Early Microstructural and Metabolic Changes
following Controlled Cortical Impact Injury in Rat:
A Magnetic Resonance Imaging and Spectroscopy Study
Su Xu,1,3Jiachen Zhuo,1,3Jennifer Racz,2Da Shi,1,3Steven Roys,1,3Gary Fiskum,2,3and Rao Gullapalli1,3
Understanding tissue alterations at an early stage following traumatic brain injury (TBI) is critical for injury
management and limiting severe consequences from secondary injury. We investigated the early microstructural
and metabolic profiles using in vivo diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy
(1H MRS) at 2 and 4h following a controlled cortical impact injury in the rat brain using a 7.0 Tesla animal MRI
system and compared profiles to baseline. Significant decrease in mean diffusivity (MD) and increased fractional
anisotropy (FA) was found near the impact site (hippocampus and bilateral thalamus; p<0.05) immediately
following TBI, suggesting cytotoxic edema. Although the DTI parameters largely normalized on the contralateral
side by 4h, a large inter-individual variation was observed with a trend towards recovery of MD and FA in the
ipsilateral hippocampus and a sustained elevation of FA in the ipsilateral thalamus (p<0.05). Significant re-
duction in metabolite to total creatine ratios of N-acetylaspartate (NAA, p=0.0002), glutamate (p=0.0006), myo-
inositol (Ins, p=0.04), phosphocholine and glycerophosphocholine (PCh+GPC, p=0.03), and taurine (Tau,
p=0.009) were observed ipsilateral to the injury as early as 2h, while glutamine concentration increased mar-
ginally (p=0.07). These metabolic alterations remained sustained over 4h after TBI. Significant reductions of Ins
(p=0.024) and Tau (p=0.013) and marginal reduction of NAA (p=0.06) were also observed on the contralateral
side at 4h after TBI. Overall our findings suggest significant microstructural and metabolic alterations as early as
2h following injury. The tendency towards normalization at 4h from the DTI data and no further metabolic
changes at 4h from MRS suggest an optimal temporal window of about 3h for interventions that might limit
secondary damage to the brain. Results indicate that early assessment of TBI patients using DTI and MRS may
provide valuable information on the available treatment window to limit secondary brain damage.
Key words: controlled cortical impact; diffusion tensor imaging; early changes; MR spectroscopy; traumatic brain
et al., 2008). TBI occurs when an external mechanical force or
pressure force (as in the case of blast injury) traumatically
injures the brain. The primary injury is characterized by acute
biochemical and cellular changes that contribute to continu-
ing neuronal damage and lead to permanent or temporary
impairment of physical, cognitive, emotional, and behavioral
functions. The biochemical and cellular changes in neurons
and glia after TBI are complex and dynamic. The patho-
raumatic brain injury (TBI) is a major cause of death
and disability worldwide (Langlois et al., 2006; Maas
physiology typically begins with mechanical trauma to the
brain (the primary injury), followed rapidly by increased
vascular permeability, altered ionic balance, oxidative stress,
excitotoxic damage, inflammation, and mitochondrial dys-
function leading to further cell death and injury (secondary
injury) (Hovda et al., 1991; Ishige et al., 1988; Kawamata et al.,
1995; Lenzlinger et al., 2001; Morganti-Kossmann et al., 2002;
Roberson et al., 2006 Xiong et al., 1997; Yang et al., 1985).
Diffusion tensor imaging (DTI) and proton magnetic reso-
nance spectroscopy (1H MRS) have recently emerged as
powerful approaches for characterizing the microstructural
and metabolic responses after TBI. Studies have shown that
1Department of Diagnostic Radiology and Nuclear Medicine,2Department of Anesthesiology and the Center for Shock Trauma and
Anesthesiology Research (STAR),3Core for Translational Research in Imaging @ Maryland (C-TRIM), University of Maryland School of
Medicine, Baltimore, Maryland.
JOURNAL OF NEUROTRAUMA 28:2091–2102 (October 2011)
ª Mary Ann Liebert, Inc.
diffusion-weighted imaging (DWI) and DTI are highly sen-
sitive to tissue microstructure change and axonal damage
associated with TBI (Arfanakis et al., 2002; Bazarian et al.,
2007; Huisman et al., 2003; Mac Donald et al., 2007a,b; Mayer
et al., 2010; Rutgers et al., 2007). In addition to quantifying the
average diffusion coefficient of water in the brain, DTI mea-
surements can determine the preferential direction of water
diffusion within axons. This information can be used to gen-
erate and visualize both normal and abnormal white matter
fiber tracts. DTI parameters include mean diffusivity (MD),
which measures the average water diffusion within the brain
tissue, and fractional anisotropy (FA), which measures the
degree of diffusion anisotropy present within axons. The
values of FA range from 0 to 1, with highly organized white
matter tracts exhibiting higher anisotropy compared to the
rest of the brain tissue because of the directionally restrictive
structure of bundled axons. Alterations of MD reflect patho-
logical changes in the brain tissue due to changes in the dif-
fusion characteristics of the intra- and extracellular water
compartments, including restricted diffusion and water ex-
change across permeable boundaries (Gass et al., 2002).
Change in the FA is indicative of the structural integrity of the
tissue. More specifically, the FA change is due to the dispro-
portional change in the diffusion along the neuronal axons
(axial diffusivity, ka) and the diffusion perpendicular to the
axons (radial diffusivity, kr). Axial diffusivity is believed to be
sensitive to axonal integrity and radial diffusivity reflects
myelin integrity (Song et al., 2002, 2003). Previous TBI studies
on both humans and animals have shown altered MD (Alsop
2009), and FA in white matter regions (Arfanakis et al., 2002;
Bazarian et al., 2007; Huisman et al., 2004; Mac Donald et al.,
2007a,b; Mayer et al., 2010; Wilde et al., 2006; Wozniak et al.,
2007) anywhere from 24h to several days after injury.
In contrast to the structural information offered by DTI
regarding brain integrity after TBI, high-resolution in vivo
proton magnetic resonance spectroscopy provides comple-
mentary information and assesses metabolic irregularities
following injury. Several of the metabolites detected by1H
MRS are highly sensitive to the pathology that contributes to
TBI, including hypoxia or ischemia, bioenergetic dysfunction,
and inflammation. A majority of clinical TBI studies have
consistently found decreases in the neuronal marker N-acet-
ylaspartate (NAA) in the brains of TBI patients (Govind et al.,
2010; Govindaraju et al., 2004; Macmillan et al., 2002; Marino
et al., 2007; Ross et al., 1998). A decrease in NAA (or NAA to
total creatine ratio [NAA/tCr], NAA to choline ratio [NAA/
Cho]) has been observed within the first 24h of injury (Hol-
shouser et al., 1997, 2000; Ross et al., 1998), and can remain
depressed as long as 8 days after TBI (Brooks et al., 2000;
Condon et al., 1998; Holshouser et al., 2005; Ross and Bluml,
2001). While reports of changes in other cerebral metabolites
have been less consistent (Brooks et al., 2000; Cecil et al., 1998;
Choe et al., 1995; Friedman et al., 1999; Garnett et al., 2000;
Marino et al., 2007; Ross et al., 2001; Shutter et al., 2004; Wild
et al., 1999; Yeo et al., 2006; Yoon et al., 2005), a few studies on
human TBI have shown that the changes in NAA, lactate
(Lac), and choline (Cho) are predictive of neurologic outcome
1–25 days post-injury (Brooks et al., 2000; Friedman, 1998,
1999; Garnett et al., 2000; Ross et al., 1998; Signoretti et al.,
2002). However, none of these clinical studies addressed the
very early changes following TBI.
MRS studies on animal models of TBI have improved our
understanding of the metabolic events underlying the injury
process and have supported the interpretation of clinical MRS
data. As shown in human TBI studies, the metabolic changes
in TBI brain may occur over weeks to months following TBI,
and these changes may persist for several years post-injury in
humans (Brooks et al., 2000; Friedman 1999; Ross et al., 1998).
Previous in vivo1H MRS studies in various rat model injuries
also indicated a time evolution of TBI (Lescot et al., 2010;
Schuhmann et al., 2003; Vagnozzi et al., 2007). Schuhmann
and colleagues (2003) showed that tCr, NAA, glutamate
(Glu), and Cho concentrations significantly decreased during
the first 24h, and then started to increase at 7 days in a con-
trolled cortical impact (CCI) model. At the same time, Lac
increased and reached its peak at 7 days after TBI. In a com-
bined DWI and1H MRS study on a lateral fluid percussion
injury model, Lescot and associates (2010) reported low ADC
values in the brain, which correlated with decreased NAA/
tCr and increased Lac level at 24h after TBI. However, un-
derstanding the microstructural and neurochemical changes
at very early stages following injury may help in elucidating
molecular pathophysiology and determining the time win-
dow available for treatment. Further, previous studies have
paid very little attention to changes in the brain tissue far
removed from injury, for example, in the contralateral hemi-
Experimental models of animal TBI are useful for under-
standing the cerebral microstructure and metabolic mecha-
nisms of brain cell death and neurologic impairment that
occur in the various forms and intensities of human TBI
(Dixon et al., 1988; Finnie and Blumbergs, 2002; Gennarelli,
1994; Lighthall et al., 1989; Morales et al, 2005; Park et al.,
and a measurable, controllable impact velocity and cortical
compression and translates to a repeatable injury with little
variability that can be studied over time (Dixon et al., 1988;
Hall et al. 2005; Lighthall et al., 1989; Meaney et al., 1994). The
features of this model include constant injury reproduction
with cortical contusion and distal axonal injury that exhibit
cognitive, memory, and motor deficits mimicking human TBI
(Chen et al., 1996; Dixon et al., 1991; Tang et al., 1997). We
report here for the first time the acute cerebral microstructural
and1H MRS in vivo using a 7.0 Tesla scanner.
CCI TBI model
Adult male Sprague-Dawley rats (n=8, 250–350g) were
subjected to left parietal controlled cortical impact injury
(Robertson et al., 2006). TBI was performed using the con-
trolled cortical impact device (Pittsburgh Precision Instru-
ments, Pittsburgh, PA) as previously described (Dixon et al.,
1991). Briefly, after being initially anesthetized with 4% iso-
flurane, the rat was maintained at 2% isoflurane, and the left
parietal bone was exposed via a midline incision after posi-
tioning it in a stereotactic frame. A high-speed dental drill
(Henry Schein, Melville, NY) was used to perform a left-sided
craniotomy that was centered 3.5mm posterior and 4mm
lateral to bregma. A 5mm round impactor tip was accelerated
to 5m/sec with a vertical deformation depth of 1.0mm and
impact duration of 50msec consistent with mild injury. The
2092XU ET AL.
bone flap was immediately replaced with dental acrylic and
the scalp incision was closed with silk. After the surgery, the
animal was allowed to recover for about 1.5h, after which it
was transported to MRI for the 2 and 4h imaging session. The
experimental protocol was approved by the Committee for
the Welfare of Laboratory Animals of the University of
In vivo DTI and1H MRS
All experiments were performed on a Bruker Biospec 7.0
Tesla 30cm horizontal bore scanner (Bruker Biospin MRI,
Ettlingen, Germany). The scanner is equipped with a BGA12S
gradient system capable of producing 400mT/m pulse gra-
dients in each of the three orthogonal axes and interfaced to a
Bruker Paravision 5.0 console. A Bruker four-element
in ananimalchamber usingagas mixtureofO2(1L/min) and
isoflurane (3%; IsoFlo, Abbot Laboratories, North Chicago,
IL). The animal was then placed prone in a Bruker animal bed
and the RF coil was positioned and fixed over the brain. The
animal bed was moved to the center of the magnet. At the rest
of the experiment, the animals were under 1–2% isoflurane
anesthesia and 1L/min oxygen administration. A MR com-
patible small-animal monitoring and gating system (SA In-
struments, Stony Brook, New York) was used to monitor the
animal respiration rate and body temperature. The animal
body temperature was maintained at 36–37?C with warm
water circulating through the mouse bed. The total duration
of the MR imaging and spectroscopic experiment was ap-
proximately 2h at each time point. The animal was under
until the completion of the 4h imaging session.
A three-slice (axial, mid-sagittal, and coronal) scout using
fast low-angle shot magnetic resonance imaging (FLASH)
(Frahm et al., 1986; Haase et al., 1986) was obtained to localize
the rat brain. A fast shimming procedure (Fastmap) was used
to improve the B0homogeneity within a region of the object
(Gruetter et al., 1993). Both proton density- and T2-weighted
images were obtained using a 2-D rapid acquisition with re-
laxation enhancement (RARE) sequence (Hennig et al., 1986)
(repetition time/effective echo time (TR/TEeff1/TEeff2)=
5500/18.9/56.8msec, echo train length=4, matrix size=256·
256, slice thickness=1mm, number of averages=2) covering
view [FOV])=3.0·3.0cm2)) and the axial (FOV=3.0·3.2cm2)
plane for anatomic reference. Diffusion tensor images were
acquired with single shot spin-echo echo-planar imaging (EPI)
sequence in the coronal plane. Diffusion sensitive gradients
were applied in 30 non-collinear directions at b=1000s/mm2.
Five additional images at b=0s/mm2were also acquired. The
acquisition parameters were FOV of 3.0·3.0cm2at a matrix
resolution of 128·128, TR/TE of 6000/50msec, slice thickness
of 1mm for a total of 24 slices, and two averages and covered
the same area as the coronal structural acquisitions. Each rat
wasscannedat three timepoints:beforetheinjuryandat 2and
4h after TBI.
shims over the voxel of interest were accomplished with the
Fastmap procedure. At a TE of 20msec, the shimming pro-
1H MRS, adjustments of all first- and second-order
1H metabolite resonance (0.023–0.03ppm). This allowed for a
good separation of the glutamate (2.35ppm) and glutamine
by variable power radiofrequency (RF) pulses with optimized
relaxation delays (VAPOR) (Tka ´c et al., 1999). Outer volume
suppression combined with point-resolved spectroscopy
(PRESS) sequence (Price and Arata, 1996) from a 3·3·3mm3
voxel was used for signal acquisition, with TR/TE=2500/
20msec, spectral bandwidth=4kHz, number of data points=
2048, number of averages=300. The voxel covered the imme-
diate pericontusional zone, all layers of the hippocampus, and
the superior thalamic structures. MRS data were acquired im-
mediately following the DTI acquisition at each time point in
both the pericontusional and the corresponding contralateral
voxels (Fig. 3).
Maps of MD and FA were generated offline using FDT
(FMRIB’s Diffusion Toolbox, Oxford, United Kingdom). Re-
gions of interest (ROIs) were drawn manually on three con-
tiguous slices using ImageJ v1.38x (Wayne Rasband, National
Institutes of Health, Bethesda, MD). Regional measures of
MD, FA, ka(axial diffusivity, ka=k1) and kr(radial diffusivity,
kr=(k2+k3)/2) values were obtained from the corpus callo-
sum (CC) and both the ipsilateral and contralateral side of the
injury from the hippocampus (hip_ips, hip_con), thalamus
(tha_ips, tha_con), cortex (cor_ips, cor_con), the olfactory
(of_ips, of_con), and fimbria of the hippocampus (fi_ips, fi_
con), as illustrated in Figure 1.
1H MRS data was fitted using the LC-Model package
(Provencher, 2001), and only metabolites with standard de-
viations (SD) % £20 were included for further analysis.
Comparisons of the DTI and MRS parameters were per-
injured) and at each time point using one way repeated
analysis of variance (ANOVA) followed by paired t-tests
adjusted for multiple comparisons using Bonferroni correc-
tion. Statistical significance was defined as p<0.05.
DTI and1H MRS at 2 hours after injury
The T2-weighted MR images demonstrated distinct, but
heterogeneous lesion at the location of the injury in the left
cortical region of the brain at 2h following CCI (Fig.1A,B,
yellow lines). Although the parameters for CCI injury in-
duction were the same for all the animals, we observed some
variability in the extent of bleeding and edema formation
between individual animals (Immonen et al., 2009; McIntosh
et al., 1989; Smith et al., 1997). Despite the presence of blood
products at the site of the injury, the quality of the DT images
was not compromised, as exemplified by the FA and MD
images shown in Figure 1D and E, respectively.
Figure 2 shows the average MD, FA, ka, and krvalues from
the 11 ROIs shown in Figure 1. In the ipsilateral side, the most
obvious alterations in the DTI parameters were in the cortical
region where all four DTI parameters were altered signifi-
cantly. In this region, the MD (p=0.004), ka(p=0.018), and kr
(p=0.002) were significantly decreased while FA (p=0.002)
increased. The ipsilateral hippocampus also underwent sig-
nificant changes where the MD (p=0.01), ka(p=0.02), and kr
EARLY DTI AND MRS CHANGES FOLLOWING TBI2093
(p=0.009) were significantly decreased. A significant increase
in FA (p=0.003) for the ipsilateral thalamus was observed
that was mainly driven by a decrease in kr(p=0.067). The
olfactory region, a remote area from the location of the injury
in the cortex, showed a marginal decrease in ka(p=0.062). It
should be noted that the two white matter–dominated re-
gions, the corpus callosum and fimbria of the hippocampus,
which are close to the injury site, showed little alterations in
the DTI parameters at 2h following injury. Brain regions
(hip_con); thalamus ipsilateral (tha_ips) and contralateral (tha_con); cortex ipsilateral (cor_ips) and contralateral (cor_con);
olfactory ipsilateral (of_ips) and contralateral (of_con); fimbria of hippocampus ipsilateral (fi_ips) and contralateral (fi_con);
and corpus callosum (cc). Data are expressed as mean–standard deviation.1p<0.05; *p<0.01. Cross marks indicate a sig-
nificant difference between 2 and 4h.
Regional MD, FA, ka, and krvalues at 2 and 4h after TBI for hippocampus ipsilateral (hip_ips) and contralateral
ROIs (black contours). (A) T2-weighted axial slice. (B) T2-weighted coronal slices showing the placement of the ROIs. (C)
Coronal FA and MD maps. ROI 1 and 2, hippocampus; 3 and 4, thalamus; 5 and 6, cortex; 7 and 8, olfactory; 9 and 10, fimbria
of hippocampus; 11, corpus callosum.
Representative MR images of a rat at 2h after TBI showing the extent of the injury (white contours) and the specific
2094 XU ET AL.
in DTI parameters, including a significant increase in FA in
the thalamus (p=0.029), marginal decrease in kain the ol-
factory region (p=0.055), and significant decrease in krin the
Coronal anatomic images along with the spectroscopic
voxel locations in the pericontusional and contralateral re-
gion, along with the corresponding spectra from an animal,
are shown in Figure 3. The in vivo1H spectra demonstrate
excellent spectral resolution and sensitivity both at the peri-
contusional zone and the contralateral sides. At 2h after in-
jury, the metabolites in the pericontusional zone, including
Glu/tCr (p=0.0006), PCh+GPC/tCr (p=0.03), Ins/tCr
(p=0.04), NAA/tCr (p=0.0002), and Tau/tCr (0.009) were
significantly reduced compared to the baseline, while Gln/
tCr was marginally increased (p=0.07) (Fig. 4). The contra-
lateral zone also exhibited several biochemical changes after
TBI but the changes were milder compared to the pericontu-
sional region (Fig. 5). There was a significant reduction in
NAA/tCr (a 6.1% reduction in contralateral zone versus a
29.4% reduction in the pericontusional zone).
DTI and1H MRS at 4 hours after injury
At 4h after injury, the DTI parameters near the pericontu-
sional cortical region maintained the same levels as those at
2h after TBI (Fig. 2). While a marginal recovery of MD
(p=0.054) and kr(p=0.07) was seen in the ipsilateral hippo-
campus in comparison to the 2h time point, overall these
parameters were still reduced compared to the baseline. A
further increase in the FA (p=0.04) and a reduction in kr
(p=0.02) were observed in the ipsilateral thalamus compared
to the baseline. Axial diffusivity, ka, which was decreased at
the 2h point normalized to baseline levels in the olfactory
region. The DTI parameters for corpus callosum did not un-
dergo any further change compared to the 2h point. How-
ever, the fimbria of the hippocampus showed significant
decrease in both the MD (p=0.02) and kr(p=0.01) compared
to the baseline. On the contralateral side, the DTI parameters
demonstrated a recovery to the baseline level by 4h.
Most of the metabolites including Glu/tCr (p=0.008),
PCh+GPC/tCr (p=0.009), Ins/tCr (p=0.01), NAA/tCr
(p=0.0002), and Tau/tCr (p=0.0039) continued to be de-
pressed at 4h following injury compared to the baseline (Fig.
4). In addition, a significant reduction of GABA/tCr (p=0.02)
was observed by this time compared to the baseline (Fig. 4).
The Gln/tCr ratio exhibited more variability compared to the
levels observed at 2h (Fig. 4). Despite these few changes
compared to baseline, no significant changes were noted in
the ratios of GABA/tCr, Gln/tCr, Glu/tCr, Ins/tCr, NAA/
tCr, Tau/tCr, and PCh+GPC/tCr between 2 and 4h follow-
ing TBI, which indicates that most significant metabolic
alterations occur within 2h after the injury in the pericontu-
sional zone. In the contralateral side, significant alterations
of Ins/tCr (p=0.024) and Tau/tCr (p=0.013) were observed
in the hippocampus. Although, the changes in these metab-
olites were not as sizeable as in the pericontusional zone, the
reductions of Ins/tCr (a 14.8% reduction in contralateral zone
versus a 16.6% reduction in the pericontusional zone) and
Tau/tCr (a 15.2% reduction in contralateral zone vs. a 12.6%
reduction in the pericontusional zone) were statistically sig-
nificant at 4h.
Both experimental and human studies have shown the
existence of a temporal window of metabolic vulnerability of
TBI (Tavazzi et al., 2007; Vagnozi et al., 2007). However, these
experiments were based on repeat concussions on animals,
concluding that repeat injuries within 3 days would lead to
profound changes in mitochondrial-related mechanism in
4h after injury on both the pericontusional and contralateral sides. GABA, c-aminobutyric acid; Glu, glutamate; Gln, glu-
tamine; GPC, glycerophosphorylcholine; Lac, lactate; Ins, myo-inositol; NAA, N-acetylaspartate; PCh, phosphorylcholine;
Tau, taurine; +Cr, total creatine. M1, M2, M3 and M4 are macromolecules.
Localized in vivo1H spectra and corresponding voxel location depicted on the anatomic image of a TBI rat at 2 and
EARLY DTI AND MRS CHANGES FOLLOWING TBI 2095
animal models, which could be easily detected based on re-
duction in NAA. While these are very important studies to
understand the effects of repeat TBIs, we hypothesized that a
knowledge of the very early changes in the metabolic profiles
and microstructural changes following a single impact injury
will have a profound impact on effective management of the
injury in the acute stage and lead to the development of
neuroprotective agents to reverse or contain the damage from
the injury. Because of the sensitivity of DTI and1H MRS to
microstructural and metabolic changes in vivo, we choose to
examine the early changes in the parameters derived from
these techniques using a reproducible model of TBI.
We observed decreasedMD,ka,andkrand anincreased FA
as early as 2h in a variety of regions immediately following
TBI, consistent with cytotoxic edema and inflammatory re-
sponse to the injury. These results agree with the findings
from the human TBI studies performed during the acute stage
both at the whole brain and regional level (Bazarian et al.,
injury in CCI TBI rat brains. Data are expressed as mean–standard deviation.1p<0.05; *p<0.01;#p<0.001.
Comparison of the neurometabolic levels in the pericontusional zone before injury (baseline) and at 2 and 4h after
injury in CCI TBI rat brain. Data are expressed as mean–standard deviation.1p<0.05; *p<0.01;#p<0.001.
Comparison of the neurometabolic levels in the contralateral zone before injury (baseline) and at 2 and 4h after
2096 XU ET AL.
2007; Buki and Povlishock, 2006; Chu et al., 2010; Mayer et al.,
2010; Wilde et al., 2008). As expected, ipsilateral cortex was
most affected due to direct impact, followed by ipsilateral
hippocampus, olfactory, thalamus, and fimbria of the hippo-
campus. Consistent with the findings from Mac Donald and
colleagues (2007a,b), we found that the DTI parameters of the
contralateral hippocampus was also altered but to a lesser
degree compared to the ipsilateral hippocampus. We also
report here for the first time the involvement of the contra-
lateral olfactory cortex and thalamus, suggesting that the in-
jury may lead to disruption in olfaction and executive
function (Halbauer et al., 2009; Onyszchuk et al., 2009; Si-
gurdardottir et al., 2010). In these regions, a decrease in axial
diffusivity was accompanied by a stronger decrease in radial
diffusivity that contributed to a decrease in MD and an in-
crease inFA.Mayerandcolleagues (2010)found increased FA
in a prospective study of mild TBI patients in the semi-acute
stage and argued that the mechanical forces from TBI result in
the stretching ofaxons and related structures, which alters the
function of the gated ion channels, resulting in changes in
water homeostasis. Other factors such as high viscosity from
cell debris, elevated lipid content within area of necrosis, and
decreased water content within myelin sheaths can also limit
increased FA and decreases in MD, ka, and krsuggests re-
duced extracellular space and highly restricted water diffu-
sion consistent with cytotoxic edema.
1H MRS, which covered all layers of the hippocampus and
the superior thalamic structures, revealed reductions of Tau/
osmolytes that are regulated in the brain and are believed to
be located primarily in glia and absent in the neurons (Brand
et al., 1993; Dutton et al., 1991; Lang et al., 1998; Verbalis
and Gullans, 1991). Histopathological evidence of astrocyte
damage in cortex and hippocampus has been reported in rats
as early as 30min following TBI (Zhao et al., 2003). Current
of organic compounds following swelling, including amino
acids (Kimelberg and Mongin, 1998; Law, 1996; Verbalis and
Gullans, 1991) and amino acid derivatives, such as Tau and
NAA (Olson and Kimelberg, 1995; Phillis et al., 1998; SaAˆn-
chez-Olea et al., 1993, 1996; Sager et al., 1997; Taylor et al.,
1995). Clinical studies have reported decreased levels of var-
ious metabolites, most notably Ins, in a number of patients on
whom plasma osmolarity was lowered (Cooper and Wyatt,
2000; HaE`ussinger et al., 1994; Videen et al., 1995). The re-
ductions of Tau and Ins found in this study may reflect local
gain of water, resulting in hypo-osmolality in the vicinity of
the impact at the very early stage after injury (Arieff, 1987;
Videen et al., 1995). In such situations it has been shown that
Ins and Tau are reduced in brain tissue (Bothwell et al., 2001;
Silver et al., 2006). It should be noted that in addition to Tau
and Ins, NAA, Glu, and GABA are also osmotically active
molecules (Bothwell et al., 2001). To date most clinical and
experimental animal studies have largely shown an increase
in Ins following injury. However the increase in Ins was ob-
served in the sub-acute to chronic stages following TBI and
these changes have been attributed to osmolality change due
to increased astrocytic activity (Schuhmann et al., 2003; Zhao
et al., 2003). Schuhmann and colleagues (2003) observed a
that eventually rose above the baseline by approximately 31%
and 44%, respectively, by 7 days following CCI. Although the
earlydecreases inInsandTauwerenot explained,theauthors
interpreted the late increase in Ins to be due to increased glial
content/glial proliferation. Overall the changes in these me-
tabolites most likely reflect the local tissue osmolality at the
very early stages following TBI.
The considerably dynamic decrease in the NAA/tCr dur-
ing the first 4h after TBI in this study agrees with the obser-
(Schuhmann et al., 2003). We found that the most severe drop
of NAA occurred at 2–4h after the injury in the pericontu-
sional zone. In addition, a much smaller but statistically sig-
nificant decrease of NAA was observed in the contralateral
side, suggesting a global NAA disturbance. Although sharp
decrease of NAA immediately after TBI has not yet been fully
understood, it may be due to impaired NAA synthesis in the
mitochondria (Bates et al., 1996; De Stefano et al., 1995; Lescot
et al., 2010; Schuhmann et al., 2003; Signoretti et al., 2008). The
nervous system-specific metabolite NAA is synthesized from
aspartate and acetyl-coenzyme A, relies on ATP, through the
action of L-aspartate N-acetyltransferase in mitochondria or
through the cleaving of N-acetyl-aspartyl-glutamate by N-
acetylated-a-linked-amino dipeptidase, along with glutamate
(Baslow, 2003). Therefore, the synthesis and catabolism of
NAA are related to mitochondrial integrity. Our previous
mitochondrial respiration study (Robertson et al., 2006)
showed mitochondrial dysfunction as early as 1h after CCI
TBI in the pericontusional zone. Several investigations have
furnished strong evidence to support the view that initial
NAA reduction reflects dysfunctional neurons suffering en-
ergetic impairment after TBI (Schuhmann et al., 2003; Sign-
oretti et al., 2001).
The decrease of Glu/tCr in close proximity to the injury at
the early stage of CCI agreed with a similar study by Schu-
mann and associates (2003). In addition, we found a local
reduction of GABA and an accumulation of Gln. At a TE of
20msec and at a field strength of 7 Tesla, the resonance of Glu
(2.35ppm) and Gln (2.45ppm) were well resolved, yielding
reliable quantification of the metabolites with the coefficient
of variation for these metabolites as low as 4–12% for Glu and
8–16% for Gln as reported from LC-Model’s processing of
spectra. The injury-induced alterations in concentrations of
the excitatory neurotransmitter Glu and the inhibitory neu-
rotransmitter GABA may indicate an imbalance in excitatory
and inhibitory activity in the hippocampal region at the very
early stage of TBI, and therefore may further contribute to the
neurological dysfunction caused by TBI. At present, it is
thought that the neurotransmission process is completed
Glu from presynaptic terminals to transport primarily to as-
trocytes, where it is converted to Gln via the Gln synthetase
pathway. The Gln is released back to the neurons, where Glu
is regenerated via phosphate-dependent glutaminase, a mi-
tochondrial enzyme. GABA is synthesized by decarboxyl-
ation of Glu by glutamic acid decarboxylase. It is possible that
the mitochondrial dysfunction caused by the very early stage
of Glu, with a compensatory reduction of GABA and an ac-
cumulation of Gln. Further studies with electrophysiological
correlation may provide more insight into the disruption of
the Glu-Gln cycle.
EARLY DTI AND MRS CHANGES FOLLOWING TBI 2097
The significant decrease of PCh+GPC/tCr at 4h after TBI
also agrees with other reports (Schuhmann et al., 2003; Viant
et al., 2005). As a metabolic marker of myelin and cellular
membrane density and integrity (i.e., phospholipid synthesis
and degradation), the decrease of PCh+GPC in the initial
stage of trauma is possibly a result of membrane degradation
in the hippocampus area. Histopathological studies have
shown evidence of astrocyte damage in hippocampus in rats
as early as 30min following TBI (Zhao et al., 2003).
The current study did not find a significant increase in the
levels of lactate in the mild CCI TBI model. Although, most of
out of the eight rats exhibited a dramatic increase of Lac,
coupled with a more severe drop in NAA and Glu although
the conventional MRI showed similar levels of injury as that
of other rats. While the discrepancy in this one rat is unclear,
the presence of Lac has been related to multiple factors, in-
cluding the increased energy demand to restore the ionic
balance (Kawamata et al., 1995) and the disordered mito-
chondrial dysfunction (Ishige et al., 1988; Unterberg et al.,
1988; Yang et al., 1985) at the initial stage of the injury. Future
studies with histological verification will need to be per-
formed in order to understand the subtle changes that may be
responsible for the ischemic conditions far from the injury, as
evidenced from increased lactate from the hippocampus and
the thalamic region.
In the current study, the1H MRS data was normalized to
the resonance intensity of tCr, as this is present relatively
equally in all brain cells and tends to be stable (Arnold and
Matthews, 1996). However, Schumann and colleagues (2003)
observed a decrease in tCr concentration during the first 24h
under similar experimental conditions as those used in this
study. While absolute quantification of tCr was not possible
with our data, a reduction in the concentration of tCr would
imply that the observed reductions in concentration of vari-
ous metabolites (except for Gln) are an underestimate and
hence the changes are more significant, which does not alter
the overall conclusion from this study. Although it is possible
of increased creatine, to our knowledge there are no studies
that have reported an increase in the concentration of Cr fol-
lowing TBI. Nevertheless, this underscores the importance of
monitoring the absolute concentrations of the metabolites for
an unbiased estimate. Taken together, these findings of this
study indicate that the knowledge of alterations in cerebral
metabolites and microstructural changes as early as 2h post-
injury by MRS and DTI, respectively, have the potential to
have an impact on the management of TBI patients.
Despite dramaticimprovements inthemanagement ofTBI,
to date there is no effective treatment available to patients,
and morbidity and mortality remain high (Hall et al., 2010;
Meyer et al., 2010; Stein et al., 2011). At present, there are no
of TBI patients (Hall et al., 2010). The majority of post-trau-
matic neurodegeneration is due to secondary pathochemical
and pathophysiological cascades that occur during the first
few minutes, hours, or days following the injury, which ex-
acerbate the damaging effects of the primary injury. The re-
cently reported clinical trials on acute TBI patients used a 4h
or longer window of treatment (Hall et al., 2010; Stein et al.,
2011), which may not be optimum given that we see changes
as early as 2h in the rat model. Although both DTI and MRS
have been studied at the sub-acute and chronic stages among
the TBI population, the combined use of these techniques
during the early and sub-acute stages has been limited. Early
techniques may provide insights into the progression of the
pathophysiology from the injury that may provide insights
into the optimum time window for treatment and also pro-
vide the much-needed predictive value in determining out-
comes. Given that the combined use of MRS and DTI is
sensitive in detecting damage in areas that conventional MRI
early evaluation of patients whose CT and conventional MRI
are occult when the clinical status of the patient dictates oth-
erwise. Of particular note is that the DTI and MRS data can
complement each other as they assess different aspects of
brain parenchyma and appear to be more sensitive compared
to the conventional MR imaging techniques used to assess
trauma and therefore can be very helpful in the assessment of
novel therapeutic strategies (Signoretti et al., 2008; Tollard
et al., 2009).
This study for the first time demonstrates that the combi-
nation of information from
changes in metabolic and microstructural changes in vivo as
early as 2h following CCI in rat brain. The microstructural
and neurochemical changes were observed within 2h fol-
lowing injury in the cortex, hippocampus, and thalamus. In
addition, changes in the microstructural environment and
neurochemistry extended beyond the site of injury to the
contralateral hippocampus and thalamus. The tendency to-
wards normalization of tissue changes as indicated by DTI
and no further metabolic changes at 4h as determined by
MRS indicates the existence of a temporal window of about 2
to 3h for planning interventions that might limit secondary
damage to the brain.
1H MRS and DTI can detect
This study was supported in part by grants from the Na-
tional Institutes of Health (1S10RR019935).
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Rao Gullapalli, Ph.D.
Core for Translational Research in Imaging
@ Maryland (C-TRIM)
Department of Diagnostic Radiology and Nuclear Medicine
University of Maryland School of Medicine
22 South Greene Street
Baltimore, MD 21201
2102 XU ET AL.