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1.  Spinal muscarinic receptors are activated during low or high frequency TENS-induced antihyperalgesia in rats 
Neuropharmacology  2003;45(8):1111-1119.
Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin—carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 μg), non-selective muscarinic antagonist atropine (30 μg) or one of the muscarinic subtype antagonists: pirenzepine (M1, 10 μg), methoctramine (M2, 10 μg), 4-DAMP (M3, 10 μg), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M1 and M3 muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.
PMCID: PMC2746650  PMID: 14614954
Electrical nerve stimulation; Cholinergic; Muscarinic; Inflammation; Pain; Spinal cord
2.  Release of GABA and activation of GABAA in the spinal cord mediates the effects of TENS in rats 
Brain research  2007;1136(1):43-50.
Transcutaneous electrical nerve stimulation (TENS) is a commonly utilized non-pharmacological, non-invasive treatment for pain. GABA is a neurotransmitter in the dorsal horn of the spinal cord that mediates analgesia locally, and also through activation of supraspinal sites. TENS reduces hyperalgesia through activation of receptor-mediated pathways at the level of the spinal cord, and supraspinally. The current study tested the hypothesis that either high or low frequency TENS applied to the inflamed knee joint increases GABA in the spinal cord dorsal horn and activates GABA receptors spinally. We utilized microdialysis to sample the extracellular fluid before, during and after TENS and analyzed GABA in dialysates with high performance liquid chromatography. We analyzed the extracellular GABA concentrations in animals with and without knee joint inflammation induced by intra-articular injection of kaolin and carrageenan. We further tested if spinal blockade of GABA receptors prevents the antihyperalgesia produced by TENS in rats with joint inflammation. We show that high frequency TENS increases extracellular GABA concentrations in the spinal cord in animals with and without joint inflammation. The increases in GABA do not occur in response to low frequency TENS, and there are no increases in glycine in response to low or high frequency TENS. However, the reduction in primary hyperalgesia by both high and low frequency TENS is prevented by spinal blockade of GABAA receptors with bicuculline. Thus, high frequency TENS increases release of GABA in the deep dorsal horn of the spinal cord, and both high and low frequency TENS reduce primary hyperalgesia by activation of GABAA receptors spinally.
PMCID: PMC2746639  PMID: 17234163
Pain; Electrical stimulation; Microdialysis; Bicuculline; Hyperalgesia
3.  Joint manipulation reduces hyperalgesia by activation of monoamine receptors but not opioid or GABA receptors in the spinal cord 
Pain  2003;106(1-2):159-168.
Joint manipulation has long been used for pain relief. However, the underlying mechanisms for manipulation-related pain relief remain largely unexplored. The purpose of the current study was to determine which spinal neurotransmitter receptors mediate manipulation-induced antihyperalgesia. Rats were injected with capsaicin (50 μl, 0.2%) into one ankle joint and mechanical withdrawal threshold measured before and after injection. The mechanical withdrawal threshold decreases 2 h after capsaicin injection. Two hours after capsaicin injection, the following drugs were administered intrathecally: bicuculline, blocks γ-aminobutyric acid (GABAA) receptors; naloxone, blocks opioid receptors; yohimbine blocks, α2-adrenergic receptors; and methysergide, blocks 5-HT1/2 receptors. In addition, NAN-190, ketanserin, and MDL-72222 were administered to selectively block 5-HT1A, 5-HT2A, and 5-HT3 receptors, respectively. Knee joint manipulation was performed 15 min after administration of drug. The knee joint was flexed and extended to end range of extension while the tibia was simultaneously translated in an anterior to posterior direction. The treatment group received three applications of manipulation, each 3 min in duration separated by 1 min of rest. Knee joint manipulation after capsaicin injection into the ankle joint significantly increases the mechanical withdrawal threshold for 45 min after treatment. Spinal blockade of 5-HT1/2 receptors with methysergide prevented, while blockade of α2-adrenergic receptors attenuated, the manipulation-induced antihyperalgesia. NAN-190 also blocked manipulation-induced antihyperalgesia suggesting that effects of methysergide are mediated by 5-HT1A receptor blockade. However, spinal blockade of opioid or GABAA receptors had no effect on manipulation induced-antihyperalgesia. Thus, the antihyperalgesia produced by joint manipulation appears to involve descending inhibitory mechanisms that utilize serotonin and noradrenaline.
PMCID: PMC2732015  PMID: 14581123
Joint manipulation; Serotonin; Noradrenaline; Pain; Capsaicin; Spinal cord
5.  Increased response of muscle sensory neurons to decreases in pH after muscle inflammation 
Neuroscience  2010;170(3):893-900.
Acid sensing ion channels (ASIC) are found in sensory neurons, including those that innervate muscle tissue. After peripheral inflammation there is an increase in proton concentration in the inflamed tissue, which likely activates ASICs. Previous studies from our laboratory in an animal model of muscle inflammation show that hyperalgesia does not occur in ASIC3 and ASIC1 knockout mice. Therefore, in the present study we investigated if pH activated currents in sensory neurons innervating muscle are altered after induction of muscle inflammation. Sensory neurons innervating mouse (C57/Bl6) muscle were retrogradely labeled with DiI. Two weeks after injection of DiI, mice were injected with 3% carrageenan to induce inflammation (n=8; 74 neurons) or pH 7.2 saline (n=5; 40 neurons, control) into the gastrocnemius muscle. 24h later sensory neurons from L4–L6 DRG were isolated and cultured. The following day the DRG neuron cultures were tested for responses to pH by whole-cell patch-clamp technique. Approximately 40% of neurons responded to pH 5 with an inward rapidly desensitizing current consistent with ASIC channels in both groups. The mean pH-evoked current amplitudes were significantly increased in muscle sensory neurons from inflamed mice (pH 5.0, 3602 ± 470 pA) in comparison to controls (pH 5.0, 1964 ± 370 pA). In addition, the biophysical properties of ASIC-like currents were altered after inflammation. Changes in ASIC channels result in enhanced responsiveness to decreases in pH, and likely contribute to the increased hyperalgesia observed after muscle inflammation.
PMCID: PMC2939292  PMID: 20691768
inflammation; protons; DRG neurons; carrageenan; pain; voltage clamp
6.  Plasma endogenous enkephalin levels in early systemic sclerosis: clinical and laboratory associations 
Clinical and experimental rheumatology  2010;28(2 Suppl 58):S7-11.
Met- and leu-enkephalins are endogenous opioid neuropeptides with potent analgesic, vasoactive, immunomodulatory and anti-apoptotic properties. We hypothesised that clinical or immunological variables of early systemic sclerosis (SSc) might be correlated to plasma enkephalin levels.
Plasma samples were collected at study entry of the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS) cohort (early SSc, n=116). Plasma met-enkephalin and leu-enkephalin levels (ug/ml) were measured by high performance liquid chromatography (HPLC) and correlated to clinical and laboratory parameters in the GENISOS database. Statistical analyses were performed by nonparametric Wilcoxon rank sum tests and Pearson correlation coefficients.
Significantly lower plasma met-enkephalin levels were associated with anti-topoisomerase-I seropositivity (6+8.3 vs. 14.9+22.8 ug/ml, p=0.02). Plasma leu-enkephalin levels were significantly higher in SSc patients with digital pulp loss (95.6+130 vs. 64.9+101 ug/ml, p=0.02). Lower mean plasma met-enkephalin levels and inversely higher leu-enkephalin levels were noted in SSc patients with Raynaud’s phenomena (p=NS).
The associations of plasma enkephalin levels to immunologic or clinical pathologies may underscore their vasogenic or fibrogenic significance and potential as therapeutic targets in early SSc.
PMCID: PMC3192018  PMID: 20576209
Met-enkephalin; leu-enkephalin; opioids; vasculopathy; scleroderma; neurotransmitter
7.  ASIC1 and ASIC3 Play Different Roles in the Development of Hyperalgesia Following Inflammatory Muscle Injury 
Acid-sensing ion channels (ASICs) respond to acidosis that normally occurs after inflammation. We examined the expression of ASIC1, ASIC2, and ASIC3 mRNAs in lumbar DRG neurons before and 24h after carrageenan-induced muscle inflammation. Muscle inflammation causes bilateral increases of ASIC2 and ASIC3, but not ASIC1 (neither ASIC1a nor ASIC1b) mRNA, suggesting differential regulation of ASIC1 versus ASIC2 and ASIC3 mRNA. Similar mRNA increases were observed following inflammation in knockout mice: ASIC2 mRNA increases in ASIC3−/− mice; ASIC2 and ASIC3 mRNAs increase in ASIC1−/− mice. Prior behavioral studies in ASIC3−/− mice showed deficits in secondary hyperalgesia (increased response to noxious stimuli outside the site of injury), but not primary hyperalgesia (increased response to noxious stimuli at the site of injury). In this study, we show that ASIC1−/− mice surprisingly do not develop primary muscle hyperalgesia, but develop secondary paw hyperalgesia. In contrast and as expected, ASIC3−/− mice develop primary muscle hyperalgesia, but do not develop secondary paw hyperalgesia. The pharmacological utility of the non-selective ASIC inhibitor A-317567, given locally, was tested. A-317567 reverses both primary and the secondary hyperalgesia induced by carrageenan muscle inflammation. Thus, peripherally located ASIC1 and ASIC3 play different roles in the development of hyperalgesia after muscle inflammation.
PMCID: PMC2943154  PMID: 20015700
ASIC; muscle inflammation; hyperalgesia; DRG neurons; carrageenan; A-317567
Neuroscience  2009;163(4):1233-1241.
Transcutaneous electric nerve stimulation (TENS) is widely used for the treatment of pain. TENS produces an opioid-mediated antinociception that utilizes the rostroventromedial medulla (RVM). Similarly, antinociception evoked from the periaqueductal grey (PAG) is opioid-mediated and includes a relay in the RVM. Therefore, we investigated whether the ventrolateral or dorsolateral PAG mediates antinociception produced by TENS in rats. Paw and knee joint mechanical withdrawal thresholds were assessed before and after knee joint inflammation (3% kaolin/carrageenan), and after TENS stimulation (active or sham). Cobalt chloride (CoCl2; 5 mM) or vehicle was microinjected into the ventrolateral periaqueductal grey (vlPAG) or dorsolateral periaqueductal grey (dlPAG) prior to treatment with TENS. Either high (100 Hz) or low (4 Hz) frequency TENS was then applied to the inflamed knee for 20 min. Active TENS significantly increased withdrawal thresholds of the paw and knee joint in the group microinjected with vehicle when compared to thresholds prior to TENS (P<0.001) or to sham TENS (P<0.001). The increases in withdrawal thresholds normally observed after TENS were prevented by microinjection of CoCl2 into the vlPAG, but not the dlPAG prior to TENS and were significantly lower than controls treated with TENS (P<0.001). In a separate group of animals, microinjection of CoCl2 into the vlPAG temporarily reversed the decreased mechanical withdrawal threshold suggesting a role for the vlPAG in the facilitation of joint pain. No significant difference was observed for dlPAG. We hypothesize that the effects of TENS are mediated through the vlPAG that sends projections through the RVM to the spinal cord to produce an opioid-mediated analgesia.
PMCID: PMC3955259  PMID: 19576962
pain; TENS; hyperalgesia; opioid; inflammation; analgesia
9.  Examining Sex Differences in Knee Pain: The Multicenter Osteoarthritis Study 
To determine whether women experience greater knee pain severity than men at equivalent levels of radiographic knee osteoarthritis (OA).
Design and Methods
A cross-sectional analysis of 2712 individuals (60% women) without knee replacement or a recent steroid injection. Sex differences in pain severity at each KL grade were assessed by knee using VAS scale and WOMAC with and without adjustment for age, analgesic use, BMI, clinic site, comorbid conditions, depression score, education, race, and widespread pain (WSP) using generalized estimating equations. Effect sizes (Cohen’s d) were also calculated. Analyses were repeated in those with and without patellofemoral OA (PFOA).
Women reported higher VAS pain at all KL grades in unadjusted analyses (d=0.21–0.31, p<0.0001–0.0038) and in analyses adjusted for all covariates except WSP (d=0.16–0.22, p<0.0001–0.0472). Pain severity differences further decreased with adjustment for WSP (d=0.10–0.18) and were significant for KL grade ≤2 (p=0.0015) and 2 (p=0.0200). Presence compared with absence of WSP was associated with significantly greater knee pain at all KL grades (d=0.32–0.52, p<0.0001–0.0008). In knees with PFOA, VAS pain severity sex differences were greater at each KL grade (d=0.45–0.62, p=0.0006–0.0030) and remained significant for all KL grades in adjusted analyses (d=0.31–0.57, p=0.0013–0.0361). Results using WOMAC were similar.
Women reported greater knee pain than men regardless of KL grade, though effect sizes were generally small. These differences increased in the presence of PFOA. The strong contribution of WSP to sex differences in knee pain suggests that central sensitivity plays a role in these differences.
PMCID: PMC4180745  PMID: 24999111
sex differences; knee pain; knee osteoarthritis
10.  Fatigue-enhanced hyperalgesia in response to muscle insult: induction and development occur in a sex-dependent manner 
Pain  2013;154(12):2668-2676.
Chronic muscle pain affects 20–50% of the population, is more common in women than men, and is associated with increased pain during physical activity and exercise. Muscle fatigue is common in people with chronic muscle pain, occurs in response to exercise and is associated with release of fatigue metabolites. Fatigue metabolites can sensitize muscle nociceptors which could enhance pain with exercise. Using a mouse model we tested whether fatigue of a single muscle, induced by electrical stimulation, resulted in enhanced muscle hyperalgesia and if the enhanced hyperalgesia was more pronounced in female mice. Muscle fatigue was induced in combination with a sub-threshold muscle insult (2 injections of pH 5.0 saline) in male and female mice. We show that male and female mice, fatigued immediately prior to muscle insult in the same muscle, develop similar muscle hyperalgesia 24h later. However, female mice also develop hyperalgesia when muscle fatigue and muscle insult occur in different muscles, and when muscle insult is administered 24 hours after fatigue in the same muscle. Further, hyperalgesia lasts significantly longer in females. Finally, muscle insult with or without muscle fatigue results in minimal inflammatory changes in the muscle itself, and sex differences are not related to estradiol (ovariectomy) or changes in brainstem activity (pNR1). Thus, the current model mimics muscle fatigue-induced enhancement of pain observed in chronic muscle pain conditions in the human population. Interactions between fatigue and muscle insult may underlie the development of chronic widespread pain with an associated female predominance observed in human subjects.
PMCID: PMC3957416  PMID: 23906552
11.  Acid-sensing ion channel 3 expressed in type B synoviocytes and chondrocytes modulates hyaluronan expression and release 
Annals of the rheumatic diseases  2009;69(5):903-909.
Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process.
To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH.
Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3−/− mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH.
In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3−/− FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3−/− FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases ASIC3−/− FLS compared to ASIC3+/+ FLS.
ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.
PMCID: PMC3476728  PMID: 19933746
12.  Differential roles of neurokinin 1 and neurokinin 2 receptors in the development and maintenance of heat hyperalgesia induced by acute inflammation 
British Journal of Pharmacology  1997;120(7):1263-1273.
Following induction of acute inflammation by intraarticular injection of kaolin and carrageenan into the knee joint in rats, there was a significant decrease in the withdrawal latency to radiant heat applied to the paw (i.e. heat hyperalgesia), an increased joint circumference and increased joint temperature.A neurokinin1 (NK1) receptor antagonist (CP-99,994, 10 mM) had no effect on the paw withdrawal latency when it was administered spinally through a microdialysis fibre before the induction of inflammation. Pretreatment with a NK2 receptor antagonist (SR48968, 1 mM) administered spinally through the microdialysis fibre prevented the heat hyperalgesia from developing in the early stages of the inflammation.Post-treatment through the microdialysis fibre with the NK1 receptor antagonist (0.0110 mM) was effective in reversing the heat hyperalgesia. In contrast, post-treatment spinally with the NK2 receptor antagonist (0.011 mM) had no effect on the heat hyperalgesia. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.Pretreatment systemically with the NK1 receptor antagonist (30 mg kg−1) had no effect on the heat hyperalgesia or pain-related behaviour ratings where 0 is none and 5 is non weight bearing and complete avoidance of limb contact. Pretreatment with a NK2 receptor antagonist (10 mg kg−1) systemically prevented the heat hyperalgesia and pain-related behaviour ratings from developing in the early stages of the inflammation. The inactive stereoisomers of NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.Post-treatment systemically with either the NK1 (0.130 mg kg−1) or the NK2 (0.110 mg kg−1) receptor antagonist resulted in a dose-dependent reversal of the heat hyperalgesia. Pain-related behaviour ratings were reduced by post-treatment only with the NK1 receptor antagonist. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the behavioural responses.Direct pretreatment of the knee joint with either the NK1 (30 mg) or the NK2 (10 mg) receptor antagonist prevented the heat hyperalgesia from developing without affecting joint swelling. The inactive stereoisomers of the NK1 receptor antagonist, CP100,263, or the NK2 receptor antagonist, SR48965, administered at the same doses, had no effect on the joint inflammation or the heat hyperalgesia.There appears to be a differential role for the spinal tachykinin receptors in the development and maintenance of the heat hyperalgesia associated with acute joint inflammation. The NK2 receptors appear to be activated early in the development of the heat hyperalgesia and NK1 receptors are involved in the maintenance of the heat hyperalgesia.Peripherally, both NK1 and NK2 receptors are involved in the development of heat hyperalgesia and pain-related behaviour ratings induced by acute inflammation.
PMCID: PMC1564603  PMID: 9105701
Pain; joint; arthritis; substance P; neurokinin A; microdialysis; dorsal horn; spinal cord; inflammation; neurokinin receptors
13.  Joint inflammation is reduced by dorsal rhizotomy and not by sympathectomy or spinal cord transection. 
Annals of the Rheumatic Diseases  1994;53(5):309-314.
OBJECTIVES--To investigate the role of primary afferents, sympathetic postganglionic efferents and descending systems on the central control of peripheral inflammation. METHODS--Acute inflammation was induced by intra-articular injection of kaolin and carrageenan into the knee joint cavity of the rat. Before the induction of the arthritis, a unilateral dorsal rhizotomy, a chemical (phentolamine) and/or surgical sympathectomy, or a spinal transection was performed. Joint inflammation (joint circumference and thermographic readings) and behavioural signs were assessed. RESULTS--Only arthritic animals with a dorsal rhizotomy showed a significant reduction of the inflammatory response compared with control arthritic animals. No significant differences in the inflammatory response occurred following sympathectomy or spinal transection. The animals who received sympathectomy showed similar behavioural manifestations to the arthritic animals. CONCLUSIONS--The central terminals of primary afferents are important in the development of acute joint inflammation since dorsal rhizotomy attenuated the inflammatory response in the knee joint. The sympathetic nervous system is not involved in the acute inflammatory phase of this arthritis model. The central processes controlling acute inflammation involve a local spinal circuit since spinal cord transection at T9 has no effect on the inflammation.
PMCID: PMC1005329  PMID: 8017984

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