The beta-lactam antibiotic, ceftriaxone, inhibits
development of opioid-induced hyperalgesia in mice
Zhijun Chen, Yuke Tian
Department of Anesthesiology of Tongji Hospital，Tongji Medical College,
Huazhong University of Science and Technology
Abstract: The glutamate transporter GLT-1 is primarily responsible for
glutamate clearance in the spinal cord. Beta-lactam antibiotics can
attenuate nuropathic pain by promoting GLT-1 expression and function
in the CNS. The present study was undertaken to test the hypothesis that
ceftriaxone, a representive beta-lactam antibiotic, prevent development
of opioid-induced hyperalgesia in mice. Opioid-induced hyperalgesia
was produced by repeated morphine administration in mice. Ceftriaxone
(200mg/kg, i.p. × 7 days) injected once daily inhibited mechanical
allodynia and heat hyperalgesia induced by repeated morphine
administration(OIH). Moreover, intraperitoneal administration of
ceftriaxone inhibited down-regulation of spinal GLT-1 expression
induced by OIH. These results suggest that beta-lactam antibiotics
inhibit the development of opioid-induced hyperalgesia by upregulating
spinal GLT-1 expression.
Introduction
Glutamate is the predominant excitatory neurotransmitter in the
central nervous system. Glutamate is normally rapidly cleared from the
synaptic cleft by five high-affinity, sodium-dependent glutamate
transporters (EAAT1-5)[1, 7, 10, 16, 20]. EAAT2, also defined as
glutamate-transporter subtype 1 (GLT-1) in rodents, expressed primarily
on astrocytes, which is responsible for 90% of extracellular glutamate
uptake in the central nervous system (CNS). Animal models demonstrate
that GLT-1 dysfunction contributes to a number of clinical disorders,
including neuropathic pain [13, 21], opioid torelance[22], opioid
dependence and withdrawal[14, 15]. Therefore, GLT-1 plays a major
role in terminating synaptic transmission and protecting neurons from
glutamate neurotoxicity.
Opioids are a class of the most effective analgesics for treating
moderate to severe pain. However, chronic morphine not only leads to
tolerance and dependence, but also results in nociceptive enhancement
called opioid-induced hyperalgesia (OIH)[5]. Although hyperalgesia
associated with opioid use has been reported clinically for decades, the
underlying mechanism of OIH remains unclear. Therefore clinincal
treatment of OIH has been very difficult so far.
Rothstein et al. [19] discovered that the beta-lactam class of
antibiotics can greatly and selectively promote GLT-1 expression and
function in the CNS. And animal studies demonstate that beta-lactam
antibiotics can attenuate nuropathic pain[9], morphine torelance[18] and
dependence[17]. Therefore this study tested the hypothesis that
ceftriaxone, a representive beta-lactam antibiotic, would inhibit
development of opioid-induced hyperalgesia in mice.
Materials and Methods
Animals
Experiments were performed on male ICR mice (25±5 g, Harlan
Laboratories, Indianapolis, IN). Animals were provided food and water
ad libitum with a 10:14 h light:dark cycle. Mice were randomly divided
into experimental groups according to a computer generated
randomization list. All animal experiments were carried out in
accordance with the International Association for the Study of Pain
(IASP) and the National Institute of Health Guidelines for the handling
and use of laboratory animals after approval by the University of Illinois
Institutional Animal Care and Use Committee.
Drugs and administration
Morphine sulfate was obtained from the National Institute on Drug
Abuse (Rockville, MD) and injected subcutaneously. Ceftriaxone was
purchased from Sigma (St. Louis, MO) and injected intraperitoneally.
To induce OIH, mice were treated subcutaneously according to a
previously published schedule [4, 11]. Mice received 20 mg/kg
morphine sulfate (twice per day, s.c.) for 3 consecutive days and two
more injections of 40 mg/kg morphine sulfate on day 4. Mice from both
groups were injected once daily with either ceftriaxone (200 mg/kg) or
equal volume of saline, beginning 3 days before the start of morphine
injection. Mechanical and thermal sensitivities were tested before and
after morphine treatment.
Mechanical allodynia
Mechanical sensitivity was determined using calibrated von Frey
filaments (Stoelting, Wood Dale, IL) as previously described [2, 3, 12,
23]. These tests were performed before ceftriaxone injection to obtain
the baseline mechanical sensitivity and daily post-morphine injection by
the same researcher. The up-down paradigm was used to determine 50%
probability of paw withdrawal threshold [2, 3, 6, 12].
Thermal hyperalgesia
The paw withdrawal latencies to radiant heat were tested using a
plantar tester (model 7372, UGO BASILE, VA, Italy) as described
previously[3, 8, 12, 23]. Mice were placed in a transparent cage on a
glass floor. The radiant heat source was focused on the central portion of
the plantar surface of the left hindpaw and paw withdrawal latencies
were recorded when the heat source was automatically turned off as a
result of paw withdrawal. A cut-off time of 20 s was applied in order to
prevent tissue damage.
Western blotting analysis
The lumbar sections of the spinal cord were quickly dissected from
euthanized mice and frozen in acetone-dry ice solution and stored -80 °C,
or immediately processed for western blotting analysis as previously
described [3, 12, 24]. Briefly, tissues were homogenized in RIPA buffer
in the presence of phosphatase inhibitors (10mM sodium fluoride,
10mM sodium pyrophosphate, and 1 mM sodium orthovanadate and 1
μM okadaic acid) and protease inhibitors (0.05 mg/ml bestatin, 0.05
mg/ml leupeptin, 0.05 mg/ml pepstatin, and 0.1 mg/ml
phenylmethylsulfonylfluoride). The homogenates were incubated on a
rotator for 1 h and centrifuged (45,000 × g) for 1 h at 4 °C. Protein
content in the supernatant was determined by a modified Bradford
method (Pierce Biotechnology, Rockford, IL). Samples were then
separated by SDS-PAGE and electrotransferred onto PVDF membrane
for western blotting analyses. Antibodies including a rabbit anti-GLT1antibody (1:1000; Cell Signaling Technology, Beverly, MA), and a
mouse anti-β-actin antibody (1:10,000, Sigma); and corresponding HRP
conjugate secondary antibodies (Amersham, Piscataway, NJ) were used.
The enhanced chemiluminescence signals (Pierce Biotechnology) were
captured by a ChemiDoc imaging system and analyzed using the
Quantity One program (BioRad, Hercules, CA). Ratios of the optical
densities of GLT-1 to those of β-actin were calculated for each sample.
Statistical analysis
A two-way repeated-measures ANOVA was used to determine
differences among groups. Student–Newman–Keuls test was used as a
post-hoc test. Statistical significance was established at the 95%
confidence limit.
Results
Intraperitoneal administration of ceftriaxone inhibited development
of OIH in mice
Four days of subcutaneous morphine administration by intermittent
injections significantly increased mechanical and thermal sensitivities
compared with saline-treated mice(Fig. 1). Intraperitoneal preventive
administration of ceftriaxone, given once daily for seven days beginning
3 days before the start of chronic morphine injection, before the
development of hyperalgesia, significantly increased the mechanical
withdrawal threshold and thermal withdrawal latency, as compared with
OIH mice in the corresponding time points observed (Fig. 1A, B). The
effect was evident from postinjection day 1 and continued until
postinjection day 7 (Fig. 1A, B). However, the intraperitoneal
administration of saline in the same protocol had no effect on the
decreased mechanical withdrawal threshold and thermal withdrawal
latency induced by OIH (Fig. 1A, B). The results suggested that the
preventive administration of ceftriaxone could prevent the development
of mechanical allodynia and thermal hyperalgesia induced by OIH.
Intraperitoneal administration of ceftriaxone inhibited downregulation of spinal GLT-1 expression induced by OIH
Western blotting analysis showed that spinal GLT-1 protein expression
obviously decreased in the OIH mice (Fig. 2). Repeated intraperitoneal
administration of ceftriaxone significantly prevented the downregulation of GLT-1 protein expression induced by OIH on postinjection
day 7 (Fig. 2). The preventive effect of ceftriaxone on the downregulation of GLT-1 after OIH was coincident in time course with its
preventive effect on hyperalgesia behaviors induced by OIH.
Discussion
In the present study we testes the hypothesis hat repeated
intraperitoneal administration of ceftriaxone can inhibit the development
of hyperalgesia. We found that ceftriaxone (200mg/kg, i.p. × 7 days)
inhibited opioid-induced mechanical allodynia, heat hyperalgesia and
downregulation of spinal GLT-1 expression.
As we know, the glutamate transporter plays a major role in the
maintenance of glutamate homeostasis. Of the five glutamate
transporters, GLT-1 (EAAT2) is responsible for 90% of glutamate
uptake in the central nervous system (CNS). Prior studies have shown
that the development of hyperalgesia was accompanied by the downregulation of GLT-1 protein and its uptake of glutamate in many pain
models such as CCI[21] and spinal nerve transection[25]. In this study
we found that the development of opioid-induced hyperalgesia was also
associated to the down-regulation of GLT-1 protein in the spinal cord.
A recent report published in nature [19] indicated that the beta-lactam
class of antibiotics increased GLT-1 expression, functional and
biochemical activity in the brain and spinal cord. Because beta-lactam
antibiotics are the most widely used antibiotics in the world, which
cause no known side effects clinically, they may be useful in the
management of GLT-1 disfunction. Hu et al [9] reported that systemic
treatment with ceftriaxone prevented and reversed CCI-induced
mechanical allodynia and heat hyperalgesia. Prior studies have shown
that repeated intraperitoneal treatment with ceftriaxone limits the
development of tolerance to repeated morphine injections as well as
alleviating naloxone-induced morphine withdrawal and inhibits
development of morphine physical dependence[17, 18]. The present data
show that intraperitoneal ceftriaxone pre-treatment inhibited opioidinduced mechanical allodynia and heat hyperalgesia.
In summary, we found that ceftriaxone inhibited development of OIH
by upregulating GLT-1 expression in lumbar spinal cord in mice. Downregulation of spinal GLT-1 is involved in the generation of OIH. These
findings highlight the possibility of a new clinical strategy to prevent
OIH.
A
Saline+Saline
Saline+MS
Cef+MS
Paw withdrawal
threshold (g)
1.5
1.0
0.5
0.0
*** ***
*** ***
Cef 0.2g/kg i.p.
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11
Days
Paw withdrawal latency
(s)
B
Saline+Saline
Saline+MS
Cef+MS
12
10
8
6
4
Cef 0.2g/kg i.p.
*** *** ***
***
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11
Days
Fig. 1 The effect of intraperitoneal administration of Cef on mechanical/thermal withdrawal
latency of OIH mice. The horizontal black bars represent the period of Cef administration. The
downward arrows indicate the time point which mice received saline or morphine sulfate (day 1–
3: 20 mg/kg; day 4: 40 mg/kg; twice daily, s.c.). Data are expressed as mean±SEM *p<0.05,
**p<0.01, ***p<0.001, compared with the saline-treated group; n=5 for each group.
GLT-1
-actin
Normalized OD of
GLT-1/beta-actin
1.5
#
1.0
*
0.5
0.0
Saline+Saline
Saline+MS
Cef+MS
Fig. 2 OIH-induced down-regulation and Cef-induced up-regulation of GLT-1 expression in
lumbar spinal cord. Data are expressed as mean±SEM, *p<0.05, compared with the salinetreated group; #p<0.05, compared with the morphine-treated group; n=4 for each group.
Reference
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
J.L. Arriza, S. Eliasof, M.P. Kavanaugh, S.G. Amara, Excitatory amino acid transporter 5, a retinal
glutamate transporter coupled to a chloride conductance, Proc Natl Acad Sci U S A 94 (1997)
4155-4160.
S.R. Chaplan, F.W. Bach, J.W. Pogrel, J.M. Chung, T.L. Yaksh, Quantitative assessment of tactile
allodynia in the rat paw, J Neurosci Methods 53 (1994) 55-63.
Y. Chen, F. Luo, C. Yang, C.M. Kirkmire, Z.J. Wang, Acute inhibition of Ca2+/calmodulindependent protein kinase II reverses experimental neuropathic pain in mice, J Pharmacol Exp
Ther 330 (2009) 650-659.
Y. Chen, C. Yang, Z.J. Wang, Ca2+/calmodulin-dependent protein kinase II alpha is required for
the initiation and maintenance of opioid-induced hyperalgesia, J Neurosci 30 38-46.
L.F. Chu, M.S. Angst, D. Clark, Opioid-induced hyperalgesia in humans: molecular mechanisms
and clinical considerations, Clin J Pain 24 (2008) 479-496.
W.J. Dixon, Efficient analysis of experimental observations, Annu Rev Pharmacol Toxicol 20
(1980) 441-462.
W.A. Fairman, R.J. Vandenberg, J.L. Arriza, M.P. Kavanaugh, S.G. Amara, An excitatory aminoacid transporter with properties of a ligand-gated chloride channel, Nature 375 (1995) 599-603.
K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and sensitive method for measuring
thermal nociception in cutaneous hyperalgesia, Pain 32 (1988) 77-88.
Y. Hu, W. Li, L. Lu, J. Cai, X. Xian, M. Zhang, Q. Li, L. Li, An anti-nociceptive role for ceftriaxone in
chronic neuropathic pain in rats, Pain 148 284-301.
Y. Kanai, M.A. Hediger, Primary structure and functional characterization of a high-affinity
glutamate transporter, Nature 360 (1992) 467-471.
D.Y. Liang, G. Liao, J. Wang, J. Usuka, Y. Guo, G. Peltz, J.D. Clark, A genetic analysis of opioidinduced hyperalgesia in mice, Anesthesiology 104 (2006) 1054-1062.
F. Luo, C. Yang, Y. Chen, P. Shukla, L. Tang, L.X. Wang, Z.J. Wang, Reversal of chronic
inflammatory pain by acute inhibition of Ca2+/calmodulin-dependent protein kinase II, J
Pharmacol Exp Ther 325 (2008) 267-275.
S. Maeda, A. Kawamoto, Y. Yatani, H. Shirakawa, T. Nakagawa, S. Kaneko, Gene transfer of GLT-1,
a glial glutamate transporter, into the spinal cord by recombinant adenovirus attenuates
inflammatory and neuropathic pain in rats, Mol Pain 4 (2008) 65.
T. Ozawa, T. Nakagawa, Y. Sekiya, M. Minami, M. Satoh, Effect of gene transfer of GLT-1, a
glutamate transporter, into the locus coeruleus by recombinant adenoviruses on morphine
physical dependence in rats, Eur J Neurosci 19 (2004) 221-226.
T. Ozawa, T. Nakagawa, K. Shige, M. Minami, M. Satoh, Changes in the expression of glial
glutamate transporters in the rat brain accompanied with morphine dependence and naloxoneprecipitated withdrawal, Brain Res 905 (2001) 254-258.
G. Pines, N.C. Danbolt, M. Bjoras, Y. Zhang, A. Bendahan, L. Eide, H. Koepsell, J. Storm-Mathisen,
E. Seeberg, B.I. Kanner, Cloning and expression of a rat brain L-glutamate transporter, Nature
360 (1992) 464-467.
S.M. Rawls, D.A. Baron, J. Kim, beta-Lactam antibiotic inhibits development of morphine physical
dependence in rats, Behav Pharmacol 21 161-164.
S.M. Rawls, M. Zielinski, H. Patel, S. Sacavage, D.A. Baron, D. Patel, Beta-lactam antibiotic
reduces morphine analgesic tolerance in rats through GLT-1 transporter activation, Drug Alcohol
Depend 107 261-263.
[19]
[20]
[21]
[22]
[23]
[24]
[25]
J.D. Rothstein, S. Patel, M.R. Regan, C. Haenggeli, Y.H. Huang, D.E. Bergles, L. Jin, M. Dykes
Hoberg, S. Vidensky, D.S. Chung, S.V. Toan, L.I. Bruijn, Z.Z. Su, P. Gupta, P.B. Fisher, Beta-lactam
antibiotics offer neuroprotection by increasing glutamate transporter expression, Nature 433
(2005) 73-77.
T. Storck, S. Schulte, K. Hofmann, W. Stoffel, Structure, expression, and functional analysis of a
Na(+)-dependent glutamate/aspartate transporter from rat brain, Proc Natl Acad Sci U S A 89
(1992) 10955-10959.
B. Sung, G. Lim, J. Mao, Altered expression and uptake activity of spinal glutamate transporters
after nerve injury contribute to the pathogenesis of neuropathic pain in rats, J Neurosci 23 (2003)
2899-2910.
Y.H. Tai, Y.H. Wang, J.J. Wang, P.L. Tao, C.S. Tung, C.S. Wong, Amitriptyline suppresses
neuroinflammation and up-regulates glutamate transporters in morphine-tolerant rats, Pain 124
(2006) 77-86.
L. Tang, Y. Chen, Z. Chen, P.M. Blumberg, A.P. Kozikowski, Z.J. Wang, Antinociceptive
pharmacology of N-(4-chlorobenzyl)-N'-(4-hydroxy-3-iodo-5-methoxybenzyl) thiourea, a highaffinity competitive antagonist of the transient receptor potential vanilloid 1 receptor, J
Pharmacol Exp Ther 321 (2007) 791-798.
L. Tang, P.K. Shukla, L.X. Wang, Z.J. Wang, Reversal of morphine antinociceptive tolerance and
dependence by the acute supraspinal inhibition of Ca(2+)/calmodulin-dependent protein kinase
II, J Pharmacol Exp Ther 317 (2006) 901-909.
V.L. Tawfik, M.R. Regan, C. Haenggeli, M.L. Lacroix-Fralish, N. Nutile-McMenemy, N. Perez, J.D.
Rothstein, J.A. DeLeo, Propentofylline-induced astrocyte modulation leads to alterations in glial
glutamate promoter activation following spinal nerve transection, Neuroscience 152 (2008)
1086-1092.