This study investigated combining deep brain stimulation (DBS), PTEN suppression, and Schwann cell transplantation as a treatment for spinal cord injury in rats. Rats received either DBS, PTEN suppression via AAV-shPTEN injection, or a combination of both, along with Schwann cell transplantation at the injury site. Locomotor activity was assessed over 8 weeks and showed no significant differences between groups. Preliminary immunohistochemistry results found that the combination of PTEN suppression and DBS resulted in higher volumes of white matter and serotonin compared to other groups. This suggests the combined approach may produce better tissue repair than individual methods alone. Further studies are needed to confirm these findings.

1. Introduction Methods
Results
Funding from National Institutes of Health NINDS NS083064!
There are approximately 12,000 spinal cords injuries (SCI) that occur in the United States each year, many of
which result in paralysis. Currently, there are several promising methods to improve symptoms of a SCI.
Previous work has shown that electrical deep brain stimulation (DBS) of the nucleus raphe magnus (NRM)
permanently improves various parameters of sensorimotor performance, while increasing the density of myelinated
fibers in white matter, as well as increasing serotonergic terminals in the gray matter. Furthermore, DBS was found
to restore levels of cAMP after injury.
Suppression of PTEN, a negative regulator of mTOR, activates this downstream pathway causing an improvement
in CNS axon regeneration after SCI. Re-formed synapses have also been observed. Despite the beneficial effects
of this suppression, regenerated axons still often fail to cross the lesion site.
Schwann cells (SC) transplanted into the lesion after injury provide protection, support axon regeneration along with
myelination, and ultimately improve function. Both PTEN suppression and cAMP have been shown to increase the
growth of implanted SCs.
Although these methods have seen positive results individually, a combined approach may offer a more effective
treatment. In this study, immunohistochemistry will be used to see if combining groups with both DBS and PTEN
suppression along with SCs produces histologically better tissue with more serotonin and white matter than either
group alone. A complete transection model is used, to allow unequivocal determination of fibers crossing the lesion.
Brainstem Injection
Following baseline BBB training, injections were made into the
brainstem of the rat with either AAV-shPTEN-GFP, in order to
delete PTEN, or AAV-shRNA again luciferase (AAV-shLuc-GFP).
GFP was used as a marker for descending axons to the spinal
cord.
Immunohistochemistry
Rats were perfused and the
entire brain and spinal cord
were extracted. This was kept
in 4% PFA temporarily and 12%
sucrose for long term storage.
The brain and spinal cord were
then embedded in gelatin serial
sagittal cryostat sections (20
µm thick) were mounted onto
slides.
Stimulator Implantation and Operation
A tungsten microelectrode integral with an epoxy-encapsulated
electronic stimulator (Figure 2) was stereotaxically positioned in
the NRM of mice under isofluorane anesthesia. The stimulator
delivered 30 µA, 1 ms, 8 Hz cathodal pulses for 5-minute
periods alternated with rest for 12 hours daily. A reed switch
allowed inactivation or parameter selection by pulsed magnetic
field. Stimulation was given for 14 days.
Injury and SC Transplant
Rats weighing 160-180 g received a complete transection at
thoracic vertebrae T8. The SCs were suspended in a 1:1 mix of
DMEM, F12 and matrigel. Lentiviral vectors encoding for
mCherry were used as a marker in order to later view the cells.
SCs were then injected into the transection site immediately
following the SCI.
Baseline BBB
8 week post Injury
Last BBB
Brainstem
Injection
Electrode Implant &
Stimulation
Stimulation
Ends
Euthanasia
SCI & Schwann cell
transplant
Day 1 BBB!
COMBINATORIAL STRATEGIES TO TREAT SPINAL CORD
INJURY
K. Purdy, Y.S. Lee, L. Freer, S.S. Sloley, A. Vitores, K. Park, M.B. Bunge, I.D. Hentall
The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, 33136
BBB Testing
Locomotor activity was assessed in adult female Fischer rats
using the Basso, Beattie, and Bresnahan (BBB) scale. Baseline
testing was done prior to the injury day and immediately
following. BBB data was collected once each week until
euthanasia.
CTRL_CTRL + - -
CTRL_DBS + + -
PTEN_CTRL + - +
PTEN_DBS + + +
SCs DBS PTEN
One set of spinal cord tissue was stained with the primary antibody
Serotonin produced in rabbit (5-HT; 1:500) coupled with a Goat 405
Anti- Rabbit secondary (1:250). A second set was stained with
FluoroMyelin in order to visualize white matter.
Stereo Investigator software was used to contour the spinal cord
sections and the Cavalieri estimator was used to estimate the
amount of white matter present. Confocal microscope images were
taken of the sections stained for serotonin rostral to the injury.
!
A
Figure 3. BBB scoring post injury over 8 weeks. (CTRL_DBS: n = 7,
CTRL_CTRL: n = 9, PTEN_DBS: n = 6, PTEN_CTRL: n = 5)
Above (Figure 3): Starting the day after injury, the rats’
locomotor activity was assessed for 8 weeks. Despite
initial increases, there was no statistical significance
between groups.
Figure 4. Immunofluorescent confocal images of stained spinal cord tissue. (n=1) !
Above (Figure 4): Images were taken rostral to injury site. Serotonin is represented in blue and axons present are shown in green. (A) Staining
for tissue that only received SC transplant. (B, C) A slight increase of 5-HT in tissue that received only DBS or PTEN removal.
(D) There appears to be an elevated amount of 5-HT present in rats the underwent both PTEN suppression and DBS.
Figure 5.
Calculated
volume of
white matter
found in the
tissue, in a 2
mm length
rostral to the
injury.
Right (Figure 5): The volume of white matter present was
calculated using software that analyzed the FluoroMyelin
staining. PTEN and DBS had a higher amount, and the
control had the lowest ratio of white matter. (n=2)
Experimental Timeline!
Figure 1. Stimulator set up and implantation
Figure 2. Set up of the different test groups.
A plus (+) indicates that the treatment was
received.!
PTEN_DBS
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
CTRL_CTRL CTRL_DBS PTEN_CTRL PTEN_DBS
RatioofWhiteMatter:Total
Group
White Matter Volume
1 2 3 4 5 6 7 8 9 10 11
Discussion
1. No effect of treatment was seen in the
behavioral findings.
2. Our preliminary results indicate that the
combination of PTEN and DBS had a higher
volume ratio of white matter.
3. There appears to be a higher volume of 5-HT
in the PTEN_DBS tissue than in other groups,
along with longer and better organized axons.
Further studies will need to confirm these
findings.
A
CTRL_CTRL
B
CTRL_DBS
D
PTEN_DBS
C
PTEN_CTRL
CTRL_CTRL
A
PTEN_DBS
B
Figure 6. High resolution
immunofluorescent confocal
images of stained tissue.
Left (Figure 6): Images
taken rostral to injury
site, marked in a white
dashed line. (A) Staining
revealed clusters of
axons with little 5-HT.
(B) PTEN and DBS
treated tissue showed
increased axon growth
and 5-HT.
Figure 7. Direct confocal
image of the injury site.