The Journal of Neuroscience, January 15, 2002, 22(2):446–454
Repetitive Mild Brain Trauma Accelerates A Deposition, Lipid
Peroxidation, and Cognitive Impairment in a Transgenic Mouse
Model of Alzheimer Amyloidosis
Kunihiro Uryu,1* Helmut Laurer,2* Tracy McIntosh,2 Domenico Praticò,3 Daniel Martinez,1 Susan Leight,1
Virginia M.-Y. Lee,1 and John Q. Trojanowski1
Departments of 1Pathology and Laboratory Medicine, 2Neurosurgery, and 3Pharmacology, Center for Neurodegenerative
Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283
Traumatic brain injury (TBI) increases susceptibility to Alzheimer’s disease (AD), but it is not known how TBI contributes to
the onset or progression of this common late life dementia. To
address this question, we studied neuropathological and behavioral consequences of single versus repetitive mild TBI
(mTBI) in transgenic (Tg) mice (Tg2576) that express mutant
human A precursor protein, and we demonstrate elevated
brain A levels and increased A deposition. Nine-month-old
Tg2576 and wild-type mice were subjected to single (n ⫽ 15) or
repetitive (n ⫽ 39) mTBI or sham treatment (n ⫽ 37). At 2 d and
9 and 16 weeks after treatment, we assessed brain A deposits
and levels in addition to brain and urine isoprostanes generated
by lipid peroxidation in these mice. A subset of mice also was
studied behaviorally at 16 weeks after injury. Repetitive but not
single mTBI increased A deposition as well as levels of A and
isoprostanes only in Tg mice, and repetitive mTBI alone induced cognitive impairments but no motor deficits in these
mice. This is the first experimental evidence linking TBI to
mechanisms of AD by showing that repetitive TBI accelerates
brain A accumulation and oxidative stress, which we suggest
could work synergistically to promote the onset or drive the
progression of AD. Additional insights into the role of TBI in
mechanisms of AD pathobiology could lead to strategies for
reducing the risk of AD associated with previous episodes of
brain trauma and for preventing progressive brain amyloidosis
in AD patients.
Senile plaques (SPs) and neurofibrillary tangles are the neuropathological hallmarks of Alzheimer’s disease (AD). SPs are
formed by fibrillar A peptides derived from amyloid precursor
proteins (APPs) through sequential proteolytic cleavage of APP
by - and ␥-secretases generating diverse species of A with
differential propensities to fibrillize and deposit in SPs (Saido,
1998; Mills and Reiner, 1999; Selkoe, 1999; Nunan and Small,
2000; Bayer et al., 2001). The 42– 43 amino acid long A variants
known as A(x-42/43) are thought to be the most amyloidogenic
and critical in the onset and progression of AD amyloidosis,
although the exact mechanisms underlying AD pathogenesis are
incompletely understood. However, multiple mutations in the
APP and presinilin-1 and -2 genes have been shown to be pathogenic for familial AD (FAD; Price and Sisodia, 1998; Selkoe,
1999; Bayer et al., 2001). These mutations are thought to cause
FAD by increasing production of A(x-42/43) followed by accelerated AD amyloidosis (Jarrett and Lansbury, 1993; Felsenstein
et al., 1994; Lannfelt et al., 1994; Hardy, 1997; Bayer et al., 2001).
Despite remarkable progress in understanding the genetics of
FAD, less is known about genetic and epigenetic risk factors for
sporadic AD (SAD) although ⬃90% of AD cases are sporadic,
and the incidence of SAD will continue to increase as life expectancy expands in coming decades (Martin, 1999). The apolipoprotein E ⑀4 allele is the most well documented genetic risk factor for
SAD (Wisniewski et al., 1994), and traumatic brain injury (TBI)
is the most robust environmental AD risk factor (Heyman et al.,
1984; Mortimer et al., 1985; Guo et al., 2000; Plassman et al.,
2000). Although recurrent TBI is thought to cause dementia
pugilistica in career boxers, a mechanistic link between TBI and
the induction or acceleration of AD has not yet been elucidated
(Murai et al., 1998). This may reflect limitations in the available
animal models used in previous studies (Nakagawa et al., 1999,
2000) of TBI.
In this article, we have used a recently developed mild TBI
(mTBI) mouse model that does not require craniotomy and
produces minimal structural brain damage (Laurer et al., 2001),
and we use this model to determine whether single versus repetitive mTBI augments disease in a transgenic (Tg) mouse model
(Tg2576) of AD-like amyloidosis (Hsiao et al., 1996). Accordingly, we conducted studies to examine the effects of single or
repetitive mTBI on cognition and motor behavior as well as on
the onset and progression of amyloidosis in Tg and wild-type
(WT) mice at 2 d and 9 and 16 weeks after mTBI. We also
monitored levels of isoprostanes, markers of lipid peroxidation
(L PO), because of evidence linking oxidative damage and AD
pathobiology (Praticò et al., 2000a, 2001). Significantly, these
studies provide the first experimental evidence implicating
Received Aug. 1, 2001; revised Oct. 3, 2001; accepted Nov. 2, 2001.
This work was supported by grants from the National Institutes of Health (National Institute on Aging; Head Injury Center Grant P50-NS08803). We thank
Takeda Chemical Industries, Ltd., for providing antibodies, Dr. K. Hsiao for Tg2576
mice, Sanjay Kasturi for technical assistance, and colleagues in the Departments of
Pathology and Laboratory Medicine, Center for Neurodegenerative Disease Research, and the Head Trauma Center for assistance and advice regarding the studies
described here.
*K.U. and H.L. contributed equally to this work.
Correspondence should be addressed to Dr. John Q. Trojanowski, Center for Neurodegenerative Disease Research, Hospital of University of Pennsylvania/Maloney,
Third Floor, Philadelphia, PA 19104-4283. E-mail: trojanow@mail.med.upenn.edu.
Copyright © 2002 Society for Neuroscience 0270-6474/02/220446-09$15.00/0
Key words: Alzheimer’s disease; amyloid plaques; brain
injury; head trauma; APP mice; oxidative stress; cognitive
function
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
TBI in mechanisms of AD by augmenting brain A accumulation and L PO.
MATERIALS AND METHODS
Animals and surgical procedure
Tg APP695swe (Tg2576) mice and W T littermates were used in this
study. At 9 months of age, both Tg and W T mice were subjected to mTBI
as described previously (Laurer et al., 2001). Briefly, mice were anesthetized with sodium pentobarbital (65 mg / kg of body weight, i.p.); ointment was applied to their eyes to protect vision during surgery; and the
mice were placed on a heating pad to maintain body temperature
throughout the surgical procedures. All animals were mounted in a
stereotactic frame; a skin incision was performed to expose the skull; and
the mice remained in the stereotactic apparatus while subjected to mTBI
using a pressure-driven instrument that is mechanically identical to a
previously described controlled cortical impact device (Dixon et al.,
1991; Smith et al., 1995) with minor modifications that have also been
described previously (Laurer et al., 2001). The impounder was rigidly
mounted at an angle of 20° from vertical, and because the depth and
duration of the impact were kept constant, head movements were minimal while delivering the load. The procedure was completed with the
closure of the incision using 4-0 silk sutures. The animals were removed
from the stereotactic frame and placed in a heated cage, and after
recovery from anesthesia (as evidenced by ambulation), they were returned to their home cages.
At 24 hr after the first mTBI, selected animals were reanesthetized as
described above, and these mice were then subjected to a second mTBI
in the same location over the left parietotemporal region. Sham-treated
animals also were anesthetized and placed in the stereotactic frame; the
skull was exposed, and the skin incision was sutured closed without brain
injury on 2 consecutive days, thereby following exactly the surgical
procedures of repetitive mTBI. All of these procedures were performed
in strict accordance with the National Institutes of Health Guide for the
Care and Use of Laborator y Animals, and they were approved by the
Institutional Animal C are and Use Committee of the University of
Pennsylvania.
Neurobehavioral analysis
Assessment of cognitive f unction. The testing paradigm for evaluation of
cognitive f unction using the Morris water maze (MW M) has been
described in detail previously (Fox et al., 1998; Pierce et al., 1998;
Bareyre et al., 2000). Briefly, the MW M is a circular pool 1 m in
diameter, painted white inside (Morris et al., 1982). The water (16 –18°C)
is made opaque by adding nontoxic, water-soluble white coloring. To test
for mTBI-induced learning impairments, animals received no training in
the MW M before injury and were trained to locate a stationary, submerged platform (0.5 cm below the surface) using external cues starting
at 16 weeks after injury. The essential feature of the MW M is that mice
can escape from the water onto the platform after being placed randomly
at one of four sites in the pool. Latencies of four trials/d were recorded
and averaged to obtain a measurement for the performance of each
animal on a given day. Animals were tested for their ability to learn the
visuospatial task in the MW M over an 8-d period that began 16 weeks
after mTBI. Notably, previous studies to adapt the MW M to mice (see
below) indicate that TBI does not cause changes in swim speed or visual
acuity that influence latencies in the mouse version of the MW M, and
this also was documented in the MW M studies here.
Motor f unction. The composite neuroscore (NS), which includes a
battery of motor tests, was obtained for mice before performing MW M
evaluations as described previously for TBI in rats (McIntosh et al., 1987,
1989) after modifications for use in mice (Murai et al., 1998; Raghupathi
et al., 1998; Nakamura et al., 1999; Scherbel et al., 1999). The NS
measures the following tasks: (1) forelimb flexion response during suspension by the tail, (2) resistance to lateral pulsion, and (3) response of
the hindlimb and toes (hindlimb flexion) when raised by the tail. Each
animal was scored by an investigator blinded to the injury status of the
animal using a scaling system ranging from 4 (preinjury control status) to
0 (af unctional).
Histological and immunohistochemical analysis
Tg2576 and W T littermate male and female mice used in this study
received sham injury or single or repetitive mTBI, and the mice were
killed 2 d or 9 or 16 weeks thereafter. Each experimental group consisted
of five or six mice, except for the single mTBI 9 and 16 week post-mTBI
J. Neurosci., January 15, 2002, 22(2):446–454 447
groups (n ⫽ 0 and 3, respectively) and the sham and repetitive mTBI
groups at 2 d after treatment (n ⫽ 3 for both of these cohorts). After the
study of living mice was concluded, they were lethally anesthetized and
perf used intracardially with PBS (0.1 M), pH 7.4, followed by phosphatebuffered 4% paraformaldehyde. Brains and spinal cords were removed,
post-fixed overnight, sliced into 2-mm-thick coronal slabs, and embedded
in paraffin in a frontal to occipital series of blocks, and the blocks were
cut in a near serial array of 6-m-thick coronal sections for analysis.
The histology and location of the mTBI site were examined by hematoxylin and eosin (H&E) as well as by Gomori’s iron staining, and the
distribution as well as the burden of A deposits were demonstrated by
immunostaining with the 4G8 anti-A 17–24 monoclonal antibody. Before quantitative analysis, initial comparisons of several other well characterized antibodies, i.e., rabbit polyclonal antibodies 2332 and 2333
(both of which recognize multiple species of A) and mouse monoclonal
antibodies 6E10 (anti-A 1–17), BA27 [anti-A(x-40)], and BA05 [antiA(x-42)] were undertaken, and 4G8 was chosen for determining the A
burden because of its robust signal and optimal results for quantitative
analysis. In addition, reactive astrocytes were visualized by an antibody
to glial fibrillary acidic protein (GFAP; Dako, C arpinteria, CA). Finally,
characteristic features of SPs also were identified in Tg mouse brains with
and without mTBI by immunohistochemistry using anti-ubiquitin antibodies (Chemicon, Temecula, CA) and thioflavin-S staining.
Paraffin sections were subjected to immunohistochemistry as described
previously (Murai et al., 1998; Nakagawa et al., 1999, 2000). Briefly, sections
were deparaffinized in xylene, hydrated in a series of ethanol and deionized
water, and subjected to an antigen retrieval step by immersing sections in
88% formic acid for 60 min before immunohistochemistry for A. Sections
were washed in water, and endogenous peroxidases were quenched using a
freshly prepared mixture of methanol (150 ml) plus hydrogen peroxide
(33%, 30 ml). The immunohistochemistry procedures have been described
previously (Nakagawa et al., 1999), and the avidin–biotin complex method
was used according to the instructions of the vendor (Vector Laboratories,
Burlingame, CA). Negative controls included the application of the same
immunohistochemistry protocol to sections, except preimmune serum was
applied instead of primary antibody.
Image analysis
Brain sections immunostained with 4G8 from Tg2576 and W T littermate
mice that survived for 2 d and 9 and 16 weeks after the last treatment
(including sham and single and repetitive mTBI) were used for quantitative analysis, but the sections from the W T mice showed no amyloid
deposits as reported previously (Hsiao et al., 1996).
Coronal brain sections from levels between the habenula nucleus and
the posterior commissure of Tg mice subjected to sham or single or
repetitive mTBI at longer postoperative survival intervals were subjected
to quantitative analysis. For injured animals, the sections selected for
analysis were subjacent to the mTBI site, as demonstrated by the presence of small iron deposits (resulting from minor blood vessel damage
and release of red blood cells) revealed by Gomori’s iron stain, and
equivalent sections from sham-treated Tg mice were subjected to quantitative analysis. Eight sections from each animal, with 4G8 immunostaining but without a counterstain, were used for quantitative image
analysis as described previously (Nakagawa et al., 1999, 2000).
Light microscopic images from the somatosensory cortex (SSC), perihippocampal cortex (PHC), and hippocampus (HP) from both ipsilateral
(left) and contralateral (right) hemispheres to the mTBI site were captured from eight series of sections using a Nikon Microphot-F X A microscope with a 4⫻ objective lens. Using a personal computer, each
image was opened with image analysis software (Image Pro-plus; Media
C ybernetics, Inc., Silver Spring, MD). Manual editing was then performed to eliminate nonspecific signals (e.g., blood vessels and staining
artifacts). The areas occupied by A-immunoreactive products in the
regions of interest were measured, and the total area occupied by the
outlined structures was measured to calculate (1) the total area with
selected immunoreactive products and (2) the percentage of the area
occupied by immunoreactive products over the outlined anatomical area
in the image.
Sandwich A ELISA
For quantitation of A brain levels, Tg2576 and W T littermate male and
female mice at 16 weeks postinjury survival times (n ⫽ 4 – 6) were
perf used transcardially with PBS containing 0.01% of the antioxidant
butylated hydroxytoluene (BHT). The cohort size for these studies was
determined on the basis of previous studies of APP Tg mice using this
448 J. Neurosci., January 15, 2002, 22(2):446–454
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
ELISA (Murai et al., 1998; Nakagawa et al., 1999, 2000). The brain was
removed, and each right and left cerebral cortex, hippocampus, and
cerebellum were collected in individual test tubes, weighed, and frozen
immediately with dry ice. Sequential extraction of samples was performed with high-salt buffer and formic acid to measure soluble and
insoluble brain A(x-40) and A(x-42/43). The right and left cerebral
cortices, hippocampus, and cerebellum were serially extracted in highsalt Re-assembly buffer (0.1 M Tris, 1 mM EGTA, 0.5 mM MgSO4, 0.75 M
NaC l, and 0.02 M NaF, pH 7.0) containing a protease inhibitor mixture
(pepstatin A, leupeptin, N-tosyl-L-phenylalanine chloromethyl ketone,
N␣-p-tosyl-L-lysine chloromethyl ketone, and soybean trypsin inhibitor,
each at 1 g /ml in 5 mM EDTA). Tissue was chopped into small pieces
and then sonicated with 10 burst pulses (level 3) of a Fisher Scientific
(Pittsburgh, PA) F60 Sonic Dismembrator. Homogenates were centrif uged at 100,000 ⫻ g for 1 hr at 4°C. Supernatants were removed, and
pellets were resuspended in 70% formic acid and resonicated and centrif uged at 100,000 ⫻ g for 1 hr at 4°C. Supernatants were removed and
diluted 1:20 with 1 M Tris base. The supernatant obtained in each
extraction step was normalized to the original wet weight of the tissue
sample and analyzed separately by ELISA. To do this, samples were
diluted in buffer EC [0.02 M sodium phosphate, 0.2 M EDTA, 0.4 M NaC l,
0.2% BSA, 0.05% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 0.4% Block-ace (Dainippon; Suita, Osaka, Japan), and
0.05% sodium azide, pH 7.0] and analyzed using Ban50 monoclonal
antibody (mAb) to capture and BA-27 and BC -05 mAbs as reporters to
detect A1– 40 and A1– 42/43, respectively, as described previously
(Iwatsubo et al., 1994; Suzuki et al., 1994; Wang et al., 1999; Nakagawa
et al., 2000).
Isoprostane analysis
Urine was collected from Tg and W T mice into plastic tubes containing
BHT after various periods of survival (9, 12, and 16 weeks after injury;
n ⫽ 4 – 6) from each injury group. Samples were spiked with a fixed
amount of internal standard (d4-8,12-iso-iPF2␣-V I) extracted on a C18
cartridge column. The eluted fraction was purified by thin-layer chromatography and finally assayed by negative ion chemical ionization gas
chromatography and mass spectrometry, as described previously (Praticò
et al., 1998, 2001; Praticò, 1999). Urine aliquots (0.1 ml) were used for
measurement of creatinine levels by a commercially available standardized automated colorimetric assay (Sigma, St. L ouis, MO). Levels were
expressed as nanograms per milligram of creatinine. Finally, aliquots of
brain tissue samples obtained for the A ELISA were used for isoprostane analysis, including samples of cerebral cortex, hippocampus, and
cerebellum collected 9 and 16 weeks after injury by the procedures
described here. All the assays were performed without knowledge of the
age or mTBI status of the mice.
Statistical analysis
Data for A deposits, levels of isoprostanes (i.e., 8,12-iso-iPF2␣-V I), and
A-40 and A-42 concentrations from the studies described above were
expressed as mean ⫾ SEM. Areas of amyloid deposition and isoprostane
and A levels were assessed by ANOVA and subsequently by Student’s
unpaired two-tailed t test, taking into consideration survival time and
type of injury for each animal. Results in the tests for neurological motor
f unction are nonparametric data and were compared using a Kruskal –
Wallis ANOVA by ranks. Data obtained in the MW M are parametric
data and are given as mean ⫾ SEM. These data were analyzed using a
four-way ANOVA for overall effects (genotype, injury status, gender, and
time) followed by multiple two-way ANOVAs for additional comparisons
between particular groups within a given genotype. Significance was set
at p ⬍ 0.05.
RESULTS
Neurobehavioral analysis
All mice underwent the MWM test, and all were able to swim
without any sign of functional motor impairment. Sham WT
animals (n ⫽ 14) demonstrated the ability to learn the visuospatial task with decreasing latencies to find the platform 16 weeks
after injury (Fig. 1 A). WT animals subjected to either single (n ⫽
20) or repetitive mTBI (n ⫽ 19) demonstrated a similar ability to
learn the new visuospatial task with latencies that were not
significantly different from those of WT sham animals (Fig. 1 A),
Figure 1. Data from behavioral and motor tests in WT and Tg2576 mice.
A, B, Data from the MWM test in WT and Tg mice. Trials were made in
consecutive 8 d sessions at 16 weeks after injury. C, Motor function tests
performed at 16 weeks after injury. Rep, Repetitive.
indicating that neither single nor repetitive mTBI influenced
learning ability assessed in the chronic postinjury period in WT
animals.
Sham Tg animals (n ⫽ 9) demonstrated the ability to learn a
visuospatial task with decreasing latencies to find the platform 16
weeks after surgery (Fig. 1 B). Tg animals subjected to single
mTBI (n ⫽ 15) demonstrated a similar ability to learn the new
visuospatial task with latencies that were not significantly different from those of Tg sham animals, indicating that single mTBI
did not result in cognitive dysfunction at 16 weeks after injury
(ANOVA, p ⫽ 0.225; Fig. 1 B). In contrast, Tg animals subjected
to repetitive mTBI (n ⫽ 13) demonstrated an altered ability to
learn a visuospatial task with latencies that were significantly
increased relative to sham-injured Tg mice (ANOVA, p ⫽
0.0158) as well as to single mTBI Tg mice (ANOVA, p ⫽ 0.0116),
indicating that repetitive mTBI resulted in a significant cognitive
dysfunction at 16 weeks after injury (Fig. 1 B). This interpretation
is supported by evidence that neither WT nor Tg sham animals
showed any motor impairments 16 weeks after surgery, and that
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
J. Neurosci., January 15, 2002, 22(2):446–454 449
neither single nor repetitive mTBI led to deficits in motor function in WT or Tg mice 16 weeks after trauma (Fig. 1C). Thus,
impaired motor performance cannot account for the MWM
performance deficits in the repetitive mTBI Tg mice described
above.
Histology
No histopathological changes or evidence of cell loss after single
mTBI injury were reported previously in the ipsilateral cortex,
hippocampal CA3, or dentate hilus up to 8 weeks after injury of
WT mice (Laurer et al., 2001), and we extended these observations to the effects of mTBI and sham treatment on the Tg mice
described here (Fig. 2). Notably, after single mTBI at 16 weeks
after injury, there was no significant cortical neuropathology at
the impact site (Fig. 2 E), and the same was true of the shamtreated mice (Fig. 2C). However, single mTBI resulted in minimal
iron deposits subjacent to the meninges at the impact site that
were not evident at low-power magnification (Fig. 2 E), but repetitive mTBI caused mild edema of the cortical surface at the injury
site 2 d after mTBI (Fig. 2 A) and more evident iron deposits at
16 weeks after mTBI (Fig. 2G), but there was no other evidence
of pathology by H&E staining, and there was no indication that
CA3 and other hippocampal areas were altered by single or
repetitive mTBI.
GFAP staining
For Tg and WT mice subjected to repetitive mTBI, there was
evidence of early and mild reactive gliosis detected by GFAP
staining 2 d after the injury in cortex subjacent to the impact site,
and a few GFAP-positive reactive astrocytes also were seen in the
subjacent hippocampus (Fig. 2 B). At 9 weeks after surgery, these
reactive astrocytes were limited to the surface of the cortex below
the impact site after repetitive mTBI (data not shown), but 16
weeks after sham treatment, there was no evidence of cortical
gliosis (Fig. 2C,D), whereas single mTBI mice showed very mild
astrocytosis and GFAP staining predominantly in the white matter after 16 weeks (Fig. 2 F). Repetitive mTBI induced GFAPpositive reactive astrocytes confined to the impact site of both
WT and Tg mice, and Figure 2, G and H, shows representative
images of this in the Tg2576 mice. Apart from the impact site,
infrequent reactivate astrocytes were found primarily in the white
matter in the WT mice, and this was the case with the Tg mice,
which also showed reactive astrocytes around SPs in addition to
variable amounts of gliosis in the cortex, subcortical white matter,
corpus callosum, and hippocampus.
Amyloid deposition
A deposition was detectable in the cerebral cortex and hippocampus in the Tg2576 mice at 9 months of age and thereafter
as reported previously (Hsiao et al., 1996; Takeuchi et al., 2000).
Between 2 d and 9 weeks after injury, the burden and distribution
pattern of A deposits were scattered and infrequent in all groups
of Tg mice, whereas there were no A deposits in any of the WT
mice. There was a mild increase in the A burden in repetitive
mTBI mice at 9 weeks after injury, but only at 16 weeks (i.e., in
12-month-old Tg mice) was the A burden increased in both
single and repetitive mTBI mice relative to sham-treated Tg mice
(Fig. 3). These amyloid deposits were detectable in selected brain
regions, i.e., the olfactory bulb, all cortical regions, including the
frontal, cingulate, and perihippocampal cortex, as well as the
hippocampus, but not in areas such as the striatum, thalamus,
cerebellum, brainstem, and spinal cord. Amyloid deposition visualized by 4G8 immunohistochemistry in sham-treated and single
Figure 2. Histological sequelae of single or repetitive (Rep) mTBI and
sham treatment in Tg2576 mice. H&E staining with Gomori’s iron stain
(A, C, E, G) and GFAP staining (B, D, F, H, insets) are shown. Single and
repetitive mTBI resulted in no or very mild damage at the impact site in
the brain at 2 d (A, B), and 16 weeks ( C–H) after the injury. Each inset
indicates a high-power view of the rectangular area shown in the images
in B, D, F, and H. Sham treatment resulted in no overt damage (C, D). A,
B, Repetitive mTBI (2 d after TBI). C, D, Sham (16 weeks after treatment). E, F, Single mTBI (16 weeks after TBI). G, H, Repetitive mTBI
(16 weeks after TBI).
or repetitive mTBI mice also was confirmed using both
thioflavin-S staining and ubiquitin immunohistochemistry (data
not shown).
To determine the effects of mTBI on progressive amyloid
deposition at various periods after injury, the area occupied by
4G8-immunopositive deposition in the SSC, PHC, and HP both
ipsilateral and contralateral to the impact site was analyzed (Fig.
4). The study groups consisted of sham-treated mice, repetitive
mTBI mice (2 d and 9 and 16 weeks after TBI), and single mTBI
mice (16 weeks after surgery). Comparison of the burden of A
positive deposits between the side of the impact and the contralat-
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
450 J. Neurosci., January 15, 2002, 22(2):446–454
Figure 3. Amyloid deposition in Tg2576 mice with sham or repetitive
(Rep) mTBI (B, D) with 4G8 immunohistochemistry at 9 (A, C) and 16 (B,
D) weeks after mTBI. SPs increased in an age-dependent manner in both
sham and injured mice, but the largest number of A-positive SPs are
seen in the 16 week postrepetitive mTBI mice ( D).
hemispheres of each anatomical region revealed that single mTBI
(average ⫽ 0.93%; ANOVA, F ⫽ 12.04; p ⫽ 0.046) and repetitive
mTBI (average ⫽ 1.57%; ANOVA, F ⫽ 6.25; p ⫽ 0.025) accelerated amyloid deposition by 16 weeks after trauma compared
with the sham group (Fig. 4 A), although the differences between
sham treatment versus repetitive mTBI at 9 weeks were not
significant. Because gender effects on amyloid burden were shown
to be significant (Callahan et al., 2001), we performed analyses on
the subgroup of male Tg2576 mice, and the outcome of these
analyses showed that the burden was higher in Tg2576 female
versus male mice. Most notably, however, there was a significant
difference only at 16 weeks after injury (single mTBI, average ⫽
0.93%; ANOVA, F ⫽ 12.04; p ⫽ 0.046; repetitive mTBI, average ⫽ 1.36%; ANOVA, F ⫽ 6.64; p ⫽ 0.032) in comparison with
sham-treated Tg mice. Finally, further analysis of these data
demonstrated that all regions (i.e., SSC, PHC, and HP) showed
trends with respect to the effect of mTBI on amyloid burden (Fig.
4 B), and by 16 weeks after injury, both single and repetitive
mTBI mice accumulated a 4- to 10-fold higher A burden than
the sham-treated Tg2576 group.
A ELISA
To independently assess the burden of brain A40 and A42,
ELISA was used to analyze brain ipsilateral and contralateral to
the hemisphere subjected to sham treatment or single versus
repetitive mTBI at 16 weeks after surgery. As additional controls,
right and left cerebellar hemispheres were collected from each
Tg2576 mouse and analyzed individually. ELISA analysis showed
that the cerebral cortex consistently had the most A40 and
A42, the hippocampus had smaller amounts of both peptides
than the cortex, whereas the cerebellum consistently had the least,
and the concentrations of A40 or A42 did not differ between
either hemisphere in all regions examined (Fig. 5). In single
mTBI Tg2576 mice, concentrations of A40 and A42 were
increased in the soluble fraction, but this group failed to show a
significant difference in the A40 levels compared with the shamtreated Tg2576 mice, although there was a significant increase in
A42 concentrations (Fig. 5A). Indeed, neither insoluble A40
nor A42 in single mTBI Tg2576 mice showed a significant
difference in comparison with cortices from sham-treated mice
(Fig. 5B). However, in the repetitive mTBI group, A40 and
A42 levels in both the soluble and insoluble fractions of neocortex from all Tg2576 mice were significantly higher than in the
sham-treated Tg2576 mouse cortex (Fig. 5).
Isoprostane analysis
Figure 4. Average percentages of the area occupied by A in three brain
areas of interest, including the PHC, SSC, and HP. A, Data from the total
number of mice in each group, including male and female for all regions
of interest. B, Data in A plotted for each region (n ⫽ 4 – 6). *p ⬍ 0.05 in
comparison with sham treatment. Rep, Repetitive.
eral side revealed that all three regions analyzed from both
hemispheres showed a comparable accumulation of A of the
Tg2576 mice, and this was true across all groups of Tg mice
throughout the time points analyzed.
Averaging the value of amyloid burden between right and left
8,12-iso-iPF2␣-VI levels in urine and brain from selected WT and
Tg mice were measured to assess the extent of oxidative stress
after mTBI or sham treatment (Fig. 6). Urinary 8,12-iso-iPF2␣-VI
levels in the sham group revealed a slight nonsignificant increase
in this isoprostane isomer because of aging, but there was a
significant difference in single mTBI Tg mice at 12 weeks after
injury (single mTBI, ANOVA, p ⫽ 0.0292; F ⫽ 6.13 compared
with the sham group), although this no longer was noticeable at
16 weeks after injury (Fig. 6 A). In contrast, the repetitive mTBI
group revealed high levels of 8,12-iso-iPF2␣-VI as early as 9 weeks
after injury (repetitive mTBI, ANOVA, p ⬍ 0.0001; F ⫽ 92.69
compared with the sham group), which were maintained until 16
weeks after injury (12 weeks after TBI, ANOVA, p ⬍ 0.0001; F ⫽
85.62; 16 weeks after TBI, p ⬍ 0.0001, F ⫽ 165.6 compared with
the sham group), and these levels were higher than in the single
mTBI Tg2576 mice (Fig. 6 A).
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
J. Neurosci., January 15, 2002, 22(2):446–454 451
Figure 5. Concentration of brain A as determined by sandwich ELISA. A1– 40 and A1– 42 peptides were measured
in soluble ( A) and insoluble ( B) fractions of cortex (CTX ),
hippocampus (HP), and cerebellum (CBL) from sham and
single and repetitive (Rep) mTBI Tg2576 mice (n ⫽ 4 – 6).
*p ⬍ 0.05; **p ⬍ 0.001 in comparison with sham injury.
Analyses of the cortex, hippocampus, and cerebellum from the
sham and injured Tg2576 mice (Fig. 6 B) revealed that the cerebral cortex and hippocampus, but not the cerebellum, showed
significantly higher 8,12-iso-iPF2␣-VI levels in both single and
repetitive mTBI groups at 16 weeks after surgery (cortex, single
vs sham ANOVA, p ⫽ 0.027; F ⫽ 14.16; repetitive vs sham
ANOVA, p ⫽ 0.0006; F ⫽ 21.30; hippocampus, single vs sham
ANOVA, p ⫽ 0.0256; F ⫽ 6.48; repetitive vs sham ANOVA, p ⫽
0.0002; F ⫽ 26.93). Interestingly, 8,12-iso-iPF2␣-VI levels in the
cortex were significantly higher than levels in the hippocampus in
all treatment groups (Fig. 6 B).
DISCUSSION
The present study shows for the first time that there is a clear
positive correlation between episodes of TBI and increased amyloid deposition in a Tg mouse model of AD amyloidosis. Specifically, we demonstrated that repetitive mTBI in the Tg2576 mice
resulted in a significant acceleration of amyloid deposition by
image analysis of immunohistochemically stained brain sections
and increased A40 and A42 production and accumulation in
soluble and insoluble brain homogenates by a sensitive A
ELISA. Moreover, these findings also were associated with a
significantly greater impairment in cognitive function in MWM
tests and elevated brain and urinary levels of an isoprostane
isomer (i.e., 8,12-iso-iPF2␣-VI) that is a well characterized and
reliable index of oxidative stress (Praticò et al., 1998, 2000a;
Praticò and Delanty, 2000). Thus, these data provide the first
compelling mechanistic linkage between previous episodes of
TBI and subsequent A amyloidosis as well as cognitive impairment and LPO similar to that observed in living AD patients.
Furthermore, these data strongly support previous epidemiological studies implicating TBI as one of the most robust environmental risk factors for AD.
Both the image analysis and ELISA studies here establish that
AD-like amyloidosis, including A deposition and increasing
levels of insoluble A, was accelerated by both single and repetitive mTBI. This is consistent with previous studies in deceased
acute human head trauma victims, which demonstrated a positive
correlation between TBI and A deposits (Roberts et al., 1994),
but it contrasts with previous animal model studies that did not
show similar correlations, although TBI causes acute and chronically progressive neurodegenerative changes in the brains of WT
and Tg animals expressing WT or mutant human APP (Smith et
al., 1997; Murai et al., 1998; Pierce et al., 1998; Nakagawa et al.,
1999, 2000). The explanation for these discrepancies among the
current and previous studies of animal models of TBI and AD is
not clear, but these differences might be attributable to the fact
that TBI induced a selective loss of neurons that secrete A in Tg
mice generated by using neuron-specific promoters to drive WT
or mutant APP transgene expression or to one or more species
differences between the brains of humans and other mammals.
Alternatively, the effects reported here suggest that mild TBI may
more closely model the types of injuries in humans that predispose individuals who survive episodes of head trauma to develop
AD later in life.
For this reason, we developed a mild TBI mouse model to
elucidate the role of head trauma in Tg mice that have been
shown to develop an age-dependent AD-like amyloidosis attributable to expression of a double APP mutation found in Swedish
FAD patients (Hsiao et al., 1996). This strategy also enabled us to
dissect out differences in the effect of single versus repetitive
mTBI in the Tg2576 mouse model of AD amyloidosis. Indeed,
the present study demonstrated some similarities and differences
in histological and pathological sequelae of mTBI between Tg
mice with single versus repetitive mTBI. For example, both injuries resulted in a trend toward acceleration of amyloid production
and deposition 9 weeks after mTBI and beyond, whereas there
452 J. Neurosci., January 15, 2002, 22(2):446–454
Figure 6. Concentration of 8,12-iso-iPF2␣-VI in urine ( A) and brain ( B).
Urine was collected at 9, 12, and 16 weeks after TBI or sham treatment.
Brain tissue was collected from hippocampus (HP), cortex (CTX ), and
cerebellum (CBL) at 16 weeks after mTBI or sham treatment. All samples
came from Tg2576 mice. Rep, Repetitive.
were differences in the extent of histological damage produced by
these injuries, because single mTBI induced milder pathology in
the neocortex compared with repetitive mTBI, consistent with
initial studies of this new model of TBI (Laurer et al., 2001).
Furthermore, single injury transiently increased LPO to a lesser
extent than the more sustained and greater effect on oxidative
stress that followed repetitive mTBI in the Tg mice. Moreover,
there were different functional consequences of single versus
repetitive mTBI such that impaired cognitive function occurred
exclusively in Tg mice subjected to repetitive mTBI at 16 weeks
after injury. Finally, it is highly noteworthy that repetitive mTBI
augmented key pathological features found in the brains of AD
patients, whereas single mTBI failed to do so, and this is consistent with previous epidemiological studies suggesting that the
more severe the brain injury, the greater the possibility of developing AD (Plassman et al., 2000). Because there is no similar
linkage of A amyloidosis to other forms of brain damage, including stroke, it appears that TBI has unique effects on A
metabolism, clearance, or both. However, there is growing evidence to suggest that the risk of developing neurodegenerative
disease is enhanced by repetitive brain injury, and although a
single head injury might not be sufficient to result in functional
impairments, the cumulative effects of multiple traumatic insults
to the brain could lead to CNS dysfunction and degeneration
(Gentleman et al., 1993; Geddes et al., 1999).
Consistent with previous reports (Nunomura et al., 2000;
Praticò et al., 2000a, 2001), the present study supports the notion
of a close linkage between elevated levels of brain oxidative stress
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
and the development of a neurodegenerative disorder as well as
amyloid pathology, but our study is the first to show that LPO is
enhanced by TBI and intimately linked to increased amyloid
deposition and accumulation in an experimental model. However,
although there are no comparable data on isoprostanes beyond
those presented here on the effects of TBI in WT rodents, the
assessment of isoprostane isomers such as 8,12-iso-iPF2␣-VI can
be exploited to analyze the extent of LPO in AD and in animal
models of this disorder (Praticò et al., 2001). Indeed, in our
previous studies of Tg2576 mice, we showed that isoprostane
levels started increasing a few months (i.e., at ⬃4 months) before
amyloid plaques appeared in brain parenchyma (i.e., at ⬃9
months) of these mice, and here we showed that the isoprostane
levels were significantly higher in the Tg2576 mice subjected to
repetitive mTBI compared with sham-treated Tg mice at 9 weeks
after surgery and well before there was significant augmentation
of amyloid deposition at 16 weeks after mTBI. The reasons for
differences in the levels of isoprostanes in the single versus
repetitive mTBI results here in the Tg mice are unclear, but they
probably reflect the greater extent of cellular stress or blood–
brain barrier damage in the repetitive mTBI experiments. A
number of other reports support the hypothesis that oxidative
damage is mechanistically involved in AD brain degeneration
(Markesbery and Carney, 1999; Praticò and Delanty, 2000).
Moreover, our previous study of the same line of Tg mice (Praticò
et al., 2001) and other studies of aging Down’s syndrome patients
(Praticò et al., 2000b) suggest that increasing oxidative stress
precedes amyloid deposition in brain. Consistent with these findings, our data here demonstrated that mTBI produced a rapid
increase in LPO in the Tg2576 mice, as reflected by measures of
brain and urinary 8,12-iso-iPF2␣-VI, and this was paralleled by an
increase in the production and accumulation of A in brain.
Because the effects of TBI extend beyond the site of impact and
evolve over many months after an episode of TBI, it is plausible
that these diffuse and sustained effects of TBI may be mediated in
part by ongoing LPO induced by TBI.
To date, numerous studies have suggested that oxidative stress
promotes amyloid aggregation and fibril formation in vivo
(Yanagisawa et al., 1995; Koppaka and Axelsen, 2000). From this
point of view, high isoprostane levels could indicate that the
oxidative damage promotes or reflects A fibrillization, in addition to its putative role in promoting APP processing to favor
production of amyloidogenic A peptides. It also has been suggested that fibrillar A could increase oxidative stress (Lorenzo
and Yankner, 1994; Hensley et al., 1995; Yatin et al., 1999). Thus,
it is plausible that the higher levels of oxidative stress caused by
mTBI in the Tg2576 mice could promote APP processing to
generate more A, thereby augmenting A fibrillization and fibril
aggregation into SPs. Subsequently, as A fibril formation, aggregation, and deposition continue, this might serve to further
increase LPO and the overall levels of oxidative stress in brain.
However, additional studies are clearly needed to elucidate the
precise cascade of events that link oxidative stress to A amyloidosis and brain degeneration in AD. Moreover, although this
and other models of AD amyloidosis do not fully recapitulate the
complete AD phenotype, and there are numerous other differences in the motor and cognitive abilities in mice and humans,
animal models of AD brain pathology provide important experimental systems for elucidating mechanisms of A- and TBIinduced neurodegeneration.
In summary, we tested the hypothesis that brain injury is an
environmental risk factor for AD, and we provided critical ex-
Uryu et al. • Head Trauma and Alzheimer Amyloidosis
perimental evidence in support of this notion. Although there
have been dramatic advances in understanding the etiology of
FAD, ⬃90% of AD is sporadic, and it is clear that distinctively
different initiating events cause SAD in contrast to genetic mutations that cause FAD. Although multiple genetic and epigenetic
factors might act synergistically to predispose individuals to develop SAD (Kurochkin and Goto, 1994; Qiu et al., 1998;
Chesneau et al., 2000; Iwata et al., 2000; Vekrellis et al., 2000),
the experimental data presented here add considerable credibility
to previous indirect epidemiological evidence linking head
trauma to mechanisms of AD.
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