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Author Manuscript
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Published in final edited form as:
Exp Neurol. 2010 April ; 222(2): 211–218. doi:10.1016/j.expneurol.2009.12.029.

Functional Electrical Stimulation Helps Replenish Progenitor
Cells in the Injured Spinal Cord of Adult Rats
Daniel Becker1, Devin S. Gary1, Ephron S. Rosenzweig2, Warren M. Grill3, and John W.
McDonald1,*
1International Center for Spinal Cord Injury, Hugo Moser Research Institute, Department of
Neurology, Johns Hopkins School of Medicine and Kennedy Krieger Institute, 707 North Broadway,
Suite 518; Baltimore, MD 21205
2Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093
3Departments of Biomedical Engineering, Neurobiology, and Surgery, Duke University, Durham,

NC

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Abstract
Functional electrical stimulation (FES) can restore control and offset atrophy to muscles after
neurological injury. However, FES has not been considered as a method for enhancing CNS
regeneration. This paper demonstrates that FES dramatically enhanced progenitor cell birth in the
spinal cord of rats with a chronic spinal cord injury (SCI). A complete SCI at thoracic level 8/9 was
performed on 12 rats. Three weeks later, a FES device to stimulate hindlimb movement was implanted
into these rats. Twelve identically-injured rats received inactive FES implants. An additional control
group of uninjured rats were also examined. Ten days after FES implantation, dividing cells weremarked with bromodeoxyuridine (BrdU). The ‘cell birth’ subgroup (half the animals in each group)
was sacrificed immediately after completion of BrdU administration, and the ‘cell survival’ subgroupwas sacrificed 7 days later. In the injured ‘cell birth’ subgroup, FES induced an 82-86 % increase incell birth in the lumbar spinal cord. In the injured ‘cell survival’ subgroup, the increased lumbar
newborn cell counts persisted. FES doubled the proportion of the newly-born cells which expressed
nestin and other markers suggestive of tripotential progenitors. In uninjured rats, FES had no effecton cell birth/survival. This report suggests that controlled electrical activation of the CNS may
enhance spontaneous regeneration after neurological injuries.

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Keywords
cell birth; exercise; neural activity; spinal cord injury; regeneration; rehabilitation; stem cell

*To whom correspondence should be addressed: John W. McDonald, MD, PhD, Hugo W. Moser Research Institute at Kennedy Krieger,
International Center for Spinal Cord Injury, Department of Neurology, Johns Hopkins School of Medicine, 707 North Broadway, Suite
518, Baltimore, MD 21205, Tel: (443) 923-9210, Fax: (443) 923-9215, mcdonaldj@kennedykrieger.org.
#Work described in this manuscript has been carried out at Washington University School of Medicine in St. Louis, MO.
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Page 2Introduction
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Electrical stimulation is being used as a therapeutic tool to promote recovery of function
following nervous system injury and disease. Examples of clinical application of electricalstimulation include cochlear implants for restoring hearing, stimulation of lower motor neurons
to restore breathing and hand grasp, and deep brain stimulation to treat the symptoms of
Parkinson's disease (McDonald et al., 2002b). Electrical stimulation has also been used to offset
the disuse atrophy associated with paralysis (de Abreu et al., 2009).
Functional electrical stimulation (FES) assisted ergometry is currently being applied in theclinical setting for persons with spinal cord injury (SCI). There are numerous publications
demonstrating practical benefits from FES in persons with SCI including: increased muscle
mass (Hjeltnes et al., 1997; Scremin et al., 1999), improvements in bone density (Frotzler etal., 2009), enhanced cardiovascular function (Faghri et al., 1992), improved bowel function(Johnston et al., 2005), decreased spasticity (Daly et al., 1996) and reductions in bladderinfection rate (Kutzenberger et al., 2005). In addition, electrical stimulation could someday be
used in combination with other therapies to enhance functional recovery from SCI. One
example is the use of electrical stimulation to facilitate partial-body-weight-supported walking
in experimental animals following a spinal cord injury (Lavrov et al., 2008).

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A growing body of evidence suggests that electrical stimulation can promote peripheral andcentral nervous system repair following injury. In a model of rat femoral nerve transection andrepair, electrical stimulation promoted BDNF release from motor neurons and enhanced
preferential motor reinnervation across the distal nerve stump. This stimulation paradigm also
promoted functional recovery following femoral nerve repair (Ahlborn et al., 2007). In a similar
model, electrical stimulation restored the specificity of sensory axon regeneration into the
cutaneous branch of the femoral nerve. In addition, electrical stimulation promoted theexpression of growth associated protein-43 and enhanced the number of regenerating sensory
axons in the femoral nerve across the distal stump (Geremia et al., 2007). In a model of dorsal
column transection (level T8), electrical stimulation promoted regeneration of CNS axons fromdorsal root ganglia in a mechanism that likely involves cAMP signaling (Udina et al., 2008).
Finally, electrical stimulation applied to the cortical pyramids in rats enhanced synapse
formation in the spinal cord during development and following corticalspinal tract injury(Brus-Ramer et al., 2007; Takuma et al., 2002).

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This paper assesses whether FES-induced patterned activity in the chronically injured spinal
cord can enhance cellular indices of CNS regeneration. Neural activity plays a critical role innervous system development and plasticity as shown by activity-dependent gene expression(Sgambato et al., 1997), modification of synaptic strength (Daoudal et al., 2003), synapse
elimination (Zhou et al., 2004), cell survival (Jagasia et al., 2009), myelination (Coman et al.,2005), and perhaps new cell birth (McDonald et al., 2002a; Tongiorgi 2008).
Because of the dramatic reduction in neural activity below the injury level (Edgerton et al.,2001), our hypothesis is that increasing neural activity with FES may aid regeneration (Grillet al., 2001; McDonald et al., 2002a). To test this hypothesis, we provided patterned neuralactivity to the caudal spinal cord of chronically-injured rats. Our injury model was a complete
two segment resection of the spinal cord at T8/9, which eliminated both ascending anddescending activity across the lesion. Three weeks after injury, we implanted a 2-channel FES
system with leads attached to the peroneal nerves. This induced stepping-like movements of
the hindlimbs and patterned neural activation in the lumbar spinal cord in the area commonly
associated with the central pattern generator for locomotion (Lavrov et al., 2008). We thenmonitored the birth and survival of neural progenitor cells, which are present in the adult spinal

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cord, and can generate neural cells (Horky et al., 2006; Horner et al., 2000; Ishii et al., 2001;
McTigue et al., 1998; McTigue et al., 2001; Tripathi et al., 2007).

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Materials and Methods
Subjects, animal care and surgery
Thirty six adult female Long Evans rats (275 ± 25g; Simonsen, Gilroy, CA) were separated
into six groups. These animals were housed (12:12 h light:dark cycle) and treated in accordancewith the Laboratory Animal Welfare Act, and with Guidelines / Policies for Rodent Survival
Surgery (Animal Studies Committee of Washington University in St. Louis). All animals
received FES implants. ‘Short term’ refers to analysis 15 days after FES device implantation,
2h after the last BrdU injection; ‘long term’ refers to analysis 22 days after FES device
implantation, 7 days after the last BrdU injection. The six groups were:
Cell birth:
1.

SCI, NO FES activation, short-term survival (n = 6)

2.

SCI, FES activation, short-term survival (n = 6)
Cell survival:

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3.

SCI, NO FES activation, long-term survival (n = 6)

4.

SCI, FES activation, long-term survival (n = 6)
Uninjured controls:5.

NO SCI, NO FES activation, short-term survival (n = 6)

6.

NO SCI, FES activation, short-term survival (n = 6)

All SCI animals were fitted with 10.5 cm plastic collars to prevent autophagia (Ejay, Glendora,CA) and had their bladders expressed 3 times a day or until recovery of reflex urination. All
rats were handled for 5 minutes per day and housed individually with absorbent bedding(ALPHA-Dri™, Shepherd, Kalamazoo, MI).
Spinal cord injury

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Rats subjected to SCI (groups 1-4) were anesthetized (75 mg/kg Ketaset®, 0.5 mg/kg
Domitor®, i.p.) and laminectomy was performed at T8-T10. A 1-mm section of the spinal cord
at T9 was removed through a dural slit, using a BARON® suction tube (Roboz, Rockville,
Maryland). The dural opening was covered with fascia, and the muscle and overlying skin wereclosed with layered sutures. Anesthesia was reversed with Antisedan® 1 mg/kg. This ‘suction
ablation’ injury model helps maintain the integrity of the dura and major blood vessels, andtherefore produces less bleeding and secondary ischemia than traditional blade transection
injury.
Stimulator and electrode implantation
All animals were anesthetized as described above. Twenty-one days after SCI in groups 1-4,
a 2-channel battery-powered electrical stimulator (Dr. J.C. Jarvis, University of Liverpool)(Salmons et al., 1991) was implanted into each animal (Figure 1A). In groups 5-6, an FES
device was implanted into each of the uninjured rats. To insert the FES device, an incision wasmade in the skin of the lower back at the midline from L1 to L5. A subcutaneous pocket was
created around the incision and the FES device was inserted into it. A 0.5 cm incision wasmade on the lateral aspect of both legs overlying the common peroneal nerve. The stainlesssteel wire stimulating electrodes were tunneled bilaterally underneath the skin from the

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stimulator site to the incision in the leg. The wire electrodes were sutured into the tibialis
anterior muscle, adjacent to the common peroneal nerve. The return electrode was sutured near
the midline at the L4 paraspinal muscles. Intraoperative test stimulation was performed toensure that the peroneal nerve was being activated during stimulation, as indicated byalternating flexion of the right and left hindlimbs.
Electrical stimulation paradigm
Three days after FES implantation the devices were activated in groups 2, 4, and 6. The FES
unit was activated three times daily, for 1 hour at a time, between the hours of 8:00 AM and5:00 PM (Figure 1B). The FES pattern consisted of 1 s stimulation of one common peroneal
nerve followed by 1 s of rest; then the other common peroneal nerve was stimulated for 1 s
followed by 1 s of rest and the cycle was repeated. Stimulus pulses were monophasic, 3 V, 200
ȝs long, and were delivered at 20 Hz. This configuration of stimulation preferentially activateslarge myelinated fibers within the common peroneal nerve (Gorman et al., 1983; Grill et al.,1996), and produced alternating flexion of the hindlimbs that crudely approximated bilateralstepping.
Bromodeoxyuridine injection paradigm

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Beginning ten days after FES implantation, rats received daily injections of BrdU (50 mg/kg
i.p.) for five consecutive days. BrdU labels new cells by incorporating into replicating DNA(Dolbeare 1996; Horner et al., 2000). To determine the amount of cell birth, groups 1, 2, 5, and6 were sacrificed at the completion of BrdU injections. To determine the ability of newborn
cells to survive in vivo, groups 3 and 4 were sacrificed 7 days after the completion of BrdU
injections.
Tissue processing and immunohistochemistry
Rats were perfused intracardially with 0.1M PBS for 5 min followed by 4% paraformaldehyde(Sigma) for 15 min. We selected every 6th section (40 ȝm frozen) from spinal cord levels C2,
T1, T7, T11, L1, and L5 for anti-BrdU immunohistochemistry (Horner et al., 2000). The
sections were incubated in 2 N HCl for 60 min at 37°C, transferred to 0.1M borate buffer (pH8.5) for 20 min, and rinsed with PBS. Nonspecific labeling was blocked with 0.1% BSA in
0.1% Triton X-100/PBS for 60 min. A mouse monoclonal anti-BrdU antibody (1:600; Roche,
Mannheim, Germany) was incubated with the tissue overnight at 4°C. Then the tissue wastreated with a CY3-conjugated secondary antibody (1:2000; Jackson, West Grove, PA) in 2%
normal goat serum (NGS) for 60 min.

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To co-label the BrdU+ cells, we fixed the sections with 4% paraformaldhyde for 30 min after
applying the CY3-conjugated antibody. Sections were permeabilized, as needed, with 0.1%
Triton-X-100 for 60 min and blocked with 2% NGS for 60 min. Primary antibodies diluted in
2% NGS were applied to the sections for 2 h. Antibodies included: rabbit anti-NG2 (1:250,
Chemicon, Temecula, CA), mouse anti-Nestin (1:8, Developmental Studies Hybridoma Bank
- DSHB), rabbit anti-GFAP (1:4, Diasorin, Stillwater, MN), mouse anti-APC-CC1 (1:20,
Oncogene, Cambridge, MA), mouse anti-ED1 (1:100, Serotec, Raleigh, NC), mouse antiOX42 (1:100, Serotec), sheep anti-Glut-1 (1:30, Biodesign, Saco, MN), rat anti-CD31 (1:20,
BD Pharmingen, Lexington, KY), mouse anti-NeuN (1:200, Chemicon), mouse anti-TUJ1(1:200, Babco, Richmond, CA), guinea pig anti-doublecortin (1:3000, Chemicon), and mouseanti-PSA-NCAM (1:8, DSHB). Secondary antibodies (1:300, Molecular Probes, Eugene, OR)
were conjugated to Alexa 488. Primary and secondary control slides were included with eachstain series to control for secondary cross-reactivity and non-specific labeling. Note thatinterpretation of immunological phenotypic assessment in vivo should be tempered by the factthat such analyses have largely been deciphered in vitro.

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In a separate procedure, slices from three rats from each of the cell birth groups were stainedwith Hoechst 33342 (1:100,000, Molecular Probes, Eugene, OR) as an index of overall celldensity.
Confocal microscopy and quantification of BrdU- and neural phenotype-positive cells
At each spinal level sampled, we quantified the number of 1) BrdU-labeled cells as an index
of ‘newly born cells’; 2) Hoechst 33342-labeled nuclei as an index of ‘total cells’; and 3) BrdUlabeled cells co-labeled with the phenotypic markers noted above. Scanning laser confocal
microscopy was used to identify doubly-labeled cells. Object counts in 40 ȝm frozen sections
were completed using an unbiased optical fractionator procedure (Peterson 1999; West et al.,1991), with the assistance of a semiautomatic stereology system (Stereoinvestigator™,
Microbrightfield Inc., Brattleboro, VT) driving a Ludl X-Y motorized stage (Ludl Electronics
Products, Ltd., Hawthorn, NY). Fluorescent Images were acquired with a Magnafire® camera.
Rough boundaries of each cord cross-section were drawn under low magnification to delimit
the optical fractionator areal sampling fraction. Samples were then marked under higher
magnification (20×, UplanApo, 0.8 N.A.). The software randomly superimposed a sampling
grid with an unbiased counting frame (x = 122 ȝm, y = 110 ȝm) and moved in a raster pattern
to provide 12-15 counting frames within the cross-section of the cord. The top of the section
was marked and a guard focus height of 2 ȝm was set in the software. The optical thickness ofeach image was approximately 0.7 ȝm and the total optical disector height was 20 ȝm.

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Cells meeting the sampling criteria were marked interactively during the session. For each
BrdU+ or Hoechst+ cell, the complete cell nucleus was followed through the z-axis, and only
cells with a well-circumscribed, labeled nucleus and/or immunopositive cell body contained
entirely within the counting volume frame were considered positive. Based on the numbers ofmarked cells and the volume of the slices, the software calculated the total numbers anddensities of BrdU+ nuclei and Hoechst+ nuclei. Six slices per spinal level were quantified per
animal in each procedure. Each animal's mean value was then calculated, and the final N usedin each statistical analysis.
To avoid the confounds associated with immunolabeling cells within and immediately
surrounding the SCI site, we performed our analyses on spinal levels more distant from theinjury, where tissue necrosis and immune cell infiltrates are minimized, and where the total
numbers of cells and cord circumference are unchanged from uninjured conditions.
Statistical analysis

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Comparison between experimental groups was conducted by two-way ANOVA using
SigmaStat® and applying Tukey post-hoc tests. Comparisons were made for the total numberof BrdU+ cells and phenotypic markers between groups and within groups by spinal cord level.
For all analyses significance was accepted at p <0.05 with Bonferroni correction for multiple
tests.

Results
Cell birth groups
FES was associated with an increase in cell birth in the lumbar spinal cord (Table 1; Figure2A). There was no significant effect of FES on cell birth above the site of injury (Table 1;
Figure 2A). There was no effect of FES on cell birth in uninjured control rats (Table 1, andSupplement Figure 2).
Most of the new cells expressed markers associated with neural progenitor cells and glial cells,
especially in segments distant from the site of injury (C2, T1, L1, and L5). This is in stark

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contrast to the primarily inflammatory cells that reside at the injury level (McTigue et al.,2001). FES had no effect on the percentage of BrdU+ cells expressing GFAP or NG2 above
or below the injury (Figures 3 and 4). However, FES almost doubled the percentage of BrdU
+ cells expressing nestin at level L5 (Figure 5). To distinguish between BrdU+/nestin+
progenitors and proliferating postmitotic astrocytes, we also tested for GFAP expression. The
majority (>98%) of BrdU+/nestin+ cells did not express GFAP, which is consistent withtripotential or bipotential progenitor identity and not dividing astrocytes (Figure 6).
To examine the possibility that other non-neural cell types contributed to the total cell birth/
survival measured in this study, we assessed the presence of tissue macrophages, microglia,
and endothelial cells using antibodies to ED1, OX-42, and CD31 / Glut-1, respectively.
Characteristic cellular anatomy was also considered in these examinations (Figure 6). Colabeling of BrdU and these cellular markers was uncommon in uninjured groups. In the injured
groups, ED-1, OX-42, and CD31/Glut-1 co-labeled cells were largely associated with the injury
site. Less than 2% of total BrdU-labeled cells double-labeled with ED1 or OX-42 and <1.5%labeled with CD31/Glut-1. There were no substantial differences in these inflammationassociated markers between injured groups with or without FES.

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The qualitative distribution pattern of BrdU+ cells did not change with FES. Consistent withprevious reports (Horner et al., 2000), BrdU+ cells were most frequently found in white matter,occasionally found in gray matter, and rarely found surrounding the central canal.
FES had no effect on total cell density as measured by Hoechst labeling (average density acrosslevels, cells/mm3 ± SEM; Control: 72540 ± 5382; FES: 72873 ± 4089; Supplement Figure 1).
These values are comparable to a previous stereological electron-microscopy study in thefemale Wistar rat (Bjugn et al., 1993). This same study found no significant differences in celldensity as a function of spinal level, which is consistent with our observations (cells/mm3 ±SEM; C2, 66262 ± 2835; T1, 71363 ± 6248; T7, 71261 ± 2660; T11, 72585 ± 3237; L1, 80507± 6881; L5, 74260 ± 6553). This suggests that the selective sampling of tissue at sites distantfrom the injury successfully reduced the influence of inflammation and tissue loss on our cellcounts.
Cell survival groups
The FES-associated increase in newly-born cells in the lumbar spinal cord persisted in the cellsurvival groups (Table 1; Figure 2B). However, there was a mean loss of 28% of BrdU+ cells,
relative to the cell birth groups, independent of FES treatment (Table 1). Thus, FES did not
improve cell survival, but the FES-treated rats still had a larger pool of newborn cells than did
the untreated rats.

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The cell survival groups had fewer NG2+ newborn cells and more APC+ newborn cells than
did the cell birth groups. At T1, for example, percentages of newborn cells that were NG2+
decreased from 49±3% to 32±8%. APC+ newborn cells at T1 increased from 3±1% to 27±3%,
suggesting that NG2+ oligodendrocyte precursors may have matured into APC+
oligodendrocytes. This putative differentiation into APC+ cells was not dependent on FES.
We did not find any evidence that newborn cells had differentiated into neurons, despite ourcareful search using antibodies to early or late neuronal markers (TUJ1, PSA-NCAM,
doublecortin, and NeuN).
General observations
The injury itself resulted in a persistent increase, relative to uninjured control rats, in new cellbirth throughout the spinal cord (see Table 1; mean BrdU+ cells/mm3 ± SEM: cell birth, 2363
± 339; cell survival, 1700 ± 119; uninjured control, 778±34). Although BrdU might integrate
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into apoptotic cells during DNA repair (Cooper-Kuhn et al., 2002), adult cells induced toundergo necrotic or apoptotic death do not have detectable levels of BrDU (Magavi et al.,2002). Consistent with this observation, BrdU+ cells in our study did not co-label with antiactivated caspase-3 antibody, which marks apoptotic cells (data not shown). Therefore, BrdU
+ nuclei represented dividing cells or recently divided cells.

Discussion

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Our study suggests that FES can be used to enhance at least one common index of cellular
regeneration. Twelve days of hindlimb FES, initiated 24 days after a complete T9 SCI, induced
an 82-86 % increase in cell birth in the lumbar spinal cord. Taking into account the two-hourbioavailability of BrdU following i.p. injection, this translates to a minimum of 110,000 extra
cells per lumbar spinal level over the 5 days of BrdU labeling. This increase occurred
selectively in the lumbar spinal cord, the area expected to be activated by peroneal nerve
stimulation based on known anatomy (Rivero-Melian 1996; Swett et al., 1985) and previous
c-Fos expression studies (Jasmin et al., 1994). Although the effect of FES was primarily onprogenitor proliferation rather than survival, the increase in newly-born cells persisted for atleast seven days after cessation of the BrdU labeling procedure. There was no significant
difference in the total number of nuclei in the spinal cord across injury groups. This is an
expected outcome because BrdU labeled cells represent 2-4% of total nuclei and this study was
not powered to detect this effect. It is possible that cell death mitigates these results and thatFES may enhance cell survival. Although survival of BrdU labeled cells trended greater in the
injured FES group, this was not significant. Assessment of this potential benefit warrants future
study.
Phenotypic assessments
The spinal cord has a resident progenitor cell pool (Horner et al., 2000; Tripathi et al., 2007).
Phenotypic assessment of the newly born cells suggests that FES helps replenish this pool. In
the cell birth groups, the newborn cells expressed nestin, NG2, GFAP, and APC, findings mostconsistent with neural progenitors and glial cells. In addition, FES doubled the proportion of
newborn cells that were nestin+. We used triple labeling (BrdU/GFAP/nestin) and confocal
microscopy (Figure 6) to rule out the possibility that the increased nestin expression was due
to reactive astrocytosis caused by SCI or FES (Lin et al., 1995). Furthermore, our major findings
are at spinal segments distant from the injury site, making reactive gliosis an unlikely confound.

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In the cell survival groups, there was a decrease in the newborn NG2+ progenitor pool and acorresponding increase in the newborn APC+ oligodendrocyte population. This observation isconsistent with the interpretation of glial maturation from progenitors to oligodendrocytes.
Combined with the FES-associated increase in cell birth, this suggests that FES increased thenumber of putative oligodendrocytes in the spinal cord.
The number of BrdU-labeled microglia, macrophages, or endothelial cells was small and did
not substantially contribute to the increased cell birth/survival seen at L1 and L5, levels distantfrom the T8/9 injury site (Carlson et al., 1998). This conclusion is consistent with the very low
contribution (<1.5 %) of microglia and endothelial cells to total BrdU-labeled cells in thenormal spinal cord (Horner et al., 2000).
We found no evidence of new neurons in the spinal cord, consistent with work in the intact
adult spinal cord (Horner et al., 2000). It is possible that the survival period in this study was
insufficient to detect new neuron differentiation (Gage et al., 1995; Kuhn et al., 1996), and thelarge nuclear size of motor neurons may make detection of BrdU labeling difficult compared
to background labeling. It is also possible that the injured spinal cord lacks factors needed forneuronal differentiation. This speculation stems from observations that spinal cord progenitors
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can differentiate into neurons in culture or when transplanted into the rodent hippocampus(Shihabuddin et al., 2000) and that the spinal cord can support neural differentiation fromtransplanted embryonic stem cell-derived neural progenitors (Liu et al., 2000; McDonald etal., 1999).
Are the effects of FES on cell birth mediated by global mechanisms?
The complete transection model used in this study minimizes trans-lesion neuronal activity. In
addition, peroneal nerve stimulation is expected to enhance neural activity selectively in the
lumbar cord (Rivero-Melian 1996; Swett et al., 1985). This situation allowed us to differentiate
between global and local effects of FES. FES enhanced cell birth selectively in the lumbosacral
spinal cord, suggesting that the proliferative effects of FES were not mediated by global
mechanisms.
Relationship to previous studies and implications for studies of regeneration and recovery
of function

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A previous study of intact animals showed that new cell birth occurs in the spinal cord of rats,
but differentiation is normally limited to glial cells (Horner et al., 2000). The numbers ofnewborn cells observed in our uninjured rats are similar to the values obtained in that study. It
has also been shown that CNS injury temporarily enhances new cell birth (McTigue et al.,2001). Our study demonstrates that cell proliferation remains increased for nearly a month after
SCI and is present at sites distant from the injury.

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Several lines of evidence also suggest that electrical activity, in the form of action potentials
down the axon, plays a necessary role in oligodendrocyte development. Barres et al. performed
optic nerve transection in post-natal day 8 rats and post-natal day 12 mice, which led to a
reduction in oligodendrocyte progenitor cell proliferation and survival. To determine theimpact of neuronal activity in this experimental model, the authors performed experiments
utilizing the Wlds mutant mice which show delayed Wallerian degeneration (by approximately
2 weeks). In these experiments the delayed degeneration phenotype rescued oligodendroglial
death but not the decrease in proliferation; suggesting that oligodendroglial proliferation but
not survival is dependent on neuronal activity (Barres et al., 1993; Barres et al., 1999). Although
there is some controversy, evidence exists that electrical activity in the axon can promote
myelination during development. Demerens et al. showed that intravitreous injection of TTX
in post-natal day 4 rats led to a significant decrease in myelinating oligodendrocytes by day 6.
However, TTX had no impact on myelination when administered on day 5. Likewise, TTX
application to mixed CNS cultures at distinct time-points, suppressed the appearance ofmyelinating segments (Demerens et al., 1996). In addition to work demonstrating the necessityof action potentials in promoting myelination, additional studies have supplied evidence that
action potentials are sufficient to enhance myelination. Fields and colleagues have shown thatinducing action potentials in dorsal root ganglion neurons, via electrical stimulation, canpromote myelination in Schwann cells and oligodendrocytes (Fields 2006). Taken together,
these findings suggest that electrical activity in axons is an important aspect of the developmentof oligodendroglia and may promote myelination.
In intact animals, exercise can lead to enhanced birth/survival of hippocampal dentate granuleneurons (Kempermann et al., 2000). Furthermore, passive cycling exercise after SCI in rats
attenuates muscle atrophy and increases the birth of muscle satellite cells (Dupont-Versteegden
et al., 1999). Taken together with the results of this study, the evidence suggests that
optimization of neural activity is important for optimizing spontaneous regeneration.
Determining whether increased cell birth and survival translate to recovery of function will
require additional analyses in models of incomplete injury. Our data suggest that the neural

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Page 9activity associated with FES may enhance markers of regeneration in addition to the presumed
motor training effects and physical integrity benefits.

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Implications for recovery after SCI
This report provides the first demonstration that FES can enhance cell generation in the injured
adult CNS. Both passive movement and FES-induced movement should produce patterned
sensory feedback to the spinal cord, provided lower motor neuron function is intact. However,in the clinic, the substantial physical benefits of resistive active (FES) exercise compared topassive limb movements make FES strategies a more pragmatic clinical treatment superior topassive strategy; physical integrity benefits include bones, muscles, and the cardiovascularsystems (Daly et al., 1996; Faghri et al., 1992; Frotzler et al., 2009; Johnston et al., 2005;
Scremin et al., 1999). New clinical data suggest that there is a possibility for recovery offunction in chronic SCI patients with FES treatment (Johnston et al., 2008; McDonald et al.,2002a; Szecsi et al., 2009; Wilder et al., 2002). The dramatic effect of FES on cell birth in the
injured spinal cord may partially mediate such recovery. The results reported here demonstrate
the need for further studies of activity-based recovery/restoration therapies in animal models
and for individuals with SCI, given that the physical benefits of active FES exercise aresufficient rationale for treatment initiation. These results raise the exciting possibility that inaddition to the physical and rehabilitative benefits of FES, FES may offer a pragmatic approach
to augmentation of spontaneous repair and perhaps recovery of neurological function.

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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
We thank L. Sage and D. Lenihan for editorial assistance, B. Jones for animal care, and A. Hansen for animal careand tissue processing. Supported by grants from the Barnes-Jewish Hospital and Barnes-Jewish Hospital Auxiliary
Foundations (St. Louis MO), Washington University School of Medicine (St. Louis, MO), Sam Schmidt Foundation(Las Vegas, NV), the Jack Orchard and ALS Hope Foundations (St. Louis, MO), and NIH NINDS NS 45023, 40520,
39577 (JWM), State of Maryland Department of Health and Mental Hygiene Spinal Cord Injury Grant FHA077-002(JWM), as well as the Johnson & Johnson Focused Giving Program (WMG).

Abbreviations
SCI

Spinal cord injury

FES

Functional electrical stimulation

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Figure 1. Experimental design

Schematic diagrams of (A) the sites of injury and FES device implantation, and (B) the 43-day
experimental design. In uninjured control groups (Supplement Figure 2), no SCI was induced,
but other event timings were identical.

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Figure 2. FES promoted new cell birth in the damaged spinal cord

BrdU-labeled cells were counted in six animals per group at six levels in coronal spinal cord
sections using stereological methods (optical fractionator). Two-way ANOVA demonstrated
effects of treatment group and spinal level (p<0.05). (A) FES induced a selective increase incell birth that was confined to the lower lumbar spinal cord, segments predicted to experience
increased activity from the patterned peroneal nerve stimulation. (B) This effect persisted in
the cell survival group 7 days later (*p < 0.05; **p < 0.001, FES vs. control).

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Figure 3. FES did not affect the proportion of BrdU+ cells that were also NG2+

NG2+/BrdU+ cell distribution (arrows; A, T1; B, L5) and morphology (C-E) in the cell birthgroups was consistent with glial progenitor cells. Panels C-E show NG2 staining (C; green),
BrdU staining (D, red), and their co-localization (E). There were more NG2+/BrdU+ cells
above the injury level in both groups (two-way ANOVA; 47 ± 3% at T1 vs. 33 ± 3% at L5 incontrol animals, *p < 0.05; 51 ± 3% at T1 vs. 39 ± 3% at L5 in FES-treated animals, **p <0.001). Scale bar (A,B) = 50 ȝm, (C-E) = 10 ȝm.

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Figure 4. FES did not affect the proportion of BrdU+ cells that were also GFAP+

GFAP+/BrdU+ cell distribution (arrows; A, T1; B, L5) and morphology (C-E) in the cell birthgroups was consistent with astrocytes. Panels C-E show GFAP staining (C; green), BrdU
staining (D, red), and their co-localization (E). There was no difference in the number of GFAP+/BrdU+ cells above and below the injury (44 ± 4% at T1 vs. 40 ± 3% at L5 in control animals;
46 ± 3% at T1 vs. 39 ± 3% at L5 in FES-treated animals). Scale bar (A,B) = 50 ȝm, (C-E) =10 ȝm.

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Figure 5. FES selectively doubled the proportion of BrdU+ cells that were also nestin+This increase (**p < 0.001) was only observed below the injury level, in spinal segments
predicted to experience increased activity from the patterned stimulation. Nestin+/BrdU+ cell
distribution (arrows; A, T1; B, L5) and morphology (C-E). Panels C-E show nestin staining(C; green), BrdU staining (D, red), and their co-localization (E). Some of the nestin+ cells
showed morphologic characteristics of reactive astrocytes (A, asterisk) but these were rare atlevels distant from the injury. As with NG2 (Figure 3), but in contrast to GFAP (Figure 4),
there were more nestin+/BrdU+ cells above the injury level in both groups (two-way ANOVA,
24 ± 2% at T1 vs. 10 ± 3% at L5 in control animals, *p < 0.001; 27 ± 2% at T1 vs. 19 ± 2% at
L5 in FES-treated animals, **p < 0.001). Scale bar (A,B) = 50 ȝm, (C-E) = 10 ȝm.

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Figure 6. BrdU+/nestin+ cells exhibited the distribution, morphology, and staining specificity
consistent with adult-derived bi- or tri-potential progenitor cells(Horner et al., 2000; Shihabuddin et al., 2000). Confocal microscope images of ventrolateralwhite matter of an FES-treated rat in the cell birth group. Nestin immunoreactivity shown inblue, BrdU labeling shown in red. A, D, and G show overlays of nestin and BrdU; B, E, andH show overlays of BrdU and other markers; C, F, and I show triple overlays. (A-C) Nestin
immunoreactivity (blue) was found predominantly in white matter (L5 shown). Less than 2%of nestin+/BrdU+ cells co-labeled with GFAP (green, arrowhead). (D-F) A few BrdU+/nestin+ cells also expressed NG2 (green, arrowhead). These triple-labeled cells were distinguishedfrom other BrdU+/nestin+ cells by their pial location and their morphologies (L5 shown). (GI) Macrophages, labeled with ED1 (green), were located within and surrounding the injury site
(T11 shown). We did not identify a substantial number of NG2+/ED-1+, nestin+/ED1+ cells.
(J-L) BrdU+ oligodendrocytes. Representative examples of the distribution pattern and cell
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morphology of BrdU+/APC-CC1+ cells (arrowheads) located at levels above (J, C2) andbelow (K, L5) the injury. These features are most consistent with oligodendrocytes, as is thefact that the number of BrdU+/APC-CC1+ cells increased substantially in the cell survivalgroups (see text). (L) Single confocal section of a BrdU+/APC-CC1+ cell. Scale bar (A-K) =50 ȝm, (L) = 10 ȝm.

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Table 1

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ᒚ± SEM116

ᒚ± SEM

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160

21

772
94

Cell Count

Volume mm3×10-2

Cells per mm3

ᒚ± SEM

Uninjured controls

1413

19

Volume mm3×10-2

Cells per mm3

270

Cell CountSCI Cell survival groups

1559

Cells per mm3

21

332

Cell Count

Volume mm3×10-2

No
FES

SCI Cell birth groups

C2

140

789

22

172

102

1521

20

307

195

1948

22

425

FES

141

833

13

110

114

1371

15

200

328

2092

13

273

No
FES

T1

106

699

14

98

122

1544

15

226

229

2295

14

326

FES

93

861

12

100

203

2338

11

261

317

2961

10

307

No
FES

T7

95

767

12

95

146

2437

11

276

477

2917

12

336

FES

158

799

13

101

165

1808

10

187

354

2963

12

346

No
FES

T11

102

936

13

120

158

1893

13

247

159

2724

12

314

FES

127

947

15

141

88

1315

14

184

370

1952

14

270

No
FES

L1

87

733

16

116

97

1856

17

316

920

3555

15

495

FES

85

608

20

119

110

1156

20

227

225

1188

19

226

No
FES

L5

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Effect of FES (shaded values) on BrdU+ cell counts in injured and uninjured rats

83

586

21

123

122

1748

19

328

415

2201

20

440

FES

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