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Departments of
* Anatomy and Cell Biology,
Surgery (Neurosurgery),
Pathology, and
§ College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK, Canada
1Correspondence: Department of Anatomy and Cell Biology, 107 Wiggins Road, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada. E-mail: juurlink{at}duke.usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: central nervous system • functional recovery • neurotrauma • oxidative stress • paraplegia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. One primary mechanism of action
of methylprednisolone is thought to be the scavenging of lipid peroxyl
radicals, which inhibits the lipid peroxidation cascade (3
, 4)
. Inhibiting oxidative stress should inhibit activation of
nuclear factor kappa B (NF
B) (5)
, thereby inhibiting
activation of proinflammatory gene expression. Indeed,
methylprednisolone does inhibit inflammatory responses after spinal
cord injury (6)
. However, the improvement seen with
methylprednisolone treatment is modest (7)
and may be
associated with adverse side effects (8)
.
Oxidative stress, particularly lipid peroxidation cascades, and
associated inflammation are major secondary mechanisms whereby physical
trauma results in spinal cord injury (9
10
11
12)
. Minimizing
oxidative stress should decrease post-traumatic inflammation since
oxidative stress activates proinflammatory mechanisms (5)
.
The quenching of lipid peroxyl radicals, thought to be a major
mechanism of action by methylprednisolone (3
, 4)
, is only
a small part of the cell’s total antioxidant defense mechanisms
(12
, 13)
. Since the tripeptide glutathione (GSH) plays
many roles in the minimization of oxidative stress, we reasoned that
promotion of GSH synthesis would be an effective way to decrease
post-traumatic oxidative stress and thereby promote retention of tissue
integrity and function after spinal cord trauma. GSH is involved in the
scavenging of both inorganic and organic peroxides, the sources of
strong oxidants; scavenging of 4-hydroxyalkenals, strong oxidants
derived from lipid peroxides; scavenging of 
-oxo-aldehydes, strong
oxidants that are increased under conditions of oxidative stress; and
the regeneration of vitamin E from the vitamin E radical (reviewed in
refs 5
, 13
).
GSH is synthesized in a two-step process, with the first reaction being
the ligation of glutamate with cysteine forming L-
-glutamyl-cysteine
and the second step being the ligation of glycine to
L--glutamyl-cysteine, thereby forming GSH. The first step is
rate-limiting, with cysteine being the rate-limiting amino acid
(14)
. Two compounds that have been used to increase
intracellular cysteine are N-acetylcysteine and
L-2-oxothiazolidine-4-carboxylate (OTC). N-acetylcysteine
does not cross the blood–brain barrier (15)
whereas OTC
does (16)
, and it has been shown to increase brain
cysteine (16)
and GSH (17)
contents.
Furthermore, an in vitro model of spinal cord injury has
demonstrated that OTC but not N-acetylcysteine could
increase neuronal GSH and increase survival (18)
.
The objectives of the experiments described in this article were to test the hypothesis that increasing spinal cord GSH content by the administration of OTC after spinal cord injury would be an efficacious way to reduce oxidative stress and thereby minimize tissue damage and maximize functional retention. We demonstrate that the data collected after administration of OTC after spinal cord trauma support our hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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320 g) were obtained from Charles
River Canada and housed in a temperature-regulated animal facility,
exposed to a 12 h light/dark cycle and with free access to food
and water. Animals were treated in accordance with the guidelines of
the Canadian Council on Animal Care. Before surgery, the rats received
the analgesic buprenorphine-hydrochloride (Reckitt Colman Ltd.)
subcutaneously (s.c.) at a dose of 0.05 mg/kg and then every 12 h
for the first 72 h after surgery. Anesthesia was performed with
halothane (5% induction and 1% maintenance). Laminectomy was
performed aseptically at T5-T6 to expose the spinal cord. A 5 s
clip compression injury was induced at the T6 spinal cord level using a
modified Kerr-Lougheed aneurysm clip (Walsh Manufacturing, Oakville,
Ontario) with a calibrated force of 50 g as described
previously (19
, 20)
. For delivery of drug or vehicle, a
sterile catheter was placed s.c. running from the dorsum to enter the
peritoneal cavity ventrally; this was done before the laminectomy was
performed. Thirty minutes after spinal cord compression, OTC (Sigma,
St. Louis, Mo.) or saline vehicle was delivered intraperitoneally and
then every 12 h for the next 5 days. The initial dose of OTC was
12 mmol/kg body weight with maintenance doses of 4 mmol/kg body weight.
Animals were closely observed for behavior, eating, and drinking as
well as weight loss for the first 12 h and thereafter at a minimum
of three times daily. Manual bladder expressions were performed three
times daily.
Measurements of oxidative stress
A total of 54 animals was used to determine the onset of
oxidative stress after spinal cord injury. We used GSH level, protein
carbonyl content and glutathione reductase activity as markers of
oxidative stress with spinal cord tissue isolated at 1 h, 3 h, 6 h, 24 h, and 48 h after injury. Rats were
transcardially perfused with cold saline, vertebral columns were cooled
with liquid nitrogen, and the after spinal cord segments were isolated:
C3, T3, T5, T6, T7, T9, and L4. Spinal cord segments were frozen in
liquid nitrogen and stored at -80°C until analyzed.
GSH was measured using 5,5'-dithio-bis(2-nitrobenzoic)
acid-derivatization [DTNB], followed by high-performance liquid
chromatography (HPLC) and UV detection (21)
. Briefly,
tissues were homogenized in 5% sulfosalicylic acid containing 0.2 mM
EDTA and sonicated (3x5 s, with intermittent cooling). The homogenates
were centrifuged at 10,000 g for 15 min and supernatants
were collected, derivatized, and analyzed using the Shimadzu
reversed-phase HPLC with ultraviolet detection. We used a final
concentration of 3500 nmol DTNB per reaction for a total volume of 1
ml. Data were collected digitally with Shimadzu Ezchrom Version 3.2
chromatography software. GSH content in rat spinal segments was
expressed in mol/g wet weight.
Protein carbonyl content was determined spectrophotometrically using
2,4-dinitrophenylhydrazine (DNPH) to trap carbonyls with the adduct
formed measured at 360 nm (22)
. Results are expressed as
pmol of DNPH incorporated per mg of protein calculated using a molar
absorption coefficient of 22,000 M-1
cm-1 for aliphatic hydrazones.
Glutathione reductase was calculated by measuring the disappearance of
NADPH (Sigma) in the presence of oxidized-glutathione based on the
procedure described by Eklow et al. (23)
, as we have done
previously (24)
.
Measurement of functional recovery
For behavioral studies, 39 animals were randomly assigned to one
of three groups: 1) animals that had no surgery (5 animals);
2) animals with surgery and spinal cord compression that
received OTC (17 animals); and 3) animals that received
surgery and spinal cord compression that received the saline vehicle
only (17 animals). One of the saline-treated and four of the
OTC-treated animals were killed because of bladder complications. One
saline- and one OTC-treated animal were each killed because of
complications arising from the surgery.
Three behavioral tests were used: the angle board method
(25)
, a modified Tarlov score (26)
, and the
Basso-Beattie-Bresnahan (BBB) open field locomotor rating scale
(27)
. The inclined plane is a behavioral task that
assesses the animal’s ability to maintain its position on a rubber
corrugated board; this board was raised at 5° increments. The maximum
angle at which an animal can support its weight for 5 s is the
capacity angle.
A semiquantitative assessment of hind limb function during open field
walking was performed using the Tarlov scale as modified by Tariq and
colleagues (26)
. Spontaneous activity of hind limb was
scored as follows: 0 = total paraplegia of hind limbs; 1 = no
spontaneous movements but responds to hind limb pinch; 2 =
spontaneous movement; 3 = able to support weight but unable to
walk; 4 = walks with gross deficit; 5 = walks with mild
deficit on broad flat surface, 6 = able to walk on broad flat
surface and support weight on a 1.8 cm wide ledge; 7 = walks on a
ledge.
Procedures for the open field training and testing of the BBB score as
described in Basso et al. (27)
were acquired only part way
through the study; hence, BBB testing was done on only 11
saline-treated and 7 OTC-treated animals. The scale has 22 levels that
range from 0 (= total paralysis) to 21 (= normal locomotion). Briefly,
rats were gentled and adapted to the open field during the fifth week
postsurgery and animals were tested at 6 wk The final open field tests
were videotaped for some rats.
Spinal cord histomorphometry
After 6 wk of behavioral evaluation, the spinal cords were fixed
by transcardial perfusion with 0.03 M phosphate-buffered saline (pH
7.4) containing 1% sodium nitrite in rats anesthetized with halothane,
followed by perfusion (100 ml/100 g) with either 4% formaldehyde
(freshly prepared from paraformaldehyde) or FAM solution (formaldehyde,
acetic acid, and methanol in 1:1:8 ratio) and left in the same fixative
for 5 days. Spinal cord segments were isolated from each animal,
embedded in paraffin, cut into 15 µm thick sections, stained for
myelin with Luxol fast blue, and counterstained with cresyl violet.
Spinal cord tissue staining deep blue was interpreted as being intact
white matter. Area of white matter was quantified using Northern
Eclipse software and expressed in pixels.
Chemicals
All chemicals used for the analytical procedures were obtained
from Sigma.
Statistics
Data are expressed as means ± SE. Where
SEMs are not visible, they are smaller than the symbols.
Statistical analyses were performed with the program InStat.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). At T6, the site of injury, GSH content was reduced to less than half
of the normal within 1 h, with immediately adjacent segments
having almost as large a drop in GSH. Over the next 48 h there
were further decreases in spinal cord GSH, with sites as distant as C3
and L4 experiencing significant decreases. Laminectomy without spinal
cord crush caused a small (10%) drop in GSH at T6 but had no effect on
other levels of the spinal cord (results not presented). The large
decrease in GSH at the site of injury and adjacent segments was
correlated with a significant decrease in activity of glutathione
reductase 24 h post-trauma in spinal cord segments T3 to T9
(Fig. 2
). Protein carbonyls were significantly increased in spinal cord levels
T3-T9 examined 24 h after injury; the site of injury and
immediately adjacent segments experienced the greatest increase in
protein carbonyls (Fig. 3
).
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Animals weighed 328.3 ± 4.2 g prior to surgery. The weight of saline-treated animals 6 wk after surgery increased to 403.9 ± 9.5 g while the weight gain of OTC-treated animals was significantly greater (P=0.0337, two-tailed Student’s t test) at 434.3 ± 9.3 g.
Administration of OTC after injury increased spinal cord GSH
(Fig. 4
) and resulted in higher glutathione reductase activity (Fig. 2)
and
decreased protein carbonyl content (Fig. 3)
compared to animals given
the saline vehicle.
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Six weeks after spinal cord crush in saline-treated animals, much of
the T6 spinal cord was occupied by a large cystic cavity with no gray
matter evident and less than 1% of white matter remaining (Fig. 5
). Administration of OTC did not result in gray matter sparing at T6,
but a peripheral rim of white matter was observed at T6 in all spinal
cords examined from animals that were administered OTC (Fig. 5)
. The
amount of white matter retained varied from 10.5 to 26% of the control
value (Fig. 6
).
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Spinal cord trauma caused a decrease in angle board capacity angle from
59° to 28° (Fig. 7
); during the 6 wk recovery in saline-treated animals this increased to
32° whereas in OTC-treated animals this increased to 42° (Fig. 7)
.
Trauma decreased the Tarlov score from 7 to 1 (Fig. 8A
). Over the next 6 wk, saline-treated animals increased
their Tarlov score to 2.4 whereas OTC-treated animals increased to 4.1.
BBB scores for 6 wk recovery are given in Fig. 8B
. The
behavior of 5 animals that underwent laminectomy without spinal cord
crush was examined after 1 wk. The Tarlov score was 6.93 ± 0.06
in these animals, which was not significantly different from the 7.0
before the laminectomy, and capacity angle was 57.2 ± 1.0, which
was not significantly different from the 58.2 ± 0.3 prior to the
laminectomy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was chosen because it
has been demonstrated to give consistent results (20)
. The
damage caused by the 50 g force aneurysm clip is severe and
resulted in permanent paraplegia with little recovery of hind limb
function, as has been described previously (20)
. This is
in keeping with the histological picture showing cystic T6 spinal cord
segment with almost no white matter remaining. Trauma caused loss of
bladder control, but after 3 wk both saline-treated and OTC-treated
animals regained bladder control. More OTC-treated animals (four
animals) were lost due to bladder problems than saline-treated animals
(one animal). This was correlated with regain of skeletal muscle
function, which enabled animals to actively resist bladder expression;
hence, we do not believe this was a direct adverse effect of OTC, but
yet another indicator of better recovery in the OTC-treated animals.
Oxidative stress
Three criteria indicated that spinal cord trauma resulted in
severe oxidative stress being experienced by the tissue directly
traumatized as well as segments immediately adjacent to the traumatized
T6 segment. These were decreases in tissue GSH, decreases in tissue
glutathione reductase activity, and increases in protein carbonyl
content. These criteria were chosen for the following reasons:
1) oxidative stress consumes GSH either by oxidation or
through formation of adducts with electrophilic compounds (5
, 13)
; 2) protein carbonyls are formed after exposure
to a variety of oxidants (28
29
30)
and thus serves as a
good overall index of oxidative stress experienced by a tissue; and
3) glutathione reductase is enzymatically inactivated by a
number of oxidants (31
32
33)
and, thus, affords an
effective functional index of tissue oxidative stress.
Within 1 h there were significant decreases in GSH in spinal
segments T5 to T9, with the greatest decrease seen at the site of
injury and immediately adjacent segments. Over the next 2 days, and
particularly between 24 and 48 h, GSH levels were significantly
decreased not only from T5-T9, but also throughout the spinal cord from
spinal segments C3 to L4. This may be due to the oxidative stress that
accompanies the prominent inflammatory changes seen after spinal cord
trauma (34
35
36
37)
. Other laboratories have shown that within
less than 1 h of spinal cord trauma there is activation of NFB,
which persists for at least 72 h (34)
, and by 24 h there is massive neutrophil infiltration and the presence of large
numbers of activated microglia/macrophages at the site of injury as
well as adjacent sites (35)
. Our preliminary observations
(not presented) also demonstrate marked inflammatory changes at 24 h postinjury. The role of these inflammatory leukocytes in secondary
injury is indicated by the significantly increased retention of
function after spinal cord trauma if neutrophils (36)
or
macrophages (37)
are depleted. That oxidative stress is
also severe at the site of injury and immediately adjacent segments is
supported by the significant decrease in activity of glutathione
reductase in the spinal cord segments T3 to T9 and by the increase in
protein carbonyl content 24 h after spinal cord crush.
The GSH that disappeared from the tissue could not be recovered as
oxidized glutathione (GSSG). Preliminary experiments using sodium
borohydride to reduce glutathiyl-protein adducts from injured spinal
cord suggest that GSSG produced formed glutathiyl-protein adducts. This
agrees with other studies demonstrating that glutathiyl-protein adducts
are formed during oxidative stress (38
39
40)
. Such adducts
cannot be reduced by glutathione reductase; hence, de novo
synthesis of GSH becomes critical in the tissue’s antioxidant
mechanisms. De novo synthesis of GSH is what administration
of OTC facilitates. Work in other laboratories has demonstrated that
tissue cysteine levels peak 4 h after administration of OTC
(16)
. This suggests that GSH levels should peak 4 h
after administration of OTC. In an environment where there is oxidative
stress, GSH is being consumed due to antioxidant activities; therefore,
rises in GSH after administration of OTC would be expected to be
limited. Nevertheless, we found measurable increases in spinal cord GSH
in OTC-treated animals relative to saline-treated controls; these GSH
increases were associated with an overall decrease in oxidative stress
experienced by the spinal cords as determined by measuring glutathione
reductase activities and protein carbonyl contents. We conclude that
administration of OTC significantly reduced oxidative stress after
spinal cord trauma.
Tissue and functional sparing
The decrease in oxidative stress after OTC administration was
associated with sparing of white matter at T6 as indicated by Luxol
fast blue staining. Luxol fast blue is a classical myelin stain. Six
weeks after injury the stained myelin will be associated with intact
axons. Hence, Luxol fast blue staining is an indirect measure of intact
axons, with cross-sectional area of staining being correlated with
total number of intact axons. The validity of measuring Luxol fast
blue-stained spinal cord as a measure of intact white matter is
indicated by the positive correlation between this measure and the
functional scores. However, additional studies are required that
involve counts of retrogradely and anterogradely labeled neurons
similar to that performed by Fehlings and Tator (41)
. In
the animals measured, the average amount of white matter spared was
between 10.5 and 26% in comparison to less than 1% in the
saline-treated animals. Although this is a modest amount, the crush
insult was severe. No gray matter was retained at T6; this is similar
to the earlier findings of Khan and Griebel (20)
. Sparing
of white matter allowed significant functional recovery with animals
attaining BBB scores ranging from 11 to 14 in comparison to a score of
5.3 with saline-treated animals. Previous research by Fehlings and
Tator has demonstrated that as little as 12% of axons retained after
injury allows considerable locomotory function as determined by
measuring inclined plane capacity angles (41)
.
Significance
There are several possible approaches for therapeutic intervention
after spinal cord injury. One approach is to try to repair the damage,
for example, by promoting axon regeneration (42)
or
through introduction of embryonic stem cells that differentiate and
possibly re-establish some of the disrupted circuitry
(43)
; these are technically challenging approaches.
A second approach is to minimize secondary mechanisms of tissue damage after spinal cord injury. Prevention of damage is a much more efficacious treatment of spinal cord injury than to attempt repair once damage has occurred. Other than the very modest sparing effect of methylprednisolone, so far there is no effective way of diminishing secondary damage after spinal cord injury. The findings with methylprednisolone indicate that minimizing oxidative stress can have beneficial therapeutic consequences. Since there are many pathways that cause oxidative stress and methylprednisolone acts on only a few of these pathways, any intervention that can act on more than one pathway has the potential to be highly effective in minimizing oxidative stress after a perturbation. As outlined at the beginning of this article, GSH is involved with many pathways that minimize oxidative stress. The promotion of de novo GSH synthesis is especially critical since the glutathione that becomes oxidized after spinal cord trauma forms glutathiyl-protein adducts and, thus, is no longer available to be reduced back to GSH. In principle, therefore promoting GSH synthesis should be an effective way to minimize oxidative stress and thus minimize secondary damage. Indeed, this is what we find.
In summary, we demonstrate that administration of the cysteine prodrug
L-2-oxothiazolidine-4-carboxylate, which has shown few adverse effects
in numerous small human clinical trials (44
45
46)
, can
effectively minimize oxidative stress and tissue damage after spinal
cord trauma, resulting in significant retention of function. Our data
suggest that promoting GSH synthesis may be an effective therapeutic
approach for treating spinal cord injury in humans. In the research
described in this paper we have examined only one dosing regimen, based
on the data presented in Anderson and Meister (16)
and
Mesina et al. (17)
, and a therapeutic intervention
starting at only one time point after injury. We have not yet examined
for any interactions with methylprednisolone, the only approved
intervention after spinal cord injury in humans. It is clear that
before clinical trials can begin, a dose response study must be
performed, the therapeutic window identified, and whether there are
synergistic or antagonistic interactions with methylprednisolone must
be determined. Some of these studies are under way, and preliminary
data indicate that significant therapeutic effects are seen with doses
as low as 1 mmol/kg body weight.
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ACKNOWLEDGMENTS |
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Received for publication April 11, 2000.
Revision received July 7, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  J. D. Golding, S. T. R. MacDonald, B. H. J. Juurlink, and B. W. C. Rosser The effect of glutamine on locomotor performance and skeletal muscle myosins following spinal cord injury in rats J Appl Physiol, October 1, 2006; 101(4): 1045 - 1052. [Abstract] [Full Text] [PDF]
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 X. Sun, A. Y. Shih, H. C. Johannssen, H. Erb, P. Li, and T. H. Murphy Two-photon Imaging of Glutathione Levels in Intact Brain Indicates Enhanced Redox Buffering in Developing Neurons and Cells at the Cerebrospinal Fluid and Blood-Brain Interface J. Biol. Chem., June 23, 2006; 281(25): 17420 - 17431. [Abstract] [Full Text] [PDF]
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 L. Wu, M. H. Noyan Ashraf, M. Facci, R. Wang, P. G. Paterson, A. Ferrie, and B. H. J. Juurlink Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system PNAS, May 4, 2004; 101(18): 7094 - 7099. [Abstract] [Full Text] [PDF]
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