Intracerebroventricular administration of riluzole prevents morphine ...

Pharmacological Reports 

2010, 62, 664–673 

ISSN 1734-1140 

Copyright © 2010 

by Institute of Pharmacology 

Polish Academy of Sciences 

Intracerebroventricular administration of riluzole 

prevents morphine-induced apoptosis in the 

lumbar region of the rat spinal cord 

Kambiz Hassanzadeh 1,4 , Bohlool Habibi-asl 2,4 , Leila Roshangar 5 , 

Mahboob Nemati 2,6 , Masood Ansarin 4 , Safar Farajnia 3,4 

Department of Physiology and Pharmacology, Faculty of Medicine, Kurdistan University of Medical Sciences, 

Pasdaran Street, Sanandaj, Iran, Zip Code: 66177–13446 

Department of Pharmacology and Toxicology, ! Biotechnology Research Center, " Drug Applied Research Center, 

# $ Department of Anatomy and Histology, Faculty of Medicine, Department of Drug and Food Control, Faculty of 

Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran 

Correspondence: Safar Farajnia, e-mail: farajnias@tbzmed.ac.ir 

Abstract: 

Opiates are the most effective drugs for pain relief. However, the repeated use of opiates induces tolerance to their analgesic effects. 

It has been shown that this morphine-induced tolerance is associated with apoptosis in the central nervous system. The aim of this 

study is to evaluate the effects of intracerebroventricular (icv) administration of riluzole, an anti-glutamatergic drug, on morphineinduced 

apoptosis in the lumbar region of the rat spinal cord. Animals were given daily injections of morphine and vehicle, morphine 

and riluzole, or riluzole alone. Nociception was assessed using a hot plate apparatus, and apoptosis was assessed using the in situ terminal 

deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The levels of anti-apoptotic factors 

Bcl-2 and HSP 70 and the pro-apoptotic agent caspase-3 were evaluated using immunoblotting. The glutamate concentration in the 

lumbar spinal cord was measured with high performance liquid chromatography (HPLC). The results indicate that the icv administration 

of riluzole attenuated morphine tolerance and reduced the number of TUNEL positive cells. Immunoblotting revealed that 

the levels of the selected anti-apoptotic agents were greater in the treatment groups compared to the controls. Furthermore, the results 

demonstrated that the administration of riluzole can attenuate the morphine-induced elevation of glutamate in the lumbar spinal 

cord. In conclusion, icv administration of riluzole attenuated morphine-induced tolerance to analgesia and apoptosis in addition to 

preventing the morphine-induced increase of glutamate in the lumbar spinal cord of rats. 

Key words: 

apoptosis, lumbar spinal cord, morphine, riluzole, tolerance 

Introduction 

The neurobiological mechanisms underlying the development 

of opiate tolerance are multifaceted and 

only partially understood. Glutamate and N-methyl- 

D-aspartate receptors (NMDARs) play an important 

role in different forms of behavioral and neural plasticity, 

including tolerance, sensitization, and physical 

dependence, that arise from long-term treatment with 

different drugs of abuse [17, 22]. Numerous studies 

have demonstrated that the administration of a variety 

664 Pharmacological Reports, 2010, 62, 664–673

Riluzole prevents morphine-induced apoptosis in the rat spinal cord 

Kambiz Hassanzadeh et al. 

of NMDA receptor antagonists can inhibit the development 

of opiate tolerance and dependence [2, 12, 13, 

37–39]. It has also been shown that the activation of 

NMDARs can lead to neurotoxicity under many circumstances 

[30, 32]. For instance, peripheral nerve 

injury has been shown to activate spinal cord 

NMDARs, which results in intractable neuropathic 

pain and neuronal cell death via apoptosis [23, 41]. In 

addition, it has been shown in vivo that neuronal 

apoptosis occurs in the dorsal horn of the rat spinal 

cord after chronic morphine treatment [24]. Mao et al. 

demonstrated that prolonged morphine administration 

induced an upregulation of pro-apoptotic elements, 

such as Bax and caspase-3, and a downregulation of 

the anti-apoptotic oncoprotein Bcl-2 in the dorsal 

horn of the spinal cord. Importantly, the upregulation 

of caspase-3 and Bax was inhibited when morphine 

was co-administered with the noncompetitive NMDAR 

antagonist MK-801, thereby supporting a link between 

NMDAR activation and the intracellular changes 

of caspase-3 and Bax in response to a prolonged morphine 

administration [24]. Interestingly, we recently 

demonstrated that both systemic and intracerebroventricular 

(icv) administration of riluzole – 2-amino- 

6-(trifluoromethoxy)benzothiazole, can attenuate the 

morphine-induced tolerance to analgesic effects [14, 

15]. Riluzole is an anti-glutamatergic agent that indirectly 

interferes with glutamate responses without interacting 

with any known binding site on NMDA, kainate, 

or AMPA glutamate receptors [8]. It has been 

shown in both in vivo and in vitro models that riluzole 

confers neuroprotection during both spinal cord and 

cortical injury/ischemia [16, 21], a beneficial effect in 

various neurodegenerative diseases [1, 9]. In this study 

we investigate the effect of riluzole on morphineinduced 

apoptosis in the lumbar section of the rat spinal 

cord after morphine tolerance. 

Guide for the Care and Use of Laboratory Animals 

(National Institutes of Health Publication No. 85–23, 

revised 1985) and were approved by the research and 

ethics committee of the Tabriz University of Medical 

Sciences. 

Intracerebroventricular cannula implantation 

Rats were anesthetized with an intraperitoneal (ip) injection 

of sodium pentobarbital (50 mg/kg) (Merck, 

Germany) and stereotaxically implanted with a 23- 

gauge stainless steel guide cannula into the lateral 

cerebral ventricle (–0.8 mm posterior, –1.3 mm midline 

to lateral and 3.5 mm ventral with respect to the 

bregma) [28]. A 30-gauge stainless steel dummy cannula 

was placed into the guide cannula to maintain the 

patency. After surgery, the animals were given a 7-day 

recovery period. During recovery, animals were habituated 

to the testing protocol, including being transferred 

to the experimental environment, handled, 

weighed, and restrained on the test platform for 1 min 

with a gentle removal and replacement of the dummy 

cannula twice daily. The animals were also habituated 

to the hot plate apparatus. 

Verification of cannula placement 

At the end of all behavioral experiments, methylene 

blue solution (5 µl, icv) was injected into the animals, 

which were then euthanized with pentobarbital and 

decapitated. The brain of each animal was removed 

and sliced along the coronal plane to verify placement 

of the guide cannula by distribution of the methylene 

blue in the ventricles. Only data from those animals 

that displayed a uniform distribution of methylene 

blue in the ventricles were considered for statistical 

analysis. In total, six animals were discarded due to 

incorrect placement of the guide cannula. 

Materials and Methods 

Animals 

Male Wistar rats weighing 250–300 g were used. The 

animals were housed in a temperature-controlled environment 

(24 ± 0.5°C) and kept on a 12 h light/dark 

cycle (light on 08:00) with free access to food and water. 

All experiments were in accordance with the 

Drug treatment 

Morphine sulfate (Darupakhsh, Iran) (10 mg/kg, ip) 

was dissolved in sterile 0.9% normal saline and injected 

using 1-ml insulin syringes. Riluzole (Sigma- 

Aldrich, Inc.) (20, 40, and 80 µg/10 µl) was dissolved 

with 1% Tween 80 in sterile 0.9% normal saline and 

infused (icv) with a Hamilton syringe. In addition, 

two groups of animals were given either vehicle or riluzole 

(80 µg/10 µl) alone without morphine. The volume 

of infusion was 10 µl given at a rate of 10 µl/min. 

Pharmacological Reports, 2010, 62, 664–673 665

Induction of tolerance to the analgesic effect of 

morphine 

Nociception was assessed with a hot plate apparatus. 

Animals were placed on a hot plate (55 ± 0.5°C) [10] 

and the latency until the rat licked its hind paw was 

recorded. A cutoff time of 40 s was imposed to prevent 

tissue damage [29]. Hot plate response latencies 

(s) are expressed as a percentage of the maximal possible 

effect (%MPE) using the equation below: 

Post - drug latency – Baseline latency 

% MPE = 

× 100 

Cutoff value – Baseline latency 

The baseline latency (BL) was determined once per 

day for each rat prior to the daily injection of morphine 

(10 mg/kg). Morphine was then injected, and 

20 min later the animals were given either riluzole or 

vehicle. The post-drug latency (LT) was measured after 

10 min, a total of 30 min after the original morphine 

injection. The percent maximal possible effect 

(%MPE) was then calculated for that day. The experiments 

were repeated until there was no significant difference 

in %MPE between the vehicle- or drug-treated 

groups and the saline group. 

Tissue preparation 

For the in situ TUNEL assay, different groups of animals 

(n = 6) were given the same treatment regimen 

as the behavioral groups. On the ninth day (one day 

after tolerance was observed in the control group), the 

animals were euthanized with pentobarbital 2 h after 

the last dose of vehicle or riluzole, and their lumbar 

spinal cords were immediately enucleated and fixed 

in 10% (w/v) formaldehyde (Merck, Germany) with 

the pH adjusted to 7.0 with NaOH. The tissue was 

then embedded in paraffin. In order to evaluate the 

possible association between morphine tolerance and 

apoptosis, one group was given morphine and riluzole 

at a dose of 80 µg/10 µl, which was the most effective 

dose for preventing morphine tolerance in behavioral 

studies. On the 14 th day, one day after the onset of behavioral 

tolerance in this group, the lumbar spinal 

cords of the rats were removed 2 h after the last dose 

of vehicle or treatment and prepared as described 

above. 

Detection of apoptotic cells 

After the tissue was fixed and embedded into the paraffin, 

3 µm sections were cut with a microtome (Leitz, 

Germany) and the TUNEL assay was carried out using 

the in situ Cell Death Detection kit (Roche Applied 

Science Cat # 11 684 817 910) according to the 

manufacturer’s instructions. Briefly, the paraffin was 

removed and the sections were rehydrated and pretreated 

with proteinase K (Roche, Germany) for 

30 min at 37°C. The sections were then exposed to the 

TUNEL reaction mixture containing terminal deoxynucleotidyl 

transferase and nucleotides, including 

fluorescein isothiocyanate-labeled dUTP (37°C). After 

1 h, the preparations were incubated for 30 min at 

37°C with a peroxidase conjugated anti-fluorescein 

antibody. Finally, the reaction product was visualized 

by incubation for 15 min at room temperature with 

3,3-diaminobenzidine tetrahydrochloride (DAB) substrate, 

followed by counterstaining with methylene 

blue. Apoptotic cells throughout the tissue section 

were intensely stained (brown) by the TUNEL treatment 

and analyzed with a Zeiss Axiovert 100 light 

microscope (100× objective) in 30 fields. 

Western blot analysis of Bcl-2, HSP 70 and 

caspase-3 

For western blotting, rats (n = 6) were rapidly (< 1 min) 

sacrificed, and lumbar spinal cord segments were removed, 

cleaned, and frozen in liquid nitrogen. Subsequently, 

tissue samples were homogenized in lysis 

buffer (500 mM Tris-HCl, pH 7.4, 150 mM NaCl, 

EDTA 0.5 mM, n-octyl--D-glucopyranoside 1.5% w/v) 

containing a complete Protease Inhibitor Cocktail 

(Roche Cat # 04 693 132 001). The protein quantification 

for each loading lane was estimated by an absorbance 

protein assay (280 nm). Lysates (20 µg protein) 

were resolved with 12% SDS-PAGE for Bcl2 

and HSP 70 and 15% SDS-PAGE for caspase-3. Next, 

the lysates were transferred to a polyvinylidene 

difluoride (PVDF) membrane (Millipore, Bedford, 

MA), blocked with 5% milk, and incubated overnight 

at 4°C with the following primary antibodies: caspase-3 

(18–20 kDa), Abcam, Cat # ab2302; Bcl-2 (26–29 kDa), 

Abcam, Cat # ab16904; HSP 70 (70 kDa), Abcam, Cat 

# ab6535; and -actin (42 kDa), Abcam, Cat # 8226. 

Subsequently, the PVDF membranes were incubated 

for 1 h at room temperature with an HRP-conjugated 

secondary antibody. The blots were then visualized 




with the enhanced chemiluminiscence (ECL) detection 

kit/system for 1 min and exposed to Hyperfilm 

(Roche) for 30 s to 5 min. Finally, the developed films 

were scanned, and the density of the immunoreactive 

bands was measured using Image J software and normalized 

to the bands of the internal control (-actin 

was the loading control). Differences in the image 

density were compared by a one-way analysis of variance 

(ANOVA) (multiple groups) followed by Tukey’s 

test. 

High performance liquid chromatography 

(HPLC) analysis of neurotransmitter glutamate 

To quantify the amount of glutamate, HPLC was employed. 

Tissue samples from rats (n = 6) were homogenized 

and protein determination was performed. 

Subsequently, the homogenates were spun at 4°C for 10 

min at 10,000 × g. The chromatograph was a KNAUER 

(Berlin, Germany) HPLC instrument. The system included 

a quaternary pump, a RF-551 fluorescence detector 

(FLD), and an auto sampler (Spark, Triatlon), 

which was controlled by Chromgate software. The 

analytical column used was a reversed-phase Hypersil 

ODS column (250 × 4.0 mm, 5 µm particle size). For 

the chromatographic separation, the mobile phases 

consisted of 8% acetonitrile in 12.5 mM phosphate 

buffer at pH 7.2, with an o-phthalaldehyde (OPA) derivative 

of glutamate eluted at a gradient flow rate of 

1 ml/min for 8 min and 2 ml/min for 12 min. The detection 

was carried out at 330 nm and 460 nm as the 

excitation and emission wavelengths, respectively. 

Glutamic acid was used as the standard. Solutions of 

0.75, 1.5, 3, 6 and 12 µg/ml glutamic acid were injected 

into the HPLC instrument and the resulting 

calibration curve was plotted. A 2.5% solution of supernatant 

in distilled water was used for the derivatization 

and determination of glutamate. The level of 

glutamate was calculated by comparing peak areas 

with those of standards, and the values are expressed 

as µmol/100 mg protein [26]. 

Data analysis 

Data obtained from the hot plate tests are expressed as 

the mean %MPE ± SEM. The histological data from 

the lumbar spinal cord sections were averaged and are 

expressed as the mean ± SEM. Data from the western 

blots are expressed as % of the control, and data from 

the HPLC are expressed as the mean ± SEM. The Student’s 

t-test was used to compare the means of two 

groups and a one-way analysis of variance (ANOVA) 

followed by Tukey’s test was used to compare the 

means of multiple treatment groups; p-values of less 

than 0.05 were considered to be significant. 

Fig. 1. Effect of daily icv injection of riluzole (20, 40 and 80 µg/10 µl) on the tolerance to the analgesic effect of morphine. Each bar represents 

the mean %MPE ± SEM for 8 rats. The Student’s t-test was used to compare the statistical differences between each treatment group and the 

saline group. A one-way ANOVA followed by Tukey’s test was used to analyze the statistical significance between treatments and the control 

group.Ap-value of less than 0.05 was considered significant for all analyses. *p< 0.05; ** p < 0.01; *** p < 0.001 when compared to the saline 

group. #p < 0.001 when compared to the control. The arrow represents the onset of morphine tolerance. M = morphine, Rilu = riluzole 


Results 

Riluzole attenuated morphine-induced 

tolerance to the analgesic effect 

As shown in Figure 1, the daily administration of morphine 

(10 mg/kg, ip) for 8 days produced tolerance to 

the antinociceptive effects in the control group that received 

morphine (10 mg/kg, ip) and 1% Tween 80 in 

0.9% saline (10 µl/rat, icv). Daily administration of 

80 µg/10 µl riluzole significantly delayed this morphine 

tolerance and shifted the onset of tolerance from 

day 8 in the control group to day 13 (p < 0.001), whereas 

lower concentrations of riluzole (20 and 40 µg/10 µl) did 

not delay the morphine tolerance significantly. 

TUNEL staining 

The TUNEL method was used to identify apoptotic 

cells. In the control group (morphine, ip and 1% 

Tween 80 in 0.9% normal saline, icv), the number of 

TUNEL-positive cells in the lumbar spinal cord was 

significantly higher than vehicle-treated animals (Fig. 

2, p < 0.01), indicating an increased background level 

of apoptotic activity in the morphine-treated animals. 

The average number of TUNEL positive cells was 

significantly reduced in the lumbar spinal cord of the 

riluzole-treated groups compared to those of the control 

group. There was a significant difference in the 

number of TUNEL-positive cells between animals 

that received vehicle or riluzole without morphine 

versus those that received morphine in the control 

group (p < 0.05). Animals treated with the highest 

dose of riluzole (80 µg/10 µl) had fewer TUNELpositive 

cells than those that received lower doses. 

Changes in the Bcl-2, HSP 70 and caspase-3 

content of lumbar spinal cord 

Western blot analysis showed that after the onset of 

morphine tolerance, the anti-apoptotic factors, Bcl-2 

(p < 0.01) and HSP 70 (p < 0.001), decreased significantly, 

whereas the amount of pro-apoptotic protein, 

caspase-3, was increased (p < 0.05) (Figs. 3–5). There 

were no significant differences in the amount of Bcl-2 

and caspase-3 between control and after tolerance 

(AT) groups. Furthermore, the results indicate that the 

amount of HSP 70 in the AT group was greater than 

that of the controls. The administration of riluzole 

(80 µg/10 µl, icv) with morphine (ip) for 9 days induced 

a significant up-regulation of Bcl-2 (p < 0.05), 

whereas the 40 and 80 µg/10 µl doses of riluzole significantly 

increased the amount of HSP 70 (p < 0.001). 

However, these doses did not affect the level of 

caspase-3 in the lumbar spinal cord, as shown in the 

corresponding western blots of Figure 5. 

Effect of riluzole on glutamate concentration in 

the lumbar spinal cord of morphine treated rats 

As shown in Table 1, chronic administration of morphine 

(10 mg/kg, ip) for 9 days induced a significant 

increase in the glutamate concentration in the lumbar 

spinal cord of morphine tolerant rats (p < 0.001). These 

findings show that the administration of riluzole (40 

and 80 µg/10 µl) after morphine attenuated the increase 

of the glutamate concentration in the related 

group (p < 0.01). 

Discussion 

The cellular mechanisms underlying the development 

of morphine tolerance remain controversial. Previous 

studies have indicated that certain addictive drugs, 

Tab. 1. Concentration of glutamate in the lumbar spinal cord of rats 

Treatment 

Glutamate concentration 

[µmol/100 mg protein] 

S + 1% Tween 80 138.50 ± 6.34*** 

M + 1% Tween 80 216.71 ± 8.26 

M + Rilu (20 µg/10 µl/rat) 190.09 ± 8.71* 

M + Rilu (40 µg/10 µl/rat) 179.37 ± 5.11** 

M + Rilu (80 µg/10 µl/rat) 183.80 ± 5.27** 

Rilu (80 µg/10 µl/rat) 187.92 ± 5.45** 

Each datum represents the mean + SEM. One-way ANOVA followed 

by Tukey’s test was used to analyze the statistical significance. 

A p- value < 0.05 was considered significant; * p < 0.05, ** p < 0.01, 

*** p < 0.001 when compared to control (M + 1% Tween 80). S = saline, 

M = morphine, Rilu = riluzole 




Fig. 2. A: Tissue sections from rat lumbar 

spinal cord were prepared and assayed 

with the In Situ Cell Death Detection 

Kit, POD. Slides were counterstained 

with methylene blue. Apoptotic 

cells scattered throughout the tissue 

section were intensely stained 

(brown) by the TUNEL treatment. Slides 

were analyzed with a light microscope 

(100´ objective). (a) S + 1% Tween 80, 

(b) morphine + 1% Tween 80, (c) morphine 

+ riluzole (20 µg/10 µl), (d) morphine 

+ riluzole (40 µg/10 µl), (e) morphine 

+ riluzole (80 µg/10 µl), (f) riluole 

(80 µg/10 µl). B: Quantification of apoptotic 

cells. The data represent the mean 

± SEM number of apoptotic (TUNELpositive) 

cells in 30 fields, which were 

counted at a magnfiication of 100´ with 

a light microscope. A one-way ANOVA 

followed by Tukey’s test was used to 

analyze the statistical significances. 

A p-value of less than 0.05 was considered 

significant for all analyses. * p 

< 0.05; ** p < 0.01; *** p < 0.001 when 

compared to the control group (M + 1% 

Tween 80). S = saline, M = morphine, 

Rilu = riluzole, AT = after tolerance 

Average Number of 

Apoptotic Cells 

*** 

* * ** 

*** 

such as morphine, can induce apoptosis in cultured 

neuronal cell lines and human cells [33, 34]. It has 

also been demonstrated that in vivo neuronal apoptosis 

occurs in the rat spinal cord dorsal horn after 

chronic morphine treatment, which has been associated 

with the expression of activated caspase-3 and 

involves the activation of the mitogen-activated protein 

kinase (MAPK) pathway [24]. In this study we 

found that the chronic administration of morphine significantly 

increased the amount of apoptotic cells in 

the lumbar spinal cord. This finding is in accordance 

with previous reports, which indicated that prolonged 

exposure to morphine induces apoptotic cell death in 

the dorsal horn regions of the spinal cord. Since this 

region is critically involved in opiate-mediated analgesia, 

it is believed that the apoptosis in this area contributes, 

at least in part, to the behavioral manifestation 

of morphine tolerance [24]. We also demonstrated 

that the administration of riluzole (80 µg/10 µl) significantly 

decreased the development of this tolerance by 

shifting the onset of tolerance from the 8 th day in the 

control group to the 13 th day. These results are in line 

with our previous study, which indicated that there 

was a significant right-shift of the dose-response 

curve and the ED50 of morphine for animals that received 

morphine and riluzole (80 µg/10 µl) compared 

to those that received morphine and saline [14]. Furthermore, 

it has been demonstrated that both morphine 

tolerance and the associated neuronal apoptosis 

share a common cellular mechanism. The NMDAR is 

thought to be important for apoptosis and tolerance 

because MK-801 or memantine, the NMDAR antagonists, 

were able to block both [18, 24]. Activation of 

NMDARs, on the other hand, initiated intracellular 

pathways of apoptotic cell death. It has been suggested 

that multiple intracellular mechanisms may be 

involved in the NMDAR-mediated apoptotic changes. 

Our results show that riluzole, as an anti-glutamatergic 

agent, can prevent morphine-induced apoptosis and 

significantly attenuate the average number of TUNEL 

positive cells (p < 0.01). After the development of tolerance 

in the group that received morphine and 

80 µg/10 µl of riluzole an increase in the number of 

apoptotic cells was observed, similar to the one seen 


Fig. 3. Effect of icv riluzole (20, 40 and 80 µg/10 µl) 

on morphine-induced changes in intracellular 

Bcl-2. A: Western blots illustrate down-regulation 

of the Bcl-2 protein (26kDa) in rats receiving 

morphine (M + 1% Tween 80) daily for 9 days 

compared to the corresponding S + 1% Tween 

80 group. Riluzole (80 µg/10 µl) increased the 

amount of Bcl-2 protein in combination with 

morphine. B: The statistical analysis showed 

differences among the different groups in the 

gray density obtained for the western blot 

bands of Bcl-2 in the lumbar spinal cord. 

A one-way ANOVA followed by Tukey’s test was 

used to analyze the statistical significances. 

A p-value of less than 0.05 was considered significant 

for all analyses. * p < 0.05, ** p < 0.01 

compared to the corresponding control group. 

S = saline, M = morphine, Rilu = riluzole, AT = 

after tolerance 



HSP 70. A: Western blots illustrate the downregulation 

of the HSP 70 protein in rats receiving 

morphine (M + 1% Tween 80) daily for 9 

days compared to the corresponding S + 1% 

Tween 80 group. Riluzole (40, 80 µg/10 µl) increased 

the amount of HSP 70 protein. B: The 

statistical analysis showed differences among 

the different groups in the gray density obtained 

for the western blot bands of HSP 70 in 

the lumbar spinal cord. A one-way ANOVA followed 

by Tukey’s test was used to analyze the 

statistical significances. Ap-value of less than 

0.05 was considered as significant for all analyses. 

*** p < 0.001 compared to the corresponding 

control group. S = saline, M = morphine, 

Rilu = riluzole, AT = after tolerance 



caspase-3. A: Western blots illustrate up-regulation 

of the caspase-3 protein (19 kDa) in rats 

receiving morphine (M + 1% Tween 80) daily for 

9 days compared to the corresponding S + 1% 

Tween 80 group. Riluzole did not change the 

amount of caspase-3 protein. B: The statistical 

analysis showed differences among the different 

groups in the gray density obtained for the 

western blot bands of caspase-3 in the lumbar 

spinal cord. A one-way ANOVA followed by 

Tukey’s test was used to analyze the statistical 

significances. Ap-value of less than 0.05 was 

considered as significant for all analyses. * p < 0.05 

compared to the corresponding control group. 

S = saline, M = morphine, Rilu = riluzole, AT = 

after tolerance 




in control conditions. This means that by the time behavioral 

tolerance was observed in the control and 

treatment groups, the apoptotic process had already 

developed. Other studies have demonstrated that 

chronic treatment with morphine, both toleranceeducing 

and dependence-educing, is associated with 

a differential modulation in two key brain proteins involved 

in the regulation of the programmed cell 

death. Specifically, there is an up-regulation of the 

pro-apoptotic Fas receptor and the intracellular proapoptotic 

elements, such as Bax and caspase-3, and 

there is a moderate down-regulation of the antiapoptotic 

oncoprotein Bcl-2 [5, 24]. The results of 

this study are consistent with others that indicate 

a morphine-induced decrease in the content of Bcl-2. 

On the other hand, our results show that 80 µg/10 µl 

of riluzole increases the level of Bcl-2 in the lumbar 

spinal cord when administered with morphine. Previous 

studies have demonstrated that Hsp70 proteins 

participate in protein folding and transport, the refolding 

of denatured proteins, and protection from apoptosis 

[31]. Hsp70 rescues cells from the apoptotic/necrotic 

death that normally occurs after heat shock, exposure 

to tumor necrosis factor , oxidative stress, 

ceramide, anti-cancer drugs, radiation, or nitric oxide 

[4, 11, 25]. Hsp70 was reported to inhibit heat shockinduced 

apoptosis downstream of cytochrome C release 

but upstream of caspase-3 activation [20]. Our 

results, in agreement with those indicating that morphine 

significantly decreased Hsp70 levels in rat neurons 

[7], show that the Hsp 70 content of lumbar spinal 

cord decreased after chronic morphine administration 

in morphine-tolerant rats. Furthermore, our results 

demonstrate that the administration of riluzole 

(40 and 80 µg/10 µl) significantly increased the 

amount of Hsp 70 (p < 0.001). This parallel increase 

in two anti-apoptotic factors, Bcl-2 and Hsp 70, could 

represent a part of the neuroprotective mechanisms of 

this drug. Riluzole did not affect the morphineinduced 

increase in the spinal levels of caspase 3. 

Nevertheless, this does not rule out the possibility that 

riluzole can effect caspase 3 activity. 

Riluzole is known as a neuroprotective agent. In 

cerebrocortical nerve terminals, riluzole has been reported 

to inhibit glutamate release by reducing the 

Ca 2+ influx through P/Q type Ca 2+ channels, and involves 

in G protein signaling mechanism sensitive to 

pertussis toxin. Importantly, the excessive release and 

accumulation of glutamate is associated with an increased 

level of intracellular calcium and plays an important 

role in CNS injury and neurodegenerative diseases 

[40]. 

Our results demonstrate that morphine increases 

the level of glutamate in the lumbar spinal cord and 

that riluzole (40 and 80 µg/10 µl) significantly attenuates 

this morphine-induced increase (p < 0.01). Although 

the measurement of the glutamate content in 

tissue is not specific enough to indicate the glutamate 

induced excitotoxicity, these results are in accordance 

with others who reported that chronic morphine administration 

can increase glutamate release in the 

CNS [3, 17, 22, 37–40] and that high concentrations 

of riluzole inhibit glutamate release. It has also been 

shown that riluzole can attenuate excitatory amino 

acid receptor activation and decrease the excitability 

of the postsynaptic cell membrane [6]. In addition to 

being an anti-glutamatergic agent, riluzole also has 

antioxidant effects and can protect dopaminergic neurons 

against oxidative stress by reducing lipid peroxidation 

and ATP consumption [19, 27, 35, 36]. 

In conclusion, we found that riluzole can attenuate 

morphine-induced tolerance and apoptosis, as well as 

decrease the morphine-induced elevation in glutamate 

concentration in the lumbar spinal cord of rats. However, 

further studies are required to clarify the mechanisms 

underlying the riluzole effects on morphineinduced 

tolerance. 

Acknowledgment: 

This research was supported by Drug Applied Research Center of 

Tabriz University of Medical Sciences grant. 

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Received: 

October 20, 2009; in revised form: January 19, 2010.

Intracerebroventricular administration of riluzole prevents morphine ...

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