Saranya devi Relief of Hypersensitivity after Nerve Injury from ALN Systemic Donepezil Involves Spinal and γ-Aminobutyric Acid Mechanisms Spinal ACh and GABA Mechanisms for Donepezil Analgesia

Masafumi Kimura, M.D.,* Ken-ichiro Hayashida, D.V.M., Ph.D.,† James C. Eisenach, M.D.,‡ KIMURA ET AL. Shigeru Saito M.D.,§ Hideaki Obata, M.D.‖

XX ABSTRACT What We Already Know about This Topic

• Donepezil increases lev- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 10.1097/ALN.0b013e318277a81c Background: Evoking spinal release of acetylcholine (ACh) els and is used for treatment of produces antinociception in normal animals and reduces • Increases in spinal acetylcholine levels have been implicated in the relief of neuropathic pain hypersensitivity after nerve injury, and some studies suggest January that ACh-mediated analgesia relies on γ-aminobutyric acid (GABA)-ergic signaling in the spinal cord. In this study, the authors tested the spinal mechanisms underlying the anti- What This Article Tells Us That Is New 118 hypersensitivity effects of donepezil, a central nervous sys- • Donepezil increased spinal acetylcholine levels and γ- tem–penetrating , in a rat model of aminobutyric acid levels to reduce nociceptive responses after nerve injury and represents a potential therapeutic pathway to neuropathic pain. reduce pain after nerve injury Methods: Male Sprague-Dawley rats were anesthetized, and L5 spinal nerve ligation was performed unilaterally. With- A antagonist; and CGP 35348 (30 μg), a γ-aminobutyric drawal threshold to a paw pressure test was measured before acid receptor type B antagonist. ACh and GABA concen- and after intraperitoneal administration of donepezil, with trations in the microdialysates from the spinal dorsal horn or without intrathecal antagonists for cholinergic and GAB- were increased after intraperitoneal donepezil treatment Aergic receptors. Microdialysis studies in the ipsilateral dor- (1 mg/kg) in both normal and spinal nerve ligation rats. sal horn of the lumbar spinal cord were also performed to Conclusions: Systemic administration of donepezil reduces measure extracellular ACh and GABA. hypersensitivity after nerve injury by increasing extracellu- Results: Donepezil increased the withdrawal threshold in lar ACh concentration, which itself induces GABA release spinal nerve ligation rats but not in normal rats. The anti- in the spinal cord. Activation of this spinal cholinergic–­ hypersensitivity effect of donepezil (1 mg/kg) in spinal GABAergic interaction represents a promising treatment for nerve ligation rats was reduced by intrathecal pretreatment neuropathic pain. with atropine (30 μg), a muscarinic receptor antagonist; mecamylamine (100 μg), a nicotinic receptor antagonist; bicuculline (0.03 μg), a γ-aminobutyric acid receptor type ERIPHERAL nerve injury can result in neuropathic P pain that is not alleviated by conventional pain reliev- ers. Currently, the most common drugs for treating neuro- * Graduate Student, § Professor of Anesthesiology, ‖ Associate Professor of Anesthesiology, Department of Anesthesiology, Gunma pathic pain are antidepressants and calcium channel α2-δ University Graduate School of Medicine, Maebashi, Japan. † Assis- ligands such as gabapentin and pregabalin.1 These drugs rely tant Professor of Anesthesiology, ‡ FM James, III Professor of Anes- in part on spinal cholinergic mechanisms for analgesia,2,3 thesiology and Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina. which suggests that cholinergic pathways may be critical in Received from the Department of Anesthesiology, Gunma Uni- the therapeutic targeting of neuropathic pain. versity Graduate School of Medicine, Maebashi, Japan. Submitted Cholinesterase inhibitors such as and for publication March 15, 2012. Accepted for publication August 30, 4–6 2012. This work was supported by a grant (Grant-In-Aid for Scien- produce analgesia to acute pain in humans. tific Research 20591823, to Dr. Obata) from the Ministry of Educa- A recent study demonstrated that the oral administration tion, Culture, Sports, Science and Technology, Tokyo, Japan, and of donepezil, an approved cholinesterase inhibitor for the grants (NS57594 to Dr. Eisenach and DA27690 to Dr. Hayashida) from the National Institutes of Health, Bethesda, Maryland. Timothy symptomatic treatment of Alzheimer dementia, produced an J. Brennan, Ph.D., M.D., served as Handling Editor. antihypersensitivity effect in rats after nerve injury but not in Address correspondence to Dr. Obata: Department of Anesthe- normal animals; this involved a spinal mechanism.7 Although siology, Gunma University Graduate School of Medicine, 3-39-22, Showa, Maebashi, Japan 371–8511. [email protected]. the detailed mechanism is not fully understood, manipula- Information on purchasing reprints may be found at www.anesthe- tions that increase acetylcholine (ACh) release in the spinal siology.org or on the masthead page at the beginning of this issue. cord produce analgesia via both muscarinic and nicotinic Anesthesiology’s articles are made freely accessible to all readers, for 8,9 personal use only, 6 months from the cover date of the issue. receptors. Cholinergic signaling is an attractive target for Copyright © 2012, the American Society of Anesthesiologists, Inc. Lippincott analgesia because it may be involved in endogenous inhibition Williams & Wilkins. Anesthesiology 2013; 118:173–80 of pain10 and may be augmented under painful conditions.7,11

Anesthesiology, V 118 • No 1 173 January 2013 Spinal ACh and GABA Mechanisms for Donepezil Analgesia

In the current study, we tested whether the analgesic effi- receptor antagonist, CGP 35348 (10 and 30 μg). Saline cacy of donepezil was increased in the presence of chronic or antagonist solution was administered intrathecally neuropathic injury compared with the normal state because 15 min before intraperitoneal donepezil injection. The of a greater effect on release of ACh concentration in the spi- doses of the antagonists were selected according to previous nal cord. Although previous studies suggest that stimulation studies7,8,19,20 and our preliminary studies. For intrathecal of spinal muscarinic and nicotinic ACh receptors increases administration, drugs were dissolved in saline in a volume γ-aminobutyric acid (GABA) release to inhibit pain trans- of 5 μl and injected in the L5-6 intervertebral space using mission in the spinal cord,12–16 this has never been directly a 30-gauge needle. Donepezil was a gift from Eisai Co. tested. We therefore tested the behavioral reliance of done- (Tokyo, Japan). Other drugs were purchased from Sigma pezil-mediated antihypersensitivity on activation of GABA Co. (St. Louis, MO). Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 receptors and whether donepezil-induced increases in ACh would increase GABA release in the spinal cord. Microdialysis Studies ACh Measurement. Microdialysis studies were performed Materials and Methods in normal rats and those 2–3 weeks after SNL surgery in a Surgical Preparation manner as previously described.21 Anesthesia was induced by The study was approved by the Animal Care and Use Com- intraperitoneal injection with urethane (1.2–1.5 g/kg) and mittees of the Gunma University Graduate School of Medi- maintained with 0.5% isoflurane in 100% oxygen through a cine (Maebashi, Japan) and Wake Forest University School of nose cone. The left femoral vein was cannulated for transfu- Medicine (Winston-Salem, North Carolina). Male Sprague- sion of saline at a rate of 1 ml/h. For intraperitoneal injection Dawley rats (250 g) were used in all experiments. Microdi- of donepezil, a 24-gauge intravenous catheter was inserted alysis study for GABA release was performed at Wake Forest into the intraperitoneal space. Rectal temperature was main- University, and other experiments were performed at Gunma tained at 37–38°C by a heating pad placed beneath the ani- University. The animals were housed under a 12-h light– mal. The L3–L5 level of the spinal cord was exposed by a dark cycle, with food and water available ad libitum. Spinal thoracolumbar laminectomy, and the rat was placed in a ste- nerve ligation (SNL) surgery was performed as previously reotaxic apparatus. The microdialysis probe (OD = 0.22 mm, described.17 The animals were anesthetized with inhaled iso- ID = 0.20 mm, length = 2 mm, AI-8-02, Eicom Co., Kyoto, flurane in oxygen, and the right L5 spinal nerve was tightly Japan) was inserted from just lateral to the dorsal root and ligated with 5-0 silk and cut just distal to the ligature as pre- advanced at a 30° angle to a depth of 2 mm using a micro- viously described. The wound was closed, and the animals manipulator (model WR-88; Narishige, Tokyo, Japan). The were allowed to recover for 2 weeks. Animals were singly microdialysis probe was perfused with Ringer’s solution (147 M M M housed after surgery for the remainder of the experiment. m NaCl, 4 m KCl, 2.3 m CaCl2) at a constant flow rate For behavioral studies, each rat was used two to three times (1 μl/min) using a syringe pump (ESP-64, Eicom Co.). After at 4- to 5-day intervals. In total, 82 SNL rats were used. 120 min of constant perfusion, 30-min perfusate fractions were collected into an auto injector (EAS-20, Eicom Co.) Behavioral Testing After two consecutive samples were collected to determine Withdrawal threshold to pressure applied to the hindpaw, the basal ACh concentrations in the dialysate, saline (0.5 ml) expressed in grams, was measured using an analgesimeter or donepezil (1.0 mg/kg) was administered intraperitoneally (37215, Ugo Basile, Comerio, Italy) as previously described.18 through an indwelling catheter. The device applies increasing pressure to the hindpaw until Samples (30 μl) were automatically injected and ana- the animal withdraws the paw. The pressure is immediately lyzed for the ACh concentration using high-performance released, and the pressure at withdrawal measured in grams. liquid chromatography with electrochemical detection by A cutoff of 250 g was used to prevent potential tissue injury. an HTEC-500 analyzing system (Eicom Co.). The chro- The experimenter was blinded to the treatment group. All matographic conditions were as follows: the mobile phase M animals were trained for 3 days with this apparatus before consisted of 50 m KHCO3 containing 300 mg/l sodium baseline values were recorded. 1-decanesulfonate (pH = 8.5) and 50 mg/l ethylenediami- netetraacetic acid-2Na. The column was an EICOMPAC Drugs and Their Administration AC-GEL (2.0 mm  150 mm; Eicom Co.). A glassy carbon Drug testing was performed 2–3 weeks after nerve ligation. working electrode (WE-3G, Eicom Co.) was used with a Rats received intraperitoneal donepezil (0.3, 0.6, and flow rate of 0.15 ml/min. The detector voltage was 0.45V, 1.0 mg/kg). Antagonist studies were performed using the and the detector temperature was 33.0°C. The retention muscarinic receptor antagonist, atropine (30 μg); the time for ACh was 12.4 min. The detection limit of the ACh nicotinic receptor antagonist, mecamylamine (100 μg); the assay in the current study was 10 fg per injection (informa- γ-aminobutyric acid type A receptor antagonist, bicuculline tion from Eicom Co.), and the interassay coefficient of varia- (0.01 and 0.03 μg); and the γ-aminobutyric acid type B tion at 29 ng (200 fmol) per injection was 12.3%.

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GABA Measurement. Microdialysis studies were performed to measure spinal GABA release using the same condition for ACh microdialysis. Samples were kept at −80°C until GABA was measured. GABA content in the microdialy- sates was measured by high-pressure liquid chromatography with electrochemical detection by an HTEC-500 analyzing system (Eicom co.). GABA in the samples was derivatized with 2-mercaptoethanol and ο-phthaldialdehyde (4 mM) in 0.1 M carbonate buffer (pH = 9.5). The ο-phthaldialdehyde derivatives were then separated on the column (3.0 mm × Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 150 mm, SC-5ODS, EICOM) at 30°C, using a mobile phase that consisted of 50 mM phosphate buffer (pH = 2.8) and methanol (1:1 vol/vol) containing 5 mg/ml ethylenedi- aminetetraacetic acid-2Na at a flow rate of 0.5 ml/min. The limit of detection for GABA in the current study was 1.5 pg per injection (10 µl), and the interassay coefficient of varia- tion at 100 pg per injection was 7.2%. Rotarod Test. Sedation and motor coordination were tested using the accelerating rotarod (ENV577; Med Associates Inc., St. Albans, VT) in normal and SNL rats. The rats were required to walk against the motion of a rotating drum with the speed accelerating from 4 to 40 rpm over 300 s. The time on the rod from the start of acceleration until the animal fell from the drum onto the counter-trip plate was recorded before and 15 min after intraperitoneal administration of donepezil (1.0 mg/kg). A 300-s cutoff was used. One train- ing period per day was performed for 2 days before the drug Fig. 1. Effect of IP DNP on the withdrawal threshold to pres- treatment, and the animals were acclimated to the device and sure applied to the hindpaw in rats with spinal nerve ligation (A) habituated to handling to minimize stress during testing. and normal rats (B). Withdrawal thresholds are expressed as Statistics. The statistical analysis was conducted using Sig- the mean ± SD for six rats in each group. *P < 0.05 compared maPlot 12 (Systat Software Inc., San Jose, CA). Data were with SAL-treated group at each time point (Student–Neu- normally distributed (Shapiro–Wilk test) and are presented man–Keuls post hoc test after two-way repeated-measures ANOVA). DNP = donepezil; IP = intraperitoneal; SAL = saline. as the mean ± SD. Time-course data from the behavioral and microdialysis studies were analyzed using a two-way repeated-measures ANOVA. When significant differences in each group, P < 0.05 by Student–Neuman–Keuls post hoc were observed, Student–Newman–Keuls post hoc tests were test). In contrast, withdrawal threshold in normal animals performed for between-group comparisons and comparisons did not differ between saline and donepezil (1.0 mg/kg) at each time point. Other data were analyzed using a paired groups (fig. 1B). On the basis of these results, we selected the or unpaired t test. A P value less than 0.05 was defined as donepezil dose of 1.0 mg/kg for the following experiments. statistically significant. Intrathecal pretreatment with the muscarinic receptor antagonist atropine (30 μg) completely blocked the antihy- Results persensitivity effect of donepezil (n = 6 in each group, P = Behavioral Studies 0.002 by Student–Neuman–Keuls post hoc test after statis- Intraperitoneal administration of donepezil (0.3, 0.6, and tically significant interaction in two-way repeated-measures 1.0 mg/kg) produced antihypersensitivity effects in SNL ANOVA; fig. 2A). The nicotinic receptor antagonist meca- rats (n = 6 in each group, P < 0.001 by two-way repeated- mylamine (100 μg) also inhibited the effect of donepezil measures ANOVA, fig. 1A). Withdrawal thresholds (n = 6 in each group, P = 0.002 by Student–Neuman–Keuls were higher in groups treated with 0.6 and 1.0 mg/kg of post hoc test after statistically significant interaction in two- donepezil compared with the saline-treated group (P < 0.05 way repeated-measures ANOVA; fig. 2B). Neither cholin- by Student–Neuman–Keuls post hoc test). The peak was ergic antagonist alone affected the withdrawal threshold observed 15 min after donepezil administration in both the compared with saline. 0.6 and 1.0 mg/kg groups, but the duration of the effect was Intrathecal pretreatment with the γ-aminobutyric acid longer in the 1.0 mg/kg group than in the 0.6 mg/kg group type A receptor antagonist bicuculline (0.03 μg) and the (120 min . 30 min) compared with the saline group (n = 6 γ-aminobutyric acid type B receptor antagonist CGP 35348

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Fig. 2. Time course of the effects of intrathecal pretreatment Fig. 3. Time course of the effects of IT, pretreatment with BIC, with ATR, a muscarinic acetylcholine receptor antagonist (A) a γ-aminobutyric acid type A (GABAA) receptor antagonist (A), and MEC, a nicotinic acetylcholine receptor antagonist (B), on and CGP 35348 (CGP), a γ-aminobutyric acid type B (GABAB) the antihypersensitivity effect of intraperitoneal DNP (1.0 mg/ receptor antagonist (B), on the antihypersensitivity effect of IP kg). SAL, atropine (30 μg), or mecamylamine (100 μg) was DNP (1.0 mg/kg). SAL, bicuculline (0.01 and 0.03 μg), or CGP administered intrathecally 15 min before the intraperitoneal 35348 (10 and 30 μg) was administered intrathecally 15 min injection of donepezil. Data are expressed as the mean ± SD before the intraperitoneal injection of donepezil. Data are ex- for six rats in each group.*P < 0.05 compared with the saline pressed as the mean ± SD for six rats in each group. *P < + donepezil–treated group at each time point (Student–Neu- 0.05 compared with saline + donepezil–treated group at each man–Keuls post hoc test after two-way repeated-measures time point (Student–Neuman–Keuls post hoc test after two- ANOVA). ATR = atropine; DNP = donepezil; IP = intraperito- way repeated-measures ANOVA). BIC = bicuculline; DNP = neal; IT = intrathecal; MEC = mecamylamine; SAL = saline. donepezil; IP = intraperitoneal; IT = intrathecal; SAL = saline.

(30 μg), neither of which affected the withdrawal threshold The intraperitoneal administration of donepezil (1 mg/ when administered alone, attenuated the antihypersensitivity kg) did not affect the rotarod performance time in normal effect of donepezil in SNL rats (n = 6 in each group, P < 0.05 and SNL rats (the rotarod performance time before and by Student–Neuman–Keuls post hoc test after statistically sig- 15 min after donepezil was 136.6 ± 33.1 and 141.3 ± 31.2 s nificant interaction in two-way repeated-measures ANOVA; in normal rats and 137.3 ± 51.4 and 135.0 ± 48.9 s in SNL fig 3). A previous study showed that intrathecal administration rats, respectively, n = 6 in each group). No adverse behavioral of bicuculline and another γ-aminobutyric acid type B receptor effects, such as sedation or agitation, were observed with any antagonist phaclofen produced tactile allodynia and thermal of the treatments. hyperalgesia in normal rats.19 In the current study, however, intrathecal administration of bicuculline (0.03 μg) and CGP 35348 (30 μg) alone did not affect the withdrawal threshold Microdialysis Studies in normal rats (the withdrawal threshold before and 30 min The baseline ACh concentrations in spinal cord dorsal after bicuculline was 148.3 ± 9.3 and 147.5 ± 8.8 g, respectively, horn microdialysates before drug injection did not differ and before and 30 min after CGP 35348 were 141.7 ± 8.2 and between SNL (2.67 ± 0.40 pg/30 μl) and normal rats 140.0 ± 16.7 g, respectively, n = 6 in each group). (3.05 ± 0.45 pg/30 μl). In the saline-treated normal and

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SNL rats, ACh concentrations in the dialysates did not change over time. In the donepezil-treated normal and SNL rats, ACh concentrations in the dialysates increased within 30 min, peaked at 1 h with approximately 1,800– 2,200% of the baseline value, and remained increased for 2 h after injection compared with the saline-treated group (n = 6 in each group, P < 0.05 by Student–Neuman– Keuls post hoc test after statistically significant interaction in two-way repeated-measures ANOVA; fig. 4). The donepezil-induced ACh increase did not differ between Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 normal and SNL rats. Baseline GABA concentrations in spinal cord dorsal horn microdialysates before drug injection were lower in SNL rats (23.7 ± 3.3 pg/30 μl) than in normal rats (40.0 ± 4.5 pg/30 μl; an unpaired t test, P = 0.012). Although a slight decrease in GABA concentrations in the dialysates was observed over time, the intraperitoneal injection of saline did not affect the Fig. 5. Microdialysis for spinal γ-aminobutyric acid (GABA) GABA concentration compared with the baseline. In the release. Normal rats or rats with SNL received IP, SAL, or donepezil-treated groups, GABA concentrations increased (DNP; 1 mg/kg). Data are presented over time as a percent- over time after drug injection and were significantly different age of the baseline. All values represent the mean ± SD for six rats in each group. *P < 0.05 and #P < 0.05 compared with from that after saline treatment in the SNL and normal rats saline-treated group in normal and SNL rats, respectively, at (n = 6 in each group, P < 0.05 by Student–Neuman–Keuls each time point (Student–Neuman–Keuls post hoc test after post hoc test after statistically significant interaction in two- two-way repeated-measures ANOVA). DNP = donepezil; IP = way repeated-measures ANOVA; fig 5). intraperitoneal; SNL = spinal nerve ligation; SAL = saline.

Discussion effect in SNL rats without inducing any adverse effects. Con- In the current study, the intraperitoneal administration of sistent with behavioral studies using cholinergic and GAB- donepezil produced a dose-dependent antihypersensitivity Aergic antagonists, direct measurements of ACh and GABA in the spinal dorsal horn with microdialysis revealed that donepezil increased the ACh and GABA concentrations. Although the analgesia provided by donepezil was specific for hypersensitivity after SNL, the increases in spinal ACh and GABA were similar in normal and SNL rats. Therefore, other mechanisms may be involved in the enhanced efficacy of donepezil for neuropathic pain. Previous animal studies show that cholinesterase inhibitors produce analgesia by supraspinal,22 spinal,23 and peripheral24 mechanisms, and oral administration of donepezil has previ- ously been shown to reduce hypersensitivity after SNL in rats via an action on spinal muscarinic receptors.7 Consistent with these findings, we showed that intraperitoneal administration of donepezil produced reduced hypersensitivity in a manner reversed by intrathecal pretreatment with atropine. Nicotinic receptors in the spinal cord have been shown to be involved in nociceptive transmission,25,26 and we confirmed in the current study a partial role for nicotinic receptors in the antihypersen- Fig. 4. Microdialysis for spinal acetylcholine increase. Normal sitivity effect of donepezil. Consistent with our study, previ- rats or rats with SNL received IP, SAL, or (DNP; 1 mg/kg). Data ous studies showed that intrathecal nicotinic receptor are presented over time as a percentage of the baseline. All increased withdrawal thresholds measured by paw pressure values represent the mean ± SD for six rats in each group. test in SNL animals.14,27 In contrast, a previous study dem- *P < 0.05 and #P < 0.05 compared with SAL-treated group in normal and SNL rats, respectively, at each time point onstrated that antiallodynic effect from orally administered 7 (Student–Neuman–Keuls post hoc test after two-way repeated- donepezil is not affected by mecamylamine in SNL rats. In measures ANOVA). DNP = donepezil; IP = intraperitoneal; normal animals, nicotinic receptors have no or little role on SNL = spinal nerve ligation; SAL = saline. thermal antinociception from cholinesterase inhibition.28,29

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These results suggest that nicotinic receptors may contribute the dysfunction of GABAergic inhibition in the spinal cord to only mechanical hyperalgesia in neuropathic pain. likely contributes to neuropathic pain,48,49 and several lines We hypothesized that donepezil would cause a greater of evidence suggest that stimulation of the spinal GABAergic release of ACh in the spinal cord in SNL rats than in normal system produces analgesia in neuropathic pain.19,50–52 Con- rats because cholinergic analgesia is augmented in neuro- sistent with these observations, our microdialysis study sug- pathic pain, as indicated by the current behavioral study and gests that donepezil inhibited SNL-induced hypersensitivity others.7 In our microdialysis studies, however, the peak levels by inducing spinal GABA release, despite the decrease of and the time course of the ACh increase after donepezil treat- baseline GABA concentration in SNL rats compared with ment were similar between normal and SNL rats. Further- normal rats. Although the peak level and time course of the more, the basal levels of ACh were not significantly different GABA increase after donepezil were similar between normal Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 between normal and SNL rats. Therefore, other mechanisms and SNL rats, donepezil may produce antihypersensitivity may contribute to the increased potency of donepezil against by activating inhibitory GABAergic neurons to re-establish hypersensitivity after nerve injury. For example, inhibi- the inhibitory tone that had been reduced by nerve injury. tory M2 muscarinic receptors have been reported to play Although donepezil may have some inhibitory effects for an essential role in cholinergic analgesia,25,30,31 and a recent pain in patients using opiates,53 there is no previous study study demonstrated that peripheral nerve injury upregulates that reported relief of neuropathic pain in patients treated M2 muscarinic receptors in primary sensory afferent neu- with donepezil. The doses of donepezil used in the current rons.32 In addition, intrathecal administration of nicotinic study were slightly greater than doses that were effective for receptor agonists inhibits hypersensitivity after nerve injury cognitive responses in animals.54,55 Therefore, this discrepancy but does not produce analgesia in normal animals.14,27 These might be related to dosage. The spinal cholinergic system is findings suggest that peripheral nerve injury enhances the heavily involved in α2-adrenoceptor–mediated analgesia for efficacy of ACh action in the spinal cord and that this plas- neuropathic pain. As such, activation of α2-adrenoceptors, ticity, rather than a change in ACh release in injury state, is which inhibits ACh release in the normal state, facilitates used by donepezil to provide enhanced analgesia after nerve ACh release after nerve injury via a G-protein–mediated injury compared with the normal condition. mechanism.56 Several antidepressants (e.g., tricyclic antide- Extensive evidence indicates a close functional relationship pressants and serotonin and noradrenaline reuptake inhibi- between spinal cholinergic and GABAergic mechanisms.33,34 tors) and calcium channel α2-δ ligands (e.g., gabapentin and Stimulation of M2, M3, and M4 muscarinic receptors pregabalin) that are routinely used in the treatment of neuro- results in presynaptic GABA release in the spinal cord and pathic pain1 rely on spinal α2-adrenoceptors and cholinergic γ-aminobutyric acid type B receptor–mediated analgesia.13,16,35 mechanisms for analgesia in neuropathic pain.2,3 The cur- Stimulation of spinal nicotinic receptors has been reported rent study builds on previous observations to strengthen the to produce presynaptic GABA release and γ-aminobutyric rationale for clinical trials examining the benefit from add-on acid type A receptor–mediated analgesia.14,15,27,36 However, therapy to these agents with donepezil.57 no previous study measured GABA concentrations in In summary, systemic administration of donepezil the spinal cord after stimulation of cholinergic receptors. reduces hypersensitivity after peripheral nerve injury in Our microdialysis experiments clearly demonstrate that rats by increasing the concentrations of ACh and GABA intraperitoneal administration of donepezil increased GABA in the spinal dorsal horn. The greater effect of donepezil on concentrations in the spinal cord. Furthermore, intrathecal behavior in the presence of nerve injury did not depend on administration of GABA receptor antagonists produced a dose- increased interstitial concentrations of ACh in the spinal related suppression of donepezil-mediated antihypersensitivity cord in the injured state. Rather, there was a dependence of at doses that themselves failed to alter withdrawal threshold the behavioral effect of donepezil on activation of GABA in normal and SNL rats. Although we did not identify the receptors in the spinal cord, and donepezil administration specific cholinergic receptor that is involved in this activity, was associated with increased release of GABA in the spi- our data suggest that the systemic administration of donepezil nal cord. These results further our understanding regarding increases extracellular ACh concentrations in the spinal cord the spinal mechanisms by which cholinesterase inhibitors to induce subsequent GABA release, and that GABA release produce analgesia in neuropathic states and strengthen is essential to the antihypersensitivity effect of donepezil after the rationale for their clinical application alone and in peripheral nerve injury. combination with antidepressants and gabapentin-like Decreases in spinal GABA immunoreactivity and the compounds. GABA-synthesizing glutamic acid decarboxylase References have been reported after nerve injury.37–41 Electrophysiologic studies confirmed that primary afferent-evoked inhibitory 1. O’Connor AB, Dworkin RH: Treatment of neuropathic pain: An overview of recent guidelines. Am J Med 2009; 122(10 postsynaptic currents in the spinal dorsal horn were reduced Suppl):S22–32 40,42,43 after nerve injury. Although controversy exists with 2. Obata H, Saito S, Koizuka S, Nishikawa K, Goto F: The regard to the actual loss of GABAergic interneurons,44–47 monoamine-mediated antiallodynic effects of intrathecally

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administered milnacipran, a serotonin noradrenaline reup- a postoperative pain model in rats. J Pharmacol Exp Ther take inhibitor, in a rat model of neuropathic pain. Anesth 2010; 334:1059–65 Analg 2005; 100:1406–10 22. Fujimoto JM, Rady JJ: Intracerebroventricular physostig- 3. Hayashida K, Parker R, Eisenach JC: Oral gabapentin acti- mine-induced analgesia: Enhancement by , beta- vates spinal cholinergic circuits to reduce hypersensitivity funaltrexamine and nor-binaltorphimine and antagonism by after peripheral nerve injury and interacts synergistically dynorphin A (1-17). J Pharmacol Exp Ther 1989; 251:1045–52 with oral donepezil. Anesthesiology 2007; 106:1213–9 23. Yaksh TL, Dirksen R, Harty GJ: Antinociceptive effects of 4. Weinstock M, Davidson JT, Rosin AJ, Schnieden H: Effect of intrathecally injected cholinomimetic drugs in the rat and physostigmine on morphine-induced postoperative pain and cat. Eur J Pharmacol 1985; 117:81–8 somnolence. Br J Anaesth 1982; 54:429–34 24. Dussor GO, Helesic G, Hargreaves KM, Flores CM: Cholinergic 5. Lauretti GR, Hood DD, Eisenach JC, Pfeifer BL: A multi-cen- modulation of nociceptive responses in vivo and neuropep- tide release in vitro at the level of the primary sensory neu-

ter study of intrathecal neostigmine for analgesia following Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 ron. Pain 2004; 107:22–32 vaginal hysterectomy. Anesthesiology 1998; 89:913–8 25. Cordero-Erausquin M, Marubio LM, Klink R, Changeux JP: 6. Hood DD, Mallak KA, Eisenach JC, Tong C: Interaction Nicotinic receptor function: New perspectives from knock- between intrathecal neostigmine and epidural clonidine in out mice. Trends Pharmacol Sci 2000; 21:211–7 human volunteers. Anesthesiology 1996; 85:315–25 26. Vincler M: Neuronal nicotinic receptors as targets for novel 7. Clayton BA, Hayashida K, Childers SR, Xiao R, Eisenach JC: analgesics. Expert Opin Investig Drugs 2005; 14:1191–8 Oral donepezil reduces hypersensitivity after nerve injury 27. Young T, Wittenauer S, Parker R, Vincler M: Peripheral nerve by a spinal muscarinic receptor mechanism. Anesthesiology injury alters spinal nicotinic acetylcholine receptor pharma- 2007; 106:1019–25 cology. Eur J Pharmacol 2008; 590:163–9 8. Pan HL, Chen SR, Eisenach JC: Intrathecal clonidine 28. Naguib M, Yaksh TL: Antinociceptive effects of spinal cholin- alleviates allodynia in neuropathic rats: Interaction with esterase inhibition and isobolographic analysis of the inter- spinal muscarinic and nicotinic receptors. Anesthesiology action with mu and alpha 2 receptor systems. Anesthesiology 1999; 90:509–14 1994; 80:1338–48 9. Paqueron X, Li X, Bantel C, Tobin JR, Voytko ML, Eisenach JC: 29. Chiari A, Tobin JR, Pan HL, Hood DD, Eisenach JC: Sex dif- An obligatory role for spinal cholinergic neurons in the anti- ferences in cholinergic analgesia I: A supplemental nico- allodynic effects of clonidine after peripheral nerve injury. tinic mechanism in normal females. Anesthesiology 1999; Anesthesiology 2001; 94:1074–81 91:1447–54 10. Eisenach JC, Detweiler DJ, Tong C, D’Angelo R, Hood DD: Cerebrospinal fluid norepinephrine and acetylcholine con- 30. Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin centrations during acute pain. Anesth Analg 1996; 82:621–6 J, Grinberg A, Sheng H, Wess J: Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout 11. Bouaziz H, Tong C, Eisenach JC: Postoperative analgesia mice. Proc Natl Acad Sci USA 1999; 96:1692–7 from intrathecal neostigmine in sheep. Anesth Analg 1995; 80:1140–4 31. Bernardini N, Reeh PW, Sauer SK: Muscarinic M2 receptors inhibit heat-induced CGRP release from isolated rat skin. 12. Baba H, Kohno T, Okamoto M, Goldstein PA, Shimoji K, Neuroreport 2001; 12:2457–60 Yoshimura M: Muscarinic facilitation of GABA release in substantia gelatinosa of the rat spinal dorsal horn. J Physiol 32. Hayashida KI, Bynum T, Vincler M, Eisenach JC: Inhibitory (Lond) 1998; 508 (Pt 1):83–93 M2 muscarinic receptors are upregulated in both axotomized and intact small diameter dorsal root ganglion cells after 13. Li DP, Chen SR, Pan YZ, Levey AI, Pan HL: Role of presyn- peripheral nerve injury. Neuroscience 2006; 140:259–68 aptic muscarinic and GABA(B) receptors in spinal glutamate release and cholinergic analgesia in rats. J Physiol (Lond) 33. Todd AJ: Immunohistochemical evidence that acetylcholine 2002; 543(Pt 3):807–18 and glycine exist in different populations of GABAergic neu- 14. Rashid MH, Ueda H: Neuropathy-specific analgesic action of rons in lamina III of rat spinal dorsal horn. Neuroscience intrathecal nicotinic agonists and its spinal GABA-mediated 1991; 44:741–6 mechanism. Brain Res 2002; 953:53–62 34. Mesnage B, Gaillard S, Godin AG, Rodeau JL, Hammer M, 15. Genzen JR, McGehee DS: Nicotinic modulation of GABAergic Von Engelhardt J, Wiseman PW, De Koninck Y, Schlichter R, synaptic transmission in the spinal cord dorsal horn. Brain Cordero-Erausquin M: Morphological and functional charac- Res 2005; 1031:229–37 terization of cholinergic interneurons in the dorsal horn of the mouse spinal cord. J Comp Neurol 2011; 519:3139–58 16. Zhang HM, Li DP, Chen SR, Pan HL: M2, M3, and M4 receptor subtypes contribute to muscarinic potentiation of GABAergic 35. Chen SR, Pan HL: Spinal GABAB receptors mediate antinoci- inputs to spinal dorsal horn neurons. J Pharmacol Exp Ther ceptive actions of cholinergic agents in normal and diabetic 2005; 313:697–704 rats. Brain Res 2003; 965:67–74 17. Kim SH, Chung JM: An experimental model for peripheral 36. Takeda D, Nakatsuka T, Papke R, Gu JG: Modulation of inhib- neuropathy produced by segmental spinal nerve ligation in itory synaptic activity by a non-alpha4beta2, non-alpha7 sub- the rat. Pain 1992; 50:355–63 type of nicotinic receptors in the substantia gelatinosa of 18. Randall LO, Selitto JJ: A method for measurement of analge- adult rat spinal cord. Pain 2003; 101:13–23 sic activity on inflamed tissue. Arch Int Pharmacodyn Ther 37. Castro-Lopes JM, Tavares I, Coimbra A: GABA decreases in 1957; 111:409–19 the spinal cord dorsal horn after peripheral neurectomy. 19. Malan TP, Mata HP, Porreca F: Spinal GABA(A) and GABA(B) Brain Res 1993; 620:287–91 receptor pharmacology in a rat model of neuropathic pain. 38. Ibuki T, Hama AT, Wang XT, Pappas GD, Sagen J: Loss of Anesthesiology 2002; 96:1161–7 GABA-immunoreactivity in the spinal dorsal horn of rats 20. Sokal DM, Chapman V: Inhibitory effects of spinal with peripheral nerve injury and promotion of recovery by on spinal dorsal horn neurones in inflamed and neuropathic adrenal medullary grafts. Neuroscience 1997; 76:845–58 rats in vivo. Brain Res 2003; 987:67–75 39. Eaton MJ, Plunkett JA, Karmally S, Martinez MA, Montanez K: 21. Obata H, Kimura M, Nakajima K, Tobe M, Nishikawa K, Changes in GAD- and GABA- immunoreactivity in the spinal Saito S: Monoamine-dependent, opioid-independent anti- dorsal horn after peripheral nerve injury and promotion of hypersensitivity effects of intrathecally administered mil- recovery by lumbar transplant of immortalized serotonergic nacipran, a serotonin noradrenaline reuptake inhibitor, in precursors. J Chem Neuroanat 1998; 16:57–72

Anesthesiology 2013; 118:173-80 179 Kimura et al. Spinal ACh and GABA Mechanisms for Donepezil Analgesia

40. Moore KA, Kohno T, Karchewski LA, Scholz J, Baba H, Woolf gradient in spinal lamina I neurons as a mechanism of neu- CJ: Partial peripheral nerve injury promotes a selective loss ropathic pain. Nature 2003; 424:938–42 of GABAergic inhibition in the superficial dorsal horn of the 49. Janssen SP, Truin M, Van Kleef M, Joosten EA: Differential spinal cord. J Neurosci 2002; 22:6724–31 GABAergic disinhibition during the development of painful 41. Yoshizumi M, Parker RA, Eisenach JC, Hayashida K: peripheral neuropathy. Neuroscience 2011; 184:183–94 Gabapentin inhibits γ-amino butyric acid release in the locus 50. Hwang JH, Yaksh TL: The effect of spinal GABA receptor coeruleus but not in the spinal dorsal horn after peripheral agonists on tactile allodynia in a surgically-induced neuro- nerve injury in rats. Anesthesiology 2012; 116:1347–53 pathic pain model in the rat. Pain 1997; 70:15–22 42. Scholz J, Broom DC, Youn DH, Mills CD, Kohno T, Suter MR, Moore KA, Decosterd I, Coggeshall RE, Woolf CJ: Blocking 51. Eaton MJ, Martinez MA, Karmally S: A single intrathecal caspase activity prevents transsynaptic neuronal apoptosis injection of GABA permanently reverses neuropathic pain and the loss of inhibition in lamina II of the dorsal horn after after nerve injury. Brain Res 1999; 835:334–9 peripheral nerve injury. J Neurosci 2005; 25:7317–23 52. Jergova S, Hentall ID, Gajavelli S, Varghese MS, Sagen J: Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/118/1/173/259568/0000542-201301000-00030.pdf by guest on 28 September 2021 43. Yowtak J, Lee KY, Kim HY, Wang J, Kim HK, Chung K, Chung Intraspinal transplantation of GABAergic neural progeni- JM: Reactive oxygen species contribute to neuropathic pain tors attenuates neuropathic pain in rats: A pharmacologic by reducing spinal GABA release. Pain 2011; 152:844–52 and neurophysiological evaluation. Exp Neurol 2012; 44. Polgár E, Hughes DI, Riddell JS, Maxwell DJ, Puskár Z, Todd 234:39–49 AJ: Selective loss of spinal GABAergic or glycinergic neurons 53. Slatkin NE, Rhiner M: Treatment of opiate-related sedation: utility is not necessary for development of thermal hyperalgesia in of the cholinesterase inhibitors. J Support Oncol 2003; 1:53–63 the chronic constriction injury model of neuropathic pain. 54. Ogura H, Kosasa T, Kuriya Y, Yamanishi Y: Donepezil, a cen- Pain 2003; 104:229–39 trally acting inhibitor, alleviates learning 45. Polgár E, Gray S, Riddell JS, Todd AJ: Lack of evidence for deficits in hypocholinergic models in rats. Methods Find Exp significant neuronal loss in laminae I-III of the spinal dorsal Clin Pharmacol 2000; 22:89–95 horn of the rat in the chronic constriction injury model. Pain 55. Yamaguchi Y, Higashi M, Matsuno T, Kawashima S: Ameliorative 2004; 111:144–50 effects of azaindolizinone derivative ZSET845 on scopolamine- 46. Polgár E, Hughes DI, Arham AZ, Todd AJ: Loss of neurons induced deficits in passive avoidance and radial-arm maze from laminas I-III of the spinal dorsal horn is not required for learning in the rat. Jpn J Pharmacol 2001; 87:240–4 development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci 2005; 25:6658–66 56. Hayashida K, Eisenach JC: Spinal α 2-adrenoceptor-mediated 47. Polgár E, Todd AJ: Tactile allodynia can occur in the spared analgesia in neuropathic pain reflects brain-derived nerve nerve injury model in the rat without selective loss of GABA growth factor and changes in spinal cholinergic neuronal or GABA(A) receptors from synapses in laminae I-II of the function. Anesthesiology 2010; 113:406–12 ipsilateral spinal dorsal horn. Neuroscience 2008; 156:193–202 57. Hayashida K, Eisenach JC: Multiplicative interactions 48. Coull JA, Boudreau D, Bachand K, Prescott SA, Nault F, Sík to enhance gabapentin to treat neuropathic pain. Eur A, De Koninck P, De Koninck Y: Trans-synaptic shift in anion J Pharmacol 2008; 598:21–6

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