Intraperitoneal Alpha-Lipoic Acid to prevent neural damage after crush injury to the rat sciatic nerve.

Abstract

Crush injury to the sciatic nerve causes oxidative stress. Alfa Lipoic acid (a-LA) is a neuroprotective metabolic antioxidant. This study was designed to investigate the antioxidant effects of pretreatment with a-LA on the crush injury of rat sciatic nerve.
Forty rats were randomized into four groups. Group I and Group II received saline (2 ml, intraperitoneally) and a-LA (100 mg/kg, 2 ml, intraperitoneally) in the groups III and IV at the 24 and 1 hour prior to the crush injury. In groups II, III and IV, the left sciatic nerve was exposed and compressed for 60 seconds with a jeweler's forceps. In Group I (n = 10), the sciatic nerve was explored but not crushed. In all groups of rats, superoxide dismutase (SOD) and catalase (CAT) activities, as well as malondialdehyde (MDA) levels were measured in samples of sciatic nerve tissue.
Compared to Group I, Group II had significantly decreased tissue SOD and CAT activities and elevated MDA levels indicating crush injury (p < 0.05). In the a-LA treatment groups (groups III and IV), tissue CAT and SOD activities were significantly increased and MDA levels significantly decreased at the first hour (p < 0.05) and on the 3rd day (p < 0.05). There was no significant difference between a-LA treatment groups (p > 0.05).
A-LA administered before crush injury of the sciatic nerve showed significant protective effects against crush injury by decreasing the oxidative stress. A-LA should be considered in the treatment of peripheral nerve injuries, but further studies are needed to explain the mechanism of its neuroprotective effects.

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BioMed Central
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Journal of Brachial Plexus and
Peripheral Nerve Injury
Open Access
Research article
Intraperitoneal Alpha-Lipoic Acid to prevent neural damage after
crush injury to the rat sciatic nerve
Mehmet Senoglu*1, Vedat Nacitarhan2, Ergul Belge Kurutas3,
Nimet Senoglu4, Idris Altun1, Yalcin Atli3 and Davut Ozbag5
Address: 1Department of Neurosurgery, Kahramanmaras Sutcu Imam University Faculty of Medicine, Kahramanmaras, Turkey, 2Department of
Physical Medicine and Rehabilitation, Kahramanmaras Sutcu Imam University Faculty of Medicine, Kahramanmaras, Turkey, 3Department of
Biochemistry, Kahramanmaras Sutcu Imam University Faculty of Medicine, Kahramanmaras, Turkey, 4Department of Anaesthesiology and
Reanimation, Kahramanmaras Sutcu Imam University Faculty of Medicine, Kahramanmaras, Turkey and 5Department of Anatomy,
Kahramanmaras Sutcu Imam University Faculty of Medicine, Kahramanmaras, Turkey
Email: Mehmet Senoglu* - mehmetsenoglu@hotmail.com; Vedat Nacitarhan - nacitarhan@ttnet.net.tr;
Ergul Belge Kurutas - ekurutas@hotmail.com; Nimet Senoglu - nimetsenoglu@hotmail.com; Idris Altun - idrisaltun46@hotmail.com;
Yalcin Atli - atliyalcin@gmail.com; Davut Ozbag - davutozbag@hotmail.com
* Corresponding author
Abstract
Objective: Crush injury to the sciatic nerve causes oxidative stress. Alfa Lipoic acid (a-LA) is a
neuroprotective metabolic antioxidant. This study was designed to investigate the antioxidant
effects of pretreatment with a-LA on the crush injury of rat sciatic nerve.
Methods: Forty rats were randomized into four groups. Group I and Group II received saline (2
ml, intraperitoneally) and a-LA (100 mg/kg, 2 ml, intraperitoneally) in the groups III and IV at the 24
and 1 hour prior to the crush injury. In groups II, III and IV, the left sciatic nerve was exposed and
compressed for 60 seconds with a jeweler's forceps. In Group I (n = 10), the sciatic nerve was
explored but not crushed. In all groups of rats, superoxide dismutase (SOD) and catalase (CAT)
activities, as well as malondialdehyde (MDA) levels were measured in samples of sciatic nerve
tissue.
Results: Compared to Group I, Group II had significantly decreased tissue SOD and CAT activities
and elevated MDA levels indicating crush injury (p < 0.05). In the a-LA treatment groups (groups
III and IV), tissue CAT and SOD activities were significantly increased and MDA levels significantly
decreased at the first hour (p < 0.05) and on the 3rd day (p < 0.05). There was no significant
difference between a-LA treatment groups (p > 0.05).
Conclusion: A-LA administered before crush injury of the sciatic nerve showed significant
protective effects against crush injury by decreasing the oxidative stress. A-LA should be
considered in the treatment of peripheral nerve injuries, but further studies are needed to explain
the mechanism of its neuroprotective effects.
Published: 25 November 2009
Journal of Brachial Plexus and Peripheral Nerve Injury 2009, 4:22doi:10.1186/1749-7221-4-22
Received: 1 September 2009
Accepted: 25 November 2009
This article is available from: http://www.jbppni.com/content/4/1/22
© 2009 Senoglu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Introduction
The rat sciatic nerve is a well-established preparation for
studying peripheral nerve injuries. Focal crush injury
causes axonal interruption but preserves the connective
sheaths (axonotmesis). As regards this type of injury,
nerve regeneration is usually successful [1].
The increased formation of reactive oxygen species (ROS)
and decreased antioxidant defense is defined as oxidative
stress, which is widely recognized as an important feature
of many diseases. Superoxide dismutase (SOD), and cata-
lase (CAT) are cellular antioxidants, which protect cells
from oxidative stress. Lipid peroxidation (LPO) is one of
the most important expressions of oxidative stress
induced by ROS. Malondialdehyde (MDA) is an indicator
of lipid peroxidation, and increases in various diseases
[2].
Alpha-Lipoic acid (a-LA) is a powerful lipophilic antioxi-
dant in vitro and in vivo, which plays a pivotal role as
cofactor in many mitochondrial reactions, readily
absorbed from the diet and can easily cross the blood
brain barrier [3].
It is known to act as scavenger of many reactive oxygen
species and to interact with other antioxidants such vita-
min C and vitamin E, resulting in their regeneration. Due
to its antioxidant activity, a-LA has been proposed as a
treatment for oxidative disorders of the nervous system
that involve free radicals since it exerts a profound neuro-
protective effect in experimental models of stroke, trauma,
degenerative disorders of the CNS and diabetes [3].
Administration of a-LA to rodents has been demonstrated
to reduce the damage that occurs after ischemia-reper-
fusion injuries in the cerebral cortex [3], heart [4,5] and
peripheral nerve [6], and after injection of NMDA into the
striatum [7]. However, to our knowledge, the effects of a-
LA on crush injury have not been investigated in the Eng-
lish literature [3-7].
The increased formation of ROS and decreased antioxi-
dant defense is defined as oxidative stress, which is widely
recognized as an important feature of many diseases.
SOD, and CAT are cellular antioxidants, which protect
cells from oxidative stress. LPO is one of the most impor-
tant expressions of oxidative stress induced by ROS. MDA
is an indicator of lipid peroxidation, and increases in var-
ious diseases [2].
The purpose of this study was to investigate the effects of
a-LA on sciatic nerve injury by measurement of SOD and
CAT activities, as well as MDA level in sciatic nerve crush
injury model in rats.
Materials and methods
Animals and Surgery
This prospective, experimental, sham-control study was
performed in the animal laboratory of the Kahraman-
maras Sutcu Imam University, Faculty of Medicine.
Female Sprague-Dawley rats were obtained from Experi-
mental Research Laboratory of Sutcu Imam University
Faculty of Medicine. The experimental design was
approved by the Ethics committee of KSU. Rats were fed
with standard rat diet routinely, however they were
deprived of food for 12 h prior to the first operation. All
rats had free access to standard rat chow and tap water.
Forty adult female Sprague-Dawley rats (200-250 grams)
were used in this study. Rats were randomly divided into
four groups including one sham, one control and two
treatment groups.
Group I - (Sham group) Normal adult female rats (Non-
crush): Non-crush group, no intervention was made, sim-
ply sciatic nerve samples were taken.
Group II - (Control group) 60 seconds of sciatic crush was
performed and then sciatic nerve samples were taken at
the 1st hour.
Group III - Crush-a-LA group (1 hr): 100 mg/kg intraperi-
toneal a-LA injection was done 24 and 1 hour before crush
injury. Sixty seconds of crush was performed. Sciatic nerve
samples were taken at the 1st hour.
Group IV - Crush-a-LA group (3rd day): 100 mg/kg intra-
peritoneal a-LA injection was done 24 and 1 hour before
crush injury. Sixty seconds of crush was performed. Sciatic
nerve samples were taken on the 3rd day.
Groups I and II received saline (2 ml, intraperitoneally).
However, groups III and IV received saline plus a-LA (100
mg/kg, 2 ml, intraperitoneally [MEDA Pharma GmbH &
Co. KG]) [8] at 1 h and 24 h before the crush injury. We
aimed to investigate the early effects of antioxidant ther-
apy with a-LA. In Group I, sciatic nerve was explorated but
not crushed. Sciatic nerve injury was induced in groups II,
III and IV. Briefly stated, exploration was conducted under
anesthesia with intraperitoneal pentobarbital (50 mg/kg),
while body temperature was maintained by using a heat-
ing blanket at 35-37°C. The sciatic nerve was exposed in
the mid-gluteal region through biceps muscle dissection
under an operating microscope, crushed by a #4 Jeweler's
forceps at the mid-point for 60 seconds, then unclamped.
The site of crush was marked with a 5-0 suture tied in sur-
rounding muscle. The operated animals were allowed and
survived. The nerves were re-exposed under the operating
microscope one hour later in groups I, II and III, and 3

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days later in Group IV and the nerve tissue was harvested.
One-centimeter-long sciatic nerve segments centered on
the lesion site points were collected for biochemical anal-
yses. No prophylactic antibiotics were given. The experi-
mental model was very well tolerated. No animal died
during the operations.
Preparation of tissue homogenates
Tissue samples were immediately excised, weighed, per-
fused with 1.15% ice-cold KCl, minced, then homoge-
nized in five volumes (w/v) of the same solution, using a
Heidolph 50110 R2R0 homogenizer. Antioxidant
enzymes and MDA assays were performed on the superna-
tant preparation in a Sorvall RC-2B centrifugation of the
homogenate at 14.000 rpm for 30 min at +4°C.
Evaluation of biochemical parameters
CAT activities were determined by measuring the decrease
in hydrogen peroxide concentration at 230 nm by the
method of Beutler [9]. Assay medium consisted of 1 M
Tris HCl-5 mM Na2EDTA buffer solution (pH 8.0), 1.0 M
phosphate buffer solution (pH 7.0), and 10 mM H2O2.
CAT activity was expressed as U/mg protein.
SOD activity was measured according to the method
described by Fridovich [10]. This method employs xan-
thine and xanthine oxidase to generate superoxide radi-
cals which react with p-iodonitrotetrazolium violet (INT)
to form a red formazan dye which was measured at 505
nm. Assay medium consisted of the 0.01 M phosphate
buffer, CAPS (3-cyclohexilamino-1-propanesulfonicacid)
buffer solution (50 mM CAPS, 0.94 mM EDTA, saturated
NaOH) with pH 10.2, solution of substrate (0.05 mM
xanthine, 0.025 mM INT) and 80 U/L xanthine oxidase.
SOD activity was expressed as U/mg protein.
LPO level in the tissue samples was expressed as MDA. It
was measured according to procedure of Ohkawa et al
[11]. The reaction mixture contained 0.1 ml of sample,
0.2 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of
20% acetic acid and 1.5 ml of 0.8% aqueous solution of
TBA. The mixture pH was adjusted to 3.5 and volume was
finally made up to 4.0 ml with distilled water and 5.0 ml
of the mixture of n-butanol and pyridine (15:1, v/v) were
added. The mixture was shaken vigorously. After centrifu-
gation at 4000 rpm for 10 min, the absorbance of the
organic layer was measured at 532 nm. The results of
MDA were expressed as nmol/mg protein.
Assay of Protein levels
The protein concentration of the tissue was measured in
digital Spectronic-20 spectrophotometer by the method
of Lowry [12].
Statistical analysis
All variables were expressed as medians, mean ± standard
deviation with the range. Data were analyzed using Mann-
Whitney U-test. Differences were considered significant
when the probability was less than 0.05. All data were
entered and processed by an SPSS 9.05 for Windows sta-
tistical package.
Results
Oxidative parameter results are presented in Table 1.
Results of the antioxidants levels in all groups are pre-
sented in Figures 1, 2, 3.
Compared with sham group (Group I), tissue SOD and
CAT activities decreased and MDA level elevated signifi-
cantly in the control group (Group II), which indicated
crush injury (p < 0.05). After a-LA treatment tissue SOD
and CAT activities increased and MDA level decreased sig-
nificantly, at the first hour (Group III, p < 0.05) and on the
3rd day (Group IV, p < 0.05). However, there were no sig-
nificant differences between treatment groups (Groups III
and IV, p > 0.05).
Discussion
In the present study, we investigated antioxidant effects of
a-LA on sciatic nerve which was subjected to 60 seconds of
crush injury, based on differences observed in biochemi-
cal parameters measured. We observed that a-LA had anti-
oxidant effects on injured sciatic nerve and these effects
were similar at the first hour and on the 3rd day.
Sciatic nerve MDA (nmol/mg protein) levels in all groups
Figure 1
Sciatic nerve MDA (nmol/mg protein) levels in all
groups.

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The pathophysiology of the crush injury has not been
fully understood, and it has been debated whether the
ischemia, secondary to compression, or the mechanical
deformation of nerve fibers per se is the more significant
etiologic factor [13].
Nerve injury may depend on the length of time of crush
insult. During a pilot study of our research, we showed
that especially 60-second compression caused injury in
sciatic nerve. After the injury due to the tissue destruction,
free oxygen radicals increase and cause tissue damage
[14,15].
Normal cell functions and integrity of cell structures may
be broken via considerable reactivity of ROS. The organ-
ism has enzymatic (e.g. superoxide dismutase, catalase,
glutathione peroxidase) and non-enzymatic (e.g. vitamin
C, vitamin E) antioxidant mechanisms that work as scav-
engers for the harmful ROS. Radical-scavenging antioxi-
dants are consumed by the increased free radical activity.
Oxidative stress can be defined as an increase in oxidants
and/or a decrease in antioxidant capacity. Although deter-
mination of either oxidants or antioxidant components
alone may give information about the oxidative stress,
determination of oxidants along with antioxidants is
more useful in this context. Therefore, oxidants and anti-
oxidant capacity should be measured simultaneously to
assess oxidative stress more accurately. In addition, the
total plasma LPO level, as an indicator of oxidative stress,
reflects the redox balance between oxidation and anti-oxi-
dation. In addition, excess amounts of ROS generated in
inflamed tissues can cause injury to host cells and also
induce DNA damage and mutations [16]. And, oxidative
DNA damage has been suggested to play an important
role in the development of cancer [17]. In several studies,
increase in oxidative components or decrease in antioxi-
dants or both have been reported in subjects with either
acute or chronic various diseases [18-21].
Antioxidant activity is a relative concept: it depends on the
kind of oxidative stress and the kind of oxidizable sub-
strate (e.g., DNA, lipid, protein). To control oxidative
processes, biological systems have been equipped with
several antioxidant mechanisms. Antioxidant enzymes
such as SOD and CAT are concerned with the removal of
superoxide anion and peroxide. An imbalance between
oxidative and antioxidative processes results in oxidative
stress. Drugs can intervene in oxidative processes as anti-
oxidants and delay or prevent their damaging effects. A-LA
acid is an example of an existing drug therapeutic effect of
which has been related to its antioxidant activity. Many
experimental results show that both lipoic acid and Dihy-
drolipoic acid (DHLA) can improve the antioxidant
capacity of tissue against different forms of oxidative
stress. Hence, some physicians started to administer lipoic
acid to patients with liver cirrhosis, mushroom poisoning,
heavy metal intoxication and diabetic polyneuropathy
[22].
The SOD-CAT system provides the first defense against
oxygen toxicity. SOD catalyzes the dismutation of the
superoxide anion radical to water and hydrogen peroxide,
which is detoxified by the CAT activity. Usually a simulta-
neous induction response in the activities of SOD and
Sciatic nerve CAT (U/mg protein) levels in all groups
Figure 2
Sciatic nerve CAT (U/mg protein) levels in all groups.
Sciatic nerve SOD (U/mg protein) levels in all groups
Figure 3
Sciatic nerve SOD (U/mg protein) levels in all groups.

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CAT is observed when an exogen antioxidant is applied
[23]. In the present study, the activities of SOD and CAT
were also found to be high in sciatic tissue of rats in
groups III and IV. The activity of SOD was reported to be
higher in various diseases by several workers [19,21] indi-
cating high production of superoxide anion radical. Since
CAT levels were detected high in sciatic tissue of rats in
groups III and IV, this may be attributed to high produc-
tion of peroxide radicals. Increased SOD and CAT activi-
ties in in those groups may be a response against oxidative
stress.
The extent of LPO is determined by the balance between
the production of oxidants and the removal and scaveng-
ing of those oxidants by antioxidants. Therefore, lipid per-
oxidation has been extensively used as a marker of
oxidative stress [24]. Antioxidants are potential candi-
dates for prevention or treatment of oxidative damage and
free radical injury [5,25].
In this study, tissue MDA levels increased and CAT and
SOD activities decreased significantly in the control group
compared with sham group showing crush injury. After a-
LA treatment, groups III and IV had significantly higher
tissue SOD and CAT activities and lower MDA levels than
control group showing antioxidative effects. However,
these antioxidant effects were similar in treatment groups
(groups III and IV) showing that preventive antioxidant
effects of a-LA took place in the early phase. This finding
was concordant with the finding of decreased oxidative
injury (i.e. decreased MDA levels in nerve tissue) seen in
pretreated groups, which was confirmed by biochemical
parameters. Oxidative stress is a mechanism of nerve
injury but likely not the major mechanism, and that ther-
apeutic strategy for neuroprotection from crush injury
should not be based on antioxidants alone.
Further clinical and laboratory investigations focusing on
specific mechanism of antioxidant effects of a-LA, and its
therapeutic effects in peripheral nerve injury is warranted.
Abbreviations
SOD: superoxide dismutase; CAT: catalase; MDA:
malondialdehyde; a-LA: Alfa Lipoic acid; CNS: Central
Nervous System; ROS: reactive oxygen species; LPO: Lipid
peroxidation; DHLA: Dihydrolipoic acid.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MS and NS designed the study and drafted the manu-
script. MS and IA performed experimental operations.
EBK, YA and DO had specimen collection of this experi-
mental study. VN and performed the statistical analysis.
All authors read and approved the final manuscript.
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Table 1: The activities of antioxidant enzymes and MDA levels in four groups.
MDA (nmol/mg prt)CAT (U/mg prt) SOD (U/mg prt)
Mean ± SDMedian
(min-max)
Mean ± SDMedian
(min-max)
Mean ± SDMedian
(min-max)
Group I
(sham)
1.64 ± 1.051.49
(0.48-3.90)
152.18 ± 75.29 150.30
(55.27-339.72)
193.21 ± 46.38 194.60
(111.06-276.40)
Group II
(control)
6.02 ± 4.504,10
(3.25-16.80)
76.43 ± 50.4364.57
(24.38-157.75)
84.58 ± 23.8379,00
(50.86-121.96)
Group III
Crush-a-LA group (1 hr)
1.84 ± 0.831,90
(0.25-3.39)
114.14 ± 25.99 112.81
(73.49-151.86)
128.12 ± 54.60125.82
(70.45-217.56)
Group IV
Crush-a-LA group
(3rd day)
2.18 ± 0.982.12
(0.60-3.90)
122.89 ± 77.63 98.82
(76.21-339.72)
108.21 ± 35.56103.93
(54.40-172.01)
Values were expressed as mean ± SD and median and range. Group I (Control), Group II (Sham), Group III and IV (Treatment).