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Introduction
Traumatic spinal cord injury (SCI) includes primary and secondary injury mechanisms. Impact injury to the spinal cord
causes mechanical damage to neuronal and vascular tissue, which is referred as primary
damage[1,2]. Many pathological changes seen after SCI include edema, electrolyte changes, loss of energy metabolism, biochemical changes, altered blood
flow and changes in microvascular permeability, which could give rise to irreversible neural tissue
injury[3_8], and are called secondary injury. Primary impact to the spinal cord causes activation of membrane phospholipases and lipases, hydrolysis
of membrane phospholipids, production of biologically active eicosanoids, and peroxidation of lipids with the formation of
oxygen free radicals. A lot of experimental studies have been performed to show the complex pathophysiological mechanisms
of secondary injury after SCI[9,10].
Previous experimental studies showed that methylprednisolone (MP) treatment had a benefical effect on functional
recovery after SCI[11_13]. Although MP treatment improves neurological recovery after experimental
SCI, the underlying mechanism is not fully understood. Previous reported experimental data point to MP preventing lipid peroxidation, free
radical formation and edema[14].
Resveratrol (3, 4', 5 trihydroxystilbene) is a naturally occurring phytoalexin present in high concentrations in the skin and
seeds of grapes[15]. It occurs naturally in a trans-
or cis isoform of resveratrol are correct
isoform[16,17]. Several studies have shown the antioxidative effect of resveratrol via
prevention of lipid peroxidation[18,19]. Additionally, it has been reported that resveratrol has several biologic effects
such as anti-platelet activity[20], estrogenic
activity[21], anti-inflamatory activity attributed to cyclooxgenase
inhibition[22,23]. Resveratrol also stimulates nitric oxide (NO) production in endothelial cells and has a vasodilatory effect on blood
vessels[24].
The purpose of the present study was to investigate neurobehavioral and histological recovery, and evaluate
biochemical responses to the treatment of experimental SCI in rats with resveratrol, MP and both resveratrol and
MP.
Materials and methods
The experimental protocol was evaluated and approved by the Ethics Review Committee of Inonu University,
Faculty of Medicine. Adult male Wistar albino rats weighing 200_250 g (221.5±28) and aged 14_16 weeks (15±0.9) were used
in this study. The rats were housed in a temperature- controlled room (22_25 ºC). Water and food were given
ad libitum. Before surgery, all rats were tested and a normal motor function was found. The animals were anesthetized by an
intraperitoneal injection of 10 mg/kg xylasine (Bayer Birlesik Alman
Ýlaç Fabrikalari TAS, Istanbul, Turkey) and 50 mg/kg ketamine
hydrochloride (Parke Davis, Ýstanbul, Turkey). A rectal probe was inserted and the animals were positioned on a
thermistor-controlled heating pad. Under sterile surgical conditions, and following a midline skin incision at T5_12 and paravertebral
muscle dissection, spinous processes and laminar arcs of T7_10 were removed with the assistance of a surgical microscope.
The dura was left intact. Weight-drop trauma modeling was performed for all the
animals[25]. The animals were subjected to
an impact of 50 g/cm to the dorsal surface of the spinal cord. The force was
applied via a stainless steel rod (3-mm diameter tip, weighing
5 g) that was rounded at the surface. The rod was dropped vertically through a 10-cm guide tube that was positioned
perpendicular to the center of the spinal cord. After the trauma, the muscles and incision were sutured with 5_0 vicryl suture
(Ethicon, Manifacturer Johnson and Johnson Intl, Lenneke Marelaan, Belgium).
Animals were randomized into six groups, each having 15 rats. Group 1 underwent laminectomy alone. Group 2
underwent laminectomy followed by SCI and received no medication. Group 3 underwent laminectomy followed by SCI and
received resveratrol. Group 4 underwent laminectomy followed by SCI and received MP. Group 5 underwent laminectomy
followed by SCI and received resveratrol and MP. Group 6 underwent laminectomy followed by SCI and received ethanol.
Resveratrol (Sigma Chemicals, St Louis, MO, USA) was prepared freshly by dissolving in 50% ethanol and was
diluted in physiological saline (2%). A 100 mg/kg single dose of resveratrol to group 3; a 30 mg/kg single dose of MP
(Mustafa Nevzat Ilac Sanayi AS) to the group 4; a 100 mg/kg single dose of resveratrol and a 30 mg/kg single dose of MP to
group 5; and 1 mL ethanol (2%) to group 6 were administered intraperiteonally immediately after trauma.
Each group of rats was then subdivided into two subgroups, one of which was killed at the 24 h for biochemical analysis
(6 rats in each group), and the other at the 6th week for neurobehavioral and histopathological assessment (9 rats in each
group) following trauma.
All rats in the 6-week group received gentamycin twice daily during the first 3 d as prophylaxis against urinary tact
infection. Bladders were emptied manually twice a day during this period. In case of mortality in any group, additional rats
were assigned to ensure a minimum of six rats per each study sub-group. Post traumatic neurological recovery was recorded
weekly during six weeks for the rats in the 6-week group. Either after 24 h or 6 weeks, all the animals were killed and 1-cm spinal
cord samples were removed for biochemical analysis and histopathological examination of the injured spinal cord area.
Behavioral assessment
Motor function score We used a motor function
scale[26] that was a slight modification of the motor score defined by Gale
et al[27]. This motor function scale is scored between 0 and 6 points as follows; 0 point for if there is no movement of the hind
limbs; 1 point for only perceptible movement; 2 points for only visible joint movements; 3 points for hind limb movement but
can not support bodyweight; 4 points for movement with supporting bodyweight; 5 points for walking with mild deficit; 6
points for normal walking. The animals were allowed to move freely in an open field
(0.7 m×0.9 m), and were observed for at least 1 min. An observer who was blinded to the groups observed and
recorded the movements in the hip, knee and ankle joints.
Inclined plane score Rivlin and Tator's angle board test, which consisted of measuring the maximum angle at which an
animal can support its weight on an inclined plane measured in 0 to 90 degrees, was used in this
study[28]. The animals were placed transversely on the inclined plane and the highest angle a rat could maintain for 5 s was recorded by the same blinded
observer. And the highest angle for each rat was described as the capacity angle for that rat.
Histopathological assessment Six weeks after SCI, 6 rats from each group were deeply anesthetized with sodium
pentobarbital, and intracardiac perfusion was performed with isotonic saline for 5 min, followed by 10% formaldehyde for 5
min. After perfusion, spinal cords were removed immediately and immersed in 10% formaldehyde for one week. The spinal
cords, which contained the contusion epicenter, were embedded in parafin. Each block was serially sectioned horizontally at
5 µm. Sections were stained with hematoxylin and eosin (H×E). The slides were viewed under a light microscope to study the
structural changes.
Quantitative histopathological evaluation of the spinal lesion was conducted for each sample by light microscopy.
Photographs of spinal cord speciemens were taken under microscopy, and these images were exported to a computer for
analysis. The border of the lesion was drawn and the percentage of lesion areas of each sample was
measured[29] using an image analysis system (Leica Micros Imaging Solutions, Cambridge, UK).
Biochemical analysis Six rats from each group were killed for biochemical analysis 24 h after injury. The injured spinal
cord samples were immediately frozen and stored in a -70 °C freezer for assays of malondialdehyde (MDA), glutathione
(GSH), nitric oxide (NO) levels and xanthine oxidase activity.
Lipid peroxidation measurements
The level of lipid peroxides in traumatized spinal cord tissue were measured as
thiobarbituric acid-reactive material and determined using the method of Mihara and
Uchiyama[30]. MDA has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red species absorbing at 535 nm. The assay
procedure for lipid peroxide in spinal cord tissue was set up as follows. Tissues were homogenized in 10 volumes
(w/v) of cold 1.5% KCl. Half a milliliter (0.5 mL) of homogenate was mixed with 3 mL of 1%
H3PO4 and 1 mL 0.6% thiobarbituric acid.
The mixture was then heated in boiling water for 60 min. After cooling, the color was extracted into 4 mL
n-butanol and the absorbance was recorded at 535 nm and 520 nm. Using tetramethoxypropane as the standard, tissue lipid peroxidate levels
were calculated as nanomole per gram of wet tissue.
Glutathione level
measurements GSH levels were measured using the method of
Elman[31]. GSH reacted with 5,5-dithiobis-2-nitrobenzoic acid resulting in the formation of a product that has a maximal absorbance at 410 nm. The results are
expressed as nanomoles per gram wet tissue.
Nitrite/nitrate measurements
NO levels were measured as total nitrite with the spectrophotometric Greiss reaction,
because total nitrite is an index of endogenous nitric oxide
production[32,33]. The procedure was partly adapted from the
method discribed by Ozbek et
al[34]. Results were reported as per gram wet tissue.
Xanthine oxidase (XO) activity
measurements XO activity was determined spectrophometrically according to the
method of Prajda and Weber based on the formation of uric acid from xanthine, which increases absorbance at 292 nm
(eM 9.2×103)
[35]. One unit of activity was defined as 1 mmol of uric acid formed per min, and data are presented as U/mg protein.
Statistical analysis Data were analyzed using an SPSS 13.0 for Windows program on a computer. Results were expressed
as mean±SEM. All parameters were tested with Shapiro-Wilks test, and distribution was not normal
(P>
0.05). Therefore, comparison among groups was tested with Kruskal-Wallis test and Bonferroni Mann-Whitney U-test was
used for dual comparison. P<0.05 was considered statistically significant.
Results
Mortality Some rats from the 6-week group died after spinal cord injury before completion of the protocol. Three of 9 rats
in group 2 (33.3%), 2 of 9 rats in group 3 (22.2%), 1 of 9 rats in group 4 (11.1%), and 2 of 9 rats in group 5 (22.2%) died within
the first 2 weeks of the study. The proportions among the 6-week groups varied widely at the end of study. For equalizing
the rat number in each group, 1 rat from group 3, 2 rats from group 4, 1 rat from group 5, and 3 rats from group 6 were randomly
excluded from the study.
Behavioral assessment
Motor function score Trauma caused a significant impairment in motor function score as compared to the control group
values (Figure 1). A significant recovery rate was observed in the rats treated with MP, resveratrol and MP+resveratrol
(P<0.05). When treatment groups were compared at the 6th week, resveratrol and the combined treatment group revealed a
significantly higher motor improvement rate than the MP treatment group
(P<0.05). However there was no significant
difference between the resveratrol and combined treatment group
(P>0.05).
Inclined plane score Trauma produced a highly significant decrease in angle score, which then gradually recovered over
a period of 6 weeks in the rats treated with resveratrol, MP and MP+resveratrol
(P<0.05) (Figure 2). Groups 3 and 5 revealed
significantly more improvement rates for the capacity on the inclined angle board than group 4
(P<0.05).
Histopathological assessment Spinal cord sections obtained from sham-surgery animals were
normal. The epicenter of the injured spinal cord obtained from trauma and vehicle groups showed characteristic necrosis, wide demye-lination, and
cavitation of white matter in the posteromedial regions of the spinal cord (Figure 3A, 3E). Treatment groups (groups 3,4,5)
showed lower cavitation than group 2 and 6 and these groups are correct of white matter in the posteromedial regions of the
spinal cord (Figure 3B_3D).
The injured area was selected and calculated as a percentage of the whole spinal cord area (Figure 4). Lesion area in the
spinal cord in group 2 and 6 were calculated as 27% and 26.6%, respectively. Treatment with resveratrol, MP and resveratrol+MP
significantly reduced the contused areas (19.3%, 20.4%, and 18.8%, respectively)
(P<0.05). There was no significat difference
between the treatment groups (P>0.05).
Biochemical analysis
Lipid peroxidation levels
Trauma and vehicle groups were found to produce a significant elevation in lipid peroxidation
levels. Groups 3_5 revealed significantly lower MDA levels than groups 2 and 6
(P<0.05). However group 3 revealed significantly lower MDA levels than group 4
(P<
0.05), group 5 showed significantly lower MDA levels when compared to groups 3 and 4
(P<0.05) (Figure 5).
Glutathione levels GSH levels were estimated in both control, trauma, vehicle and treatment groups (Figure 6). Levels of
reduced glutathione in trauma and vehicle groups were significantly lower than the control group
(P<0.05). Groups 3_5 revealed significantly higher GSH levels than groups 2 and 6
(P<0.05). Groups 3 and 5 revealed significantly higher GSH
levels when compared to group 4 (P<
0.05). There was no significant difference between groups 3 and 5
(P>0.05).
Nitrite/nitrate levels Trauma caused significant elavation in NO levels
(P<0.05). Treatment with resveratrol, MP and
resveratrol+MP significantly reduced NO levels
(P<0.05). Combined treatment with resveratrol and MP caused significantly
lower NO levels than resveratrol and MP treatment groups
(P<0.05). Similiar to the combined treatment group, the resveratrol
treatment group revealed lower NO levels than the MP group
(P<0.05) (Figure 7).
Xanthine oxidase (XO) levels
XO levels significantly elevated in the trauma and vehicle groups when compared to the
control group (P<0.05). Treatment with MP, resveratrol or combined treatment with MP and resvertarol significantly
decreased the XO levels (P<0.05). When all treatment groups were compared to each other, groups 3 and 5 revealed
significantly lower XO levels than group 4
(P<0.05). There was no significant difference between group 3 and 5
(P>0.05) (Figure 8).
Discussion
In the present study, we have demonstrated neuroprotec-tive effects of resveratrol after SCI. Our study confirmed a
previous study[36] that resveratrol reduced lipid peroxidation and showed that resveratrol had a benefical effect on long term
neurological and histopathological outcome after SCI.
Traumatic spinal cord injury proceeds through a series of sequential events, beginning with primary injury and followed
by secondary injury. Tissue repair, and some degree of neurological recovery after injury depend on the severity of primary
injury. Primary mechanical injury may cause neuronal tissue damage and loss of axons, rupture of arterioles and venules
causing hemorrhage and blockage of axoplasmic transport. The pathological processes involved in secondary injury include
free radical generation, intracellular influx of calcium, ischaemia and
edema[17,37,38].
Previous studies showed that high-dose MP had a neuro-protective effect immediately after SCI. The reduction of lipid
peroxidation has been postulated to be the major neuroprotective mechanism of
MP[14,39,40]. It suppresses the breakdown of
membranes by inhibiting lipid peroxidation and hydrolysis at the site of
injury[5,41]. Young recommended that all experimental
SCI studies should be compared with treatment agonist
MP[42]. So, we used MP in this study to compare aganist resveratrol.
Reactive oxygen species (ROS) consist of oxygen free radicals and associated entities that include superoxide free
radicals, hydrogen peroxide, singlet oxygen, nitric oxide (NO), and peroxynitrite. The production of ROS can lead to cell
injury through cell membrane lipid destruction and can
diffuse intracellularly and cause mitochondrial enzyme damage and cleavage of
DNA[43,44]. Recently, resveratrol was found
to be a highly potent antioxidant that could inhibit free radical generation in the brain, spinal cord, heart, kidney, liver, red cell
membrane, and so on[19,36,44_49]. It has been shown that it inhibits lipid
peroxidation[50], and prevents apoptotic cell death
induced by oxidative stress[51,52]. It has been postulated that resveratrol could supress mitochondria-induced production of
ROS in rat brain[53], protect DNA from oxidative damage in stroke-prone hypertensive
rats[54], and could inhibit neuronal loss
after ischemia/reperfusion injury in
gerbils[44]. Direct neuroprotective effect of resveratrol against oxidative stress has been
demonstrated in PC12 cells[55]. After SCI, blood flow in the spinal cord is reduced, microcirculation is disturbed and
microvascular permeability increases, which leads to spinal cord
edema[26]. Kaplan et al showed that prophylactic use of resveratrol
reduced neurologic injury and provided clinical improvement by attenuating the inflamatory milieu in the rabit spinal cord
ischemia/reperfusion model[56]. Yang
et al showed that resveratrol reduced spinal cord edema rates by 11.5% and improved
the energy metabolism system 48 h after
SCI[36].
It is known that lipid peroxidation products increase soon after
injury[4,57]. Free oxygen radical-mediated lipid
peroxi-dation has been increasingly suggested to be an important factor in posttraumatic neuronal degeneration. MDA is a main
breakdown product of LP in the CNS and is formed from the breakdown of polyunsaturated fatty acids, serving as a
convenient index for determining the extent of lipid peroxi-dation. In the present study, tissue lipid peroxidation was
evaluated by measuring the thiobarbituric acid reactive substances. Recent studies showed that MDA levels in the injured
spinal cord had similar values within 24
h[38]. So, we assessed MDA levels of all groups at the 24 h post-injury. Our study
showed that resveratrol treatment was more effective than MP treatment and combined treatment with resveratrol and MP
had an additional protective effect on lipid peroxidation levels. The effect of MP and resveratrol on lipid peroxidation is on
a different route. MP, a glucocorticoid with potent anti-inflammatory properties, induces synthesis and the release of
anti-inflammatory peptides. This activity plays a major role in its neuroprotective effect and requires receptor
activation[58]. Resveratrol is known as a free radical scavenger. Additional neuroprotection on lipid peroxidation with a combined treatment
may be explained by the synergistic or additive effect of resveratrol and MP in the acute phase of injury.
The potential harmful effect of oxidative stress is controlled by cellular antioxidant defense mechanisms including
enzymatic defense systems and non-enzymatic defense systems such as reduced glutathione (GSH). Glutathione is an
endogenous antioxidant found in all animal cells. It reacts with the free radicals and can protect cells from singlet oxygen, hydroxide
radical, and superoxide radical damage. Decreased levels of reduced glutathione has been reported after excessive
generation of free radicals and described as a result of compensatory mechanism to combat increased oxidative
stress[6,59]. In the present study a decreased level of glutathione was seen after SCI. This result may be secondary to raised MDA levels. All
the treatment groups were found to have significantly elevated GSH levels. Similar to lipid peroxidation, resveratrol treatment
resulted in higher GSH levels than MP treatment, but combined treatment with both agents did not reveal additional
neuroprotection.
Oxygen free radicals can be generated by a variety of sources at the cellular level. An important source of oxygen free
radicals is known to be xanthine oxidase (XO), which can be formed from xanthine dehydrogenase (XD) either reversibly (via
oxidation or blockage of its thiol groups) or irreversibly (via limited proteolysis) in pathological
conditions[60]. In a normal tissue, XD, which is the native form of the enzyme, catalyzes the conversion of hypoxanthine to xanthine and uric acid
through the utilization of NAD+ as its electron acceptor. Indeed, this reaction does not lead to the production of oxygen free
radicals or the generation of hydrogen peroxide
(H2O2). However, XO is also able to catalyze the reduction of oxygen in the
pathological states leading to the formation of superoxide
(O2¯· ) and
H2O2 where the reaction is considered to play a crucial
role in the proposed mechanism of oxidative
injury[60,61].
Nitric oxide (NO) is a free radical gas molecule that is produced from
L-arginine by the catalytic action of nitric oxide
synthases (eNOS, iNOS, and nNOS) [62]. In physiological levels, NO participates in a variety of physiological processes
consisting of neurotransmission and regulation of the blood vessel
wall[63]. But increased NO, especially associated with
oxidative stress, is a harmful condition for tissue. In the present study, both GSH, XO, and NO levels have been used in order
to show the severity of spinal cord injury and the effectiveness of pharmacological agents on oxidative stress. It is shown
that resveratrol is more effective on both GSH, XO, and NO levels than MP. Combined treatment revealed more neuroprotective
effects only for NO levels.
The neurological deficits following spinal cord trauma may increase during the first hours up to the first week following
injury. Behavioral tests are important tools to assess the outcome of experimental spinal cord injuries, including
spontaneous functional improvement over time, and the
effects of different treatments. Many experimental and clinical studies have focused on secondary injury mechanisms in an
effort to improve neurological outcome following spinal cord injury. A reliable test protocol suitable for the injury model used
is essential to evaluate functional recovery after spinal cord
injury[27,28,64,65]. The inclined plane is a test known to be sensitive
and reliable in evaluating different degrees of spinal cord injury. This test has been validated for rats with compression
injuries as well as contusive
injuries[27,28]. A significant slower recovery was observed in all of the
injured rats treated with MP, resveratrol or combined treatment for motor function score and inclined angle board capacity.
When each group was evaluated by itself, statistically significant differences were found between the first and the sixth
weeks for the all groups. However, significant differences in the amounts of recovery rates were found among them. At the
end of observation it can be stated that the effect of resveratrol on neurological recovery is better than MP, and the combined
treatment of both drugs did not contribute to an increased rate of recovery.
Yang et al demonstrated that both resveratrol and MP had a protective effect on axons, neurons, myelin and subcellular
organelles[36]. But their experimental design comprised the acute phase of the SCI (48 h maximum). The present experimental
results suggest that single dose of 100 mg/kg resveratrol administration could effectively reduce the
lesion area after SCI. When resveratrol treatment was compared to MP treatment, at the end of the 6 weeks, resveratrol did
not have a more remarkable protective effect on the
lesion area. Combined treatment with resveratrol and MP did not provide an additional effect on the lesion area. From our
findings, we can propose that resveratrol strongly
improves the histopathological outcome following spinal cord injury. Yang
et al used single doses of 50 mg/kg and 100
mg/kg resveratrol in their study where they suggested that resvera-trol had a stronger effect in improving the energy metabolism
system and inhibiting the LP than MP at a dose of 100 mg/kg. In our study we used a 100 mg/kg single dose of resveratrol and
a 30 mg/kg single dose of MP[38]. Our results showed that treatment with resveratrol revealed better results for both MDA,
GSH, NO, and XO levels than MP treatment in the acute stage of experiment. And also that resveratrol treatment revealed
better results for neurobe-havioral recovery in the chronic stage of the experiment. The interesting finding in this study is
that, in spite of better biochemical results in the resveratrol treatment group in the acute stage of the study, all the treatment
groups revealed similiar histopathological recovery at the chronic stage. It could be stated that combined treatment with
both drugs doesn't give significantly better results than seperate using of them for the chronic stage of SCI.
In conclusion, resveratrol treatment is more effective than MP treatment for the prevention of lipid peroxidation after
spinal cord injury in the acute stage of SCI. The anti-oxidative effect of MP and resveratrol is by different routes. These
different anti-oxidative mechanisms provide additional protection in the acute stage. Although there was better neurobehavioral
recovery in the resveratrol treatment group, each agent caused similiar histopathological recovery in the chronic stage. So,
we can claim that resveratrol is an agent at least as effective as MP on secondary damage after SCI. Although they prevent
secondary damage through different mechanisms, combined use does not provide extra protection in the chronic stage of
SCI.
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