Spinal Serotonin 1A Receptor Contributes to the Analgesia of Acupoint Catgut Embedding by Inhibiting Phosphorylation of the N-Methyl-d-Aspartate Receptor GluN1 Subunit in Complete Freund's Adjuvant-Induced Inflammatory Pain in Rats

Acupoint catgut embedding (ACE) is a widely used traditional Chinese medicine method to manage various diseases, including chronic inflammatory pain. We sought to assess the possible analgesic effects of ACE in comparison with electroacupuncture (EA) and to study the analgesic mechanisms of ACE in a rat model of inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the hind paw of rats. The von Frey, radiant heat, and gait analysis tests were performed to evaluate the analgesic effects of ACE and EA, and Western blot and immunohistochemistry assays were carried out to determine the molecular mechanisms of ACE. ACE treatments were administered every 4 days or every week with different acupoints (ipsilateral, contralateral, or bilateral ST36 and GB30 acupoints). The most effective ACE strategy for attenuating the nocifensive response induced by CFA injection was performing ACE once a week at ipsilateral ST36 in combination with GB30. EA treatment every other day at ipsilateral ST36 and GB30 showed comparable analgesic effects. ACE inhibited the increased activation of the GluN1 subunit of the N-methyl-d-aspartate receptor and the subsequent Ca²⁺-dependent signals (CaMKII, ERK, and CREB) that take place in response to CFA. The effects of ACE were similar to intrathecal injection of vilazodone (a serotonin 1A receptor [5-HT_1AR] agonist) and were blocked by WAY-100635 (a 5-HT_1AR antagonist). In summary, we show that ACE attenuates CFA-induced inflammatory pain in rats by activating spinal 5-HT_1AR and by inhibiting the phosphorylation of GluN1, thus, inhibiting the activation of Ca²⁺-dependent signaling cascades.

Endogenous Anandamide and Cannabinoid Receptor-2 Contribute to Electroacupuncture Analgesia in Rats

Acupuncture is widely used clinically to treat acute and chronic pain conditions, but the mechanisms underlying its effect are not fully understood. Although endocannabinoids are involved in modulation of nociception in animal models and in humans, their role in acupuncture analgesia has not been assessed. In this report, we determined the effect of electroacupuncture (EA) on the level of anandamide in the skin tissue and the role of cannabinoid CB1 and CB2 receptors in the analgesic effect of EA in an animal model of inflammatory pain. Inflammatory pain was induced by local injection of complete Freund's adjuvant (CFA) into the hind paw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified with von Frey filaments. The anandamide concentration in the skin tissue was measured by using high-performance liquid chromatography. EA, applied to GB30 and GB34, at 2 and 100Hz significantly reduced thermal hyperalgesia and mechanical allodynia induced by CFA injection. Compared with the sham group, EA significantly increased the anandamide level in the inflamed skin tissue. Local pretreatment with a specific CB2 receptor antagonist, AM630, significantly attenuated the antinociceptive effect of EA. However, the effect of EA was not significantly altered by AM251, a selective CB1 receptor antagonist. These findings suggest that EA potentiates the local release of endogenous anandamide from inflamed tissues. Activation of peripheral CB2 receptors contributes to the analgesic effect of EA on inflammatory pain.PerspectiveThis study shows that electroacupuncture increases the anandamide level in inflammatory skin tissues, and CB2 receptors contribute to the analgesic effect of electroacupuncture in a rat model of inflammatory pain. This information improves our understanding of the mechanisms involved in the analgesic effect of acupuncture.     

鞘内注射抗生长抑素血清对佐剂性关节炎大鼠电针镇痛及中枢β-EP水平的影响

目的观察鞘内注射抗生长抑素血清（ASSS）对佐剂性关节炎（AA）大鼠痛阈、电针（EA）镇痛及中枢β-内啡肽（-βEP）水平的影响,探讨其可能的镇痛机制。方法以AA大鼠为炎症痛模型,以痛阈为指标观察鞘内注射ASSS对痛阈和EA镇痛的影响;以放免法测定AA大鼠下丘脑、脊髓中-βEP含量,观察鞘内注射ASSS及EA镇痛对其影响。结果鞘内注射ASSS可显著降低AA大鼠痛阈,并使EA镇痛效应明显降低;EA镇痛可提高下丘脑和脊髓-βEP含量,鞘内注射ASSS可使中枢-βEP水平进一步显著升高。结论内源性生长抑素在痛觉调制中起重要作用,有显著的镇痛效果并与增强EA镇痛作用密切相关;其镇痛机制可能与中枢-βEP水平相关。     

Acupuncture analgesia in a new rat model of ankle sprain pain

The lack of suitable experimental animal models for persistent pain showing clear acupuncture analgesia, has been the major stumbling block in the investigation of the physiological mechanisms of acupuncture analgesia. The present study developed a new rat model of ankle sprain pain and the effect of electroacupuncture (EA) on this model was examined. A common source of persistent pain in humans is the lateral ankle sprain. To model this condition, the rat's right ankle was bent repeatedly, overextending lateral ligaments, for 4 min under halothane anesthesia. The rat subsequently showed swelling of the ankle and a reduced stepping force of the affected limb for the next several days. The reduced stepping force of the limb was presumably due to a painful ankle since systemic injection of morphine produced temporary improvement of weight bearing. EA was applied to the SI-6 acupuncture point on the contralateral forelimb for 30 min under halothane anesthesia. After the termination of EA, behavioral tests measuring stepping force were periodically conducted during the next 4h. EA produced a 40% recovery in the stepping force of the sprained foot lasting for at least 2h. The magnitude of this improvement was equivalent to that obtained after a systemic injection of 2mg/kg of morphine and this improvement of stepping pressure was interpreted as an analgesic effect. The analgesic effect was specific to the acupuncture point since (1). the analgesic effect on the ankle sprain pain model could not be mimicked by EA applied to a nearby point, LI-4 and (2). EA applied to the SI-6 point was not effective in the knee arthritis pain model. The analgesic effect could not be blocked by systemic injection of opioid antagonists naloxone or naltrexone. These data suggest that EA produces a potent analgesic effect on the ankle sprain pain model in the rat. This analgesic effect is produced by applying EA to a site remote from the painful area in a stimulus point-specific way. The present study provides a powerful experimental animal model that can be used for investigating the unique physiological mechanisms involved in acupuncture analgesia.     

N-Methyl-d-aspartate receptor (NMDAR) independent maintenance of inflammatory pain

Following peripheral inflammation, NMDA receptor (NMDAR) activation in spinal cord dorsal horn neurons facilitates the generation of pain in response to low threshold inputs (allodynia) and signals the phosphorylation of protein kinase C (pPKC) and extracellular signal-regulated kinase 2 (pERK2). Intraplantar complete Freund’s adjuvant (CFA) induces inflammatory nociception (allodynic pain) at 24 hours (h) with a concurrent increase in neuronal pPKCγ and pERK2 but not glial pERK2. These effects are attenuated in a spatial knockout of the NMDAR (NR1 KO) confined to SCDH neurons. Although glia and proinflammatory cytokines are implicated in the maintenance of inflammatory pain and neuronal activation, the role of NMDARs and neuronal–glial–cytokine interactions that initiate and maintain inflammatory pain are not well defined. In the maintenance phase of inflammatory pain at 96 h after CFA the NR1 KO mice are no longer protected from allodynia and the SCDH expression of pPKCγ and pERK2 are increased. At 96 h the expression of the proinflammatory cytokine, IL-1β, and pERK2 are increased in astrocytes. Intrathecal IL-1 receptor antagonist (IL-1ra), acting on neuronal IL-1 receptors, completely reverses the allodynia at 96 h after CFA. Deletion of NMDAR-dependent signaling in neurons protects against early CFA-induced allodynia. Subsequent NMDAR-independent signaling that involves neuronal expression of pPKCγ and the induction of pERK2 and IL-1β in activated astrocytes contributes to the emergence of NMDAR-independent inflammatory pain behavior at 96 h after CFA. Effective reduction of the initiation and maintenance of inflammatory pain requires targeting the neuron–astrocyte–cytokine interactions revealed in these studies.     

Dynorphin: important mediator for electroacupuncture analgesia in the spinal cord of the rabbit

Intrathecal injection of 12 nmol of dynorphin elicited marked analgesia as measured by tail flick latency, the effect being about 20 times more potent than with morphine. This analgesic effect could be reversed by naloxone at a dose 1.5-fold higher than that needed to reverse morphine analgesia. Intrathecal injection of anti-dynorphin antibody blocked electroacupuncture (EA) analgesia by 77%, the effect lasting for at least 4 h. In rabbits made tolerant to EA analgesia by long-term EA stimulation, intrathecal injection of dynorphin no longer exhibited an analgesic effect. No analgesia was noticed when dynorphin (10 nmol) was injected into the periaqueductal grey (PAG) of the rabbit, nor was EA analgesia blocked by anti-dynorphin antibody injected into PAG. These results suggest that dynorphin reduces nocifensive responses in the spinal cord and may play an important role in mediating EA analgesia at the spinal level.     

From Peripheral to Central: The Role of ERK Signaling Pathway in Acupuncture Analgesia

Despite accumulating evidence of the clinical effectiveness of acupuncture, its mechanism remains largely unclear. We assume that molecular signaling around the acupuncture needled area is essential for initiating the effect of acupuncture. To determine possible bio-candidates involved in the mechanisms of acupuncture and investigate the role of such bio-candidates in the analgesic effects of acupuncture, we conducted 2 stepwise experiments. First, a genome-wide microarray of the isolated skin layer at the GB34-equivalent acupoint of C57BL/6 mice 1 hour after acupuncture found that a total of 236 genes had changed and that extracellular signal–regulated kinase (ERK) activation was the most prominent bio-candidate. Second, in mouse pain models using formalin and complete Freund adjuvant, we found that acupuncture attenuated the nociceptive behavior and the mechanical allodynia; these effects were blocked when ERK cascade was interrupted by the mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) inhibitor U0126 (.8 μg/μL). Based on these results, we suggest that ERK phosphorylation following acupuncture needling is a biochemical hallmark initiating the effect of acupuncture including analgesia.PerspectiveThis article presents the novel evidence of the local molecular signaling in acupuncture analgesia by demonstrating that ERK activation in the skin layer contributes to the analgesic effect of acupuncture in a mouse pain model. This work improves our understanding of the scientific basis underlying acupuncture analgesia.     

Electroacupuncture reduces the expression of proinflammatory cytokines in inflamed skin tissues through activation of cannabinoid CB2 receptors

Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it is not clear how CB2R activation contributes to the antinociceptive effect of EA. The major proinflammatory cytokines, such as tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and IL‐6, are involved in inflammatory pain. Here we determined the effects of CB2R activation and EA on the expression level of IL‐1β, IL‐6 and TNF‐α in inflamed skin tissues. Inflammatory pain was induced by injection of complete Freund's adjuvant into the left hindpaw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified using von Frey filaments. The mRNA and protein levels of IL‐1β, IL‐6 and TNF‐α in inflamed skin tissues were measured using real‐time polymerase chain reaction and Western blot, respectively. Local injection of the selective CB2R agonist AM1241 or EA applied to GB30 and GB34 significantly reduced thermal hyperalgesia and mechanical allodynia induced by tissue inflammation. The specific CB2R antagonist AM630 significantly attenuated the antinociceptive effect of EA. Furthermore, EA or AM1241 treatment significantly decreased the mRNA and protein levels of IL‐1β, IL‐6 and TNF‐α in inflamed skin tissues. In addition, pretreatment with AM630 significantly reversed the inhibitory effect of EA on these cytokine levels in inflamed skin tissues. Our results suggest that EA reduces inflammatory pain and proinflammatory cytokines in inflamed skin tissues through activation of CB2Rs.     

Electroacupuncture-induced analgesia in a rat model of ankle sprain pain is mediated by spinal alpha-adrenoceptors

In a previous study, we showed that electroacupuncture (EA) applied to the SI-6 point on the contralateral forelimb produces long-lasting and powerful analgesia in pain caused by ankle sprain in a rat model. To investigate the underlying mechanism of EA analgesia, the present study tested the effects of various antagonists on known endogenous analgesic systems in this model. Ankle sprain was induced in anesthetized rats by overextending their right ankle with repeated forceful plantar flexion and inversion of the foot. When rats developed pain behaviors (a reduction in weight-bearing of the affected hind limb), EA was applied to the SI-6 point on the contralateral forelimb for 30 min under halothane anesthesia. EA significantly improved the weight-bearing capacity of the affected hind limb for 2h, suggesting an analgesic effect. The alpha-adrenoceptor antagonist phentolamine (2mg/kg, i.p. or 30 microg, i.t.) completely blocked the EA-induced analgesia, whereas naloxone (1mg/kg, i.p.) failed to block the effect. These results suggest that EA-induced analgesia is mediated by alpha-adrenoceptor mechanisms. Further experiments showed that intrathecal administration of yohimbine, an alpha(2)-adrenergic antagonist, reduced the EA-induced analgesia in a dose-dependent manner, whereas terazosin, an alpha(1)-adrenergic antagonist, did not produce any effect. These data suggest that the analgesic effect of EA in ankle sprain pain is, at least in part, mediated by spinal alpha(2)-adrenoceptor mechanisms.     

Electro-acupuncture attenuates behavioral hyperalgesia and selectively reduces spinal Fos protein expression in rats with persistent inflammation.

This study examined the effect of electro-acupuncture (EA) on persistent inflammatory hyperalgesia in a rat model. Inflammation and hyperalgesia were induced by injecting complete Freund's adjuvant (CFA) into one hindpaw of the rat. Hyperalgesia was determined by a decrease in paw withdrawal latencies (PWL) to a noxious thermal stimulus. EA was applied bilaterally at the acupuncture point Huantiao (G30) at the rat's hindlimbs. EA-treated rats (n = 11) had significantly longer PWLs as compared with placebo control rats (n = 7) in the inflamed paw at 2.5 hours and 5 days after injection of CFA (P <.05) and longer PWLs as compared to sham control rats (n = 9) at 2.5 hours (P >.05). Paw edema was significantly reduced in EA-treated rats versus placebo controls at 24 hours after inflammation (P <.01). Inflammation-induced spinal Fos expression in the medial half of laminae I-II in EA-treated rats versus placebo rats (n = 5 per group) was significantly reduced (P <.01). These data showed that EA delayed the onset and facilitated the recovery of inflammatory hyperalgesia and suppressed the inflammation-induced spinal Fos expression in neurons (laminae I-II) involved in receiving noxious stimulation. This rat model of persistent pain and inflammation seems to be an ideal animal model for studying the effect of acupuncture.     

Electroacupuncture Alleviates Mechanical Allodynia in a Rat Model of Complex Regional Pain Syndrome Type-I via Suppressing Spinal CXCL12/CXCR4 Signaling

Complex regional pain syndrome (CRPS) results in chronic and excruciating pain in patients. Conventional therapies lack effectiveness, rendering it one of the most difficult to treat neurological conditions.. Electroacupuncture (EA) is an effective alternative therapy for pain relief. Here, we investigated whether EA exerts analgesic effect on a rat model of CRPS type-I (CRPS-I) and related mechanisms. The rat chronic postischemic pain (CPIP) model was established to mimic CRPS-I. 100Hz EA exerted robust and persistent antiallodynic effect on CPIP model compared with 2 Hz EA or sham EA. EA markedly suppressed the overexpression of CXCL12/CXCR4 in spinal cord dorsal horn (SCDH) of CPIP model, leading to substantial decrease in neuronal and glial cell activities in SCDH. Pharmacological blocking CXCR4 mimicked EA-induced antiallodynic effect and related cellular events in SCDH, whereas exogenous CXCL12 abolished EA's effect. CXCR4 signaling resulted in ERK activation in SCDH, contributing to mechanical allodynia of CPIP model rats, whereas EA markedly reduced ERK activation. Therefore, we demonstrated that EA interferes with CXCL12/CXCR4 signaling in SCDH and downstream ERK pathway to exert robust antiallodynic effect on an animal model of CRPS-I. Our work suggests that EA may be a potential therapeutic option for CRPS-I in clinic.PerspectiveOur work identified that EA exerts robust antiallodynic effect on an animal model of CRPS-I, via mechanisms involving inhibition of CXCL12/CXCR4 signaling. EA further attenuates downstream neuronal and glial cell activation and ERK pathway in SCDH. This work suggests that EA may be a potential therapeutic option for CRPS-I management in clinic.     

硬膜外持续镇痛与静脉持续镇痛用于开胸术后患者的镇痛效果观察

目的 探讨硬膜外持续镇痛与静脉持续镇痛方法用于开胸手术的镇痛效果和安全性.方法 选择60例ASA Ⅰ～Ⅱ级行开胸手术的患者随机分为硬膜外镇痛组(EA)和静脉镇痛组(IA).记录两组患者术后4 h、12 h的镇痛评分、术后使用镇痛药物的情况、监测动脉血气的pH、PCO:和SpO2情况.结果 应用硬膜外持续镇痛与静脉持续镇痛均可取得满息的镇痛效果,EA组患者安静与活动时的疼痛评分低于IA组(P＜0.05);术后动脉血气分析显示两组患者的pH、PCO2和SpO2差异无统计学意义.结论 对于开胸手术,应用硬膜外持续镇痛与静脉持续镇痛均可取得满意的镇痛效果.硬膜外持续镇痛效果优于静脉持续镇痛,两种不同镇痛方式对术后患者酸碱平衡,PCO2和SpO2的影响无明显差异.     

Spinal mitochondrial-derived peroxynitrite enhances neuroimmune activation during morphine hyperalgesia and antinociceptive tolerance

Treatment of severe pain by morphine, the gold-standard opioid and a potent drug in our arsenal of analgesic medications, is limited by the eventual development of hyperalgesia and analgesic tolerance. We recently reported that systemic administration of a peroxynitrite (PN) decomposition catalyst (PNDC) or superoxide dismutase mimetic attenuates morphine hyperalgesia and antinociceptive tolerance and reduces PN-mediated mitochondrial nitroxidative stress in the spinal cord. These results suggest the potential involvement of spinal PN signaling in this setting; which was examined in the present study. PN removal with intrathecal delivery of manganese porphyrin-based dual-activity superoxide/PNDCs, MnTE-2-PyP⁵⁺ and the more lipophilic MnTnHex-2-PyP⁵⁺, blocked hyperalgesia and antinociceptive tolerance in rats. Noteworthy is that intrathecal MnTnHex-2-PyP⁵⁺ prevented nitration and inactivation of mitochondrial manganese superoxide dismutase. Mitochondrial manganese superoxide dismutase inactivation enhances the superoxide-to-PN pathway by preventing the dismutation of superoxide to hydrogen peroxide, thus providing an important enzymatic source for PN formation. Additionally, intrathecal MnTnHex-2-PyP⁵⁺ attenuated neuroimmune activation by preventing the activation of nuclear factor kappa B, extracellular-signal-regulated kinase and p38 mitogen activated protein kinases, and the enhanced levels of proinflammatory cytokines, interleukin (IL)-1β and IL-6, while increasing anti-inflammatory cytokines, IL-4 and IL-10. The role of PN was further confirmed using intrathecal or oral delivery of the superoxide-sparing PNDC, SRI-110. These results suggest that mitochondrial-derived PN triggers the activation of several biochemical pathways engaged in the development of neuroinflammation in the spinal cord that are critical to morphine hyperalgesia and tolerance, further supporting the potential of targeting PN as an adjunct to opiates to maintain pain relief.     

'Knock-down' of spinal CB1 receptors produces abnormal pain and elevates spinal dynorphin content in mice

Recent studies demonstrate the possible existence of tonic modulatory control of nociceptive input mediated by spinal cannabinoid receptors (CB1). Accordingly, it is predicted that a reduction in the spinal CB1 receptors may enhance sensitivity to sensory stimuli and a decrease in spinal antinociceptive potency to cannabinoid agonists. An antisense oligodeoxynucleotide (ODN) specific to the CB1 receptor was used to 'knock-down' CB1 receptors in the lumbar spinal cord and dorsal root ganglia by the local, repeated intrathecal (i.th.) administration of the ODN. This treatment resulted in a decrease in lumbar spinal CB1 receptor expression accompanied by a decrease in the response thresholds to both innocuous tactile and noxious thermal stimuli. The antinociceptive action of the CB1 agonist, WIN 55,212-2, by i.th. administration was also significantly attenuated after treatment with the antisense ODN. Similar treatment using a mismatch control ODN had no effect on receptor protein or on sensory thresholds. The effects of the antisense ODN treatment on sensory thresholds were fully reversed after discontinuation of the ODN injection. The antisense ODN treated rats also showed a significant increase in lumbar spinal dynorphin A. Acute i.th. injection of MK-801 or an antidynorphin antiserum blocked the antisense ODN-induced tactile and thermal hypersensitivity. These data support the possibility of endogenous inhibitory cannabinoid tone to limit spinal afferent input of thermal and tactile stimuli. Lifting of this inhibitory tone through a 'knock-down' of spinal CB1 receptors apparently lowers the thresholds for sensory input, as reflected by the actions of MK-801 to block tactile and thermal hypersensitivity. The increased spinal dynorphin may act to further promote afferent outflow and abnormal pain because sequestration of spinal dynorphin with antiserum also reverses the manifestations of abnormal pain following knock-down of CB1 receptors.     

A psychophysical analysis of acupuncture analgesia

A psychophysical analysis of acupuncture analgesia was carried out in which low back pain patients made visual analogue scale (VAS) responses both to their chronic pain and to different levels of experimental heat pain (5 sec exposures to 43–51 °C) before and after electroacupuncture. VAS (sensory) responses to chronic pain, direct temperature matches to chronic pain, and VAS (sensory) responses to experimental pain were reduced in an internally consistent manner 1–2 h after treatment. The magnitude of this analgesic response was similar for dermatomes within the region of chronic pain and acupuncture needle placement (lower back) as well as for dermatomes remote from needle placement and chronic pain (forearm). Individual patients manifested either (1) a central-inhibitory pattern of analgesia wherein experimental and chronic pain in back regions and experimental pain in forearm regions were reduced, or (2) an origin-specific pattern wherein only the chronic low back pain was reduced. Patients tested several days after treatment all manifested the latter pattern of analgesia. VAS sensory and VAS affective analgesic responses to electroacupuncture treatment showed a delayed onset (1–24 h) to maximum effect and a duration of 10–14 days. Cumulative sensory and affective analgesic effects were observed at the end of 4 months of biweekly acupuncture treatments. The results of this analysis reveal the unique spatial and temporal properties of electroacupuncture analgesia and the extent to which it is mediated by at least two different mechanisms.     

Electroacupuncture suppresses capsaicin-induced secondary hyperalgesia through an endogenous spinal opioid mechanism

Central sensitization, caused either by tissue inflammation or peripheral nerve injury, plays an important role in persistent pain. An animal model of capsaicin-induced pain has well-defined peripheral and central sensitization components, thus is useful for studying the analgesic effect on two separate components. The focus of this study is to examine the analgesic effects of electroacupuncture (EA) on capsaicin-induced secondary hyperalgesia, which represents central sensitization. Capsaicin (0.1%, 20 μl) was injected into the plantar side of the left hind paw, and foot withdrawal thresholds in response to von Frey stimuli (mechanical sensitivity) were determined for both primary and secondary hyperalgesia in rats. EA (2 Hz, 3 mA) was applied to various pairs of acupoints, GB30–GB34, BL40–BL60, GV2–GV6, LI3–LI6 and SI3–TE8, for 30 min under isoflurane anesthesia and then the effect of EA on mechanical sensitivity of paw was determined. EA applied to the ipsilateral SI3–TE8, but to none of the other acupoints, significantly reduced capsaicin-induced secondary hyperalgesia but not primary hyperalgesia. EA analgesic effect was inhibited by a systemic non-specific opioid receptor (OR) antagonist or an intrathecal μ- or δ-OR antagonist. EA analgesic effect was not affected by an intrathecal κ-OR antagonist or systemic adrenergic receptor antagonist. This study demonstrates that EA produces a stimulation point-specific analgesic effect on capsaicin-induced secondary hyperalgesia (central sensitization), mediated by activating endogenous spinal μ- and δ-opioid receptors.     

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1 day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.     

Inflammation-Induced Shift in the Valence of Spinal GABA-A Receptor–Mediated Modulation of Nociception in the Adult Rat

The objective of this study was to assess the impact of persistent inflammation on spinal γ-aminobutyric acid-A (GABA-A) receptor–mediated modulation of evoked nociceptive behavior in the adult rat. Nocifensive threshold was assessed with von Frey filaments applied to the dorsal surface of the hind paw. The GABA-A receptor agonist muscimol, the antagonist gabazine, the benzodiazepine receptor agonist midazolam, and antagonists PK11195 and flumazenil were administered spinally in the presence and absence of complete Freund's adjuvant (CFA)-induced inflammation. In naive rats, muscimol increased and gabazine decreased nociceptive threshold. After CFA, the effects of these compounds were reversed: Low doses of muscimol exacerbated the inflammation-induced decrease in nociceptive threshold and gabazine increased nociceptive threshold. Midazolam increased nociceptive threshold both in the presence and absence of inflammation. Flumazenil but not PK11195 blocked the analgesic effects of midazolam. These findings indicate that inflammation-induced changes in GABA-A signaling are complex and are likely to involve several distinct mechanisms. Rectifying the changes in GABA-A signaling may provide effective relief from hypersensitivity observed in the presence of inflammation.

Perspective

An inflammation-induced shift in spinal GABA-A receptor signaling from inhibition to excitation appears to underlie inflammatory pain and hypersensitivity. Use of GABA-A receptor selective general anesthetics in association with therapeutic interventions may be contraindicated. More importantly, rectifying the changes in GABA-A signaling may provide effective relief from inflammatory hypersensitivity.     

Higher doses of intraoperative analgesia are associated with lower levels of persistent pain and less analgesic consumption six months after total hip arthroplasty

Background

Persistent postoperative pain is a major health problem affecting nearly 30% of all patients undergoing total hip arthroplasty. Previous studies have demonstrated an association between the intensity of acute postoperative pain and persistent pain, but this association might be an epiphenomenon of insufficient intraoperative analgesia. In this study, we investigated the association between the intraoperative level of analgesia and the persistent postoperative pain 6 months after surgery.

Methods

We investigated 110 patients undergoing primary total hip arthroplasty under total intravenous general anaesthesia in a prospective cohort study. A highly standardized surgical and a standardized anaesthetic procedure were performed to reduce variability and psychosocial influences were investigated to adjust for confounders. Acute postoperative pain was controlled using patient‐controlled analgesia pumps. Postoperative pain intensities and analgesic requirements were monitored for 6 months following surgery.

Results

Of 105 patients included in the analysis, 32% continued using daily pain medication 6 months after surgery and reported a median pain level of 4/10. Multivariate analyses confirmed that the amount of intraoperative analgesia is a significant predictor of regular analgesic use and pain intensity 6 months after surgery.

Conclusions

Higher levels of intraoperative analgesia are associated with lower levels of persistent pain and less analgesic consumption 6 months after total hip arthroplasty. Persistent pain may be attributable to intraoperative nociception, which is likely not adequately assessed and suppressed using current clinical measures.

Significance

Our study suggests that lower doses of intraoperative analgesia are associated with higher levels of persistent postoperative pain. Persistent pain may be caused by intraoperative nociception, which is likely not adequately suppressed using current clinical standard analgesic measures.