Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1

Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle-induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long-lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non-selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.     

HCN2 channels account for mechanical (but not heat) hyperalgesia during long-standing inflammation

There is emerging evidence that hyperpolarization-activated cation (HCN) channels are involved in the development of pathological pain, including allodynia and hyperalgesia. Mice lacking the HCN isoform 2 display reduced heat but unchanged mechanical pain behavior, as recently shown in preclinical models of acute inflammatory pain. However, the impact of HCN2 to chronic pain conditions is less clear and has not been examined so far. In this report, we study the role of HCN2 in the complete Freund’s adjuvant inflammation model reflecting chronic pain conditions. We used sensory neuron–specific as well as inducible global HCN2 mutants analyzing pain behavior in persistent inflammation and complemented this by region-specific administration of an HCN channel blocker. Our results demonstrate that the absence of HCN2 in primary sensory neurons reduces tactile hypersensitivity in chronic inflammatory conditions but leaves heat hypersensitivity unaffected. This result is in remarkable contrast to the recently described role of HCN2 in acute inflammatory conditions. We show that chronic inflammation results in an increased expression of HCN2 and causes sensitization in peripheral and spinal terminals of the pain transduction pathway. The contribution of HCN2 to peripheral sensitization mechanisms was further supported by single-fiber recordings from isolated skin–nerve preparations and by conduction velocity measurements of saphenous nerve preparations. Global HCN2 mutants revealed that heat hypersensitivity—unaffected in peripheral HCN2 mutants—was diminished by the additional disruption of central HCN2 channels, suggesting that thermal hyperalgesia under chronic inflammatory conditions is mediated by HCN2 channels beyond primary sensory afferents.     

Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia after muscle inflammation

Acid-sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3−/− mice. ASIC3−/− mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild-type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild-type mice prevented development of hyperalgesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR-ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO-K1 cells transfected with mASIC3 cDNA in culture, the miR-ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH-evoked currents and had no effect on protein expression or acidic pH-evoked currents of ASIC1a. When miR-ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan-induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miR-ASIC3. In CHO-K1 cells co-transfected with ASIC1a and ASIC3, miR-ASIC3 reduced the amplitude of acidic pH-evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3-containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild-type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.     

Spinal neurokinin3 receptors mediate thermal but not mechanical hyperalgesia via nitric oxide

Although intrathecally administered senktide, an agonist at the neurokinin3 receptor, attenuates withdrawal responses to noxious stimuli in the restrained animal, senktide increases motor neuron activity in spinal cords of neonatal rats and facilitates the electrically-evoked nociceptive flexor reflex in the adult rat. The present study examined the effects of intrathecal administration of senktide on withdrawal responses to noxious thermal and mechanical stimuli in awake, unrestrained, adult rats. Intrathecal administration of senktide (10 nmol) in chronically catheterized rats did not alter the responses elicited by a noxious mechanical stimulus (508 mN, von Frey monofilament). Conversely, intrathecal senktide (10 nmol) induced thermal hyperalgesia, indicated by decreased withdrawal latency to radiant heat. Thermal hyperalgesia peaked 20-26 min following drug injection and returned to normal within 30 min. SR 142801 (60 nmol), a non-peptide neurokinin3 receptor antagonist, inhibited the senktide-induced hyperalgesia, providing further support that the effect of senktide is mediated by neurokinin3 receptors. Pretreatment with N(G)-nitro-L-arginine methyl ester (30 nmol), a nitric oxide synthase inhibitor, blocked the effect of senktide, indicating that senktide-induced thermal hyperalgesia is also mediated by the production of nitric oxide. Intrathecal senktide produced vasodilation and increased skin temperature in the hind paw. Intravenous hexamethonium, a ganglionic nicotinic receptor antagonist, similarly increased paw temperature without decreasing withdrawal latency to radiant heat. Thus, the increased skin temperature associated with intrathecal senktide was insufficient to account for the thermal hyperalgesia observed. Collectively, the present work demonstrates that NK3 receptors mediate thermal but not mechanical hyperalgesia through a pathway that involves the production of NO.     

Heat, but not mechanical hyperalgesia, following adrenergic injections in normal human skin

The development of adrenergic sensitivity in nociceptors has been suggested as a mechanism of neuropathic pain. We sought to determine if nociceptors in the skin of normal subjects exhibit adrenergic sensitivity. We investigated the effects of intradermal administration of norepinephrine, phenylephrine, and brimonidine on heat pain sensitivity. Norepinephrine and phenylephrine (in concentrations ranging from 0.1 to 10 microM by factors of 10), brimonidine (at 0.01-1 microM), and saline were injected (30 microl volume) in a random, double-blind manner to different sites on the volar surface of the forearm in ten subjects. Before and after the injections, heat testing was performed with a non-contact laser thermal stimulator. Heat pain threshold was measured by means of a 'Marstock' technique in which subjects pressed a reaction time key when they perceived that a slowly increasing heat stimulus (1 degrees C/s ramp from a 36 degrees C base) was painful. In addition, the subjects used magnitude estimation techniques to rate the intensity of pain to a suprathreshold heat stimulus (47 degrees C, 2 s). Mechanical testing was done using 200-microm diameter probes attached to calibrated weights that provided forces over the range of 16-512 mN. The intradermal injections of norepinephrine, phenylephrine and brimonidine produced little evoked pain. However, a dose-dependant decrease in heat pain threshold, but not mechanical pain threshold, was observed. At the highest drug dose injected, all three adrenergic compounds produced a significant decrease in heat pain threshold compared to the saline injection. A significant increase in response to the suprathreshold heat stimulus was also found. One possible explanation for this apparent heat hyperalgesia is that the decrease in perfusion due to the localized vasoconstriction may alter the heat response. However, in control studies we found that the non-adrenergic vasoconstrictors, angiotensin II and vasopressin did not produce heat hyperalgesia at doses that produced comparable decreases in blood flow. In addition, occlusion of blood flow with a blood pressure cuff did not lead to heat hyperalgesia. Thus, the heat hyperalgesia observed with the adrenergic agonists is not due to a decrease in perfusion associated with the injection. These results indicate that alpha(1)- and alpha(2)-adrenoceptor-mediated mechanisms may play a role in sensitization of nociceptors to heat stimuli in normal skin.     

Body fat and skeletal muscle mass,but not body mass index,are associated with pressure hyperalgesia in young adults with patellofemoral pain

BackgroundYoung adults with patellofemoral pain (PFP) have a high prevalence of being overweight or obese, which is associated with impaired lower limb function and muscle weakness. However, the impact of being overweight or obese on pain sensitivity has not been explored.ObjectivesWe investigated the association between body fat, skeletal muscle mass, and body mass index (BMI) with pressure hyperalgesia and self-reported pain in young adults with PFP.Methods114 adults with PFP (24 ± 5 years old, 62% women) were recruited. Demographics and self-reported pain (current and worst knee pain intensity in the previous month - 0–100 mm visual analog scale) were recorded. Body fat and skeletal muscle mass were measured using bioelectrical impedance. Pressure hyperalgesia was measured using a handheld algometer (pressure pain threshold) at three sites: center of patella of the painful knee, ipsilateral tibialis anterior, and contralateral upper limb. The association between body fat, skeletal muscle mass, and BMI with pressure hyperalgesia and self-reported pain were investigated using partial correlations and hierarchical regression models (adjusted for sex, bilateral pain, and symptoms duration).ResultsHigher body fat and lower skeletal muscle mass were associated with local, spread, and widespread pressure hyperalgesia (ΔR²=0.09 to 0.17, p ≤ 0.001; ΔR²=0.14 to 0.26, p<0.001, respectively), and higher current self-reported pain (ΔR²=0.10, p<0.001; ΔR²=0.06, p = 0.007, respectively). Higher BMI was associated with higher current self-reported pain (ΔR²=0.10, p = 0.001), but not with any measures of pressure hyperalgesia (p>0.05).ConclusionHigher body fat and lower skeletal muscle mass help to explain local, spread, and widespread pressure hyperalgesia, and self-reported pain in people with PFP. BMI only helps to explain self-reported pain. These factors should be considered when assessing people with PFP and developing their management plan, but caution should be taken as the strength of association was generally low.     

Joint mobilization reduces hyperalgesia associated with chronic muscle and joint inflammation in rats.

Joint mobilization is a common treatment used by healthcare professions for management of a variety of painful conditions, including inflammatory joint and muscle pain. We hypothesized that joint mobilization would reduce the bilateral hyperalgesia induced by muscle and joint inflammation. Mechanical hyperalgesia was measured by examining the mechanical withdrawal threshold of the rat's paw before and after induction of inflammation with 3% carrageenan (gastrocnemius muscle) or 3% kaolin/carrageenan (knee joint), and for 1 hour after knee joint mobilization. The mobilization consisted of rhythmically flexing and extending the knee joint to the end of range of extension while the tibia was simultaneously moved in an anterior to posterior direction. A bilateral decrease in mechanical withdrawal thresholds occurred 1, 2, and 4 weeks after inflammation of the knee joint or muscle. In animals with muscle inflammation, mobilization of the knee joint increased the mechanical withdrawal threshold bilaterally when given 1, 2, or 4 weeks after inflammation. However, in animals with knee joint inflammation, mobilization of the knee joint at 4 weeks increased the mechanical withdrawal threshold but had no effect when administered 1 or 2 weeks after inflammation. Therefore, joint mobilization reduces hyperalgesia induced by chronic inflammation of muscle and joint. PERSPECTIVE: This article shows that unilateral joint mobilization reduces bilateral hyperalgesia induced by chronic muscle or joint inflammation. Understanding the pain conditions in which mobilization produces an analgesic effect should assist the clinician in selecting appropriate treatment techniques. The bilateral effect suggests that central mechanisms could mediate the analgesia.     

Role of ASIC3 in the primary and secondary hyperalgesia produced by joint inflammation in mice

The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 −/− mice and examined the primary (response to tweezers) and secondary hyperalgesia (von-Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 −/− mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 −/− mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene-related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co-localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.     

Pre-emptive ring-block with bupivacaine prevents the development of thermal hyperalgesia, but not sustained mechanical hyperalgesia, in rat tails exposed to ultraviolet A light.

We investigated the role of the C-fiber barrage in the development of hyperalgesia in rat tails exposed to ultraviolet A (UVA)-light exposure by pre-emptively blocking C-fiber activation with the local anesthetic bupivacaine. Thirty minutes before UVA-light exposure, male Sprague-Dawley rats were given subcutaneous injections, in the base of the tail, of either saline or bupivacaine (1 mL of .5%). Thermal hyperalgesia was assessed daily from day 1 to day 10 after UVA-light exposure by measuring response latency to noxious heat (tail immersion in 49 degrees C water). Injection of bupivacaine completely prevented the development of thermal hyperalgesia (P < .05, main effect of group, 2-way analysis of variance). Primary mechanical hyperalgesia was assessed daily from day 1 to day 14 after UVA-light exposure by measuring aversive behavior responses to a punctate pressure applied to the tail with a von Frey anesthesiometer. The rats given bupivacaine developed hyperalgesia to the mechanical challenge that persisted for 14 days (P < .05, main effect of time, 2-way analysis of variance) and was identical to the hyperalgesia developed by the rats given saline. We concluded that the C-fiber barrage is involved in the development of thermal hyperalgesia but not sustained primary mechanical hyperalgesia, induced by exposing rats' tails to UVA light. PERSPECTIVE: UVA-light exposure of the rat tail causes prolonged hyperalgesia to noxious thermal and mechanical challenges. We have demonstrated that the C-fiber barrage is involved in the development of sustained thermal hyperalgesia, but not mechanical hyperalgesia, caused by exposure of the rat tail to UVA light.     

Thiamine, pyridoxine, cyanocobalamin and their combination inhibit thermal, but not mechanical hyperalgesia in rats with primary sensory neuron injury

Neuropathic pain after nerve injury is severe and intractable, and current drugs and nondrug therapies offer substantial pain relief to no more than half of affected patients. The present study investigated the analgesic roles of the B vitamins thiamine (B1), pyridoxine (B6) and cyanocobalamin (B12) in rats with neuropathic pain caused by spinal ganglia compression (CCD) or loose ligation of the sciatic nerve (CCI). Thermal hyperalgesia was determined by a significantly shortened latency of foot withdrawal to radiant heat, and mechanical hyperalgesia was determined by a significantly decreased threshold of foot withdrawal to von Frey filaments stimulation of the plantar surface of hindpaw. Results showed that (1) intraperitoneal injection of B1 (5, 10, 33 and 100 mg/kg), B6 (33 and 100 mg/kg) or B12 (0.5 and 2 mg/kg) significantly reduced thermal hyperalgesia; (2) the combination of B1, B6 and B12 synergistically inhibited thermal hyperalgesia; (3) repetitive administration of vitamin B complex (containing B1/B6/B12 33/33/0.5 mg/kg, for 1 and 2 wk) produced long-term inhibition of thermal hyperalgesia; and (4) B vitamins did not affect mechanical hyperalgesia or normal pain sensation, and exhibited similar effects on CCD and CCI induced-hyperalgesia. The present studies demonstrate effects of B vitamins on pain and hyperalgesia following primary sensory neurons injury, and suggest the possible clinical utility of B vitamins in the treatment of neuropathic painful conditions following injury, inflammation, degeneration or other disorders in the nervous systems in human beings.     

Endogenous kappa-opioid receptor systems inhibit hyperalgesia associated with localized peripheral inflammation

Peripheral inflammation evokes functional and biochemical changes in the periphery and spinal cord which result in central sensitization and hypersensitivity. Inhibitory control systems from the rostral ventromedial medulla (RVM) are also activated. The present study investigates whether endogenous kappa-opioid receptor (KOPr) systems contribute to these neuroadaptations. Inflammation was induced by intraplantar injection of complete Freund’s adjuvant (CFA) into one hindpaw. Mechanical and thermal thresholds were determined using the Von Frey and radiant heat tests, respectively. KOPr gene deletion in mice or systemic administration of the long-acting KOPr antagonist, norbinaltorphimine (norBNI) significantly exacerbated mechanical and thermal hypersensitivity of the ipsilateral, inflamed paw. Thermal and mechanical thresholds of the non-inflamed, contralateral hindpaw were unaffected by CFA treatment. However, gene deletion as well as norBNI treatment resulted in mechanical, but not thermal hypersensitivity of the non-inflamed paw. Similar results were obtained when norBNI was administered intrathecally or into the RVM in rats. These data demonstrate a previously unrecognized role of endogenous KOPr systems in inhibiting hyperalgesia during inflammation. Furthermore, they demonstrate that decreased KOPr activity in either the spinal cord or RVM not only enhances mechanical and thermal hyperalgesia of the inflamed limb but also leads to an unmasking of mechanical hyperalgesia at a site remote from inflammation. The differential effects of KOPr antagonism on mechanical versus thermal thresholds for the non-inflamed paw support the notion that distinct neuroanatomical or neurochemical mechanisms modulate the processing of thermal versus mechanical stimuli.     

In vivo evidence for apoptosis, but not inflammation in the hindlimb muscle of neuropathic rats

Loose ligation of the rat sciatic nerve (chronic constriction injury (CCI) model) provokes signs and symptoms like those observed in complex regional pain syndrome (CRPS) patients. Neurogenic inflammation is a purported cause of neuropathic pain despite inconsistent evidence to support this hypothesis. To clarify this issue, we examined effects of CCI on microcirculation, inflammatory cell-cell interaction and cell integrity in muscle tissue using intravital fluorescence microscopic, molecular and immunohistochemical techniques. CCI-rats, but not sham-operated animals developed symptoms of neuropathic pain and oedema on the ipsilateral hindpaw. Despite signs of neuropathic pain, high resolution in vivo multifluorescence microscopy revealed physiological values for functional capillary density, leukocyte-endothelial cell interaction and microvascular permeability in muscle tissue of CCI-animals, similarly as found in controls, indicating absence of perfusion failure and inflammatory cell reaction. However, CCI-animals represented with marked apoptosis of bisbenzimide-stained muscle tissue cells, as given by in vivo fluorescence microscopic assessment of cell death-associated condensation, fragmentation and/or crescent-shaped formation of their nuclear chromatin. Apoptosis was further confirmed by increased caspase 3 protein levels and positive terminal deoxyuridine nick end labeling histochemistry. The present study demonstrates that sciatic nerve ligation-induced neuropathy causes cell apoptosis without concomitant inflammation-associated microcirculatory dysfunction in muscle tissue. Beside the well-known pattern of neuropathic pain, the CCI-model has now additionally been shown to reflect the response of muscle tissue to impaired innervation, i.e. prompting muscle cells to undergo non-inflammatory apoptotic cell death. This finding deserves further investigation in that apoptosis may contribute to neuropathic pain conditions like CRPS.     

Therapeutic administration of nitric oxide synthase inhibitors reverses hyperalgesia but not inflammation in a rat model of polyarthritis

Nitric oxide (NO) has been postulated to play a role in pain as well as in inflammation. In the present studies, the effects of NO synthase (NOS) inhibitors on both pain and inflammation were examined in a rat model of polyarthritis. Female Lewis rats were injected intraperitoneally (i.p.) with peptidoglycan/polysaccharide (PG/PS) or saline to induce arthritis. Hind paw volume, response latency to thermal nociceptive stimulus and mechanical threshold were measured daily for the next 35 days. Paw inflammation, thermal hyperalgesia and mechanical allodynia developed in all rats that received PG/PS compared to saline. On day 19 (chronic inflammation phase), rats were given either N(G)-nitro-L-arginine methyl ester (L-NAME, non-selective NOS inhibitor, 100 mg/l), L-N (6)-(1-iminoethyl) lysine (L-NIL, selective inducible NOS inhibitor, 10 mg/l) or no drug in drinking water. By day 21, L-NAME treatment reversed the thermal hyperalgesia completely and this effect remained until day 35. Similarly, L-NIL treatment reversed thermal hyperalgesia from days 24 to 34. Neither treatment affected mechanical allodynia. Paw volume was not different between PG/PS treated and PG/PS plus L-NAME treated rats. However, the PG/PS plus L-NIL treatment produced an increase in paw volume greater than did PG/PS alone. Other rats were treated with PG/PS plus the antiinflammatory agent indomethacin (days 19-35). Indomethacin treatment reversed all the measured parameters, although the reversal of mechanical allodynia was only partial. These results suggest that NO is involved in thermal, but not mechanical sensory pathways and that the selective inhibition of inducible NOS activity exacerbates established inflammation.     

Secondary heat, but not mechanical, hyperalgesia induced by subcutaneous injection of bee venom in the conscious rat: effect of systemic MK-801, a non-competitive NMDA receptor antagonist

Subcutaneous (s.c.) administration of bee venom into the plantar surface of one hind paw in rats has been found to produce an immediate single phase of persistent spontaneous nociceptive responses (continuously flinching, licking or lifting the injected paw) for 1-2 h accompanied by a 72-96 hour period of primary heat and mechanical hyperalgesia in the injection site and a spread of heat, but not mechanical, hyperalgesia in the non-injected hind paw (Chen et al., 1999b). To gain insight into the underlying mechanisms of the bee venom-induced hyperalgesia in particular, we further identified a heat, but not mechanical, hyperalgesia in an area (paw pad) distant from the injection site induced by s.c. injection of bee venom into the posterior leg 0.8-1.2 cm proximal to the heel measured by paw withdrawal reflex to radiant heat or von Frey monofilament stimuli in conscious rats. In the bee venom-treated hind limb, however, significant reduction in both thermal latency and mechanical threshold of withdrawal reflex was identified for a period of more than 96 h in the heel with a similar characteristic to the primary heat and mechanical hyperalgesia identified in the injection site previously. The time course of the heat hyperalgesia identified in the paw pad of the bee venom-treated side was shorter and lasted for less than 48 h, which was in parallel with the reduction in thermal latency of the withdrawal reflex identified in the non-injected hind paw. Moreover, pre- or post-treatment with a single dose of MK-801 (0.01 mg/kg, i.p.), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, completely blocked the occurrence, and reversed the established process of the heat hyperalgesia identified in either the bee venom-treated or non-treated paw pads, while the same treatments with the drug did not produce any influence upon the development and maintaining of the heat and mechanical hyperalgesia identified in the heel of the injected hind limb. Taken together with our previous results following s.c. intraplantar bee venom injection, we conclude that: (1) in addition to the well-identified primary heat and mechanical hyperalgesia in the injection site and its adjacent area, s.c. bee venom is also able to produce a secondary heat hyperalgesia in a region distant from the injection site which has a similar characteristic to the contralateral heat hyperalgesia; (2) NMDA receptors are involved in either development or maintenance of the secondary and the contralateral heat hyperalgesia, but without any role in those processes of the primary heat and mechanical hyperalgesia; (3) the secondary heat hyperalgesia seen in the injected hind limb is likely to share the same neural mechanisms with that identified in the non-injected side via co-activation of NMDA receptors.     

P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain.

The present study was undertaken to determine the role of P2X3 receptor (P2X3R) on heat hyperalgesia in a newly developed rat model of trigeminal neuropathic pain. The unilateral infraorbital nerve (IoN) was partially ligated by 6-0 silk. To assess heat sensitivity, a vibrissal pad (VP) was placed on a hot plate and the latency until the rats withdrew their head was measured. Mechanical sensitivity of VP was also assessed by the use of von Frey filament. Both heat and mechanical hyperalgesia were observed at the VP ipsilateral to the IoN ligation. The latency to heat stimuli was prolonged after subcutaneous administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X1,2,3,5,7,1/5,2/3R antagonist) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP, P2X1,3,2/3,1/5R antagonist). The latency was shortened after administration of alpha,beta-methylene ATP (alpha,beta-meATP, P2X1,3,2/3R agonist), although no changes appeared after administration of beta,gamma-methylene-L-ATP (beta,gamma-me-L-ATP, P2X1R agonist). The protein gene product-9.5 and calcitonin gene-related peptide immunoreactive nerve fibers significantly decreased in the VP skin of ipsilateral to the IoN ligation. In the ipsilateral trigeminal ganglion, the number of P2X3-immunoreactive neurons significantly increased in the small cell group. In this study, we developed an experimental model of trigeminal neuropathic pain by partial ligation of IoN, which produced heat and mechanical hyperalgesia in the VP. Pharmacological and immunohistochemical studies revealed that the P2X3R plays an important role in the heat hyperalgesia observed in this model. PERSPECTIVE: The study describes the development of a novel model of trigeminal neuropathic pain. Heat hyperalgesia in this model was inhibited by peripheral injection of P2XR antagonists. The results suggest that P2X3R is a potential target for development of a novel therapy for trigeminal neuropathic pain.     

Mice lacking acid‐sensing ion channels (ASIC) 1 or 2, but not ASIC3, show increased pain behaviour in the formalin test

Extracellular acidification is a component of the inflammatory process and may be a factor driving the pain accompanying it. Acid-sensing ion channels (ASICs) are neuronal proton sensors and evidence suggests they are involved in signalling inflammatory pain. The aims of this study were to (1) clarify the role of ASICs in nociception and (2) confirm their involvement in inflammatory pain and determine whether this was subunit specific.This was achieved by (1) direct comparison of the sensitivity of ASIC1, ASIC2, ASIC3 and TRPV1 knockout mice versus wildtype littermates to acute thermal and mechanical noxious stimuli and (2) studying the behavioural responses of each transgenic strain to hind paw inflammation with either complete Freund’s adjuvant (CFA) or formalin.Naïve ASIC1^?/? and ASIC2^?/? mice responded normally to acute noxious stimuli, whereas ASIC3^?/? mice were hypersensitive to high intensity thermal stimuli. CFA injection decreased mechanical and thermal withdrawal thresholds for up to 8 days. ASIC2^?/? mice had increased mechanical sensitivity on day 1 post-CFA compared to wildtype controls. TRPV1^?/? mice had significantly reduced thermal, but not mechanical, hyperalgesia on all days after inflammation. Following formalin injection, ASIC1^?/? and ASIC2^?/?, but not ASIC3^?/? or TRPV1^?/?, mice showed enhanced pain behaviour, predominantly in the second phase of the test.These data suggest that whilst ASICs may play a role in mediating inflammatory pain, this role is likely to be modulatory and strongly dependent on channel subtype.     

Low frequencies, but not high frequencies of bi-polar spinal cord stimulation reduce cutaneous and muscle hyperalgesia induced by nerve injury

Spinal cord stimulation (SCS) is an established treatment for neuropathic pain. However, SCS is not effective for all the patients and the mechanisms underlying the reduction in pain by SCS are not clearly understood. To elucidate the mechanisms of pain relief by SCS, we utilized the spared nerve injury model. Sprague–Dawley rats were anesthetized, the tibial and common peroneal nerves were tightly ligated, and an epidural SCS lead implanted in the upper lumbar spinal cord. SCS was delivered daily at one of 4 different frequencies (4 Hz, 60 Hz, 100 Hz, and 250 Hz) at approximately 85% of motor threshold 2 weeks after nerve injury for 4 days. Mechanical withdrawal threshold of the paw and compression withdrawal threshold of the hamstring muscles were measured before and after SCS on each day. All rats showed a decrease in withdrawal threshold of the paw and the muscle 2 weeks after nerve injury. Treatment with either 4 Hz or 60 Hz SCS significantly reversed the decreased withdrawal threshold of the paw and muscle. The effect was cumulative with a greater reversal by the fourth treatment when compared to the first treatment. Treatment with 100 Hz, 250 Hz or sham SCS had no significant effect on the decreased withdrawal threshold of the paw or muscle that normally occurs after nerve injury. In conclusion, SCS at 4 Hz and 60 Hz was more effective in reducing hyperalgesia than higher frequencies of SCS (100 Hz and 250 Hz); and repeated treatments result in a cumulative reduction in hyperalgesia.     

TRPV1 is important for mechanical and heat sensitivity in uninjured animals and development of heat hypersensitivity after muscle inflammation

Inflammatory thermal hyperalgesia is principally mediated through transient receptor potential vanilloid 1 (TRPV1) channels, as demonstrated by prior studies using models of cutaneous inflammation. Muscle pain is significantly different from cutaneous pain, and the involvement of TRPV1 in hyperalgesia induced by muscle inflammation is unknown. We tested whether TRPV1 contributes to the development of mechanical and heat hypersensitivity of the paw in TRPV1(-/-) mice after muscle inflammation. Because TRPV1(-/-) mice lack TRPV1 at the site of inflammation (muscle) and at the testing site (paw), we do not know whether TRPV1 is important as a mediator of nociceptor sensitization in the muscle or as a heat sensor in the paw. Using recombinant herpesviruses, we reexpressed TRPV1 in TRPV1(-/-) mice in primary afferents innervating skin, muscle, or both to determine which sites were important for the behavioral deficits. Responses to repeated application of noxious mechanical stimuli to the hind paw were enhanced in TRPV1(-/-) mice; this was restored by reexpression of TRPV1 into skin. Withdrawal latencies to noxious heat were increased in TRPV1(-/-) mice; normal latencies were restored by reexpression of TRPV1 in both skin and muscle. Heat hypersensitivity induced by muscle inflammation did not develop in TRPV1(-/-) mice; mechanical hypersensitivity was similar between TRPV1(-/-) and TRPV1(+/+) mice. Heat hypersensitivity induced by muscle inflammation was restored by reexpression of TRPV1 into both muscle and skin of TRPV1(-/-) mice. These results suggest that TRPV1 serves as both a mediator of nociceptor sensitization at the site of inflammation and as a heat sensor at the paw.