NIH Public Access Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

NIH-PA Author Manuscript

Published in final edited form as: Anesthesiology. 2014 December ; 121(6): 1311–1319. doi:10.1097/ALN.0000000000000422.

Cannabinoid Receptor Type 1 Antagonist, AM-251, Attenuates Mechanical Allodynia and Thermal Hyperalgesia after Burn Injury Masashi Ueda, M.D., Ph.D., Hajime Iwasaki, M.D., Shuxing Wang, M.D., Ph.D., Eri Murata, B.S., K.Y. Trudy Poon, M.S., Jianren Mao, M.D., Ph.D., and J. A. Jeevendra Martyn, M.D., F.R.C.A., F.C.C.M. Department of Anesthesiology, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA, Shriners Hospitals for Children® – Boston, Massachusetts, and Harvard Medical School, Boston, Massachusetts (M.U., H.I., S.W., E.M., K.Y.T.P, J.M., J.A.J.M.) and Department of Anesthesiology, Fukui University, Fukui, Japan (EM)

NIH-PA Author Manuscript

Abstract Background—Burn injury causes nociceptive behaviors, and inflammation-related pathologic pain can lead to glial cell activation. Hypothesis tested was that burn injury activates glial cells, and cannabinoid receptor 1 (CB1R) antagonist, AM251, will decrease burn pain. Methods—Anesthetized rats received 0.75cm2 third degree burn on dorsal hind paw. Vehicle or AM251 30 μg intrathecally (older rats, n = 6/group) or, either vehicle, 0.1 or 1.0 mg/kg intraperitoneally (younger rats, n = 6/group), started immediate postburn, was administered for 7 days. Mechanical allodynia and thermal hyperalgesia were tested on ventral paw for 14 days. Microglial and astroglial activity were assessed by immunocytochemistry.

NIH-PA Author Manuscript

Results—Allodynia, observed on burn side from day 1–14, was significantly (p < 0.05) attenuated by intrathecal and intraperitoneal AM251 (1 mg/kg) starting from 3 to 14 days. Hyperalgesia, observed from day 3–12, was completely (p < 0.05) reversed by intrathecal and intraperitoneal AM251 (1 mg/kg). AM251 0.1 mg/kg had no effect. Microglial activity (n = 3/time point) increased (p < 0.05) 18.5 ± 7.5 and 12.3 ± 1.6 (mean +/− SD) fold at 7 and 14 days, respectively. Astroglial activity (n = 4/time point) increased 2.9 ± 0.3 fold at day 7 only. Glial activities were unaltered by AM251. Conclusion—AM251 inhibited nociceptive behaviors after burn even beyond 7-day period of administration. Although many studies have documented the utility of CB1R agonists, this study indicates that endogenous cannabinoids may have an unexpected pronociceptive effect during development of burn pain, explaining why CB1R antagonist, AM251 improves nociceptive behaviors. The decreased nociception with AM251 without altering glial activity indicates that AM251 acts further downstream of activated glial cells.

Corresponding Author: J. A. Jeevendra Martyn, M.D., F.R.C.A., F.C.C.M., Harvard Medical School, Masschusetts General Hospital, Department of Anesthesiology, 51 Blossom St, Room 206, Boston, MA 02114, [email protected], Phone: (617) 726-8807, Fax: (617) 371-4821. COI: The authors declare no competing interests.

Ueda et al.

Page 2

INTRODUCTION NIH-PA Author Manuscript NIH-PA Author Manuscript

The endocannabinoid system refers to a group of neuromodulatory lipids and their receptors that are involved in many physiological processes including pain sensation, appetite, and memory.1–4 Pain-related cannabinoid receptors are of two types, cannabinoid receptor 1 and 2 (CB1R and CB2R), and are located in the central and peripheral nervous systems.3–6 Exogenous cannabinoid agonists have been used to attenuate pain responses in several pain models including inflammatory and neuropathic pain.7,8 More specifically, the CB1R together with N-methyl D-aspartate (NMDA) receptor induce analgesia, although the mechanism underlying this interaction is unclear.8 Thus, CB1R agonist ligands via CB1R influence neuronal excitability by a variety of mechanisms, and these effects are relevant to pain perception and behavior. 6–8 Recent reports, however, showed that global (whole body) CB1R deficient mice, or mice lacking CB1R in dorsal horn inhibitory interneurons were protected from capsaicin-induced mechanical sensitization.9,10 Moreover, preadministration of rimonabant, a CB1R antagonist, resulted in decrease of hyperalgesic and allodynic areas in a human model; there were no changes, however, in the area of acute pain.10 Therefore, an exogenous CB1R antagonist seems to have beneficial effects in pathologic-state-induced chronic nociceptive behaviors. Based on these latter studies,9,10 it seems that endogenous cannabinoids may induce disinhibition of nociceptive transmission in the spinal cord and dorsal horn during pathologic state-induced pain.

NIH-PA Author Manuscript

Allodynia and hyperalgesia are concomitant features of burn injury in humans and is seen both at the site of burn, and at areas in close proximity to the injury.11,12 This pain in humans is exaggerated during procedures (e.g., dressing changes).11,13 Management of burn pain in the perioperative period and in intensive care unit has been challenging.14–15 Burn injury-induced pain probably has inflammatory and neuropathic components. Although previous studies in rodent pain models have documented alterations in expression of NMDA, protein kinase C γ, and/or opioid receptors,16–18 the basic mechanisms underlying burn-induced pain and its treatment have received very little attention. Recent studies on nonburn induced neuropathic pain conditions suggest that astroglia and microglia are activated in pathologic pain states.3–6 Although ~50% of astroglia and ~80% of microglia in rat spinal cord have immunoreactivity to CB1R, as to how endogenous cannabinoids and exogenous cannabinoid ligands modulate pain, and the activity of astroglia and microglia in burn injury-induced pain remains unknown. In this study, using rat hindpaw burn injury model of thermal hyperalgesia and mechanical allodynia,16–18 we tested the hypothesis that burn injury is associated with increased activity of glial cells in the spinal cord, and that administration of CB1R antagonist, AM-251, will beneficially modulate the burn injuryassociated hyperalgesia and allodynia.

MATERIALS AND METHODS Experimental animals and drugs Adults male Sprague Dawley rats of age 8–9 weeks weighing 300–350 gm, or younger male Sprague Dawley rats of age 4–5 weeks weighing 140–150 gm were used. Adult and younger animals were tested because of our interest in both adult and pediatric burn pain. Rats were housed in individual cages in one of the Massachusetts General Hospital, Boston, Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 3

NIH-PA Author Manuscript

Massachusetts animal care facilities, which was artificially illuminated from 7:00 A.M. to 7:00 P.M., with water and food pellets available ad libitum. The protocol was approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. CB1R antagonist, AM-251, was purchased from Sigma-Aldrich (St. Louis, MO). AM-251 was dissolved in dimethyl sulphoxide (DMSO) (70%). For all in vivo nociceptive behaviorstudies, we chose a sample size of n = 6 for each group based on prior experience in this model.16–18 Burn injury

NIH-PA Author Manuscript

The model has been previously described by us,16–18 and consists of a localized burn injury to the dorsum of the hind paw with demonstration of nociceptive behaviors on the plantar aspect of paw. Prior to infliction of burn injury, the rats were anesthetized with pentobarbital 50 mg/kg. A third-degree burn injury was produced by immersion of only the dorsum of hindpaw into 85°C water for 12 s. The burn-injured area was limited to an area of approximately 0.75 cm2 by pressing the dorsum of hindpaw firmly into the hot water through a holed plastic template. This third degree burn injury most reliably produced postinjury nociceptive behaviors including thermal hyperalgesia and mechanical allodynia in both older and younger rats.16–18 Sham injury was produced by immersing the dorsal part of the right hindpaw into a warm water bath (35°C) for 12 s. Intrathecal catheter implantation One week before burn injury to adult rats, a polyethylene-10 catheter was implanted intrathecally under sevoflurane anesthesia administered via a commercial vaporizer with exit port attached to a hollow tube, which in turn was connected to a funnel that was applied to the snout of the rat. The catheter was inserted into each adult rat at the level of lumbar 1–2 as described by Yaksh and Rudy.19 Those animals that exhibited neurological deficits after intrathecal catheter implantation were excluded from the experiments. AM-251 or vehicle (DMSO) was delivered via the intrathecal catheter in a total volume of 10 μl followed by a saline flush (10 μl). Although catheters were inserted one week before burn, AM251 or vehicle administration was started after burn injury (Day 0). Following recovery from anesthesia, the rats were returned to their cages.

NIH-PA Author Manuscript

Nociceptive behavioral testing Animals were habituated to the behavioral test environment for two consecutive days prior to burn injury. The adult animals were tested for thermal hyperalgesia and mechanical allodynia preburn and at 1, 3, 5, 7, 10, 12, and 14 days postburn. Nociceptive behaviors in the younger rats were tested on same days except day 10 and 12 after burn. For testing thermal hyperalgesia, each plantar surface of a rat’s hindpaw on the ipsilateral and contralateral surface was exposed to a beam of radiant heat through a transparent perplex glass surface as described by Hargreaves et al.20 The withdrawal latency was averaged from at least two trials separated by 2-min intervals, and the cutoff was at 20 s to avoid tissue damage. Mechanical allodynia was determined on ipsilateral and contralateral sides on the same days described above. For examining mechanical allodynia, each rat was placed on a metal mesh

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 4

NIH-PA Author Manuscript

floor covered with a plastic box ( 15 × 15 × 18 cm), and allowed to habituate for 30 min. Mechanical stimulation resulting from the bending force of a von Frey filament was applied to the planter surface of each hindpaw. Each trial consisted of five applications of a von Frey filament given every 4 s, and the cutoff force was 10 gm. Brisk foot withdrawals (at least three times of five applications) in response to von Frey filament stimulation were considered positive. Depending on the initial response, subsequent filaments were applied in the order of either descending or ascending force to determine the threshold force.21,22 Drug treatment

NIH-PA Author Manuscript

The older burn- and sham-burned rats received either CB1R antagonist (AM-251 30 μg) or vehicle DMSO intrathecally daily for 7 days via the previously implanted intrathecal catheter. The dose of intrathecal A251 was based on previously reported dose of AM251 administered directly into the central venous system.23 The administration was started immediate postburn (day 0). The choice of administration of AM251 versus vehicle to rats was not randomized. The withdrawal threshold to thermal and mechanical stimulation was examined on the ipsilateral and contralateral hindpaw for 14 days after burn or sham burn injury. In view of the difficulty in placing intrathecal catheter in the younger rats, one of three therapeutic regimens (1 mg/kg, or 0.1 mg/kg or vehicle once daily, administered intraperitoneally) was started after the burn and continued for 7 days. These doses were based on prior reports of parenteral administration of AM251 to rodents.24,25 In order to avoid the acute effect of AM-251 or vehicle, the injections of AM251 were performed only after behavioral tests. Another set of younger naïve animals received AM251 (1.0 mg/kg) or vehicle intraperitoneally, and nociceptive behaviors tested on the hind paw at 0–7 days. Immunohistochemistry

NIH-PA Author Manuscript

Immunostaining with glial fibrillary acid protein (GFAP) and ionized calcium-binding adapter molecule-1 (Ibal) as markers of astroglial and microglial activation, respectively, was performed in a separate group of young rats at 3, 7, and 14 days after burn. The effect of treatment with AM-250 or vehicle on the expression of astroglia (using GFAP as a marker26) and of microglia (using Ibal as a marker27) was studied on the burn-injured and contralateral sides. For harvesting of spinal cord segments, the rats were anesthetized with pentobarbital, and the chest was opened. A needle was inserted through the heart into the ascending aorta, which was perfused with saline (200 ml via infusion pump), followed by 4% paraformaldehyde (300 ml via infusion pump). Spinal cord lumbar segments were then excised, post fixed overnight, and kept 3 days in 20% sucrose. The spinal cord samples were sectioned with a cryostat (25 μm thick). All sections for immunohistochemical procedure were treated under the same conditions on the same day to minimize the between-group variability. Sections were blocked with 3% bovine serum in 0.3% Triton X-100 for 1 h at room temperature and incubated over night at 4°C with primary antibody (GFAP from Abcam, Inc., Cambridge, MA; microglial, Iba-1 from Chemicon/Millipore, Billerica, MA 1:2,000 dilution.). The sections were then incubated for 1 h at room temperature with the corresponding fluorotein isothiocyanate (FITC) or cyanine 3- (CY3-) conjugated secondary antibody (1:800 dilution; Chemicon/Millipore). For analyses of immunostained specimens, the sections were randomly selected and images scanned using a Nikon fluorescence microscope. Images of dorsal horn regions were then captured with a CCD Spot camera Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 5

(Diagnostic Instruments, Inc., Sterling Heights, MI) as described previously.22,28 Photoshop program ( Adobe, San Jose, CA) was used for quantitation of expression of Iba1 and GFAP.

NIH-PA Author Manuscript

Statistical Analyses

NIH-PA Author Manuscript

Each group consisted of unique treatment strata (vehicle or AM21) and age strata (older or younger rats). Each rat served as its own control when ipsilateral and contralateral sides were tested at the different time-points of testing. All data were analyzed by two-way ANOVA using the method of generalized linear model with repeated measures. Statistical significance was declared at p < 0.05, repeated measure was accounted for the fitted dataset. The effect of treatment with AM251 or no treatment was examined on the contralateral side using pain response as the outcome. If no statistical significant result was observed, the untreated contralateral side was selected as the baseline (comparator) for further assessment over time. Subsequent analyses were focused on assessing the treatment effect, where the burned (ipsilateral) side was examined and the untreated ipsilateral side was used as comparator. Post-hoc analyses were performed when significant main effect was observed. Subsequent analysis by study day on treatment effect was further examined. These were analyzed in the same manner as the primary analyses. Statistical significance was declared with corresponding Bonferroni-adjusted alpha value by the number of pairs (two-sided test). In addition, comparisons on pain responses between older and younger rats were made. All the analyses were performed using SAS® version 9.3 (Cary, NC). The fold change in glial activities is reported as mean +/− SD. The persons performing the animal or immunohistochemistry experiments were not blinded to the experimental condition of the preparation.

RESULTS Nociceptive Behaviors

NIH-PA Author Manuscript

Older Rats: After placement of intrathecal catheters, six rats demonstrated some form of neurological deficits. These rats were excluded from study and not given burn or sham injury. No rats were lost after initiation of burn or sham burn injury. There were no differences in nociceptive responses among vehicle-treated contralateral and AM251-treated contralateral sides of burned-injured mice (two measurements/rat, 6 rats/group at each measured day). The adult burned rats receiving vehicle treatment showed significant (p < 0.05) mechanical allodynia (Von Frey testing) on the ipsilateral side of burn compared to contralateral side at all experimental days (starting at day 1 and persisting up to day14, fig. 1). In the adult burned rats receiving AM-251 intrathecally, mechanical allodynia that was seen at day 1 postburn was significantly attenuated by day 3. This beneficial effect of AM251 on allodynia was maintained until day 14 after burn even after termination of AM-251 on day 7 (fig. 1). When thermal hyperalgesia (thermal withdrawal response) was tested in adult rats, statistically significant (p < 0.05) nociceptive behavior was observed in the vehicle group on the ipsilateral compared to contralateral side of burn at day 3, 5, 7, 10, and 12; thermal hyperalgesia had spontaneously recovered by day 14 (fig. 2). In the rats receiving AM251

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 6

intrathecally, thermal hyperalgesia was completely reversed on the ipsilateral side of burned rats at all times that had previously demonstrated thermal hyperalgesia (fig. 2).

NIH-PA Author Manuscript NIH-PA Author Manuscript

Younger Rats: A group of younger naïve animals (two measurements/rat, four rats/group) that received 1 mg/kg AM251 showed no differences in pain responses or gait compared to vehicle treated animals. In other words, AM251 had no beneficial or harmful effects on pain response or gait in nonpathologic states. Statistical difference was not observed in the nociceptive responses in the vehicle-treated contralateral side and AM251-treated (1 mg/kg or 0.1 mg/kg) contralateral sides. In the rats with burn receiving intraperitoneal vehicle treatment (two measurements/rat, six rats/group for each time period), significant (p < 0.05) mechanical allodynia was observed on the ipsilateral side throughout the experimental period (day 1–14, fig. 3). In the group receiving 1 mg/kg intraperitoneally (two measurements/rat, six rats/time period), mechanical allodynia was attenuated at day 1, 3, 5, 7, and 14 days after burn compared to contralateral side (fig. 3). Thus, the beneficial effects of AM251 (1mg/kg I.P.) persisted up to day 14 post-burn, even though the drug was stopped at day 7 after burn. The younger rats receiving 0.1mg/kg AM-251 (two measurements/rat, six rats for each time period) demonstrated allodynia on the ipsilateral side, comparable to that of the burned-injured rats in the vehicle group, throughout the observation period of 1– 14 days (fig. 3). When thermal hyperalgesia was tested in the younger rats, the ipsilateral burn side showed exaggerated nociceptive responses at 3, 5, and 7 days (fig. 4). The thermal hyperalgesia exhibited at day 3, 5, and 7 had spontaneously reversed to normal by day 14 (fig. 4). The hyperalgesic responses observed previously at day 3, 5, and 7 were completely reversed by AM-251 (1 mg/kg) treatment (fig. 4). The rats receiving 0.1 mg/kg AM-251 continued to demonstrate thermal hyperalgesia on the ipsilateral side at day 3, 5, and 7 (fig. 4). Immunoreactivity

NIH-PA Author Manuscript

The immunoreactivity studies were performed only in the younger rats at day 3, 7, and 14 days after burn. The Iba1 immunoreactivities between sham burn and contralateral side of burn were not different (n = 3 for burn, n = 3 for sham burn for each time point). After burn injury, the dorsal horn immunoreactivity of Iba1 (microglial activity) on the ipsilateral side significantly (p < 0.05) increased 18.5 +/− 7.5 and 12.3 +/− 1.6 fold compared to contralateral dorsal horn at 7 and 14 days, respectively. The expression of Iba1 between 7 and 14 days did not differ. Representative fluorescence images for each time period demonstrating the activation of Ibal is shown (fig. 5). The immunoreactivity of GFAP reflecting astroglial activity (n = 4 for burn, n = 4 for sham burn at each time point of 3, 7, and 14 days) showed no differences in activities between sham burn and contralateral side of burn. The astroglial activity (GFAP expression) on the ipsilateral side relative to contralateral side showed a 2.9 ± 0.3 fold increase (p < 0.05) at day 7 only (fig. 6). In view of the increased microglial and astroglial activities at day 7 following burn and improvement of nociceptive behaviors when treated with AM251 at day 7, we examined if treatment with AM251 would alter glial activities. The administration of AM-251 to the burned-injured animals did not change the activities (intensities) of Ibal on the ipsilateral and contralateral side when examined at day 7 after burn (fig. 7). Similarly, although the (astroglial) activity

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 7

NIH-PA Author Manuscript

was increased at day 7 after burn, the administration of AM251 (1.0 mg/kg for 7 days) did not change the expression of GFAP on burned, contralateral and also in naïve mice) when compared to vehicle treatment (fig. 6).

Discussion

NIH-PA Author Manuscript

The present study demonstrates that: (1) burn injury to adult rats leads to hyperalgesic and allodynic responses on the ipsilateral side of burn compared to the contralateral side; (2) postburn intrathecal treatment of adult rats with the CB1R antagonist AM-251 results in partial or complete reversal of the altered nociceptive behaviors during and beyond the period of infusion; (3) younger rats with burn injury showed nociceptive responses similar to that of adult animals; (4) interaperitoneal treatment of young rats with AM-251 started immediately after burn also attenuated or reversed the nociceptive (hyperalgesia and allodynia) behaviors induced by burn injury; (5) burn injury results in increase in activity of microglia at day 7 and 14 after burn evidenced by increased Ibal immunoreactivity; (6) The astroglial activity, reflected by GFAP expression, was upregulated only at day 7 after burn, (7) reversal of the nociceptive behaviors was not associated with quantitative changes in the activities of microglia or astroglia; (8) in all rats, the contralateral nociceptive responses were not affected by the administration of AM-251, when compared to preburn and untreated contralateral side nociceptive responses. Although many studies have documented the utility of cannabinoids agonists to attenuate pain, the results of this study indicate that endogenous cannabinoids may have an unexpected pronociceptive effect during development of burn-induced pain. This may explain why inhibition of endogenous CB1R activity by an antagonist, AM-251, results in improvement of nociceptive behaviors.

NIH-PA Author Manuscript

Immunoreactivities of astroglia (by fluorescent GFAP) and microglia (by fluorescent Ibal) were examined at 3, 7, and 14 days after burn. Increased immunoreactivity of microglia was observed at day 7 and 14 following burn. The increased immunoreactivities of microglia following burn injury is consistent with the observations of others that immunoreactivity of microglia are increased in neuropathic syndromes and/or inflammation of central nervous system.28–30 The activity of astroglia was upregulated at day 7 after burn. Nociceptive behaviors, however, were seen at day 3 although astroglial activity was not increased at this time. Although allodynia was observed at day 14, the hyperalgesia had reverted to normal by this time in association with normalization of the increased astroglial activity observed at day 7. This disparate relationship between astroglial expression and pain behaviors has been observed previously in other pain syndromes.31,32 There can be a dissociation between pain and microglial activation also.33,34 Once increased glial activity occurs, the altered activity can persist even after recovery from nociceptive pain behaviors, as observed for microglia and hyperalgesia at day 14.31–34 Thus, altered glial activities may reflect the presence or persistence of a pathologic state rather than reflection of a type of nociceptive behavior. CB1R antagonist, AM-251, has been shown to directly inhibit the increase of glial calcium levels, which can lead to glutamate release and activation of NMDA receptors.35 Thus, inhibition of endogenous C1BR by AM-251 may decrease the activation of calcium-induced activation of NMDA receptors. There is a usually, but not always, a close relationship before glial cell activation and cytokine levels in pathologic pain conditions.36,37 In fact, localized

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 8

NIH-PA Author Manuscript

burn injury, similar to the model described in this study, resulted in activation of cytokines.38 Spinal intrathecal injection antisense oligonucleotides for gp130, a receptor subunit shared by members of the interleukin-6 family of cytokines, attenuated burn-induced pain.38 In view of the close relationship between glial cell expression and cytokine levels,32–33 and the lack of changes in glial expression after administration of AM251, cytokine expression with and without AM251 treatment was not examined in the current study. In our study, the reversal or attenuation of the nociceptive behaviors in the absence of changes in glial cell activities suggests that the actions of AM-251 may not be at the glial level, but further downstream. Pharmacological examples of reversal of deleterious effects of activated signaling proteins without altering their expression has been described previously following burn injury and other pathologic states.39–42 For example, inhibitors of glycogen synthase kinase-3β and inducible nitric oxide synthase reversed the deleterious actions of these signaling proteins without altering their expression.39–42 In other words, the deleterious effects of activated proteins can be modulated without altering their expression but by altering their downstream signaling effects.

NIH-PA Author Manuscript NIH-PA Author Manuscript

AM-251 was effective during intrathecal and intraperitoneal routes. These beneficial effects on nociception had a latent period of onset (2 days), but persisted after termination of AM-251 administration at 7 days. From a clinical perspective, the ability to reverse nociceptive behaviors when administered by interperitoneal route is practically more relevant to the burned patient where intrathecal administration may be more restricted because of the infection potential when drugs are administered into the central nervous system of immunocompromized burned patients. The utility of agonists of CBIR for pain treatment has been documented in many previous studies. As observed in this study, if the use of CB1R antagonists proves to be useful in additional studies, this could be a new strategy to modulate development of acute or chronic burn pain or even burn-induced hyperalgesia. There are several ongoing clinical trials on the use of oral CB1R antagonists to treat various pathologic states (NCT00603109, NCT00588731). These clinical trials are testing the utility of CBIR antagonist for non-pain conditions such as appetite, weight control and cognitive dysfunction. Based on efficacy and safety reports from these trials, the potential use of endocannabinoid antagonists to treat burn-induced pain in humans would be of great interest to burn care physicians and related medical personnel. Thus CBIR antagonists, would be a novel, previously unreported, application to treat pain, specifically burn pain. This bench observation in rats possibly may have almost immediate application to the bedside, provided those clinical trials enumerated above provide proof of safety in humans.

Acknowledgments Supported in part by a grant from the National Institutes of Health GM P50-2500 Project I to JAJM.

References 1. Velayudhan L, Van Diepen E, Marudkar M, Hands O, Suribhatla S, Prettyman R, Murray J, Baillon S, Bhattacharyya S. Therapeutic potential of cannabinoids in neurodegenerative disorders: A selective review. Curr Pharm Des. 2014; 20:2218–30. [PubMed: 23829360]

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 9

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

2. Robson PJ. Therapeutic potential of cannabinoid medicines. Drug Test Anal. 2014; 6:24–30. [PubMed: 24006213] 3. Diezi M, Buclin T, Kuntzer T. Toxic and drug-induced peripheral neuropathies: Updates on causes, mechanisms and management. Curr Opin Neurol. 2013; 26:481–8. [PubMed: 23995278] 4. Yuan S, Burrell BD. Nonnociceptive afferent activity depresses nocifensive behavior and nociceptive synapses via an endocannabinoid-dependent mechanism. J Neurophysiol. 2013; 110:2607–16. [PubMed: 24027102] 5. Zogopoulos P, Vaileiou I, Patsouris E, Theocharis SE. The role of endocannabinoids in pain modulation. Fundam Clin Pharmacol. 2013; 27:64–80. [PubMed: 23278562] 6. Arevalo-Martin A, Garcia-Ovejero D, Sierra-Palomares Y, Paniagua-Torija B, Gonzalez-Gil I, Ortega-Gutierrez S, Molina-Holgado E. Early endogenous activation of CB1 and CB2 receptors after spinal cord injury is a protective response involved in spontaneous recovery. PLoS One. 2012; 7:e49057. [PubMed: 23152849] 7. McDowell TS, Wang ZY, Singh R, Bjorling D. CB1 cannabinoid receptor agonist prevents NGFinduced sensitization of TRPV1 in sensory neurons. Meurosci Lett. 2013; 551:34–8. 8. Palazzo E, Marabese I, de Novellis V, Oliva P, Rossi F, Berrino L, Rossi F, Maione S. Metabotropic and NMDA glutamate receptors participate in the cannabinoid-induced antinociception. Neuropharmacology. 2001; 40:319–26. [PubMed: 11166324] 9. Nadal X, La Porta C, Andreea Bura S, Maldonado R. Involvement of the opioid and cannabinoid systems in pain control: New insights from knockout studies. Eur J Pharmacol. 2013; 716:142–57. [PubMed: 23523475] 10. Pernia-Andrade AJ, Kato A, Witchi R, Nyllas R, Katona I, Fruend T, Watanabe M, Filitz J, Koppert W, Schuttler J, Ji G, Neugebauer V, Marsicano G, Lutz B, Horacio V, Zellhofer HU. Spinal endocannabinoids and CB1, receptors mediate C-fiber-induced heterosynaptic pain plasticity. Science. 2009; 325:760–4. [PubMed: 19661434] 11. Osgood PF, Szyfelbein SK. Management of burn pain in children. Pediatr Clin North Am. 1989; 36:1001–13. [PubMed: 2569179] 12. Atchison NE, Osgood PF, Carr DB, Szyfelbein SK. Pain during burn dressing change in children: Relationship to burn area, depth and analgesic regimens. Pain. 1991; 47:41–5. [PubMed: 1771092] 13. Lilleso J, Hammer NA, Pedersen JL, Kehlet H. Effect of peripheral morphine in a human model of acute inflammatory pain. Br J Anaesth. 2000; 85:228–32. [PubMed: 10992829] 14. Trupkovic T, Kinn M, Kleinschmidt S. Analgesia and sedation in the intensive care of burn patients; Results of a European survey. J Intensive Care Med. 2011; 26:397–407. [PubMed: 21257635] 15. Agarwal V, O’Neil PJ, Cotton BA, Pun BT, Haney S, Thompson J, Kassebaum N, Shintani A, Guy J, Ely EW, Pandharipande P. Prevalence and risk factors for development of delirium in burn intensive care unit patients. J Burn Care Res. 2010; 31:706–15. [PubMed: 20647937] 16. Wang S, Zhang L, Ma Y, Chen L, Tian Y, Mao J, Martyn JAJ. Nociceptive behavior following hindpaw burn injury in young rats: Response to systemic morphine. Pain Med. 2011; 12:87–98. [PubMed: 21143761] 17. Wang S, Lim G, Yang L, Zeng Q, Sung B, Martyn JAJ, Mao J. A rat model of unilateral hindpaw burn injury: Slowly developing rightwards shift of the morphine dose-response curve. Pain. 2005; 116:87–95. [PubMed: 15936884] 18. Song, Li; Wang, S.; Zuo, Y.; Chen, L.; Martyn, JAJ.; Mao, J. Midazolam exacerbates morphine tolerance and morphine-induced hyperactive behaviors in young rats with burn injury. Brain Res. 2014; 1564:52–61. [PubMed: 24713351] 19. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav. 1976; 17:1031–6. [PubMed: 14677603] 20. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988; 32:77–88. [PubMed: 3340425] 21. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988; 33:87–107. [PubMed: 2837713]

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 10

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

22. Mao J, Mayer DJ, Hayes RL, Price DD. Spatial patterns of increased spinal cord membrane-bound protein kinase C and their relation to increases in 14C-2-deoxyglucose metabolic activity in rats with painful peripheral mononeuropathy. J Neurophysiol. 1993; 70:470–81. [PubMed: 8410149] 23. Sink KS, Segovia KN, Nunes EJ, Collins LE, Vemuri VK, Thakur G, Makriyannis A, Slamone JD. Intracerebroventricular administration of cannabinoid CB1 receptor antagonists AM251 and AM4113 fails to alter food-reinforced behavior in rats. Psychopharmacology (Berl). 2009; 206:223–32. [PubMed: 19588124] 24. Mathes CM, Ferrara M, Rowland NE. Selection of a palatable dietary option is not preferentially reduced by cannaboinoid CB1 receptor antagonist AM251 in femail C57B1/6J mice. Pharmacol Biochem Behav. 2009; 94:119–23. [PubMed: 19660493] 25. Bialuk I, Winnicka MM. AM251, cannabinoids receptors ligand, improves recognition memory in rats. Pharmacol Rep. 2011; 63:670–9. [PubMed: 21857077] 26. Dahl D, Crosby CJ, Sethi JS, Bignami A. Glial fibrillary acidic (GFA) protein in vertebrates: Immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies. J Comp Neurol. 1985; 239:75–88. [PubMed: 3900154] 27. Li K, Tan YH, Light AR, Fu KY. Different peripheral tissue injury induces differential phenotypic changes of spinal activated microglia. Clin Dev Immunol. 2013; 2013:901420. [PubMed: 23818916] 28. Molander C, Xu Q, Grant G. The cytoarchitectonic organization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord. J Comp Neurol. 1984; 230:133–41. [PubMed: 6512014] 29. Juknat A, Pietr M, Kozela E, Rimmerman N, Levy R, Gao F, Coppola G, Geshwind D, Vogel Z. Microarray and pathway analysis reveal distinct mechanisms underlying cannabinoid-mediated modulation of LPS-induced activation of BV-2 microglial cells. PLoS One. 2013; 8:e61462. [PubMed: 23637839] 30. Perego C, Fumagalli S, De Simoni MG. Three-dimensional confocal analysis of microglia/ macrophage markers of polarization in experimental brain injury. J Vis Exp. 2013:79. 31. Ducourneau VR, Dolique T, Hachem-Delaunay S, Miraucourt LS, Amadio A, Blaszczyk L, Jacquot F, Ly J, Devoize L, Oliet SH, DAllel R, Mothet JP, Nagy F, Fenelon VS, Voisin DL. Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers. Pain. 2014; 155:275–91. [PubMed: 24120461] 32. Ru-Rong J, Temugin B, Maiken N. Glia and pain: Is chronic pain a gliopathy? Pain. 2013; 154:S10–S28. [PubMed: 23792284] 33. Colburn RW, DeLeo JA, Rickman AJ, Yeager MP, Kwon P, Hickey WF. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J Neuroimmunol. 1997; 79:163–75. [PubMed: 9394789] 34. Leinders M, Knaepen L, De Kock M, Sommer C, Hermans E, Deumens R. Up-regulation of spinal microglia Iba-1 expression persists after resolution of neuropathic pain hypersensitivity. Neurosci Lett. 2013; 554:146–50. [PubMed: 24021808] 35. Navarrete M, Araque A. Endocannabinoids mediate neuron-astrocyte communication. Neuron. 2008; 57:883–93. [PubMed: 18367089] 36. Hanisch UK. Microglia as a source and target of cytokines. Glia. 2002; 40:140–55. [PubMed: 12379902] 37. Guo W, Wang H, Watanabe M, Shimizu K, Zou S, LaGraize SC, Wei F, Dubner R, Ren K. Glialcytokine-neuronal interactions underlying the mechanisms of persistent pain. J Neurosci. 2007; 27:6006–18. [PubMed: 17537972] 38. Summer GJ, Romero-Sandoval EA, Bogen O, Dina OA, Khasar SG, Levine JD. Proinflammatory cytokines mediating burn-injury pain. Pain. 2008; 135:98–107. [PubMed: 17590515] 39. Yasukawa T, Tokunago E, Ota H, Sugita H, Martyn JAJ, Kaneki M. S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance. J Biol Chem. 2005; 280:7511–8. [PubMed: 15632167] 40. Kaneki M, Fukishima Y, Shinozaki S, Fukaya M, Habiro M, Shimizu N, Chang K, Yasuhara S, Martyn JAJ. iNOS inhibitor, L-NIL, reverses burn-induced glycogen synthase kinase-3β activation in skeletal muscle of rats. Metabolism. 2013; 62:341–6. [PubMed: 22995863]

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 11

NIH-PA Author Manuscript

41. Tanioka T, Tamura Y, Fukaya M, Shinozaki S, Mao J, Kim M, Shimizu N, Kitamura T, Kaneki M. Inducible nitric-oxide synthase and nitric oxide donor decrease insulin receptor substrate-2 protein expression by promoting proteosome-dependent degradation in pancreatic beta-cells: Involvement of glycogen synthase kinase-3beta. J Biol Chem. 2011; 286:29388–96. [PubMed: 21700708] 42. Kaneki M, Shimizu N, Yamada D, Chang K. Nitrosative stress and pathogenesis of insulin resistance. Antioxid Redox Signal. 2007; 9:319–29. [PubMed: 17184170]

NIH-PA Author Manuscript NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 12

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 1. Intrathecal AM-251 attenuates mechanical allodynia of burn injury

NIH-PA Author Manuscript

Mechanical allodynia using Von Frey Filaments was tested preburn, and at 1–14 days postburn. Burn injury resulted in significant allodynia in the vehicle group throughout the observation period on the ipsilateral compared to contralateral side. Intrathecal AM-251 treatment, started immediately after burn and continued daily for 7 days attenuated the allodynia starting at 3 days. The beneficial effect of AM251 lasted up to 14 days beyond the infusion period of AM251.

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 13

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 2. Intrathecal AM-251 completely reverses thermal hyperalgesia of burn

Following hind paw burn, thermal hyperalgesia was measured preburn, and at 1–14 days postburn using beam of radiant heat. On vehicle treated ipsilateral (burn) side, hyperalgesia was induced at day 3, 5, 7 10, and 12, and spontaneously recovered by day 14. Intrathecal AM-251 treatment, started immediately after burn, and continued for 7 days after burn completely reversed the hyperalgesia even after termination of drug administration at day 7.

NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 14

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 3. Intraperitoneal AM-251 to younger rats attenuates mechanical allodynia

Following hind paw burn, mechanical allodynia was observed as early as day 1 after burn and persisted even at day 14 on the untreated ipsilateral side of burn. The administration of AM-251 (1 mg/kg or 0.1 mg/kg daily) was started immediately postburn and continued up to day 7. The dose of 1.0 mg/kg of AM-251 attenuated the allodynia at 3, 5, 7, and 14 days. The administration of 0.1 mg/kg AM-251 showed no beneficial effects on mechanical alloydynia.

NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 15

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 4. Interperiotneal AM-251 to younger rats reverses thermal hyperalgesia

Following hind paw burn injury, thermal hyperalgesia was measured using beam of radiant heat on the ipsilateral and contralateral sides. Hyperalgesic nociceptive behaviors were not observed at day 1 but observed at days 3, 5, and 7 postburn on the vehicle treated ipsilateral (burn) side. The hyperalgesia spontaneously reversed by day 14 after burn. Treatment with 1 mg/kg AM-251 completely reversed the development of hyperalgesia. The hyperalgesia persisted in the groups AM-251 0.1 mg/kg had no beneficial effect on hyperalgesia.

NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 16

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 5. Burn injury increases ionized calcium-binding protein-1 (Ibal) expression in microglia on the ipsilateral side

Using Ibal antibody, microglial activity was determined at 3, 7, and 14 days after burn (n = 3 for each of burn and sham burn). Microglial activity did not differ between sham-burn and contralateral side of burn. At day 3, a diffuse 2–3 fold (nonsignificant) increase in microglial activity was observed; at day 7 and 14, microglial activity (indicated by arrows) was significantly (p < 0.05) increased 18.5 ± 7.5 and 12.5 ± 1.6 fold, respectively, on the dorsal horn of the ipsilateral side compared to contralateral side. Scale bars indicate 200μm

Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 17

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 6. Increased glial fibrillary acidic protein (GFAP) expression at 7 days after burn is unaltered by treatment with AM251

Atroglial activity on the spinal cord dorsal horn was determined using fluorescent GFAP antibody at day 7. Groups studied (n = 4/group) included sham-burn treated with vehicle (Sham + Veh), ipsilateral side of burn treated with vehicle (Burn + Veh), contralateral side to burn treated with vehicle (Contra + Veh), ipsilateral to burn treated with AM-251 (Burn + AM-251), contralateral to burn treated with AM-251 (Contra + AM-251). Scale bar for 6A and B indicate 100 μm and for 6Ca, 6Cb, 6Da, 6Db indicate 25 μm. Burn injury significantly increases astroglia activity 2.9 ± 0.3 fold (p < 0.05)on the ipsilateral side compared to contralateral side at day 7. Treatment with AM-251 does not change the activity/expression of astroglia on the ipsilateral side compared to vehicle treatment of ipsilateral side (n = 4/ group).

NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Ueda et al.

Page 18

NIH-PA Author Manuscript NIH-PA Author Manuscript

Figure 7. Increased ion calcium-binding adaptor molecule-1 (Ibal) expression in microglia of the spinal cord dorsal horn is unaltered by the administration of AM251

Activity of microglia with and without AM251 in spinal dorsal horn at level of lumbar 1–2 was studied at day 7 after burn using specific fluorescent tag, Iba1. Groups studied included sham burn animals treated with vehicle (Sham + Veh), ipsilateral side of burn treated with vehicle (Burn + Veh), contralateral side to burn treated with vehicle (Contra + Veh), burn injured side treated with AM-251 (Burn + AM-251), contralateral side to burn treated with AM-251 (Contra + AM-251). Scale bars for 7A and B are 100 μm and for 7ca, 7cb, 7Da, and 7DB are 25 μm. Burn injury, relative to contralateral side, activates microglia on the ipsilateral side. Treatment with AM-251 did not alter activation of microglia on the ipsilateral side of burned mice compared to ipsilateral side treated with vehicle (n = 3/ group).

NIH-PA Author Manuscript Anesthesiology. Author manuscript; available in PMC 2015 December 01.

Cannabinoid receptor type 1 antagonist, AM251, attenuates mechanical allodynia and thermal hyperalgesia after burn injury.

Burn injury causes nociceptive behaviors, and inflammation-related pathologic pain can lead to glial cell activation. This study tested the hypothesis...
872KB Sizes 0 Downloads 6 Views