856 Biol. Pharm. Bull. 39, 856–862 (2016) Vol. 39, No. 5 Regular Article Effects of Adjuvant Analgesics on Cerebral Ischemia-Induced Mechanical Allodynia Wataru Matsuura, Shinichi Harada, and Shogo Tokuyama* Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kobe Gakuin University; 1–1–3 Minatojima, Chuo-ku, Kobe 650–8586, Japan. Received December 21, 2015; accepted January 27, 2016 Central post-stroke pain (CPSP), a potential sequela of stroke, is classified as neuropathic pain. Al- though we recently established a CPSP-like model in mice, the effects of adjuvant analgesics as therapeutic drugs for neuropathic pain in this model are unknown. Hence, the aim of the present study was to assess the usefulness of our model by evaluating the effects of adjuvant analgesics used for treating neuropathic pain in this mouse model of CPSP. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanical allodynia was measured after BCAO using the von Frey test. The mechanical allodynia was significantly increased on day 3 after BCAO compared with that during the pre-BCAO assessment. BCAO-induced mechanical allodynia was significantly decreased by intraperito- neal injections of imipramine (a tricyclic antidepressant), mexiletine (an antiarrhythmic), gabapentin (an an- tiepileptic), or a subcutaneous injection of morphine (an opioid receptor agonist) compared with that follow- ing vehicle treatment in BCAO-mice. By contrast, milnacipran (a serotonin and norepinephrine reuptake in- hibitor), paroxetine (selective serotonin reuptake inhibitor), carbamazepine (antiepileptic), and indomethacin (nonsteroidal anti-inflammatory drug) did not affect the BCAO-induced mechanical allodynia. Our results show that BCAO in mice may be useful as an animal model of CPSP. In addition, BCAO-induced mechanical allodynia may be suppressed by some adjuvant analgesics used to treat neuropathic pain. Key words central post-stroke pain; global ischemia; allodynia; neuropathic pain Pain may occur with actual substantial or even potential may be useful as a mouse model of CPSP. In the clinic, CPSP histologic lesions, producing uncomfortable sensory and emo- is usually treated with adjuvant analgesics, such as antidepres- tional experiences, according to the definition by the Inter- sants, antiepileptogenics and narcotic analgesics, based on the national Association for the Study of Pain. One type of pain, treatment of neuropathic pain or surgery.4,9–12) However, the neuropathic pain, is caused by dysfunctional peripheral or effect of adjuvant analgesics on the BCAO-induced pain is central nerves and results in the appearance of allodynia and unclear. hypersensitivity to pain.1,2) Cerebral stroke patients may expe- In the present study, to determine the usefulness of our rience several types of pain, including musculoskeletal pain, BCAO-induced mouse model of CPSP, we evaluated the thera- headache, and neuropathic central post-stroke pain (CPSP). peutic potential of clinically relevant neuropathic pain drugs Among the different types of neuropathic pain, CPSP is an as adjuvant analgesics in this model. especially intractable condition to treat. CPSP is associated with various symptoms, such as burning and lancinating pain, MATERIALS AND METHODS and the incidence of CPSP after cerebral stroke is reportedly 1–14%.3,4) In clinical studies, the risk factors associated with Animals All experimental procedures were approved by CPSP are the degree of brain infarction, sexual dimorphism, the ethics committee for animals at Kobe Gakuin University alcohol intake, depression anamnesis, statin use, dyslipidemia, (approval number: 14–21). The experiments were performed diabetes, and peripheral vascular disorder.5) The pathogenic on male ddY mice (5 weeks old, 25–30 g) obtained from SLC mechanisms for CPSP and neuropathic pain appear similar at (Shizuoka, Japan). The animals were housed at 23–24°C with the point of excitation of primary afferent fibers and for epi- a 12-h light–dark cycle (lights on at 8:00 a.m.). Food and water static or central excitation or suppression pathway disorders. were available ad libitum. The present study was conducted in Recently, we established a CPSP-like animal model by ex- accordance with the Guiding Principles for the Care and Use perimentally inducing focal (middle cerebral artery occlusion of Laboratory Animals approved by the Japanese Pharmaco- model) or global cerebral ischemia (bilateral carotid artery oc- logical Society. clusion model; BCAO).6,7) In our BCAO model of global isch- Animal Model of Global Cerebral Ischemia Transient emia, pain thresholds were significantly decreased in response global cerebral ischemia was induced by occlusion of the BCA to mechanical and thermal stimulation to the hind limbs com- in mice as described previously.7,13) Briefly, mice were anes- pared with those of sham-operated animals. These decreased thetized with pentobarbital (60 mg/kg). The rectal temperature pain thresholds were associated with ischemic neuronal dam- was maintained at 37±0.5°C with a heating blanket (FH-100; age. In a previous study, we showed that the regulation of Unique Medical, Osaka, Japan) equipped with a small animal BCAO-induced CPSP may involve alterations in the signaling heat controller (ATC-101B; Unique Medical). The bilateral of astrocyte free fatty acid receptor 1, also known as GPR40, common carotid arteries were occluded for 30 min using Su- a receptor of long-chain fatty acids.8) Thus, this BCAO model gita standard aneurysm clips (Mizuho Ikakogyo Co., Ltd., * To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan Vol. 39, No. 5 (2016) Biol. Pharm. Bull. 857 Tokyo, Japan). Sham-operated mice were subjected to the Effect of Antiepileptogenic Drugs on the Mechanical Al- same procedure as above without occlusion of the BCA. The lodynia on Day 3 after Global Cerebral Ischemia On day final number of mice used for each drug treatment is stated in 3 after BCAO, the increase in the number of escape behaviors the figure legends. was completely blocked by the administration of the calcium Drugs Imipramine hydrochloride (Sigma-Aldrich, St. channel α2–δ ligand gabapentin (30 mg/kg) in a dose-depen- Louis, MO, U.S.A.), milnacipran hydrochloride (Toronto dent manner [Fig. 2A, drug×time interaction: F(15, 90)=8.82, Research Chemicals, Toronto, Canada), gabapentin (Sigma- p<0.01, drug effect: F(3, 90)=111.19, p<0.01, time effect: F(5, Aldrich), morphine hydrochloride (Takeda Pharmaceutical 90)=42.05, p<0.01] but not by the sodium channel inhibitor Company Limited, Osaka, Japan) and mexiletine hydrochlo- carbamazepine (20 mg/kg) [Fig. 2B, drug×time interaction: ride (Sigma-Aldrich) were dissolved in saline. Carbamazepine F(10, 24)=5.54, p<0.01, drug effect: F(2, 24)=387.40, p<0.01, (Sigma-Aldrich) was dissolved in saline with 1% Tween 20 time effect: F(5, 24)=26.15, p<0.01]. and 2% dimethyl sulfoxide. Paroxetine hydrochloride (Toronto Effect of an Antiarrhythmic Drug on the Mechanical Research Chemicals) was dissolved in saline containing 0.75% Allodynia on Day 3 after Global Cerebral Ischemia The methanol. Indomethacin (Sigma-Aldrich) was dissolved in 1% BCAO-induced mechanical allodynia was dose-dependently carboxymethyl cellulose sodium. and significantly suppressed (30 mg/kg) by treatment with the Drug Administration The BCAO-mice were treated sodium channel blocker mexiletine (10 or 30 mg/kg) [Fig. 3, with a single injection (intraperitoneal; i.p.) of imipramine drug×time interaction: F(15, 72)=13.81, p<0.01, drug effect: (5, 20 mg/kg), milnacipran (30 mg/kg), paroxetine (40 mg/kg), F(3, 72)=57.16, p<0.01, time effect: F(5, 72)=40.14, p<0.01]. gabapentin (10, 30 mg/kg), carbamazepine (20 mg/kg), mexi- Effect of a Narcotic Analgesic on the Mechanical Al- letine (10 or 30 mg/kg), indomethacin (10 mg/kg), or vehicle lodynia on Day 3 after Global Cerebral Ischemia The (0.1 mL/10 g body weight); morphine (1 or 10 mg/kg) was ad- strong opioid analgesic morphine (1 or 10 mg/kg) dose- ministered subcutaneously (s.c.). The doses of all drugs were dependently and significantly decreased the BCAO-induced selected based on the results of previous reports.14,15) mechanical allodynia [Fig. 4, drug×time interaction: F(15, Assessment of Mechanical Allodynia Mechanical allo- 72)=11.57, p<0.01, drug effect: F(3, 72)=46.61, p<0.01, time dynia was evaluated using von Frey filaments (Neuroscience effect: F(5, 72)=25.41, p<0.01]. Inc., Tokyo, Japan) as previously described.8) Mice were Effect of a Nonsteroidal Anti-inflammatory Drug placed on a 5×5 mm wire mesh grid floor, covered with an (NSAID) on the Mechanical Allodynia on Day 3 after opaque cup to avoid visual stimulation, and allowed to adapt Global Cerebral Ischemia The NSAID indomethacin for 2–3 h prior to testing. The von Frey filament was then ap- (10 mg/kg) had no effect on the BCAO-induced mechani- plied to the middle of the plantar surface of the hind paw with cal allodynia [Fig. 5, drug×time interaction: F(10, 48)=4.51, a weight of 0.4 g. On the indicated days, withdrawal responses p<0.01, drug effect: F(2, 48)=83.43, p<0.01, time effect: F(5, following hind paw stimulation were measured 10 times, and 48)=21.47, p<0.01]. mechanical allodynia was defined as an increase in the num- ber of withdrawal responses to the stimulation. On day 3 after DISCUSSION BCAO, the von Frey test was performed on the BCAO mice at 10, 20, 30, and 60 min after each drug treatment. CPSP can be defined as one of the central neuropathic Statistical Analysis All data were analyzed using two- pain conditions occurring after cerebral stroke in which the way ANOVA followed by Tukey’s test, and are presented as affected areas are those body parts corresponding to cerebro- the mean±standard error of the mean (S.E.M.).