Pain Mechanisms

Section Editors: Tony L. Yaksh/Quinn H. Hogan

Central Administration of Minocycline and Riluzole

Prevents Morphine-Induced Tolerance in Rats
Bohlool Habibi-Asl, PharmD, PhD BACKGROUND: Long-term exposure to opiates induces tolerance to the analgesic
effect. The neurobiological mechanism of this phenomenon is not completely clear.
Kambiz Hassanzadeh, PharmD In this study, we evaluated the effects of central administration of minocycline (a
tetracycline derivative) and riluzole (an antiglutamatergic drug) on morphine-
induced tolerance in rats.
Mohammad Charkhpour, PharmD, METHODS: Groups of rats received daily morphine (10 mg/kg, IP) in combination
PhD with saline (10 ␮L/rat, intracerebroventricular [ICV]) or 1% Tween 80 (10 ␮L/rat,
ICV) or minocycline (60, 120, and 240 ␮g/10 ␮L per rat, ICV) or riluzole (20, 40, 80
␮g/10 ␮L per rat, ICV). Nociception was assessed using hotplate apparatus
(55°C ⫾ 0.5°C). Hotplate latency was recorded when the rat licked its hindpaw.
Baseline latencies were determined once per day for each rat, then morphine (10
mg/kg) was injected. After 20 min, the above-mentioned drugs were administered
and postdrug latency was measured 10 min after the injection of drugs or vehicles.
RESULTS: Results showed that ICV administration of minocycline and riluzole
delayed morphine-induced tolerance. Morphine tolerance was complete after 8
days in the control groups but was complete in the groups treated with minocycline
(120 ␮g/10 ␮L per rat) and riluzole (80 ␮g/10 ␮L per rat) on the 13th day. In
addition, our results showed that minocycline and riluzole increased the total
analgesic effect of morphine (area under the curve of the percentage of maximal
possible effect values).
CONCLUSION: The effects of minocycline on nitric oxide and the glutamatergic system
and the effect of riluzole on the glutamate system are potentially important
mechanisms in delaying morphine-induced tolerance.
(Anesth Analg 2009;109:936 –42)

O pioids such as morphine are the most widely used

drugs for the alleviation of moderate to severe chronic
antinociceptive effect of morphine and morphine
physical dependence.
pain. Systemically administered morphine produces Using behavioral studies, we and others have
antinociception via actions at both spinal and su- shown that a variety of NMDA receptor antagonists
praspinal sites.1 have the ability to inhibit the development of opiate
Repeated use of opioids induces tolerance that results tolerance and dependence.2– 8
in a loss of drug effect or the requirement for escalating Other studies have shown that activation of
doses to produce pain relief. The neurobiological mecha- NMDARs can lead to neurotoxicity under many cir-
nisms of the development of opioid tolerance are multi- cumstances.9 For instance, peripheral nerve injury has
faceted and only partially understood. been shown to activate spinal cord NMDARs, which
There are several lines of evidence that suggest that results in not only intractable neuropathic pain but
N-methyl-d-aspartate (NMDA) glutamate receptors also neuronal cell death because of apoptosis.10 –12
(NMDARs) are involved in the plasticity that arises There are also several lines of evidence which
from long-term administration of morphine.2–5 This suggest that activation of NMDARs leads to toxic
idea was suggested by Trujillo and Akil2 who reported calcium influx, which activates numerous enzymes,
including neuronal nitric oxide (NO) synthase (NOS).
that the NMDA receptor antagonist, MK-801 (dizo-
NO is able to further increase excitotoxicity by enhanc-
cilpine), inhibited the development of tolerance to the
ing glutamate release from presynaptic neurons and
inhibiting glial glutamate transporters.13–15
From the Department of Pharmacology and Toxicology, Faculty Minocycline, a semisynthetic tetracycline deriva-
of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
tive, is able to provide neuroprotection against global
Accepted for publication April 13, 2009.
ischemia in gerbils and focal brain ischemia in rats.16,17
Address correspondence and reprint requests to Kambiz Has-
sanzadeh, Department of Pharmacology and Toxicology, Faculty of Another study strongly suggested that minocycline
Pharmacy, Tabriz University (Medical Sciences), Tabriz 51664- acts at an earlier plasmalemmal step by limiting
14766, Iran. Address e-mail to hassanzadehk@tbzmed.ac.ir. glutamate release and the ensuing [Ca2⫹] elevation in
Copyright © 2009 International Anesthesia Research Society target neurons. Minocycline may prevent the activa-
DOI: 10.1213/ane.0b013e3181ae5f13
tion of this [Ca2⫹]-dependent intracellular pathway,
936 Vol. 109, No. 3, September 2009
thus inhibiting neuronal death. A decrease in neuronal Verification of Cannula Placement
excitability, together with a marked decrease in gluta- At the end of all experiments, methylene blue
mate release, may explain the cytoprotective proper- solution (5 ␮L/rat, ICV) was injected into the cannula
ties of minocycline.18 –20 and the animals were killed by an overdose of ether
Many studies have indicated that the protective effect followed by decapitation. The brain of each animal
of minocycline was associated with reduced activation of was dissected out and cut in the coronal plane to
inducible NOS and interleukin-1b-converting enzyme, verify the placement of the guide cannula and distri-
which are mainly expressed by microglia.19 bution of methylene blue in the ventricles. Only data
Riluzole is the only proven effective medicine for from animals that showed uniform distribution of
amyotrophic lateral sclerosis (ALS) because it has been methylene blue in the ventricles were considered for
demonstrated to delay the time of death in ALS statistical analysis. In all, six animals were discarded
patients.20 Riluzole is an antiglutamatergic drug, because the placement of the guide cannula was
which interferes with responses mediated by excita- incorrect.
tory amino acids, even though it does not interact with
any known binding site on NMDA, kainate, or Drug Treatment
␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Morphine sulfate (Sigma-Aldrich; Sigma-Aldrich,
(AMPA) glutamate receptors.20 In addition, it has been Germany) (10 mg/kg, daily) was dissolved in double
shown that coadministration of riluzole with morphine distilled water and was injected IP using 1-mL insulin
decreased the intensity of the withdrawal syndrome, syringes. Minocycline (Sigma-Aldrich) (60, 120, and 240
reflecting a reduction in physical dependence.21 ␮g/10 ␮L per rat) was dissolved in double distilled
In view of these data, both minocycline and riluzole water and infused ICV using a Hamilton microsy-
have a common mechanism of action on the glutama- ringe. Riluzole (Sigma-Aldrich) (20, 40, and 80 ␮g/10
tergic system; therefore, in this study, we evaluated ␮L per rat) was dissolved (1% Tween 80 in sterile 0.9%
the effect of intracerebroventricular (ICV) administra- normal saline) and infused ICV using a Hamilton
tion of minocycline and riluzole on morphine toler- syringe. Dizocilpine (Sigma-Aldrich) (1 ␮g/10 ␮L per
ance in rats. rat) was dissolved in double distilled water and
infused ICV using a Hamilton microsyringe. Two
METHODS control groups were included, which received either
Animals morphine, IP ⫹ 1% Tween 80 in saline normal 0.9%,
ICV or morphine, IP ⫹ distilled water 0.9%, ICV.
Male Wistar rats (Razi Institute, Tehran, Iran) weigh-
Volume of infusion was 10 ␮L at a rate of 10 ␮L/min
ing 250 –300 g (eight animals in each group) were used in
in each rat.
this study. They were kept in a temperature-controlled
room (24°C ⫾ 0.5°C) and maintained on a 12-h light/ Assessment of Nociception
dark cycle (light on 08:00 am) with free access to food Nociception was assessed using the hotplate appa-
and water. All experiments were executed in accordance ratus (55°C ⫾ 0.5°C).23 Hotplate latency was recorded
with the Guide for the Care and Use of Laboratory when the rat licked its hindpaw. A cut-off time (40 s)
Animals (National Institutes of Health Publication No. was imposed to prevent tissue damage.24 Hotplate
85-23, revised 1985) and were approved by the research response latencies (s) are expressed as the percentage
and ethics committee of Tabriz University of Medical of maximal possible effect (%MPE) using the equation
Sciences. below:
Intracerebroventricular Cannula Implantation Post-drug latency (s) ⫺ Baseline latency (s)
Rats were anesthetized with sodium pentobarbital %MPE ⫽ ⫻ 100
Cut-off value (s) ⫺ Baseline latency (s)
(50 mg/kg, IP) (Merck, Germany) and a stainless steel
guide cannula (23 gauge) was implanted stereotaxi- Baseline latency was determined once per day for
cally into the lateral cerebral ventricle (coordinates: each rat, before daily injection of morphine (10 mg/kg).
⫺0.8 mm posterior, ⫺1.3 mm midline to lateral, and After 20 min, the drugs were administered and the
3.5 mm ventral) with respect to bregma.22 postdrug latency was measured 10 min after the injec-
A stainless steel guide (30 gauge) was placed into tion of drugs or vehicles (30 min after the injection of
the guide cannula as a dummy cannula to maintain morphine). The %MPE was then calculated for that day.
patency. After surgery, a recovery period of 7 days The experiments continued until there was no significant
was allowed before experiments. During the recovery difference in %MPE between the vehicle- or drug-treated
period, animals were habituated to the testing envi- groups and the saline group.
ronment including transfer to the experimental room
and twice daily handling, weighing, restraining on the Evaluation of the Global Analgesic Effect
platform for 1 min, and gently removing and replac- To evaluate the global analgesic effect and to allow
ing the dummy cannula. Animals were also habitu- a comparison of the effects from different behavioral
ated to the hotplate apparatus and testing started after tests, the area under the curve (AUC) of the %MPE
the recovery period of 7 days in all groups. was calculated. To calculate the AUC, the trapezoidal

Vol. 109, No. 3, September 2009 © 2009 International Anesthesia Research Society 937
 Figure 2. Effect of daily ICV injections of minocycline (60,
Figure 1. Analgesic effect of daily administration of mor- 120, and 240 ␮g/10 ␮L per rat) on tolerance to the analgesic
phine (10 mg/kg, IP) in combination with distilled water (10 effect of morphine. Each bar represents the mean of percent-
␮L/rat) or 1% Tween 80 in normal saline (10 ␮L/rat). age of maximal possible effect (%MPE) ⫾ sem for eight rats.
Developed tolerance to the analgesic effect of morphine was One-way analysis of variance (ANOVA) followed by
complete on the 8th day when there were no significant Tukey’s test were used to analyze the statistical signifi-
differences in percentage of maximal possible effect (%MPE) cances. P values ⬍0.05 were considered to be significant in
between the control groups and the saline group. Each bar all analyses. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 when
represents the mean of %MPE ⫾ sem for eight rats. M ⫽ compared with the control group. Arrow represents the day
morphine; DW ⫽ distilled water; Mino ⫽ minocycline; of morphine tolerance. M ⫽ morphine; DW ⫽ distilled
Rilu ⫽ riluzole. water; Mino ⫽ minocycline.

0.9% or distilled water. The analgesic effect of mor-

rule was used. The AUC (1–13 days) was calculated
phine decreased on the 8th day compared with the 1st
using the trapezoidal rule from the observed values.
day and, because there were no significant differences
Evaluation of Tolerance Induction between the control groups and the saline-treated
To evaluate the induction of tolerance, groups of animals on Day 8, this was considered the day of
rats received either saline or morphine (10 mg/kg, IP) morphine tolerance initiation.
⫹ saline or morphine (10 mg/kg, IP) ⫹ minocycline
Evaluation of the Effect of Minocycline on Morphine-
(the most effective dose) or morphine (10 mg/kg, IP)
Induced Tolerance to the Analgesic Effect
⫹ riluzole (the most effective dose) for 8 days, then on
Minocycline delayed the onset of morphine-
the 9th day (1 day after morphine tolerance in the
induced tolerance. Minocycline (60, 240, and 120
control group) several doses of morphine (10, 25, 50,
␮g/10 ␮L per rat) delayed morphine tolerance for 4, 4
and 100 mg/kg, IP) were administered to generate
and 5 days, respectively (Fig. 2). Analysis of the AUC
analgesic dose-response curves. Morphine antinoci-
of hotplate latency (Table 1), which allowed evalua-
ceptive 50% effective dose (ED50) values for each of the
tion of the global effect, showed that repeated treat-
drug groups were derived using linear regression of
ment with minocycline before morphine enhanced the
%MPE of the morphine dose. Differences in the ED50
effectiveness of morphine. On the other hand, mino-
estimations were determined using the confidence
cycline (120 ␮g/10 ␮L per rat) had the greatest AUC of
interval method at P ⫽ 0.05.25
%MPE (289.3) compared with the control group
Data Analysis (177.5) or minocycline (60 ␮g/10 ␮L per rat) (264.7) or
Data are expressed as the mean of %MPE ⫾ sem of minocycline (240 ␮g/10 ␮L per rat) (234.7) (Table 1).
eight rats per group. One-way analysis of variance In addition, the results in Figure 3 show a significant
followed by Tukey’s test were used to analyze statis- shift to the right in the dose-response curve for
tical significance in multiple comparisons. P values animals who received morphine (10 mg/kg) ⫹ saline
⬍0.05 were considered to be significant in all analyses. (10 ␮L/rat) compared with those receiving saline (10
*P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 indicate a ␮L/rat) or morphine (10 mg/kg) ⫹ minocycline (120
significant difference as compared with the saline ␮g/10 ␮L per rat). A significant shift to the right in
group for that day. The AUC1–13 data were analyzed ED50 in the control group (88.64) compared with saline
by one-way analysis of variance. (29.5) or morphine (10 mg/kg) ⫹ minocycline (120
␮g/10 ␮L per rat) (61.1) treated animals was also seen.
RESULTS Evaluation of the Effect of Riluzole on Morphine-Induced
Induction of Tolerance to the Antinociceptive Effect Tolerance to the Analgesic Effect
of Morphine Riluzole (20, 40, and 80 ␮g/10 ␮L per rat) also
As shown in Figure 1, daily administration of delayed morphine tolerance. Riluzole (20 and 40
morphine (10 mg/kg, IP) induced tolerance to the ␮g/10 ␮L per rat) delayed morphine tolerance for 4
antinociceptive effect of this drug in both the control days; however, the results indicated that riluzole (80
groups which received 1% Tween 80 in saline normal ␮g/10 ␮L per rat) decreased the development of
938 Minocycline and Riluzole Decreased Morphine Tolerance ANESTHESIA & ANALGESIA
Table 1. The Global Analgesic Effect of Morphine in the Control and Treatment Groups During 13 Days of Experimentation
Minocycline AUC sem Riluzole AUC sem
Control mino 177.5 4.2 Control rilu 156 3.4
60 ␮g/10 ␮L per rat 264.7* 3.7 20 ␮g/10 ␮L per rat 216† 3.1
120 ␮g/10 ␮L per rat 289.3* 2.6 40 ␮g/10 ␮L per rat 197‡ 2.4
240 ␮g/10 ␮L per rat 234.7* 2.9 80 ␮g/10 ␮L per rat 261* 3.4
Dizo 1 ␮g/10 ␮L per rat 363.8* 4.1
Area under the curve (AUC) of percentage of maximal possible effect (%MPE) was calculated for each group for 13 days. To calculate the AUC, the trapezoidal rule was used. One-way analysis
of variance followed by Tukey’s test was used to analyze the differences between the control and treatment groups.
Mino ⫽ Minocycline; Rilu ⫽ Riluzol; Dizo ⫽ Dizocilpine.
P values less than 0.05 were considered to be significant in all analyses. *P ⬍ 0.001; †P ⬍ 0.01; ‡P ⬍ 0.05 when compared with the control group.

Figure 4. Effect of daily ICV injections of riluzole (20, 40, and

80 ␮g/10 ␮L per rat) on tolerance to the analgesic effect of
Figure 3. Hotplate responses and percent of maximal pos- morphine. Each bar represents the mean of percentage of
sible effect (%MPE) to various morphine doses (10, 25, 50, maximal possible effect (%MPE) ⫾ sem for eight rats.
and100 mg/kg, IP) administered on Day 9 after 8 days of One-way analysis of variance (ANOVA) followed by
continuous ICV drug infusion. Each point represents the Tukey’s test were used to analyze the statistical signifi-
mean ⫾ sem of eight rats. Different morphine doses admin- cances. P values ⬍0.05 were considered to be significant in
istered on Day 9 demonstrated a significant shift to the right all analyses. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍ 0.001 when
in the dose-response curve and antinociceptive ED50 values compared with the control group. Arrow represents the day
in animals treated with morphine ⫹ saline compared with of morphine tolerance. M ⫽ morphine; Rilu ⫽ riluzole.
those receiving saline or morphine ⫹ minocycline (120
␮g/10 ␮L per rat) or morphine ⫹ riluzole (80 ␮g/10 ␮L per
rat). Comparison of the Effect of Minocycline or Riluzole
with Dizocilpine on Attenuation of Morphine
Tolerance Development
morphine tolerance and could delay morphine toler- To examine the possible mechanism involved in
ance for 5 days (Fig. 4). Riluzole (80 ␮g/10 ␮L per rat) morphine-induced tolerance, we tested the effect of
had the greatest AUC of %MPE (261) compared with ICV administration of dizocilpine (1 ␮g/10 ␮L per rat)
the control group (156) or riluzole (20 ␮g/10 ␮L per (a noncompetitive NMDA receptor antagonist), be-
rat) (216) or riluzole (40 ␮g/10 ␮L per rat) (197) (Table cause this agent has well-established effects on opioid
1). Figure 3 shows a significant shift to the right in the tolerance and was used as a positive control in this
dose-response curve for animals that received mor- study. As shown in Figure 6, dizocilpine attenuated
phine (10 mg/kg) ⫹ saline (10 ␮L/rat) compared with morphine tolerance and delayed tolerance initiation
those receiving saline (10 ␮L/rat) or morphine (10 for 6 days at this dose. Dizocilpine (1 ␮g/10 ␮L per
mg/kg) ⫹ riluzole (80 ␮g/10 ␮L per rat). Further- rat) did not have a significant analgesic effect, thus the
more, a significant shift to the right in ED50 in the effects of this drug are not related to analgesia.
control group (88.64) was observed when compared
with saline (29.5) and morphine (10 mg/kg) ⫹ riluzole DISCUSSION
(80 ␮g/10 ␮L per rat) (66.8) treated animals. Tolerance is a behavioral adaptation to the pro-
longed use of opioid drugs, such as morphine. The
Evaluation of the Analgesic Effects of Minocycline cellular mechanism underlying the development of
and Riluzole morphine tolerance remains controversial. In the cur-
Administration of the most effective doses of mino- rent study, we investigated the effect of minocycline
cycline (120 ␮g/10 ␮L per rat) or riluzole (80 ␮g/10 and riluzole on morphine-induced tolerance to the
␮L per rat) on morphine-induced tolerance did not analgesic effect. The main findings of this study indi-
have a significant analgesic effect and there were cate that ICV administration of minocycline and ri-
significant differences between the administration of luzole can prevent the development of morphine
these drugs and saline (Fig. 5). tolerance.

Vol. 109, No. 3, September 2009 © 2009 International Anesthesia Research Society 939
Figure 5. Analgesic effects of daily ICV injections of dizo- Figure 6. Effects of daily ICV injections of the most effective
cilpine (1 ␮g/10 ␮L per rat), minocycline (120 ␮g/10 ␮L per doses of minocycline (120 ␮g/10 ␮L per rat) and riluzole (80
rat), riluzole (80 ␮g/10 ␮L per rat), and saline (10 ␮L/rat). ␮g/10 ␮L per rat) were compared with dizocilpine (1 ␮g/10
Each bar represents the mean of percentage of maximal ␮L per rat) used as a positive control. Each bar represents the
possible effect (%MPE) ⫾ sem for eight rats. One-way mean of percentage of maximal possible effect (%MPE) ⫾ sem
analysis of variance (ANOVA) followed by Tukey’s test for eight rats. M ⫽ morphine; DW ⫽ distilled water; Mino ⫽
were used to analyze the differences between the saline and minocycline; Rilu ⫽ riluzole; Dizo ⫽ dizocilpine.
treatment groups. P values ⬍0.05 were considered to be
significant in all analyses. Mino ⫽ minocycline; Rilu ⫽
riluzole; Dizo ⫽ dizocilpine. completely prevented NMDA toxicity and the preced-
ing activation and proliferation of microglial cells.
These results support the notion that microglial activa-
Several studies have indicated that repeated admin- tion contributes to excitotoxic neuronal death, which can
istration of opiates can activate the NMDA receptor be inhibited by antiinflammatory compounds, such as
through the G-protein associated with the opioid minocycline.18 The mechanism underlying the role of
receptor and/or intracellular mechanisms.26,27 This glial cells in the effects of morphine on naive mice is
opiate-related activation of NMDARs may initiate unclear. It is possible that morphine acts directly on
subsequent intracellular changes, such as the produc- microglia, triggering alterations in their morphology,
tion of NO and/or the activation of protein kinase C metabolism, and function.36 Furthermore, a recent
(PKC). Both NO and PKC have been shown to be study indicated that systemic administration of mino-
critical in the development of morphine tolerance.28,29 cycline attenuated morphine antinociceptive toler-
Previous studies have shown that minocycline, a ance.37 Mika et al.37 concluded that the effect of
semisynthetic tetracycline derivative, exhibits neuro- minocycline on morphine tolerance is related to mi-
protective effects against neuronal damage in animal croglia. Their results provide evidence that systemic
disease models.18,30 –33 Our results showed that mino- administration of minocycline in mice influences mor-
cycline (120 ␮g/10 ␮L per rat) postponed morphine phine’s effectiveness and delays the development of
tolerance for 5 days and minocycline (60 and 240 morphine tolerance by attenuating microglial activa-
␮g/10 ␮L per rat) shifted morphine tolerance from the tion and its markers.
8th to the 12th day (Fig. 2). On the other hand, the total According to the above-mentioned studies and our
analgesic effect of morphine (AUC of %MPE) signifi- findings, it is concluded that the effect of minocycline
cantly increased in all treatment groups (Table 1). in this study is possibly related to its neuroprotective
Although minocycline (120 ␮g/10 ␮L per rat) delayed property, its effect on preventing glutamate release,
morphine tolerance more than other doses, there was and its inhibition of microglial cells and NOS.
no significant difference among the three doses in Figure 4 showed that riluzole decreased tolerance
their effects on morphine tolerance. Furthermore, the to the analgesic effect of morphine. Riluzole is the only
results shown in Figure 5 demonstrate that the doses proven effective medicine for ALS and was demon-
of minocycline administered in this study did not strated to delay the time of death in these patients.38,39
have an analgesic effect. Therefore, the action of This is thought to result from the neuroprotective
minocyline in preventing morphine tolerance is not effect of riluzole, which has a complex mechanism of
related to its analgesic effect. Several reports attribute action involving several effects: inhibition of voltage-
the neuroprotective effects of minocycline to various dependent sodium channels,40,41 high-voltage-activated
intracellular signaling pathways, including antioxi- calcium and potassium channels,41 and inhibition of
dant systems,34 inhibition of NOS19 blockade of in- PKC, suggesting involvement in antioxidative pro-
flammatory responses,35 prevention of the activation cesses.42 Our results showed that riluzole (20, 40, and 80
of Ca2⫹-dependent intracellular pathways, and a ␮g/10 ␮L per rat) delayed morphine tolerance. As
marked decrease in glutamate release.20 shown in Figure 4, low doses of riluzole (20 and 40
In addition, another study showed that NMDA- ␮g/10 ␮L per rat) only delayed morphine tolerance
induced neuronal death involved proliferation and for 2 days and 1 day, respectively; however, riluzole
activation of microglial cells and that minocycline (80 ␮g/10 ␮L per rat) delayed morphine tolerance for
940 Minocycline and Riluzole Decreased Morphine Tolerance ANESTHESIA & ANALGESIA
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942 Minocycline and Riluzole Decreased Morphine Tolerance ANESTHESIA & ANALGESIA