Decreased Resting Functional Connectivity after Traumatic Brain Injury in the Rat

Abstract

Traumatic brain injury (TBI) contributes to about 10% of acquired epilepsy. Even though the mechanisms of post-traumatic epileptogenesis are poorly known, a disruption of neuronal networks predisposing to altered neuronal synchrony remains a viable candidate mechanism. We tested a hypothesis that resting state BOLD-fMRI functional connectivity can reveal network abnormalities in brain regions that are connected to the lesioned cortex, and that these changes associate with functional impairment, particularly epileptogenesis. TBI was induced using lateral fluid-percussion injury in seven adult male Sprague-Dawley rats followed by functional imaging at 9.4T 4 months later. As controls we used six sham-operated animals that underwent all surgical operations but were not injured. Electroencephalogram (EEG)-functional magnetic resonance imaging (fMRI) was performed to measure resting functional connectivity. A week after functional imaging, rats were implanted with bipolar skull electrodes. After recovery, rats underwent pentyleneterazol (PTZ) seizure-susceptibility test under EEG. For image analysis, four pairs of regions of interests were analyzed in each hemisphere: ipsilateral and contralateral frontal and parietal cortex, hippocampus, and thalamus. High-pass and low-pass filters were applied to functional imaging data. Group statistics comparing injured and sham-operated rats and correlations over time between each region were calculated. In the end, rats were perfused for histology. None of the rats had epileptiform discharges during functional imaging. PTZ-test, however revealed increased seizure susceptibility in injured rats as compared to controls. Group statistics revealed decreased connectivity between the ipsilateral and contralateral parietal cortex and between the parietal cortex and hippocampus on the side of injury as compared to sham-operated animals. Injured animals also had abnormal negative connectivity between the ipsilateral and contralateral parietal cortex and other regions. Our data provide the first evidence on abnormal functional connectivity after experimental TBI assessed with resting state BOLD-fMRI.

Full text

Decreased Resting Functional Connectivity after
Traumatic Brain Injury in the Rat
Asht Mangal Mishra
1,5
, Xiaoxiao Bai
1
, Basavaraju G. Sanganahalli
3,5
, Stephen G. Waxman
1,6
,
Olena Shatillo
8
, Olli Grohn
7
, Fahmeed Hyder
3,4,5
, Asla Pitka
¨nen
8,9
, Hal Blumenfeld
1,2,5
*
1Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2Department of Neurosurgery, Yale University
School of Medicine, New Haven, Connecticut, United States of America, 3Department of Diagnostic Radiology, Yale University School of Medicine, New Haven,
Connecticut, United States of America, 4Department of Biomedical Engineering, Yale University School of Medicine, New Haven, Connecticut, United States of America,
5Core Center for Quantitative Neuroscience with Magnetic Resonance, Yale University, New Haven, Connecticut, United States of America, 6Center for Neuroscience and
Regeneration Research, West Haven, Connecticut, United States of America, 7Biomedical NMR research group, Biomedical Imaging Unit, University of Eastern Finland,
Kuopio, Finland, 8Department of Neurobiology, A. I. Virtanen Institute of Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 9Department of Neurology,
Kuopio University Hospital, Kuopio, Finland
Abstract
Traumatic brain injury (TBI) contributes to about 10% of acquired epilepsy. Even though the mechanisms of post-traumatic
epileptogenesis are poorly known, a disruption of neuronal networks predisposing to altered neuronal synchrony remains a
viable candidate mechanism. We tested a hypothesis that resting state BOLD-fMRI functional connectivity can reveal
network abnormalities in brain regions that are connected to the lesioned cortex, and that these changes associate with
functional impairment, particularly epileptogenesis. TBI was induced using lateral fluid-percussion injury in seven adult male
Sprague-Dawley rats followed by functional imaging at 9.4T 4 months later. As controls we used six sham-operated animals
that underwent all surgical operations but were not injured. Electroencephalogram (EEG)-functional magnetic resonance
imaging (fMRI) was performed to measure resting functional connectivity. A week after functional imaging, rats were
implanted with bipolar skull electrodes. After recovery, rats underwent pentyleneterazol (PTZ) seizure-susceptibility test
under EEG. For image analysis, four pairs of regions of interests were analyzed in each hemisphere: ipsilateral and
contralateral frontal and parietal cortex, hippocampus, and thalamus. High-pass and low-pass filters were applied to
functional imaging data. Group statistics comparing injured and sham-operated rats and correlations over time between
each region were calculated. In the end, rats were perfused for histology. None of the rats had epileptiform discharges
during functional imaging. PTZ-test, however revealed increased seizure susceptibility in injured rats as compared to
controls. Group statistics revealed decreased connectivity between the ipsilateral and contralateral parietal cortex and
between the parietal cortex and hippocampus on the side of injury as compared to sham-operated animals. Injured animals
also had abnormal negative connectivity between the ipsilateral and contralateral parietal cortex and other regions. Our
data provide the first evidence on abnormal functional connectivity after experimental TBI assessed with resting state BOLD-
fMRI.
Citation: Mishra AM, Bai X, Sanganahalli BG, Waxman SG, Shatillo O, et al. (2014) Decreased Resting Functional Connectivity after Traumatic Brain Injury in the
Rat. PLoS ONE 9(4): e95280. doi:10.1371/journal.pone.0095280
Editor: Maxim Bazhenov, University of California, Riverside, United States of America
Received October 9, 2013; Accepted March 25, 2014; Published April 18, 2014
Copyright: ß2014 Mishra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) R01 NS049307 (HB), NIH R01 NS066974 (HB), Epilepsy Foundation Postdoctoral Research
and Training Award ID: 123505 (AMM), Yale QNMR Pilot study (AMM), P30 NS052519 (FH), the Betsy and Jonathan Blattmachr family (HB), the Academy of Finland
(AP, OG), and by the Sigrid Juselius Foundation (AP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hal.blumenfeld@yale.edu
Introduction
Traumatic brain injury (TBI) is a common brain insult as every
21 seconds, one person in the USA sustains a TBI, and it is
estimated that TBI annually affects about 1.7 million Americans
(http://www.cdc.gov/injury/about/focus-tbi.html). White matter
and axonal injury are common pathologic sequela of TBI [1].
Importantly, injury-related breakdown of the neuronal networks
associates with a wide spectrum of functional impairments,
including compromised somatomotor, cognitive, and behavioral
performance [1]. Based on previous studies, network abnormalities
apparently also contribute to the development of other co-
morbidities, particularly post-traumatic epilepsy (PTE) [2].
Assessment of the development, severity, and progression of
abnormalities in connectivity between the brain regions following
TBI could pinpoint the at-risk patients for functional deficits, and
guide targeting of therapeutic interventions for those who benefit
most.
Blood oxygenation level dependent (BOLD) functional mag-
netic resonance imaging (fMRI) has enabled the mapping of
different brain networks in health and disease [3,4]. BOLD-fMRI
at rest measures remotely distributed brain networks fluctuating
synchronously in the brain. Resting functional connectivity was
first used to reveal motor brain networks [5]. For more than 15
years, resting functional connectivity has been used to study
different neuronal networks such as those involved in motor,
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sensory, visual, memory, language, or cognitive processing [6,7]
Recently it has also been used to study epilepsy in humans [8–11]
as brain regions involved in BOLD-fMRI during seizures can be
used as seed regions for analysis of resting functional connectivity
[8,10,12]. So far, resting state BOLD-fMRI has not been applied
in experimental models of TBI, even though it could be expected
to reveal network abnormalities not easily detectable with other
methodologies in vivo.
We hypothesize that local cortical damage caused by TBI
results in a widespread disruption of cortical and subcortical
networks, which associates with functional impairments, particu-
larly the development of increased seizure susceptibility. The
consequent PTE develops as progressive fragmentation of
functional and structural connectivity. Therefore, resting BOLD-
fMRI could provide us clues about changes in neuronal networks
that are critical for post-traumatic epileptogenesis. To address
these hypotheses, we assessed network changes by resting BOLD-
fMRI in rats, in which post-traumatic epileptogenesis was induced
with lateral fluid-percussion injury (FPI), an established model of
human PTE [13,14]. We focused on brain areas known to be
injured by the impact and recently suggested to be involved in
post-traumatic epileptogenesis, including the parietal cortex,
hippocampus, and thalamus [15–17]. Our goal was to find a
noninvasive method for assessment of the network changes during
post-TBI epileptogenesis that could ultimately be applied to studies
of human PTE.
Material and Methods
Animals
Adult male Sprague-Dawley rats (Harlan Netherlands B.V.,
Horst, the Netherlands) were used. The animals were housed in
individual cages under standard conditions (cage size 53. 32.5.
20 cm, 12 h light/12 h dark rhythm, lights off at 7 p.m., room
temperature 2161uC, and humidity 50–60%). Water and food
were available ad libitum. Procedures related to the induction of
TBI were approved by the Animal Ethics Committee of the
Provincial Government of Southern Finland, and carried out in
accordance with the guidelines of the European Community
Council Directives 86/609/EEC. All experimental procedures
were in full compliance with Yale University Institutional Animal
Care and Use Committee protocols approved in agreement with
the National Institutes of Health.
Induction of lateral fluid-percussion brain injury
TBI was induced by lateral fluid-percussion injury (FPI) as
described previously [13,18]. Briefly, rats were anesthetized with
intraperitoneal injection (i.p.) of a cocktail, containing sodium
pentobarbital (582 mg/kg), chloral hydrate (60 mg/kg), magne-
sium sulphate (127 mg/kg), propyleneglycol (43%), and ethanol
(11.7%). Then, they were inserted into a stereotaxic frame with
lambda and bregma at the same horizontal level. A midline scalp
incision was made and a 5-mm circular piece of parietal bone was
removed over the left cortex, midway between the lambda and
bregma and midway between the sagittal suture and temporal
ridge. Care was taken to leave the dura intact. A plastic female
Luer-Lock connector was secured in the craniotomy with Vetbond
adhesive (3M, St. Paul, MN, USA). The connector was anchored
with dental acrylate to a screw placed in the skull rostral to the
bregma. Animals were placed on heating pads while anesthetized
to maintain the normothermic temperature. Ninety minutes
(90 min) after injection of anesthetic, the rat was attached to the
fluid percussion device (AmScien Instruments, Richmond, Vir-
ginia, USA) to produce TBI (pressure level 3.460.01 atm).
Animals were removed from the device, and thereafter, dental
cement, screw, and Luer-Lock connector were removed, and scalp
was sutured. Sham-operated animals underwent surgery but were
not injured. Mean weight of animals at the time of TBI or sham-
operation was 350611 g.
Survival BOLD–fMRI
Survival MRI was performed at four months post-TBI. On each
imaging day we scanned a pair of animals, that is, a sham-
operated rat was scanned in the morning and a rat with lateral FPI
in the afternoon and vice versa (randomly counterbalanced)
(between 9:00 am and 6:00 pm). For imaging, rats were positioned
prone in a specially designed plastic holder with the head fixed and
bregma positioned at the center of the quadrature coil. The animal
was then inserted into the magnet with its head positioned at the
isocenter of the magnet. Electroencephalogram (EEG) was
recorded simultaneously with BOLD–fMRI, using a pair of 1-
mm diameter carbon-filament electrodes (WPI, Sarasota, FL). The
purpose of the EEG recording during the MRI was to detect
epileptiform activity that would interfere with the ‘resting state’
needed for the MRI analysis. To minimize MRI signal distortion,
the carbon filaments were placed between the scalp and the
surface of the skull in the frontal and occipital areas and secured to
the skin with tissue glue (3M Vetbond, 3M Animal Care Products,
MN) [19]. The exposed portion of each electrode crossed the
midline from left to right in the coronal plane. The EEG signals
were acquired in differential mode between the two electrodes,
amplified (6100), and filtered (1–30 Hz) using a Model 79D Data
Recording System (Grass Instruments Co., Quincy, MA). EEG
signals were digitized and recorded (sampling rate 1 KHz) using a
CED Micro 1401 and Spike 2 software (Cambridge Electronic
Design, Cambridge, UK). All MRI data were acquired using a 9.4
T Bruker horizontal bore (16–cm internal diameter) spectrometer
(Agilent Technologies), equipped with passively shielded shim/
gradient coils (47.5 G/cm) operating at 400.5 MHz for protons. A
quadrature (162) coil with two 2.1 cm loops was used as
transmitter and receiver. To optimize the homogeneity of the
static magnetic field, the system was shimmed before each
experiment using global manual shimming. 3% isoflurane in
air/oxygen (70%/30%) was used as induction anesthesia. There-
after, rats received a bolus of dexmedetomidine (i.p., 0.3 mg/kg;
Domitor, Pfizer, Karlsruhe, Germany) and isoflurane was
discontinued after 5 min. At 15 min after bolus injection, a
continuous infusion of dexmedetomidine (0.1 mg/kg/h; 1 ml/h,
i.p.) was started. During subsequent animal preparation and MRI
imaging, animals were breathing spontaneously on room air.
Rectal temperature was monitored and maintained at ,37.5uCby
a water-circulated heating pad (Model #TP3E, Gaymar
Industries, Inc., NY). At the end of the MRI experiments,
dexmedetomidine was antagonized by atipamezole (0.1 mg/kg,
i.p.; Antisedan, Pfizer, Karlsruhe, Germany).
Anatomical images for each animal were acquired with 12
interlaced slices in the coronal plane using fast spin echo multi
slice, with repetition time (TR) 4000 ms; echo time (TE) 48 ms;
flip angle = 40–55u; field of view (FOV) 25.6625.6 mm; 2566256
matrix size; in–plane resolution of 98698 mm; and slice thickness
1000 mm, without gap. BOLD–fMRI data were obtained in the
same planes as anatomical images. We used single-shot spin echo
echo planar imaging (SE–EPI). SE–EPI data were acquired with
the following parameters: TR, 2000 ms, TE, 34 ms; excitation flip
angle 90u; inversion flip angle 180u; FOV, 25.6625.6 mm, 64664
matrix size; in-plane resolution, 3906390 mm; and slice thickness
1000 mm. All SE–EPI experiments were acquired with 12 slices.
The 12 slices were acquired over 2000 ms, followed by a 2s pause
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before the next image onset so that EEG could readily be
interpreted during data acquisition. Time between onset of
consecutive image acquisitions was therefore 4s. We acquired
150 images per experimental run, resulting in a total imaging time
of 600 s for one experimental run and four runs were acquired per
animal. Sixteen dummy scans occurred before the receiver was
turned on. Dummy scans were used to ensure that the proton spin
system was in a steady state before data were collected.
EEG recording
Electrode implantation. A week after survival MRI (i.e., 4.5
months after lateral FPI), sham-operated and injured rats were
anesthetized with ketamine (100 mg/kg), xylazine (10 mg/kg), and
acepromazine (1 mg/kg), and then, inserted in a stereotactic frame
(David Kopf Instruments, Tujunga, CA) for implantation of
stainless steel electrodes (Part #MS333/3-A, tripolar, uncut
untwisted 0.005; Plastics One Inc., Roanoke, VA; internal control
#8LMS3333XXXE, pedestal height: 8 mm). To provide a good
electrical contact before wrapping around skull screws, the
polyimide insulation was scraped off from the ends of the
recording electrodes, exposing stainless steel wire up to 10 mm
from the tip but leaving insulation intact proximally, as verified
under the microscope. Small burr holes [using Micro Drill Steel
Burrs, 2.3 mm shaft diameter, 44 mm overall length; item #
19007-14, Fine Science Tools (USA), Inc.] were made in the skull
without damaging the dura and electrodes were secured to the
skull using stainless steel screws (Plastics One, Part #0-80X1/16,
internal control #8L010121201F with shaft length = 1.60 mm,
head diameter = 2.50 mm, shaft diameter = 1.57 mm). EEG
recording electrodes were implanted in the ipsilateral (left) frontal
(AP +2.0, ML +2.0 mm) and contralateral parietal cortex (AP -6.0,
ML +2.0 mm). A ground electrode was placed in the midline over
the cerebellum. An anchoring screw without electrode was placed
contralateral to craniotomy at an equal distance (ML -2.0 mm)
between the coronal suture and the lambdoidal suture. Dental
acrylic (Cat #1255710; Henry Schein Inc, Indianapolis, IN; Lang
Jet Denture Repair Acylic) was used to fix the electrode pedestal to
the skull. Level of anesthesia was monitored by respiration, heart
rate, glabrous skin perfusion, and responses to foot pinch.
EEG monitoring. One week after recovery from electrode
implantation, awake and freely-moving sham-operated and
injured rats underwent a 1-h baseline EEG recording (between
9:00 a.m. and 6:00 p.m). Recordings were done via a tripolar cable
(Plastics One, Inc., Catalog#335-340-3 0-SPR 80CMtripolar)
and a commutator (Plastics One, Inc., Catalog#
8BSL3CXCOMMT) connected to a Grass CP 511 amplifier
(Grass- Telefactor, Astro Med, Inc., West Warwick, RI). Band pass
frequency filter settings were 1–300 Hz. Signals were digitized at a
sampling rate of 1 KHz with an NI USB-6008 A/D converter and
LabView 7.1 software (National Instruments, Austin, TX), and
analyzed using Spike 2 (Cambridge Electronic Design, Cam-
bridge, UK).
Pentylenetetrazol (PTZ) test
Right after the baseline EEG monitoring, sham-operated and
injured rats were exposed to a PTZ test to assess seizure
susceptibility as described before [13]. Briefly, PTZ (1,5-penta-
methylenetetrazol, Santa Cruz Biotechnology, Inc, USA) was
dissolved in sterile 0.9% sodium chloride (final concentration of
12.5 mg PTZ/ml 0.9% sodium chloride), and administered at a
dose of 25 mg/kg (i.p.). Each rat received a single injection of PTZ
solution. Following the PTZ injection, rats were placed separately
into cages where they could move freely, and EEG was recorded
for 60 minutes after PTZ administration. Behavior was monitored
by an observer. The time of the occurrence of epileptiform
behavioral events was recorded and scored according to a
modified Racine’s scale (1 = twitching, freezing, 2 = myoclonic
jerks of one forelimb; 3 = bilateral forelimb clonus; 4 = forelimb
clonus with rearing; 5 = tonic–clonic convulsion).
Analysis of EEG data
EEG signals acquired during survival EEG–fMRI were first
processed using Spike 2 software and magnetic field-induced
artifacts were reduced as described previously [19–21]. Thereafter,
EEGs were screened for occurrence of electrographic interictal
epileptiform discharges (IEDs) in EEG acquired during fMRI as
well as before and after PTZ administration. IED was defined as a
high–amplitude rhythmic discharge containing a burst of slow
waves, spike-wave and/or polyspike-wave components and lasting
.1s.
After PTZ administration, latency to the first IED was
calculated. Start and end times of all IEDs were marked in the
EEG files manually using Spike 2 software and a script provided
by CED (Cambridge, U.K.). Intervals containing artifact were also
marked and excluded from the analysis. Number of IEDs was
calculated during the 60 min after PTZ administration. Based on
the evolution of IEDs seen after PTZ injection, we divided this
60 min in three 20 min time segments, 1). 0–20 min, 2). 21–
40 min, and 3). 41–60 min. Then we performed paired t-test
between IEDs in these three 20 minutes time segments in FPBI
and in sham rats.
Histologic analysis
Fixation and cryoprotection. One hour after the PTZ-test,
rats were deeply anesthetized with sodium pentobarbital (Euthasol,
100–150 mg/kg i.p.) and perfused intracardially with 0.9%
sodium chloride followed by 4% cold paraformaldehyde (PFA)
in 0.1 M phosphate buffered saline (PBS) (pH = 7.4). After
perfusion, brains were postfixed in 4% PFA in PBS at +4uC for
1 h. Before cryopreservation, brains were immersed in 0.02 M
potassium phosphate buffered 20% glycerol at +4uC for at least
36 h. Thereafter, brains were blocked placed on dry ice for 15
minutes, and then stored at 270uC until histologic processing.
Tissue processing. The brains were sectioned in the coronal
plane (30 mm, 1-in-5 series) with a sliding microtome. The first
series of sections was stored in 10% formalin at room temperature
and the remaining four series in a cryoprotectant tissue-collecting
solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium
phosphate buffer) at 220uC until stained.
Histologic stainings. The first series of sections was stained
for thionin to identify the cytoarchitectonic boundaries of different
brain areas as well as the distribution and severity of tissue
damage.
An adjacent 1-in-5 series of sections was stained for calcium
using the Alizarin red method [22] to determine calcium
deposition. In brief, sections were mounted on gelatinized glass
slides and immersed in 2% Alizarin Red (w/v, distilled water
pH 4.1 to 4.3; Merck, Darmstadt, Germany) for 30 s followed by a
rinse in distilled water. Sections were quickly dehydrated with
acetone and xylene and mounted in Depex (VWR International
Ltd, Poole, UK).
A possible role for iron accumulation in the pathophysiological
events after mild TBI has been suggested recently [23]. Therefore,
to stain iron deposition in the brain, one series of sections was
stained with Perls’ Prussian Blue [24]. Sections were hydrated with
distilled water and incubated in solution containing 1% HCl and
1% potassium hexacyanoferrate (II)-trihydrate (Merck, Germany)
for 15 min at room temperature. To visualize the anatomic
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structures of the brain, the sections were counterstained with
Mayer’s hematoxylin (Sigma-Aldrich).
Myelinated fibers were stained with gold chloride solution. For
staining, coronal sections were mounted on gelatinized slides and
dried at 37uC. Mounted sections were incubated at room
temperature in the dark for 11–14 h in a 0.2% gold chloride
solution (HAuCl4.3H2O, G-4022 Sigma) made in 0.02 M sodium
phosphate buffer (pH 7.4) containing 0.9% NaCl. The slides were
then washed twice in 0.02 M sodium phosphate buffer in 0.09%
NaCl (4 min each), and placed in a 2.5% sodium thiosulfate
solution for 5 min. After 3 washes (10 min each) in the buffer
solution, sections were dehydrated through an ascending series of
ethanol, cleared in xylene, and cover-slipped with DePeX
mounting medium (BDH, Laboratory Supplies, Dorset, UK).
Resting BOLD-fMRI correlation analysis
Resting BOLD–fMRI connectivity analysis was performed
using in-house programs written in MATLAB 7.1. Although rats
were paralyzed during experiments, all fMRI series were first
screened for movement artifacts using a movie function and
center–of–mass analysis, restricted to voxels within the brain
boundaries, to ensure that all runs exhibited movement of less than
20% of a pixel in either the x or y direction as described previously
[19,25].
The data were next band-pass filtered (0.01,f,0.08 Hz) to
remove low–frequency drift and reduce the influence of high–
frequency noise. For fMRI analysis four pairs of regions of
interests (ROIs) were made: bilateral frontal and parietal cortex,
hippocampus, and thalamus (Fig. 1A, B).
ROIs were individually drawn on each rat’s MRI data. We
selected these brain regions because they are known to be involved
in posttraumatic epileptogenesis [15,17]. ROI size was 6 voxels in
frontal cortex and in hippocampus, 7 voxels in parietal cortex, and
12 voxels in the thalamus. ROI size was different in different
regions in order to quantitate maximum fMRI signals available
from each brain regions at the same time to avoid signal from non-
brain area. For individual rats, a mean time course was calculated
for each ROI by averaging the time courses of all voxels within the
ROI. We then computed the Pearson’s correlation coefficient
between mean time courses of each possible pair of ROIs.
Subsequently, these correlation coefficients(r) were converted to z-
scores by Fisher’s z transform z(r) = 0.5 ln[(1+r)/(12r)] [26]. To
normalize for differences in number of images, each z score was
divided by the square root of variance, calculated as !1/(n23),
where n is the degrees of freedom defined as the number of image
acquisitions within each epoch.
For group statistical analysis, we compared z scores for each
ROI pair between the sham-operated and injured rats. To
accomplish this, we performed one–way ANOVA followed by
Tukey’s Honestly Significant Difference (HSD) method for post hoc
pair-wise comparisons to assess the group differences.
Results
There was no acute mortality in 8 rats randomized to the sham
group in the 48 hours following sham injury. Two of 8 sham-
operated animals later died before MRI. One of the 13 rats
randomized to the TBI group died during anesthesia before injury.
From the 12 remaining animals randomized to lateral FPI group,
3 (25%) died during the first 48 h corresponding to moderate-
severe injury severity. From the remaining 9 TBI animals, 2 died
before MRI. Results are therefore from 6 sham operated and 7
TBI animals. On the day of fMRI mean weights of sham operated
animals (495631 gm, mean 6SD) were not different (p = 0.63)
from TBI animals (488617 gm).
Spontaneous epileptiform activity in EEG
Survival BOLD-fMRI was performed at 4 months after lateral
FPI or sham injury. No IEDs were detected during survival fMRI
runs in sham-operated or injured rats. Also, no IEDs were
detected in the 1-h baseline EEG recordings performed before
PTZ test. Thus the baseline EEGs obtained in the same animals
approximately two weeks apart (during survival fMRI and prior to
PTZ) were similar and did not show spontaneous IEDs.
Decreased latency to the first seizure and increased
number of IEDs in rats with TBI
EEG recordings after PTZ administration revealed frequent
IEDs in all rats. Post-PTZIEDs were associated with twitching and
freezing, corresponding to a score of 1 on the modified Racine’s
scale. In rats with TBI, the latency to the 1
st
IED was shorter than
that in the sham-operated animals (207640 s and 4426101 s,
p = 0.04) (Fig. 2A). In 5 of 7 the injured rats, the latency to the 1
st
spike was less than -1SD of the control mean. The total number of
IEDs was higher in injured rats as compared to that in sham-
operated animals (223630 vs.118631per 60 min, p = 0.03)
(Fig. 2B). Interestingly, the frequency of IEDs in injured rats
(n = 7) increased already during the first 20 min post-PTZ,
becoming significant during 21–40 min (p = 0.01) and 41–
60 min (p = 0.04) post-PTZ as compared to sham-operated
Figure 1. Decreased functional connectivity in TBI rats vs.
controls. Two coronal MRI slices (A, B) from a rat with lateral FPI (note
the tissue loss in the left hemisphere) demonstrating the brain regions
of interest (ROIs) superimposed on coronal BOLD-fMRI images used for
resting BOLD–fMRI signal correlation analysis. Four ROIs were made in
each hemisphere (8 ROIs total): frontal cortex, parietal cortex,
hippocampus, and thalamus (L, left; R, right). Panel B shows
schematically using arrows the significant differences in connectivity
(1) between the ipsilateral and contralateral parietal cortices and (2)
between the ipsilateral parietal cortex and hippocampus in the rats
with lateral FPI and sham-operation. Statistical significance: *, p,0.05.
(C) Example of resting functional connectivity in a sham-operated
control rat. Pearson correlation values are shown using the left parietal
cortex ROI (B) correlated to the whole brain. Some positive correlation is
seen with the contralateral parietal cortex and ipsilateral hippocampus.
(D) Example of the same analysis for the left parietal cortex ROI in a TBI
animal shows reduced connectivity. Warm colors represent increases
and cool colors decreases in connectivity; scale bars are for Pearson
correlation with display threshold = 0.2. Voxels lying outside the brain
or in CSF are not shown.
doi:10.1371/journal.pone.0095280.g001
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animals (n = 6) (Fig. 3). In 7 of 7 rats, the total number of IEDs
was higher than +1SD of the control mean.
Decreased resting functional connectivity in rats with TBI
Rats with TBI had lower resting fMRI correlation coefficients
than sham-operated rats (Table 1). An example of resting fMRI
connectivity in a sham-operated control compared to a TBI rat is
shown in Figure 1 C, D. At the group level, rats with TBI had
significantly decreased correlation coefficients between the left and
right parietal cortices as well as ipsilateral to injury between the
parietal cortex and hippocampus as compared to sham-operated
animals (Table 1, Fig. 1B). Injured rats also had abnormal
negative connectivity values between the left and right parietal
cortex and other brain regions, not seen in control animals
(Table 1). We attempted to correlate the functional connectivity
in individual animals to the seizure susceptibility with PTZ
administration, however the sample size was too small to
demonstrate a significant relationship (data not shown).
Histology
Histologic findings were qualitatively similar in all 7 injured
animals available for analysis and they are summarized in Fig. 4.
Iron deposits were present in all injured animals and were
observed ipsilaterally in several white matter bands, including the
external capsule at the level of the cortical lesion (Fig. 4E, G) and
medial aspects of fimbria. Iron deposits were seen ipsilaterally in
the corpus callosum above the septal end of the hippocampus in all
injured rats, and they extended contralaterally in 4 of 7 animals
(Fig. 4C). In tissue parenchyma, patches of iron deposits were
present in all injured rats in the subgranular region of the infra
pyramidal blade of the temporal dentate gyrus (Fig. 4F). In 2 of 7
injured rats we also found them in the parasubiculum (Fig. 4I).
Occasionally they were also seen ipsilaterally in the dorsal midline
thalamic area and bilaterally in the ventral brain stem.
Calcium deposits were exclusively located in the ipsilateral
thalamus, most typically in the ventroposterior nucleus or in the
area dorsomedial or dorsolateral to it (Fig. 4H).
As reported previously [27], myelin staining revealed reduced
fiber density in layer VI of the perilesional cortex (Fig. 4A) and in
stratum lacunosum moleculare of the CA1 of the ipsilateral
hippocampus (Fig 4J) in all injured rats.
Discussion
The present study was designed to test a hypothesis that resting
state BOLD-fMRI functional connectivity can reveal network
abnormalities in brain regions that are connected to the lesioned
cortex, and that these changes associate with functional impair-
ment, particularly epileptogenesis. To our knowledge these are the
first data reporting abnormalities in functional connectivity in
experimental TBI using resting state BOLD-fMRI. In fact, there
are no previous functional connectivity studies published in human
TBI either.
A selective set of pathways show impaired functional
connectivity after experimental TBI which associate with
seizure susceptibility
Our previous studies have shown that up to 80% rats with
moderate to severe lateral FPI develop increased seizure suscep-
tibility, and up to 50% of the animals express spontaneous seizures
Figure 2. (A) Latency to the first interictal epileptiform discharge (IED) and (B) Number of IEDs in rats with lateral FPI or sham-operation. Note a
decrease in latency to the 1
st
IED (p = 0.03) and an increase in IEDs (p = 0.03) in injured rats (n = 7) as compared to sham-operated animals (n = 6).
Abbreviations: SD, standard deviation.
doi:10.1371/journal.pone.0095280.g002
Figure 3. Evolution of interictal epileptiform discharges (IEDs)
over time after administration of pentylenetetrazol (25 mg/kg
body weight) in rats with lateral FPI (n = 7) and sham-operated
animals (n = 6). Abbreviations: SD, standard deviation.
doi:10.1371/journal.pone.0095280.g003
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Table 1. Correlation coefficients of resting BOLD-fMRI signals and their significances from different brain regions in sham-operated rats (n =6) and in rats with traumatic brain
injury (TBI) (n = 7) and comparison between the two groups.
Significance (p values)
comparing correlation
coefficients from different
ROIs: sham-operated rats
vs. rats with TB
Frontal Cx - R HC - R Parietal Cx - R Thalamus - R Frontal Cx - L HC – L Parietal Cx – L
Thalamus - L 0.36 0.74 0.41 0.55 0.74 0.32 0.23
Parietal Cx - L 0.99 0.11 0.03 0.16 0.88 0.03
HC - L 0.99 0.57 0.06 0.41 0.26
Frontal Cx - L 0.50 0.68 0.16 0.32
Thalamus - R 0.57 0.70 0.09
Parietal Cx - R 0.30 0.17
HC - R 0.55
Correlation (r)
values in different
ROIs in sham-
operated rats
Thalamus - L 0.024 0.076 -0.005 0.230 0.091 0.248 0.070
Parietal Cx - L 0.111 0.135 0.187 0.160 0.034 0.185
HC - L 0.044 0.114 0.106 0.199 0.017
Frontal Cx - L 0.163 0.045 0.076 0.058
Thalamus- R 0.042 0.135 0.046
Parietal Cx - R 0.106 0.174
HC - R 0.029
Correlation (r)
values in different
ROIs in rats with TBI
Thalamus - L 0.119 0.050 0.074 0.186 0.123 0.192 -0.048
Parietal Cx - L 0.110 0.018 0.019 0.032 0.055 -0.08
HC - L 0.045 0.065 -0.033 0.160 0.122
Frontal Cx - L 0.073 0.074 -0.024 0.149
Thalamus - R 0.098 0.101 -0.061
Parietal Cx - R 0.012 0.011
HC - R -0.036
Abbreviations: Cx, cortex; L, left; R, right. Values in boldface were significantly different (p,0.05) in sham-operated and injured rats.
doi:10.1371/journal.pone.0095280.t001
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in a 1-year follow-up [13,28]. The present study confirms these
observations by showing that 100% of animals with TBI induced
with lateral FPI 5 months earlier had increased IEDs and 71% had
a reduced latency to the 1st epileptiform spike in PTZ test,
indicating increased seizure susceptibility. Analysis of resting state
BOLD–fMRI functional connectivity in the same animals showed
decreased connectivity between the ipsilateral and contralateral
parietal cortex, between the ipsilateral parietal cortex and
ipsilateral hippocampus as well as negative connectivity between
the ipsilateral and contralateral parietal cortex with other regions.
Moreover, histologic analysis revealed abnormalities in pathways
mediating the connectivities, including corpus callosum, capsula
externa, and perforant pathway. Regarding the abnormal
connectivity between the ipsilateral and contralateral parietal
cortex, an obvious candidate for the cause is the lesion in the
corpus callosum which was present in each animal with lateral
FPI. However, the explanation for an abnormal connectivity
between the parietal cortex and ipsilateral hippocampus is
apparently polysynaptic, and could include abnormalities in
several ipsilateral white matter bands including the external
Figure 4. Summary of pathological findings in animals with lateral FPI. Three thionin-stained whole brain coronal sections on the right
show the approximate location, from which panels A-K were taken. Rostro-caudal level of sections corresponds to the level of MRI slices (A) Myelin-
stained section demonstrating a reduced thickness of white matter (fiber bundle between the open arrows) and in particular, reduction in the density
of fibers in layer VI (black arrow) of the S1 region ipsilaterally. (B) Note a thicker white matter band contralaterally and abundance of myelinated fibers
in layer VI. The pattern of staining is comparable to that in sham-operated controls (not shown). (C) Perl-stained sections showing iron deposits in
corpus callosum bilaterally (open arrows). (D) Iron deposits in plexus choroideus (open arrows). (E) Iron deposits in cortical lesion and deep portion of
the cortex near white matter band. (F) Iron deposits in the subgranular region of the dentate gyrus. Dashed line indicates the border between the
granule cell layer and the hilus. (G) Iron deposits in subcortical white matter on the lesion side. (H) Alizarin-stained calcifications in the ipsilateral
thalamus (open arrow). (I) Iron deposits in the parasubiculum. (J) Myelin staining from the ipsilateral hippocampus showing a reduced density of
fibers in the stratum lacunosum-moleculare of the CA1 (open arrow) compared to contralateral hippocampus. Contralaterally (K) the pattern of
staining is comparable to that in sham-operated controls (not shown). Abbreviations: g, granule cell layer; m, molecular layer; l-m, stratum lacunosum
molecular of CA1; p, pyramidal cell layer. Scale bars equals in panels A–F, H–K 100 mm.
doi:10.1371/journal.pone.0095280.g004
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capsule which were also present in all injured animals. Other
physiological changes such as alterations in axonal function
[29,30] could also potentially contribute to reduced connectivity
even in the absence of obvious white matter structural changes.
Desynchronization has been reported during the preictal state
preceding pharmacologically-induced epileptic seizures. Under
those circumstances it is characterized by a biphasic network
dynamics with an early desynchronization phase followed by a late
resynchronization phase, in which the activity and synchronization
of the network gradually increase [31]. Though we did not directly
address the possible role of early desynchronization in the
development of seizures, our findings of decreased resting
functional connectivity, which may be secondary to structural
changes induced by the lateral FPI support the concept of
dissociation of brain network synchrony during epileptogenesis
[32].
A previous study in rats with lateral FPI at 8 months after brain
injury showed decreased cerebral blood flow (CBF) and increased
vessel density in the perilesionalcortex [17]. The hippocampus,
however, showed bilateral decreases in CBF but no change in
vessel density. Therefore, in addition to histologic lesions, the
reduced cortical and hippocampal CBF may explain both the later
seizure susceptibility and decreased connectivity of ipsilateral
parietal cortex to ipsilateral hippocampus. Because ipsilateral and
contralateral hypoperfusion in the cortex as well as in the
hippocampus can persist for several months after lateral FPI, they
could contribute an overall decrease in resting functional
connectivity observed in the present study [15].
In addition to epileptogenesis, impaired functional connectivity
could contribute to other post-TBI co-morbidities. The different
stages of working memory have been associated with poor
interhemispheric coherence on EEG in the frontal and temporal
regions in patients with mild TBI [33]. In moderate TBI patients,
Gupta and colleagues [34] have shown that impaired structural
connectivity measured through diffusion tensor imaging (DTI)
fractional anisotropy and radial diffusivity correlated with
neuropsychological test scores. Follow-up upon recovery in these
DTI indices were associated with recovery in neurocognitive
deficits [34]. Our findings provide evidence for bilateral network
abnormalities in TBI in brain regions which are necessary for
working memory.
Methodological considerations
Our study was performed under anesthesia, while posttraumatic
epileptogenesis studies using fMRI in human PTE patients could
be done in the awake state. Throughout all imaging sessions,
anesthesia dosage was kept constant, and a continuous rate of
infusion of dexmedetomidine anesthesia greatly enhances our
ability to obtain stable and reliable MRI measurements in the
rodent model [35–37], without blocking primary somatosensory
cortex-evoked potentials [38]. Although ideally the measurements
should be repeated in the awake state, the differences between
groups are unlikely to be caused entirely by anesthesia. Awake
MRI experiments are feasible in rodent models, but considerably
more technically challenging, since it is necessary to train and
habituate each animal for a prolonged period [39,40].
The resting functional connectivity approach offers a number of
advantages in studying chronic changes in posttraumatic epilepto-
genesis. The relatively slow time scale of neurovascular events
measured by resting BOLD–fMRI connectivity provides a window
into disease mechanisms over longer time scales. In contrast to
EEG, resting BOLD–fMRI allows the detection of signals in the
whole brain simultaneously.
Furthermore, resting state BOLD–fMRI can be used to assess
basal functional connectivity in both superficial and deep brain
networks by calculating temporal correlations of BOLD–fMRI
signals between remote brain areas during the resting state [5,41].
This method has been shown to provide enough sensitivity to
reveal altered functional connectivity in language networks in
temporal lobe epilepsy [10] and also in generalized spike-wave
epilepsy [8,9]. Our BOLD–fMRI findings expand these data by
showing that after TBI there is a dissociation of short-range as well
as long-range brain network connectivity in injured rats with
increased seizure susceptibility. In further work with a larger
sample size it may be possible to investigate relationships between
seizure susceptibility and resting functional connectivity in
individual animals.
Conclusion
We found a decreased connectivity between the ipsilateral and
contralateral parietal cortex and between the parietal cortex and
hippocampus on the side of injury in rats with lateral FPI as
compared to sham-operated animals. We also found abnormal
negative connectivity in rats with TBI between the ipsilateral and
contralateral parietal cortex and other regions. Our study using
resting state BOLD-fMRI functional connectivity provides the first
proof-of-concept evidence that this methodology can be applied
for identification of mechanisms and biomarkers for posttraumatic
epileptogenesis, which can be used to monitor the progression of
epileptogenesis and antiepileptogenic efficacy of treatments.
Acknowledgments
We thank Jarmo Hartikainen, Heli Myo¨ha¨nen, Alexendra Gribizis,
Harrison Bai, Xiaoxian Ma, Dr. Bei Wang, and Dr. Azeet Narayan for
their assistance during experiments, the engineering staff of the Magnetic
Resonance Research Center (?http://mrrc.yale.edu) including Peter
Brown and Scott McIntyre and Quantitative Neuroscience with Magnetic
Resonance (?http://qnmr.yale.edu) for hardware maintenance and radio
frequency probe construction.
Author Contributions
Conceived and designed the experiments: AMM XB BGS SGW OG FH
AP HB. Performed the experiments: AMM XB BGS SGW OS AP.
Analyzed the data: AMM XB SGW OS OG AP. Contributed reagents/
materials/analysis tools: AMM XB BGS FH AP HB. Wrote the paper:
AMM XB AP HB.
References
1. Blennow K, Hardy J, Zetterberg H (2012) The neuropathology and
neurobiology of traumatic brain injury. Neuron 76: 886–899. Available:
http://www.ncbi.nlm.nih.gov/pubmed/23217738.
2. Pitka¨ nen A, Bolkvadze T, Immonen R (2011) Anti-epileptogen esis in rodent
post-traumatic epilepsy models. Neurosci Lett 497: 163–171. Available: http://
www.ncbi.nlm.nih.gov/pubmed/21402123.
3. D’Esposito M, Deouell LY, Gazzaley A (2003) Alterations in the BOLD fMRI
signal with ageing and disease: a challenge for neuroimaging. Nat Rev Neurosci
4: 863–872. Available: http://www.ncbi.nlm.nih.gov/pubmed/14595398.
4. Bassett DS, Bullmore ET (2009) Human brain networks in health and disease.
Curr Opin Neurol 22: 340–347. Available: http://www.pubmedcentral.nih.gov/
articlerender.fcgi?artid= 2902726&tool = pmcentrez&rendertype = abstract.
5. Biswal B, Yetkin FZ, Haughto n VM, Hyde JS (1995) Functional connectivity in
the motor cortex of resting human brain using echo-planar MRI. Magn Reson
Med 34: 537–541. Available: http://www.ncbi.nlm.nih.gov/pubmed/8524021.
6. Biswal BB, Mennes M, Zuo X-N, Gohel S, Kelly C, et al. (2010) Toward
discovery science of human brain function. Proc Natl Acad Sci U S A 107:
4734–4739. Available: http://www.pubmedcentral.nih.gov/articlerender.
fcgi?artid = 2842060&tool = pmcentrez&rendertype = abstract.
Resting Functional Connectivity in Brain Injury
PLOS ONE | www.plosone.org 8 April 2014 | Volume 9 | Issue 4 | e95280

7. Fox MD, Raichle ME (2007) Spontaneous fluctua tions in brain activity observed
with functional magnetic resonance imaging. Nat Rev Neurosci 8: 700–711.
Available: http://www.ncbi.nlm.nih.gov/pubmed/17704812.
8. Bai X, Guo J, Killory B, Vestal M, Berman R, et al. (2011) Resting functional
connectivity between the hemispheres in childhood absence epilepsy.Bai, X., Guo,
J., Killory, B., Vestal, M., Berman, R., Negishi, M., … Blumenfeld, H. (2011).
Resting functional connectivity between the hemispheres in childhood absence.
Neurology 76: 1960–1967. Av ailable: http://www.pubmedcentral. nih.gov/
articlerender.fcgi?artid= 3109878&tool = pmcentrez&rendertype = abstract.
9. Killory BD, Bai X, Negishi M, Vega C, Spann MN, et al. (2011) Impaired
attention and network connectivity in childhood absence epilepsy. Neuroimage
56: 2209–2217. Available: http://www.pubmedcentral.nih.gov/articlerender.
fcgi?artid = 3105167&tool = pmcentrez&rendertype = abstract.
10. Waites AB, Briellmann RS, Saling MM, Abbott DF, Jackson GD (2006)
Functional connectivity networks are disrupted in left temporal lobe epilepsy.
Ann Neurol 59: 335–343. Available: http://www.ncbi.nlm.nih.gov/pubmed/
16404743.
11. Lemieux L, Daunizeau J, Walker M (2011) Concepts of Connectivity and
Human Epileptic Activity. Front Syst Neurosci 5: 1–13. Available: http://www.
ncbi.nlm.nih.gov/pubmed/?term = 21472027
12. Mishra AM, Bai X, Purcaro MJ, Motelow JE, DeSalvo MN, et al. (2009)
Interictal resting functional connectivity in WAG/Rij rats: A possible biomarker
of epilepsy. Epilepsia 50: 362. Available: http://onlinelibrary.wiley.com/doi/10.
1111/j.1528-1167.2009.02377_10.x/abstract.
13. Kharatishvili I, Nissinen JP, McIntosh TK, Pitka¨nen A (2006) A model of
posttraumatic epilepsy induced by lateral fluid-percussion brain injury in rats.
Neuroscience 140: 685–697. Available: http://www.ncbi.nlm.nih.gov/pubmed/
16650603.
14. Pitka¨ nen A, Immonen RJ, Gro¨hn OH J, Kharatishvili I (2009) From traumatic
brain injury to posttraumatic epilepsy: what animal models tell us about the
process and treatment options. Epilepsia 50 Suppl 2: 21–29. Available: http://
www.ncbi.nlm.nih.gov/pubmed/19187291.
15. Hayward NMEA, Tuunanen PI, Immonen R, Ndode-Ekane XE, Pitka¨nen A, et al.
(2011) Magnetic resonance imaging of regional hemodynamic and cerebrovascular
recovery after lateral fluid-percussion brain injury in rats. J Cereb blood flow Metab
Off J Int Soc Cereb Blood Flow Metab 31: 166–177. Available: http://www.
pubmedcentra l.nih.gov/articlere nder.fcgi?artid = 3049 481&tool = pmcentrez&
rendertype = abstract.
16. Immonen R, Kharatishvili I, Gro¨ hn O, Pitkanen A (2013) MRI Biomarkers for
Post-Traumatic Epileptogenesis. J Neurotrauma: 1–23. Available: http://www.
ncbi.nlm.nih.gov/pubmed/23469770.
17. Hayward NMEA, Immonen R, Tuunanen PI, Ndode-Ekane XE, Gro¨hn O, et
al. (2010) Association of chronic vascular changes with functional outcome after
traumatic brain injury in rats. J Neurotrauma 27: 2203–2219. Available: http://
www.ncbi.nlm.nih.gov/pubmed/20839948.
18. McIntosh TK, Vink R, Noble L, Yamakami I, Fernyak S, et al. (1989)
Traumatic brain injury in the rat: characterization of a lateral fluid-percussion
model. Neuroscience 28: 233–244. Available: http://www.ncbi.nlm.nih.gov/
pubmed/2761692.
19. Mishra AM, Ellens DJ, Schridde U, Motelow JE, Purcaro MJ, et al. (2011)
Where fMRI and Electrophysiology Agree to Disagree: Corticothalamic and
Striatal Activity Patterns in the WAG/Rij Rat. J Neurosci 31: 15053–15064.
Available: http://www.ncbi.nlm.nih.gov/pubmed/22016539.
20. Englot DJ, Mishra AM, Mansuripur PK, Herman P, Hyder F, et al. (2008)
Remote effects of focal hippocampal seizures on the rat neocortex. J Neurosci
28: 9066–9081. Available: http://www.pubmedcentral.nih.gov/articlerender.
fcgi?artid = 2590649&tool = pmcentrez&rendertype = abstract.
21. Nersesyan H, Hyder F, Rothman DL, Blumenfeld H (2004) Dynamic fMRI and
EEG Recordings During Spike-Wave Seizures and Generalized Tonic – Clonic
Seizures in WAG/Rij Rats. J Cereb Blood Flow Metab 24: 589–599. Available:
http://www.ncbi.nlm.nih.gov/pubmed/?term = 15181366
22. Ma¨ kinen S, van Groen T, Clarke J, Thornell A, Corbett D, et al. (2008)
Coaccumulation of calcium and beta-amyloid in the thalamus aftertransient middle
cerebral artery occlusion in rats. J Cereb blood flow Metab Off J Int Soc Cereb
Blood Flow Metab 28: 263–268. Available: http://www.pubmedcentral.nih.gov/
articlerender.fcgi?artid= 3049469&tool= pmcentrez&rendertype= abstract.
23. Raz E, Jensen JH, Ge Y, Babb JS, Miles L, et al. (2011) Brain iron quanti fication
in mild traumatic brain injury: a magnetic field correlation study. Ajnr
Am J Neuroradiol 32: 1851–1856. Available: http://www.ncbi.nlm.nih.gov/
pubmed/21885717.
24. Karhunen H, Bezvenyuk Z, Nissinen J, Sivenius J, Jolkkonen J, et al. (2007)
Epileptogenesis after cortical photothrombotic brain lesion in rats. Neuroscience
148: 314–324. Available: http://www.ncbi.nlm.nih.gov/pubmed/17629408.
25. DeSalvo MN, Schridde U, Mishr a AM, Motelow JE, Purcaro MJ, et al. (2010)
Focal BOLD fMRI changes in bicuculline-induced tonic-clonic seizures in the
rat. Neuroimage 50: 902–909. Available: http://www.pubmedcentral.nih.gov/
articlerender.fcgi?artid = 2830359&tool = pmcentrez&rendertype = abstract.
26. Jenkins GM, Watts DG (1968) Spectral analysis and its applications. Holden-
Day Publication.
27. Niskanen J-P, Airaksinen AM, Sierra A, Huttunen JK, Nissinen J, et al. (2013)
Monitoring functional impairment and recovery after traumatic brain injury in
rats by FMRI. J Neurotrauma 30: 546–556. Available: http://www.ncbi.nlm.
nih.gov/pubmed/23259713. Accessed 22 January 2014.
28. Kharatishvili I, Immonen R, Gro O, Pitka A, Gro¨hn O, et al. (2007)
Quantitative diffusion MRI of hippocampus as a surrogate marker for post-
traumatic epileptogenesis. Brain 130: 3155–3168. Available: http://www.ncbi.
nlm.nih.gov/pubmed/?term = 18055492
29. Volman V, Ng LJ (2013) Computer modeling of mild axonal injury: implications
for axonal signal transmission. Neural Comput 25: 2646–2681. Available:
http://www.ncbi.nlm.nih.gov/pubmed/23777525.
30. Coggan JS, Ocker GK, Sejnowski TJ, Prescott SA (2011) Explaining
pathological changes in axonal excitability through dynamical analysis of
conductance-based models. J Neural Eng 8: 065002. Available: http://www.
ncbi.nlm.nih.gov/pubmed/?term = 22058273
31. Cymerblit-sabba A, Schiller Y (2010) Network Dynamics during Development of
Pharmacologically Induced Epileptic Seizures in Rats In Vivo. J Neurosci 30:
1619–1630. Available: http://www.ncbi.nlm.nih.gov/pubmed/?term= 20130172
32. El-Hassar L, Esclapez M, Bernard C (2007) Hyperexcitability of the CA1
hippocampal region during epileptogenesis. Epilepsia 48 Suppl 5: 131–139.
Available: http://www.ncbi.nlm.nih.gov/pubmed/17910593.
33. Kumar S, Rao SL, Chandramouli BA, Pillai S V (2009) Reduction of functional
brain connectivity in mild traumatic brain injury during working memory.
J Neurotrauma 26: 665–675. Available: http://www.ncbi.nlm.nih.gov/
pubmed/?term = 19331523
34. Kumar R, Saksena S, Husain M, Srivastava A, Rathore RKS, et al. (2010) Serial
changes in diffusion tensor imaging metrics of corpus callosum in moderate
traumatic brain injury patients and their correlation with neuropsychometric
tests: a 2-year follow-up study. J Head Trauma Rehabil 25: 31–42. Available:
http://www.ncbi.nlm.nih.gov/pubmed/20051898.
35. Hettinger PC, Li R, Yan J-G, Matloub HS, Cho YR, et al. (2011) Long-term
vascular access ports as a means of sedative administration in a rodent fMRI
survival model. J Neurosci Methods 200: 106–112. Available: http://www.ncbi.
nlm.nih.gov/pubmed/21726581.
36. Zhao F, Zhao T, Zhou L, Wu Q, Hu X (2008) BOLD study of stimulation-
induced neural activity and resting-state connectivity in medetomidine-sedated
rat. Neuroimage 39: 248–260. Available: http://www.ncbi.nlm.nih.gov/
pubmed/?term = 17904868
37. Weber R, Ramos-cabrer P, Wiedermann D, van Camp N, Hoehn M, et al.
(2006) A fully noninvasive and robust experimental protocol for longitudinal
fMRI studies in the rat. Neuroimage 29: 1303–1310. Available: http://www.
ncbi.nlm.nih.gov/pubmed/?term = 16223588
38. Franken ND, Van Oostrom H (2008) Evaluation of analgesic and sedative effects
of continuous infusion of dexmedetomidine by measuring somatosensory- and
auditory-evoked potentials in the rat. Vet Anaesth Analg: 424–431. Available:
http://www.ncbi.nlm.nih.gov/pubmed/?term = 18466160
39. Khubchandani M, Mallick HN, Jaganna than NR, Mohan Kumar V (2003)
Stereotaxic assembly and procedures for simultaneous electrophysiological and
MRI study of conscious rat. Magn Reson Med 49: 962–967. Available: http://
www.ncbi.nlm.nih.gov/pubmed/12704780.
40. Sachdev RNS, Champney GC, Lee H, Price RR, Pickens DR 3rd, et al. (2003)
Experimental model for functional magnetic resonance imaging of somatic sensory
cortex in the unanesthetized rat. Neuroimage19: 742–750. Available: http://www.
ncbi.nlm.nih.gov/entrez/query.fcgi?cmd = Retrieve&db = PubMed&dopt =
Citation&list_uids = 12880803.
41. Lowe MJ, Mock BJ, Sorenson JA (1998) Functional Connectivity in Single and
Multislice Echoplanar Imaging Using Resting-State Fluctuations 2. Neuroimage
132: 119–132. Available: http://www.ncbi.nlm.nih.gov/pubmed/
?term = 9558644
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