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NEUROPHARMACOLOGY
Department of Biopharmaceutical Sciences (F.L., C.Y., Y.C., P.K.S., L.T., Z.J.W.), and Cancer Center (Z.J.W.), University of Illinois, Chicago, Illinois; and Feinberg School of Medicine, Northwestern University, Chicago, Illinois (L.X.W.)
Received September 28, 2007; accepted January 3, 2008.
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Abstract |
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; McMahon et al., 1993; Willis, 2001). Central sensitization resulting from neuronal plasticity in the dorsal horn of the spinal cord is a pivotal mechanism underling the development of hyperalgesia and allodynia (Woolf, 1983; Coderre et al., 1993). However, the specific pathways leading to central sensitization remain unclear.
A number of receptors, including the N-methyl-D-aspartate (NMDA) receptors, have been found to contribute to the development of central sensitization (Woolf and Thompson, 1991; Xu et al., 1992). Activation of these receptors initiates cascades of intracellular signaling events involving Ca2+ and various protein kinases (Lin et al., 1996; Malmberg et al., 1997). For example, activation of the NMDA receptors causes Ca2+ influx, leading to changes in neuronal plasticity (Womack et al., 1988). Considerable evidence has demonstrated that Ca2+-mediated cell signaling is important in nociception (Saegusa et al., 2001; Kim et al., 2003). One action of Ca2+ is through its activation of calmodulin, which in turn triggers the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is expressed in the superficial laminae of the dorsal horn of the spinal cord and in the small to medium diameter primary sensory neurons in dorsal root ganglia, where nociceptive signals are transmitted and processed (Bruggemann et al., 2000; Carlton, 2002). The function of CaMKII in pain is somewhat mixed. Fang et al. (2002) reported that CaMKII activity [determined by phosphorylated CaMKII (pCaMKII)] was significantly increased in the spinal cord within minutes after an intradermal injection of capsaicin (Fang et al., 2002). The second phase of formalin-induced paw-licking behavior was significantly reduced in CaMKII
(T286A) mutant mice that are unable to be autophosphorylated and activated; however, mutant and wild-type mice similarly showed decreased thermal and mechanical thresholds induced by complete Freund's adjuvant (CFA) or formalin (Zeitz et al., 2004). On the contrary, it was reported that KN93, a CaMKII inhibitor, was capable of preventing the development of thermal hyperalgesia and mechanical allodynia following chronic constriction injury (CCI) (Dai et al., 2005) or inferior alveolar nerve transaction (Ogawa et al., 2005). KN93 selectively and directly binds to the CaM-binding site of CaMKII, preventing the activation of CaMKII (Sumi et al., 1991). However, KN93 was found to be ineffective when given 7-day postinjury (0.25 µg/µl/h i.t. via an Alzet pump for 7 days) to reverse established hyperalgesia and allodynia in CCI model (Dai et al., 2005). It is not known whether the lack of acute effects by KN93 was dose-related. Previously, we found that different doses of CaMKII inhibitors were required to disrupt opioid tolerance depending on the degree of opioid tolerance (Tang et al., 2006a).
In this study, we tested the hypothesis that sufficient CaMKII inhibition is capable of acutely reversing already established inflammatory pain. Acute actions of CaMKII inhibitors in CFA-induced inflammatory pain was examined. Investigating an acute action of CaMKII inhibition in chronic pain is critical not only for understanding the mechanisms but more importantly for designing useful drug therapies of chronic inflammatory pain. Most patients seek medical treatment of chronic pain after the initial nerve or tissue injuries.
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Materials and Methods |
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) and approved by the University of Illinois Institutional Animal Care and Use Committee.
CFA-Induced Inflammatory Pain. Unilateral inflammation was induced by injecting 20 µl of CFA into mice dorsal surface of the left hindpaw (i.pl.) as described previously (Tang et al., 2007). Control mice received 20 µl of saline. Mice were tested for thermal hyperalgesia and mechanical allodynia before and 24 and 72 h after CFA injection.
Drug Administration. Intrathecal injection (i.t.) was given in a volume of 5 µl by percutaneous puncture through an intervertebral space at the level of the 5th or 6th lumbar vertebra, as described previously (Hylden and Wilcox, 1980; Wang et al., 2001). Success of the i.t. injection was verified by a lateral tail-flick. The pretreatment group was given KN93 (5–30 nmol i.t.) 30 min before CFA injection. For acute reversal experiments, mice were administered with KN93 (15–45 nmol i.t.) or KN92 (45 nmol i.t.) 2 h before pain testing on days 1 and 3 post-CFA injection. Trifluoperazine was administered i.p. (0.1–0.5 mg/kg) or via gastric gavage (1 mg/kg) 2 h before pain testing on day 1 post-CFA injection. In these studies, control mice received an equal volume of saline.
Thermal Hyperalgesia. The paw-withdrawal latencies to heat stimuli were measured using a plantar tester (model 7372; Ugo Basile, Comerio, Italy) as described previously (Hargreaves et al., 1988; Wang et al., 2001; Tang et al., 2007). Mice were placed in a clear plastic cage on a glass floor. After a 30-min period of habituation, paw-withdrawal latencies to radiant heat stimulation were measured. The radiant heat source was focused on the middle portion of the plantar surface of the left hindpaw and was automatically ceased when a paw-withdrawal occurred. A cut-off time of 20 s was applied to prevent tissue damage.
Mechanical Allodynia. Mechanical allodynia was measured using calibrated von Frey filaments (Stoelting, Wood Dale, IL) as described previously (Chaplan et al., 1994; Tang et al., 2007). In brief, mice were placed into individual Plexiglas containers with a wire mesh floor and allowed to acclimate for 30 min before testing. Each von Frey filament was applied perpendicularly to the midplantar surface for 5 s or until a withdrawal response had occurred. The up-down paradigm was used to determine 50% probability of paw-withdrawal threshold (Dixon, 1980; Chaplan et al., 1994; Wang et al., 2001).
CaMKII Activity Assay. The CaMKII activity was measured based on a previously published method (Ocorr and Schulman, 1991). Mouse brain tissues containing CaMKII were extracted with the extraction buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM EGTA, 10 mM sodium pyrophosphate, 25 mM benzamidine, 20 mM soybean trypsin inhibitor, 10 mM aprotinin, 5 mM leupeptin, 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. After homogenization and centrifugation (45,000g, 60 min), 2.5-µl supernatant (10 µg of protein) was added into 22.5 µl of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM dithiothreitol, 5 mM β-glycerol phosphate, 0.2 mM sodium orthovanadate, 1 µM calmodulin, 1 mM CaCl2, 0.02 mg/ml BSA, 100 µM ATP, 0.5 µCi of [
-32P]ATP (3000 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) and 0.1 mM autocamtide-2 at 30°C for 5min. To determine the Ca2+/calmodulin-independent protein kinase activity of CaMKII, reactions were carried out under the same condition except for the presence of 1 mM EGTA and omission of CaCl2 and calmodulin. Reactions were stopped by spotting onto P81 phosphocellulose paper and immediately washed with 75 mM H3PO4 three times. Incorporation of 32P was quantified by liquid scintillation counting of the P81 paper. Percentage of inhibition of CaMKII activity was calculated (Ocorr and Schulman, 1991).
Western Blotting Analysis. Immediately after the behavior test, lumbar sections of spinal cord were quickly dissected from euthanized mice and frozen on dry ice for Western blotting analysis as described previously (Tang et al., 2006a). In brief, tissues were homogenized using a glass homogenizer in 200 µl of radioimmunoprecipitation assay buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 5 mM EDTA in phosphate-buffered saline, pH 7.4] in the presence of phosphatase inhibitors (10 mM sodium fluoride, 10 mM sodium pyrophosphate, and 1 µM okadaic acid) and protease inhibitors (0.05 mg/ml bestatin, 0.05 mg/ml leupeptin, 0.05 mg/ml pepstatin, and 0.1 mg/ml phenylmethylsulfonyl fluoride). The homogenates were incubated on a rotator at 4°C for 2 h, and the soluble fraction was separated by centrifugation (45,000g, 60 min). Protein content in the supernatant was determined by a modified Bradford method (Pierce Biotechnology, Rockford, IL). Samples (60 µg of protein) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membrane. The membrane was preblocked in 5% nonfat milk in 20 mM Tris-buffered saline, pH 7.6, with 0.1% Tween 20 and probed with a rabbit anti-(T286)pCaMKII antibody (1:1000; Promega, Madison, WI). The membrane was then washed and incubated with a donkey anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (1:1000; Amersham, Piscataway, NJ), washed, and developed using an enhanced chemiluminescence detection system (ECL; Amersham). The membrane was then stripped and reprobed with a mouse anti-β-actin antibody (1: 10,000; Sigma) followed by another incubation with anti-mouse horseradish peroxidase-conjugated secondary antibody (1:10,000;
Amersham) and developed as above. ECL signals were captured by a ChemiDoc imaging system and analyzed using the Quantity One program (Bio-Rad, Hercules, CA). Ratios of the optical densities of pCaMKII to those of β-actin were calculated for each sample.
Statistical Analysis. Comparisons between groups were analyzed using a two-way repeated measures analysis of variance. Student-Newman-Keuls test was used as a post-hoc test. Statistical significance was established at the 95% confidence limit.
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), significantly blocked the development of mechanical allodynia (Fig. 1A) and thermal hyperalgesia (Fig. 1B) (p < 0.001 compared with the CFA group, n = 8). We further examined the CaMKII activity (pCaMKII) after CFA injection by the Western blotting analysis and found that pCaMKII was significantly up-regulated by CFA [Fig. 2, lane 2, p < 0.05 compared with the control group (lane 1), n = 4]. Pretreatment with KN93 (30 nmol i.t.) prevented the CFA-induced increase of pCaMKII (Fig. 2, lane 6, p < 0.05 compared with the CFA group, n = 4). Pretreatment with CFA or CFA + KN93 (30 nmol) did not seem to significantly alter CaMKII protein expression, although there is a general trend of slight up-regulation (Fig. 3). KN93 (15 nmol) was partially effective in blocking the CFA-induced mechanical allodia but not thermal hyperalgesia (Fig. 1). At an even lower dose, KN93 (5 nmol) was not active in either assay. ED50 values for KN93 were estimated to be 15.9 (blocking allodynia) and 19.4 nmol (blocking hyperalgesia).
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Effect of Acute CaMKII Inhibition on CFA-Induced Inflammatory Pain. We next tested whether acute CaMKII inhibition could reverse established CFA-induced pain behavior. KN93 (45 nmol i.t.) reversed CFA-induced mechanical allodynia (Fig. 4A) and thermal hyperalgesia (Fig. 4B) on day 1 post-CFA injection (p < 0.001 compared with the CFA group, n = 8). KN93 at a lower dose (30 nmol) was partially effective (p < 0.01 compared either with the control or the CFA group, n = 8) in reversing CFA-induced thermal hyperalgesia but not mechanical allodynia. KN93 at an even lower dose (15 nmol) did not affect CFA-induced allodynia or hyperalgesia (Fig. 4). CFA-increased CaMKII activity was significantly attenuated by KN93 only at the higher dose (45 nmol) (Fig. 2, lane 5, p < 0.05 compared with the CFA group, n = 4) but not at the lower dose (30 nmol) (Fig. 2, lane 4, p > 0.05). Therefore, KN93 dose-dependently reversed mechanical allodynia and thermal hyperalgesia, consistent with the inhibitor's action on CaMKII activity. ED50 values were estimated to be 30.2 (antiallodynia) and 29.5 nmol (antihyperalgesia). On day 3 post-CFA, allodynia and hyperalgesia had returned in the mice that were treated CFA + KN93 (data not plotted). In these mice, another dose of KN93 (45 nmol) on day 3 was able to again reverse the allodynia and hyperalgesia (Fig. 4). Treatment with KN92 (45 nmol i.t.) did not affect CFA-induced CaMKII activation (Fig. 2, lane 3, p > 0.05 compared with the CFA group, n = 4), allodynia, or hyperalgesia (Fig. 4, p > 0.05 compared with the CFA group, n = 8). Neither KN93 nor KN92 (45 nmol i.t.) caused changes in gross behavior or nociception baseline in naive mice (Fig. 5).
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To determine a more precise time course of the action of KN93 (i.t.) in reversing CFA-induced allodynia and hyperalgesia, another series of experiments was performed in which the thermal and mechanical sensitivities were monitored for up to 24 h post-KN93 in CFA-treated mice (Fig. 6). The effect of KN93 (30 and 45 nmol) started at 30 min, the first testing time point, and peaked at 2 h in both allodynia and hyperalgesia studies. Antihyperalgesic effect of KN93 (30 and 45 nmol) lasted for at least 4 h, whereas the antiallodynic action was marginally significant at 4 h only for the highest dose (p < 0.05 compared with the CFA group, n = 8). KN93 at 15 nmol did not affect either thermal or mechanical sensitivity. At the time of the peak effect (2 h), ED50 values were estimated to be 30.3 (antiallodynia) and 28.7 nmol (antihyperalgesia). By the next day (24 h), all KN93 and CFA-treated mice achieved essentially the same thermal and mechanical sensitivities compared with the mice treated with CFA alone (p < 0.001 compared with the control, n = 8).
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). In the current study, we further verified that trifluoperazine inhibited CaMKII activity, with an EC50 = 14.4 ± 1.1 µM, in an in vitro CaMKII enzymatic assay (Fig. 7A). This drug, therefore, not only provides another test of our hypothesis but more importantly may ultimately provide a drug therapy targeting the CaMKII signaling pathway. Trifluoperazine (0.5 mg/kg i.p.) completely reversed mechanical allodynia (Fig. 8A) and thermal hyperalgesia (Fig. 8B) induced by CFA (p < 0.001 compared with the CFA group, n = 8). Correlating with its behavioral effect, trifluoperazine was found to suppress CFA-induced increase of spinal CaMKII activation (pCaMKII) in mice (Fig. 7B). The drug exhibited a partial effect in alleviating mechanical allodynia at a lower dose (0.25 mg/kg i.p.) (p < 0.05 compared with the CFA group, n = 8). At an even lower dose (0.1 mg/kg i.p.) trifluoperazine had no effect on CFA-induced hyperalgesia or allodynia (Fig. 8). Therefore, trifluoperazine was able to reverse CFA-induced inflammatory pain in a dose-dependent manner. ED50 values were estimated to be 0.3 (antiallodynia) and 0.3 mg/kg (antihyperalgesia).
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To examine the time course of the action of trifluoperazine in reversing CFA-induced allodynia and hyperalgesia, thermal and mechanical sensitivities were monitored for up to 48 h in CFA-pretreated mice that received a single dose of trifluoperazine (0.5 mg/kg i.p.) (Fig. 9). Trifluoperazine showed a rapid onset of reversing the established CFA-induced thermal hyperalgesia, even at the first time point (30 min). The antihyperalgesic effect peaked at 2 to 4 h and lasted for at least 8 h. In the mechanical sensitivity experiments, the drug not only exhibited potent antiallodynic action, it further produced analgesia (p < 0.05 compared with the control at the time of the peak effect = 4h, n = 8). The potential analgesic action was followed up in experiments in which naive mice were given a single intraperitoneal injection of trifluoperazine (0.5 mg/kg). During the first 30 to 60 min, trifluoperazine did not alter baseline mechanical or thermal nociception (Fig. 10, p > 0.05 compared with the predrug baseline, n = 8). Given that antiallodynic and anti-hyperalgesic actions of trifluoperazine had a rapid onset (30 min), these data suggest that its antiallodynic and antihyperalgesic actions do not entirely depend on its analgesia. At 2 to 4 h postinjection, trifluoperazine produced significant analgesia by itself, although the magnitude was smaller than that of antiallodynic and antihyperalgesic actions. In addition, the duration of action was shorter than that of antiallodynic and antihyperalgesic actions. At 8 h postinjection when antiallodynic and antihyperalgesic actions were still observed, trifluoperazine produced a weak but significant thermal hyperalgesic effect.
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; Ogawa et al., 2005).
The main hypothesis that CaMKII is required for the maintenance of inflammatory pain was further tested. Such studies are critical for designing new pain therapies based on the CaMKII signaling pathways, because most patients present with long-lasting abnormal pain after, not before, nerve or tissue injuries. A previous study found KN93 (given i.t. 7-day postinjury, 0.25 µg/µl/h via an Alzet pump for 7 days) to be ineffective in reversing already established neuropathic pain after CCI (Dai et al., 2005). In contrast, it was reported that KN93 (120 pmol i.t.) and myristoylautocamtide 2-related inhibitory peptide (1 nmol i.t.) effectively reversed CCI-induced neuropathic pain in mice, although the exact experimental details were not provided (Garry et al., 2003). Our data demonstrated that KN93 was effective in reversing established CFA-induced inflammatory pain. The discrepancy could have been caused by different experimental models of pain or animals used in these studies. An alternative explanation may be related to the degree of CaMKII inhibition. Compared with the dose for preventing CFA-induced inflammatory pain, a higher dose of KN93 was necessary to reverse CFA-induced hyperalgesia and allodynia. When we attempted to reverse CFA-induced hyperalgesia and allodynia with KN93 at lower doses (15–30 nmol), the drug was partially effective or not effective. As the dose was increased to 45 nmol, CFA-induced inflammatory pain was acutely reversed. The degree of CaMKII inhibition in relation to the CaMKII activity in the pain state was further supported by biochemical data indicating that CaMKII activity was up-regulated considerably in the pain state (Fig. 2, lane 2 compared with lane 1). Furthermore, KN93 at the higher dose (45 nmol; Fig. 2, lane 5), but not at the lower dose (30 nmol; Fig. 2, lane 4), was effective in reducing the up-regulated CaMKII activity. As an agent to prevent CFA-induced pain, KN93 at 30 nmol was sufficient to prevent the up-regulation of CaMKII activity (Fig. 2, lane 6 compared with lane 2). The increase in pCaMKII was largely caused by the increased activity, as CaMKII expression was not significantly increased by the pretreatment with CFA or CFA + KN93. Therefore, different doses of CaMKII inhibitors were required to produce sufficient CaMKII inhibition depending on the activity of CaMKII that was heightened in the chronic pain states. A similar observation has been made previously in the studies of opioid tolerance in which higher doses of CaMKII inhibitors were required to achieve sufficient CaMKII inhibition when the degree of opioid tolerance was increased (Tang et al., 2006a). Data from our and two other laboratories (Garry et al., 2003; Dai et al., 2005; Ogawa et al., 2005) have so far suggested an important role of CaMKII in chronic pain, which was not supported by a study employing CaMKII(T286A) mutant mice (Zeitz et al., 2004). These mice express a mutant form of CaMKII that cannot be autophosphorylated and activated. Whereas the second phase of formalin-induced paw-licking behavior was significantly reduced in CaMKII(T286A) mutant mice, wild-type and mutant mice showed similar CFA- or formalin-induced thermal and mechanical pain thresholds (Zeitz et al., 2004). The exact reason for the discrepancy is not known; however, two different approaches are used in these studies to chemically inhibit or genetically eliminate the normal function of CaMKII. Interpretation of experiments applying chemical inhibitors can be compromised by the potential lack of selectivity of the inhibitors used. On the other hand, mouse genetic mutation studies can suffer from problems association with gene manipulation, such as unmatched genetic background, compensatory changes, or other nonspecific genomic effects.
The acute action of CaMKII inhibitors in reversing established pain was further supported by the experiments employing the antipsychotic drug trifluoperazine, which is a calmodulin inhibitor and suppresses CaMKII activity (Tang et al., 2006b). Therefore, trifluoperazine not only presents a unique opportunity to test our hypothesis but more importantly may provide a drug candidate for alleviating chronic pain in clinical settings. Systemic trifluoperazine (i.p.) dose-dependently reversed mechanical allodynia and thermal hyperalgesia in CFA-treated mice. The drug was also effective given orally. These data suggested that the action of CaMKII inhibitors was not limited to spinal intervention. Trifluoperazine (i.t.) has been previously reported to produce either analgesia (at low doses) or hyperalgesia (at a high dose) in a formalin-induced inflammatory pain model (Golbidi et al., 2002). The doses we used would have been considered as "low doses", although the exact conversion was not possible due to different routes of administration. Our data suggested that the analgesia/hyperalgesia balance might also depend on the duration of treatment. However, the antiallodynic and anti-hyperalgesic actions of trifluoperazine in the current study could not be fully attributed to its analgesic action, because the former had an earlier onset, larger magnitude of effect, and longer duration of action. In fact, at 8 h postinjection when analgesia had disappeared and trifluoperazine produced a weak thermal hyperalgesic effect in naive mice, antiallodynic and antihyperalgesic actions persisted in CFA-treated mice.
How persistent CaMKII activation is achieved in pain states is puzzling, because one would probably expect desensitization of a kinase after prolonged activation. One plausible mechanism may be through the interaction of CaMKII with NMDA receptors. It has been demonstrated that CaMKII can phosphorylate NMDA receptors and enhance receptor function (McGlade-McCulloh et al., 1993; Lau and Huganir, 1995). Phosphorylation of the NMDA receptor is a key means to regulate the function of this ligand-gated ion channel, which is most permeable to Ca2+. Phosphorylation of NMDA receptors by CaMKII has been shown to enhance the NMDA receptor function, leading to the influx of Ca2+ through the channels (Kitamura et al., 1993). Therefore, activation of CaMKII as a result of inflammation and nerve injury can potentially increase the activity of the NMDA receptors leading to Ca2+ influx. Increased cytosolic Ca2+ ions bind and change the conformation of CaM, which in turn leads to the activation of more CaMKII (Strack et al., 1997). Therefore, a feed-forward loop may exist between CaMKII and the NMDA receptors in the chronic pain state.
Indeed, inflammatory injury has been shown to increase levels of glutamate in the spinal dorsal horn (Sluka and Westlund, 1992), and this was blocked by NMDA receptor antagonists (Sluka and Westlund, 1993). It has also been demonstrated that NMDA receptor antagonists administered before inflammatory or peripheral nerve injuries suppress or delay the onset of hyperalgesia and attenuate fully developed hyperalgesia when administered after the injury (Zhang et al., 1998; Dai et al., 2005). Overexpression of the NR2B subunit of the NMDA receptor causes increased inflammatory mechanical allodynia, whereas knockdown of spinal NMDA receptors with antisense oligonucleotides prevents the expression of chemically induced hyperalgesia (Garry et al., 2000; Wei et al., 2001). Therefore, CaMKII may work in concert with the NMDA receptors in the development and maintenance of the neural events leading to hyperalgesia.
In conclusion, our findings suggest the critical involvement of CaMKII in the process of maintaining and/or inducing persistent inflammatory pain. Blocking the CaMKII signaling pathway may provide a useful therapeutic target for the treatment of chronic pain. Trifluoperazine at relatively (to its antipsychotic effect) low doses was found to be highly efficacious in reversing the CFA-induced inflammatory pain. Although the drug has multiple pharmacologic effects and is not expected to be selective at CaMKII, it is approved by the United States Food and Drug Administration as an orally available antipsychotic drug that has been used in humans for many years. Therefore, we propose that trifluoperazine should be tested in clinical settings for the treatment of chronic pain.
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Footnotes |
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F.L. is a visiting fellow from the Department of Anesthesiology, Tongji Medical College, Wuhan, China.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NMDA, N-methyl-D-aspartate; CaMKII, Ca2+/calmodulin-dependent protein kinase II; pCaMKII, phosphorylated CaMKII; CFA, complete Freund's adjuvant; CCI, chronic constriction injury; KN93, [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine); KN92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; i.pl. intarplantarly.
Address correspondence to: Dr. Zaijie Jim Wang, MC865, University of Illinois, 833 South Woods Street, Chicago, IL 60612. E-mail: zjwang{at}uic.edu
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2 h postdrug injections) on day 1 post-CFA.
