Effects of Benfotiamine
and Methylcobalamin on Paclitaxel induced Peripheral
neuropathy
......................................................................................................................................................................
Kawa F. Dizaye
Chro Y. Qadir
Hawler Medical University, Iraq
Correspondence:
Dr. Kawa Dizaye. Professor of Pharmacology,
Head of Dept of Pharmacology.
Hawler Medical University, Iraq
Email: dr_kawadizaye@yahoo.com
ABSTRACT
Background: Reports indicate that
paclitaxel causes a dose-limiting distal
and symmetrical sensorimotor peripheral
neuropathy. This study was designed to evaluate
the protective effects of benfotiamine and
methylcobalamin on prevention of paclitaxel
induced peripheral neuropathy.
Methods: Twenty
four rats and twenty four mice were involved
in this study. Each animal group was allocated
to two main experimental groups [control
group (n=6) and paclitaxel model group (n=18)].
The paclitaxel model group in rats was subdivided
into 3 subgroups [paclitaxel group (6mg/kg
i.p.) for 4 weeks, paclitaxel + benfotiamine
(100mg/kg orally, daily for 8 weeks) and
paclitaxel + methylcobalamin (500µg/kg
i.p., twice weekly for 8 weeks)]. Whereas
the paclitaxel model group of mice was subdivided
into 3 sub groups [paclitaxel group (6mg/kg
i.p. for 4 weeks), paclitaxel + benfotiamine
(100mg/kg orally, daily for 6 weeks) and
paclitaxel + methylcobalamin (500µg/kg
orally, daily for 6 weeks)]. Electrophysiological
and histological investigations, as well
as a number of classical behavioural tests
of nociception were performed.
Results: Paclitaxel
administration produced significant increase
in latency, but decrease in amplitude and
conduction velocity in peripheral motor
nerves in rats. Degenerative changes of
sciatic nerve were observed in rats. The
paw withdrawal latency for heat hyperalgesia
and the tail withdrawal latency for cold
(allodynia and hyperalgesia) in mice were
significantly reduced. Benfotiamine administration
significantly ameliorated all electrophysiological
changes induced by paclitaxel in peripheral
motor nerves. Moreover benfotiamine decreased
histological changes in rat's sciatic nerve.
In mice benfotiamine administration significantly
ameliorated the reduced withdrawal latencies
for cold and hot.
Methylcobalamin administration together
with paclitaxel attenuates the reduction
in conduction velocity in rats but had no
effect on the reduced amplitude. Methylcobalamin
reduced degenerative changes in Schwann
cells but had no effect on reduced myelin
thickness. While in mice daily methylcobalamin
administration significantly reduced the
decreased withdrawal latencies for cold
and hot.
Conclusion: Benfotiamine
100mg/kg was very efficient in prevention
of sensorimotor neuropathy induced by paclitaxel,
whereas the suggested methylcobalamin (500µg/kg)
twice weekly did not sufficiently prevent
peripheral motor nerve destruction induced
by paclitaxel, while the administration
of high dose methylcobalamin every day is
efficient in removal of thermal nociception
induced during paclitaxel treatment.
Key words: Benfotiamine; Methylcobalamin;
Paclitaxel; peripheral neuropathy
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Approximately 1.5 million new
diagnoses of cancer were anticipated in 2009 in
the United States (1). Improved medical treatments
and advances in technology have allowed many people
with cancer to increase their lifespan; however,
these life-saving interventions come with many
potential risks. Chemotherapy induced peripheral
neuropathy (CIPN) is a debilitating and disabling
condition that affects approximately 3% to 7%
of patients who are treated with a single agent,
and more than 38% of patients being treated with
a combination of drugs(2).
Peripheral neuropathy is a common and potential
dose-limiting complication of cancer chemotherapy.
Involvement of the peripheral nervous system may
be in the form of purely sensory and painful neuropathy,
which occurs after therapy with cisplatin, oxaliplatin
and carboplatin, or mixed sensory-motor neuropathy
which may be accompanied by dysfunction of the
autonomic nervous system, that results after therapy
with vincristine, taxanes, suramin and other drugs
(3, 4).
Paclitaxel is one of the most effective and commonly
used anti-neoplastic drugs originally derived
from the bark of the western yew tree, Taxus brevifolia,
with activity against several tumors including
ovarian cancer not responsive to primary treatment
methods, metastatic breast cancer, Kaposi's sarcoma,
bladder, testicular, lung, and head and neck cancers
(5,6).
Paclitaxel-induced sensory disturbance is characterized
by preferential impairment of myelinated fiber
function in cancer patients.
Studies have shown that paclitaxel administration
inhibits the usual regenerative response of axons
and Schwann cells to nerve crush injuries in rodent
models (7).
There have been several in vivo and in vitro experimental
studies of taxane neurotoxicity. Cultured sensory
neurons show proliferation and aggregation of
neurotubules; application of nerve growth factor
inhibits this effect (8).
Painful peripheral neuropathy occurs with other
agents in the taxane class, as well as with chemotherapeutics
in the vinca alkaloid and platinum-complex classes.
The cause of the neuropathy and of the pain syndrome
is unknown.
This study was designed to evaluate the neuroprotective
effects of benfotiamine and methylcobalamin in
paclitaxel induced peripheral neuropathy.
Animals
The experiments were performed on 24 male albino
rats and 24 male albino mice. The rats were used
for both nerve conduction studies and nerve biopsy,
whereas the mice were used to detect the effect
of each drug on heat nociception stimuli.
Before experiment, the animals were kept in the
animal house of the college of Medicine / Hawler
Medical University. They were housed in groups
of six per cage, on sawdust, maintained on a 12h-12h
light-dark cycle. They were given food rich in
nutrient and tap water. Room temperature was maintained
at 25 C°.
Anaesthesia
The rats were anaesthetized by a combination of
Ketamine and xylazine which were injected intra-peritoneally
at a dose of 35 mg/kg, and 5mg/kg body weight
respectively (9). After six minutes a state of
anesthesia was reached. They were placed on a
heated table to maintain their body temperature
at around 37 °C.
Induction of peripheral neuropathy
Peripheral neuropathy was induced by paclitaxel-induced
peripheral neuropathy model. In the paclitaxel-induced
peripheral neuropathy model, paclitaxel (6 mg/kg)
was injected intraperitoneally once a week for
4 weeks - Days 0, 7, 14, and 21- (10, 11).
All experiments were conducted according to the
guidelines of the Hawler Medical University Research
Ethics Committee for Research Ethics Committee
Approval.
Experimental design
The rats were divided into two groups. The first
group consisted of 6 rats and served as a control
group (injected with 0.5 ml sterile saline intraperitoneally).
The second group consisted of eighteen rats which
received paclitaxel (6 mg/kg) injection intraperitoneally
(i.p.) once a week for 4 weeks) and served as
a Paclitaxel model. The second group was subdivided
into three subgroups of six rats each (first subgroup
served as a positive control that received paclitaxel
(6 mg/kg) injection, second subgroup received
benfotiamine 100 mg /kg orally, daily for eight
weeks and the third subgroup received methylcobalamin
500 µg /kg, intraperitoneally twice weekly
for eight weeks).
The mice were divided into two groups. The first
group consisted of 6 mice and served as a control
group (injected with 0.5 ml sterile saline intraperitoneally).
The second group consisted of eighteen rats that
received paclitaxel (6 mg/kg) injection i.p. once
a week for 4 weeks) and served as a Paclitaxel
model. The second group was subdivided into three
subgroups of six mice each (first subgroup served
as a positive control received paclitaxel (6 mg/kg)
i.p. injection, second subgroup received benfotiamine
100 mg /kg orally, daily for six weeks and the
third subgroup received methylcobalamin 500 µg
/kg, orally, daily for six weeks).
Motor nerve conduction studies
Electrophysiological measurements were conducted
at Hawler Teaching Hospital/Neurophysiology Unit.
The data were analyzed using (Nicolet, Madison,
WI, USA) software program.
Experimental animal nerve conduction studies were
done by using the invasive techniques, with needle
electrodes (12, 13).
Latency and Amplitude were measured. Motor Nerve
Conduction Velocity (MCV) was calculated by dividing
the distance between the stimulation point and
recording electrode by the motor latency.
Motor nerve conduction studies (MNCS) were determined
30 to 35days after last dose paclitaxel. Nerve
conduction studies were performed using standard
equipment (Nicolet, Madison, WI, USA) on anaesthetized
rats.
Tail-immersion test
Antinociception was evaluated by measuring response
latencies in cold water tail-immersion (tailflick)
assay (14, 15, and 16).
Response latencies were measured as the period
of time the animal took to respond to the thermal
stimuli. The temperature of cold water (4±1°C)
for cold hyperalgesia and (10±1°C)
for cold allodynia. Water was maintained at the
right temperature by the addition of ice cubes.
The duration of tail immersion was manually recorded
(1 sec. precision), with a cut-off time of 20
sec.
Tail-flick test was performed by gently holding
the mouse in a terry cloth towel and immersing
between 2 and 3 cm from the tip of the tail into
the water, and the response was defined as the
removal of the tail from the cold water (17).
The paw hot plate test for
hot hyperalgesia
This test consists of introducing a mouse into
an open-ended cylindrical space with a floor consisting
of a metallic plate that was heated by the electrical
current (18). The plate was heated to a constant
temperature (50±1°C) the response produced
was in the form of two behavioral components that
can be measured in terms of their reaction times,
namely paw licking and jumping. To determine latencies,
the time was recorded from start of introducing
a mouse to the occurrence of the first avoidance
response, with a cut-off time of 20 seconds.
Statistical analysis
All data are expressed as means ± standard
error of means (M ± SEM) and Statistical
analysis was carried out using statistically available
software (SPSS Version 11.5). Data analysis was
made using one-way analysis of variables (ANOVA).
Comparisons between groups were done using Duncan
test and unpaired student t-test. P< 0.05 was
considered as statistically significant.
Effects of paclitaxel, methylcobalamin
and benfotiamine on the Motor Nerve Conduction
Studies (MNCS).
Effects on latency of sciatic nerve in rats.
The mean latency of sciatic nerve in the control
group was 0.92 ms ± SE 0.086 (Table1);
it was increased significantly in paclitaxel receiving
group rats to 1.74 ms ± SE 0.087 (Table
1). In Benfotiamine and paclitaxel receiving group
the mean latency was 0.94 ms ± SE 0.074
(Table 1), While in the methylcobalamin and paclitaxel
receiving group the mean latency was 1.2 ms ±
SE 0.094 (Table 1).
Table 1: Effect of paclitaxel, paclitaxel and
methylcobalamin and paclitaxel and benfotiamine
on motor nerve conduction study (n=24)
The same letters mean that there is no
significant difference
The different letters mean there is a significant
difference at P < 0.05
Effects on amplitude of sciatic nerve in rats
The mean amplitude of sciatic nerve in the control
group was 27.5 mv ± SE 2.1 (Table 1). It
was reduced significantly in the paclitaxel receiving
group rats to 12.02 mv ± SE 0.92 (Table
1). The mean amplitude in the benfotiamine and
paclitaxel receiving group was 26.66 mv ±
SE 1.22 (Table 1). While the mean amplitude in
methylcobalamin and paclitaxel receiving group
was reduced significantly to 18.4 mv ±
SE 2.5 (Table 1).
Effects on conduction velocity of sciatic nerve
in rats
The mean conduction velocity of sciatic nerve
in the control group was 56.24 m/s ± SE
5.1 (Table 1); it was reduced significantly in
the paclitaxel receiving group rats to 29.04m/s
± SE 1.46 (Table 1). In the benfotiamine
and paclitaxel receiving group the conduction
velocity was 54.46 m/s ± SE 3.95. There
wasn't a significant change (Table 1), whereas
the conduction velocity in methylcobalamin and
paclitaxel receiving group was also reduced to
42.7 m/s ± SE 3.26 (Table 1) but better
than in the paclitaxel group.
Effects of paclitaxel, methylcobalamin
and benfotiamine on Tail thermal threshold in
mice
Tail immersion test for cold allodynia (10±2°c)
in mice
The mean withdrawal latency of cold allodynia
for all groups of mice was measured weekly and
is shown in Figure 1.
On day 28 the withdrawal latency of the control
group was 17.8 sec. ± SE 1.7 and in the
paclitaxel group was reduced significantly to
10.3 seconds. ± SE 2.6 (P=0.042). While
compared to the control group the paclitaxel and
benfotiamine receiving group was 16.5 seconds
± SE 1.6 no significant change was observed.
The paclitaxel and methylcobalamin receiving group
was 17.7 seconds. ± SE 1.7 compared to
the control group and no significant differences
were seen.
Figure 1: Tail immersion tests for cold allodynia
(10±2°c), withdrawal latencies (sec.)
measured on weekly bases for all the groups each
group with six mice
Tail immersion test (cold
hyperalgesia 4±1°c) in mice.
There was a significant reduction in the mean
withdrawal latency for cold hyperalgesia in the
paclitaxel receiving group which was 3 seconds
± SE 1.09 (P = 0.0001) compared to the
control group. Whereas in the paclitaxel and benfotiamine
receiving group it was 16 ± SE 2.5 (P =
0.46) which is non significant compared to the
control group. The mean tail withdrawal latency
in the paclitaxel and methylcobalamin receiving
group was (13.5± SE 2.9, P = 0.19), compared
to the control group where no significant change
was seen (Figure 2).
Figure 2: Tail immersion tests for cold hyperalgesia
(4 ± 1°c), withdrawal latencies (sec.)
measured on weekly bases for all the groups each
group with six mice.
Effects of paclitaxel, methylcobalamin
and benfotiamine on paw thermal threshold in mice.
The mean paw withdrawal latency in the control
group was 3 sec± SE 0.44, (Figure 3). Compared
to the control group the mean paw withdrawal latency
in the paclitaxel group was reduced significantly
to 1.5 sec ± SE 0.22 (P= 0.017) whereas
compared to the control group no significant changes
were observed in mean paw withdrawal latency for
both paclitaxel and benfotiamine and paclitaxel
and methylcobalamin groups. The mean paw withdrawal
latencies were 2.6seconds ± SE 0.24 (P=
0.45) and 2.5 ± SE 0.22 (P= 0.34) respectively.
Figure 3: The mean paw withdrawal latencies
in four mice groups, each group (n=6) 7 days after
last dose of paclitaxel
(28th day).
In vitro study:
Histopathological section of rat's sciatic nerve
stained by E &H stain.
Sciatic nerve for control group:
The histopathological results of this study showed
normal architecture of rat's sciatic nerve fibers
in the control group in which most of the nerve
fibers were equal in size, diameter with regular
thickness of myelin, continuation of myelin cell
basement membrane and normal nucleus of myelin
cell (Figure 4).
Effect of paclitaxel on sciatic nerve
The result of this study showed that 6 mg/kg paclitaxel
once a week for four weeks resulted in marked
destruction of sciatic nerve fibers in rats, as
shown in Figure (5).
Figure 4: Cross section from the SN of a normal
control specimen showing normal appearance of
the individual nerve fibers (nucleus and myelin)
of Schwann cells (arrows) (N= nucleus ,
M= myelin, NF= nerve fiber, C=capillary) E &
H stain X 400.
Figure 5: Cross section of SN from the paclitaxel
receiving group specimen showing abnormal appearance
of the individual nerve fibers (nucleus degeneration
and disruption of myelin sheath) of Schwann cells
and multiple different size nerve fibers (arrows)
(N= nucleus , M= myelin , NF= nerve fiber,) E
& H stain X 400.
Effect of paclitaxel and benfotiamine
on sciatic nerve
In this study the results showed that with daily
administration of benfotiamine 100mg/kg together
with paclitaxel and continued for four weeks after
last dose of paclitaxel preserves most nerve fibers
from destruction (Figure 6)
Effect of paclitaxel and methylcobalamin on
sciatic nerve
The result of this study showed that administration
of methylcobalamin 500 µg /kg, intraperitoneally
twice weekly together with paclitaxel in rats
for eight weeks inhibits destruction of Schwann
cells but, resulted in significant reduction of
myelin thickness in most of the cells (Figure
7).
.
Figure 6: Cross and oblique sections of SN
from paclitaxel and benfotiamine receiving group
specimen showing most nerve fibers normal, with
normal nucleus, normal myelin thickness and maintenance
of basement membrane of Schwann cells (arrows)
(N= nucleus , M= myelin , NF= nerve fiber, ONF=
oblique view nerve fiber) E & H stain X 400.
Figure 7: Cross section of SN- B (lower) -
Cross and oblique section of SN, from paclitaxel
and methylcobalamin receiving group specimen showing
marked reduction in myelin thickness and multiple
different size nerve fibers but, with maintenance
of basement membrane of Schwann cells (arrows)
(N= nucleus , M= myelin , NF= nerve fiber, ONF=
oblique view nerve fiber, P = perineurium) E &
H stain X 400.
Neurotoxic effect of paclitaxel
on peripheral nerves in rats
Peripheral neuropathy that induced by chemotherapeutic
substances such as paclitaxel, thalidomide, and
cisplatin represents beside other adverse effects
of these drugs, a major clinical problem due to
the frequency of this toxic process and the lack
of therapeutic measures to treat the resultant
disability. Furthermore, this adverse effect often
represents the dose-limiting factor in therapeutic
oncologic regimen, where higher doses might be
otherwise desirable (3,19, 20).
In this study, a motor nerve conduction study
(MNCS) of rat's sciatic nerves in the paclitaxel
group showed marked prolongation in the mean latency,
moreover there was a reduction in conduction velocity
and amplitude compared to the control group. This
pattern might explain the possibilities of axonal
degeneration and demyelination with subsequent
neuropathy. Axonal neuropathy is identified by
nerve conduction study (NCS) as a low compound
muscle action potential; pure demyelinating neuropathy
is identified by NCS as slow conduction velocity
and prolonged latency (21).
This result was in agreement with Lipton et al,
(1989) and Sahenk et al, (1994) who have concluded
that in paclitaxel-induced neuropathy, both axonal
degeneration and demyelination patterns were possible
on NCS.(22,23)
Authier et al, (2000) reported that exposure to
paclitaxel at a single dose of 16 or 32 mg/kg
did not change NCV in vitro; they concluded that
this might be due to the low sensitivity of electrophysiological
methods in early detection of neuropathy. The
same researchers reported that NCV decreased following
paclitaxel treatment 6mg/kg once a week for 5
weeks (24).
Moreover the model of paclitaxel-induced neuropathy
in mature rats, with minimal effects on general
health, by using two intravenous injections 12
mg/kg, 3 days apart, showed reduction in amplitudes
of sensory compound nerve action potentials in
the tail. Motor amplitudes were not affected,
but both motor and sensory conduction velocities
decreased. These effects persisted for at least
4 months after treatment (25).
This study also showed shortening of the tail
withdrawal latencies for cold allodynia and hyperalgesia;
there was shortening of the paw withdrawal latency
in hot plate test. This is in agreement with several
studies performed on laboratory animals in which
Paclitaxel 6 mg/kg was given intraperitoneally
(i.p.) once a week for 4 weeks which significantly
shortened the paw withdrawal latency in acetone
test compared with vehicle for cold hyperalgesia
and decreased the travelled distance compared
with vehicle in the balance beam test (11).
Flatters and Bennett, (2004) concluded that four
(i.p.) injections on alternate days of 2 mg/kg
paclitaxel induced a pronounced cold allodynia
and hyperalgesia (26).
Polomano et al, (2001) in an experimental paclitaxel-induced
painful peripheral neuropathy concluded that paclitaxel
at low doses 0.5, 1 and 2 mg/kg caused heat-hyperalgesia
and cold-allodynia, but had no effect on motor
performance (27).
The mechanism of chemotherapy-induced neuropathy
is still uncertain. Direct toxic damage to axons
and Schwann cells and disturbed cytoplasmic flow
are considered to be the main pathogenic factors
(3).
Spontaneous improvement of nerve function over
time, as observed in some animal models, suggests
involvement of components of the nerve which have
regenerative capacity, unlike neurons themselves
(25). However, involvement of the vasa nervorum
is a more attractive hypothesis since the majority
of substances causing this type of neuropathy,
i.e., paclitaxel, thalidomide, and cisplatin exhibit
antiangiogenic properties in addition to their
direct effects on tumor cells (28, 29).
Dvorak et al, (1995) and Kirchmair et al, (2005)
found that the neuropathy caused by a chemotherapeutic
drug was due to destruction of the blood supply
of the nerve, i.e., the vasa nervorum.(30,20)
Kirchmair et al. (2005) showed that cisplatin-induced
neuropathy is associated with the induction of
endothelial cell apoptosis and destruction of
the vasa nervorum and is reversed or inhibited
by the angiogenic cytokine vascular endothelial
growth factor (VEGF) (20).
The mechanism of chemotherapy-induced neuropathy
also could be due to chemotherapeutic drugs that
cause high levels of oxidative stress and are
thought to rely, in part, on using this stress
mechanism to kill cancer cells, but Perumal and
Shanthi (2005) concluded that oxidative stress
might actually reduce the overall effectiveness
of chemotherapy because oxidative stress slows
the process of cell replicationbut, during cell
replication, chemotherapy actually kills cancer
cells, therefore slower cell replication can mean
lower effectiveness of chemotherapy. One approach
to addressing this problem is the addition of
certain antioxidants at specific dosages to lessen
oxidative stress, thus making the chemotherapy
treatment more effective (31).
Cameron and Cotter (1997) in an experimental study
have shown that reactive oxygen species (ROS)
also have effects on blood vessel function, which
compromise perfusion of several organs including
peripheral nerves. That was responsible for the
earliest defects in nerve function in experimental
models and will exacerbate nerve damage by causing
further ROS-dependent ischemia-reperfusion effects
(32).
Protective effect benfotiamine on paclitaxel
neurotoxicity
The result obtained in this study from daily administration
of benfotiamine 100 mg /kg orally for eight weeks
together with paclitaxel 6 mg/kg weekly for four
weeks, showed significant decrease in latency
of sciatic nerve and subsequently an increase
in nerve conduction velocities. An increase in
amplitude of compound motor action potential reached
that of the control group. The protective effect
of benfotiamine against paclitaxel induced neuropathy
could be explained by radical scavenging property
of benfotiamine, because benfotiamine exhibited
an antioxidant effect by reducing the oxidative
stress and genomic damage caused by mitogenic
model compounds; such effect was found to be related
to the direct antioxidant effect of benfotiamine
(33).
Cameron and Cotter, (1999) in an antioxidant study
observed that oxidative stress makes a marked
contribution to the etiology of nerve dysfunction
in experimental diabetes because reactive oxygen
species (ROS) cause vascular endothelium dysfunction
which reduces NO mediated vasodilatation and increases
local vasoconstrictor production and reactivity.
This reduces nerve perfusion, causing endoneurial
hypoxia which results in conduction deficits (33).
However nitric oxide (NO) is an important vascular
target for ROS. Superoxide neutralizes NO and
the peroxynitrite formed is a source of hydroxyl
radicals that can cause endothelial damage (34,
35).
Regarding peripheral nerves, Nagamatsu et al,
(1995) and Low et al, (1997) suggested that ROS
can directly damage neurons and Schwann cells
(36,37).
Recently, a new study showed that benfotiamine
reduces superoxide and hydroxyl radical levels
in the heart of diabetic mice by inducing the
activation of pentose phosphate pathway, which
regenerates the antioxidant NADPH (38).
Cascinu et al, (2002) suggested that increased
levels of the reduced form of glutathione may
be one of the possible mechanisms to prevent neurotoxicity
because of glutathione's possible mechanism in
reducing neurotoxicity of platinum-based drugs.
Reactive oxygen species generated by platinum
drugs result in neuronal cell death. GSH, as an
ROS scavenger, may prevent such damage (39).
This result is supported by an in vitro study
in which cisplatin induced apoptosis of mouse
neurons, was prevented by pre incubation with
N-acetylcysteine, a precursor to GSH (40).
The result of this study is in agreement with
the result of a study performed on experimental
animals where NCV was normalized by benfotiamine
after three months of administration (41, 42).
Moreover in this study, compared to the control
group no significant changes were observed in
withdrawal latency of tail immersion test for
cold (allodynia and hyperalgesia) and paw withdrawal
latency in hot plate test in mice treated with
oral benfotiamine 100 mg/kg, daily for six weeks
and paclitaxel 6 mg/kg, i.p. once a week for four
weeks, This result is in agreement with Winkler
et al, (1999) who concluded that benfotiamine
is effective in large doses and even in smaller
daily dosages in treatment of painful diabetic
neuropathy(43).
Protective effects of methylcobalamin on paclitaxel
neurotoxicity
In this study, the results obtained from administration
of methylcobalamin 500 µg /kg, intraperitoneally
twice weekly and paclitaxel 6 mg/kg once a week
for four weeks, showed decrease in latency of
sciatic nerve and subsequently increase in nerve
conduction velocity in comparison to the paclitaxel
receiving group but did not reach that of the
control group.
While the amplitude of compound motor action potential
(CMAPs) was very low in sciatic nerve compared
to the control group, this may be due to insufficient
dose of methylcobalamin in this study as Yamatsu
et al, (1976) observed that daily injection of
500 µg /kg of methylcobalamin produced a
significant increase in the weight of the soleus
muscle which recovered to the extent of being
the same weight of the contra lateral 4 weeks
after the nerve-crush. These results suggest that
methylcobalamin may have an inhibitory effect
on Wallerian degeneration and also a facilitatory
effect on the neural regeneration of the crushed
sciatic nerve of rats (44). Watanabe et al, (1994)
examined the effects of ultra-high dose of methylcobalamin
on the rate of nerve regeneration in rats with
acrylamide neuropathy, using the amplitudes of
compound muscle action potentials (CMAPs) after
tibial nerve stimulation as an index of the number
of regenerating motor fibers. Those treated with
ultra-high dose showed significantly faster CMAP
recovery than saline-treated control rats, whereas
the low-dose group showed no difference from the
control (45).
Furthermore the result of this study, did not
show significant change in withdrawal latency
of tail immersion test for cold allodynia/hyperalgesia
and paw withdrawal latency in hot plate test in
mice treated with oral methylcobalamin 500 µg
/kg, daily for six weeks and paclitaxel 6 mg/kg
once a week for four weeks compared to the control
(saline treated) group. This is in agreement with
Mizukami et al, (2011) who suggested that correction
of neural oxidative stress may be attributed to
the beneficial effects of methylcobalamin (10
mg /kg every other day, intramuscularly which
is a higher dose than the dose used in this study)
in normalization of nerve conduction velocity
of diabetic nerve (46).
Histopathology
In this study histological examination by light
microscope showed features of segmental demyelination,
such as a thinning and destruction of myelin sheaths,
nucleus degradation of Schwann cells and multiple
different size cells in the paclitaxel receiving
group compared to the control group. The result
of this study was in agreement with Hashimoto
et al. (2004) who concluded that the local paclitaxel
injection showed features of segmental demyelination,
such as a marked decrease in large-diameter myelinated
nerve fibers, thinning and destruction of myelin
sheaths, and atrophy of axons (47). Furthermore
histological changes in the paclitaxel group agreed
with Kawashiri, (2009) who concluded that Paclitaxel
(6 mg/kg, i.p.) induced the decrease in the density
of myelinated fibers and the degeneration of myelinated
fibres in rat sciatic nerve(11).
In this study, light microscope histopathology
examination of sciatic nerve in a group that received
benfotiamine 100 mg /kg daily and paclitaxel 6
mg/kg weekly for four weeks, showed nerve fiber
architecture near to that of the control group,
in which most of the cell had normal nucleus,
normal myelin thickness and maintenance of basement
membrane of Schwann cells. This result might be
explained by improvement of a nerve conduction
study in this group as Raso et al, (2005) and
Mazzer et al, (2008) concluded that maintenance
of the basement membrane of Schwann cells surrounding
the original nerve fibers were intact despite
the disrupted axon enabled Schwann cells to provide
pathways to guide the regenerating axons (48,49).
After contact with the periphery is established,
the regenerating nerve fibers enlarge and myelinate
(50).
Light microscope histopathologic examination of
sciatic nerve in rats which received methylcobalamin
500 µg /kg, (i.p.) two times weekly and
paclitaxel 6 mg/kg once a week for four weeks,
showed a marked reduction in myelin thickness
and multiple different size nerve fibers but,
with maintenance of basement membrane and nucleus
of Schwann cells. This result showed that methylcobalamin
500 µg /kg, two times weekly enhanced improvement
of nerve but did not reach that of the control
group, as Yagihashi et al, (1982) observed that
methylcobalamin at high daily dose of 500 µg
/kg for 16 weeks resulted in decreased demyelination
and protection of nerve fiber density and size
in streptozotocin-diabetic rats (51).
Benfotiamine 100mg/kg was very
efficient in prevention of sensorimotor neuropathy
induced by paclitaxel. Whereas the suggested methylcobalamin
(500µg/kg) twice weekly did not sufficiently
prevent peripheral motor nerve destruction induced
by paclitaxel, while the administration of high
dose methylcobalamin every day is efficient in
removal of thermal nociception induced during
paclitaxel treatment.
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