REDUCING MALADAPTIVE SENSORY NEURONAL GROWTH TO TARGET
BELOW-LEVEL PAIN FOLLOWING SPINAL CORD INJURY
by
Blaire M. Conner
A Senior Honors Project Presented to the
Honors College
East Carolina University
In Partial Fulfillment of the
Requirements for
Graduation with Honors
by
Blaire M. Conner
Greenville, NC
May 2015
Approved by:
Sonja K. Bareiss, Ph. D., PT
Department of Physical Therapy College of Allied Health Sciences
1
I hereby declare I am the sole author of this thesis. It is the result of my own work and is
not the outcome of work done in collaboration, nor has it been submitted elsewhere as
coursework for this or another degree.
Signed:
Date: 4/28/2015
Blaire M. Conner
2
REDUCING MALADAPTIVE SENSORY NEURONAL GROWTH TO TARGET
BELOW-LEVEL PAIN FOLLOWING SPINAL CORD INJURY (SCI)
Chronic neuropathic pain is a common, debilitating consequence of spinal cord injury
(SCI). Up to 94% of the SCI population suffers from SCI pain, with over half reporting it
as their worst medical problem. Modern day methods of SCI pain management are
ineffective. Recent evidence suggests that this pain is due, in part, to aberrant outgrowth
of sensory neurons at and below the level of injury. We have previously shown that SCI
results in phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK-3β), a key
regulator of neuronal growth. The purpose of this study was to characterize the timedependent nature of SCI-induced sensory neuron outgrowth below the level of injury, and
to establish an optimal timeframe for application of a GSK-3β activator, in an effort to
block SCI-induced sprouting and the development of below-level pain. Long-Evans rats
received a dorsal horn injection of quisqualic acid (SCI) or saline (sham operated control)
and were sacrificed 1, 3, 14 and 22 days following surgery. At the designated time points,
DRGs ipsilateral to the site of injection were disassociated, cultured and analyzed for
neurite outgrowth and length. In the second experimental approach, rats received
intrathecal delivery of the GSK-3β activator (LY294002) the first 3 days after injury and
were sacrificed 14 days following surgery. Time course studies show a graded increase in
below-level growth responses following SCI. Intrathecal administration of LY294002,
initiated at the time of injury, significantly reduced below-level DRG neurite outgrowth
14 days post-SCI. Additionally, LY294002 treatment prevented the development of
below-level hyperalgesia. Based on these results GSK-3β may be involved in the
3
modulation of abnormal sensory growth responses following SCI, and might constitute a
new therapeutic target to prevent below-level SCI pain.
4
Acknowledgements:
We thank Maurice Smith, Lindsey Cannon, Morgan Rowe and Alysha Wonka for
their technical assistance. Special thanks to Sonja Bareiss Ph. D., PT for her dedication,
time served as an outstanding mentor and for making this research possible. This work
was funded by the Craig H. Neilsen Foundation, Wooten Foundation for
Neurodegenerative Disease Research, and a grant from East Carolina University
Undergraduate Research and Creative Achievement Awards.
5
Table of Contents
Introduction
7
Materials and Methods
10
Results
14
Discussion
20
Conclusions
25
References
27
6
REDUCING MALADAPTIVE SENSORY NEURONAL GROWTH TO TARGET
BELOW-LEVEL PAIN FOLLOWING SPINAL CORD INJURY (SCI)
1. Introduction:
Chronic, neuropathic pain is a significant secondary consequence of traumatic and
ischemic spinal cord injury (SCI). Up to 94% of the SCI population suffers from SCI
related pain (1), with over half reporting it as their worst medical problem (2). SCI pain is
particularly resistant to treatment, and modern day methods are unsuccessful in pain
management, forcing patients to live with debilitating pain (1-4). Below-level
neuropathic pain differs from at-level pain, defined as pain presenting greater than three
segments caudal to the level of injury (5, 6). Below-level pain can be either spontaneous
or stimulus-evoked (3), and includes sensory abnormalities such as below-level
mechanical allodynia and thermal hyperalgesia (3-5, 7, 8). The prevalence of below-level
pain is reported anywhere from 19% to 97.1% in the SCI population, averaging around
54% (1, 3, 7, 9, 10). Unfortunately, due to the complex nature of spinal cord injury
pathology, little is known of the mechanisms leading to chronic pain syndromes of SCI
patients.
Previous studies have investigated the contribution of spinal and supraspinal
mechanisms to below-level chronic pain following injury (11). However, an increasing
amount of evidence is suggesting contributions of structural plasticity, intrinsic growth,
and hyperexcitability of peripheral DRG neurons in the development of below-level SCI
pain (12-16). Studies also suggest that synaptogenesis of primary afferents and
nociceptors into the dorsal horn below the level of the lesion contribute to development
of pain (17-20). Recently, we have shown that excitotoxic quisqualic acid induced injury
7
results in outgrowth of below-level sensory neurons (13). This model reliably produces
sensory abnormalities related to below-level pain syndromes, including allodynia and
hyperalgesia (21). Although strong evidence supports the role of peripheral plasticity in
the development of below-level pain, the mechanisms responsible for this maladaptive
growth remain unclear.
One proposed mechanism involves glycogen synthase 3β (GSK-3β), an
intracellular signaling molecule that is abundant in the nervous system (22, 23). GSK-3β
is a serine/threonine kinase that is constitutively active in cells, and is inhibited by
phosphorylation of Ser-9. GSK-3β is a downstream target of many neurotrophic signaling
cascades that lead to inhibition of GSK-3β, and consequential neuronal outgrowth that
may contribute to neuropathic pain development (24-27). When active, GSK-3β acts as a
suppressant of neuronal growth and induces neurite retraction and growth cone collapse
(26, 28-30). In response to nervous system injury, an up-regulation of neurotrophic
factors such as nerve growth factor (NGF) leads to activation of phosphatidylinositol 3kinase (PI3K), which inhibits GSK-3β (25, 28, 31). The mechanisms of the PI3K-GSK3β pathway that regulate neuronal growth are fairly well studied. Although there is some
evidence that PI3K mediated inhibition of GSK-3β is involved in below-level pain
mechanisms (24, 32), what remains unclear is the precise role of PI3K-GSK-3β signaling
in peripheral DRG neurons following SCI, and its contribution to below-level sprouting
and neuropathic pain.
In this study, we completed a characterization of the time-dependent nature of
below-level sensory neurons to establish an optimal timeframe for treatment with a
pharmaceutical PI3K inhibitor and GSK-3β activator, LY294002, in an effort to block
8
SCI-induced sprouting and the development of below-level pain. We found that QUISinduced SCI results in persistent growth initiation and early (3-14 days) neurite
elongation of below-level sensory neurons following an isolated thoracic injury. We then
demonstrated that early PI3K mediated GSK-3β activation was successful in preventing
QUIS-induced sensory outgrowth and development of below-level pain.
9
1. Materials and methods
2.1. Animals and surgery for excitotoxic SCI
All experiments were evaluated and approved by the Institutional Animal Care
and Use Committee of East Carolina University. Surgical procedures and excitotoxic
injury model are previously described by Yezierski, et al.(21). In summary, male Long
Evans rats (200-225g) were anesthetized with isoflurane and prepped for surgery. An
incision was made on the posterior midline and muscle layers were removed to expose
the thoracolumbar junction. A laminectomy was performed at the levels of T11-L1, and
the dura was incised longitudinally and reflected. Intramedullary injections of volume 1.2
μl, 125 mM quisqualic acid (QUIS/injury) or 1.2 μl of phosphate buffered saline (PBS)
(Sham/control) were administered using a glass micropipette (5-10 μl tip diameter)
attached to a 10 μl Hamilton syringe. The syringe was mounted to a microinjector (Kopf
5000) connected to a micromanipulator. Injections were made unilaterally at T12 into the
dorsal horn of the spinal cord, 1000 μm below the surface, over a 60-s time interval. After
surgery, the muscle and skin incisions were sutured. For the investigation of the timedependent growth of below level sensory neurons, animals were euthanized at 1, 3, 14,
and 22 days (D) post-surgery and DRGs were isolated and prepared for culturing.
Animals were distributed in the following groups: Sham 1D (n=5), QUIS 1D (n=5),
Sham 3D (n=5), QUIS 3D (n=5), Sham 14D (n=5), QUIS 14D (n=8), Sham 22D (n=8),
QUIS 22D (n=10).
10
2.2. Intrathecal drug delivery
Immediately following saline or QUIS injection, animals receiving drug delivery
had a polyethylene catheter (PE-10 tubing) inserted subdurally into the intrathecal space
directly caudal to the level of injury. The catheter was secured by suturing the tubing to
spinal muscles along the incision. The rostral end of the catheter was tunneled under the
skin, externalized at the base of the occiput and secured with sutures and skin adhesive
(VetBond). Spinal muscle and skin incisions were closed around the catheter with staples.
Animals from both groups (Sham and QUIS) were randomly selected to receive
intrathecal delivery of LY294002 (PI3K inhibitor, 0.5µg in 10µl of vehicle) or an
equivalent volume of vehicle (10% DMSO). The drug was delivered once a day for the
first 3 days following surgery. Animals were euthanized at 14 days post-surgery and
DRGs were isolated and prepared for culturing. Animals were distributed in the
following groups: 14 day Sham vehicle (veh) n=9, QUIS (veh) n=12, QUIS (LY) n=11.
2.3. Below-level thermal pain behavior
Below-level evoked pain is characterized in this model as below-level
hyperalgesia, or a heightened sensitivity to pain in the hind-paws (21). Below-level
hyperalgesia was determined using a Hargreaves apparatus to measure hind-paw
withdrawal latencies from a noxious thermal stimulus (33). Behavioral testing was
performed as previously described (21). Briefly, animals were placed on a raised
plexiglas platform and allowed to acclimate for 15 minutes. Once acclimated, a heat
source located under one hind-paw was activated for a maximum of 30 seconds to avoid
skin damage or injury. The time is recorded until the animal exhibits a withdrawal
11
response. Three trials were performed on each hind-paw and a mean latency was
calculated for each day. Animals were assessed 7 days prior to surgery (baseline) and at 7
and 14 days post-injury for changes in thermal thresholds.
2.4. DRG cultures
At the designated survival time points (1, 3, 14, and 22 days) animals were
anesthetized with isoflurane and DRGs ipsilateral to the injection site were collected
below the lesion (L4-L5). Approximately two DRGs were isolated from both Sham and
QUIS animals and placed in Hibernate A (BrainBits, Springfield, IL) with 10% horse
serum and 100 microgram/L penicillin, 100 microgram/l streptomycin. Neurons were
cultured as previously described by Twiss et al. (34). Briefly, DRGs were rinsed in
plating media (DMEM/F12+N2+glutamine+horse serum+penicillin/streptomycin) and
disassociated mechanically and enzymatically via microsnipping and trituration, followed
by centrifugation and incubation with collagenase (Sigma, St. Louis, MO) and 0.25%
trypsin (Invitrogen, Grand Island, NY). Cells were plated at low density onto 12mm
coverslips, previously coated with poly-L-lysine and laminin, and allowed to incubate in
plating media at 37.0° C for twenty-four hours. Cell density was 142 ± 34 cells per
coverslip 24 hours after dissociation.
2.5. Morphological analysis
DRG neurons were fixed with 4% paraformaldehyde and rinsed with PBS 24
hours after plating. Neurons were permeablized with 0.2% Triton, incubated with 100
mM glycine, and blocked with 10% bovine serum albumin (BSA). Cells were stained
12
with a neuronal-specific growth marker, rabbit anti-tubulin III (Sigma, St. Louis, MO,
1:75 dilution) antibody conjugated to immunofluorescent marker, secondary antibody
Cy3 (Jackson ImmunoResearch, 1:300 dilution). Coverslips were mounted onto slides
using Pro-Long Gold anti-fade with DAPi (Invitrogen) for visualization of the nucleus.
Images were taken at 20x and 40x magnification using a Leica DM4000 microscope and
Q-imaging Retiga 2000R camera. The morphological data analysis was completed using
Image Pro Express software. The measurement feature was used to quantify the soma
size of all neurons, classified by the following parameters: small (≤30.4 μm), medium
(30.5-40.4 μm), and large (≥40.5 μm). The tracing feature was used to measure the length
of the longest neurite greater than the soma size and exceeding 25 μm. The percent of
sprouting neurons (above soma size exceeding 25 μm) and the average length of the
longest neurite were reported for each condition. DRGs from each experimental group
(sham 1D,QUIS 1D, sham 3D, QUIS 3D, sham 14D, QUIS 14D, sham 22D, QUIS 22D,
sham vehicle QUIS veh, QUIS LY) were pooled from below the level of lesion. A
minimum of 3 coverslips totaling greater than 200 neurons were analyzed from each
condition.
2.6. Statistical analysis
Statistical analysis was performed using GraphPad Prism version 5.04 (San
Diego, CA) and data were reported as a mean + SEM. Significance was set at p ≤ 0.05.
One-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for
between group comparisons or paired t-tests were used to determine differences between
the experimental means. Pearson Chi-Square analysis method was used for categorical
data (% neurons with neurites).
13
Figure 1. First experimental approach (Exp 1): tissue was harvested at 1, 3, 14, and 22
days post-surgery to assess time-dependent sensory neuron outgrowth. Second
experimental approach (Exp 2): intrathecal delivery (i.t.) of a GSK-3β activator,
LY294002 (LY), was administered for 3 days following injury. Animals were assessed
for below-level evoked pain responses (hyperalgesia) and sensory neuron outgrowth 14
days following surgery.
2. Results
3.1. Excitotoxic SCI Induces Early Outgrowth of Below-Level Sensory Neurons
Our previous reports show that QUIS-induced SCI results in enhanced DRG neurite
outgrowth at 14 days post-surgery below the level of injury (13). Similar reports of
enhanced DRG growth and hyperexcitability suggest that these peripheral changes may
be induced early after SCI (12, 14). In order to further characterize the time-dependent
outgrowth of below-level sensory neurons, we assessed DRG growth responses 1, 3, 14,
and 22 days following injury. We found that QUIS-induced SCI results in an increase of
neurons with neurites (above soma size growth exceeding 25 m) at 1 day (21%,
n=neurons with neurites/neurons, n=144/675, p<0.01), 3 days (18%, n=51/278, p<0.01),
14 days (21%, n=64/304, p<0.05), and 22 days post-surgery (27%, n=69/251, p<0.0001)
14
compared to saline-injected (sham) controls at 1 day (14%, n=50/350), 3 days (8%,
n=17/224), 14 days (9%, n=48/533) and 22 days post-surgery (8%, n=17/209) (Fig. 2B).
We also found that neurite elongation showed significant (p<0.05) differences between
sham and QUIS at 3 days post-surgery (90.8 m + 14.1, n=38; 164.8 m + 33.8, n=31)
and 14 days post-surgery (53.3 m + 6.4, n=48; 124 m + 28.5, n=64) (Fig. 2A, C).
Interestingly, enhanced growth was evident at 1 day post-surgery in both sham (189.4 m
+ 25.9, n=46) and QUIS (198.8 m + 17.1, n=136) groups (Fig. 2A, C), which may be
related to surgical induced responses. A similar trend was evident at 22 days post-surgery
(84.8 m + 12.9, n=119) (Fig. 2A, C), however differences between sham and QUIS
groups did not reach statistical significance demonstrating tapering growth effects
following spinal injury. These findings suggest that QUIS induced SCI results in early (314 days post-injury) abnormal growth responses of sensory neurons below the level of
injury, suggesting that early growth initiation and neurite elongation may be an important
contributor to below-level neuropathic pain following SCI.
A
Sham 3D
QUIS 1D
QUIS 3D
QUIS 14D
QUIS 22 D
Below Level Average Maximum Length
Below Level % Neurons with Neurites
B
C
15
Figure 2. QUIS-induced SCI promotes below-level sensory neuron outgrowth at 1, 3,
14 and 22 days post-injury. A. Representative images of cultured sensory (DRG)
neurons from Sham 3D, QUIS 1D, QUIS 3D, QUIS 14D, and QUIS 22D animals. B.
Quantification of % neurons with neurites show significant increases in neurons initiating
growth at 1, 3, 14, and 22 days post-injury, (data reported + SEM. *p< 0.05, **p<0.01,
***p<0.001, ****p<0.0001). C. Average length of the longest neurite show neurite
elongation (length) was significantly increased 3 and 14 days post-injury (mean ± SEM;
*p< 0.05). Scale bar = 50 m.
3.2. Short-Term Intrathecal Treatment with GSK-3β Activator (LY294002) Prevents
Outgrowth of Below-Level Sensory Neurons
Previous studies demonstrate that spinal cord injury induces abnormal growth
responses of DRG neurons that are associated with sensory dysesthesias and pain (12,
13). Although mechanisms responsible for injury induced growth are largely undefined,
recent reports show that GSK-3β is inhibited in the spinal dorsal horn and DRG
following central and peripheral nervous system injury (35, 36). GSK-3β is a key
regulator of neuronal growth, where inactivation leads to elongating neuronal growth (2426). In our second set of experiments, we investigated the effect of pharmaceutical GSK3β activation on preventing early outgrowth of below-level neurons after SCI. Using
LY294002, a known GSK-3 activator, we assessed growth responses 14 days following
surgery from the following groups of animals: sham vehicle (sham veh; 10% DMSO,
n=7) control, QUIS vehicle (QUIS veh; 10% DMSO, n=12), or QUIS LY294002 (QUIS
LY, 2.5 g /10 l, n=11). Consistent with our previous results, animals from QUIS veh
16
showed an increase in neurons with neurites (21%, n=64/304) compared to sham veh
controls (9 %, n=48/533, p<0.05) (Fig. 3B). Neurite elongation was also most robust in
QUIS veh (124 m + 28.5) animals compared to sham veh (52.3 m + 6.3, p<0.05)
groups (Fig. 3A, C). QUIS animals that received LY294002 daily for the first 3 days
following surgery showed significantly reduced neurite initiation (10%, n=68/686,
p<0.05) and elongation (41.3 m + 2.8, n=68, p<0.01), comparable to sham veh controls
(Fig. 3A, B, C). These data suggest that enhanced below-level neuronal growth induced
by SCI can be blocked by GSK-3β activation via LY294002 treatment.
A
Sham veh
B
QUIS veh
QUIS LY
Below Level % Neurons with Neurites
C
Below Level Average Maximum Length
Figure 3. QUIS-induced SCI animals that received LY for 3 days after injury show
sensory neuron growth responses similar to controls. A. Representative images of
cultured sensory (DRG) neurons from sham veh, QUIS veh, and QUIS LY animals. B.
17
Quantification of % neurons with neurites and C. Average length of the longest neurite.
Bar graphs show a significant decrease in neurons initiating growth and reduced neurite
length post-injury (data reported + SEM. *p< 0.05, **p<0.01, ***p<0.001, p<0.0001).
Scale bar = 50 m.
3.3. Intrathecal Delivery of the GSK-3β Activator (LY294002) Reduces Below-Level Pain
Targeted excitotoxic QUIS-induced spinal cord injury consistently produces belowlevel pain responses associated with spinal segments caudal to the lesion (21). All injured
animals develop below-level sensory abnormalities common to neuropathic pain
syndromes, independent of the at-level dysesthesias (grooming behavior) in this SCI
model (13, 21). Changes in below-level sensitivities develop 10-14 days after injury, and
once present, show no signs of reversal (21). To examine the effect of LY294002 on the
development of below-level hyperalgesia, withdrawal responses to a noxious thermal
stimulus were evaluated in QUIS veh and QUIS LY animals prior to injury (baseline), 7
and 14 days post-injury. Baseline latencies averaged 8.8 + 0.3 s for QUIS veh and 9.3 +
0.3 s QUIS LY (Fig 4). No differences in thermal thresholds were observed at 7 days
post-injury between QUIS veh (9.0 + 0.6s) and QUIS LY (9.1 + 0.4 s) (Fig 4). At 14 days
post-injury animals that received vehicle showed a trend toward decreasing latencies (7.9
+ 0.4 s) compared to baseline (p=0.06), whereas those that received intrathecal
LY294002 treatment showed no change in thermal thresholds (9.0 + 0.4 s) from baseline
(9.3 + 0.3 s) or non-injured levels (p=0.64) (Fig. 4). These results suggest that the
development of below-level pain, correlating with the reduction of peripheral outgrowth,
was prevented by early, short-term LY294002 treatment.
18
Time Points
Time in seconds
9.75
9.25
8.75
8.25
7.75
QUIS LY
LY
QUIS veh
DMSO
7.25
Baseline
7D post-SCI 14D post-SCI
Figure 4. Latency of withdrawal from a noxious thermal stimulus was similar in
animals prior to SCI (baseline). No changes in thermal thresholds were seen at 7 days
post-injury. At 14 days, animals receiving QUIS veh showed a trend toward decreasing
latencies compared to baseline (p=0.06) while those receiving LY showed no change
(p=0.64).
19
3. Discussion
In this study we demonstrated a role for PI3K-GSK-3β signaling in below-level
maladaptive outgrowth of DRG neurons following QUIS-induced spinal cord injury. Our
investigation of the time-dependent growth indicated persistent neurite initiation and
early neurite elongation (3-14 days) of sensory neurons below the level of spinal injury.
Early, short-term intrathecal drug delivery of a PI3K inhibitor and GSK-3β activator,
LY294002, reduced both growth initiation and elongation of DRG neurons to non-injured
levels and prevented the development of below-level pain after SCI. These data suggest
SCI-induced sensory outgrowth preceded the development of below-level pain, proposing
that sensory outgrowth may contribute to below-level evoked pain responses.
4.1. Peripheral Growth Responses and Below-Level Pain
Previous studies surrounding SCI pain have primarily focused on central nervous
system mechanisms of pain development (4, 8, 11, 15, 16, 19). However, the current
study along with previous results from our lab suggest that peripheral growth of DRG
neurons post-SCI contributes to the development of below-level sensory abnormalities
and pain (13). Reports by Bedi et al. support these findings, showing that intrinsic growth
of sensory neurons below the lesion is enhanced after SCI in a contusion model,
suggesting this growth is not model-specific (12). This group has also shown that SCI
induced spontaneous activity and a hyperactive state in below-level sensory neurons that
correlated with increased below-level sensitivities and hyperalgesia (14). These factors
may coincide with the documented erroneous synaptogenesis of primary afferents into
the dorsal horn, leading to amplification of pain pathways that contribute to chronic pain
20
after SCI (17-20). In support of peripheral contributions to pain, Krenz et al. reported an
increase in fiber density of myelinated afferents in the dorsal horn below the level of
spinal cord transection persisting for two weeks, and an increased area of unmyelinated,
nociceptive-labeled fibers at 2 weeks (20), suggesting that both nociceptive and nonnociceptive fibers may contribute to the onset of evoked-pain behaviors we observe at 14
days post-injury. Therefore it is possible that rerouting of various fiber types contribute to
below-level pain.
4.2. GSK-3as a Potential Regulator of Sensory Growth following SCI
Although maladaptive sensory afferent plasticity contributes to the development
of pain post-injury, the mechanisms that mediate this growth are unknown. There is
significant evidence for GSK-3in the regulation neurotrophic sensory growth through
interactions of microtubule stabilization (28), axon polarity (26), growth cone collapse
proteins (29), and other cytoskeletal substrates (27, 30). Despite GSK-3’s established
role in mediating neuronal growth, its link to pain and sprouting is undefined. Emerging
studies suggest that GSK-3is inhibited in the dorsal horn of the spinal cord following
central and peripheral nervous system injury (35, 36). Alterations in GSK-3 activity in
sensory afferent projections following nervous system injury suggest that it may play and
important function in modulating sprouting and pain. Furthermore, our reversal of SCI
induced below-level growth and evoked pain with GSK-3 activator treatment provides
further evidence to support a role for GSK-3 in sensory growth and pain. Future studies
will examine biochemical changes of GSK-3activity in the DRG and spinal cord dorsal
horn below the lesion in an effort to correlate this with altered growth.
21
4.3. Characterization of Aberrant Below-Level Sensory Growth
This study is the first to provide a detailed characterization of the time-dependent
nature of SCI-induced sensory outgrowth in segments caudal to the SCI. Here we showed
that elongating growth was significant at 3 and 14 days following SCI, with a tapering
effect observed at 22 days post-injury. Consistent with our results, Bedi et al. found that
elongating growth was robust below the lesion at 3 days post-injury compared to
controls, but no longer present at 1 month post SCI (12). Bedi et al. also demonstrated a
persistent enhancement of neurite initiation 1 month after SCI which is consistent with
the enhanced neurite initiation seen at 22 days in our study (12).
We and others have reported varying effects of growth in small nociceptive fibers,
medium proprioceptors, and large mechanoreceptive neurons post-SCI (12, 13). Bedi et
al. reports that elongating growth is observed in small and medium sized neurons at 3
days post-injury, but not large (12). However, previous studies from our lab show an
elongating response in large neurons (in addition to small and medium neurons) at 14
days post-SCI (13). This delayed growth of large neurons could be due to the temporal
nature of growth in various primary afferent populations, or the progressiveness of the
excitotoxic injury model. Future investigations of below-level growth will include these
size distinctions, which is important for determining preferential growth of sensory fibers
and their contributions to below-level pain. Sensory growth following SCI differs from
conditioning peripheral nerve injury models, showing increased neurite initiation and
arborization of small nociceptive labeled fibers, rather than an elongating response (37).
However, this may be due to phenotypic change of myelinated and unmyelinated fibers
22
after injury, differences in central vs. peripheral injury, or suggest that other mechanisms
regulate intrinsic growth state of different classifications of DRG neurons (37). Further
studies are needed to determine whether elongation versus branching is primarily
involved in the development of pain post-SCI.
We found that early, short term treatment with a PI3K inhibitor, LY294002, was
able to indiscriminately block SCI-induced growth (elongation and initiation) and prevent
the development of below-level pain. Findings by Xu et al. also show that early
intrathecal treatment (1 and 3 days post-injury) with known PI3K inhibitors can attenuate
below-level thermal pain responses following peripheral nervous system injury, however
treatment initiated at 7 days post-injury showed no improvement in pain thresholds (38),
suggesting that early intervention is necessary to potentially block elongating growth of
sensory sprouting leading to pain. Collectively, these results suggest a role for PI3Kmediated growth in early stages of neuropathic pain development, potentially attributed
to elongating growth of sensory neurons.
Interestingly, we found robust growth responses of initiation and elongation at 1
day post-injury for both sham and QUIS groups. Time course studies of peripheral nerve
injury present elongating growth at 1 day post injury and persistent growth initiation
following a conditioned lesion (39). In contrast, Bedi et al. reports that growth initiation
between sham and injury groups was similar early at 3 days after injury, but not
elongation, suggesting different mechanisms are responsible for the varying morphology
(12). A potential mechanism of growth in the sham condition may be related to incisional
or surgical trauma, causing pro-inflammatory influences on peripheral growth responses;
however the mechanisms responsible for these changes are unexplained. One study
23
suggests that skin incision causes up-regulation of axonal regenerative genes, similar to
responses seen after peripheral nerve injury, which may induce changes in structural
plasticity of DRG sensory neurons leading to pain (40). Experiments are underway to
provide comparisons between naïve, incisional, sham and QUIS groups to investigate the
possible contribution of post-surgical responses on neurite outgrowth. Further studies of
more refined time points could offer insight into a time-dependent nature of different
mechanisms regulating growth.
4.4. PI3K-GSK-3Signaling and Below-Level Pain
This study provides the first evidence for the role of PI3K-GSK-3signaling in
the development of below-level pain after SCI. Although many studies demonstrate the
role of GSK-3 pathways in mediating structural plasticity and chronic pain (26, 28-30),
emerging research suggests that GSK-3contributes to other mechanisms of pain
development as well. Reports by Weng et al. not only show that GSK-3 is expressed in
the dorsal horn, but found that altered function of GSK-3 following peripheral nerve
injury enhanced glial glutamate transporter protein expression, a mechanism that is
responsible for changes in neuronal activity leading to below-level pain (35). Conflicting
with our results, treatment with GSK-3inhibitors was shown to decrease thermal pain
responses in a peripheral nerve injury model (35). This variance in targeting GSK3(activation vs. inhibition) may be attributed to differences in drug delivery methods
(pre-emptive intraperitoneal injection), injury models (central vs. peripheral nerve) and
pain states (acute vs. chronic). It is plausible that these mechanisms targeting GSK-
24
3inhibition regulate pain in the late stage, and GSK-3activation contributes to
peripheral sprouting in early pain development.
Although PI3K-GSK-3signaling has an established role in sensory neuronal
growth, the PI3K pathway also activates inflammatory pain mechanisms that may
contribute to post-SCI pain (32). Studies have shown that NGF/capsaicin-induced PI3K
activation in sensory neurons results in inflammatory heat hyperalgesia via extracellular
protein kinase (ERK) which contributes to the onset of inflammatory pain (32). Injection
with PI3K inhibitors, LY294002 and wortmannin, successfully prevented inflammatory
thermal hyperalgesia (32). This suggests that activating GSK-3through the PI3K
pathway may also prevent inflammation to block the development of pain (32, 35).
Further studies are needed to define the various contributions of PI3K-GSK-3 signaling
relating to sprouting vs. inflammatory mediated pain.
1. Conclusion
In summary, we identified PI3K-GSK-3β signaling as a regulator of aberrant
sensory growth that contributes to the development of below-level pain following SCI.
We characterized the time-dependent growth of below-level sensory neurons and found
that QUIS-induced injury results in persistent growth initiation and early (3-14 days)
neurite elongation of sensory neurons several segments below an isolated thoracic injury.
We also showed that early, short-term GSK-3β activation with a pharmaceutical PI3K
inhibitor, LY294002, is sufficient to prevent SCI-induced growth and the onset of belowlevel thermal pain. Our findings provide information on a novel target for reducing
25
peripheral outgrowth, offering a potential therapeutic to the clinical dilemma of chronic
neuropathic pain management following spinal cord injury.
26
References
1.
Turner JA, Cardenas DD, Warms CA, McClellan CB. Chronic pain associated
with spinal cord injuries: A community survey. Archives of Physical Medicine and
Rehabilitation. 2001;82(4):501-8.
2.
Ravenscroft A, Ahmed YS, Burnside IG. Chronic pain after SCI. A patient
survey. Spinal Cord. 2000;38(10):611-4.
3.
Siddall PJ, Taylor DA, McClelland JM, Rutkowski SB, Cousins MJ. Pain report
and the relationship of pain to physical factors in the first 6 months following spinal cord
injury. Pain. 1999;81(1–2):187-97.
4.
Finnerup NB, Baastrup C. Spinal cord injury pain: mechanisms and management.
Current pain and headache reports. 2012;16(3):207-16.
5.
Siddall PJ TD, Cousins MJ. Classification of pain following spinal cord injury.
Spinal cord. 1997;35(2):69-75.
6.
Widerström-noga E, Biering-sørensen F, Bryce T, Cardenas DD, Finnerup NB,
Jensen MP, et al. The International Spinal Cord Injury Pain Basic Data Set. Spinal Cord.
2008;46(12):818-23.
7.
Finnerup NB, Johannesen IL, Sindrup SH, Bach FW, Jensen TS. Pain and
dysesthesia in patients with spinal cord injury: A postal survey. Spinal Cord.
2001;39(5):256-62.
8.
Finnerup NB, Jensen TS. Spinal cord injury pain-mechanisms and treatment.
European Journal of Neurology. 2004;11(2):73-82.
9.
Siddall PJ, McClelland JM, Rutkowski SB, Cousins MJ. A longitudinal study of
the prevalence and characteristics of pain in the first 5 years following spinal cord injury.
Pain. 2003;103(3):249-57.
10.
Nakipoglu-Yuzer GF, Atci N, Ozgirgin N. Neuropathic pain in spinal cord injury.
Pain Physician. 2013;16(3):259-64.
11.
Yezierski RP. Pain following spinal cord injury: pathophysiology and central
mechanisms. Nervous System Plasticity and Chronic Pain. 2000;129(0):429-49.
12.
Bedi SS, Lago MT, Masha LI, Crook RJ, Grill RJ, Walters ET. Spinal cord injury
triggers an intrinsic growth-promoting state in nociceptors. Journal of Neurotrauma.
2012;29(5):925-35.
13.
Bareiss SK, Gwaltney M, Hernandez K, Lee T, Brewer KL. Excitotoxic spinal
cord injury induced dysesthesias are associated with enhanced intrinsic growth of sensory
neurons. Neuroscience Letters. 2013;542(Journal Article):113-7.
14.
Bedi SS, Yang Q, Crook RJ, Du J, Wu Z, Fishman HM, et al. Chronic
spontaneous activity generated in the somata of primary nociceptors is associated with
pain-related behavior after spinal cord injury. The Journal of Neuroscience.
2010;30(44):14870-82.
15.
Deumens R, Joosten EA, J, Waxman SG, Hains BC. Locomotor dysfunction and
pain: the scylla and charybdis of fiber sprouting after spinal cord injury. Molecular
Neurobiology. 2008;37(1):52-63.
16.
Brown A, Weaver LC. The dark side of neuroplasticity. Experimental Neurology.
2012;235(1):133-41.
27
17.
Ondarza AB, Ye Z, Hulsebosch CE. Direct evidence of primary afferent sprouting
in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and
CGRP. Experimental Neurology. 2003;184(1):373-80.
18.
Christensen MD, Hulsebosch CE. Spinal cord injury and anti-NGF treatment
results in changes in CGRP density and distribution in the dorsal horn in the rat.
Experimental neurology. 1997;147(2):463-75.
19.
Christensen MD, Hulsebosch CE. Chronic central pain after spinal cord injury.
Journal of Neurotrauma. 1997;14(8):517-37.
20.
Krenz NR, Weaver LC. Sprouting of primary afferent fibers after spinal cord
transection in the rat. Neuroscience. 1998;85(2):443-58.
21.
Yezierski RP, Liu S, Ruenes GL, Kajander KJ, Brewer KL. Excitotoxic spinal
cord injury: behavioral and morphological characteristics of a central pain model. Pain.
1998;75(1):141-55.
22.
Woodgett JR. Judging a protein by more than its name: GSK-3. Sci STKE 2001,
2001:re12.
23.
Leroy K, Brion JP. Developmental expression and localization of glycogen
synthase kinase-3β in rat brain. Journal of Chemical Neuroanatomy. 1999;16(4):279-93.
24.
Dill J, Wang H, Zhou F, Li S. Inactivation of glycogen synthase kinase 3
promotes axonal growth and recovery in the CNS. The Journal of Neuroscience.
2008;28(36):8914-28.
25.
Jones DM, Tucker BA, Rahimtula M, Mearow KM. The synergistic effects of
NGF and IGF-1 on neurite growth in adult sensory neurons: convergence on the PI 3kinase signaling pathway. Journal of neurochemistry. 2003;86(5):1116-28.
26.
Jiang H, Guo W, Liang X, Rao Y. Both the Establishment and the Maintenance of
Neuronal Polarity Require Active Mechanisms: Critical Roles of GSK-3β and Its
Upstream Regulators. Cell. 2005;120(1):123-35.
27.
Seira O, Del Río JA. Glycogen synthase kinase 3 beta (GSK3beta) at the tip of
neuronal development and regeneration. Molecular Neurobiology. 2014;49(2):931-44.
28.
Zhou FQ, Jiang Z, Dedhar S, Wu YH, Snider WD. NGF-induced axon growth is
mediated by localized inactivation of GSK-3beta and functions of the microtubule plus
end binding protein APC. Neuron. 2004;42(6):897-912.
29.
Eickholt BJ, Walsh FS, Doherty P. An inactive pool of GSK-3 at the leading edge
of growth cones is implicated in Semaphorin 3A signaling. The Journal of Cell Biology.
2002;157(2):211-7.
30.
Sanchez S, Sayas CL, Lim F, Diaz-Nido J, Avila J, Wandosell F. The inhibition
of phosphatidylinositol-3-kinase induces neurite retraction and activates GSK3. Journal
of Neurochemistry. 2001;78(3):468-81.
31.
Christie KJ, Webber CA, Martinez JA, Singh B, Zochodne DW. PTEN inhibition
to facilitate intrinsic regenerative outgrowth of adult peripheral axons. The Journal of
Neuroscience. 2010;30(27):9306-15.
32.
Zhuang ZY, Xu H, Clapham DE, Ji RR. Phosphatidylinositol 3-kinase activates
ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through
TRPV1 sensitization. The Journal of Neuroscience. 2004;24(38):8300-9.
33.
Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method
for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1998;32:77-88
28
34.
Twiss JL, Smith DS, Chang B, Shooter EM. Translational control of ribosomal
protein L4 mRNA is required for rapid neurite regeneration. Neurobiology of Disease.
2000;7(4):416-28.
35.
Weng HR, Gao M, Maixner DW. Glycogen synthase kinase 3 beta regulates glial
glutamate transporter protein expression in the spinal dorsal horn in rats with neuropathic
pain. Experimental Neurology. 2014;252(0):18-27.
36.
Bareiss SK, Dugan E, Brewer KL. Activaton of GSK-3b to reduce abnormal
primary afferent outgrowth and the development of neuropathic pain following spinal
cord injury. Program No. 315.23. 2014 Neuroscience Meeting Planner. Washington, DC:
Society for Neuroscience, 2014. Online.
37.
Xu JT, Tu HY, Xin WJ, Liu XG, Zhang GH, Zhai CH. Activation of
phosphatidylinositol 3-kinase and protein kinase B/Akt in dorsal root ganglia and spinal
cord contributes to the neuropathic pain induced by spinal nerve ligation in rats.
Experimental Neurology. 2007;206(2):269-79.
38.
Lankford KL, Waxman SG, Kocsis JD. Mechanisms of enhancement of neurite
regeneration in vitro following a conditioning sciatic nerve lesion. The Journal of
Comparative Neurology. 1998;391(1):11-29.
39.
Hill CE, Harrison BJ, Rau KK, Hougland MT, Bunge MB, Mendell LM, et al.
Skin incision induces expression of axonal regeneration-related genes in adult rat spinal
sensory neurons. The Journal of Pain. 2010;11(11):1066-73.
40.
Kalous A, Keast JR. Conditioning lesions enhance growth state only in sensory
neurons lacking calcitonin gene-related peptide and isolectin B4-binding. Neuroscience.
2010;166(1):107-21.
29