Important Role In Pain Perception Research Articles

A53 FECAL BIOACTIVE PHOSPHOLIPIDS AS TRIGGERS OF INTESTINAL HYPERSENSITIVITY IN IRRITABLE BOWEL SYNDROME

Abstract Background Chronic abdominal pain is the most common complaint of patients with gastrointestinal disorders. Its treatment is of limited efficacy as the pathophysiology is poorly understood. The gut microbiome has been shown to affect host physiology, including the neural system function, and growing evidence suggests that it plays an important role in pain perception. Gut microbiota produces a large variety of molecules that can regulate pain perception, such as histamine or bioactive lipids. Phospholipid mediators (PLM) are signaling molecules linked to neurogenic pain as by-products of inflammatory processes in mammals. But it is unknown whether bacteria can produce PLM and whether they play any role in visceral hyperalgesia. Purpose To investigate whether PLM are present in stool of patients with chronic abdominal pain, and whether they have potential to induce visceral hypersensitivity. Method Stool samples from 27 patients with irritable bowel syndrome (IBS) were collected both during periods of severe and minimal/no pain. The fecal concentration of two PLM (lipid A and lipid B) was determined by ELISA. The passage of lipid A and lipid B through the intestinal barrier was determined ex vivo using Ussing chambers and in vivo by their intracolonic instillation, and their levels assessed in the serosal compartment and the serum, respectively, by ELISA. We used primary cultures of sensory neurons from the dorsal root ganglia (DRG) of conventional mice (SPF) to study the neuronal activation in vitro. Calcium mobilization in DRG sensory neurons was measured by an inverted fluorescence microscope using a fluorescent probe Fluo-4 (1mM) after stimulation with different concentration of lipid A or lipid B. Visceral sensitivity in vivo after intracolonic instillation of combined lipids A and B was evaluated by colorectal distension. Result(s) The concentration of lipid A (p=0.001) and lipid B (p=0.002) in stool was significantly higher in IBS patients when experiencing severe abdominal pain compared to periods of minimal pain. The concentration of lipid A (p=0.03; p=0.07) and lipid B (p=0.018; p=0.018) in the serosal compartment and in the serum, respectively, was higher compared to the control condition. The percentage of neurons responding to lipid A or lipid B was significantly higher compared to the control condition, at all tested concentrations. Finally, combined lipid A and lipid B induced visceral hypersensitivity within 10 minutes (p=0.0433) and 90 minutes (p=0.0020) after intracolonic instillation. Conclusion(s) Our data suggest that PLM can be found in stool of patients with abdominal pain, that they cross the colonic barrier and increase visceral sensitivity. Further studies are needed to ascertain whether gut bacteria produce these PLM and investigate the precise mechanisms by which PLM induce hyperalgesia. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Weston Family Foundation Disclosure of Interest None Declared

Whole-brain mapping of efferent projections of the anterior cingulate cortex in adult male mice.

The anterior cingulate cortex (ACC) is a key cortical region that plays an important role in pain perception and emotional functions. Previous studies of the ACC projections have been collected primarily from monkeys, rabbits and rats. Due to technological advances, such as gene manipulation, recent progress has been made in our understanding of the molecular and cellular mechanisms of the ACC-related chronic pain and emotion is mainly obtained from adult mice. Few anatomic studies have examined the whole-brain projections of the ACC in adult mice. In the present study, we examined the continuous axonal outputs of the ACC in the whole brain of adult male mice. We used the virus anterograde tracing technique and an ultrahigh-speed imaging method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR). We created a three-dimensional (3D) reconstruction of mouse brains. We found that the ACC projected ipsilaterally primarily to the caudate putamen (CPu), ventral thalamic nucleus, zona incerta (ZI), periaqueductal gray (PAG), superior colliculus (SC), interpolar spinal trigeminal nucleus (Sp5I), and dorsal medullary reticular nucleus (MdD). The ACC also projected to contralateral brain regions, including the ACC, reuniens thalamic nucleus (Re), PAG, Sp5I, and MdD. Our results provide a whole-brain mapping of efferent projections from the ACC in adult male mice, and these findings are critical for future studies of the molecular and synaptic mechanisms of the ACC and its related network in mouse models of brain diseases.

Effects of GABAA receptors in nucleus cuneiformis on the cannabinoid antinociception using the formalin test.

Nucleus cuneiformis (NC), a reticular nucleus of the midbrain, is a part of the descending pain modulatory system and therefore has an important role in pain perception. Considering the abundance of GABAA and cannabinoid receptors in the NC and also the bidirectional roles for GABA in controlling nociception, the present study examined the effects of bilateral intra-NC microinjection of different doses of the GABAA receptor agonist, muscimol, and the GABAA receptor antagonist, bicuculline, on pain modulation using formalin test. We also assessed interaction between canabinergic and GABAergic systems in the NC during this test. Rats were exposed to intra-NC microinjection of bicuculline (50,100, and 200 ng/side) or muscimol (60, 120, and 240 ng/side) and then subjected to the formalin test. In another set of experiments, the effects of muscimol (60 ng/side) or bicuculline (50 ng/side) administration 5 min before a cannabinoid receptor agonist WIN 55,212-2 (5, 10, and 20 μg/side) microinjection into NC on the formalin test were evaluated. Microinjection of bicuculline and muscimol into the NC decreased and increased pain responses, respectively, in a dose-dependent manner during both phases of the test. Microinjection of WIN 55,212-2 into the NC significantly reduced pain responses in a dose-dependent manner. Microinjection of bicuculline or muscimol in combination with WIN 55,212-2 into the NC respectively potentiated and attenuated WIN 55,212-2-induced antinociception in the formalin test. This study shows that GABA in the NC is involved in pain modulation and suggests the existence of a GABAA-mediated inhibitory system in the NC on pain control. Furthermore, it seems that the antinociceptive effect of WIN 55,212-2 in the formalin test is mediated partly by the activity of local GABAA receptors in the NC.

Potential role of brain biomarkers in primary knee osteoarthritis patients using magnetic resonance spectroscopy

BackgroundPain in osteoarthritis (OA) primarily results from tissue damage but its' intensity does not essentially parallel the extent of joint destruction or presence of active inflammation, thus suggesting the likely involvement of a central component. The mid-anterior cingulate cortex (mACC) has an important role in pain perception, intensity and progression. In OA, low mACC γ-aminobutyric acid (GABA) was associated with high pain suggesting a role of prefrontal disinhibition. Aim of the work: To investigate the role of mACC (GABA) levels in chronic knee OA (KOA) pain and determine if magnetic resonance spectroscopy (MRS) brain neurotransmitters can serve as potential biomarkers. Patients and methods: Forty-five patients with primary KOA (M/F:33/12; age:57 ± 6 years) along with 15 matched controls were recruited. Pain was assessed using Visual Analogue Scale (VAS), Pain Catastrophizing Scale (PCS) and Western Ontario McMaster Osteoarthritis (WOMAC) questionnaire. mACC (GABA) was assessed and brain MRS neurotransmitters analysed including glutamate (Glx); N-acetylaspartate (NAA), total choline (tCho) and myo-inositol. Results: MRS analysis demonstrated no metabolite differences between controls and KOA patients in GABA, Glx, NAA and tCho. Myo-inositol:Glx ratio was significantly higher in patients (1.47 ± 0.37 vs 1.1 ± 0.29; p < 0.001). mACC (GABA) negatively correlated with VAS (r = −0.86, p < 0001), PCS (r = −0.94, p < 0001) and WOMAC (r = −0.96, p < 0001) in KOA patients. Myo-inositol:Glx significantly correlated with the age (r = 0.31, p < 0.038), disease duration (r = 0.61, p < 0.0001), VAS (r = 0.4, p < 0.02), PCS (r = 0.48, p < 0.001) and WOMAC (r = 0.53, p < 0.0001). Conclusions: This work confirms the importance of mACC in central sensitization of pain and highlights a promising role of the inflammatory neurotransmitter GABA and myo-inositol:Glx ratio as mechanistic biomarkers of chronic KOA pain.

An Association of Serotonin with Pain Disorders and Its Modulation by Estrogens.

Ovarian hormones play an important role in pain perception, and are responsible, at least in part, for the pain threshold differences between the sexes. Modulation of pain and its perception are mediated by neurochemical changes in several pathways, affecting both the central and peripheral nervous systems. One of the most studied neurotransmitters related to pain disorders is serotonin. Estrogen can modify serotonin synthesis and metabolism, promoting a general increase in its tonic effects. Studies evaluating the relationship between serotonin and disorders such as irritable bowel syndrome, fibromyalgia, migraine, and other types of headache suggest a clear impact of this neurotransmitter, thereby increasing the interest in serotonin as a possible future therapeutic target. This literature review describes the importance of substances such as serotonin and ovarian hormones in pain perception and illustrates the relationship between those two, and their direct influence on the presentation of the aforementioned pain-related conditions. Additionally, we review the pathways and receptors implicated in each disorder. Finally, the objective was to stimulate future pharmacological research to experimentally evaluate the potential of serotonin modulators and ovarian hormones as therapeutic agents to regulate pain in specific subpopulations.

Endogenous Isoquinoline Alkaloids Agonists of Acid-Sensing Ion Channel Type 3.

Acid-sensing ion channels (ASICs) ASIC3 expressed mainly in peripheral sensory neurons play an important role in pain perception and inflammation development. In response to acidic stimuli, they can generate a unique biphasic current. At physiological pH 7.4, human ASIC3 isoform (hASIC3) is desensitized and able to generate only a sustained current. We found endogenous isoquinoline alkaloids (EIAs), which restore hASIC3 from desensitization and recover the transient component of the current. Similarly, rat ASIC3 isoform (rASIC3) can also be restored from desensitization (at pH < 7.0) by EIAs with the same potency. At physiological pH and above, EIAs at high concentrations were able to effectively activate hASIC3 and rASIC3. Thus, we found first endogenous agonists of ASIC3 channels that could both activate and prevent or reverse desensitization of the channel. The decrease of EIA levels could be suggested as a novel therapeutic strategy for treatment of pain and inflammation.

The relationship between back pain and schoolbag use: a cross-sectional study of 5,318 Italian students

Background ContextBack pain at a young age is considered to be predictive of chronicity. Several studies have investigated the relationship between the use of a schoolbag and back pain, although some aspects are still unclear. PurposeThe aim of this study was to evaluate back pain due to schoolbag use in terms of (1) prevalence and intensity, (2) differences between male and female pupils, and (3) predisposing factors. Study DesignThis is a cross-sectional study. Patient SampleThe sample was composed of 5,318 healthy pupils aged 6 to 19 years (classified according to three age groups: children, younger adolescents, and older adolescents). Outcome MeasuresSchoolbag-related pain was assessed by means of an ad hoc questionnaire. The intensity of pain was assessed using the Wong scale. MethodsSubjects underwent a face-to-face interview using an ad hoc questionnaire. The intensity of pain was assessed using the Wong scale. On the basis of the prevalence and intensity of back pain, we divided our population into two groups: (1) no or mild pain group and (2) moderate or severe pain group. The “schoolbag load” (ratio between schoolbag and pupil weight multiplied by 100) was calculated for each subject. ResultsMore than 60% of the subjects reported pain. Although the schoolbag load decreased from children to young and older adolescents, schoolbag-related pain significantly increased (p<.001). Girls reported significantly more frequent and more severe pain than boys. The logistic model confirmed that adolescent girls are the group at greatest risk of suffering from intense pain. The schoolbag load had a weak impact on back pain, whereas the schoolbag carrying time was a strong predictor. ConclusionsAdolescent girls have the highest risk of experiencing severe back pain, regardless of schoolbag load. This suggests that other factors (anatomical, physiological, or environmental) might play an important role in pain perception. These aspects should be investigated to plan appropriate preventive and rehabilitative strategies.

Effect of Hypovitaminosis D on Postoperative Pain Outcomes and Short-Term Health-Related Quality of Life After Knee Arthroplasty: A Cohort Study.

Vitamin D may have an important role in pain perception. Inadequate vitamin D levels are associated with suboptimal recovery after surgery. However, the effects of hypovitaminosis D on postoperative pain-related outcomes and its impact on health-related quality of life after surgery are not well understood. The objective of this study was to determine the effects of hypovitaminosis D on postoperative pain-related outcomes and health-related quality of life at 3 months after knee arthroplasty.This was a longitudinal cohort study of 191 consecutive Hong Kong Chinese patients who were given patient-controlled morphine analgesia for up to 72 hours after 214 knee arthroplasties. Serum total 25-hydroxyvitamin D (25-OHD) concentration was assessed by liquid chromatography-tandem mass spectrometry. The primary outcomes were postoperative pain intensity at rest scores (0–72 h), Western Ontario and McMaster Universities (WOMAC) osteoarthritis index (pain, stiffness and function), and moderate-to-severe persistent pain (transformed WOMAC pain score of 0–75 at 3 months after knee arthroplasty; 0, extreme pain; 100, no pain). Group differences were analyzed using generalized estimating equation models and a logistic regression model.The prevalence of preoperative hypovitaminosis D (25-OHD <50 nmol/L) was 44% (95% confidence interval [CI]: 37%–51%). There were transient higher pain intensity scores in the moderate-to-severe hypovitaminosis D (25-OHD <30 nmol/L) group compared with the sufficient vitamin D group. Vitamin D status had no effect on total WOMAC index (P = 0.22). The incidence of moderate-to-severe persistent pain was 9% (95% CI: 6%–14%). Hypovitaminosis D increased the risk of moderate-to-severe persistent pain (adjusted odds ratio 2.64, 95% CI: 1.03–6.77).Preoperative hypovitaminosis D had subtle effects on pain intensity scores in the early postoperative period and is a risk factor for moderate-to-severe persistent pain after knee arthroplasty. Hypovitaminosis D was not associated with worse health-related quality of life at 3 months after knee arthroplasty.

A Guanidinium Group Containing Dietary Supplement Inhibits ASIC3

Acid‐sensing ion channels are sodium selective channels that are sensitive to extracellular protons, specifically those following ischemia and injury. The peripheral ASIC3 subtype plays an important role in pain perception. Protons and inflammatory mediators can activate or modulate ASIC3 to produce the pain signal, suggesting that ASIC3 is a pharmacological target for pain therapy. The large extracellular domain of ASICs offers multiple sites for interacting with protons and guanidinium group containing compounds. Guanidinium compounds such as 2‐guanidine‐4‐methylquinazoline (GMQ), amiloride, and agmatine are known to modulate the electrophysiological properties of ASIC3. Here we identified a dietary supplement (DS) that shares molecular similarity to these ASIC ligands and modulate ASICs. We utilize whole‐cell patch‐clamp electrophysiology recording to determine the interaction of DS with rat ASIC3 (rASIC3), transiently expressed in CHO‐K1 cells. Our preliminary data suggests that ASIC3 peak current amplitude and steady‐state current is reduced in the presence of DS. In the absence of extracellular calcium, DS reduces the rASIC3 proton sensitivity by shifting pH‐activation profile to lower pH. Future studies will focus on determining the effect of DS on the ASIC3 window current and other ASIC3 properties to resolve the mechanism of action of the DS influence on channel activity.

GABAAreceptors are involved in the analgesic effects of morphine microinjected into the central nucleus of the amygdala

The central nucleus of the amygdala (CeA) has an important role in pain perception and analgesia. Opioid and GABAA receptors, which are both involved in pain modulation, are found in high concentration in the CeA. The present study was designed to examine the interaction of opioidergic and GABAergic systems in the CeA during modulation of acute thermal pain. In the present study, different doses of morphine (25, 50 and 100μg/rat), either alone or after 5min pretreatment with the selective GABAA receptor agonist muscimol (60ng/rat) or the selective GABAA receptor antagonist bicuculline (50ng/rat), were injected bilaterally into the CeA of each rat. Tail-flick latencies (TFL) were measured every 5min for 60min. The results revealed that microinjection of morphine into the CeA significantly increased TFL in a dose-dependent manner. Microinjection of bicuculline or muscimol in combination with morphine into the CeA increased and decreased TFL, respectively. It seems that morphine in the CeA facilitates the function of descending inhibitory systems by interacting with the activity of local GABAA receptors.

The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

The Influence of Pain Sensitivity on the Symptom Severity in Patients with Lumbar Spinal Stenosis

Background: The symptom severity of back pain/leg pain is not correlated with the severity of degenerative changes and canal stenosis in lumbar stenosis. Considering the individual pain sensitivity might play an important role in pain perception, this discordance between the radiologic findings and clinical symptoms in degenerative lumbar stenosis might originate from the individual difference of pain sensitivity for back pain and/or leg pain. Objective: To determine the relationship among the clinical symptoms, radiologic findings, and the individual pain sensitivity in the patients with degenerative lumbar spinal stenosis. Study Design: Retrospective analysis of prospectively collected data. Setting: A spine center in the department of orthopedic surgery. Methods: In 94 patients who had chronic back pain and/or leg pain caused by degenerative lumbar spinal stenosis, a medical history, a physical examination, and completion of a series of questionnaires, including pain sensitivity questionnaire (PSQ) [total PSQ and PSQ-minor], Oswestry Disability Index (ODI), Visual Analog Pain Scale (VAS) for back pain, and Short Form36 (SF-36) were recorded on the first visit. Radiologic analysis was performed using the MRI findings. The grading of canal stenosis was based on the method by Schizas, and the degree of disc degeneration was graded from T2-weighted images with the Pfirrmann classification. The correlations among variables were statistically analyzed. Results: Total PSQ and PSQ-minor were not dependent on the grade of canal stenosis after gender adjustment. VAS for leg pain and back pain was highly associated with the total PSQ and the PSQ-minor. Total PSQ and PSQ-minor were also significantly associated with ODI. Among SF36 scales, the PSQ minor had significant correlations with SF-36 such as bodily pain (BP), Roleemotional (RE), and Mental Component Summary (MCS) after control of confounding variables such as body mass index (BMI), age, and the grade of canal stenosis/disc degeneration. Total PSQ was significantly associated with the SF-36 RP, BP, and RE. Furthermore, after adjustment for gender and pain sensitivity, there was no significant association between the grade of canal stenosis and VAS for back pain/leg pain and ODI, and no correlation was found between the grade of disc degeneration and VAS for back pain/leg pain and ODI, either. Limitations: The multiple lesions of canal stenosis and/or disc degeneration and the grade of facet degeneration were not considered as a variable. Conclusion: The current study suggests that the pain sensitivity could be a determining factor for symptom severity in the degenerative spinal disease. Key words: Pain sensitivity, pain sensitivity questionnaire, lumbar spinal stenosis, visual analog pain scale, Oswestry disability index, Short Form-36

Inhibitory regulation of acid-sensing ion channel 3 by zinc

Acid-sensing ion channel 3 (ASIC3) is a proton-gated, voltage-insensitive Na + channel that is expressed primarily in peripheral sensory neurons and plays an important role in pain perception, particularly as a pH sensor following cardiac ischemia. We previously reported that ASIC3 currents are not affected by zinc at nanomolar concentrations. In this study, we examined the potential role of micromolar zinc in the regulation of ASIC3. In CHO cells expressing ASIC3, we found that ASIC3 currents triggered by dropping the pH from 7.4 to 6.0 were inhibited by pretreatment with zinc in a concentration-dependent manner; the half-maximum inhibitory concentration of zinc was 61 μM. ASIC currents activated by a relatively small drop in pH from 7.4 to 7.2 or 7.0 were also subject to inhibition by zinc. The inhibition was fast and pH independent, and occurred within a relatively narrow range of zinc concentrations between 30 and 300 μM. Further, increasing extracellular Ca 2+ concentrations from 2 to 10 mM failed to affect inhibition of ASIC3 currents by zinc. Experimentally elevating intracellular zinc levels did not affect the inhibition of ASIC3 currents by equal concentrations of extracellular zinc, and modification of cysteine or histidine residues had no effect on the inhibition of ASIC3 currents by zinc. These collective results suggest that zinc is an important regulator of ASIC3 at physiological concentrations, that zinc inhibits ASIC3 in a pH- and Ca 2+-independent manner, and that inhibition of ASIC3 currents is dependent upon the interaction of zinc with extracellular domain(s) of ASIC3.

In vivo Patch-Clamp Analysis of Response Properties of Rat Primary Somatosensory Cortical Neurons Responding to Noxious Stimulation of the Facial Skin

BackgroundAlthough it has been widely accepted that the primary somatosensory (SI) cortex plays an important role in pain perception, it still remains unclear how the nociceptive mechanisms of synaptic transmission occur at the single neuron level. The aim of the present study was to examine whether noxious stimulation applied to the orofacial area evokes the synaptic response of SI neurons in urethane-anesthetized rats using an in vivo patch-clamp technique.ResultsIn vivo whole-cell current-clamp recordings were performed in rat SI neurons (layers III-IV). Twenty-seven out of 63 neurons were identified in the mechanical receptive field of the orofacial area (36 neurons showed no receptive field) and they were classified as non-nociceptive (low-threshold mechanoreceptive; 6/27, 22%) and nociceptive neurons. Nociceptive neurons were further divided into wide-dynamic range neurons (3/27, 11%) and nociceptive-specific neurons (18/27, 67%). In the majority of these neurons, a proportion of the excitatory postsynaptic potentials (EPSPs) reached the threshold, and then generated random discharges of action potentials. Noxious mechanical stimuli applied to the receptive field elicited a discharge of action potentials on the barrage of EPSPs. In the case of noxious chemical stimulation applied as mustard oil to the orofacial area, the membrane potential shifted depolarization and the rate of spontaneous discharges gradually increased as did the noxious pinch-evoked discharge rates, which were usually associated with potentiated EPSP amplitudes.ConclusionsThe present study provides evidence that SI neurons in deep layers III-V respond to the temporal summation of EPSPs due to noxious mechanical and chemical stimulation applied to the orofacial area and that these neurons may contribute to the processing of nociceptive information, including hyperalgesia.

Defining the Voltage Sensor Properties and Pharmacology of Nav1.9

The voltage-activated sodium channel Nav1.9 is preferentially expressed in DRG neurons where it is believed to play an important role in pain perception. However, progress in revealing the gating characteristics and pharmacological sensitivities of Nav1.9 has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here we use a protein engineering approach to study the contributions of the four Nav1.9 voltage sensors to channel function. We define individual S3b-S4 paddle motifs within each voltage sensor and show that these structural motifs sense changes in membrane voltage and determine the kinetics of voltage sensor activation.

Regulatory Elements of TRPA1 Function

Transient Receptor Potential Ankyrin 1 channel, TRPA1, is a member of the TRP family and has been shown to be expressed in dorsal root ganglions, trigeminal ganglion neurons and hair cells. TRPA1 is proposed to play an important role in pain perception and temperature sensing, and might be involved as transduction channel in mechanosensation essential for the auditory response in mammals. Pungent chemicals and environmental irritants (e.g. mustard oil) are substances activating this ion channel. Specifically, activation of TRPA1 by allyl isothiocyanates occurs via covalent modification of cysteine residues within the cytoplasmic N terminus of the channel. Here we have focused on the role of TRPA1 C-terminus employing Ca2+ imaging, patch-clamp and confocal fluorescence resonance energy transfer (FRET) microscopy. A TRPA1 C-terminal deletion mutant failed to activate upon allyl isothiocyanate application despite a similar homomerization potential as the wild-type form evident from both functional assays and FRET measurements, respectively. The TRPA1 C-terminus alone exhibited multimerization potential in FRET microscopy, yet failed to function as a dominant negative species when co-expressed with wild-type TRPA1 suggesting a minor role for channel assembly. Further, a single amino acid mutation in TRPA1 C-terminus led to a substantial reduction in TRPA1 currents. With regard to a potential regulatory role of calmodulin (CaM), FRET experiments suggested its association with TRPA1 upon elevation of intracellular calcium concentrations either by allyl isothiocyanate induced stimulation of TRPA1 or ionomycin. Consistently, electrophysiological experiments revealed faster inactivation of TRPA1 in presence of over-expressed CaM. In summary our experiments point to TRPA1 C-terminus a crucial element for channel function that is additionally regulated by CaM. (supported by FWF P18169)

(616): Early dexamethasone treatment following sciatic nerve cuff implantation decreases allodynia as well as substance P in the lumbar spinal cord of rats with neuropathic pain

Pain related peptides were monitored at early stages of neuropathic pain development and dexamethasone treatment was evaluated following the implantation of a polyethylene cuff around the sciatic nerve. Sprague-Dawley male rats (n=18) weighing 300-350g were tested for touch sensitivity with von Frey filaments prior to and 3 days following cuff implantation (n=12) or sham (n=6) surgeries. Half of the cuff-implanted rats received 1mg/kg dexamethasone one hour following the surgery. Spinal cords were removed on the 4th day post-surgery and the lumbar enlargement was processed for the detection of selected peptides (substance P, neurotensin, VIP, CCK and CGRP) using liquid chromatography tandem mass spectrometry (LC/MS/MS). The sciatic nerve was collected, fixed in a mixture of formaldehyde and paraformaldehyde, and stained with hematoxylin and eosin for routine observations as well as toluidine blue (fixation with gluteraldehyde 2.5% and a cacodylate buffer (0.05M, pH 7.4) and the fixed in 1% osmium tetraoxide) for nerve diameter fibre counts with an image analysis system (PCI Compix). Except for neurotensin, all peptide concentrations were greater with neuropathic pain however only substance P and CCK (p<0.05) were significant. Following dexamethasone treatment, only substance P concentrations were less (p<0.05) than non-treated neuropathic animals and values were comparable to controls. With treatment touch sensitivity was comparable to control animals. Histopathology of nerve sections showed a decrease in C (p<0.01) and Ad (p<0.03) fibres in neuropathic animals and a near normal percentage of C fibres following dexamethasone treatment. Treated animals also showed less inflammatory reactions (fibrosis and inflammatory cells) compared to cuff implanted animals without treatment. Results suggest that dexamethasone may be used in the early stages of the treatment of neuropathic pain induced by an inflammatory traumatic processes and that sub-stance P appears to play an important role in pain perception in early stages of neuropathic pain development.

5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo.

T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. T current blockade exhibited mild voltage-dependence, suggesting that 5beta-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.

Analgesia and cyclo-oxygenase inhibitors

Non-steroidal anti-inflammatory drugs are among the most commonly used medications and they are a mainstay in the treatment of inflammatory diseases. Non-steroidal anti-inflammatory drugs are widely used to reduce pain associated with acute or chronic inflammation. Recently, a new class of inhibitors of the inducible enzyme cyclo-oxygenase-2 have become available. These inhibitors selectively target the inducible enzyme and have been shown to spare the gastrointestinal tract. While a role of cyclo-oxygenase-2 in the development of chronic inflammation has been well established, its role in pain perception is still unclear. Recent experimental data led to the hypothesis that cyclooxygenase-1 plays an important role in pain perception. This short review addresses some recent preclinical data as well as some clinical evidence showing that cyclo-oxygenase-1 is an important component of inflammatory pain.

Racial Differences in Pain Perception: a Biological Basis

Behavioral differences between the various human races have been observed (e.g. intelligence). The perception of pain is essential for human survival. Individual as well as group differences have been reported in pain behavior, suggesting that pain behavior may be genetically influenced. To develop more efficacious pain treatment, it will be important to understand the genetic mechanism of pain and to uncover any differences between the races. The presence of genetic differences will indicate the need to consider race in future pain management strategies. Key Words: nociception; pain; racial differences Racial differences in skeleto-muscular, endocrine and nervous systems have been previously described (e.g., Baker, 1974; Yanovski et al., 1996). Because phenotype and behavior shows significant heritability, they are, at least partially, genetically mediated. For example, Asian American students will academically outperform black American students, regardless of socio-economic standing (NSF, 1994). Conversely, it should be no surprise that blacks are athletically more able than Asians. Genes are thought to mediate many behaviors, ranging from aggression to self-discipline, since their level of expression markedly differ between the races and have moderate to high heritabilities (Rushton, 1995). The perception of pain is a mechanism by which humans receive bodily as well as environmental information. Although it may seem that having no pain perception maybe beneficial, in reality a dysfunctional pain perception system is deleterious. For example, patients who are congenitally insensitive to pain repeatedly inflict severe injuries upon themselves and require constant medical attention. The reduced pain perception is due to gene mutations that alter normal sensory neuron function (Mardy et al., 1999). Conversely, heritable pathologies exist that exacerbate pain perception, such that the usual stimulus is now perceived as extremely painful (references in Mogil et al., 1999a). Growing evidence indicate that genes play an important role in pain perception, just as genes may be important in other kinds of behaviors. The current paper reviews racial differences in pain perception. Evidence for the genetic basis of pain perception is reviewed in animal studies, which may have relevance to humans. Ultimately, the goal is to identify genes that are involved in normal as well as abnormal pain sensation and to devise treatment based on empirical data. Pain is defined as the sensation that accompanies impending or ongoing tissue damage. Pain detection and the response to it is crucial for survival. The diverse responses to pain range from simple to complex behaviors, from a withdrawal reflex to remembering that touching a hot stove has dire consequences. In humans, the painful sensory experience has been characterized as biphasic, the first phase being perceptual/sensory and the second phase being reactive (Bonica, 1990). This means that a pain experience can be compartmentalized into a phase where pain is detected or realized, then a phase where there is a response to pain. The response to pain integrates emotion, affect and memory. There are those who argue that environment shapes pain behavior. Environmentalists argue that individual as well as racial differences in pain perception are due to cultural background. Another force that shapes pain behavior is learning (Melzack and Wall, 1982). Pain - Nurture? Pain has a unique status in human societies. Ceremonial torture and mutilation, involving excruciating pain, are used by some societies as tests of loyalty or worthiness. Thus, high tolerance of pain, in a religious or spiritual context, is viewed as desirable. However, long-term pain arising from illness, is viewed differently. Rural Black Africans believe that illness and the accompanying pain are signs of possession by evil spirits and an inevitable facts of life. …