ORIGINAL REPORT
Effects of Lithium on Peripheral Neuropathy Induced by Vincristine in Rats
Houman Alimoradi1, Nasir Pourmohammadi2, Shahram Ejtemaei Mehr2, Gholamreza Hassanzadeh3,
Mohammad Reza Hadian4, Mohammad Sharifzadeh5, Azam Bakhtiarian2, and Ahmad Reza Dehpour2
1
2
Toxicology Research Center, Jundishapur University of Medical Sciences, Ahvaz, Iran
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3
4
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5
Department of Anatomy, Tehran University of Medical Sciences, Tehran, Iran
Brain and Spinal Injury Repair Research center, Tehran University of Medical Sciences, Tehran, Iran
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
Received: 21 Nov. 2011; Received in revised form: 12 Mar. 2012; Accepted: 15 Apr. 2012
Abstract- Vincristine (VCR) as a frequently used antimitotic agent which is commonly prescribed for wide
spectrum of neoplasm, causes mixed sensorimotor neuropathy. Several evidences show lithium could be a
neuroprotective agent, therefore to assess whether a pretreatment and at subtherapeutic dose it could prevent
the peripheral neuropathy produced by VCR, rats were treated with VCR 0.1mg/kg i.p. for 3 alternative doses
and / or lithium chloride (20mg/kg or 40 mg/kg i.p. daily from the first day to the day of sacrifice).
Erythrocyte lithium concentration (ELC) and plasma lithium concentration (PLC) were measured at the
seventh day of study and the day of scarification. After seventh day of lithium administration, PLC and ELC
reached to a steady state at subtheraputic dose and they did not significantly change at normal housing
situation. Hot plate, open field test and nerve conduction velocity were used to evaluate the sensory and
motor neuropathy. Only VCR treated rats showed behavioral, electrophysiological and histological evidences
of a mixed sensorimotor neuropathy by significant increase in hot plate latencies and a marked decrease in
total distance moved and conduction velocities in both sensory and motor nerves. Lithium at the dose of
20mg/kg and specially 40mg/kg robustly reduced the rate of mortality, general toxicity and was able to
ameliorate mixed sensorimotor neuropathy induced by VCR. These results suggest that lithium at dose of
20mg/kg and 40 mg/kg, potentially by its effects on cell survival pathways such as inhibition of glycogen
synthase kinase-3 (GSK3β), can prevent both motor and sensory components of VCR neuropathy.
© 2012 Tehran University of Medical Sciences. All rights reserved.
Acta Medica Iranica, 2012; 50(6): 373-379.
Keywords: Vincristine; Lithium chloride; Rat; Peripheral neuropathy
Introduction
VCR is a member of Vinca alkaloid family and is
indicated for the treatment of Hodgkin, non Hodgkin’s
lymphoma and particularly in pediatric cancers either by
itself or in combination with other antitumor agents
(1,2). The cytotoxicity induced by VCR is based on well
established pharmacologic properties that include,
binding to tubulins and disrupting microtubules
formation in mitotic spindles and thus preventing cell
division (3,4). Lithium which is commonly prescribed
for bipolar disorders, showed some neuroprotective
effects in central nervous system (CNS) and these
effects might be via interaction of NMDA (n-methyl daspartate) receptors, reduction in pro apoptotic proteins,
p53 and bax, increased Bcl2, cytoprotective proteins and
cell survival kinase activation (5,6). We have previously
shown that lithium is able to ameliorate sensory
neuropathy related to paclitaxel (7). Although Petrini et
al., has previously shown that lithium at therapeutic
dose is able to ameliorate VCR induced neuropathy
(8), their interventions were just two behavioral test
(hot plate and cold test) and also VCR dose was much
more than its LD50. Moreover they did not measure the
lithium level in serum or any pathologic changes in
peripheral nerves. Here we designed a comprehensive
study to show the potential neuroprotective effects
of lithium at subtherapeutic doses and as a pretreatment
on peripheral sensorymotor neuropathy induced by
VCR.
Corresponding Author. Ahmad Reza Dehpour
Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Tel.: +98 21 88973652, Fax: +98 21 66402569,E-mail: dehpour@yahoo.com
Effects of lithium on peripheral neuropathy in rats
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Materials and Methods
Animals
Experiments were performed on 250 ± 30g male
Sprague Dawley rats in pharmacology department of
Tehran University of medical sciences. Animals were
housed in a temperature and humidity controlled
environment with 12 hr light/dark cycle. Food consists
of normal rat chow and water ad libitum. This study was
performed according to the guidelines of the US national
institute of health (NIH publication no.85.23, revised
1985) guides for the care of lab animals. Rats were
randomly divided into six study groups: (1) 12 rats
received VCR 3mg/kg (VCR group); (2) 12 rats
received VCR 3mg/kg and lithium chloride
20mg/kg (VCR+ Li 20 group); (3) 12 rats received VCR
3mg/kg and lithium chloride 40mg/kg (VCR+ Li 40
group); (4) 14 rats received lithium chloride 20mg/kg
(Li 20 group); (5) 14 rats received lithium chloride
40mg/kg (Li 40 group) and finally (6) 8 rats received
saline in the same program as VCR treating animals
(saline group).
Drug administration
Vincristine sulfate (Sigma Chemical Co, St. Louis,
MO) dissolved in saline and was injected
intraperitoneally (i.p.) on three alternative days at the
dose of 0.1mg/kg (total cumulative dose =0.3mg/kg).
Lithium receiving groups took one intraperitoneal
injection of Lithium chloride (Merck, Darmstadt
dissolved in saline) in each day from the day of
beginning to the day of sacrifice at two doses of
20mg/kg or 40mg/kg.
Survival study and general toxicity
To assess the general condition of animals, they were
daily observed to compare signs of edema, cachexia,
alopecia and mortality in different groups.
Measurement of lithium in plasma and RBC
At the 7th day of study 6 rats from lithium
receiving groups were randomly chosen and
anesthetized by Pentobarbital sodium (65mg/kg) and
about 6cc blood were collected from left ventricle.
Blood samples were centrifuged at 1600g and then
PLC and ELC were determined by atomic absorption
spectrophotometer (GBC scientific pty ltd) as
previously reported (9). The same measurement was
done for all rats in lithium receiving groups before
sacrifice.
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Acta Medica Iranica, Vol. 50, No. 6 (2012)
Behavioral examinations
The effect of VCR on neuropathic pain and sensory
neuropathy was evaluated by hot plate test two times per
week from the first VCR injection to the last one.
Animals (8 in each group) were placed on a 52±0.2c
heated plate (Socrel hot-plate model DS37, Ugo Basile,
Italy) and time spent until the first episode of heat
sensitivity was including jumping, fore paw or hind paw
licking (10). Motor impairment was evaluated by open
field activity. Rats were placed into an area (diameter═
1.4 m) and locomotion within the area was tracked over
a 10 minuets using a high resolution monochrome
camera and stored and analyzed with ethovision
software (v.8) and total distance moved (cm) was
calculated (11,12).
Electrophysiological examination
After the last behavioral tests, to measure NCV
animals were anesthetized with pentobarbital sodium
(65mg/kg), body temperature was monitored and
maintained within normal limits and then motor nerve
conduction velocity (NCV) in the left sciatic was
recorded as previously described using power lab (MLT
1030/D, AD Instruments, Power Lab, Spain) and the
same stimulating and recording pin electrodes (AD
Instrument, pin) (13).
Histological and morphometric studies
After a deep general anesthesia by sodium
pentobarbital overdose (100 mg/kg i.p.), right sciatic
nerve was approached and fixed in 1.25% glutaraldehyde-1% paraformaldehyde in 0.2 M phosphate buffer
at pH 7.4. Laminectomy was then performed and the
lumbar (L4-L5) DRG (dorsal root ganglion) was
exposed and fixed in the same fixator. Semithin (0.5µm)
cross section of neurons were stained with hematoxylin
and eosin (H and E) and were used for morphometric
analysis (3 section for each animal were randomly
observed), the following measurements were obtained:
large cell (called A type) and small cell (called B type)
diameters from DRG and G-ratio (it is defined as the ratio
of the inner diameter to the outer diameter of a myelinated
axon in sciatic sections) from sciatic sections.
Statistical analysis
The results are reported as mean ± S.EM. The
statistical analyses were performed using one way
analysis of variance (ANOVA) by SPSS (v.18). Group
differences were calculated by post hoc analysis using
Tukey test. For all tests, differences with values of P <
0.05 were considered significant.
H. Alimoradi, et al.
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PLC and ELC were determined between the 7th day of
study and the day of sacrifice (P>0.05).
Figure 1. Hotplate response of rats treated with VCR with or
without Li at doses of 20/kg or 40mg/kg. Animals treated only
with VCR showed hyporalgesia after the last injection of VCR
because withdrawal latency was significantly increased
compare with saline (*P<0.001). A significant decrease in
latencies was observed between single VCR treated rats and
VCR+Li 20 group (+P<0.05) and also no significant
differences in latencies were detected between saline and
VCR+ Li 40 group (P>0.05).
Results
Survival study and general toxicity
The rate of mortality in VCR only treated rats was
around 41.6% and some marked signs of general toxicity
such as looseness and decreased in amount of stool,
alopecia, abnormal posture (consisting of foot and head
drop) and a significant decrease in body weight (P<0.05)
were observed in these animals compared with saline
group.
Lithium at the dose of 20 and 40 mg/kg improved
general condition of animals treated with VCR since
fewer symptoms that above mentioned were observed in
VCR+Li 20 group and VCR+ Li 40 group. In addition
no significant body weight changes were determined in
VCR+Li 20 group or VCR+Li 40 group versus saline
group (P>0.05).
Plasma and erythrocyte lithium concentrations
PLC and ELC at 7th day of study reached to a steady
state and these concentrations for lithium administration
at
the
dose
of
20mg/kg
(n=6)
were:
PLC=0.236±0.006mM and ELC=0.137±0.005mM and
for Li 40mg/kg (n=6): PLC=0.458±0.007mM and
ELC=0.304±0.009mM. No significant differences in
Behavioral studies
A day after the last injection of VCR a significant
thermal hypoalgesia , determined by increase in hotplate
latency, was shown in rats treated with only VCR
compared with saline group (P<0.001). Although
hotplate latencies in VCR+ Li 20 group significantly
decreased versus VCR group (P<0.05), thermal
hypoalgesia still was observed compared with saline
group (P<0.05). No significant differences in latencies
were detected VCR+Li 40 group versus saline (P>0.05;
Figure 1). Animals treated with VCR were infected with
motor impairment and gait disturbance and interestingly
they could not change their path during movement and
their spontaneous exploratory activities were reduced
(Figure 2.A). As an indicator of motor impairment, there
was a significant reduction in total distance moved by
VCR only treated animals compared with saline group
(P<0.001). Lithium repaired the gait disturbance
induced by VCR because total distance moved in
VCR+Li 20 was significantly enhanced compared with
single VCR treated group (P<0.01) and also there was
no significant difference between VCR+Li 40 and saline
group (P<0.05). No significant changes in total distance
moved were determined in lithium only treated animals
(P>0.05; Figure 2.B).
Nerve conduction velocity
There was a significant difference noticed between
sciatic nerve conduction velocity recorded from saline
and VCR group (P<0.001). Figure 3 shows that NCV
was significantly increased in VCR and lithium treated
animals (VCR+Li 20 group vs.VCR group: P<0.05;
VCR+Li 40 group vs. saline P>0.05).
Histopathologic findings
Histopathologic examinations of longitude sections
of sciatic nerve in VCR only treated rats showed a
tangible deformation, swelling and degeneration of axon
fibers (Figure 4), and also cross sections of respective
nerves showed some marked pathological changes such
as swelling in some nerve fibers, myelin sheet alteration,
and slight demyelination, axonal shrinkage, axonal
degeneration and total axon loss (Figure 5.A). Since
fewer nerve fibers showed deformation and
degeneration in VCR+Li treated groups, our study
demonstrates that lithium was robustly able to reduce
the pathological lesion related to VCR treatment (Figure
5.B and C).
Acta Medica Iranica, Vol. 50, No. 6 (2012) 375
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Effects of lithium on peripheral neuropathy in rats
Figure 2. A Spontaneous exploratory activity as measured in an open field arena is altered in rats that suffering from neuropathy. It
visually can be understood that VCR caused gait disturbance and lithium was able to ameliorate this disorder. The total distance
moved within the open field arena (diameter 1.4 meter) was assessed over 10 min and it was significantly altered in VCR group
compared with saline (*P<0.001). In VCR+ Li 20 treated rats total distance moved was significantly higher than VCR only treated
rats (+P<0.01) and there was no difference in total distance moved in VCR+ Li 40 versus saline (P>0.05).
significantly evident in single VCR treated animals by
increase in diameter of small cells and decrease in large
cells. Moreover mean diameter of large cells in VCR
group was significantly decreased versus saline group
(P<0.001) while in VCR+Li 20mg/kg it was higher than
only VCR treated group (P<0.01) and in VCR+Li 40
group it had no significant difference with saline
(P>0.05). Mean diameter of small cells in VCR group
was significantly increased versus saline (P<0.05) but
there were no significant differences in diameter of
small cells in VCR and lithium treated groups (P>0.05;
Table 2).
Figure 3. The sciatic nerve conduction velocity NCV value
recorded from VCR treated and or lithium (20mg/kg or 40
mg/kg) treated rats. *P<0.001 vs. saline, +P<0.05 vs. VCR
group
In addition as shown in table 1, there was a
significant difference in G-ratio between VCR group
and saline (P<0.001) while G-ratio was significantly
increased in VCR+Li 20 group versus VCR group
(P<0.05) and there was no significant diference between
VCR+Li 40 group and saline (P<0.05). Lastly as shown
in figure 6 prominent changes in DRG neurons were
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Table 1. Morphometeric data on sciatic nerves from study
groups, the results are reported as mean±S.EM. and n=15 for
each group (3 section from each animal). *P<0.001 vs. saline,
+P<0.05 vs. VCR group
Study groups
G ratio
Saline
VCR+ Li 20
VCR+ Li 40
VCR
Li 20
Li 40
0.42±0.02
0.34±0.01 +
0.38±0.02
0.29±0.02 *
0.40±0.01
0.41±0.01
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H. Alimoradi, et al.
Figure 4. Longitudinal section of the sciatic nerves from saline (A), VCR treated group (B) and VCR+Li 40 group (C). A notable
swelling and degeneration were observed in longitudinal section of the sciatic nerves in single VCR treated rats, but no marked
differences were shown between VCR+Li and saline.
Figure 5. Sciatic cross sections of respective groups (shown in Figure 4), axonal swelling in some nerves and remarkable
degeneration in other nerves and also demyelination were detected in VCR treated groups and lithium was able to repair these lesions
induced by VCR as fewer changes were seen in VCR+ Li 20 and VCR+ Li 40 groups (H&E × 400).
Figure 6. Dorsal root ganglion neurons of saline (A), VCR treated group (B) and VCR+Li 40 group (C), stained with H & E. In
DRG of VCR treated rats (B) there were a marked reduction in number and diameter of A type cells (large cells) and also an increase
in number and diameter of B type cells (small cells) while fewer changes were detected in VCR+Li 20 or 40. Original magnification
(a_d) × 200.
Table 2. Morphometeric data on DRG neurons from all study groups are summarized in this table, the results
are reported as mean ± S.E. and n=15 for each group (3 section from each animal). *P<0.01 vs. saline,
**P<0.001 vs. saline, +P<0.05 vs. VCR group.
Study groups
Saline
VCR+ Li 20
VCR+ Li 40
VCR
Li 20
Li 40
Large cell Diameter (µm)
43.2±1.71
36.4±1.03 +
40.1±1.82
28.0±1.06 **
39.1±1.20
40.3±1.63
Small cell Diameter (µm)
21.2±0.8
20.9±1.2
21.0±0.7
26.3±1.0 *
22.5±1.2
20.8±0.9
Acta Medica Iranica, Vol. 50, No. 6 (2012) 377
Effects of lithium on peripheral neuropathy in rats
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Discussion
Our finding shows that VCR administration at the dose
of 0.3 mg/kg induced a marked peripheral sensorimotor
neuropathy with behavioral, electrophysiological and
histological alterations. PLC and ELC were measured at
the 7th day of study (lithium pretreatment period) and at
the day of sacrifice and we found that after about 7
days, lithium in plasma and erythrocytes had a steady
state concentration and in normal housing situation they
did not significantly change (14) and these
concentrations were subtheraputic. Lithium at the dose
of 20 mg/kg and 40 mg/kg was able to ameliorate the
rate of mortality and general toxicity (8,14). Based on
the results of electrophysiological and behavioral
examinations we can safely say that lithium at doses of
20mg/kg
and
40mg/kg
significantly
reduced
sensorimotor neuropathy induced by VCR. There were
recognizable differences in DRG and sciatic neurons
between VCR and VCR + Li 20 mg/kg or 40mg/kg
groups and these findings show that lithium repaired
pathologic damages induced by VCR. Such as
axonopathy, decrease in axon’s diameter and
demyelination. As conduction velocity could be a good
indicator of axon’s function, the NCV finding and
histological analyses for study groups were parallel.
What has emerged from this study showed the same
results as Petrini et al. (9). Lithium has a wide variety of
mechanisms of action; therefore it is difficult to suggest
a possible mechanism for its neuroprotective property.
Lithium plays an important role in microtubule
dynamics and stability directly or indirectly via GSK3β
(15-18). Bhattacharyya and Wolff reported that lithium
in concentrations of 0.2–1.0 meq promotes tubulin
polymerization and also showed that lithium protects
microtubules against depolymerizing effects of
colchicine or vinblastine (15). It has been widely
accepted that lithium robustly protects some damaged
cells against excitotoxicity via factors involved in cell
survival pathway, including cAMP (cyclic adenosine
monophosphate) response element binding protein, brain
derived neurotrophic factor, and bcl2 (19, 20).
Considering what has already been expressed, the
possible mechanisms of interaction between lithium and
VCR neuropathy might be due to its effect on factors
involved in cell survival pathways as Balaei et al.,
reported the same results for the protective effect of
lithium on cardiotoxicity induced by doxorubicine (14)
or by its effect on microtubul stability.
In summary, the data presented here imply that
lithium at the dose of 20 mg/kg and 40 mg/kg
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Acta Medica Iranica, Vol. 50, No. 6 (2012)
significantly ameliorates the sensorimotor neuropathy
related to VCR. In order to employ this inexpensive,
relatively safe neuroprotective drug in chemotherapeutic
regimens that induce neuropathy, larger preclinical and
clinical studies need to be performed.
Acknowledgement
The collaboration of Dr. Khosro Abdi in PLC and ELC
detection, Ms. Maryam Rahimi Balaei and Dr. Mehrak
Javadi in expert technical assistance is gratefully
acknowledged by the authors. This study was supported
by a grant from Tehran University of Medical Sciences.
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