Neuroscience & Medicine, 2012, 3, 294-305 Published Online September 2012 (
S-Nitrosoglutathione Administration Ameliorates Cauda
Equina Compression Injury in Rats
Anandakumar Shunmugavel1, Mushfiquddin Khan1, Marcus M. Martin2, Anne G. Copay2,
Brian R. Subach2, Thomas C. Schuler2, Inderjit Singh1*
1Department of Pediatrics Medical University of South Carolina, Charleston, USA; 2The Spinal Research Foundation, The Virginia
Spine Institute, Reston, USA.
Email: *
Received May 4th, 2012; revised June 19th, 2012; accepted June 11th, 2012
Lumbar spinal stenosis (LSS) causes ischemia, inflammation, demyelination and results in cauda equina (CE) syndrome,
with pain and locomotor functional deficits. We investigated whether exogenous administration of S-nitrosoglutathione
(GSNO), an endogenous redox modulating anti-neuroinflammatory agent, hastens functional recovery in a CE com-
pression (CEC) rat model. CEC was induced in adult female rats by the surgical implantation of two silicone blocks
within the epidural spaces of L4-L6 vertebrae. GSNO (50 μg/kg body weight) was administered by gavage 1 h after the
injury, and the treatment was continued daily thereafter. GSNO induced change in the pain threshold was evaluated for
four days after the compression. Tissue analyses and locomotor function evaluation were carried out at two weeks and
four weeks after the CEC respectively. GSNO significantly improved motor function in CEC rats as evidenced by an
increased latency on rotarod compared with vehicle-treated CEC rats. CEC induced hyperalgesia was decreased by
GSNO. GSNO also increased the expression of VEGF, reduced cellular infiltration (H&E staining) and apoptotic cell
death (TUNEL assay), and hampered demyelination (LFB staining and g-ratio). These data demonstrate that admini-
stration of GSNO after CEC decreased inflammation, hyperalgesia and cell death leading to improved locomotor func-
tion of CEC rats. The therapeutic potential of GSNO observed in the present study with CEC rats suggests that GSNO is
a candidate drug to test in LSS patients.
Keywords: LSS; VEGF; Demyelination; g-Ratio; Neuroprotection
1. Introduction
Lumbar spinal stenosis (LSS) is a stable disorder with
mild to severe disability and neurological deficits. LSS is
one of the most frequent reasons for spinal surgery asso-
ciated with chronic low back pain in the elderly popula-
tion. In the US, 90 out of 100,000 persons older than 60
years undergo LSS related spinal surgery every year [1].
The socioeconomic burden due to LSS is huge and has
been increasing rapidly, despite considerable techno-
logical advances in diagnosis [2,3]. LSS compresses
cauda equina (CE) fibers over time and results in inter-
mittent claudication/pseudoclaudication [4]. Further,
CEC results in neurodegeneration and neuronal dysfunc-
tion through Wallerian-degeneration [5,6]. LSS-induced
CEC interrupts normal spinal microvascular circulation
[7,8], and leads to secondary tissue damage from hy-
poxia/ischemia injury. Secondary damage also aug-
ments inflammation-mediated neuronal damage to the CE
fibers and spinal cord. Increased synthesis of inflamma-
tory cytokines, chemokines, and matrix metallopro-
teinases (MMPs) during CEC are reported to result in
defects in motor functions [9,10]. The enhanced inflam-
matory cytokine synthesis also hampers the recovery
after CEC. Therefore, dampening the inflammatory re-
sponse following LSS/CEC by pharmacological strate-
gies will be a key approach in LSS treatment.
Decompressive laminectomy has been the most sought
surgical treatment option for LSS [11]. However, the
success rate of surgery is highly variable (between 45%
to 72%) [12]. Advances in therapeutic treatment of LSS
are limited by the complexity of the pathology involved.
In addition, there is a lack of suitable animal model to
delineate the molecular mechanisms involved in the
pathophysiology [13,14]. Recently, a rat animal model of
CEC was developed by Takenobu et al. [15] by using
silicon blocks to induce LSS. This model was further
validated by Watanabe et al. [16]. In the present study,
we followed the same animal model and evaluated the
efficacy of GSNO therapy in treating LSS.
*Corresponding author.
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats 295
GSNO, an endogenous thiol produced by the reaction
of nitric oxide (NO) with glutathione (GSH), is an effi-
cient nitrosylating agent [17]. Nitrosylation, like phos-
phorylation, plays a major role in several physiological
and pathological processes [18,19]. Pharmacologically,
GSNO protects the central nervous system (CNS) from
excitotoxicity, inflammation, and reactive oxygen species
(ROS) [20]. Our recent studies have proven GSNO to
enhance motor function recovery and neurological func-
tion in rat animal models of traumatic brain injury (TBI)
and ischemia reperfusion injury [21,22] and ameliorate
spinal cord injury (SCI) induced sexual dysfunction [23].
GSNO elicited anti-inflammatory effects were attributed
to the down regulation of NF-κB, cell adhesion mole-
cules, cytokines, and inducible NOS (iNOS) [21,22,
24-26]. Decreased expression of ICAM-1, ED1, and
MMP-9 proteins and edema has been attributed to the
enhanced integrity of blood brain barrier [21]. Further-
more, GSNO inhibits platelet activation and reduces
embolization in humans [27-31]. The neuroprotective
effect of GSNO has also been attributed to its ability to
reduce neuronal apoptosis by inhibiting caspase-3 activ-
ity [22,32]. GSNO’s antioxidant/anti-apoptotic property
is also attributed to the redox modulation [33] through
increasing the level of antioxidant GSH [34] and reduc-
ing the level of oxidant peroxynitrite [35,36].
In the present study, we tested the therapeutic poten-
tial of GSNO in treating CEC-induced by LSS in a rat
animal model. We investigated the efficacy of GSNO
therapy in locomotor functional recovery, cellular infil-
tration and apoptosis and expression of VEGF in the
spinal cord and CE fibers of CEC rats.
2. Methods
2.1. Animals and Experimental Design
Female Sprague-Dawley rats (225 - 250 g), purchased
from Harlan Laboratories (Durham, NC) were housed in
the animal facility at 12 h dark and light cycle. Ambient
temperature was maintained at 25˚C. The animal proce-
dures for the study were approved by the Institutional
Animal Care and Use Committee (IACUC) of the Medi-
cal University of South Carolina. Animals were acclima-
tized for one week before the experiments began.
The experiment consisted of three groups: sham-oper-
ated (Sham), vehicle-treated CEC animals (Vehicle), and
GSNO-treated CEC animals (GSNO). GSNO (World
Precision Instruments, Inc., Sarasota, FL; 50 µg/kg body
weight) was gavage fed to animals 1 h after LSS surgery
and every 24 h thereafter till the end of the experiment.
GSNO was freshly prepared in sterile water every day.
2.2. Cauda Equina Compression
CEC was induced following the method described by
Takenobu et al. [15]. Briefly, animals were anesthetized
with a ketamine-xylazine cocktail (80 mg/kg and 10
mg/kg body weight respectively). After confirming the
validity of anesthesia by toe pinching, the animals were
depilated on the dorsal spine line, and the spine was ex-
posed at the L3-S2 level. Two appropriate holes were
made to expose the dura overlying the spinal cord of the
L4 and L5 vertebrae. Two pieces of silicone rubber (4
mm × 1 mm × 1 mm) (Bentec Medical Inc., Woodland,
CA) were placed into the L4 and L5 epidural spaces. The
wound was irrigated with sterile phosphate buffered sa-
line (PBS) solution, and the incision was closed in layers
using polysorb 4. Sham animals underwent the same
procedure except for the placement of silicone blocks.
Animals were returned to cages, kept on a 37˚C heating
blanket overnight. The animals were closely monitored
for symptoms of hind limb paralysis and urinary bladder
incontinence. Animals that showed either hind limb pa-
ralysis or loss of tail sensation on pinching were ex-
cluded from the study.
2.3. Locomotor Function Evaluation
CEC-induced locomotor deficit was measured with ro-
tarod. Latency on a rotarod was measured as described
previously from our laboratory [21]. Animals were pre-
trained on an automated 4-lane rotarod unit supplied by
Columbus Instruments (Columbus, OH). Total time in
seconds that the animal could stay on the drum was re-
corded. Each animal was given three trials, and the mean
was presented as the final data.
2.4. Hyperalgesia and Nociception
Pain threshold of experimental animals were measured
using dynamic plantar aesthesiometer (DPA) and anal-
gesy meter (AM). DPA (Ugo Basile, Italy) is an auto-
mated version of von Frey hair analysis used to measure
changes in the development of mechanical allodynia re-
sulting from neuropathic pain [37]. Animals were accli-
matized to the DPA for about 15 minutes before re-
cording. In addition, animals were acclimated to the test-
ing area of DPA for 3 - 5 days before the actual experi-
ment. Animals were placed individually in the testing
area with a wire mesh floor. The instrument raised the
filament through the wire mesh to touch the foot and
progressively increased the force until it reached a
maximum of 20 g of force. The foot withdrawal/latency
was recorded using the software provided by the manu-
facturer. AM (Ugo Basile, Italy) was used to measure the
influence of GSNO on nociception of CEC rats following
the method described before [38]. Continuously increas-
ing pressure was applied to the dorsal surface of the hind
paws. The actual load (g) applied at the moment the ani-
mal withdrew its paw was recorded. Three trails were
made on each paw with 5-minute intertest intervals.
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats
2.5. Myelination and g-Ratio
CE fibers from sham, vehicle, and GSNO-treated ani-
mals were extracted 7 weeks after the induction of CEC.
The tissues were fixed overnight in 2% cacodylate glu-
taraldehyde and post-fixed in 2% osmium tetroxide for 2
h. Following rinsing in distilled water, the tissues were
dehydrated in ascending grades of alcohol, infiltrated
with propylene oxide and embedded at proper orientation
[39]. Thin sections of 0.5 micron were cut with Teichert
ultra microtome and stained with toluidine blue. Ultra-
thin sections of 70 nm were then cut and picked up on
copper grids. Sections were stained with uranyl acetate
and lead citrate. Ultra structural details were observed
and recorded with a JEOL 1010 Transmission electron
microscope. Myelination was determined based on the
g-ratio, calculated following the procedure of Chomiak
and Hu [40].
2.6. Histology and Immunofluorescence
Animals were sacrificed with an overdose of nembutal
(150 mg/kg body weight) and perfused transcardially
with saline followed by 4% paraformaldehyde (PFA) in
PBS, pH 7.4. Spinal cord and CE fibers were extracted as
described [41], and post-fixed overnight in PFA. After
fixation, the tissues were dehydrated in a series of alco-
hol and infiltrated with paraffin wax using Leica
TP-1020 automatic tissue processor. Tissue blocks were
sectioned (5 - 8 µm) with a Leica HM-325 rotary micro-
tome. Sections were rehydrated by passing through de-
creasing grades of ethanol (100%, 95%, 60%, 70% and
30%) and water. Sections were stained with hematoxylin
and eosin (H&E) [42] and luxol fast blue (LFB), as de-
scribed previously [43]. Cellular infiltration was quanti-
tated using NIH image J software.
For immunofluorescence studies, endogenous per-
oxidase was blocked with 3% H2O2 after deparafinization
and rehydration. To enhance immunoreactivity, sections
were pretreated with 0.1% trypsin in a 10 mmol/L citrate
buffer (pH 6.0) at near boiling for 20 minutes. After
blocking with normal goat serum, sections were incu-
bated with the primary antibodies (1:200) overnight at
4˚C. The following primary antibodies were used: rabbit
anti VEGF (1:200; abcam, San Francisco, CA) and PE-
CAM-1 (1:200; Santa Cruz Biotech, Santa Cruz, CA).
After washing with PBS (3 times, 10 min each), sections
were incubated with Alexafluor conjugated secondary
antibodies (1:100; Molecular probes, Invitrogen, Eugene,
OR) for 1 h at room temperature. Sections were again
rinsed with PBS, counterstained with DAPI (Invitrogen,
Eugene, OR) and mounted with aqueous antifade solu-
tion. Immunofluorescence was analyzed using an epif-
luorescence microscope equipped with Axio-Vision 4.5
imaging software. Intensity of myelin staining and VEGF
positive cells were quantitated using Image Pro plus 5.1
software [44].
2.7. TUNEL Assay
The terminal deoxynucleotidyl transferase-mediated bio-
tinylated UTP nick end labeling (TUNEL) assay was
performed using an Apoptag fluorescein in situ apoptosis
detection kit (Serological Corp. Norcross, GA). TUNEL
positive cells were counted in at least five microscopic
frames of each section.
2.8. Statistical Analysis
Statistical analysis was performed using Graph pad prism
3.0 software. Statistical significance was determined us-
ing Students’ t-test. Values were expressed as mean ± SD
of n determinations. p values less than 0.05 were consid-
ered statistically significant.
3. Results
3.1. GSNO Ameliorates Locomotor Dysfunction
Rats were pretrained on the rotarod for 5 days before the
experiments began. All the animals were able to walk on
rotarod for 280 ± 8 seconds on the day before the surgi-
cal induction of LSS. Animals in each group (n = 12)
were tested on rotarod for 28 days following CEC. La-
tency on rotarod of different experimental groups of rats
is given in Figure 1. On day 2 post injury, the vehicle
group showed 44.3 ± 8.5 sec latency on rotarod. With the
progression of natural healing, the walking efficiency in-
creased a little further to 58.4 ± 14.6 sec on the 28th day.
Figure 1. Effect of GSNO on CEC-mediated locomotor
dysfunction in rats. GSNO treatment showed significantly
improved motor function of the CEC rats when compared
with vehicle group from day 7 onward. Sham rats did not
show significant change in locomotor function on all the
days tested. Data are expressed as mean ± SD (n = 12 in
each group). ***p < 0.001 vs. vehicle.
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats
Copyright © 2012 SciRes. NM
infiltrated cells (H&E) on day 14 after CEC and the re-
sults are given in Figure 2. Spinal cord of the vehicle
group showed increased degree of demyelination (Figure
2(a): b & e). LFB staining in the GSNO group was com-
parable with that of the sham group (Figure 2(a): c, f &
a, d). GSNO also decreased cellular infiltration in the
spinal tissue (Figure 2(a): i) when compared to the vehi-
cle group (Figure 2(a): h). CEC induced decrease in
LFB staining intensity and number of infiltrated cells
was quantified by using Image pro plus 5.1 software and
the data are given in Figures 2(b) and 2(c). Spinal cord
of CEC vehicle rats showed significantly lesser intensity
of LFB (b) and increased number of infiltrated cells (c).
The GSNO group showed significantly improved (p <
0.001) motor function starting from day 7 (84.5 ± 22.8
seconds) to day 28 (149.7 ± 11.6 seconds) after CEC.
Sham operated animals did not show any significant
change in the walking time on rotarod on any day tested
(Figure 1). The data clearly show GSNO mediated ame-
lioration of locomotor dysfunction in CEC rats.
3.2. Gsno Decreases Demyelination and Cellular
Spinal cord sections proximal to the CEC region and CE
fibers were processed and stained for myelin (LFB) and
(b) (c)
Figure 2. Effect of GSNO on CEC-induced changes in LFB histochemistry (a-f) and cellular infiltration (g-i) of spinal cord 14
days after CEC. Myelin level in the spinal cord was significantly reduced in vehicle (b & e) compared to sham (a & d) and
GSNO rats (c & f). Corresponding histogram on quantification of LFB staining intensity was given in Figure 2(b). Increased
level of cellular infiltration was seen in spinal cord of vehicle (h) compared with sham (g) and GSNO groups (i). Photomicro-
graphs are representative of n = 5 in each group. Histogram (c) shows the quantitative difference in number of infiltrated
cells in the spinal cord of different experimental groups. **p > 0.01;***p > 0.001, vs. sham and GSNO.
S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats
A similar trend was also seen with LFB and H&E
staining of CE fibers of different experimental groups.
While the CE of vehicle group showed reduced LFB
staining (Figure 3(a): b) and increased infiltration (Fig-
ure 3(a): e), the GSNO group had LFB staining (Figure
3(a): c) and cellular infiltration (Figure 3(a): f) levels
similar to that of sham group (Figure 3(a): a & d). To
support further, the CEC induced demyelination and de-
generation, CE fibers were examined with EM 7 weeks
after the CEC (Figure 4). CE from sham animals had
compact myelin sheath (Figure 4(a): arrow). The Schwann-
like cells encircling the myelinating fiber can also be
seen (Figure 4(a): arrow head). Degenerated myelin
layer was seen with CE of vehicle rats (Figure 4(a): Ve-
hicle). Schwann-like cells were also reduced in size, and
these cells were not in close association with the neurons.
Schwann-like cells showed healthy contact with the fi-
bers also in the GSNO group, as indicated in Figure 4(a):
As the g-ratio is a reliable factor of axonal myelina-
tion, we determined the g-ratio in the CE of sham, vehi-
cle and GSNO group of rats. The g-ratio of CE fibers of
sham, vehicle and GSNO rats determined in the present
study were 0.67 ± 0.06, 0.71 ± 0.05 and 0.66 ± 0.04 re-
spectively. Vehicle group has significantly higher g-ratio
than the sham and GSNO groups. GSNO group showed a
slightly lesser g-ratio than the sham group. However, the
difference was not statistically significant (Figure 4( b)).
(b) (c)
Figure 3. Effect of GSNO on CEC-induced changes in LFB histochemistry (a-c) and cellular infiltration (d-f) of CE nerve
fiber 14 days after CEC. Myelin level of the nerve fiber was significantly reduced in vehicle (b) when compared with sham (a)
and GSNO (c) groups. Increased cellular infiltration was also observed in vehicle (e) compared with sham (d) and GSNO (f)
rats. Photomicrographs are representative of n = 5 in each group. Histograms show the quantitative difference in LFB stain-
ing intensity and number of infiltrated cells of CE of different experimental groups. Vehicle group had significantly de-
creased level of LFB staining (b) and increased number of cellular infiltration (c)) compared with sham and GSNO animals.
***p > 0.001 vs. sham and GSNO.
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats 299
(a) (b)
Figure 4. Effect of GSNO on CEC-induced degeneration of myelin (Figure 4(a)) and g-ratio of CE fibers (Figure 4(b)). CE
fibers 7 week after CEC were processed and analyzed with EM. g-ratio was calculated with axons of 2 µm thickness. CE of
vehicle animals showed remarkable degeneration of myelin (a; arrow). The myelin pattern of CE fibers of GSNO animals
was similar to that of sham animals. The Schwann-like cell morphology and its relationship to the nerve fiber were compara-
ble in sham and GSNO groups (arrow head). Electron micrographs are representative of n = 4 in each group. g-ratio of axons
of vehicle was significantly higher than sham and GSNO animals (b), indicating decreased level of myelin. *p > 0.05 vs. sham
and GSNO.
3.3. GSNO Enhances the Pain Threshold
Pain threshold was determined in both naïve (Figures 5(a)
and (b)) and CEC (Figures 5(c) and (d)) rats. The pain
threshold of naïve rats in the presence and absence of
GSNO was measured at 0, 1, 2, 4, 6 and 24 h using AM
(Figure 5(a)) and DPA (Figure 5(b)). GSNO induced
increase in the pain threshold of animals was maximum
at 4 - 6 h after administration. However, 24 h following
the treatment, the difference between the untreated and
GSNO-treated animals was insignificant.
The pain threshold of sham, vehicle, and GSNO
groups following CEC was measured in a way similar to
naïve animals using AM (Figure 5(c)) and DPA (Figure
5(d)). Sham animals did not show any changes in pain
threshold on the days measured. Vehicle rats were sig-
nificantly hypersensitive to the pain stimulus. However,
GSNO rats had thresholds comparable to baseline, indi-
cating that GSNO decreases the hyperalgesia in CEC
3.4. GSNO Increases the Expression of VEGF in
CE Fibers
The expression of VEGF was determined on the com-
pressed CE nerve fibers 14 days following CEC (Fig-
ure 6(a)). Both vehicle and GSNO groups had signifi-
cantly enhanced expression of VEGF compared with
the sham group. However, the GSNO group exhibited
remarkably increased expression of VEGF compared
with the vehicle group, as shown in Figure 6(b). VEGF
expression was seen mainly in cells, also positive for
PECAM-1 (Figure 6(c)) in the GSNO group, indicating
that GSNO-mediated amelioration of CEC injury in-
volves the participation of endothelial VEGF. PE-
CAM-1 is an integral membrane glycoprotein highly
expressed by endothelial cells [45].
3.5. GSNO Reduces the Number of TUNEL
Positive Cells in Spinal Cord
Apoptotic cell death was evaluated on days 3 and 14 af-
ter CEC using a TUNEL staining assay (Figure 7(a)).
TUNEL-positive cell counting was performed as previ-
ously described from our laboratory [21]. The spinal cord
of vehicle rats had a significantly higher number of
TUNEL-positive cells compared with the spinal cord of
sham rats. The trend of increase in number of TUNEL-
positive cells in the vehicle was similar at day 3 and 14
after the CEC. In contrast, the spinal cord of GSNO
group showed significantly reduced number of TUNEL-
positive cells on days 3 and day 14 after compression.
The data indicate GSNO possesses an anti-apoptotic ac-
tivity, as has been previously demonstrated by us in a rat
model of TBI [21]. Spinal cords from the sham group did
not show significant number of TUNEL-positive cells on
both the days tested.
4. Discussion
CEC due to narrowing of the spinal canal initiates a
complex series of secondary events, including inflamma-
tion, altered redox and cellular apoptosis [46-48]. Sec-
ondary injury ultimately leads to neurodegeneration and
associated functional deficits [4,16,49]. The present
study shows that GSNO treatment after the onset of LSS
ameliorated motor dysfunction, reduced hyperalgesia,
and inhibited tissue degeneration. Mechanistically, the
protection was offered through inhibiting cellular infil-
tration, decreasing demyelination and apoptotic cell
death, and increasing the expression of VEGF.
Locomotor deficit is the hallmark of LSS and the con-
sequent CEC [15,16]. The compression causes defects in
axonal myelin which affect the functioning of motor
neurons [15]. Therefore, improvement of motor function
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats
Figure 5. Effect of GSNO on pain threshold of naïve and
CEC rats. Naive rats were treated either with sterile water
(vehicle) or GSNO after measuring the baseline pain thres-
hold using AM (a) as well as DPA (b). Pain threshold was
measured at 1, 2, 4, 6 and 24 hours after the treatments.
GSNO-mediated increase in pain threshold was noted 4 - 6
h after administration. However, no significant effect was
seen at 24 h. Data are presented a mean ± SD for n = 8 in
each group. **p < 0.01 vs. vehicle. Effect of GSNO on pain
threshold of CE C animals was given in (c) & (d). Pai n thres-
hold was measured with AM. Base line represents the pain
threshold of rats before CEC. There was a significant dif-
ference in pain threshold between the vehicle and sham
groups on all days tested. However, no significant difference
was observed between sham and GSNO groups from day 2.
Figure 2(d) shows pain threshold of CEC rats measured
with DPA. Vehicle animals showed significantly lesser pain
threshold on all days tested compared with the base line.
Pain threshold of GSNO animals was not significantly dif-
ferent from the baseline on all days tested except day 2.
Data are presented as mean ± SD for n = 8 in each group. *p
< 0.05, **p < 0.005 vs. vehicle, #p < 0.05: vs. baseline.
remains the major yardstick of therapeutic interventions
in humans and experimental animals after LSS. In this
study, motor function deficits were observed in CEC rats
from day 2 onwards (Figure 1), indicating the validity of
the animal model to investigate therapeutic intervention
in LSS. Deficit in motor function of CEC rats observed
in the present study corroborates with the earlier report of
Watanabe et al. [16]. To test the therapeutic efficacy of
GSNO in LSS, CEC animals were administered with
GSNO (50 µg/kg body weight) 1 hour after the CEC.
The dose of GSNO used in the present study was deter-
mined based on our recent studies in rat models of TBI
[21] and SCI [50]. Also, we have shown that exogenous
administration of GSNO (50 µg/kg) did not alter the
other physiological parameters such as blood pressure in
a rat model of TBI [51].
The g-ratio is a highly reliable factor to assess the
level of axonal myelination. It has been shown previ-
ously that the g-ratio is altered during demyelinating
disease [52]. The g-ratio of CE fibers of sham, vehicle
and GSNO rats determined in the present study were
0.67 ± 0.06, 0.71 ± 0.04 and 0.66 ± 0.04 respectively.
The g-ratio of CE fibers observed in the present study is
slightly lesser than the theoretically predicted g-ratio of
spinal cord (0.79) previously reported [40]. CE fibers of
vehicle group showed significantly higher g-ratio, when
compared to vehicle and GSNO groups. The g-ratio of
GSNO group was slightly lesser than the sham group.
However, the difference was not statistically significant.
It has been well established that during the recovery
phase of demyelinating disease axons undergo an initial
hypermyelination phase and eventually revert to normal
g-ratio [52]. Therefore, the present observation proves
that GSNO helps axonal remyelination after spinal
stenosis. However, the temporal effect of GSNO on re-
myelination has to be studied in detail.
Improvement of motor function in CEC rats by GSNO
administration is the major finding of this study (Figure
1). A similar efficacy of GSNO in improving motor
function has been reported in our previous studies on SCI
and TBI animal models [21,50]; strengthening the hy-
pothesis that GSNO enhances functional recovery in
CNS injuries. Degeneration and tissue loss due to secon-
dary injury and apoptotic cell death [49] underlie motor
function deficits met with CNS diseases. We observed
the loss of myelin and increased cellular infiltration in
both the spinal cord (Figure 2) and CE fibers (Figure 3)
of vehicle animals, indicating a profound neuronal func-
tional deficit. Interestingly, the GSNO group had myelin
levels comparable to the sham animals in both spinal
cord and CE fibers (Figures 2 and 3), supporting the
neuroprotective potential of GSNO therapy. Cellular in-
filtration was also blocked by GSNO treatment (Figures
2 and 3), supporting the anti-inflammatory property of
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S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats 301
GSNO. A similar effect of GSNO on myelination and
cellular infiltration has been previously reported from our
laboratory in TBI and SCI animal models [21,50]. Fur-
ther, to support GSNO-mediated neuro-protection, CE
fibers were analyzed for myelination pattern using EM
study. While the myelination pattern and myelin levels
were similar in sham and GSNO fibers, the vehicle fibers
had significantly increased myelin degeneration and g-
ratio (Figure 4). Furthermore, GSNO treatment reduced
the number of TUNEL-positive cells in the spinal cord of
CEC animals (Figure 7), supporting the neuroprotective
potential of GSNO. The neuro-protective effect of GSNO
has already been shown against neurotoxins [35], IR in-
jury [22], and TBI [21]. The anti-apoptotic effect of
Figure 6. Effect of GSNO treatment on CEC-induced
change in VEGF expression in CE fibers 14 days after CEC.
CE fibers subjected to CEC were examined for VEGF ex-
pression by immunofluorescence staining (a). VEGF ex-
pression was elevated both in vehicle and GSNO compared
with the sham rats. However, GSNO showed increased ex-
pression of VEGF compared with vehicle group. VEGF
positive cells were counted in the CE as explained in the
material and methods section. GSNO significantly increased
the VEGF expression in the CE fibers of CEC rats (b).
Colocalization of VEGF with PECAM-1 (c) in GSNO group
indicates that VEGF was expressed mainly in endothelial
cells. Photomicrographs are representative of n = 3 in each
Figure 7. Effect of GSNO on CEC-induced apoptotic cell
death in spinal cord. Significant increase in the number of
TUNEL positive cells (determined at day 3 and 14) was
observed in the spinal cord from Vehicle compared with
sham group. GSNO significantly decreased the number of
apoptotic cells in the spinal cord of CEC rats. Photomicro-
graphs (a) are representative of n = 3 in each group. Data of
TUNEL positive cell counting (b) are expressed as mean ±
SD. *p < 0.05, **p < 0.01 vs. Sham, #p < 0.05 vs. vehicle.
GSNO is shown to be regulated by blocking the activity
of caspase-3 and NF-κB [25,32]. Previously, we have
also reported GSNO-mediated inhibition of caspase-3
activity in IR injury [22] and NF-κB activity in neural
and endothelial cells [22,26]. GSNO-mediated down
regulation of caspase-3 through NF-κB is invoked via
S-nitrosylation [25,32]. Additionally, GSNO may stimu-
late the neurorepair process through the upregulation of
growth and neurotropic factors including VEGF and
BDNF [50,51]. In this study, the increased expression of
VEGF was observed in both the GSNO and the vehicle
CE fibers (Figure 7). However, the expression was re-
markably high in the endothelial cells (PECAM-1 posi-
tive) of CE fibers of GSNO group compared with the
vehicle group (Figure 6). Increased expression of VEGF
following LSS has been reported to contribute to adap-
tive functional recovery [16]. Obviously, increased
VEGF expression in the vehicle fibers was insufficient to
achieve functional recovery in this study (Figure 6).
VEGF is regulated by the transcription factor HIF1α,
which is modulated by NO/nitrosylation [36,53]. There-
Copyright © 2012 SciRes. NM
S-Nitrosoglutathione Administration Ameliorates Cauda Equina Compression Injury in Rats
fore, GSNO as an endogenous nitrosylating agent seems
to be an ideal drug to activate VEGF through HIF1-α,
which in turn will stimulate the enhanced repair process
of the degenerating spinal cord as well as CE fibers
through angiogenesis that restores normal blood flow to
the compressed ischemic spinal tissue. Because ischemia
is a common occurrence following LSS [54], enhancing
blood flow in the compressed area should also contribute
to a speedy recovery [55-57]. Experimental drugs in-
creasing blood flow have been shown to ameliorate LSS
[58]. A study in a rat model of IR shows GSNO enhanc-
ing the cerebral blood flow and thereby improving neu-
rological functions [22]. GSNO was reported also to in-
crease the dermal blood flow in a rat model of diabetes
[59]. Therefore, GSNO-mediated protection in LSS may
occur, at least in part, through its vasculo protective effect.
Pain is a significant hallmark of pathology associated
with LSS [60]. CEC elicits pain of both neurogenic and
inflammatory origin. Despite a thorough understanding
on the mechanisms of pain, satisfactory pain-manage-
ment therapy to LSS is not yet available [60]. Moreover,
the molecular mechanisms of pain involved in LSS are
more complex and poorly understood [60,61]. In view of
this, the pain threshold was evaluated in both naïve and
CEC rats (Figure 5) using two different modalities.
GSNO treatment significantly increased the pain thresh-
old of both naïve and vehicle rats indicating that GSNO
has analgesic property in addition to neuroprotection [61].
Significant increase in tissue peroxynitrite level has been
reported after spinal compression injury [46,62]. Oxida-
tive obnoxious molecules, including peroxynitrite, are
also implicated in both neurogenic and inflammatory
pain. Neurogenic nociception originates due to NMDA
receptor activation [60]. GSNO, via S-nitrosylation,
down regulates NMDA receptors and the activity of
neuronal nitric oxide synthase (nNOS) [63,64]. GSNO
also reduces peroxynitrite-induced lipid peroxidation in
neurodegenerative diseases [33,35,65]. Further, in an
oxidative environment peroxynitrite is formed by nitric
oxide produced from nNOS as well as iNOS [66]. Induc-
tion of iNOS and other inflammatory mediators is inhib-
ited by GSNO via down regulation of the NF-κB tran-
scription factor [22,23,26]. The level of peroxynitrite in
the brain is also decreased after GSNO treatment in rat
models of IR and TBI [34,51]. All these observations
strengthen the therapeutic potential of GSNO in treating
neurogenic as well as inflammation-mediated pain in
In conclusion, exogenous administration of GSNO,
reduces neuronal degeneration, demyelination and apop-
totic cell death and also increases the pain threshold and
improves motor function in a rat model of CEC. Hence,
GSNO is a potential and ideal candidate to be evaluated
in LSS patients.
5. Acknowledgements
This work was supported by grants from The Spinal Re-
search Foundation, VA and by grants from the Betty L.
Beatty and Guy E. Beatty Foundations. The work was
also supported in part by grants NS-72511, NS-22576,
and NS-37766 and DC00422; 07506 from the NIH, CO6
RR018823 and CO6 RR0015455 from the Extramural
Research Facilities Program of the National Center for
Research Resources. The authors thank Dr. Hainan Lang,
Ph.D., (DC00422; 07506) Department of Pathology and
Laboratory Medicine for help in histology. We are
grateful to Dr. Thomas G. Smith from the MUSC Writ-
ing Center and Ms. Danielle Waulene Lowe for editing
the manuscript. Ms. Joyce Bryan and Ms. Chara Wil-
liams are acknowledged for their help in animal and re-
agent procurement and secretarial assistance.
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