Brain and Spinal Cord Interaction: Protective Effects of
Exercise Prior to Spinal Cord Injury
Fernando Gomez-Pinilla1,2*, Zhe Ying1, Yumei Zhuang2
1 Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, California, United States of America, 2 Department of Neurosurgery,
UCLA Brain Injury Research Center, University of California Los Angeles, Los Angeles, California United States of America

Abstract
We have investigated the effects of a spinal cord injury on the brain and spinal cord, and whether exercise provided before
the injury could organize a protective reaction across the neuroaxis. Animals were exposed to 21 days of voluntary exercise,followed by a full spinal transection (T7–T9) and sacrificed two days later. Here we show that the effects of spinal cord injury
go beyond the spinal cord itself and influence the molecular substrates of synaptic plasticity and learning in the brain. The
injury reduced BDNF levels in the hippocampus in conjunction with the activated forms of p-synapsin I, p-CREB and p-CaMK
II, while exercise prior to injury prevented these reductions. Similar effects of the injury were observed in the lumbar
enlargement region of the spinal cord, where exercise prevented the reductions in BDNF, and p-CREB. Furthermore, theresponse of the hippocampus to the spinal lesion appeared to be coordinated to that of the spinal cord, as evidenced bycorresponding injury-related changes in BDNF levels in the brain and spinal cord. These results provide an indication for theincreased vulnerability of brain centers after spinal cord injury. These findings also imply that the level of chronic activity
prior to a spinal cord injury could determine the level of sensory-motor and cognitive recovery following the injury. In
particular, exercise prior to the injury onset appears to foster protective mechanisms in the brain and spinal cord.
Citation: Gomez-Pinilla F, Ying Z, Zhuang Y (2012) Brain and Spinal Cord Interaction: Protective Effects of Exercise Prior to Spinal Cord Injury. PLoS ONE 7(2):e32298. doi:10.1371/journal.pone.0032298
Editor: Paul A. Adlard, Mental Health Research Institute and the University of Melbourne, Australia
Received October 19, 2011; Accepted January 24, 2012; Published February 22, 2012
Copyright: ß 2012 Gomez-Pinilla 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 award R01 NS056413 and the Craig H. Neilsen Foundation. 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: fgomezpi@ucla.edu

rodents receiving a full transection to the SC. This information issignificant to understand the potential for functional recovery of
SCI patients based on their prior lifestyle history, and has direct
applicability to evaluate prognosis in injured athletes.
Studies have been focused on molecular systems associated withthe action of brain-derived neurotrophic factor (BDNF). BDNF is
a powerful synaptic facilitator [8], and promotor of neuronalplasticity [9,10], and its production is influenced by exercise in the
hipocampus [11] and spinal cord [12]. Given that exercise has astrong influence on learning and memory ability associated withhippocampal BDNF [11], we focused this study on the
hippocampus to evaluate potential effects of SCI on the substrates
of cognitive function. We have assessed synapsin I and GAP-43
based on their involvement on the mechanisms by which BDNF
affects neuronal and synaptic plasticity. Synapsin I is a wellcharacterized member of a family of nerve terminal-specificphosphoproteins and is implicated in neurotransmitter release,axonal elongation, and maintenance of synaptic contacts [13].
BDNF stimulates the synthesis and activation of synapsin I
resulting in elevated neurotransmitter release [14]. GAP-43 has
important roles in axonal growth, neurotransmitter release[15,16], and learning and memory [17].

Introduction
Spinal cord injury (SCI) is a major cause of disability in civilian
and military environments, and involves long-lasting hardships tovictims and families. Besides the main deficits in motor and sensory
functions, cognitive and emotional disorders are common featuresin patients suffering from SCI [1]. New evidence indicates that lossof spinal cord (SC) input to the brain can promote profound
functional modifications in brain centers, such as rapid changes inthe spontaneous electrophysiological activity of neuronal networks[2]. In addition, new research is starting to reveal that lesion to thespinal cord can impact molecular systems important for synaptic
plasticity in the brain [3,4]. Accordingly, we have initiated studies todetermine whether a SCI can concert the action of molecularsystems that regulate neuronal plasticity in the hippocampus and
SC. We have focused our studies in the early injury period, as these
events can have critical implications for long-term plasticity [5]. The
results of these studies can provide critical information to startunderstanding the mechanisms by which SCI can affect an array offunctions beyond motor disorders, and can provide new clues how
to enhance functional recovery.
Exercise is considered beneficial for overall health of theorganism during homeostatic and disease conditions. A growing
body of evidence indicates that exercise provided after the onset ofa SCI counteracts some of the effects of the lesion [6,7]; however,the capacity of exercise when provided before the injury onset are
not clearly established. Accordingly, we have embarked in studies
to evaluate the protective effects of exercise in the brain and SC of
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Results
Effects of SCI and exercise on the hippocampus
BDNF. One-way ANOVA analysis showed a significant
group effect (F(4,38) = 44.578, p,0.001) for the hippocampal
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Brain and Spinal Cord Interactionlevels of BDNF. We included two intact groups, Int(0) and Int(2),in our design to control for possible effects of the two-day resting
period on BDNF levels. The Bonferroni post-hoc test indicatedthat exercise elevated levels of BDNF (143%, p,0.01) in the intact
animals sacrificed immediately after the last exercise bout (Exc/
Int(0), but promoted an increasing tendency (120% (p.0.05) in
the animals sacrificed two days after exercise Exc/Int(2),compared to sedentary control animals (Sed/Con, 100%). With
regards to the effects of spinal cord injury on the hippocampus,
there was a reduction in BDNF level in the sedentary injured
animals (Sed/SCI, 63%, p,0.01) while exercise prior to the injury(Exc/SCI, 103%) prevented this reduction, as compared to thesed/Con group. Levels of BDNF in the exercise animals exposed
to SCI (Exc/SCI) showed a decreasing tendency (p.0.05) relative
to Exc/Int(2) animals (Fig. 1A).
We performed immunohistochemistry for BDNF in order toevaluate the phenotypic expression of protein changes detectedwith western blot analysis. Qualitative analysis under lightmicroscopy showed weak BDNF labeling in the various hippocampal subregions in sedentary controls, and the intensity of thelabeling became more prominent along the CA3 and dentate
gyrus (DG) (Fig. 1B). The SCI reduced the intensity of the BDNF
labeling; however, exercise appeared to attenuate this reduction, as
verified with the western blot analysis.
Synaptic markers (Fig. 2). For the levels of p-synapsin I(Fig. 2A), there was a statistically significant difference between
groups as determined by one-way ANOVA (F(4,38) = 22.929,
p,0.01). The Bonferroni post-hoc test was used for multiple
comparisons showed that exercise increased the p-synapsin I levelsin the Exc/Int(0) group (124%, p.0.05). There was a significant
increase (137%, p,0.05) in p-synapsin I levels in the Exc/Int(2)group. SCI significantly reduced (54%, p,0.01) the levels of psynapsin I while exercise prior to the SCI counteracted theseeffects such that the p-synapsin I levels in the Exc/SCI groupremained nearby the Sed/Con group (Fig. 2A).
With regards to p-CREB (Fig. 2B), one-way ANOVA showedthat there was a significant group effect (F(4,38) = 23.112, p,0.01).
The Bonferroni post-hoc test for multiple comparisons revealed
that exercise increased p-CREB levels significantly in both intact

groups [Exc/Int(0): 131%, p,0.01; Exc/Int(2): 137%, p,0.01].
SCI reduced the levels of p-CREB in sedentary animals (Sed/SCI:
79%, p,0.05) while exercise prevented this decrease such that pCREB remained nearby the control levels (Exc/SCI: 102%).
Furthermore, p-CREB levels of Exc/SCI group were significantly
lower than Exc/Int(2) group (137% vs. 102%, p,0.01; Fig. 2B).
With regards to p- CaMKII, one-way ANOVA showed asignificant group effect (F(4, 38) = 25.086, p,0.01; Fig. 2C). The
Bonferroni post-hoc test for multiple comparisons indicated thatexercise increased the p-CaMKII levels in the Exc/Int(0) group(144%, p,0.01) and the Exc/Int(2) group (143%, p,0.01), as
compared to Sed/Con group (Fig. 2C). SCI reduced the levels of
p-CaMKII in the Sed/SCI group (79%, p,0.05) as compared to
the Sed/Con group, while exercise prevented these reductions(Exc/SCI: 99%, Fig. 2C). SCI counteracted the effects of exercise
as compared to Exc/Int(2).
With regards to GAP-43, one-way ANOVA analysis showed asignificant group effect (F(4,38) = 16.077, p,0.01). The Bonferroni
post-hoc test for multiple comparisons showed that exercise
increased the levels of GAP-43 in both Exc/Int(0) and Exc/
Int(2) groups (140% and 136% respectively, p,0.01), as comparedto the Sed/Con group. There was an increasing tendency in the
Sed/SCI group (116%, p.0.05). SCI did not alter the levels of
GAP-43 in exercise animals (Exc/SCI: 89%, p.0.05), as
compared to Sed/Con (Fig. 2D); however the level of GAP-43
were decreased in exercise animals with SCI (Exc/SCI) as
compared to Exc/Int(2) animals (p,0.01) (Fig. 2D).

Effects of SCI and exercise on the spinal cord
BDNF. To evaluate the effects of exercise prior to SCI in the
spinal cord, we measured BDNF protein levels in the lumbar
enlargement region (Fig. 3). One-way ANOVA showed asignificant group differences (F(4, 38) = 27.024, p,0.01). The
Bonferroni post hoc test showed that BDNF levels were increasedin the Exc/Int(2) group (143%, p,0.01) while there was anincreasing tendency (116%, p.0.05) in Exc/Int(0), compared to
the Sed/Con group (Fig. 3A). With regards to the effects of the
injury, SCI reduced BDNF levels to 72% (p,0.01) of Sed/Con in

Figure 1. Effects of SCI and exercise on BDNF levels in the hippocampus. (A) Exercise elevated levels of BDNF in intact animals sacrificed on
the last exercise day (Exc/Int0) or two days after ceasing exercise (Exc/Int2). SCI reduced BDNF level and exercise prior to SCI prevented this reduction.
*p,0.05, **p,0.01. Representative western blot bands for BDNF are shown for each animal group. (B) Microphotographs of representative
immunohistochemical sections showing localization of BDNF in the hippocampus. SCI reduced BDNF immunolabeling (white arrows, Sed/SCI) mainly
affecting the CA 3 region, but exercise prior to the SCI prevent these effects (black arrows, Exc/SCI).
doi:10.1371/journal.pone.0032298.g001

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Figure 2. Effects of SCI and exercise on plasticity markers in the hippocampus: (A) p- synapsin I; (B) p-CREB; (C) p-CaMKII; (D) GAP43. Exercise increased protein levels of these markers in intact animals sacrificed on the last exercise day (Exc/Int(0)) or two days after ceasing exercise(Exc/Int(2)). Exercise prior to SCI prevented the effects of the injury. *p,0.05, **p,0.01. Representative western blot bands are shown for eachexperimental group.
doi:10.1371/journal.pone.0032298.g002

Con group. SCI reduced p-CREB levels in sedentary animals(Sed/SCI, 62%, p,0.01) and exercise prevented these effects(Exc/SCI, 88%, p.0.05).
With regards to the levels of p-CaMK II, we found a significant
group difference (F(4,38) = 24.674, p,0.01) by one-way ANOVA.
The Bonferroni post-hoc test performed for multiple comparisons
showed that exercise increased the levels of p-CaMK II in the
Exc/Int(0) group and the Exc/Int(2) group to 142% (p,0.01) and
143% (p,0.01), respectively (Fig. 4C). The levels of p-CaMK II
after SCI in the sedentary group (Sed/SCI) were reduced to 74%(p,0.05), while exercise prior to injury prevented the injury
reduction (Exc/SCI: 93%) such that the p-CaMK II levels
remained nearby the Sed/Con animals (Fig. 4C).
For the levels of GAP-43, one-way ANOVA analysis showed asignificant group difference (F(4, 38) = 19.417, p,0.01). Exercise
did not change the levels of GAP-43 in the Exc/Int(0) (95%,p.0.05) and Exc/Int(2) (113%, p.0.05) animals (Fig. 4D). The
levels of GAP-43 were decreased in the Sed/SCI (64%, p,0.01)
and the Exc/SCI (61%, p,0.01) groups as compared to the Sed/
Con group (Fig. 4D).

the Sed/SCI group, while exercise prior to the SCI (Exc/SCI)
counteracted this reduction (Fig. 3A).
We performed BDNF immunohistochemistry in the SC lumbar
enlargement region in order to illustrate the phenotypic expression
for changes in proteins levels quantified using western blot analysis.
Qualitative analysis using light microscopy showed that BDNF was
localized to motoneurons in the ventral horn and their axonal
processes coursing through the white matter. There was a relative
decrease in BDNF immunostaining in the SC of lesioned mice
(Fig. 3B) while exercise appeared to preserve the immunostaining(Fig. 3B).
Synaptic markers. We measured the protein levels of thesame synaptic markers in the spinal cord lumbar enlargement
region as in the hippocampus. For the levels of p-synapsin I, oneway ANOVA showed a significant group effect (F(4,38) = 33.084,
p,0.01). The Bonferroni post-hoc test indicated that exercise
elevated the p-synapsin I levels in the Exc/Int(0) group (123%,
p,0.05) while no significant changes were found in Exc/Int(2)group (Fig. 4A). SCI reduced the p-synapsin I levels to 64%(p,0.01) and 53% (p,0.01) in Sed/SCI and Exc/SCI groups,
respectively (Fig. 4A).
There was a significant group difference for the levels of pCREB (F(4,38) = 30.533, p,0.01) assessed with an one-way
ANOVA (Fig. 4B). The multiple comparisons were carried out
by the Bonferroni post-hoc test. The levels of p-CREB weresignificantly higher in the Exc/Int(0) group (131%, p,0.01) and in
the Exc/Int(2) group (125%, p,0.05), as compared to the Sed/

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Coordinated changes in BDNF in the hippocampus vs.
spinal cord
Given the effects of the SCI on the hippocampus and SC, wesought to determine whether these effects could be coordinated.
Accordingly, we performed a linear regression analysis forindividual rats to evaluate the possibility of a correlation between

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Figure 3. Effects of SCI and exercise on BDNF levels in the lumbar region of the spinal cord. (A) Exercise elevated levels of BDNF in intact
animals sacrificed on the last day of exercise (Exc/Int(0)) or two days after ceasing exercise (Exc/Int(2)). SCI reduced levels of BDNF while exercise
counteracted these effects (Exc/SCI). Representative western blot bands displaying BDNF are shown for each animal group. **P,0.01.
(B)Microphotographs of representative immunohistochemical sections showing localization of BDNF in the hippocampus. SCI reduced BDNF
immunolabeling (Sed/SCI) mainly affecting motoneurons (white arrows), but exercise prior to the SCI prevented these effects (black arrows; Exc/SCI).
doi:10.1371/journal.pone.0032298.g003

levels of BDNF in the hippocampus Vs the SC. Results showed ahigh correlation in animals that had received the SCI (R = 0.727,
p,0.01, Fig. 5), suggesting the possibility that the SCI coordinatesa corresponding response in the brain and SC. Results showed nostatistical correlation for exercise and sedentary intact animals(R = 0.262, P.0.05).

Figure 4. Effects of SCI and exercise on plasticity markers in the lumbar region of the spinal cord: (A) p-synapsin I; (B) p-CREB; (C) pCaMKII; (D) GAP-43. Exercise increased protein levels of these markers in intact animals, which were sacrificed on the last exercise day (Exc/Int(0))
or two days after ceasing exercise (Exc/Int(2)). Exercise prior to SCI prevented the effects of the injury. *p,0.05, **p,0.01. Representative western
blot bands are shown for each experimental group.
doi:10.1371/journal.pone.0032298.g004

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turn, in agreement with our results, Fumagalli et al [4] havereported reductions of BDNF mRNA in the hippocampus of rats
24 h after an impact induced SCI. Based on the role of the
hippocampus in the processing of information important forlearning and memory and emotions, our results suggest thepossibility that SCI increases the vulnerability to emotional andcognitive disorders. This possibility is in agreement with clinicalstudies showing that SCI reduces the quality of life of patients andoften is associated with increased propensity for anxiety anddepression [1].
In turn, it has recently been reported that damage to the brain
can also influence the brain stem and SC centers. Specifically,
concussive brain injury in humans, which is known to compromise
cognitive performance and other brain functions, has also been
associated with gait deficits [19]. Gait deficiency likely involves
multiple centers in the brain and spinal cord, and theiridentification requires further studies. Animal models of concussive
injury, which is known to affect hippocampal-dependent cognition, have been shown to reduce levels of BDNF and associatedmolecules in the SC, in addition to the hippocampus [20].
Accordingly, this reciprocal interaction between the hippocampus
and SC can provide a platform from which to start understandinga wide range of cognitive and motor disorders observed after SCI
or brain injury.

Figure 5. Proportional changes in the hippocampus and SC
after SCI. Linear regression analysis for individual rats showed acorrelation between levels of BDNF in the hippocampus Vs the SC(R = 0.727, **p,0.01). Results showed no statistical correlation forexercise and sedentary intact animals (R = 0.262, *p.0.05, data notshown).
doi:10.1371/journal.pone.0032298.g005

Discussion

Effects of SCI on the expression of neurotrophins in the
spinal cord

We have found that a complete SC transection can impact
select molecular systems that support neuronal function andplasticity in the brain and SC. The fact that SCI reduced levels of
BDNF and related proteins in the hippocampus indicates that thebrain is also affected by the SCI pathology. Our results provide
evidence for a coordinated response of the brain and spinal cord to
SCI. Interestingly, exercise provided before the injury onset
showed a protective action in the brain as well as in the SC as
evidenced by its counteractive effects on the injury-related
reductions of most of the molecular systems under study. These
results emphasize the capacity of exercise to recruit molecular
adaptations that can enhance plasticity in the brain and spinal
cord.

Our results showing that SCI reduced levels of BDNF in the
lumbar SC region may be the results of various mechanisms. It is
posible that a reduction in BDNF is caused by an interruption of
suprasegmental input necessary for maintaining levels of BDNF. It
is also feasibly that the injured neurons and their projections fromthe peri-injury region may reduce supply of BDNF. In addition,
production of BDNF may be affected by the release of growth
inhibitors molecules such as myelin-associated glycoproteins [21]after the injury, which are known to exert inhibitory effects on
BDNF levels and function [22]. Thus, it is possible that a
combination of factors may play a role in the injury-related
reduction in SC BDNF plasticity.
Based on the results showing that the SCI affected levels of
BDNF in the brain and SC, we sought to determine whether the
injury could somehow coordinate a response in the hippocampus
and SC. Accordingly, we performed linear regression analysis tocorrelate levels of BDNF in the brain and SC in intact and SCI
animals. We found that levels of hippocampal BDNF werecorrelated to SC BDNF levels in animals that had received SCI,
while no correlation was observed in intact animals. These results
seem to emphasize the possibility that the lesion concerts a
response in the brain and SC.

Effects of SCI on the expression of neurotrophins in thebrain
Our results show that SCI reduced levels of BDNF and related
plasticity markers in the hippocampus, and indicate that the SC
contributes to maintain BDNF levels in the brain. In addition,
based on the fact that the reduction of BDNF was accompanied bychanges in proteins related to the function of BDNF on synaptic
plasticity, it is assumable that these molecular adjustments havefunctional consequences. The studies were focused on the acuteperiod following the injury based on the critical role of this period
for injury recovery [5]. In particular, it is known that molecular
activity occurring during this period is crucial for the functional
capacity of the SC for learning a motorsensory task [18]. Given
the active role of BDNF on synaptic plasticity, it is likely that
reductions in BDNF during this period may impair synaptictransmission that is crucial for normal hippocampal function. It
has recently been reported that SCI promotes neurite sprouting
and elevations of synapsin I in the hindbrain of the lamprey [3].
However, there are several differences with the current study
including species, timing of the effect, and brain regions involved.
Changes in sprouting were observed after 1 week, which isinterpreted by the authors as a compensatory reaction to the injury[3], in an animal species characterized by exuberant plasticity. In
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Effects of exercise
Exercise elevated levels of BDNF and related molecules in the
SC and hippocampus of intact animals as previously described[12,23]. Interestingly, intact exercise animals that were allowed torest for 2 days before the sacrifice showed higher levels of BDNF in
the SC, but not in hippocampus, than those animals sacrificed
immediately after exercise. These results may indicate thatexercise increases axonal transport of BDNF from brain centers.
Interestingly, the SCI reduced SC levels of BDNF in both theexercise and sedentary animals, suggesting that the SCI may
disrupt the axonal transport. In turn, hippocampal BDNF levels in
SCI animals were maintained nearby intact levels after exercise.
Exercise also showed important effects in counteracting a SCIrelated reduction in p-synapsin I in the hippocampus, which is in
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agreement with the close described association between BDNF
and synapsin I [24].
The concurrent effects of exercise on the brain and spinal cord
are significant to better understand the neural pathways by which
exercise can affect the brain. It is known that exercise is an
important promotor of BDNF-related plasticity in the hippocampus and other brain structures; however, the interaction between
supraspinal and infraspinal centers during exercise remains
elusive. Although running wheel exercise heavily relies onneuromuscular work, the influence of hindlimbs and rearlimbs
activity on the brain has received insufficient attention. The factthat an injury at the thoraciic SC level reduced the action of
exercise in hippocampal plasticity emphasizes the influence of thehindlimb activity on brain plasticity.

Materials and Methods
The experiments were performed in accordance with theUnited States National Institutes of Health Guide for the Care andUse of Laboratory Animals. All procedures were approved by the
UCLA Chancellor’s Animal Research Committee.

Exercise paradigm
The experiments were performed in male C57BL/6 mice(Charles River Laboratories International, Inc.). The mice
approximately 10 weeks of age were housed individually instandard polyethylene cages in an environmentally controlled
room (22–24uC) with a 12 h light/dark cycle. Animals were either
exposed to cages provided with running wheel (exercise) or regular
cages (sedentary). The cages equipped with running wheels(diameter = 12 cm, width = 5 cm, Mini Mitter, Bend, Oregon,
USA) allowed animals to exercise ad libitum. Running revolutions
were recorded using VitalViewer software (Mini Mitter, Bend,
Oregon, USA). After completing a 3-week period, exercise animals
were divided into three groups: animals sacrificed the morning of
the last day of exercise (Exc/Int(0)); animals sacrificed 2 days after
the last exposure to exercise (Exc/Int(2)); animals received a fulltransection lesion into the SC and sacrificed two days after the
injury (Exc/SCI) (Fig. 6). Sedentary rats received a full transection
lesion into the SC and were sacrificed two days later (Sed/SCI)(Fig. 6). Sedentary animals without injury were used as the main
control group (Sed/Con) (Fig. 6). The average running distance
between three groups was not significantly different (Exc/
Int(0):8.6360.36 km/day; Exc/Int(2): 8.6960.69 km/day; Exc/
SCI: 8.9960.49 km/day).

Downstream BDNF effectors
To evaluate the possible functional implications for changes in
the neurotrophin system resulting from SCI, we measured thelevels of various molecules that have a recognized interaction
with BDNF and are important for synaptic plasticity. Our results
showed that SCI reduced levels of all the molecular markers
examined in the hippocampus, with the exception of GAP-43.
These results emphasize the importance of the SC activity on
maintaining brain function and plasticity, i.e, BDNF and synaptic
markers in the hippocampus. Exercise showed a clear elevation in
p-CREB, p-CaMKII, and GAP-43 in the hippocampus of intact
rodents, and the SCI counteracted the effects of exercise. It isinteresting that changes in p-synapsin I followed closely the
BDNF pattern in the hippocampus. The coordinated response
between BDNF and synapsin I may be explained by theestablished association between these two molecular systems in
the hippocampus. BDNF phosphorylates synapsin I to induce themitogen-activated protein kinase signaling pathway which, inturn, modulates neurotransmitter release [14]. In addition, we
have been able to reduce the exercise-induced increase insynapsin I mRNA in the hippocampus by blocking BDNF action
using the tyrosine kinase receptor blocker K252A [23].
Therefore, the change in synapsin I in the present study is alikely indicator of modified synaptic plasticity associated to thefunction of BDNF.
Similar to BDNF, synapsin I levels were down-regulated in the
SC following SCI. Exercise was not sufficient to maintain levels of
p-synapsin I elevated in the SC of SCI rats, which suggests that the
2-day rest period may have weaken the effects of exercise. Indeed,
exercise was able to elevate levels of p-synapsin I in intact animals
at day 0 but not after the 2-day rest period. Exercise in the SC was
effective in counteracting a reduction in p-CREB.

LesionBriefly, the mice were pre-sedated with buprenorphine(0.05 mg/kg body weight, s.c.) and mice were maintained in a
deep anesthetic state throughout the surgery with isoflurane gas(1.5–2.5% isoflurane mixed with 100% O2 at a flow rate of .4 L/
minute via face mask). A complete SC transection at a midthoracic level (T7–T9) was performed under aseptic conditions. A
laminectomy was made in the vertebral column in the midthoracic region (,T7–T9), the dura opened and the SC exposed.
The spinal cord was lifted with a curved probe and completelytransected at the T8–T9 junction. The surgical areas were lavaged
and wounds closed with sutures. All animals were continuallymonitored until the tissue collection time.

Tissue preparation and protein determination
Animals were sacrificed in the early morning. Brain and SC
tissues were dissected, frozen on dry ice, and stored at 270uC until
further use. Hippocampus and SC lumbar enlargement regions
were homogenized in a freshly prepared lysis buffer (137 mM
NaCl, 20 mM Tris-HCl pH 8.0, 1% NP-40, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 1 mg/mL
leupeptin, 0.5 mM sodium vanadate). Homogenates were centrifuged at 12,000 g for 20 min to remove insoluble material. The
supernatants were collected into clean 1.5 ml tubes and stored at
270uC. The total protein concentration of homogenates wasdetermined with a MicroBCA kit (Pierce, Rockford, IL, USA),using BSA as a standard.

Conclusions and functional implications
Results emphasize the impact of SCI on molecular systems that
support neuronal function and plasticity in the brain and spinal
cord. In particular, exercise was able to counteract the injuryrelated reductions in BDNF, CaMKII, and CREB in the
hippocampus, and exercise prior the injury appeared to normalize
these reductions. These findings suggest that an active lifestyle may
confer the CNS with the capacity to defend against neurologicaland cognitive weaknesses after SCI. In particular, our results may
be significant to understand the influence of pre-injury conditions
such as lifestyle factors on the capacity of patients for healing
following neurological damage. The fact that pre-injury conditions
can have an impact on the degree of recovery may also help
explain the high degree of individual variability in the patient
response to treatment.
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Western blot
A total of 25 mg of protein from each sample was used forwestern blot experiment. Sample proteins were separated on 10%
SDS-polyacrylamide gels. After electrophoresis, the proteins weretransferred onto PVDF membranes and nonspecific binding was
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Figure 6. Schematic diagram of the experimental design showing the timing for the applications of exercise (Exc) and spinal cord
injury (SCI) in the various experimental groups.
doi:10.1371/journal.pone.0032298.g006

blocked with 5% nonfat dry milk in Tris-buffered saline containing
0.05% Tween-20 (TBST).The membranes were incubated with a
primary antibody overnight at 4uC, followed by a secondary
antibody for 1 hr at room temperature. The following primary
antibodies were used: BDNF (1:1000, Santa Cruz Biotechnology),
phospho-synapsin I (1:1000, Cell Signaling), phospho-CREB(1:1000, Millipore), phospho CaMKII (1:1000, Santa Cruz
Biotechnology), GAP-43 and actin (1:1000, Santa Cruz Biotechnology). The secondary antibodies used were anti-goat or antirabbit IgGHRP (1:10,000, Santa Cruz Biotechnology). Afterrinsing with buffer (0.1% Tween-20 in TBS), the immunocomplexes were visualized by chemiluminescence using the Amersham
ECL Plus Western Blotting Detection kit (GE Healthcare BioSciences, Piscataway, NJ, USA) according to the manufacturer’s
instructions. The film signals were digitally scanned using a HP
Scanner (HP Scanjet 3970) and quantified with NIH Image
software. Actin was utilized as an internal control for sample
loading, and each blot was normalized to its corresponding actin
value.

ized using a Zeiss Axio Imager HI microscope with the ApoTome
Slider Axioimaging system.

Statistical analyses
A total of 43 mice were used for western blot assays (Sed/
Con: n = 10; Exc/Int(0): n = 10; Exc/Int(2): n = 5; Sed/Sci:n = 11; Exc/SCI: n = 7). Results of western blot analysis wereexpressed as a mean percent of intact animals maintained under
sedentary conditions (Sed/Con) 6 standard error of the mean
(SEM). We had two groups of intact animals that were exposed
to exercise: animals sacrificed the morning of the last day of
exercise (Exc/Int(0)), and animals sacrificed 2 days after the last
exposure to exercise (Exc/Int(2)). The rationale for the Exc/
Int(2) group was to provide a reference point to evaluate theeffects of the two inactive days between the last day of exercise
and sacrifice.
All statistical analyses were done by statistic software SPSS 16.0.
Protein data were analyzed by one-way ANOVA. Post hoc tests
were conducted using Bonferroni comparisons. A linear regression
analysis was performed to assess correlation between levels of
BDNF in hippocampus Vs SC. Group differences were consideredsignificant when p,0.05.

Immunohistochemistry
Fixed brain tissues (n = 4/group; 4% w/v paraformaldehyde)
were sliced in the sagittal plane (25 um) and spinal cord tissues(n = 4/group) were sliced in the transverse plane (25 um), collected
free floating in PBS, and processed for BDNF immunohistochemistry, as previously described [25]. A 1:1,000 dilution was used forthe rabbit polyclonal anti-BDNF antisera (BDNF antibody waspurchased from Millipore). Immunostained sections were visual-

Author Contributions
Conceived and designed the experiments: FGP ZY. Performed theexperiments: YMZ. Analyzed the data: ZY YMZ. Wrote the paper: FGP
YZ.

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