Sinyoung Kang

Simultaneous Disruption of Mouse ASIC1a, ASIC2 and ASIC3 Genes Enhances Cutaneous Mechanosensitivity

Three observations have suggested that acid-sensing ion channels (ASICs) might be mammalian cutaneous mechanoreceptors; they are structurally related to Caenorhabditis elegans mechanoreceptors, they are localized in specialized cutaneous mechanosensory structures, and mechanical displacement generates an ASIC-dependent depolarization in some neurons. However, previous studies of mice bearing a single disrupted ASIC gene showed only subtle or no alterations in cutaneous mechanosensitivity. Because functional redundancy of ASIC subunits might explain limited phenotypic alterations, we hypothesized that disrupting multiple ASIC genes would markedly impair cutaneous mechanosensation. We found the opposite. In behavioral studies, mice with simultaneous disruptions of ASIC1a, -2 and -3 genes (triple-knockouts, TKOs) showed increased paw withdrawal frequencies when mechanically stimulated with von Frey filaments. Moreover, in single-fiber nerve recordings of cutaneous afferents, mechanical stimulation generated enhanced activity in A-mechanonociceptors of ASIC TKOs compared to wild-type mice. Responses of all other fiber types did not differ between the two genotypes. These data indicate that ASIC subunits influence cutaneous mechanosensitivity. However, it is unlikely that ASICs directly transduce mechanical stimuli. We speculate that physical and/or functional association of ASICs with other components of the mechanosensory transduction apparatus contributes to normal cutaneous mechanosensation.

Effect of capsaicin treatment on nociceptors in rat glabrous skin one day after plantar incision

&NA; Dilute capsaicin produces a differential effect on incision‐related pain behaviors depending upon the test; it reduces heat hyperalgesia and guarding pain but not mechanical hyperalgesia. This suggests that common mechanisms for heat hyperalgesia and guarding pain occur, and distinct mechanisms exist for mechanical hyperalgesia. The purpose of the present study was to evaluate the effect of capsaicin treatment on the activity of cutaneous nociceptors sensitized by incision to understand the mechanisms for the selective action of dilute capsaicin on incisional pain. We compared the effect of 0.05% capsaicin vs. vehicle treatment on pain behaviors after incision and on the activity of nociceptors from these same rats using the in vitro glabrous skin–nerve preparation. Immunohistochemical expression of protein gene product 9.5 (PGP9.5), neurofilament 200, calcitonin gene related peptide (CGRP) and isolectin B4 (IB4) in skin was also evaluated 1 week after 0.05% capsaicin infiltration. Infiltration of 0.05% capsaicin decreased CGRP and IB4/PGP9.5‐immunoreactivity of nociceptors in skin. The same dose of capsaicin that inhibited heat hyperalgesia and guarding behavior interfered with chemo‐ and heat sensitivity of C‐fibers. Neither mechanical hyperalgesia nor mechanosensitivity of nociceptors was affected by capsaicin, suggesting that the concentration of capsaicin used in this study did not cause fiber degeneration. These results demonstrate that nociceptors desensitized by capsaicin contribute to heat hyperalgesia and guarding pain after plantar incision. These putative TRPV1‐expressing C‐fibers are sensitized to heat and acid after incision, and the transduction of heat and chemical stimuli after plantar incision is impaired by dilute capsaicin.

Chemosensitivity and Mechanosensitivity of Nociceptors from Incised Rat Hindpaw Skin

Background:The authors have demonstrated a decrease in pH in the incisional wound environment, suggesting a possible contribution of low pH to postsurgical pain. In this study, the authors characterized the acid-responsiveness of nociceptors innervating the plantar aspect of the rat hind paw 1 day after plantar incision and compared this to plantar skin from unincised control rats. Methods:Using the rat glabrous in vitro skin-tibial nerve preparation, afferent nerve activities from single mechanosensitive nociceptors were recorded. Differences in mechanosensitivity, spontaneous activity, and chemosensitivity of units were evaluated. For chemosensitivity, acid-responsiveness of nociceptors to lactic acid (pH 5.5 to 6.5) was studied. Results:C-fibers showed dose-dependent, sustained responses to lactic acid. A greater proportion of C-fibers from 2 mm or less from the incision was activated by pH 6.0 lactic acid (52.9%) compared to control (14.3%). Total evoked potentials during acid exposure were greater in C-fibers innervating 2 mm or less from the incision compared to those in unincised skin. The prevalence of acid responses and total evoked potentials during acid exposure in C-fibers innervating more than 2 mm from the incision were not different from control. Few A-fibers responded to lactic acid, with a range of pH 5.5 to 6.5 in both incision and control groups. Increased spontaneous activity and mechanosensitivity were also evident. Conclusions:C-fibers in the vicinity of the incision showed qualitatively and quantitatively greater chemosensitivity to pH 6.0 lactic acid compared to control. This change was localized to 2 mm or less from the incision, suggesting increased chemosensitivity of nociceptive C-fibers 1 day after plantar incision.

Wound hypoxia in deep tissue after incision in rats.

Our previous studies using rat models of incisional pain have shown that tissue lactate levels increase and pH decreases for several days after incision, suggesting the presence of an ischemic‐like condition. The purpose of this study was to evaluate the time course and the extent of tissue hypoxia that develops in incised muscle and skin. We directly measured oxygen tension at several time points after incisions of the gastrocnemius muscle, the paraspinal skin, and the plantar hindpaw in anesthetized rats using an oxygen‐sensitive microelectrode. In vivo hypoxia of the incised tissues was also evaluated immunohistochemically using a hypoxia marker, pimonidazole hydrochloride. To minimize intersubject variability, unincised contralateral tissues were used as a control. Tissue oxygen tension was decreased in both skeletal muscle and skin compared with control, for several days after incision. When measured directly, oxygen tension decreased immediately and remained low for several days after incisions. Pimonidazole immunostaining revealed hypoxic areas in incised muscle and skin for several days. By postoperative day 10, tissue oxygen tension recovered to that of control tissue. These results support the evidence that a hypoxic condition is present in deep tissue after incisions and that an ischemic‐like mechanism may contribute to postoperative pain.

Antinociceptive synergy between the cannabinoid receptor agonist WIN 55,212-2 and bupivacaine in the rat formalin test.

BACKGROUND:The analgesic interaction between cannabinoids and local anesthetics has not been investigated. We sought to determine the nature of the interaction between the intrathecal cannabinoid receptor agonist (WIN 55,212-2) and bupivacaine using the formalin test. METHODS:Lumbar intrathecal catheters were implanted in male Sprague-Dawley rats. After intrathecal administration of WIN 55,212-2, bupivacaine, or their combination, 50 &mgr;L of 5% formalin was injected subcutaneously into the hindpaw. Dose–response curves were established and the respective ED50 (50% effective dose) values were determined for each agent alone. Fixed-ratio combinations of WIN 55,212-2 and bupivacaine were tested for combined antinociceptive effects in the formalin test and an isobolographic analysis was performed to characterize the pharmacologic interaction of both drugs. RESULTS:Intrathecally administered WIN 55,212-2, bupivacaine, or their combination produced a dose-dependent decrease in the number of flinches during Phase 1 and 2 of the formalin test. Isobolographic analysis revealed a synergistic interaction between intrathecal WIN 55,212-2 and bupivacaine in both phases of the formalin test. In combination, the ED50 value was significantly smaller than the theoretical additive value (P < 0.05). CONCLUSIONS:These results demonstrate that intrathecally coadministered WIN 55,212-2 and bupivacaine provide synergistic antinociceptive interaction in both phases of the formalin test.

Muscle Reactive Oxygen Species (ROS) Contribute to Post-Incisional Guarding via the TRPA1 Receptor.

Background Deep tissues and their afferents have unique responses to various stimuli and respond to injury distinctively. However, the types of receptors and endogenous ligands that have a key role in pain after deep tissue incision are unknown. TRPA1 has been shown to mediate pain-related responses in inflammation- and nerve injury-induced pain models. We hypothesized that TRPA1 has an important role in pain behaviors after deep tissue incision. Methods The effect of various doses of intraperitoneal (i.p.) TRPA1 antagonist, HC-030031, on pain behaviors after skin + deep tissue incision of the rat hind paw was measured. In vivo reactive oxygen species (ROS)-imaging and hydrogen peroxide (H2O2) levels after incision were also evaluated. Separate groups of rats were examined for H2O2-evoked pain-related behaviors after injections into the deep tissue or the subcutaneous tissue. Results Guarding pain behavior after skin + deep tissue incision was decreased by i.p. HC-030031. However, HC-030031 did not affect mechanical or heat responses after incision. Treatment either before or after incision was effective against incision-induced guarding behavior. ROS increased after skin + deep tissue incision in both the incised muscle and the skin. Tissue H2O2 also increased in both skin and muscle after incision. H2O2 injection produced pain behaviors when injected into muscle but not after subcutaneous injection. Conclusions This study demonstrates that TRPA1 antagonist HC-030031 reduced spontaneous guarding pain behavior after skin + deep tissue incision. These data indicate that TRPA1 receptors on nociceptors are active in incised fascia and muscle but this is not evident in incised skin. Even though endogenous TRPA1 agonists like ROS and H2O2 were increased in both incised skin and muscle, those in skin do not contribute to nociceptive behaviors. This study suggests that endogenous TRPA1 ligands and the TRPA1 receptor are important targets for acute pain from deep tissue injury.

Conditioned place preference and spontaneous dorsal horn neuron activity in chronic constriction injury model in rats.

Abstract Patients with neuropathic pain commonly present with spontaneous pain, in addition to allodynia and hyperalgesia. Although evoked responses in neuropathic pain models are well characterized, determining the presence of spontaneous pain is more challenging. We determined whether the chronic constriction injury (CCI) model could be used to measure effects of treatment of spontaneous pain, by evaluating dorsal horn neuron (DHN) spontaneous activity and spontaneous pain-related behaviors. We measured conditioned place preference (CPP) to analgesia produced by sciatic nerve block with bupivacaine in rats with established CCI. We undertook another CPP experiment using hind paw incision. We also examined DHN spontaneous activity in CCI rats. Although CCI produced nocifensive responses to mechanical stimuli, CPP to analgesic nerve block was not evident 14 days after injury: Compared with baseline (314 ± 65 seconds), CCI rats did not show a preference for the bupivacaine-paired chamber after conditioning (330 ± 102 seconds). However, sciatic nerve block after hind paw incision produced CPP on postoperative day 1, serving as a positive control. The proportion of spontaneously active DHNs (33%) was not significantly increased in CCI rats compared with the sham (21%). The median rate of spontaneous activity in the CCI group (12.6 impulses per second) was not different from the sham group (9.2 impulses per second). Also, there was no change in DHN spontaneous activity after sciatic nerve block with bupivacaine. Our findings suggest that CCI as a neuropathic pain model should not be used to measure effects of treatment of spontaneous pain driven by the peripheral input.

Evaluation of leukemia inhibitory factor (LIF) in a rat model of postoperative pain.

UNLABELLED Postoperative pain remains a significant problem despite optimal treatment with current pharmaceutical agents. In an effort to provide better postoperative pain control, there is a need to understand the factors that contribute to the development of pain after surgery. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine released from tissues after injury. We hypothesized that LIF expression in skin, muscle, and dorsal root ganglion (DRG) would increase after plantar incision. The mRNA and protein expression of LIF and LIF receptor (LIF-R) were measured after plantar incision in the rat. Pain behaviors, immunohistochemistry, and C-fiber heat responses to LIF were also studied. LIF expression increased after incision in skin and muscle, and LIF-R was present in large and small DRG neurons. LIF administration to the hindpaw increased pain behaviors, a process that was reversed by anti-LIF. However, LIF and anti-LIF treatment at the time of incision did not augment or ameliorate pain behaviors. LIF treatment activated the second messenger system, JAK-STAT3, in cultured DRG neurons, but failed to alter spontaneous activity or heat responses in C-fiber nociceptors. In conclusion, LIF is not a target for postoperative analgesia; LIF may be important for skin and muscle repair and regeneration after incision. PERSPECTIVE This article highlights an incision pain model for the study of factors involved in nociception. The study demonstrates that LIF in is an unlikely target for novel early postoperative analgesics.

Hydrogen Peroxide Induces Muscle Nociception via Transient Receptor Potential Ankyrin 1 Receptors

Background: H2O2 has a variety of actions in skin wounds but has been rarely studied in deep muscle tissue. Based on response to the transient receptor potential ankyrin 1 antagonists after plantar incision, we hypothesized that H2O2 exerts nociceptive effects via the transient receptor potential ankyrin 1 in muscle. Methods: Nociceptive behaviors in rats (n = 269) and mice (n = 16) were evaluated after various concentrations and volumes of H2O2 were injected into the gastrocnemius muscle or subcutaneous tissue. The effects of H2O2 on in vivo spinal dorsal horn neuronal activity and lumbar dorsal root ganglia neurons in vitro were evaluated from 26 rats and 6 mice. Results: Intramuscular (mean ± SD: 1,436 ± 513 s) but not subcutaneous (40 ± 58 s) injection of H2O2 (100 mM, 0.6 ml) increased nociceptive time. Conditioned place aversion was evident after intramuscular (–143 ± 81 s) but not subcutaneous (–2 ± 111 s) injection of H2O2. These H2O2-induced behaviors were blocked by transient receptor potential ankyrin 1 antagonists. Intramuscular injection of H2O2 caused sustained in vivo activity of dorsal horn neurons, and H2O2 activated a subset of dorsal root ganglia neurons in vitro. Capsaicin nerve block decreased guarding after plantar incision and reduced nociceptive time after intramuscular H2O2. Nociceptive time after intramuscular H2O2 in transient receptor potential ankyrin 1 knockout mice was shorter (173 ± 156 s) compared with wild-type mice (931 ± 629 s). Conclusions: The greater response of muscle tissue to H2O2 may help explain why incision that includes deep muscle but not skin incision alone produces spontaneous activity in nociceptive pathways.

Deep tissue incision enhances spinal dorsal horn neuron activity during static isometric muscle contraction in rats

Translational correlates to pain with activities after deep tissue injury have been rarely studied. We hypothesized that deep tissue incision causes greater activation of nociception-transmitting neurons evoked by muscle contraction. In vivo neuronal activity was recorded in 203 dorsal horn neurons (DHNs) from 97 rats after sham, skin-only, or skin + deep muscle incision. We evaluated DHN responses to static, isometric muscle contractions induced by direct electrical stimulation of the muscle. The effect of pancuronium on DHN response to contractions was also examined. Approximately 50% of DHNs with receptive fields in the hindpaw were excited during muscle contraction. One-second .5- and 1.0-g muscle contractions produced greater DHN activity after skin + deep muscle incision (median [interquartile range], 32 [5-39] impulses, P = .021; and 36 [26-46] impulses, P = .006, respectively) than after sham (6 [0-21] and 15 [8-32] impulses, respectively). Neuromuscular blockade with pancuronium inhibited the muscle contractions and DHN activation during electrical stimulation, demonstrating contraction-induced activation. The greater response of spinal DHNs to static muscle contraction after skin + deep muscle incision may model and inform mechanisms of dynamic pain after surgery. PERSPECTIVE: Completion of various activities is an important milestone for recovery and hospital discharge after surgery. Skin + deep muscle incision caused greater activation of nociception-transmitting DHNs evoked by muscle contraction compared with skin-only incision. This result suggests an important contribution of deep muscle injury to activity-evoked hyperalgesia after surgery.