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Published in final edited form as:
Neuroscience. 2008 September 9; 155(4): 1070–1078. doi:10.1016/j.neuroscience.2008.06.057.

BDNF-Exercise Interactions in the Recovery of Symmetrical
Stepping After a Cervical Hemisection In RatsZhe Ying1, Roland R Roy1,3, Hui Zhong1, Sharon Zdunowski1, V Reggie Edgerton1,3, andFernando Gomez-Pinilla1,2
1Department of Physiological Science, Los Angeles 90095, California, USA
2Department of Neurosurgery, UCLA Brain Injury Research Center, Los Angeles 90095, California,

USA
3Brain Research Institute, Los Angeles 90095, California, USA

Abstract
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Clinical evidence indicates that motor training facilitates functional recovery after a spinal cord injury(SCI). Brain-derived neurotrophic factor (BDNF) is a powerful synaptic facilitator and likely plays
a key role on motor and sensory functions. Spinal cord hemisection decreases the levels of BDNF
below the injury site, and exercise can counteract this decrease (Ying et al., 2005). It is not clear,
however, whether the exercise-induced increases in BDNF play a role in mediating the recovery of
locomotion after a SCI. We performed a lateral cervical (∼C4) hemisection in adult rats. Seven days
after hemisection, the BDNF inhibitor trkB IgG was injected into the cervical spinal cord below thelesion (∼C5-C6). Half of the rats were exposed to voluntary running wheels for 14 days. Locomotor
ability was assessed by determining the symmetry between the contralateral (unaffected) vs. theipsilateral (affected) forelimb at the most optimum treadmill speed for each rat. Sedentary andexercised rats with BDNF inhibition showed a higher level of asymmetry during the treadmill
locomotion test than rats not treated with the BDNF inhibitor. In hemisected rats, exercise normalized
the levels of molecules important for synaptic function, such as cyclic AMP response element binding
protein (CREB) and synapsin I, in the ipsilateral cervical enlargement, whereas the BDNF blocker
lessened these exercise-associated effects. The results indicate that BDNF levels play an importantrole in shaping the synaptic plasticity and in defining the level of recovery of locomotor performanceafter a SCI.

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Keywords
spinal cord injury; locomotion; TrkB IgG; synaptic plasticity; neurotrophin
Although neurotrophins have been identified as molecular systems with the potential toenhance spinal cord repair, most of the strategies to induce motor recovery after a spinal cord
injury (SCI) have involved the addition of exogenous neurotrophins into the CNS. These
strategies, however, do not address the intrinsic potential of the neural system to produce
neurotrophins that could have an important effect on the recovery of stepping after a SCI. Given

Corresponding author: Fernando Gomez-Pinilla, Dept. of Physiological Science, UCLA, Los Angeles, CA 90095-1527, Phone/fax: (310)206-9693, fgomezpi@ucla.edu.
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the capacity of voluntary exercise to induce endogenous neurotrophins in the spinal cord (Yinget al., 2003), we hypothesized that neurotrophins play a crucial role in the effects of exercise
on the recovery of motor function after a SCI.

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A large number of studies have shown the potential of motor training to promote functional
recovery after a SCI (Edgerton et al., 2004, Frigon and Rossignol, 2006), yet the specificmechanisms and molecular systems involved remain largely unidentified. It is known thatphysical activity increases the expression of select neurotrophins, such as brain-derived
neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), in the intact and injured spinal cord
(Gomez-Pinilla et al., 2002, Ying et al., 2003). The role of these neurotrophins in promoting
synaptic plasticity or functional recovery after a SCI, however, remains unclear. In particular,
BDNF is a powerful modulator of neuronal excitability and synaptic transmission (Lu andFigurov, 1997, Kafitz et al., 1999), two processes that are crucial for functional recovery after
an injury. The well-described involvement of BDNF in promoting neuronal excitability andsynaptic modification suggests that it could play a supporting role in determining the level offunctional recovery after a SCI. For example, in addition to promoting axonal growth (Bregman
et al., 1997), BDNF delivered to the injured spinal cord can facilitate step-like oscillations inadult rats after a mid-thoracic contusion or complete spinal cord transection when the hindlimbs
are suspended. In addition, when BDNF was administered continuously over a 4-week period,
an open field motor score in complete spinal rats was significantly higher in treated than nontreated rats during weekly testing (Jakeman et al., 1998).
A further question is “through what mechanisms might BDNF operate to alter the course offunctional recovery after a SCI”? Several of the downstream molecular systems that canmediate the action of BDNF on synaptic plasticity have been identified. BDNF affects thesynthesis (Wang et al., 1995) and phosphorylation (Jovanovic et al., 1996) of synapsin I.
Synapsin I is a member of a family of nerve terminal-specific phosphoproteins involved inneurotransmitter release (Jovanovic et al., 2000), axonal elongation, and maintenance ofsynaptic contacts (Brock and O'Callaghan, 1987). The transcription factor cyclic AMP
response element binding protein (CREB) is required for various forms of memory including
spatial learning (Silva et al., 1998) and appears to play a role in neuronal resistance to insult
in conjunction with BDNF (Walton et al., 1999). CREB is characterized by its ability tomodulate gene expression encoding BDNF and cell survival in the CNS (Tao et al., 1998, Ying
et al., 2002). GAP-43 is present in growing axon terminals and has an important role in axonal
growth, neurotransmitter release (Oestreicher et al., 1997), and learning and memory(Routtenberg et al., 2000).

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In the present study, we investigated the role of BDNF and some of its downstream molecularregulators in mediating the effects of exercise on the recovery of motor function after a SCI.
We used a specific molecule, i.e., trkB IgG, to block the function of BDNF during voluntary
exercise in rats hemisected at a cervical level. We hypothesized that BDNF modulation induced
by exercise plays a critical role in facilitating the recovery of motor function following a SCI.
The results, in general, support this hypothesis.

Exprimental Procedures
Animals and General Procedures
Male Sprague-Dawley rats at 2 months of age (n=34, Charles River, San Diego, CA) werehoused singly in standard polyethylene cages. After one week of acclimation, the animals wereassigned randomly to either a sedentary control group (Con, n=5) or one of four hemisectedgroups, i.e., sedentary (Sed) or exercised (Ex) with saline (Sal) injection, (Sed/Sal, n=8, Ex/
Sal, n=8), Sed or Ex with BDNF-inhibitor (IgG) injection (Sed/IgG, n=8, Ex/IgG, n=5). All
rats were housed in standard polycarbonate cages (10.25″ × 18.75″ × 8″) individually during
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the experimental period. Rats in the exercise groups were housed in standard cages equippedwith running wheels beginning one week after the cervical hemisection. The vivarium room
was maintained at 26 ± 1°C, with 40% humidity and a 12:12 h light: dark cycle. All rats were
supplied with rat chow and water ad libitum. The studies were approved by the UCLA
Chancellor's Animal Research Committee and followed the American Physiological Society
Animal Care Guidelines.
Cervical Hemisection Procedures

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All surgeries and injections were performed under aseptic conditions. The rats received ananalgesic (Buprenex, 1.0 mg/kg, s.c.) 45 min prior to surgery. Surgery was performed with therats anesthetized deeply with isoflurane gas (1.0 to 2.5%) via facemask and placed on a watercirculating heating pad maintained at 37°C to prevent hypothermia. A longitudinal dorsal
midline skin incision was made over the spinal column from ∼C2 to C6 and the muscles
overlying the vertebral column were reflected. A partial laminectomy was performed
approximately between vertebral levels C4 and C6. The dura mater was incised longitudinallyand lidocaine hydrochloride (1%; 2 or 3 drops) was applied at the hemisection site (∼C4). The
right one-half of the spinal cord was isolated using a specifically designed probe and thentransected using microscissors. Small cotton balls were used to separate the cut ends of thehemicord to assure a complete hemisection. Gelfoam was inserted between the cut ends of thehemicord. The paravertebral muscles and fascia surrounding the spinal column were sutured
using 4-0 Dexon and the skin incision was sutured using 4-0 Ethilon. The rats were allowed
to fully recover from anesthesia in an incubator (27°C) and were given lactated Ringers solution(5 ml, s.c.). PolyFlex (G.C. Hanford Manufacturing Co., Syracuse, NY), a general antibiotic,
was administered (100 mg/kg, s.c., twice daily) during the first 3 days of recovery.
Inhibitor Preparation and Cervical Spinal Cord Injection Procedures

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Recombinant Human TrkB/Fc Chimera (TrkB IgG) was used to block the BDNF action by
subtracting it from the media. It was in powder form when purchased from R&D systems, Inc.,
Mineapolis, MN. Sterile PBS containing 0.1% of bovine serum albumin (BSA) was added to
the vial to prepare a stock solution of 100 ug/mL. Saline was used as a standard control for
microspheres injection. Microspheres were used as the vehicle for drug insertion to spinal cord.
We prepared microspheres (Lumaflour, New York, NY) using the methods described by(Riddle et al., 1997) and used in our laboratory (Vaynman et al., 2004). These proceduresconsisted of coating the microspheres with each solution via passive absorbency by incubating
overnight at 4°C with a 1:5 mix of microspheres to trkB IgG or saline. The morning after
coating the microspheres, the solution was centrifuged at 14,000g for 30 min and themicrospheres were resuspended in sterile water at a 10% concentration. After one week ofrecovery from the surgery, the hemisected rats were assigned randomly into either a sedentary
or an exercise group. Under the same surgical conditions as described above, a partial
laminectomy was performed between C5 and C7 to expose the dorsal spinal cord. One half ofthe rats in each group were injected with trkB IgG (Sed/IgG and Ex/IgG groups) and the otherone-half with the same volume of a standard control saline solution (Sed/Sal and Ex/Sal
groups). For these injections, the vertebral column was stabilized on a stereotaxic apparatus
using clamps on the spinal processes at C2 and C9. TrkB IgG (5 ug/ul) or saline coated withmicrospheres were injected bilaterally at C5-C6 (0.6 mm lateral to midline, 1.5 mm below theduramater for the first injection, and 0.8 mm below the duramater for the second injection)using a Hamilton syringe in a volume of 2 μl over a 15 min period. For the next 14 days, therats in the exercised groups were housed in cages with voluntary running wheels, whereas the
non-exercised rats were housed in same type of cages but without running wheels. A fifth groupof rats served as a sedentary control group, i.e., no surgery, no exercise, and no injections. TrkB
IgG fusion proteins mimic the TrkB receptor and abolish BDNF function by sequestering free

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BDNF. We have successfully used TrkB IgG embedded in microspeheres to block BDNF
action for up to 14 days after the injection (Vaynman et al., 2004).

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Voluntary Exercise Procedures
The rats in the exercise groups (Ex/Sal and Ex/IgG) were placed in standard cages equippedwith running wheels that rotated against a resistance of 100 g (Ying et al., 2005). Sedentaryrats (Con, Sed/Sal, and Sed/IgG) were left undisturbed in their home cages. The numbers ofwheel revolutions were monitored and recorded by computer).
Biochemical Analyses

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The day after the last exercise session (14 days after the BDNF inhibitor or saline treatment),
all rats were decapitated in the early morning. The cervical spinal cord enlargement (C3-C6)
ipsilateral to the lesion was dissected rapidly, frozen on dry ice, and stored at −70° C until
processed. We used the RNA STAT-60 kit (TEL-TEST, Friendswood, TX) and the
manufacturer's protocol for total RNA isolation. The mRNAs for BDNF, NT-3, Synapsin I,
GAP-43, and CREB were measured in the total RNA obtained from individual animals by
TaqMan real time quantitative RT-PCR using the ABI PRISM 7700 Sequence detection system(Applied Biosystems), and TaqMan EZ RT-PCR Core reagents (Applied Biosystems). Thissystem directly detects the increase in fluorescence of a dye-labeled DNA probe specific foreach factor under study plus a probe specific for the glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene used as an endogenous control. The sequences of probes,
forward and reverse primers (Integrated DNA Technologies, Coralville, IA) were: BDNF:(5′AGTCATTTGCGCACAACTTTAAAAGTCTGCATT-3′), forward: (5′GGACATATCCATGACCAGAAAGAAA-3′), reverse: (5′GCAACAAACCACAACATTATC GAG-3′); NT-3: (5′TGACCGACAAGTCCTCAGCCATTGAC-3′); forward: (5′TGTGACAGTGAGAGCCTGTGG-3′), reverse: (5′-TGTAACCTGGTGTCCCCGAA-3′);synapsin I (5′CATGGCACGTAATGGAGACTACCGCA3′), forward: (5′CCGCCAGCTGCCTTC-3), reverse: (5′-TGCAGCCCAATGACCAAA-3′); GAP-43:(5′–
CTCATAAGGCTGCAACCAAAATTCAGGCT-3′), forward: (5′GATGGTGTCAAACCGGAGGAT-3′), reverse: (5′CTTGTTATGTGTCCACGGAAGC-3′); CREB: (5′-CATGGCACGTAATGGAGACTACC
GCA-3′); forward: (5′-CCGCCAGCATGCCTTC-3′), reverse: (5′TGCAGCCCAATGACCAAA-3′). The RT-PCR reaction conditions were for 2 min at 50°C
as an initial step to activate uracil glycosylase (UNG), followed by 30 min at 60°C as reverse
transcription and completed by UNG-deactivation at 95°C for 5 min. The 40 cycles of the twostep PCR-reaction conditions were 20 s at 94°C and 1 min at 62°C.

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Assessment of Quadrupedal Locomotor Ability
Prior to the hemisection surgery all rats were acclimated to running on a treadmill at speeds
ranging from 6 to 30 cm sec-1 for 10-15 min on two occasions. Thirteen days after treatment
with the inhibitor or saline injections, all rats were tested for quadrupedal locomotor capability.
The SIMI Motion Analysis System (zFlo; Quincy, Massachusetts) was used to collect videofrom 4 synchronized cameras at 100frames/sec. The forelimbs were shaved bilaterally andretroreflective markers were placed on bony landmarks at the shoulder, elbow, wrist, and tipof the 5th metacarpal-phalange. During the testing, the rats were stepped on a treadmill
beginning at 3 cm/sec and the speed was increased by ∼3.5 cm/sec until the rat could no longer
maintain that speed. The maximum speed of stepping was considered that speed at which the
rat could successfully complete 10 consecutive steps. All other measures related to thekinematics of stepping of the forelimbs were determined from the steps performed at the
maximum speed and normalized to a distance of 100 cm on the treadmill. Each step was

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considered weight-bearing or oscillating based on visual inspection of the video, i.e., a stepwas considered oscillating when the forelimb was actively moved forward and backward
regardless of the level of weight-bearing. The evaluators were blind to the group assignment.
The percent time in stance for each step cycle was determined as the time from toe-on to toeoff the treadmill. The number of weight-bearing steps and oscillating steps, and the stance
duration relative to step period are reported as the ratio between the right (non-hemisected)
and left (hemisected) sides. In addition, the proportion of steps that were weight-bearing on
the hemisected side is reported. The angle of the wrist relative to the movement forward at aplane perpendicular to the treadmill through the shoulder marker was used to indicate themaximum forward reaching position of both forelimbs.
Statistical Analyses

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For quantification of RT-PCR results, fluorescent signal intensities were plotted against the
number of PCR cycles on a semi logarithmic scale. The amplification cycle at which the firstsignificant increase of fluorescence occurred was designed as threshold cycle (Ct). The process
was automatically carried out by ABI sequence detector software version 1.6.3. GAPDH wasused as an endogenous control to standardize the amount of sample RNA. Measurements of
BDNF, synapsin I, CREB, NT-3, Gap-43 mRNA for individual animals were normalized withcorresponding GAPDH values. Statistical analysis was performed using GraphPad Prism 4.01
software (GraphPad Software, San Diego, CA). Overall differences in the biochemical andfunctional data were determined using analysis of variance (ANOVA) and individual groupdifferences were determined using Tukey's post-hoc tests. Statistical differences wereconsidered significant at p < 0.05. The functional measures are presented as right (hemisectedside)/left (contralateral side) ratios to show the degree of symmetry between the two forelimbs,
whereas the biochemical data are expressed as percent of sedentary control values for graphic
clarity. Values are reported as mean ± standard error of the mean (SEM) for 5 to 8 rats pergroup.

Results
The gait patterns of the forelimbs changed dramatically immediately after the cervicalhemisection. Although the locomotor capability improved with time, observable deficits inforelimb function remained throughout the experimental period. Multiple measures of thekinematics of the forelimbs were examined to gain a reasonably comprehensive understandingof the level and nature of the recovery that had occurred among the different groups of rats.
Effects of BDNF Blocking on Locomotor Performance

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The trajectory of the forelimbs ipsilateral (right) and contralateral (left) to the lesion wasassessed to critically evaluate the stepping performance. The level of impairment in the step
trajectories was greatest in the ipsilateral limbs of the rats treated with the BDNF blocker. This
impairment was reflected in the differences in the shape and the consistency of the trajectoryof the wrist during approximately 5 consecutive steps between the ipsilateral and contralateralforelimbs. Examples of the trajectories of the forelimb during the best stepping observed ineach group are illustrated in Figure 1. In general the symmetry of the Ex/Sal rats was moresimilar to that observed in control rats compared to the IgG-treated groups. Clearly, the mostmarked asymmetry in the trajectories was observed in the two groups receiving IgG. The swing
phase of one step cycle from the left and right forelimbs from one rat for each group is illustrated
as a stick diagram in the middle of Figure 1. The specific step cycle illustrated as a stick diagram
is shown in each trajectory plot as a bold line. These illustrations provide a readily visible
perception of the movement deficiencies that typically characterized each group of rats. As
shown in Figure 2F, the amount of forward region of the forelimb was compromised the mostin the right forelimb (lesioned side) of animals treated with the inhibitor.

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The maximum speed at which a rat can generate successful and consistent stepping serves as
one general measure of the neural control of forelimb function. There were no significant
differences in the maximum speed of quadrupedal locomotion among the groups, although the
Sed/IgG group had the slowest mean maximum speed of locomotion (Fig. 2A). The level ofsymmetry in the movement of the two forelimbs expressed as the ratio of the right limb
(ipsilateral to the hemisection)/left limb (contralateral to the hemisection) was assessed using
a series of parameters. There were no group differences in the ratio of the duration of the stancephase of a step relative to the cycle period duration (Fig. 2B). However, the ratio of the numberof backward and forward step-like motions (ratio of oscillating steps) during the locomotor
test was closer to unity in the rats not receiving the inhibitor compared to the IgG treated rats,
with the value being significantly lower in the Ex/IgG and in the Sed/IgG than in the Sed/Sal
and in the Ex/Sal groups (p< 0.05) (Fig. 2C). The Ex/Sal group showed a trend (p = 0.19) tohave the best performance. This measure included all oscillating movements independent of
whether there was any weight bearing in the limbs.

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The number of weight-bearing steps provided similar results, with the only significant
difference being a lower value in the Ex/IgG than the Ex/Sal group (Fig. 2D). The Ex/Sal group
showed a trend to have a better performance compared to the Sed/Sal group (p= 0.29). Figure
2E shows the proportion of weight-bearing steps on the hemisected side (right side), the Ex/
IgG group showed a marginally significant decrease compare to Sed/sal group (P<0.055). The
most discriminating feature of the forelimb movement was the distance that the wrist was
extended beyond an imaginary vertical line through the shoulder during the swing phase of thestep cycle. This distance was shorter on the lesioned than the non-lesioned side in both groups
treated with the inhibitor(Fig. 1F). Again, the Ex/Sal group showed a tendency (p=0.13) for
improvement compared to the Sed/Sal group on the lesioned side, and there was a significantly
lower value on the lesioned side in the Ex/IgG compared to the Ex/Sal group.
The general conclusion from these results on stepping symmetry is that the lesion limited theability of the affected limb to execute alternating swing-stance phases, to bear weight, and thedegree to which the forelimb could reach forward before initiating the next stance phase. In
addition, the level of impairment of these features, in general, was greatest in the rats treatedwith the BDNF blocker.
Effects of Exercise and BDNF Blocking on BDNF-Mediated Plasticity

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BDNF mRNA levels in the cervical hemicord ipsilateral to the cervical hemisection in Sed/
Sal rats decreased to 82% of Con (intact group) values (p<0.05), (Fig. 3A). After two weeks
of exercise, the BDNF mRNA levels in the Ex/Sal rats were increased to Con values (99% of
Con values). In the rats injected with the BDNF inhibitor, the levels of BDNF mRNA werereduced to 72% (Sed/IgG, p<0.05) and 81% (Ex/IgG, p<0,05) of Con values: these values also
were lower than in the Ex/Sal group. Synapsin I is a downstream effector for the action of
BDNF on synaptic plasticity. We found that synapsin I mRNA levels were significantly lower
in Sed/Sal than Con rats (71% of Con, p<0.05)), but similar (91% of Con) to Con in the Ex/
Sal rats (Fig. 3B). In rats receiving the inhibitor, the levels of synapsin I mRNA were reduced
to 80% (Sal/IgG, p<0.05) and 70% (Ex/IgG, p<0.05) of Con values. In addition, the levels in
the Ex/IgG group were significantly (P<0.05) lower than in the Ex/Sal group. The effects ofexercise were abolished by the BDNF inhibitor. We assessed the levels of BDNF inhibition
on another member of the neurotrophin family, i.e., NT-3, that has an important role in spinal
cord plasticity. NT-3 mRNA levels after the hemisection (Sed/Sal) were reduced to 67% of
Con values (p<0.05) (Fig. 3C). With exercise (Ex/Sal) the levels of NT-3 mRNA remained at
Con levels (91% of Con). NT-3 mRNA levels in sedentary with inhibitor rats (Sed/IgG) wereat the control levels (92% of Con), whereas these levels in the Ex/IgG group were reduced to
72% of Con value.

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CREB mRNA levels were determined based on the involvement of CREB in BDNF-mediatedneuronal plasticity (Barde, 1994, Acheson et al., 1995, Hardingham and Bading, 2002,
Hardingham et al., 2002). The levels of CREB mRNA in the Sed/Sal rats were reduced to 77%
of Con values (p<0.05) (Fig. 3D). In contrast, CREB mRNA levels in the Ex/Sal rats (107%of Con) were maintained at Con values, however, the values were elevated relative to theinjured group without exercise (Sed/Sal). The CREB mRNA levels in Sed/IgG and Ex/IgG
groups were 85% and 71% (P<0.05) of Con, respectively. Levels of GAP-43 were measured
to have an estimate of the interventions on axonal growth events. GAP-43 mRNA levels in
Sed/Sal rats were 91% of Con values (Fig. 3E). In contrast, the GAP-43 mRNA levels in Ex/
Sal rats (115%) were significantly (P<0.05) higher than Con values. The levels of GAP-43 inboth the sedentary and exercised rats treated with the inhibitor were similar to Con. The
GAP-43 values were significantly higher in the Ex/Sal than the Sed/Sal and Ex/IgG groups.
Relationships Between Motor Performance and the Levels of the Markers of Plasticity

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There was a positive relationship between the number of total wheel revolutions and the BDNF
mRNA levels in the Ex/Sal group (Fig. 4A, r = 0.71, p<0.05). Treatment using the BDNF
inhibitor trkB IgG reduced the correlation coefficient to r= 0.51, p=0.3772 (Fig. 4A). Therealso was a positive relationship between the ratio (R/L) of weight-bearing steps (the mostsensitive measure of stepping ability) and the levels of Synapsin I (Fig. 4B, r = 0.56, p<0.05),
and GAP-43 (Fig. 4D, r = 0.40, p<0.05) across all groups. Interestingly, the correlation between
the ratio of weight-bearing steps and either CREB mRNA (Fig. 4C, r = 0.34), BDNF mRNA
(Fig. 4E, r= 0.29), or NT-3 mRNA (Fig. 4F, r= 0.06) were not significant.

Discussion
Given the actions of BDNF on neuronal excitability and synaptic function and findings thatthe level of physical activity is an important modulator of BDNF in the spinal cord, we
examined whether the activity-mediated modulation of BDNF plays a central role indetermining the level of recovery of motor function following a SCI. We examined the effectsof cervical lateral hemisection on plasticity-associated molecules that are modulated as afunction of BDNF, in the periphery of the injured area. In addition, adult rats were injectedwith a BDNF blocker and exposed to voluntary running wheel exercise for 14 days. Blocking
BDNF abrogated the counteractive effects of exercise on the locomotor deficits. BDNF
blocking also limited the effects of exercise in counteracting the decrease in synaptic plasticityrelated molecules observed after the hemisection. These results indicate that BDNF plays animportant role in mediating the effects of exercise on the recovery of locomotion after a SCI.
Exercise and Locomotor Performance in the Injured Spinal Cord

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The interlimb coordination of the forelimbs of sedentary and exercised, hemisected rats had
some degree of asymmetry, and the level of asymmetry was greater in the inhibitor-treatedrats. The high level of asymmetry in both the Sed/IgG and Ex/IgG groups suggests that BDNF
inhibition was sufficient to limit spontaneous and exercise-induced motor recovery. Based on
the number of wheel revolutions, it appears that the level of exercise was similar in the Ex/Sal
and Ex/IgG groups. Some caution is warranted in the interpretation of these data because thenumber of wheel revolutions does not necessarily represent the use of the affected forelimb:e.g., the rats could run effectively in the wheel by running tripedally, i.e., not using the affected
forelimb. However, it has been observed previously that wheel running after a SCI improves
locomotor recovery and stimulates serotonergic fiber growth (Engesser-Cesar et al., 2007).
Accordingly, it is possible that the effects of exercise on spinal cord injured rats can be enhanced
using a more severe training paradigm, i.e., treadmill training.

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The ability of exercise to up-regulate BDNF in the spinal cord based on the amount of wheel
running (Fig. 4A) is consistent with exercise promoting CNS plasticity. The reduction in BDNF
levels observed after hemisection was normalized when the rats were allowed to run in a wheel.
It is clear that the level of wheel running was sufficient to increase the levels of BDNF andNT-3 mRNA relative to sedentary rats (Fig. 3A and 3C). This exercise effect also was evident
for several molecules that are modulated by BDNF and linked to synaptic plasticity such as
synapsin I (Fig. 3B), CREB (Fig. 3D), and GAP-43 (Fig. 3E). In contrast, the inhibitor
abolished the increase in the markers of synaptic plasticity and, in most cases, these marker
levels were somewhat lower in Ex/IgG than Sed/IgG rats. For example, blocking of BDNF
action abrogated the ability of exercise to maintain normal levels of synapsin I mRNA after
injury. BDNF phosphorylates synapsin I primarily through the trkB receptor to induce themitogen-activated protein kinase signaling pathway that modulates neurotransmitter release(Jovanovic et al., 2000). In addition to the effects of TrkB IgGs on blocking BDNF unction,
we cannot discard the possibility that the TrkB IgG intervention may have some detrimental
effects on the recovery from stepping not directly associated with BDNF blocking. Exercise
normalized the depressed levels of CREB mRNA after the hemisection, and the inhibition of
BDNF abolished the effects of exercise on CREB mRNA levels. CREB is one of the bestcharacterized transcription factors in the CNS and can be modulated by BDNF (Finkbeiner etal., 1997) CREB is phosphorylated by BDNF at the transcription regulatory site, and CREB
can feed-back on BDNF by regulating its gene transcription via a calcium-dependent
mechanism (Finkbeiner et al., 1997). CREB is required for several forms of memory (Silva etal., 1998) and appears to play a role in neuronal resistance to insult (Walton et al., 1999) these
are likely two basic components of functional recovery after a SCI. BDNF blocking in the
hippocampus using a similar trkB-IgG treatment has been shown to reduce the hippocampal
increase in synapsin I mRNA and CREB mRNA associated with exercise (Vaynman et al.,2004), consistent with the interpretation that BDNF may modulate synapsin I and CREB during
exercise.

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We have previously shown that exercise attenuates the decrease in GAP-43 observed after a
spinal cord hemisection (Ying et al., 2005). The present results provide new evidence that
BDNF may be involved in the effects of exercise on GAP-43 as the BDNF blocker abolishedthe effects of exercise on GAP-43. Given the role of GAP-43 on axonal growth,
neurotransmitter release, and learning and memory, it is likely that exercise-related increases
in GAP-43 can be associated with maintaining synaptic function (Oestreicher et al., 1997).
Indeed, a potential action of BDNF on synaptic plasticity associated with exercise would likely
involve mechanisms that support neurite outgrowth. BDNF also could facilitate locomotor
recovery through other mechanisms. It can stimulate locomotor-like activity in adult rats byincreasing the excitability of spinal locomotor networks (Jakeman et al., 1998). BDNF also
can affect muscle excitability. For example, it has been reported that BDNF stimulates therelease of acetylcholine at the neuromuscular synapse of cultured myocytes resulting in thepotentiation of spontaneous twitching (Kleiman et al., 2000).
It is notable that the biochemical markers of synaptic plasticity in the Ex/IgG rats were
somewhat lower than those of Sed/IgG rats (Fig. 3B, D, and E) and that this apparent exercise
effect on biochemical markers corresponds to consistently poorer stepping performance in the
Ex/IgG rats (Fig. 1C and D). This relationship can be seen more clearly by the correlations
between the behavioral performance and the levels of the biochemical markers (Fig. 4). The
correlation ratios suggest that there is a significant link between the levels of several mRNA's
that mediate plasticity and the recovery in weight-bearing stepping after a cervical hemisection.
Although several of these correlations are significant, it is apparent that there are othermodulatory factors that affect recovery in these hemisected rats. One possible explanation is

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NIH-PA Author Manuscriptthat the Ex/IgG rats indeed not only had less potential for mediating changes because of reduced
levels of BDNF (and NT-3), but when exercised there also was a reinforcement of an alreadyexisting asymmetry by practicing asymmetric stepping.
BDNF and NT-3 Interaction

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Spinal cord hemisection also reduced levels of NT-3 mRNA while exercise returned NT-3
mRNA to nearly normal levels. We have previously shown that a thoracic spinal cordhemisection does not affect levels of NT-3 in the lumbar enlargement (Ying et al., 2005). These
results appear to indicate that the rostrocaudal level of the injury or distance from the injury
site are important factors for the NT-3 modulation. It is interesting that exercise elevated theexpression of NT-3 in both of these studies, suggesting that exercise can affect injured tissue
as well as tissue distant from the injury site. It is intriguing that blocking BDNF maintained
NT-3 mRNA to near control levels in spinal cord hemisected rats. It is even more intriguingthat blocking BDNF abolished the effects of exercise on NT-3 elevation. These observations
may reflect a compensatory mechanism, as it is known that BDNF interacts with NT-3 for theirown expression regulation (Ullal et al., 2007). It is known that in spite of the high affinity of
trkB receptors for BDNF, these receptors can also be activated by NT-3. It is also significant
that trkB signalling can contribute to NT-3 activation. A study performed on axotomized adultrat rubrospinal neurons showed that application of BDNF in conjunction with NT-3 had a morepotent effect on neuronal survival than administration of either one alone. (Novikova et al.,2000).

Conclusions
Our results show that a spinal cord hemisection lesion reduced locomotor performance in rats,
and that voluntary exercise counteracted this impairment in locomotor ability. In addition,
blocking the BDNF action with a specific inhibitor abrogated the action of exercise inameliorating the behavioral deficit. These results are consistent with BDNF playing an activerole in mediating the improvement in stepping that occurs with appropriate rehabilitative
training after a SCI. It is, however, crucial to determine how the action of BDNF interfaces
with other factors and molecular signaling mechanisms activated by exercise to have acomprehensive outlook of the effects of exercise-induced BDNF on functional recovery.

Acknowledgments
this work was supported by NIH NS50465 (FGP), NS16333 (VRE), and Craig H. Nelson Foundation.

References
NIH-PA Author Manuscript

Acheson A, Conover JC, Fandl JP, DeChiara TM, Russell M, Thadani A, Squinto SP, Yancopoulos GD,
Lindsay RM. A BDNF autocrine loop in adult sensory neurons prevents cell death [see comments].
Nature 1995;374:450–453. [PubMed: 7700353]
Barde YA. Neurotrophins: a family of proteins supporting the survival of neurons. Prog Clin Biol Res
1994;390:45–56. [PubMed: 7724649]
Bregman BS, McAtee M, Dai HN, Kuhn PL. Neurotrophic factors increase axonal growth after spinal
cord injury and transplantation in the adult rat. Exp Neurol 1997;148:475–494. [PubMed: 9417827]
Brock TO, O'Callaghan JP. Quantitative changes in the synaptic vesicle proteins synapsin I and p38 andthe astrocyte-specific protein glial fibrillary acidic protein are associated with chemical-induced injury
to the rat central nervous system. J Neurosci 1987;7:931–942. [PubMed: 3106588]
Edgerton VR, Tillakaratne NJ, Bigbee AJ, de Leon RD, Roy RR. Plasticity of the spinal neural circuitry
after injury. Annu Rev Neurosci 2004;27:145–167. [PubMed: 15217329]

Neuroscience. Author manuscript; available in PMC 2010 January 12.

Ying et al.

Page 10

NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Engesser-Cesar C, Ichiyama RM, Nefas AL, Hill MA, Edgerton VR, Cotman CW, Anderson AJ. Wheelrunning following spinal cord injury improves locomotor recovery and stimulates serotonergic fiber
growth. Eur J Neurosci 2007;25:1931–1939. [PubMed: 17439482]
Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME. CREB: a major
mediator of neuronal neurotrophin responses. Neuron 1997;19:1031–1047. [PubMed: 9390517]
Frigon A, Rossignol S. Functional plasticity following spinal cord lesions. Prog Brain Res 2006;157:231–
260. [PubMed: 17167915]
Gomez-Pinilla F, Ying Z, Roy RR, Molteni R, Edgerton VR. Voluntary exercise induces a BDNFmediated mechanism that promotes neuroplasticity. Journal of Neurophysiology 2002;88:2187–2195.
[PubMed: 12424260]
Hardingham GE, Bading H. Coupling of extrasynaptic NMDA receptors to a CREB shut-off pathway isdevelopmentally regulated. Biochim Biophys Acta 2002;1600:148–153. [PubMed: 12445470]
Hardingham GE, Fukunaga Y, Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by
triggering CREB shut-off and cell death pathways. Nat Neurosci 2002;5:405–414. [PubMed:
11953750]
Jakeman LB, Wei P, Guan Z, Stokes BT. Brain-derived neurotrophic factor stimulates hindlimb steppingand sprouting of cholinergic fibers after spinal cord injury. Exp Neurol 1998;154:170–184. [PubMed:
9875278]
Jovanovic JN, Benfenati F, Stow YL, Sihra S, Sanghera JS, Pelecch SL, Greengard P, Czernik AJ.
Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate sinapsyn I-actin
interactions. Proc Natl Acad Sci USA 1996;93:3679–3683. [PubMed: 8622996]
Jovanovic JN, Czernik AJ, Fienberg AA, Greengard P, Sihra TS. Synapsins as mediators of BDNFenhanced neurotransmitter release. Nat Neurosci 2000;3:323–329. [PubMed: 10725920]
Kafitz KW, Rose CR, Thoenen H, Konnerth A. Neurotrophin-evoked rapid excitation through trkB
receptors. Nature 1999;401:918–921. [PubMed: 10553907]
Kleiman RJ, Tian N, Krizaj D, Hwang TN, Copenhagen DR, Reichardt LF. BDNF-Induced potentiationof spontaneous twitching in innervated myocytes requires calcium release from intracellular stores.
J Neurophysiol 2000;84:472–483. [PubMed: 10899220]
Lu B, Figurov A. Role of neurotrophins in synapse development and plasticity. Rev Neurosci 1997;8:1–
12. [PubMed: 9402641]
Oestreicher AB, De Graan PN, Gispen WH, Verhaagen J, Schrama LH. B-50, the growth associatedprotein-43: modulation of cell morphology and communication in the nervous system. Prog
Neurobiol 1997;53:627–686. [PubMed: 9447616]
Riddle DR, Katz LC, Lo DC. Focal delivery of neurotrophins into the central nervous system using
fluorescent latex microspheres. Biotechniques 1997;23:928–934. 936–927. [PubMed: 9383561]
Routtenberg A, Cantallops I, Zaffuto S, Serrano P, Namgung U. Enhanced learning after genetic
overexpression of a brain growth protein. Proc Natl Acad Sci U S A 2000;97:7657–7662. [PubMed:
10861025]
Silva AJ, Kogan JH, Frankland PW, Kida S. CREB and memory. Annu Rev Neurosci 1998;21:127–148.
[PubMed: 9530494]
Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME. Ca2+ influx regulates BDNF transcription
by a CREB family transcription factor-dependent mechanism. Neuron 1998;20:709–726. [PubMed:
9581763]
Vaynman S, Ying Z, Gomez-Pinilla F. Hippocampal BDNF mediates the efficacy of exercise on synaptic
plasticity and cognition. Eur J Neurosci 2004;20:2580–2590. [PubMed: 15548201]
Walton M, Connor B, Lawlor P, Young D, Sirimanne E, Gluckman P, Cole G, Dragunow M. Neuronal
death and survival in two models of hypoxic-ischemic brain damage. Brain Res Brain Res Rev
1999;29:137–168. [PubMed: 10209230]
Wang T, Xie K, Lu B. Neurotrophins promote maturation of developing neuromuscular synapses. J
Neurosci 1995;15:4796–4805. [PubMed: 7623111]
Ying SW, Futter M, Rosenblum K, Webber MJ, Hunt SP, Bliss TV, Bramham CR. Brain-derived
neurotrophic factor induces long-term potentiation in intact adult hippocampus: requirement for ERK
activation coupled to CREB and upregulation of Arc synthesis. J Neurosci 2002;22:1532–1540.
[PubMed: 11880483]
Neuroscience. Author manuscript; available in PMC 2010 January 12.

Ying et al.

Page 11

NIH-PA Author Manuscript

Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F. Voluntary exercise increases neurotrophin-3 and its
receptor TrkC in the spinal cord. Brain Res 2003;987:93–99. [PubMed: 14499950]
Ying Z, Roy RR, Edgerton VR, Gomez-Pinilla F. Exercise restores levels of neurotrophins and synaptic
plasticity following spinal cord injury. Exp Neurol 2005;193:411–419. [PubMed: 15869943]

Abbreviations
BDNF

brain-derived neurotrophic factor

CNS

central nervous system

CREB

cyclic AMP response element binding protein

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

SCI

spinal cord injury

UNG

uracil glycosylase

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Figure 1.

Examples of the trajectory of the ipsilateral (right) and contralateral (left) forelimbs during
stepping on a treadmill at the maximum speed for a representative rat in each group are shown.
Note that the highest level of asymmetry is present in the rats in the two groups receiving the
BDNF blocker. Stick figures of the swing phase of the shoulder (S), elbow (E) and wrist (W)of the left and right forelimb for one-step cycle for one rat from each group is illustrated in the
middle columns of the figure. The step cycle represented in the stick figure from the multiple
steps illustrated as wrist trajectories is marked with a bold line. The displacement values
represent absolute positions in space, but the multiple trajectories were transposed so that the
beginning of the stance phase was positioned consistently relative to the other step cycles.
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Therefore, the absolute vertical distance of the trajectory only approximates the relative
distance of the wrist to the surface of the treadmill belt. Group abbreviations are the same as
in Fig. 1.

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Figure 2.

The measures of locomotor performance are shown for each hemisected group: (A) maximum
speed of treadmill locomotion; (B) ratio of percent stance per step cycle; (C) ratio of oscillating
steps (weight-bearing plus non-weight bearing steps); (D) ratio of weight-bearing steps; (E)proportion of weight-bearing steps on the hemisected side (right side); and (F) the distance thatthe wrist was extended beyond the vertical line through the shoulder during the swing phaseof the step cycle for both the right and left forelimbs. All ratios are expressed as right limb (R,ipsilateral to the hemisection)/left limb (L, contralateral to the hemisection). Sed, sedentary;
Sal, saline, Ex, exercise: IgG, BDNF blocker. Values are mean ± SEM. * and +, significant
group difference and right-left difference, respectively, at P < 0.05.
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Figure 3.

Relative levels of mRNAs for BDNF (A), synapsin I (B), NT-3 (C), CREB (D), and GAP-43(E) in the cervical region of the ipsilateral hemicord in sedentary (Sed/Sal, Sed/IgG) andexercised (Ex/Sal, Ex/IgG) hemisected rats that received saline or trkB IgG treatment. Data
are presented as a percent of sedentary intact control rats. mRNA levels were measured using
realtime RT-PCR and corrected for equivalent levels of total mRNA using a GAPDH mRNAprobe in the same assay solution. Values are means ± SEM for 5-8 rats/group. * P < 0.05.

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Figure 4.

The relationship between BDNF mRNA levels and total wheel revolutions for each rat in the
Ex/Sal and Ex/IgG groups is shown in (A). The relationships between the levels of Synapsin
I, CREB, GAP-43, BDNF and NT-3 mRNA levels and the ratio (ipsilateral/contralateral to thehemisection) of weight-bearing steps are shown in (B), (C), (D), (E), and (F), respectively.
Group abbreviations are the same as in Fig. 1. * p < 0.05.

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