Contralateral Hindpaw Research Articles

Assessment of the anti-allodynic efficacy of a glycine transporter 2 inhibitor relative to pregabalin and duloxetine in a rat model of prostate cancer-induced bone pain.

The pathobiology of prostate cancer-induced bone pain (PCIBP) is underpinned by both inflammatory and neuropathic components. Here, we used a rat model of PCIBP to assess the analgesic efficacy of a glycine transporter 2 (GlyT2) inhibitor (N-(6-((1,3-dihydroxypropan-2-yl)amino)-2-(dimethylamino)pyridin-3-yl)-3,5-dimethoxy-4-(4-(trifluoromethyl)phenoxy) benzamide) relative to two clinically available adjuvant drugs that are recommended for the relief of neuropathic pain, viz, pregabalin and duloxetine. PCIBP was induced in male Wistar Han rats following intra-tibial injection (ITI) of rat prostate cancer (AT3B) cells into the left tibia. Sham-rats received an ITI of heat-killed AT3B cells. PCIBP rats with fully developed mechanical allodynia in the ipsilateral hindpaws as assessed using von Frey filaments, received single oral (p.o.) bolus doses of the GlyT2 inhibitor (3-30mg/kg), pregabalin (3-100mg/kg), duloxetine (3-100mg/kg), or vehicle. Baseline paw withdrawal thresholds (PWTs) were determined in the ipsilateral (injured side) and contralateral hindpaws immediately prior to dosing and at scheduled times for 3h post dosing in individual animals. Single oral bolus doses of the GlyT2 inhibitor (3-30mg/kg) evoked partial pain relief at the doses tested in the ipsilateral hindpaws of PCIBP rats without any discernible behavioural side effects. By contrast, single oral bolus doses of pregabalin at 10-100mg/kg evoked dose-dependent and complete alleviation of mechanical allodynia. By comparison, single oral bolus doses of duloxetine at doses up to 100mg/kg lacked efficacy. Oral administration of this GlyT2 inhibitor evoked partial pain relief in PCIBP rats and did not evoke central nervous system side effects in contrast to GlyT2 inhibitors reported by others.

Monitoring of Stimulus Evoked Murine Somatosensory Cortex Hemodynamic Activity With Volumetric Multi-Spectral Optoacoustic Tomography.

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson’s correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.

Red nucleus IL-6 mediates the maintenance of neuropathic pain by inducing the productions of TNF-α and IL-1β through the JAK2/STAT3 and ERK signaling pathways.

We previously reported that interleukin (IL)-6 in the red nucleus (RN) is involved in the maintenance of neuropathic pain induced by spared nerve injury (SNI), and exerts a facilitatory effect via Janus-activated kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) and extracellular signal-regulated kinase (ERK) signal transduction pathways. The present study aimed at investigating the roles of tumor necrosis factor-α (TNF-α) and IL-1β in RN IL-6-mediated maintenance of neuropathic pain and related signal transduction pathways. Being similar to the elevation of RN IL-6 three weeks after SNI, increased protein levels of both TNF-α and IL-1β were also observed in the contralateral RN three weeks after the nerve injury. The upregulations of TNF-α and IL-1β were closely correlative with IL-6 and suppressed by intrarubral injection of a neutralizing antibody against IL-6. Administration of either the JAK2 antagonist AG490 or the ERK antagonist PD98059 to the RN of rats with SNI remarkably increased the paw withdrawal threshold (PWT) and inhibited the up-regulations of local TNF-α and IL-1β. Further experiments indicated that intrarubral injection of exogenous IL-6 in naive rats apparently lowered the PWT of the contralateral hindpaw and boosted the local expressions of TNF-α and IL-1β. Pretreatment with AG490 could block IL-6-induced tactile hypersensitivity and suppress the up-regulations of both TNF-α and IL-1β. However, injection of PD98059 in advance only inhibited the upregulation of IL-1β, but not TNF-α. These findings indicate that RN IL-6 mediates the maintenance of neuropathic pain by inducing the productions of TNF-α and IL-1β. IL-6 induces the expression of TNF-α through the JAK2/STAT3 pathway, and the production of IL-1β through the JAK2/STAT3 and ERK pathways.

Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors

Lemairamin (also known as wgx-50), is isolated from the pericarps of the Zanthoxylum plants. As an agonist of α7 nicotinic acetylcholine receptors (α7nAChRs), it can reduce neuroinflammation in Alzheimer’s disease. This study evaluated its antinociceptive effects in pain hypersensitivity and explored the underlying mechanisms. The data showed that subcutaneous lemairamin injection dose-dependently inhibited formalin-induced tonic pain but not acute nociception in mice and rats, while intrathecal lemairamin injection also dose-dependently produced mechanical antiallodynia in the ipsilateral hindpaws of neuropathic and bone cancer pain rats without affecting mechanical thresholds in the contralateral hindpaws. Multiple bi-daily lemairamin injections for 7 days did not induce mechanical antiallodynic tolerance in neuropathic rats. Moreover, the antinociceptive effects of lemairamin in formalin-induced tonic pain and mechanical antiallodynia in neuropathic pain were suppressed by the α7nAChR antagonist methyllycaconitine. In an α7nAChR antagonist-reversible manner, intrathecal lemairamin also stimulated spinal expression of IL-10 and β-endorphin, while lemairamin treatment induced IL-10 and β-endorphin expression in primary spinal microglial cells. In addition, intrathecal injection of a microglial activation inhibitor minocycline, anti-IL-10 antibody, anti-β-endorphin antiserum or μ-opioid receptor-preferred antagonist naloxone was all able to block lemairamin-induced mechanical antiallodynia in neuropathic pain. These data demonstrated that lemairamin could produce antinociception in pain hypersensitivity through the spinal IL-10/β-endorphin pathway following α7nAChR activation.

Low frequency electroacupuncture alleviates neuropathic pain by activation of spinal microglial IL-10/β-endorphin pathway

Electroacupuncture produces analgesia in chronic pain patients and animal models of pain hypersensitivity. The current study aims to illustrate the mechanisms underlying electroacupuncture-attenuated neuropathic pain. Neuropathic rats, induced by tight ligation of L5/L6 spinal nerves, markedly reduced mechanical thresholds in the ipsilateral hindpaws relative to the contralateral hindpaws. Low frequency (2 Hz) electroacupuncture stimulation for a period of 20 min alleviated neuropathic pain in the ipsilateral hindpaws of neuropathic rats in a time-dependent manner. The same electroacupuncture treatment also stimulated spinal gene and protein expression of IL-10 and β-endorphin but not dynorphin A, measured by real-time quantitative PCR and ELISA kits. Intrathecal injection of the specific IL-10 antibody in neuropathic rats completely blocked electroacupuncture-increased spinal expression of β-endorphin, but the β-endorphin antibody failed to alter electroacupuncture-stimulated spinal IL-10 expression. Using a double fluorescence immunostaining technique, we observed that electroacupuncture stimulated spinal IL-10 and β-endorphin expression in microglia but not in neurons or astrocytes in the spinal dorsal horn of neuropathic rats. Pretreatment with intrathecal injection of the microglial inhibitor minocycline, specific IL-10 antibody and β-endorphin antiserum (but not the dynorphin A antibody), or selective μ-opioid receptor antagonist CTAP (but not κ- or δ-opioid receptor antagonist) completely blocked electroacupuncture-induced attenuation of neuropathic pain. These results suggest that low frequency electroacupuncture alleviates neuropathic pain through stimulation of the spinal microglial expression of IL-10 and subsequent expression of β-endorphin.

Repeated Testing With the Hypertonic Saline Assay in Mice for Screening of Analgesic Activity.

In vivo animal assays are a cornerstone of preclinical pain research. An optimal stimulus for determining the activity of potential analgesics would produce responses of a consistent magnitude on repeated testing. Intraplantar (i.pl.) injection of hypertonic saline (HS) in mice produces robust nociceptive responses to different analgesics, without evidence of tissue damage. Here, we investigated whether the nociceptive response is changed by repeating the injection at different times and sites in a mouse and whether it is attenuated by morphine. We conducted randomized and blinded experiments to assess responses to repeated i.pl. 10% HS in female CD-1 mice. An injection of HS was followed by a second injection into the same hind paw at 4 hours, 24 hours, or 7 days. A separate group of mice each received i.pl. injections at 5, 10, and 15 days. In 2 independent experiments, 30 minutes after initial HS injections in the ipsilateral hind paw, mice received HS injection into the contralateral hind paw or ipsilateral forepaw. The ability of morphine to block the nociceptive responses was examined by injecting morphine at 5-day intervals. Repeated injection of HS did not alter the responses at 4 hours (84 vs 75 seconds; mean difference [95% CI], -9 [-40 to 23]; P = .6), 24 hours (122 vs 113 seconds; -6 [-24 to 12]; P = .5), or 7 days (112 vs 113 seconds; -0.3 [-12 to 11]; P = .95) or at multiple injections (day 0, 122 seconds vs day 5, 121 seconds; -0.3 [-28 to 27], P > .99; day 10, 118 seconds; 2.5 [-36 to 41], P = .99; day 15, 119 seconds; 2 [-36 to 38], P = .99). A previous hind paw injection did not change the responses of the contralateral hind paw (right, 93 seconds versus left, 96 seconds; -3 [-20 to 13], P = .7) or of the ipsilateral forepaw (forepaw after HS, 146 seconds versus forepaw after 0.9% saline, 149 seconds; -3 [-28 to 22], P = .8). Morphine dose-dependently attenuated HS responses (control, 94 seconds vs 4 mg/kg, 66 seconds; 29 [-7 to 64], P = .12; vs 10 mg/kg, 27 seconds; 67 [44-90], P < .0001; 4 vs 10 mg/kg, 67 [44-90], P = .03). The repetition of i.pl. HS produces consistent reproducible responses without tissue damage. This results in efficient, rapid detection of analgesic activity, reducing the number of animals required.

A newly identified nociresponsive region in the transitional zone (TZ) in rat sensorimotor cortex

The primary somatosensory cortex (S1) comprises a number of functionally distinct regions, reflecting the diversity of somatosensory receptor submodalities innervating the body. In particular, two spatially and functionally distinct nociceptive regions have been described in primate S1 (Vierck et al., 2013; Whitsel et al., 2019). One region is located mostly in Brodmann cytoarchitectonic area 1, where a subset of neurons exhibit functional characteristics associated with myelinated Aδ nociceptors and perception of 1st/sharp, discriminative pain. The second region is located at the transition between S1 and primary motor cortex (M1) in area 3a, where neurons exhibit functional characteristics associated with unmyelinated C nociceptors and perception of 2nd/slow, burning pain. To test the hypothesis that in rats the transitional zone (TZ) – which is a dysgranular region at the transition between M1 and S1 – is the functional equivalent of the nociresponsive region of area 3a in primates, extracellular spike discharge activity was recorded from TZ neurons in rats under general isoflurane anesthesia. Thermonoxious stimuli were applied by lowering the contralateral forepaw or hindpaw into a 48–51 °C heated water bath for 5–10 s. Neurons in TZ were found to be minimally affected by non-noxious somatosensory stimuli, but highly responsive to thermonoxious skin stimuli in a slow temporal summation manner closely resembling that of nociresponsive neurons in primate area 3a. Selective inactivation of TZ by topical lidocaine application suppressed or delayed the nociceptive withdrawal reflex, suggesting that TZ exerts a tonic facilitatory influence over spinal cord neurons producing this reflex. In conclusion, TZ appears to be a rat homolog of the nociresponsive part of monkey area 3a. A possibility is considered that this region might be primarily engaged in autonomic aspects of nociception.

Peripheral Kappa Opioid Receptor (KOR)‐Mediated Antinociception Requires G Protein‐Gated Inward Rectifying Potassium (GIRK) Channels

Targeting peripherally expressed kappa opioid receptors (KOR) is a promising approach to treat inflammatory pain that would be devoid of CNS related adverse effects. KOR are known to couple to Gi‐protein‐mediated signaling pathways involved in the suppression of neuronal firing and nociception. In this study, we sought to determine the involvement of Gi protein coupled inward rectifying potassium (GIRK) channels in peripheral KOR‐mediated antinociceptive behavior in rats. Rats were injected intraplantarly (i.pl., 50ul) with PGE2 (0.3ug) with or without the KOR agonist, U50488 (0.1ug) and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. Treatment with U50488 completely reduced the thermal allodynia produced by PGE2. Consistent with activation of Gi proteins by KOR, we found that the reduction of PGE2‐evoked thermal allodynia by U50488 was lost completely 24h after i.pl. injection of pertussis toxin. To determine if GIRK channels mediated the antinociceptive response to KOR, we measured the effect of U50488 on PGE2‐evoked thermal allodynia 15 minutes after i.pl. injection of either vehicle or tertiapin Q (TPNQ), an inhibitor that blocks channels containing GIRK1 and GIRK2 subunits. Administration of TPNQ had no effect on either baseline PWL or PGE2‐evoked thermal allodynia, however, U50488‐mediated reduction of PGE2‐evoked thermal allodynia was lost after TPNQ administration. We next tested if direct activation of GIRK channels by i.pl. injection of the GIRK activator, ML297, could produce antinociceptive effects. ML297 (33ug) produced a robust analgesic effect (+5 sec above baseline) compared to vehicle treated animals as well as prevented PGE2‐evoked thermal allodynia. To further corroborate the importance of GIRK channels in peripheral KOR mediated antinociception, we tested effects of siRNA‐mediated knockdown of GIRK2 channel subunits following local administration of GIRK2 Accell‐siRNA (Dharmacon) into the hindpaw. After two daily i.pl. injections of GIRK2 siRNA, neither baseline PWL nor PGE2‐evoked thermal allodynia were altered, however, U50488‐mediated reduction in PGE2‐evoked thermal allodynia was lost. We verified knockdown of the GIRK2 subunit in hindpaws by RT‐qPCR and immunohistochemistry. Compared to the contralateral (CONTRA) hindpaw, GIRK2 mRNA was decreased significantly in the ipsilateral (IPSI) hindpaw. Further, there was no difference between IPSI and CONTRA hindpaws in the immunofluorescence for PGP9.5, a marker used to identify neuronal processes, or DAPI, a cell nuclei marker. By contrast, GIRK2 immunofluorescence was significantly decreased in IPSI hindpaws compared to CONTRA hindpaws suggesting that the expression of GIRK2 was indeed reduced. Together these results suggest that peripherally mediated antinociception in response to activation of KOR is due, in large part, to activation of GIRK channels.Support or Funding InformationThis work is supported by NIH/NIDA RO1 DA 038645 and NIH/NIGMS RO1 GM 106035.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aleurites moluccanus and its main active constituent, the flavonoid 2″-O-rhamnosylswertisin, in experimental model of rheumatoid arthritis

Ethnopharmmacological relevanceAleurites moluccana is used in folk medicine to treat pain, fever, asthma, hepatitis, gastric ulcer and inflammatory process in general, and the nut oil had been topically applied to treat arthritis and other joint pain, however the seeds are classified as toxic for oral use. AimFaced with the need for new alternative to treat the symptoms and modify rheumatoid arthritis (RA) the aim of this work was to evaluate the effects of A. moluccanus’ leaves dried extract in rats and mice submitted to complete Freund adjuvant (CFA)-induced RA. Material and methodsWistar Rats and Swiss mice were submitted to CFA-induced RA in the right hindpaw. They received A. moluccanus extract (orally; p.o.), dexamethasone (subcutaneously), 2″-O-rhamnosylswertisin (p.o.) or vehicle (p.o.), from the 14th day after the CFA injection for up to 8 days. The mechanical hypersensitivity was evaluated using the von Frey filaments and the paw-oedema was measured using a plethysmometer. The rats’ injected hindpaw was used to perform the histological analysis. ResultsA. moluccanus was able to significantly reduce the mechanical hypersensitivity in both ipsi- and contralateral hindpaws of mice injected with CFA, in a dose dependent manner. Furthermore, the paw-oedema was progressively reduced by A. moluccanus. Similar results were obtained for the positive-control drug dexamethasone and the isolated compound 2″-O-rhamnosylswertisin. Besides the effects mentioned above, the extract was also effective to repair the joint damage in CFA-induced RA rats, including reduction of fibrosis, cartilage degradation and bone erosion scores. ConclusionThese results together with the literature data reinforce the anti-hypersensitivity and anti-inflammatory activity of A. moluccanus extract. Part of the observed effects is due to the presence of the compound 2″-O-rhamnosylswertisin. The fact that the extract acted as a disease modifier point this herbal product as a promisor and safe tool to treat RA and other associated chronic diseases.

The impact of anxiety on chronic musculoskeletal pain and the role of astrocyte activation.

Anxiety and depression are associated with increased pain responses in chronic pain states. The extent to which anxiety drives chronic pain, or vice versa, remains an important question that has implications for analgesic treatment strategies. Here, the effect of existing anxiety on future osteoarthritis (OA) pain was investigated, and potential mechanisms were studied in an animal model. Pressure pain detection thresholds, anxiety, and depression were assessed in people with (n = 130) or without (n = 100) painful knee OA. Separately, knee pain and anxiety scores were also measured twice over 12 months in 4730 individuals recruited from the general population. A preclinical investigation of a model of OA pain in normo-anxiety Sprague-Dawley (SD) and high-anxiety Wistar Kyoto (WKY) rats assessed underlying neurobiological mechanisms. Higher anxiety, independently from depression, was associated with significantly lower pressure pain detection thresholds at sites local to (P < 0.01) and distant from (P < 0.05) the painful knee in patients with OA. Separately, high anxiety scores predicted increased risk of knee pain onset in 3274 originally pain-free people over the 1-year period (odds ratio = 1.71; 95% confidence interval = 1.25-2.34, P < 0.00083). Similarly, WKY rats developed significantly lower ipsilateral and contralateral hind paw withdrawal thresholds in the monosodium iodoacetate model of OA pain, compared with SD rats (P = 0.0005). Linear regressions revealed that baseline anxiety-like behaviour was predictive of lowered paw withdrawal thresholds in WKY rats, mirroring the human data. This augmented pain phenotype was significantly associated with increased glial fibrillary acidic protein immunofluorescence in pain-associated brain regions, identifying supraspinal astrocyte activation as a significant mechanism underlying anxiety-augmented pain behaviour.

Spatial and Temporal Heterogeneities of Capillary Hemodynamics and Its Functional Coupling During Neural Activation.

The cerebral vascular system provides a means to meet the constant metabolic needs of neuronal activities in the brain. Within the cerebral capillary bed, the interactions of spatial and temporal hemodynamics play a deterministic role in oxygen diffusion, however, the progression of which remains unclear. Taking the advantages of high-spatiotemporal resolution of optical coherence tomography capillary velocimetry designed with the eigen-decomposition statistical analysis, we investigated intrinsic red blood cell (RBC) velocities and their spatiotemporal adjustment within the capillaries permeating mouse cerebral cortex during electrical stimulation of contralateral hind paw. We found that the mean capillary transit velocity (mCTV) is increased and its temporal fluctuation bandwidth (TFB) is broadened within hind-paw somatosensory cortex. In addition, the degree to which the mCTV is increased negatively correlates with resting state mCTV, and the degree to which the TFB is increased negatively correlates with both the resting state mCTV and the TFB. In order to confirm the changes are due to hemodynamic regulation, we performed angiographic analyses and found that the vessel density remains almost constant, suggesting the observed functional activation does not involve recruitment of reserved capillaries. To further differentiate the contributions of the mCTV and the TFB to the spatiotemporally coupled hemodynamics, changes in the mCTV and TBF of the capillary flow were modeled and investigated through a Monte Carlo simulation. The results suggest that neural activation evokes the spatial transit time homogenization within the capillary bed, which is regulated via both the heterogeneous acceleration of RBC flow and the heterogeneous increase of temporal RBC fluctuation, ensuring sufficient oxygenation during functional hyperemia.

Injecting NMDA and Ro 25-6981 in insular cortex induce neuroplastic changes and neuropathic pain-like behaviour.

Neuropathic pain is associated with abnormal sensitivity of the central nervous system. Although the mechanism underlying the development of sensitization remains to be fully elucidated, recent studies have reported that neuroplastic changes in the pain circuitry may be involved in hypersensitivity associated with neuropathic pain. However, it is difficult to investigate such phenomena in existing animal pain model. Therefore, in this study, we developed a novel animal model - the circuit plasticity reconstruction (CPR) model - to mimic central sensitization associated with neuroplastic changes. NMDA and Ro 25-6981 were injected into the right insular cortex of Sprague-Dawley rats, while electrical stimulation was delivered to the contralateral hind paw. Mechanical allodynia was tested by von Frey test with up-down method, and neuroplastic changes were confirmed by PSA-NCAM-positive immunostaining. The mechanical withdrawal threshold of the left hind paw decreased beginning 1day after CPR modelling and persisted until day 21 comparing to the modified CPR 1 (mod-CPR 1) group (CPR: 91.68±1.8%, mod-CPR 1: 42.71±3.4%, p<0.001). In contrast, mod-CPR 2 surgery without electrical stimulation did not induce mechanical allodynia. Immunostaining for PSA-NCAM also revealed that neuroplastic changes had occurred in the CPR group. Our results demonstrated that CPR modelling induced neuroplasticity within the insular cortex, leading to alterations in the neural circuitry and central sensitization. This article represents that the CPR model can mimic the neuropathic pain derived by neuroplastic changes. Our findings indicate that the CPR model may aid the development of novel therapeutic strategies for neuropathic pain and in elucidating the mechanisms underlying pain induced by central sensitization and neuroplastic changes.

Interactions of peripheral endothelin-1 and nerve growth factor as contributors to persistent cutaneous pain.

Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.

Neuropathic Pain Causes Pyramidal Neuronal Hyperactivity in the Anterior Cingulate Cortex.

The anterior cingulate cortex (ACC) is thought to be important for acute pain perception as well as the development of chronic pain after peripheral nerve injury. Nevertheless, how ACC neurons respond to sensory stimulation under chronic pain states is not well understood. Here, we used an in vivo two-photon imaging technique to monitor the activity of individual neurons in the ACC of awake, head restrained mice. Calcium imaging in the dorsal ACC revealed robust somatic activity in layer 5 (L5) pyramidal neurons in response to peripheral noxious stimuli, and the degree of evoked activity was correlated with the intensity of noxious stimulation. Furthermore, the activation of ACC neurons occurred bilaterally upon noxious stimulation to either contralateral or ipsilateral hind paws. Notably, with nerve injury-induced neuropathic pain in one limb, L5 pyramidal neurons in both sides of the ACC showed enhanced activity in the absence or presence of pain stimuli. These results reveal hyperactivity of L5 pyramidal neurons in the bilateral ACC during the development of neuropathic pain.

Linagliptin attenuates chronic post-ischemia pain: Possible anti-inflammatory and anti-oxidant mechanisms

Complex regional pain syndrome (CRPS) is a debilitating neurologic disorder with an interlinked and yet incompletely defined pathogenesis. Treatment options remain a therapeutic challenge. Linagliptin is one of the dipeptidyl peptidase-4 (DPP-4) inhibitors which are used for the treatment of diabetes mellitus. Apart from the improvement of glycemic control, accumulating evidence points to the beneficial effects of DPP-4 inhibitors in a wide array of conditions. Herein, the present study was outlined to investigate the antinociceptive effect of linagliptin in acute pain conditions, and in an animal model of CRPS.A prolonged hind paw ischemia reperfusion (I/R) injury to the left hind paw was done to induce chronic post-ischemia pain (CPIP) in rats. Allodynia and hyperalgesia were assessed in both ipsilateral and contralateral hind paws at different time points. At the end of the experiment, markers of oxidative stress and inflammatory cytokines were assayed in paw skin samples.The results showed that linagliptin had no effect on acute nociception. On the other hand, linagliptin treatment, for seven days, ameliorated hyperalgesia, mechanical and cold allodynia, and attenuated oxidative and inflammatory markers in CPIP rats.In conclusion, linagliptin was able to ameliorate aberrant pain behavior induced by prolonged hind paw ischemia. These effects can be attributed, at least partially, to the reduction of inflammatory cytokine levels and restore oxidant/antioxidant balance in the CPIP model. Hence, linagliptin pleiotropic effects open a new horizon to further investigate its role in the treatment of inflammatory and chronic painful conditions, especially in diabetic patients.

Repeated electroacupuncture treatment attenuated hyperalgesia through suppression of spinal glial activation in chronic neuropathic pain rats

BackgroundCumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown.MethodsA total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4–5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot.ResultsFollowing CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P < 0.05), and the mechanical PT of the contralateral hind-paw was considerably decreased from the 6th day on after surgery (P < 0.05). CCI also significantly upregulated the number of Iba-1 labeled microgliacytes and the fluorescence intensity of glial fibrillary acidic protein (GFAP) -labeled astrocyte in the superficial laminae of DHs on bilateral sides (P < 0.05). After repeated EA, the mechanical and thermal PTs at bilateral hind-paws were significantly relieved (P < 0.05). The increased of number of microgliacytes was markedly suppressed by 2 days’ EA intervention, and the average fluorescence intensity was suppressed by 2 weeks’ EA. The expression of GFAP protein were down-regulated by 1 and 2 weeks’ EA treatment, respectively (P < 0.05).ConclusionsRepeated EA can relieve neuropathic pain and mirror-image pain in chronic neuropathic pain rats, which is probably associated with its effect in downregulating glial cell activation of the lumbar spinal cord, the microgliacyte first and astrocyte later.

Decreased spontaneous activity and altered evoked nociceptive response of rat thalamic submedius neurons to lumbar vertebra thrust.

The thalamus is a central structure important to modulating and processing all mechanoreceptor input destined for the cortex. A large number of diverse mechanoreceptor endings are stimulated when a high velocity low amplitude thrust is delivered to the lumbar spine during spinal manipulation. The objective of this study was to determine if a lumbar thrust alters spontaneous and/or evoked nociceptive activity in medial thalamic submedius (Sm) neurons. Extracellular recordings were obtained from 94 thalamic Sm neurons in 54 urethane-anesthetized adult Wistar rats. Spontaneous activity was recorded 5min before and after an L5 control (no thrust) and thrust (85% rat body weight; 100ms) procedure. In a subset of responsive nociceptive-specific neurons, mean changes in noxious-evoked response (10-s pinch with clip; 795g) at three sites (tail, contra- and ipsilateral hindpaw) were determined following an L5 thrust. Mean changes in Sm spontaneous activity (60s bins) and evoked noxious response were compared using a mixed model repeated measures ANOVA with Bonferroni post hoc t tests and paired t tests, respectively. Compared to control, spontaneous Sm activity decreased 180-240s following the lumbar thrust (p<0.005). Inhibitory evoked responses were attenuated in the contralateral hindpaw following an L5 thrust compared to control (p<0.05). No other changes in spontaneous or noxious-evoked Sm activity were found. A delayed, but prolonged suppression of spontaneous Sm activity along with changes in noxious-evoked inhibitory responses in the contralateral hindpaw following lumbar vertebra thrust suggest that thalamic submedius neurons may play a role in central pain modulation related to manual therapy intervention.

Bilateral tactile hypersensitivity and neuroimmune responses after spared nerve injury in mice lacking vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is one of the neuropeptides showing the strongest up-regulation in the nociceptive pathway after peripheral nerve injury and has been proposed to support neuropathic pain. Nevertheless, the story may be more complicated considering the known suppressive effects of the peptide on the immune reactivity of microglial cells, which have been heavily implicated in the onset and maintenance of pain after nerve injury. We here used mice deficient in VIP and the model of spared nerve injury, characterized by persistent tactile hypersensitivity. While tactile hypersensitivity developed similarly to wild type mice for the ipsilateral hindpaw, only transgenic mice showed a mirror-image tactile hypersensitivity in the contralateral hindpaw. This exacerbated neuropathic pain phenotype appeared to be mediated through a local mechanism acting at the level of the lumbar spinal cord as a distant nerve lesion in the front limb did not lead to hindpaw hypersensitivity in VIP-deficient mice. Innocuous tactile hindpaw stimulation was found to increase a neuronal activation marker in the bilateral superficial laminae of the lumbar dorsal horn of VIP-deficient, but not wild type mice, after SNI. A deeper study into the immune responsiveness to the nerve lesion also proved that VIP-deficient mice had a stronger early pro-inflammatory cytokine response and a more pronounced microglial reactivity compared to wild type controls. The latter was also observed at four weeks after spared nerve injury, a time at which bilateral tactile hypersensitivity persisted in VIP-deficient mice. These data suggest an action of VIP in neuropathic states that is more complicated than previously assumed. Future research is now needed for a deeper understanding of the relative contribution of receptors and fiber populations involved in the VIP-neuropathic pain link.

Local analgesic effect of tramadol is mediated by opioid receptors in late postoperative pain after plantar incision in rats.

Tramadol is a drug used to treat moderate to severe pain. It is known to present a peripheral effect, but the local mechanisms underlying its actions remain unclear. The role of peripheral opioid receptors in postoperative pain is not well understood. In the present study, we examined the peripheral opioid receptors to determine the local effect of tramadol in a plantar incision pain model. Rats were subjected to plantar incision and divided into four groups on postoperative day (POD) 1: SF_SF, 0.9% NaCl injected into the right hindpaw; SF_TraI, 0.9% NaCl and tramadol injected into the right hindpaw; SF_TraC, 0.9% NaCl and tramadol injected into the contralateral hindpaw; and Nal_Tra, naloxone and tramadol injected into the ipsilateral hindpaw. To determine the animals’ nociceptive threshold, mechanical hyperalgesia was measured before incision, on POD1 before treatment and at 15, 30, 45, and 60 minutes after the incision. The same procedure was repeated on the POD2. The expression levels of μ-opioid receptor (MOR) and δ-opioid receptor (DOR) were obtained through immunoblotting assays in the lumbar dorsal root ganglia (L3–L6) in naïve rats and 1, 2, 3, and 7 days after the incision. Our results showed that the plantar incision was able to cause an increase in mechanical hyperalgesia and that tramadol reversed this hyperalgesia on POD1 and POD2. Tramadol injections in the contralateral paw did not affect the animals’ nociceptive threshold. Naloxone was able to antagonize the tramadol effect partially on POD1 and completely on POD2. The DOR expression increased on POD2, POD3, and POD7, whereas the MOR expression did not change. Together, our results show that tramadol promoted a local analgesic effect in the postoperative pain model that was antagonized by naloxone in POD2, alongside the increase of DOR expression.

Antinociceptive action of botulinum toxin type A in carrageenan-induced mirror pain.

"Mirror pain" or mirror-image pain (MP) is pain opposite to the side of injury. Mechanism and frequency in humans are not known. There is no consent on therapy. Here we report that unilaterally injected botulinum toxin type A (BT-A) has bilateral effect in experimental MP, thus deserves to be investigated as therapy for this condition. We examined the localization of BT-A's bilateral antinociceptive action in MP induced by 3% carrageenan intramuscular injection in Wistar rats. BT-A was applied peripherally (5U/kg), into ipsilateral or contralateral hind paw pad (i.pl.) and centrally (1U/kg), at spinal (intrathecally, i.t.) or supraspinal (intracisternally, i.c.) level. Additionally, we examined the involvement of central opioid and GABAergic systems, as well as the contribution of peripheral capsaicin-sensitive neurons to BT-A's bilateral antinociceptive effect. Ipsilateral i.pl. and i.t. BT-A reduced the bilateral mechanical sensitivity to von Frey filaments, while contralateral i.pl. and i.c. treatments had no effect on either tested side. Bilateral antinociceptive effect of ipsilateral i.pl. BT-A was prevented by μ-opioid antagonist naloxonazine (1.5μg/10μl) and GABAA antagonist bicuculline (1μg/10μl) if applied at the spinal level, in contrast to supraspinal application of the same doses. Local treatment of sciatic nerve with 2% capsaicin 5days following BT-A i.pl. injection caused desensitization of sciatic capsaicin-sensitive fibers, but did not affect bilateral antinociceptive effect of BT-A and the presence of cleaved SNAP-25 at the spinal cord slices. Present experiments suggest segmental actions of peripheral BT-A at spinal level, which are probably not solely dependent on capsaicin-sensitive neurons.