Introduction
Brachial plexus injuries in adults are commonly caused by auto or motorcycle accidents.
The treatment of this type of injury consists of nerve repair and nerve grafting for
extraforaminal nerve root or trunk injury, and of neurotization or nerve transfer
for nerve roots avulsion; however, the outcome of brachial plexus reconstruction and
the restoration of shoulder and elbow function are often poor in spite of the sophistication
of the various methods used[[1],[2]]. The death of a major proportion of the innervating neuronal pool is likely to
be the most fundamental neurobiological barrier to functional restitution because
survival is an essential prerequisite for regeneration[[3]]. Currently the primary aim of management of root avulsion of the brachial plexus
is motor recovery. However, 80-90% of motoneurons have been shown to die after avulsion[[4],[5]], complicating this goal. Immediate repair or nerve grafting offers some degree
of protection to the motoneurons but is clinically limited, so there remains a need
for medical approaches to maintain the viability of the injured motoneurons.
The Ginkgo biloba extract EGb761 is a standardized mixture of active substances obtained
from green leaves of the Ginkgo biloba tree, composed of 24% flavonoid glycosides
and 6% terpenoids[[6],[7]]. EGb761 has been reported to be a potent free radical scavenger and many studies
have demonstrated that the compound affects hemodynamics, metabolism, and hemorrheology.
Additionally, EGb761 has antioxidant properties and transmitter/receptor effects in
the brain, spinal cord, peripheral nervous system, retina, vestibulocochlear apparatus
and cardiovascular system[[8],[9],[10]]. The mechanism of EGb761 action in the central nervous system (CNS) is relatively
well-studied, and the main effect it exerts seems to be related to its antioxidant
properties. Recently, in vitro studies have shown that EGb761 has a protective effect
against neuronal apoptotic death[[11],[12]] and an inhibitory effect on the expression of inducible nitric oxide synthase (iNOS)
and nitric oxide (NO) production [[13]]. Animal experiments have also shown that EGb761 can prevent neuronal damage after
brain ischemia through the inhibition of NOS [[14]]. However, its potential effect in patients suffering from spinal cord injury (SCI)
is still unknown. In our previous studies, de novo expression of neuronal NOS (nNOS)
was observed in injured motoneurons, and the time course and density of nNOS expression
both correlated well with the severity of motoneuron death following brachial root
avulsion, in which the oxidant peroxynitrite played an important role [[15],[16]]. This raises the question of whether EGb761 has a similar neuroprotective effect
on avulsion-injured motoneurons. In the present study, we used EGb761 to treat rats
immediately after avulsion injury. The effect of EGb761 was estimated according to
the survival of injured motoneurons. The investigation of the protective mechanism
of EGb761 was focused on the production of NO, and the activity of both nNOS and iNOS.
Our present study found that EGb761 protects motoneurons against avulsion injury and
that this neuroprotective effect was related to the reduction of both NO and NOS in
the injured spinal cord.
Materials and methods
Animals and Surgery
Adult male Sprague-Dawley rats (250-280 g) were obtained from the Laboratory Animal
Center of Sun Yat-sen University, and all procedures were approved by the Committee
for the Use of Live Animals in Teaching and Research at Sun Yat-sen University. All
rats had free access to standard rat chow and tap water. Rats were fed with standard
rat diet routinely, but were deprived of food for 12 h before the first operation.
All rats in the present study received root avulsion surgery. Spinal root avulsion
surgery followed the procedures described in our previous publications [[4],[17],[18]]. Briefly, the rats were anesthetized with intramuscular injections of ketamine
(80 mg/kg) and xylazine (8 mg/kg), and all nerve roots, including C5, C6, C7, C8 and
T1 of the right brachial plexus, were separated under an Olympus surgical microscope.
Extra-vertebral avulsion of the ventral and dorsal roots was carried out on C5, C6,
C7, C8, and T1 by pulling the nerve root out with microhemostatic forceps. The avulsed
ventral and dorsal roots together with the dorsal root ganglia were cut away from
the distal ends of the spinal nerves and examined under the microscope to confirm
the success of the surgery. All surgical instruments were appropriate for the size
of each animal. The skin was then sutured, and long-acting penicillin (3,000,000 units,
sc) was given after the surgery. The rats were allowed to recover until awake and
returned to their cages.
EGb761 treatment
Rats received daily intraperitoneal (i.p.) injections of vehicle (saline) or EGb761
(50 mg/kg body weight). EGb761 treatment was started immediately after the root avulsion
surgery. EGb761 was provided by Schwabe Pharmaceuticals (Karlsruhe, Germany). The
extract is well-characterized[[19]] and is being used in ongoing clinical trials[[20]]. EGb761 was dissolved in physiological saline and the pH adjusted to 7.4. The survival
time points were 5 days (5 d), 2 weeks (2 w), 4 weeks (4 w), 6 weeks (6 w) and 8 weeks
(8 w) post-injury. The effect of EGb761 on avulsion-induced motoneuron injury was
studied in the following experimental groups. Animals in Groups 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23 and 25 received EGb761 while those in Groups 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24 and 26 received normal saline solution (i.p.), serving
as controls. Groups 1-8 were used for determination of NO levels in the serum and
injured spinal cord at the 5 d, 2 w, 4 w, and 6 w time points. Groups 9-16 were used
for determination of cNOS and iNOS levels in injured spinal cord at the 5 d, 2 w,
4 w, and 6 w time points. Groups 17-26 were used for determination of the number of
nNOS-positive and survival motoneurons in injured C7 ventral horn at the 5 d, 2 w,
4 w, 6 w and 8 w time points. Each treatment group included six to eight rats.
Determination of NO level
24 h after the last EGb761 or saline administration, the rats were anesthetized with
a lethal dose of 10% Chloral Hydrate and 1 ml of blood was taken from the caudal vein,
after which they were sacrificed by cervical dislocation. Using dorsal laminectomy,
the spinal segments from C5 to T1 were identified and removed. The level of NO in
spinal cord and serum was determined using a NO kit (Nanjing Jiancheng Institute of
Biology and Engineering, Nanjing, China). Briefly, the method involved measuring the
levels of NO metabolites (nitrite and nitrate), which are more stable than NO. We
thus estimated the level of NO in the sample by determining total nitrate and nitrite
concentration. The rationale for this method is based on the fact that nitrate reductase
catalyzes the enzymatic conversion of nitrate to nitrite and determines total nitric
oxide concentration. This step was followed by the colorimetric measurement of nitrite
as an azo dye product of the Griess reaction. A two-step diazotization reaction occurs
during the Griess reaction, wherein acidified nitrite produces a nitrosating agent
that reacts with sulphanilic acid to produce the diazoniumion. This product is then
coupled with N-(1-naphthyl) ethylenediamine to form the chromophoric azo-derivative,
which has a peak absorbance of 550 nm. The NO level in spinal cord was expressed as
μmol/g of spinal cord protein. The NO level in serum was expressed as μmol/L of serum[[21]].
Determination of constitutive NOS (cNOS)
Rats were killed by cervical dislocation 24 h after the last EGb761 or saline administration.
Using dorsal laminectomy, the spinal segments from C5 to T1 were identified and removed[[15],[22],[23]]. Inducible NOS (iNOS) activity and total NOS activity in spinal cord were measured
with a NOS kit (Nanjing Jiancheng Institute of Biology and Engineering, Nanjing, China),
which assessed activity by measuring the conversion of L-[14C]-arginine to L-[14C]-citrulline [[24]]. The total NOS activity was determined by incubating samples (50 μL) for 15 min
at 37°C in a reaction mixture containing buffer solution and 20 μM nicotinamide adenine
dinucleotide phosphate(β-NADPH), 1 mM CaCl2, 50 μM tetrahydrobiopterin (BH4) and 1 μCi/ml L-[14C]-arginine. Inducible NOS (iNOS) activity was measured by omitting calcium and adding
1 mM EDTA to the reaction mixture (50 μL) for 60 min at 37°C. The reaction was stopped
by the addition of 1 ml of ice-chilled buffer containing 30 mM HEPES and 3 mM EDTA
(pH 5.5), after which the reaction mix was applied to Dowex AG50W-X8 columns to remove
L-[14C]-arginine. Columns were eluted two times with 0.5 ml of distilled water and L-[14C]-citrulline was quantified using a liquid scintillation spectrophotometer. cNOS
activity was computed by subtracting iNOS activity from total NOS activity. One unit
(U) of total NOS activity was defined as picomoles of L-[14C]-citrulline produced per minute per microgram protein/milliliter. The activity of
cNOS in spinal cord was expressed as U/mg of spinal cord protein.
NADPH-d histochemistry plus neutral red
At the end of each survival time (5 d, 2 w, 4 w, 6 w, 8 w), rats were anesthetized
with a lethal dose of 10% Chloral Hydrate and perfused transcardially with saline,
followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4). After perfusion, the vertebral
column was dissected, and the spinal cord was removed. The C7 spinal segment was defined
as the region between the uppermost root and lowermost root of the C7 nerve of the
contralateral spinal cord. The C7 segment of the spinal cord of each animal was removed,
fixed by immersion in fresh fixative overnight and cryoprotected in 30% (v/v) phosphate-buffered
sucrose overnight. Frozen transverse sections (40 μm) were cut and collected in 0.01
M PB. Every third section from each animal was used for NADPH-d histochemistry plus
neutral red counterstaining. We have previously shown that NADPH-d staining recognizes
NOS-containing neurons under normal conditions, and that NADPH-d labels the same population
of lesioned motoneurons as both NOS-ICC and NOS in situ hybridization[[4],[23]]. Neuronal NOS-containing neurons were stained with NADPH-diaphorase (NADPH-d) following
our previous studies [[15],[23]]. Briefly, sections were incubated at 37°C for 1 h in 10 ml of 0.1 M Tris-HCL(PH
8.0) containing 0.2% Triton X100, 10 mg NADPH (Sigma), 2.5 mg nitro-blue tetrazolium
(NBT) at 37°C for 1 h and then washed with 0.1 M PB three times. The stained sections
were mounted onto slides and counterstained with 1% neutral red (Sigma). These sections
were used to count the numbers of nNOS-positive and surviving motoneurons.
Counting of motoneurons
Approximately thirty cross sections (of 40 μm thickness a piece) of the C7 spinal
segment could be obtained from each animal, and every third section was used for NADPH-d
histochemistry plus neutral red counterstaining. In total, ten light microscopic images
of the C7 ventral horn of these sections in each animal were captured (20 × and 40
× lens) with a Lucida camera attached to a Leica DFC350FX/DMIRB microscope. Data quantification
and analysis were performed by two independent persons, both of whom were blinded
to the treatment groups and the previous studies[[4],[15],[23]]. In NADPH-d plus neutral red-stained sections, a motoneuron with a visible nucleus
in the neutral red stain was counted as a surviving cell. The number of surviving
motoneurons was quantified on both the intact side and the lesioned side of the C7
section. The number of surviving motoneurons on the contralateral intact side was
set as 100%. The surviving motoneruons on the lesioned side, including both the NADPH-d
positive and the NADPH-d-negative but neutral red-stained motoneurons, were then counted.
The number of surviving motoneurons ipsilaterally was expressed as a percentage of
the number of surviving motoneurons contralaterally in the same C7 section[[22],[25]]. The number of ipsilateral nNOS-positive motoneurons, represented by only the NADPH-d
reactive motoneurons, was expressed as a percentage of the number of surviving motoneurons
on the contralateral side of the same C7 section. The number of nNOS-positive or surviving
motoneurons of each animal was expressed as the mean of the nNOS-positive or surviving
motoneurons in the 10 serial C7 sections[[15],[23],[25]].
Statistical analysis
The statistical calculations and data handling were performed using SPSS version 16.0.
All variables were expressed as medians, mean ± standard deviation (X ± SE) with the
range. A one-way ANOVA was applied to detect differences among groups followed by
Tukey-Kramer multiple comparison tests. Differences were considered significant at
p values < 0.05.
Results
Effect of EGb761 on NO levels in the serum and injured spinal cord
Following spinal root avulsion, the levels of NO in the serum and spinal cord increased,
reaching a maximum at 2 w and then gradually descending until 6 w. The neuroprotective
effect of EGb761 against avulsion injury was closely related to a reduction in nitric
oxide production in the serum and injured spinal cord. In serum, EGb761 reduced nitric
oxide levels at 2 w and 6 w but not at 4 w ([Fig. 1A]) compared to saline controls, while NO levels in injured C6-T1 spinal segments of
treated animals were reduced at every time point ([Fig. 1B]).
Figure 1 EGb761 protects motoneurons against avulsion-induced injury in rats. EGb761 protects motoneurons against avulsion-induced injury in rats. The neuroprotective
effect of EGb761 against avulsion injury is associated with reductions in nitric oxide
production and NOS expressions in the injured spinal cord. Compared to saline controls,
EGb761 reduced nitric oxide levels in the serum at 2 w and 6 w, but not at 5 d and
4 w (Fig. A) and reduced nitric oxide levels in injured C6-T1 spinal segments at the
2 w, 4 w and 6 w time points (Fig. B). EGb761 down-regulated cNOS levels (Fig. C),
iNOS (Fig. D), nNOS levels in injured C6-T1 spinal segments (Fig. E,F), nNOS expressions
in ipsilateral ventral horn motoneurons, and the death of injured motoneurons in ipsilateral
ventral horn (Fig. H,J,L,N,P) induced by C5-T1 root avulsion injury. *P < 0.01 compared
with the ’avulsion+saline (Av)’ group at the same time point in graphic presentations
of Fig. G,I,K,M,O. Cross-sections of rat C7 segments from rats that underwent root
avulsion and were injected with saline (Fig G,I,K,M,O) or EGb761 (Fig. H,J,L,N,P).
Panels (G) and (H) were from rats surviving for 5 d after root avulsion. Panels (I)
and (J) were from rats surviving for 2 w after root avulsion. Panels (K) and (L) were
from rats surviving for 4 w after root avulsion. Panels (M) and (N) were from rats
surviving for 6 w after root avulsion. Panels (O) and (P) were from rats surviving
for 8 w after root avulsion. NADPH-d plus neutral red stain ×10 in Fig G-P.
Treatment with EGb761 regulated cNOS and iNOS activity in injured C6-T1 spinal segments
Root avulsion also resulted in a change in cNOS and iNOS activity in injured C6-T1
spinal segments. The activity of cNOS gradually increased after spinal root avulsion,
reaching a peak at 2 w, and then descended gradually until 6 w. Meanwhile, the activity
of iNOS increased gradually from the 2 w to 6 w. The activity of cNOS and iNOS were
down-regulated in animals that had been administered EGb761. Quantitative analysis
showed there were significant differences between the EGb761 treated group and the
saline control group in the activity of cNOS in injured C6-T1 spinal segments at 2
w and 4 w but not 6 w ([Fig. 1C]), while iNOS activity in injured C6-T1 spinal segments showed significant differences
at each time point ([Fig. 1D]).
Expression of nNOS in ipsilateral ventral horn motoneurons after EGb761 treatment
There is no expression of nNOS in the undamaged ventral horn motoneurons of the spinal
cord, but nNOS can be induced in motoneurons on the lesioned side following root avulsion.
The number of nNOS positive motoneurons in the ipsilateral ventral horn increased
rapidly to a peak at 2 w, and then decreased gradually at 4 w and 6 w, confirming
our previous studies[[4]]. Following spinal root avulsion, nNOS labeling was widely distributed in almost
every injured motoneuron and was evident in the somatic cytoplasm and dendrites ([Fig. 1I, K, M]), In EGb761 treated rats, expression of nNOS was significantly down-regulated. Fewer
nNOS positive neurons, exhibiting weak staining in the soma, were observed on the
lesioned side ([Fig. 1J, L, N].) compared to the saline-treated controls. Quantitative analysis of NADPH-d-stained
slides showed that the differences between the EGb761 and saline-treated control group
were significant (P < 0.001) at every time point ([Fig. 1E]). Morphologically, many surviving motoneurons were NADPH-d-positive by histochemistry
in the saline control group after injury. However, very few NADPH-d-positive motoneurons
were observed at the same time point post-injury in the EGb761-treated group, and
most of the remaining motoneurons were NADPH-d-negative.
Survival of injured motoneurons after EGb761 treatment
The loss of motoneurons in the C7 spinal segments following avulsion was apparent
at the end of 2 w, and was accompanied by the rapid appearance of nNOS positive motoneurons.
The loss of motoneurons sharply increased at the 4 w and 6 w time points([Fig 1K-N]), and the ipsilateral ventral horn showed signs of atrophy ([Fig 1O]), confirming our previous conclusions[[4]]. Quantitative analysis showed that the number of surviving motoneurons in the EGb761
treated group was higher than in the saline-treated group ([Fig. 1F]), and statistical analysis showed that the differences between the EGb761 and saline
treated groups were significant at 2 w, 4 w and 6 w (P < 0.05). Morphologically, many
of the remaining motoneurons were NADPH-d-positive in the saline control group by
6 w post-injury, while fewer NADPH-d-positive motoneurons were found at this time
point in the EGb761 treated group ([Fig. 1M-N]).
Discussion
The present study demonstrated that avulsion-induced motoneuron death was related
to changes in nitric oxide production and NOS activity in injured spinal segments.
Furthermore, we found that EGb761 prevented death of motoneurons by suppressing both
iNOS and nNOS activity, thus reducing NO production in the injured spinal cord.
Extracts of Ginkgo biloba, such as EGb761, are commonly used to increase blood circulation
and to protect the lipid portion of cellular membranes against damage induced by free
radicals [[26]]. EGb761 has also been shown to enhance cognition by increasing synaptic plasticity
in the hippocampus [[27]]. Additionally, EGb761 has been proven to have cardiovascular protective effects
in myocardial ischemia-reperfusion injury mediated through targeting NOS and NO production
in the injured central nervous system[[28]]. In accordance with our present data, pretreatment with EGb761 has been found to
attenuate up-regulation of cNOS and iNOS in the brain and have neuroprotective effects
in hyperthermic brain injury[[29]]. A previous report has also shown that inhibition of the up-regulation of NO produced
by iNOS reduced apoptosis in traumatic SCI models[[30]]. In the present study, our findings suggest that EGb761 has a neuroprotective effect
on avulsion-induced motoneuron injury by significantly attenuating avulsion-induced
up-regulation of cNOS and iNOS activities in the spinal cord and nNOS expressions
in injured motoneurons. Thus, it is possible that down-regulation of iNOS activity
by EGb761 treatment might be an effective therapy for root avulsion injury.
Previous studies have shown that all three of the NOS isoforms, nNOS, iNOS, and eNOS,
are up-regulated in the injured spinal cord[[31]]. Our present data confirmed that cNOS activity in injured spinal segments was markedly
increased, regardless of whether there was treatment with EGb761. The cNOS consists
of both nNOS and eNOS. In the present study we did not specifically quantify eNOS
activity in injured spinal segments; however, the endothelial cells of blood vessels
in the ipsilateral ventral horns were intensely stained in the NADPH-d reaction, indicating
an up-regulation of eNOS in the injured spinal cord. A consensus has been reached
that eNOS acts as a neuroprotective agent during the central nervous system injury[[32]], as high expression of eNOS in the endothelial cells of blood vessels may increase
blood flow and therefore aid in the survival of injured neurons[[33],[34]]. However, there are still debates about the role of nNOS in CNS injury. Many previous
studies have considered NO produced by the nNOS to be neuroprotective[[8]], while other studies have found NO produced by nNOS to be neurotoxic [[35]]. Kwak et al., found that nNOS was actually protective against cell death at early
stages of the injury and was constantly expressed in neurons, yet aberrant neuronal
expression of nNOS could result in the loss of its neuroprotective role [[36]]. We agree with the opinion that factors such as the concentration range of NO,
the redox state of the molecule, the cell type source, and the environment in which
the NO is produced by nNOS appear to determine the role of nNOS in the CNS[[37]]. Normally there is no nNOS labeling by immunohistochemistry in spinal ventral horn
motoneurons[[38]], but labeling is apparent in neurons located in the dorsal horn and around the
central canal [[4],[25]]. Our present study further confirmed that avulsion induced an increase in nNOS
activity in ipsilateral ventral horn motoneurons. It is possible that the increase
in nNOS activity in the first 2 weeks might play a neuroprotective role in avulsion
injury, a notion based on a number of observations. First, motoneuron loss was mild
within the first 2 weeks following avulsion with or without EGb761 treatments. Second,
our previous study showed that down-regulation of nNOS protein in injured ventral
horn motoneurons augmented the subsequent motoneuron loss[[15],[22]]. Finally, the present data further demonstrated a remarkable loss in motoneurons
beginning around 4 weeks after avulsion, a decline which was correlated not only with
increased iNOS activity but also with decreased cNOS activity in the spinal cord.
In summary, the present study showed that avulsion-induced motoneuron death was correlated
with increased iNOS activity and changes in cNOS activity in the injured spinal cord,
as well as nNOS expression in injured motoneurons. EGb761 treatment diminished avulsion-induced
motoneuron death by attenuating the avulsion-induced NO production and cNOS, iNOS
activities in the injured spinal cord and nNOS expression in injured motoneurons.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
The authors of this paper indicated in the title made substantial contributions to
the following tasks of research: initial conception and design (XC, LFL, JZ, LLW,
FLL, SL, LHZ); administrative, technical, or material support (XC, LFL, JZ, LLW, FLL,
LHZ); acquisition of data (XC, LFL, JZ, LLW, FLL, SL, LHZ); laboratory analysis and
interpretation of data (XC, LFL, JZ, LLW, FLL, SL, LHZ); drafting of the manuscript
(XC, JZ, LHZ); critical revision of the manuscript for important intellectual content
(XC, JZ, LLW, SL, LHZ). All authors read and approved the final manuscript. The views
expressed herein are those of the authors and not necessarily their institutions or
sources of support.
Cite this article as: Cheng et al., EGb761 protects motoneurons against avulsion-induced oxidative stress in rats Journal of Brachial Plexus and Peripheral Nerve Injury 2010, 5:12
