and in the photomicrographs B–E, there is no detectable upregulation of activated microglia cells in
any of the examined cerebellar regions or across any of our treatment groups (sham-no PTSD, sham-PTSD, TBI-PTSD, and TBI alone); p.0.05. Scale
bars for B-E are 1 mm.
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Figure 4. H&E, cell proliferation Ki67+ +, and neuronal differentiation DCX+ + expressions in the hippocampus of TBI and TBI-PTSD
rats. Figures 4 A–C represent quantitative data of A) total # of neurons in hippocampal CA3, B) estimated # of Ki67+ proliferating cells in the SGZ of
the DG, and C) the estimated # of DCX+ migrating cells in the SGZ of the DG. Figures 4 D–G represent H&E staining of the ipsilateral hippocampal
CA3 region in D) sham-no PTSD, E) sham-PTSD, F) TBI-PTSD, G) TBI. Figures 4 H-K represent Ki67+ immunostaining of the ipsilateral SGZ of the DG in
H) sham-no PTSD, I) sham-PTSD, J) TBI-PTSD, K) TBI. Figures 4 L–O represent DCX+ immunostaining of the ipsilateral SGZ of the DG in L) sham-no
PTSD, M) sham-PTSD, N) TBI-PTSD, O) TBI. CA3, F3,8=13.570, **p,0.0017, SGZ Ki67, F3,20=5.017, *p,0.0106, DCX, F3,20=1.959 p,0.1512. Scale bars
for D–G are 50 mm and H–O are 1 mm.
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affected by either TBI alone or by the combination of TBI and
To our knowledge, experiments using animal models of TBI
exposed to PTSD to evaluate synergistic effects in neuroinflamma-
tion and neuronal degeneration are currently lacking, representing
a major gap of knowledge on the interaction between TBI and
psychological morbidities, such as PTSD. There is one study that
assessed the influence of chronic stress on blast-related traumatic
brain injury in rats . As in the current work, these investigators
documented brain injury in response to stress and TBI. However,
as they did not include a non-stress TBI group, it cannot be
confirmed that stress exacerbated the histopathology they
observed. Thus, the issue of the conditions, brain areas and
aspects of histopathology affected by stress-TBI interactions
remains unresolved. This lack of conclusive scientific evidence
for stress-TBI interactions also highlights the complexity behind
the pathological mechanisms that link chronic head traumas and
PTSD in TBI survivors. Nonetheless, while the present observa-
tions do not show PTSD exacerbation of TBI-induced histopath-
ological deficits, these findings provide compelling results that can
be used to guide future studies on the biological bases of TBI and
its association with the development of neuropsychological
morbidities post-TBI or the worsening of pre-existing conditions.
For example, the current work focused on assessment of
histological pathology in response to PTSD and brain trauma. It
will be important in subsequent work to include an analysis of how
stress-TBI interactions are expressed at behavioral and cognitive
levels, in conjunction with histopathological assessments.
Long-term consequences of chronic TBI may be related to
increased risk for chronic neuroinflammation, neurodegenerative
diseases, executive function impairments, and the development of
neuropsychological disorders such as anxiety, dementia, depres-
sion and PTSD [5,6,13,23,28,29,30,31,32,33]. A growing body of
literature suggests that TBI and PTSD are becoming the signature
morbidities of our military personnel and veterans [5,6,25].
Clinical studies show that TBI is significantly associated with
limited functional impairments, while TBI comorbid with PTSD
and depression was significantly associated with chronic long
lasting cognitive deficits in servicemen following deployment [5,6].
In addition, animal studies show that exposure to blast injuries
induced psychological abnormalities and increments of proteins
that enhance fear responses for several months after the initial
exposure . In the clinic, patients with a history of brain injury
display neuropsychological disturbances affecting executive func-
tion, attention and memory , which may be mediated by
reduced cerebral blood flow in the thalamus, a brain structure
Figure 5. Cell proliferation Ki67+ +, and neuronal differentiation DCX+ + expression in the SVZ of the lateral ventricle. Figures 5 A–B
represent quantitative data of A) total # of Ki67+ cells representing the number of cell proliferating in the SVZ, B) the estimated # of DCX+ migrating
cells in the SVZ of the lateral ventricle. Figures 5 C–F represent Ki67+ immunostaining of the ipsilateral SVZ in C) sham-no PTSD, D) sham-PTSD, E) TBI,
F) TBI-PTSD. Figures 5 G–J represent DCX+ immunostaining of the ipsilateral SVZ of the lateral ventricle in G) sham-no PTSD, H) sham-PTSD, I) TBI, J)
TBI-PTSD. SVZ Ki67, F3,20=7.863, **p,0.0012, SVZ DCX, F3,20=0.324, ns p=0.8076. Scale bars for C–J are 50 mm.
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implicated in neurological impairments such as memory and
learning and verbal respond speed . These findings are in
agreement with the present results which depict a significant
exacerbation of activated microglia cells in dorsal thalamic nuclei
in animals exposed to TBI combined with PTSD.
The present results also showed that TBI and TBI with PTSD
produced an equal extent of hippocampal neurodegeneration.
However, PTSD alone did not alter the hippocampal CA3 survival.
Similarly, TBI and combined TBI- PTSD, but not PTSD alone,
reduced the cellular proliferative capacity in both neurogenic niches
of SGZ and SVZ, while neuronal differentiation was not significantly
impaired in any of the treatment groups. These results support
previous clinical studies, showing a lack of decrease in hippocampal
volume in TBI-PTSD patients [15,36,37,38,39,40,41,42,43]. Our
findings are in agreement with these clinical studies, whereby chronic
stress did not exacerbate the neuropathological effects of TBI alone.
However, these findings warrant further investigations in view of
previousworkdemonstrating thatstress,particularlyelevated levelsof
corticosterone, can exacerbate damage to the hippocampus in
response to metabolic insults, including hypoxia or administration of
neurotoxins . Thus, whether the reported stress-induced
exacerbation of brain damage from metabolic challenges can be
distinguished from potential stress-TBI interactions remains to be
Finally, clinical studies have found that there is a close
association between patients that suffer TBI-and a traumatic
psychological event, with decreased hippocampal neuronal
volume. In addition, these studies showed that these patients have
an increased risk for the development of more severe neuropsy-
chological disorders such as severe depression, bipolar disorder and
PTSD . There is also evidence suggesting that there is a strong
link between TBI and worsening of psychological conditions such as
depression and PTSD[15,45,46,47,48,49,50,51,52,53]. To this end,
whether hippocampal cell loss and alteration in neurogenic capacity
are associated with the development of TBI co-morbidities is not
clear. Further studies addressing the biological bases on how TBI
combined with neuropsychological stress and more severe PTSD
impairs hippocampal function, such as long-term potentiation and
memory formation, are needed.
To our knowledge, this is the first laboratory report of
histopathological characterization of TBI-PTSD. This study
reveals that the combined TBI-PTSD group displayed similar
neuroinflammation and impaired cell proliferation profiles. Other
TBI-mediated cell death events, such as oxidative stress and
apoptosis, warrant further investigations. Investigations of TBI and
its co-morbidity factors will allow a better understanding of the
disease pathology and guide treatment that will address both
primary and secondary cell death events, as well as psychological
and physical impairments.
Conceived and designed the experiments: CVB DMD PRS. Performed the
experiments: SAA SW NT KS HI DGH CVB. Analyzed the data: SAA
NT CVB. Contributed reagents/materials/analysis tools: DMD PRS YK
CVB. Wrote the paper: SAA DMD CVB. Designed psychosocial stress
paradigm: DD. Contributed analysis: PRS YK.
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