Pain Behavior In Mice Research Articles

Transient receptor potential vanilloid 1 mediates pain in mice with severe sickle cell disease

AbstractPain is the leading cause of emergency department visits, hospitalizations, and daily suffering in individuals with sickle cell disease (SCD). The pathologic mechanisms leading to the perception of pain during acute RBC sickling episodes and development of chronic pain remain poorly understood and ineffectively treated. We provide the first study that explores nociceptor sensitization mechanisms that contribute to pain behavior in mice with severe SCD. Sickle mice exhibit robust behavioral hypersensitivity to mechanical, cold, and heat stimuli. Mechanical hypersensitivity is further exacerbated when hypoxia is used to induce acute sickling. Behavioral mechanical hypersensitivity is mediated in part by enhanced excitability to mechanical stimuli at both primary afferent peripheral terminal and sensory membrane levels. In the present study, inhibition of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) with the selective antagonist A-425619 reversed the mechanical sensitization at both primary afferent terminals and isolated somata, and markedly attenuated mechanical behavioral hypersensitivity. In contrast, inhibition of TRPA1 with HC-030031 had no effect on mechanical sensitivity. These results suggest that the TRPV1 receptor contributes to primary afferent mechanical sensitization and a substantial portion of behavioral mechanical hypersensitivity in SCD mice. Therefore, TRPV1-targeted compounds that lack thermoregulatory side effects may provide relief from pain in patients with SCD.

Activation of M3 muscarinic receptors inhibits T-type Ca 2+ channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca 2+ channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1 μM reversibly inhibited T-currents by ~ 26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca 2+ channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.

Microsomal prostaglandin E synthase-1 enhances bone cancer growth and bone cancer-related pain behaviors in mice

Aims Nonsteroidal anti-inflammatory drugs are a therapeutic modality for chronic cancer pain arising from bone metastases. Chronic administration of a cyclooxygenase (COX)-2 inhibitor is effective to bone cancer-related pain. However, adverse cardiovascular effects have limited COX-2 inhibitor therapy, and elucidation of better targets for blocking prostaglandin (PG) biosynthesis is necessary. Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes isomerization of the endoperoxide PGH 2 to PGE 2. To investigate the validity of mPGES-1 as a therapeutic target, we evaluated bone cancer pain-related behaviors in mPGES-1 knockout (PGES-1−/−) mice. Main methods Lewis lung carcinoma cells (LLCCs) were injected into the intramedullary space of the femur of wild-type (WT) and PGES-1−/− mice. Pain-related behaviors were evaluated. Key findings PGES-1−/− mice exhibited reduced tumor growth in bone marrow compared to WT. The expression of pro-calcitonin gene-related peptide (CGPR) in the dorsal root ganglia of L 1–5 was significantly higher in WT mice at day 14, whereas it was unchanged in mPGES-1 mice. In the observation of pain-related behaviors, mPGES-1−/− mice exhibited significantly fewer spontaneous flinches and their onset was several days later than WT. The appearance of other pain-related behaviors in mPGES-1−/− mice was also delayed as compared to WT. LLCC-injected WT mice treated with a COX-2 inhibitor, celecoxib, exhibited similar temporal changes to mPGES1−/−. Significance The present results suggest that mPGES-1 plays a crucial role in the enhancement of bone cancer growth and bone cancer pain, and that inhibition of mPGES-1 may have clinical utility in the management of bone cancer pain.

Varying Perceived Social Threat Modulates Pain Behavior in Male Mice

We previously demonstrated that male mice display significantly reduced pain behavior on the acetic acid abdominal constriction test when confined in close proximity to a stranger male mouse. We show here the testosterone-dependence (via castration and testosterone propionate replacement) of this phenomenon, likely a form of (social) stress-induced analgesia. However, when similar male dyads are separated by vertical metal bars, allowing only partial physical contact, we find that the mice exhibit hyperalgesia, not analgesia, in response to both acetic acid injection and noxious radiant heat, relative to testing in isolation. This finding is specific to same-sex maledyads, and no change in nociceptive sensitivity is observed when males are tested in the presence of a female conspecific. We propose that pain sensitivity varies with respect to the severity of the social threat: mild social threat produces hyperalgesia and more severe social threat produces analgesia. This work highlights the importance of social threat in modulating pain behavior in a sex-specific manner. The findings add to a growing body of evidence that social factors affect pain behavior in mice, thus allowing the study of the mechanistic underpinnings of social modulation of pain in humans.

Pre-Differentiated Embryonic Stem Cells Promote Neuronal Regeneration by Cross-Coupling of BDNF and IL-6 Signaling Pathways in the Host Tissue

The mechanism of embryonic stem (ES) cell therapeutic action remains far from being elucidated. Our recent report has shown that transplantation of ES cells, predifferentiated into neuronal progenitors, prevented appearance of chronic pain behaviors in mice after experimentally induced spinal cord injury. In the current study, we tested the hypothesis that this beneficial effect is mediated by antiapoptotic and regenerative signaling pathways activated in the host tissue by transplanted ES cells. Spinal cord injury was induced by unilateral microinjections of quisqualic acid at spinal levels T12-L2. At 1 week after injury, the pre-differentiated towards neuronal phenotype ES cells were transplanted into the site of injury. Here we show that transplantation of pre-differentiated ES cells activate both brain-derived neurotrophic factor (BDNF) and interleukin-6 (IL-6) signaling pathways in the host tissue, leading to activation of cAMP/PKA, phosporylation of cofilin and synapsin I, and promoting regenerative growth and neuronal survival.

Mechanical Hypersensitivity Occurs in a Mouse Model of Sickle Cell Disease

The acute painful event is the hallmark of sickle cell disease (SCD) in humans, accounting for the majority of hospitalizations. In addition, pain managed at home is underreported in both children and adults with SCD. Vaso-occlusion is intricately linked to the development of sickle cell pain, initiating tissue damage and numerous biochemical, electrochemical, and neurologic cascades which affect nociception (the perception of pain). It has been hypothesized that repeated painful events may lead to sensitization, which can manifest as allodynia (painful response to a usually non-painful stimulus) and chronic pain. However, the mechanisms of pain in SCD remain poorly understood. The objective of this study was to evaluate nociception in a mouse model of SCD both at baseline and after acute vaso-occlusion. The Berkeley mouse model of SCD exhibits many characteristics of human SCD, including vaso-occlusion. Acute vaso-occlusive events in the Berkeley SCD mouse can be induced by exposure to short periods of hypoxia followed by reoxygenation. We compared pain behavior responses to heat and mechanical force in female Berkeley SCD (SS) mice before and after exposure to 1 hour of hypoxia (FiO2 10%) followed by 1 hour of reoxygenation in room air. Pain behavior in SS mice was also compared to that in control female HbA (AA) mice, which solely express normal human hemoglobin and are maintained on the same mixed genetic background as the SS mice. Female mice expressing both normal and sickle human hemoglobin (AS mice) comprised an additional control group. Pain behavior was assessed by two methods: the Hargreaves test of thermal (heat) nociception and the von Frey test of mechanical allodynia. The Hargreaves test is conducted by applying a radiant heat stimulus to the plantar surface of the hind paw (Hargreaves, et al, 1988). The time to withdrawal of the paw from the heat stimulus is recorded as the paw withdrawal latency. Baseline data from the Hargreaves test of 12 AA, 12 AS, and 14 SS mice showed a subtle decrease in paw withdrawal latency in SS as compared to AA or AS mice; however, the differences in paw withdrawal latency between groups were not statistically significant. Exposure to hypoxia-reoxygenation did not affect paw withdrawal latency in response to heat stimulation in any of the three groups of mice. The second test for pain behavior used was the von Frey test for mechanical hypersensitivity (i.e., allodynia). Mice are tested for response to the application of increasing and decreasing force strengths of nylon monofilaments to the plantar surface of the hind paws. The paw withdrawal threshold was determined using the up-down method of Dixon (1980). Baseline data from 12 AA, 12 AS and 12 SS mice showed a significant difference in paw withdrawal threshold between groups (p=0.05); SS mice exhibited the lowest paw withdrawal threshold, reflecting increased sensitivity to mechanical stimuli. Exposure to hypoxia-reoxygenation did not alter the paw withdrawal threshold of AA and AS mice and tended to decrease paw withdrawal threshold of SS mice, consistent with vaso-occlusion causing pain in SS mice exposed to hypoxia-reoxygenation. These results suggest that Berkeley SS mice exhibit mechanical hypersensitivity at baseline compared to AA and AS mice, perhaps reflective of increased baseline vaso-occlusion in SS as compared to control mice. Exposure to hypoxia-reoxygenation, which induces RBC sickling, acute vaso-occlusion and tissue damage in SS mice, accentuates the sensitivity of the SS mice to mechanical stimuli. The results of these ongoing studies will expand our understanding of mechanisms underlying sickle cell-associated pain behavior in mice with SCD. Translationally, this may lead to improved pain management strategies for both acute and chronic pain experienced by individuals affected by this chronic debilitating disease.

Increased production of mitochondrial superoxide in the spinal cord induces pain behaviors in mice: The effect of mitochondrial electron transport complex inhibitors

Scavengers of reactive oxygen species (ROS) have been shown to produce a strong antinociceptive effect on persistent pain, and mitochondria are suggested to be the main source of ROS in the spinal dorsal horn. To explore whether excessive generation of mitochondrial superoxide alone can induce pain, the effect of mitochondrial electron transport complex inhibitors on the development of mechanical hyperalgesia was examined in mice. Intrathecal injection of an electron transport complex inhibitor, antimycin A or rotenone, in normal mice resulted in a slowly developing but long-lasting and dose-dependent mechanical hyperalgesia. The levels of mechanical hyperalgesia after antimycin A, a complex III inhibitor, were higher than that with rotenone, a complex I inhibitor. A large increase of mitochondrial superoxide in the spinal dorsal horn and a strong antinociceptive effect of ROS scavengers, phenyl- N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were observed in antimycin A-treated mice. The study indicates that the enhanced production of spinal mitochondrial superoxide alone without nerve injury can produce mechanical hyperalgesia.

Pharmacological validation of a model of cystitis pain in the mouse

Interstitial cystitis (IC) is a chronic pelvic-perineal pain syndrome of unknown etiology that mainly targets the lower urinary tract. Pain is the most prominent feature of IC and current therapies provide limited relief. Novel treatment options for IC could be identified if more predictive animal models were available. A rat model based on administration of cyclophosphamide (CP) mimics the symptoms of IC and has been well characterized. However, experiments in mice have not consistently reported both the spontaneous and evoked pain behaviors. The current series of studies demonstrate that CP (200-400mg, i.p.) increased both spontaneous and evoked pain behaviors in mice. Additionally, clinically relevant compounds: morphine (1-10mg/kg), ketorolac (1-5.6mg/kg) and duloxetine (3-30mg/kg) all significantly reversed pain behaviors. In contrast, gabapentin (56mg/kg) had no effect. Thus, CP-induced cystitis in mice may be used to evaluate novel therapeutics for the treatment of pain due to interstitial cystitis.

Effect of Aspirin and Acetaminophen on Proinflammatory Cytokine-Induced Pain Behavior in Mice

Aspirin (ASA) is a widely used oral analgesic which acts as an inhibitor of cyclooxygenase. Acetaminophen (ACET) is also an effective analgesic and may selectively inhibit brain prostaglandin synthetase. Various proinflammatory cytokines injected into the central nervous system show pain behavior. In the present study, the effects of orally administered ASA and ACET on pain behaviors induced by various proinflammatory cytokines were examined. At a dose of 100 mg/kg, ASA or ACET did not affect the pain behavior induced by TNF-α (100 pg), IL-1β (100 pg) or IFN-γ (100 pg) administered intrathecally. However, at doses of 200 and 300 mg/kg, ASA or ACET significantly and dose-dependently attenuated pain behavior induced by TNF-α, IL-1β or IFN-γ administered intrathecally. Our results suggest that orally administered ASA and ACET produce antinociception by inhibiting the nociceptive action of TNF-α, IL-1β or IFN-γ administered intrathecally.

Spinal CK2 regulates nociceptive signaling in models of inflammatory pain

Casein kinase 2 (CK2) is a widely expressed protein kinase. Over the last several years a long list of protein substrates has evolved, many of which have proven or hypothesized roles in nociceptive signal transmission. However, CK2 has not itself been demonstrated to participate in nociception prior to this time. We set out to test the hypothesis that spinal CK2 regulates nociception using several pain models. Our first studies focused on the ability of the selective CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBBT) and 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) to reduce formalin-stimulated pain behaviors in mice. Both phases of the response to subcutaneous formalin were strongly inhibited by intrathecal administration of TBBT or DRB in dose-dependent fashion. Likewise, using the complete Freund's adjuvant (CFA) model of chronic inflammatory pain, TBBT was observed to strongly reduce mechanical allodynia. The inhibition of spinal CK2 with either inhibitor did not, however, alter withdrawal latencies in the hotplate thermal pain model while intrathecal morphine was very effective. Immunohistochemical studies demonstrated all three known CK2 subunits, α, α′ and β to be expressed in spinal cord tissue as did real-time PCR experiments. While mRNA levels for each of the subunits was transiently enhanced after formalin or CFA hindpaw injection, overall spinal cord protein levels were not elevated in a sustained fashion. Our results indicate that CK2 participates in inflammatory nociception both in the acute and chronic phases. Simple changes in the abundance of spinal CK2 subunits do not likely underlie these phenomena, however.

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We have previously shown that subcutaneous bee venom (BV) injection reduces visceral pain behavior in mice, but it is not clear which constituent of BV is responsible for its antinociceptive effect. In the present study, we now demonstrate that a water-soluble subfraction of BV (BVA) reproduces the antinociceptive effect of BV in acetic acid-induced visceral pain model. We further evaluated three different BVA subfractions that were separated by molecular weight, and found that only the BVAF3 subfraction (a molecular weight of <10 kDa) produced a significant antinociceptive effect on abdominal stretches and suppressed visceral pain-induced spinal cord Fos expression. Injection of melittin (MEL), a major constituent of BVAF3, also produced a visceral antinociception. However, melittin's antinociception was completely blocked by boiling for 10 min at 100 °C, while boiling either whole BV or BVAF3 did not prevent their antinociception. The antinociceptive effect of BVAF3 was completely blocked by intrathecal pretreatment with the α 2-adrenoceptor antagonist, yohimbine (YOH), while intrathecal pretreatment with the opioid antagonist, naloxone (NAL) or the serotonin antagonist, methysergide, had no effect. These data demonstrate that BVAF3 is responsible for the visceral antinociception of whole BV and further suggest that this effect is mediated in part by spinal α 2-adrenergic activity.

Role of μ-opioid receptors in formalin-induced pain behavior in mice

Intraplantar formalin injection is widely used as an experimental model of tonic pain. We investigated the role of endogenous μ-opioid receptor mechanisms in formalin-induced nocifensive behavior in mice. The flinching response induced by formalin (2%, 20 μl) was studied in mice with normal (wild type, n = 8) and absent (homozygous μ-opioid receptor knockout, n = 8) μ-opioid receptor levels. The flinch responses were counted every 5 min for 60 min post-formalin injection. Lumbar spinal cord (L4, 5) was harvested 2 h post-formalin injection to examine c-Fos expression using immunohistochemistry. The effects of naloxone (5 mg/kg, sc) administered 30 min before the intraplantar formalin injection on the flinching response of wild-type mice ( n = 7) were also recorded. The second-phase formalin response (10–60 min after formalin) was higher in homozygous μ-opioid receptor knockout mice compared to the wild-type mice ( P < 0.01). Naloxone administration in wild-type mice before formalin injection resulted in pain behavior similar to that observed in homozygous μ-opioid receptor knockout mice ( P > 0.05). The c-Fos expression induced by formalin injection in the knockout mice was not different from that observed in wild-type mice. Our results suggest that the endogenous μ-opioid system is activated by intraplantar formalin injection and exerts a tonic inhibitory effect on the pain behavior. These results suggest an important modulatory role of endogenous μ-opioid receptor mechanisms in tonic pain states.

Acetic acid conditioning stimulus induces long-lasting antinociception of somatic inflammatory pain

A wide variety of noxious stimuli are known to induce a powerful inhibition of pain sensation evoked at a remote region of the body. Here we show that an intraperitoneal acetic acid (AA) conditioning stimulus produces long-lasting inhibition of formalin-evoked somatic inflammatory pain behavior in mice. This novel long-lasting antinociception was completely blocked by the 5-hydroxytryptamine type 2A/2C (5-HT 2A/2C) receptor antagonists, ketanserin and ritanserin, but not by the opioid receptor antagonist, naloxone, and α-adrenergic receptor antagonists, phentolamine and yohimbine. In contrast, the 5-HT 3/4 receptor antagonist, tropisetron, significantly potentiated this long-lasting antinociception. The conditioning stimulus significantly upregulated the levels of both tryptophan hydroxylase immunoreactivity in the medulla oblongata and the 5-HT 2A/2C receptor mRNA level in the spinal cord. These results suggested that the visceral noxious stimulus caused a long-lasting augmentation of the serotonergic inhibitory system and downregulated the somatic inflammatory nociceptive transmission.

Hyperalgesia During Opioid Abstinence: Mediation by Glutamate and Substance P

Opioid-abstinence hyperalgesia (OAH) is a phenomenon characterized by thermal and mechanical hyperalgesia that occurs between intermittent doses of opioids or after the chronic administration of these drugs when administration is abruptly stopped. In these studies we attempted to determine whether the activation of spinal cord dorsal horn neurons was greater in mice with OAH than in control mice in response to the intrathecal administration of the primary neurotransmitters glutamate and substance P. After mice were treated with an established protocol consisting of the implantation of morphine pellets followed by removal after 6 days, the mice were hyperalgesic as assessed with the hotplate and Hargreaves thermal paw withdrawal assays. Mechanical allodynia was also demonstrated. The intrathecal injection of either glutamate (5-25 micro g) or substance P (0.02-0.1 nmol) caused greater pain behaviors in mice with OAH than in control mice. Likewise, it was observed that the dorsal horn regions of OAH mice had more Fos-positive nuclei after either glutamate or substance P administration than did control mice. We conclude that mice with OAH exhibit increased pain behaviors and have increased numbers of Fos-positive nuclei in response to intrathecal glutamate and substance P administration when compared with control mice. Thus, spinal sensitization to primary neurotransmitters may be responsible in part for the manifestation of OAH. Opioids are a mainstay of treatment for many types of chronic pain. These studies provide evidence that the hyperalgesia induced by chronic opioid administration may be in part to spinal neuroplastic changes.

Hyperalgesia During Opioid Abstinence: Mediation by Glutamate and Substance P

Opioid-abstinence hyperalgesia (OAH) is a phenomenon characterized by thermal and mechanical hyperalgesia that occurs between intermittent doses of opioids or after the chronic administration of these drugs when administration is abruptly stopped. In these studies we attempted to determine whether the activation of spinal cord dorsal horn neurons was greater in mice with OAH than in control mice in response to the intrathecal administration of the primary neurotransmitters glutamate and substance P. After mice were treated with an established protocol consisting of the implantation of morphine pellets followed by removal after 6 days, the mice were hyperalgesic as assessed with the hotplate and Hargreaves thermal paw withdrawal assays. Mechanical allodynia was also demonstrated. The intrathecal injection of either glutamate (5–25 μg) or substance P (0.02–0.1 nmol) caused greater pain behaviors in mice with OAH than in control mice. Likewise, it was observed that the dorsal horn regions of OAH mice had more Fos-positive nuclei after either glutamate or substance P administration than did control mice. We conclude that mice with OAH exhibit increased pain behaviors and have increased numbers of Fos-positive nuclei in response to intrathecal glutamate and substance P administration when compared with control mice. Thus, spinal sensitization to primary neurotransmitters may be responsible in part for the manifestation of OAH.

Heme oxygenase inhibitors reduce formalin-induced Fos expression in mouse spinal cord tissue

Recent work from our laboratory and others supports a role for heme oxygenase in nociception and pain of several etiologies including inflammatory, incisional and neuropathic. Since it has been observed that heme oxygenase inhibitors reduce formalin-induced pain behaviors in mice and rats, we attempted to determine if this analgesic effect was reflected in a reduction in formalin-induced spinal cord Fos expression, an index of neuronal activation. To perform these studies, it was necessary to first examine the cytoarchitecture of the mouse lumbar spinal cord so that histological sections from known segmental levels could be chosen, and Fos-positive nuclei could be assigned to established dorsal horn laminae. After documenting the segmental and laminar distribution of Fos-positive nuclei following a 5% formalin injection, we went on to determine that the heme oxygenase inhibitor tin-protoporphyrin or morphine reduced this Fos expression as analyzed using confocal fluorescence microscopy. It was also observed that mice lacking expression of heme oxygenase type 2, an isozyme of heme oxygenase found in high abundance in the spinal cord, had lowered Fos expression after the formalin injection. Additional confocal microscopy studies demonstrated widespread expression of heme oxygenase type 2 in spinal cord neuron cell bodies. Double-labeling experiments showed that a high percentage of Fos-positive nuclei identified after administration of formalin were located within heme oxygenase type 2-positive cell profiles. Our studies support the hypothesis that heme oxygenase type 2 plays a role in formalin-induced nociception. Furthermore, from these results we suggest that the heme oxygenase type 2 located in spinal cord dorsal horn neurons participates in this nociceptive pathway.

PS-17-8 Intrathecal injection of substance P before neurectomy modurates neuropathic pain behavior in mice via NMDA receptor

PS-17-8 Intrathecal injection of substance P before neurectomy modurates neuropathic pain behavior in mice via NMDA receptor