doi: 10.1111/fcp.12246 ORIGINAL ARTICLE
Neither nefopam nor acetaminophen can be used as postoperative analgesics in a rat model of ischemic stroke Maud Petraulta,b*, Sophie Gautiera,b,c, Vincent Berezowskia,d, Thavarak Ouka,b,c, Michele Bastidea,b, Olivier Petraulta,d, Regis Bordeta,b,c Inserm U1171 – Degenerative and Vascular Cognitive Disorders, Lille F-59000, France Univ. Lille, Lille F-59000, France c Department of Medical Pharmacology, CHU Lille, Lille F-59000, France d Univ. Artois, Lens F-62300, France a b
Keywords analgesia, brain ischemia, neuroprotection, pain
Received 3 June 2016; revised 6 September 2016; accepted 27 September 2016
*Correspondence and reprints:
[email protected]
ABSTRACT
Analgesics such as opioid agonists are usually not given during the postoperative phase of experimental stroke because they are susceptible to interfere with the evaluation of neuroprotective therapies. Here, we investigate the potential of acetaminophen and nefopam, two nonopioid analgesic drugs, to exert an analgesic effect without inducing neuroprotection in a murine model of ischemic stroke. We demonstrate that acetaminophen (200 mg/kg, PO) induces a significant decrease in the infarct volume, particularly in the cortex (VEHICLE: 200.1 mm3 vs. ACETAMINOPHEN: 140.9 mm3, P < 0.05), while nefopam (2, 20 or 40 mg/kg, IM), administered at the end of middle cerebral artery occlusion (MCAO), do not influence the infarct size (VEHICLE: 268.6 mm3 vs. NEFOPAM 2: 248.8 mm3, NEFOPAM 20: 250.6 mm3 and NEFOPAM 40: 215.9 mm3, P > 0.05). Moreover, we find that nefopam administration (20 mg/kg, IM) in the acute postoperative phase do not change the level of neuroprotection induced by MK801 (3 mg/kg, IV), a well-known neuroprotectant (VEHICLE: 268.6 mm3 vs. MK801: 194.4 mm3 and vs. MK801 + NEFOPAM 20: 195.2 mm3). On the other hand, although nefopam induces analgesia in healthy animals, it is not the case when administered during MCAO (behavior scores at 5 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.6, P < 0.5; IR: 0.40 vs. IR + NEFOPAM 20: 0.67, P > 0.05). Our data suggest that neither acetaminophen nor nefopam can be used as analgesic agents to meet the needs of limiting rodent pain and distress during experimental stroke surgery.
INTRODUCTION Recent guidelines in France and Europe aim at reducing the use of animals in biomedical research, as well as animal stress and pain inflicted during experimentation (Decree number 2013-118 of the official journal of the French Republic, issued on February 1st, 2013). Apart from euthanasia issues, these regulations entail an improvement of experimental anesthesia and analgesia, by means of drugs that should be compatible with each other regarding their respective primary effects, but also
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the pathophysiological paradigm for disease modeling and therapy. Analgesics such as opioid agonists are usually not given during the postoperative phase of experimental stroke because they are susceptible to have a protective effect on the ischemic brain, interfering with the evaluation of neuroprotective therapies, which now requires a growing set of behavioral testing for functional assessment. The latter is very likely to be biased if the tested animals are both already neuroprotected by the analgesic drug and still suffering pain from the surgical and anesthetic procedures. te Francßaise de Pharmacologie et de The rapeutique ª 2016 Socie Fundamental & Clinical Pharmacology 31 (2017) 194–200
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As a surgical procedure is needed for the onset of a global or focal cerebral ischemia in rodents (MCAO: middle cerebral artery occlusion), analgesia should be performed during the postoperative phase in which sensitivity to pain stimulation has been increased by the preceding MCAO [1]. Opioid agonists or nonsteroidal anti-inflammatory drugs have been previously reported to induce neuroprotection, rendering them inappropriate for use in stroke models [2,3]. As inflammation is a hallmark of ischemic stroke pathophysiology, we chose acetaminophen and nefopam as two nonopioid analgesic drugs having a low anti-inflammatory effect, which minimizes the risk of interference with the neuroprotective action of candidate substances. Acetaminophen is known to exert a central antinociceptive effect, although its mechanism of action remains elusive [4]. Nefopam has proved efficacy in reducing thermal hypersensitivity after acute or postoperative pain in rats [5] and in antagonizing N-methylD-aspartate (NMDA) receptor [6]. Because both drugs have not been challenged for neuroprotection, we examined their effect on infarct size in a rat model of cerebral ischemia, in parallel to their analgesic efficacy in the postoperative period. EXPERIMENTAL PROCEDURE Animal and drug administration All experiments were performed in accordance with the European Community legislation (2010/63/UE). The local Ethics Committee approved the experiments (CEEA no 75). All animals were adult male Wistar rats (Janvier SAS, Le Genest-St-Isle, France) weighing 280– 330 g and randomly assigned to MCAO or control groups. Animals were housed under controlled laboratory conditions, with a 12-h dark cycle, a temperature of 21 2 °C and a humidity of 60 to 70%. The animals had ad libitum access to standard chow and tap water. Three experimental protocols were conducted during this study. The first protocol aimed at studying the modulation of infarct volumes by both analgesics. For that purpose, all rats were subjected to a 60-min middle cerebral artery (MCA) occlusion followed by a 24-h period of reperfusion before sacrifice. In the first step, acetaminophen (200 mg/kg) or its vehicle (methylcellulose 0.5%) was orally administered (1 mL) to the rats at the end of MCA occlusion. In the second step, nefopam (2–20 or 40 mg/kg) or its vehicle (saline 0.9%) was administered by te Francßaise de Pharmacologie et de The rapeutique ª 2016 Socie Fundamental & Clinical Pharmacology 31 (2017) 194–200
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intramuscular (IM) route at the end of MCA occlusion. For this part of the study, eight animals were obtained in each group. In the second protocol, the effect of the neuroprotective compound MK801 was assessed when nefopam was administered or not as an analgesic procedure. For this purpose, MK801 (3 mg/kg), dose frequently used in experimental neuroprotective research [7,8], or its vehicle (saline 0.9%) was administered intravenously 3 min before MCAO to rats receiving or not nefopam (20 mg/kg, IM) at the end of the MCAO. Control rats received vehicles of MK801 (saline 0.9%) and nefopam at the same time and route. Eight animals per group were performed in this protocol. The third protocol has examined the analgesic potential of nefopam (20 mg/kg, IM) through a pain test, known as the formalin test. This test was performed on either healthy animals or ischemic animals, with four animals per group. MCA occlusion model After anesthesia with chloral hydrate intraperitoneal administration (300 mg/kg), a rectal probe was inserted and body temperature was maintained at 37 0.5 °C with a heating lamp. The ostium of the right MCA was occluded intraluminally, as previously described [9]. The right carotid arteries were exposed through a midline cervical incision and the common carotid and external carotid arteries were ligated with a silk suture. The pterygopalatine artery was exposed (by developing a plane alongside the internal carotid artery) and then ligated at its origin with a fine silk. An aneurysm clip was placed across internal carotid artery, and an arteriotomy was made in the common carotid artery stump, allowing the introduction of a 4/0 monofilament nylon suture with its tip rounded by flame heating. Once the suture was in place, the aneurysm clip on the internal carotid artery was removed. The suture was gently advanced into the internal carotid artery and passed into the intracranial circulatory system as far as in the narrow lumen at the start of the MCA. Mild resistance to this advancement indicated that the intraluminal occluder had entered the anterior cerebral artery. After 60 min, the suture was carefully removed, until its tip was blocked by a ligature placed on common carotid artery (to allow reperfusion). The animals were placed in a cage to recover from anesthesia at room temperature and were then allowed to eat and drink ad libitum.
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Infarct volume measurement Twenty-four hours after reperfusion, rats were killed with an overdose of pentobarbital (200 mg/kg, ip). Brains were rapidly removed and placed in ice-cold isopentane solution, frozen and coronally dissected into 50-lm-thick slices on a cryostat at 12 levels in 1-mm steps, according to the Paxinos and Watson stereotaxic atlas. Sections were stained by cresyl fast violet. The unstained area of the brain was defined as the infarct zone. Infarcted cortical and subcortical areas and hemispheric areas were calculated separately for each coronal slice using image analysis software (ImageJ) after digitization. Next, infarct volumes (total, cortical and subcortical) and hemispheric volumes (in mm3) were calculated by the summing the respective areas for all sections for all animals and the distance between them. Lastly, the infarct volumes were corrected for the brain edema effect using the following equation: corrected infarct volume = total infarct volume (left hemisphere volume/right hemisphere volume). Formalin test (pain test) Pain assessment was performed by formalin test for 1 h and scored for each minute period. Formalin (50 lL) was injected subcutaneously into the plantar surface of the right hind paw while the rat was restrained manually. Behaviors were observed 2 h after MCA, during 1 h. The behavior scores were determined as described by Dubuisson and Dennis [10]: 0 = normal weight bearing the injected paw; 1 = lameness during locomotion or resting the paw lightly on the floor; 2 = elevation of the injected paw so that at most the nails touch the floor; and 3 = licking or bite of the injected paw. To clarify the results, the scores were grouped into three time points: 5, 30 and 60 min after formalin injection. This test was performed on either healthy animals or ischemic animals, and their reaction was scored every minute for 1 h and pooled at 5, 30, and 60 min. Healthy animals underwent no anesthesia and no surgery and were considered as controls. The healthy + nefopam group was carried out in the same conditions, but were injected with nefopam at 20 mg/kg (IM) at 1 h before the beginning of the formalin test. Statistical analysis All values were expressed as mean standard error of the mean (SEM). Continuous variables were compared with a one-way ANOVA followed by a post hoc
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Kruskal–Wallis test if variance analysis was significant, or with a t-test analyze. A value of P < 0.05 indicated statistical significance. RESULTS Effect of acetaminophen on infarct volume Administration of acetaminophen induced a significant decrease in total and cortical infarct volumes as compared to the vehicle-treated group (total: VEHICLE: 268.6 mm3 vs. ACETAMINOPHEN: 199.4 mm3, cortical: VEHICLE: 200.1 mm3 vs. ACETAMINOPHEN: 140.9 mm3, P < 0.05). In contrast, subcortical volumes were similar in both groups (VEHICLE: 68.51 mm3 vs. ACETAMINOPHEN: 58.49 mm3, P = 0.09) (Figure 1). Mortality due to anesthesia was observed in each group, 27% in vehicle-treated group (3/11) and 20% in acetaminophen-treated group (2/10). Dose-related effect of nefopam on infarct volume At the dose of 2 or 20 mg/kg, nefopam did not induce any significant change in total, cortical or subcortical corrected volumes (total: VEHICLE: 268.6 mm3 vs. NEFOPAM 2: 248.8 mm3 and NEFOPAM 20: 250.6 mm3, P = 0.14; cortical: VEHICLE: 200.1 mm3 vs. NEFOPAM 2: 181.9 mm3 and NEFOPAM 20: 186.1 mm3, P = 0.32; subcortical: VEHICLE: 68.51 mm3 vs. NEFOPAM 2: 65.95 mm3 and NEFOPAM 20: 64.51 mm3, P = 0.53). At the dose of 40 mg/kg, a decrease was observed in the infarct volume, particularly at the cortical level, but found not significant (cortical: VEHICLE: 200.1 mm3 vs. NEFOPAM 40: 153.3 mm3, P = 0.07) (Figure 2). Following
Figure 1 Effect of acetaminophen on total, cortical and subcortical brain infarct volumes after MCAO. Volumes are corrected for edema and expressed in mm3 (Mean SEM). The means scores were significantly different according to a t-test. *P < 0.05 (n = 8 animals per group). NS, nonsignificant. te Francßaise de Pharmacologie et de The rapeutique ª 2016 Socie Fundamental & Clinical Pharmacology 31 (2017) 194–200
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these results, the dose of 20 mg/kg was chosen to achieve the following goals: (i) to verify the absence of change in the neuroprotective effect of a reference compound, (ii) to assess the analgesic potential of nefopam in our MCAO model. Mortality due to anesthesia was observed in each group, 27% in vehicletreated group (3/11) and 20% in nefopam-treated group (2 of 10 for each dose). Effect of nefopam administration on MK 801-induced neuroprotection MK801 was found neuroprotective as observed in the significant (P < 0.05) decrease in total and cortical infarct volumes, as compared to vehicle-treated rats. Administration of nefopam as an analgesic at the end of the MCAO did not change the effect of MK801 on infarct volumes. Indeed, total and cortical infarct volumes remained significantly different in MK801 + nefopam-treated rats, from the ones measured in vehicle-treated rats, and were not different from volumes measured in rats treated with MK801 alone (total: VEHICLE: 268.6 mm3 vs. MK801: 194.4 mm3 and vs. MK801 + NEFOPAM 20: 195.2 mm3, P = 0.004; cortical: VEHICLE: 200.1 mm3 vs. MK801: 135.4 mm3 and vs. MK801 + NEFOPAM 20: 137.6 mm3, P = 0.007; subcortical: VEHICLE: 68.51 mm3 vs. MK801: 59.00 mm3 and vs. MK801 + NEFOPAM 20: 57.57 mm3, P = 0.06) (Figure 3). Mortality due to anesthesia was observed in each group, 27% in vehicle-treated group (3/11) and 36% in MK801-treated group (4/12) and 38% in MK801-nefopam-treated group (5/13).
Analgesic effect of nefopam As a first step, we studied the pain felt after formalin injection for 1 h in healthy animals treated or not by nefopam. A significant difference in behavior scores was observed between healthy and healthy + nefopam groups at 5 and 30 min after formalin injection (5 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.6, P = 0.02; 30 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.75, P = 0.04) (Figure 4a). No significant difference was found at 60 min after formalin injection between both groups (HEALTHY: 2.15 vs. HEALTHY + NEFOPAM 20: 1.7, P = 0.87). In the second step, we performed the formalin test on ischemic animals (I/R) treated or not with nefopam. This test was carried out 1 h after administration of nefopam. No significant difference was observed at 5, 30 and at 60 min after formalin injection (5 min: IR: 0.40 vs. IR + NEFOPAM 20: 0.67, P = 0.5; 30 min: IR: 0.20 vs. IR + NEFOPAM 20: 1.47, P = 0.5; 60 min: IR: 1.67 vs. IR + NEFOPAM 20: 2.0, P = 0.3) (Figure 4b). No mortality was observed in this part of the study. DISCUSSION In this study, we demonstrate that acetaminophen, at the dose recommended to induce an analgesic effect in experimental animals, exerts a neuroprotective effect in the model of MCAO excluding its use as analgesic drug in stroke model. In contrast, even though nefopam induces a slight decrease at the highest dose, at the dose of 20 mg/kg, it induces neither neuroprotective effect nor change in neuroprotective effect of a
Figure 2 Effect of nefopam at the doses of 2, 20 or 40 mg/kg on brain infarct volume after MCAO. Volumes were corrected for edema and expressed in mm3 (Mean SEM); the mean scores were not significantly different according to the one-way analysis of variance. P = 0.14 for total, P = 0.32 for cortical, P = 0.53 for subcortical infarct volumes (n = 8 animals per group). NS, nonsignificant. te Francßaise de Pharmacologie et de The rapeutique ª 2016 Socie Fundamental & Clinical Pharmacology 31 (2017) 194–200
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Figure 3 Neuroprotective effect of MK801 in rats treated or not with nefopam (20 mg/kg, IM). Brain infarct volumes were determined after 1-h MCAO followed by 23 h of reperfusion, corrected for edema and expressed in mm3 (Mean SEM). MK801 was injected 3 min before MCAO (IV, 3 mg/kg). The mean scores were significantly different according to the one-way analysis of variance with Kruskal–Wallis post hoc tests. *P < 0.05 (n = 8 animals per group). NS, nonsignificant.
reference drug. Unfortunately, although nefopam induces analgesic effect on healthy animals, it induces hyperalgia after surgical procedure in comparison with untreated animals. The demonstration of the neuroprotective effect of acetaminophen finds echoes in the cardiology literature, where it is reported for antioxidant and anti-inflammatory properties [11]. In the brain, acetaminophen’s mechanism of action may involve the inhibition of central cyclo-oxygenase COX-2 and COX-3
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enzymes and the modulation of serotonergic system [12,13]. As well, acetaminophen enabled the lowering of oxidative stress-induced brain neuron damage through the inhibition of superoxide anion production, and an increased synthesis of anti-apoptotic protein Bcl-2 [14,15]. These pathways could support acetaminophen’s protective action during ischemia–reperfusion injury. Here, we used an analgesic dose of acetaminophen (200 mg/kg) [16,17], which exerted a neuroprotective effect, as visualized by a reduction in infarct volume. In our hands, this result rules out the use of acetaminophen in experimental stroke. Just as it has not neuroprotective effect per se, we find that administration of nefopam at the end of cerebral ischemia did not change the level of neuroprotection induced by MK801. It is tempting to speculate that nefopam did not interfere with the binding of MK801 to NMDA receptors in the glutamate pathway [18]. Indeed, it has been demonstrated that nefopam act on the glutamatergic pathway via modulations of calcium and sodium channels, that leads to decreased activation of postsynaptic glutamatergic receptors such as NMDA receptors [13]. To end, our observation suggests that an acute administration of nefopam at a dose of 20 mg/kg may induce postoperative analgesia without masking the pharmacological effect of a neuroprotective agent such as MK801. However, one cannot rule out a possible interaction with less potent NMDA receptor antagonist. At the dose of 20 mg/kg, nefopam is known to induce analgesia in pain models [5,6]. We chose to administrate nefopam at unique dose to as it is currently performed in neuroprotection studies. By injecting nefopam at the end of the surgery, we intended to
Figure 4 Pain assessment in healthy or ischemic rats treated or not with nefopam (20 mg/kg, IM). Pain scores (ranging from 0 to 3) were pooled at 5, 30 and 60 min after formalin injection. Formalin test was performed on healthy animals (a) and on I/R animals (b) treated or not with nefopam. The mean scores were significantly different according to the t-test. *P < 0.05 (n = 4 animals per group). NS, nonsignificant. te Francßaise de Pharmacologie et de The rapeutique ª 2016 Socie Fundamental & Clinical Pharmacology 31 (2017) 194–200
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target the most critical period in terms of pain and distress for operated rodents. To confirm the analgesic effect of nefopam in our model of cerebral ischemia, we used the formalin test at 1 h of nefopam injection. Analgesia was confirmed in healthy animals, but not maintained at the end of the test (e.g., 2 h after nefopam injection). In humans, the plasma half-life of nefopam is estimated to be 5 h, so an acute administration is likely to be sufficient to induce an analgesic period of 12 h. As drug metabolism is faster in rodents than in humans [19], the analgesic effect of nefopam may be less extensive in time. Surprisingly, the analgesic effect of nefopam disappeared in ischemic animals, with a nonsignificant hyperalgesic episode at 30 min of the formalin injection. This observation was all the more puzzling as the baseline perception of pain was much lower in ischemic animals than in healthy ones as described in a previous study [20]. This suggests that the mechanisms involved in the physiopathology of cerebral ischemia may interfere with pain processes, biasing the analgesic effect expected after nefopam injection. In conclusion, we demonstrate here that acetaminophen protects the brain tissue after MCAO, and thus should not be used to induce analgesia in rodents subjected to experimental stroke, as it may prejudice the validation of the potential of new neuroprotectants. Although an analgesic dose of nefopam neither exert neuroprotection nor interfere with the action of the neuroprotectant MK801, it can induce hyperalgia in rats after surgical procedure, which may have detrimental consequences on the functional assessments needed in stroke research [21]. Meeting the criteria for animal ethics and experimental issues remains a difficult challenge that would benefit from extended investigations, including a larger set of analgesic compounds. DISCLOSURE An abstract for this study was presented at the SFPT Annual Meeting in Nancy 19–21 April 2016 under reference PM2-034 page 43. Fundam. Clin. Pharmacol. (2016) 30 S1 1:98. REFERENCES 1 Tsai Y.C., Huang S.J., Chang C.L. et al. The influence of focal ischemic brain injury on tail-flick latency in the rat. Acta Anaesthesiol. Sin. (1994) 32 115–120.
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