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Paper 312)
Abstracts
A30 - Other (616/Paper 312) Genetic polymorphisms in GTP-cyclohydrolase gene (GCH1) do not affect acute experimental and clinical pain in humans H Kim, H Lee, R Dionne; National Institutes of Health, Bethesda, MD Candidate gene studies on the basis of biological hypotheses have been a practical approach to identify relevant genetic variation in complex traits such as pain, though it can be easily biased. Based on their roles in pain pathways, it is suggested that GTP cyclohydrolase, an enzyme that regulates the biosynthesis of the enzyme cofactor tetrahydobiopterin (BH4), is a modulator of peripheral neuropathic, inflammatory and even experimental pain in humans (Tegeder et al, Nature Med 2006). We have examined the effects of variations of loci in the human GTP cyclohydrolase gene (GCH1) on acutely induced experimental thermal/cold and clinical pain responses in humans. Normal subjects (443 females and 292 males) were genotyped for the 18 single nucleotide polymorphisms (SNPs) from GCH1. Haplotypes were generated from the GCH1 in 4 major ethnic populations. We evaluated normal European American subjects (194 females and 173 males) for cold and heat pain sensitivity and analyzed its association with the individual SNPs and haplotypes in the GCH1. Those requiring removal of third molars (N⫽177) then underwent surgical removal of 3-4 third molars and provided ratings of pain intensity every 20 minutes postoperatively for 20 –240 mins (mean ⫽122 mins) using a 10 cm visual analog scale (VAS). Unlike the report by Tegeder et al, no significant association was found between pain sensitivity and any of genetic polymorphisms including haplotypes in GCH1 in the responses to experimentally or clinically induced acute pain. The negative results between GCH1 genetic variations and pain sensitivity in our study may differ from other studies due to different populations and separate pain phenotype from different types of pain stimuli. To overcome these potential biases, whole genome association studies on pain are needed.
(617) Selective colocalization of neuropeptides and botulinum toxin induced cleaved SNAP-25 in dorsal root ganglia culture X Li; University of Georgia, Athens, GA Recently, investigators have observed the interesting phenomenon that botulinum neurotoxin (BoNT) may provide significant benefit in the reduction of certain types of pain. However, the mechanistic basis for this phenomenon is unclear, since an interaction between the terminals of the sensory nervous system and BoNTs is not well understood. Is there a direct effect of BoNT on primary sensory neurons associated with pain? In an effort to address this, a study of the neuropeptide content of sensory neurons demonstrating sensitivity to BoNT serotype A was undertaken in murine primary sensory neurons in culture. The SNARE protein SNAP-25, the intracellular target of BoNT A, was developmental expressed in embryonic dorsal root ganglia culture and cleaved by BoNT A (10-8M) in a time dependent manner. Cleavage of SNAP-25 was detected in a select population of DRG neurons by immunofluorescence, that were double-labeled with some pain-related neuropeptides, including CGRP, galanin, GABA and nociceptin, but not with substance-P and glutamate. It has previously been reported that BoNT A blocks the release of substance-P, and glutamate in the spinal dorsal horn and decreases the sensitivity of pain transduction from spinal cord to brain. Our preliminary results suggest that the release of certain neuropeptides related to pain transmission or modulation in primary sensory neurons may be selectively altered by BoNT A. Thus, a potential mechanism for pain relief by BoNT A may be mediated by toxine-induced interference of multiple pain related factors.
S5
B. Systems (Physiology, Anatomy, Animal Models) B01 - Animal Models of Chronic Pain (618) A novel approach to the use of animals in studies of pain: Development of the Canine Brief Pain Inventory (CBPI) D Brown, R Boston, J Coyne, J Farrar; University of Pennsylvania, Philadelphia, PA Novel compounds for chronic pain that appear promising in laboratory animals often fail to prove efficacious in clinical trials, suggesting a need for more predictive animal models. The spontaneous pain caused by naturally occurring diseases in companion animals (pets) requires treatment, and the study of novel therapies in these animals can provide greater insight into the potential efficacy in humans. Studies of novel analgesics in companion dogs that spontaneously develop bone cancer reveal drug effects which are not evident in rodent studies. The development of outcome pain measures for use in companion dogs that specifically correspond with outcomes routinely used in clinical research could further increase the predictive capability of these preclinical companion animal studies. The objective of this study was to develop the Canine Brief Pain Inventory (CBPI), based on the human Brief Pain Inventory (BPI), for use in the canine model of spontaneous bone cancer. The item structure, response scaling, and severity items of the BPI were maintained for the CBPI. Impact items were generated through focus groups and an expert panel; poorly performing items were removed; with the reduced set of items subject to factor analysis, reliability, and validity testing. The “severity” and “impact” factors hypothesized based on the BPI were demonstrated in the CBPI. Internal consistency of both factors was high (Cronbach’s alpha 0.95 and 0.93), as was test-retest reliability (kappa 0.73 and 0.65). Extreme groups validation against normal dogs showed significantly higher factor scores (P⬍0.001 for both) and convergent validity was demonstrated against quality of life (r⫽0.49 and 0.63). The CBPI and BPI reliably measure the same pain constructs in comparable diseases. This innovative approach to preclinical outcomes development applied to companion animals with complex behaviors and clear parallels to human disease pathogenesis, progression and symptomatology, could transform the predictive ability of preclinical pain studies.
(619) Role of TRPV1 receptors in a novel model of chronic ischemic pain R Radhakrishnan, R Nallu; Western University of Health Sciences, Pomona, CA The pain mechanism in ischemic conditions like intermittent claudication is poorly understood, partly due to the lack of suitable chronic pre-clinical models. In the present study we modified an existing model of ischemia (Orito et al., 2004) in an effort to develop a better characterized chronic ischemic pain model. Since ischemia reduces pH in the affected tissues and TRPV1 channels transduce pH changes, we also studied the role of TRPV1 in this model. Baseline heat (Hargreave’s) and mechanical (von Frey) thresholds were measured in the hindpaws of male Sprague-Dawley rats, and they were trained to run on a treadmill with an electric shock-grid. Left iliac artery of the rats was then surgically occluded and animals were allowed to recover for 48h. At 48h, 72h, and weekly from 1wk through 8wk, heat and mechanical thresholds were measured again. Animals were then placed on the treadmill and forced to run for 5 min using mild electric shock motivation. Pain behavior was measured as 1) onset time for lifting, shaking or dragging the hind limb; 2) incapacity of the animal to run on the treadmill, indicated by the number of hits on the shock-grid; 3) reduction in heat and/or mechanical threshold after treadmill test. Our results show that the onset time for exercise-induced pain was shorter, number of hits on shock-grid was higher, and the mechanical threshold was reduced (mechanical allodynia) in ligated animals compared to sham. The pain behavior was present for 3-6 weeks with peak at 48-72h. Preliminary results show that at 72h time-point, TRPV1 antagonist capsazepine (25 mg/kg.,i.p.) increased the capacity of the ligated animals to run, but did not affect the onset of pain or allodynia, indicating a role for TRPV1 receptors in this model. Studies are underway to further characterize the model and to confirm the preliminary findings.
A30 - Other (616/Paper 312) Genetic polymorphisms in GTP-cyclohydrolase gene (GCH1) do not affect acute experimental and clinical pain in humans H Kim, H Lee, R Dionne; National Institutes of Health, Bethesda, MD Candidate gene studies on the basis of biological hypotheses have been a practical approach to identify relevant genetic variation in complex traits such as pain, though it can be easily biased. Based on their roles in pain pathways, it is suggested that GTP cyclohydrolase, an enzyme that regulates the biosynthesis of the enzyme cofactor tetrahydobiopterin (BH4), is a modulator of peripheral neuropathic, inflammatory and even experimental pain in humans (Tegeder et al, Nature Med 2006). We have examined the effects of variations of loci in the human GTP cyclohydrolase gene (GCH1) on acutely induced experimental thermal/cold and clinical pain responses in humans. Normal subjects (443 females and 292 males) were genotyped for the 18 single nucleotide polymorphisms (SNPs) from GCH1. Haplotypes were generated from the GCH1 in 4 major ethnic populations. We evaluated normal European American subjects (194 females and 173 males) for cold and heat pain sensitivity and analyzed its association with the individual SNPs and haplotypes in the GCH1. Those requiring removal of third molars (N⫽177) then underwent surgical removal of 3-4 third molars and provided ratings of pain intensity every 20 minutes postoperatively for 20 –240 mins (mean ⫽122 mins) using a 10 cm visual analog scale (VAS). Unlike the report by Tegeder et al, no significant association was found between pain sensitivity and any of genetic polymorphisms including haplotypes in GCH1 in the responses to experimentally or clinically induced acute pain. The negative results between GCH1 genetic variations and pain sensitivity in our study may differ from other studies due to different populations and separate pain phenotype from different types of pain stimuli. To overcome these potential biases, whole genome association studies on pain are needed.
(617) Selective colocalization of neuropeptides and botulinum toxin induced cleaved SNAP-25 in dorsal root ganglia culture X Li; University of Georgia, Athens, GA Recently, investigators have observed the interesting phenomenon that botulinum neurotoxin (BoNT) may provide significant benefit in the reduction of certain types of pain. However, the mechanistic basis for this phenomenon is unclear, since an interaction between the terminals of the sensory nervous system and BoNTs is not well understood. Is there a direct effect of BoNT on primary sensory neurons associated with pain? In an effort to address this, a study of the neuropeptide content of sensory neurons demonstrating sensitivity to BoNT serotype A was undertaken in murine primary sensory neurons in culture. The SNARE protein SNAP-25, the intracellular target of BoNT A, was developmental expressed in embryonic dorsal root ganglia culture and cleaved by BoNT A (10-8M) in a time dependent manner. Cleavage of SNAP-25 was detected in a select population of DRG neurons by immunofluorescence, that were double-labeled with some pain-related neuropeptides, including CGRP, galanin, GABA and nociceptin, but not with substance-P and glutamate. It has previously been reported that BoNT A blocks the release of substance-P, and glutamate in the spinal dorsal horn and decreases the sensitivity of pain transduction from spinal cord to brain. Our preliminary results suggest that the release of certain neuropeptides related to pain transmission or modulation in primary sensory neurons may be selectively altered by BoNT A. Thus, a potential mechanism for pain relief by BoNT A may be mediated by toxine-induced interference of multiple pain related factors.
S5
B. Systems (Physiology, Anatomy, Animal Models) B01 - Animal Models of Chronic Pain (618) A novel approach to the use of animals in studies of pain: Development of the Canine Brief Pain Inventory (CBPI) D Brown, R Boston, J Coyne, J Farrar; University of Pennsylvania, Philadelphia, PA Novel compounds for chronic pain that appear promising in laboratory animals often fail to prove efficacious in clinical trials, suggesting a need for more predictive animal models. The spontaneous pain caused by naturally occurring diseases in companion animals (pets) requires treatment, and the study of novel therapies in these animals can provide greater insight into the potential efficacy in humans. Studies of novel analgesics in companion dogs that spontaneously develop bone cancer reveal drug effects which are not evident in rodent studies. The development of outcome pain measures for use in companion dogs that specifically correspond with outcomes routinely used in clinical research could further increase the predictive capability of these preclinical companion animal studies. The objective of this study was to develop the Canine Brief Pain Inventory (CBPI), based on the human Brief Pain Inventory (BPI), for use in the canine model of spontaneous bone cancer. The item structure, response scaling, and severity items of the BPI were maintained for the CBPI. Impact items were generated through focus groups and an expert panel; poorly performing items were removed; with the reduced set of items subject to factor analysis, reliability, and validity testing. The “severity” and “impact” factors hypothesized based on the BPI were demonstrated in the CBPI. Internal consistency of both factors was high (Cronbach’s alpha 0.95 and 0.93), as was test-retest reliability (kappa 0.73 and 0.65). Extreme groups validation against normal dogs showed significantly higher factor scores (P⬍0.001 for both) and convergent validity was demonstrated against quality of life (r⫽0.49 and 0.63). The CBPI and BPI reliably measure the same pain constructs in comparable diseases. This innovative approach to preclinical outcomes development applied to companion animals with complex behaviors and clear parallels to human disease pathogenesis, progression and symptomatology, could transform the predictive ability of preclinical pain studies.
(619) Role of TRPV1 receptors in a novel model of chronic ischemic pain R Radhakrishnan, R Nallu; Western University of Health Sciences, Pomona, CA The pain mechanism in ischemic conditions like intermittent claudication is poorly understood, partly due to the lack of suitable chronic pre-clinical models. In the present study we modified an existing model of ischemia (Orito et al., 2004) in an effort to develop a better characterized chronic ischemic pain model. Since ischemia reduces pH in the affected tissues and TRPV1 channels transduce pH changes, we also studied the role of TRPV1 in this model. Baseline heat (Hargreave’s) and mechanical (von Frey) thresholds were measured in the hindpaws of male Sprague-Dawley rats, and they were trained to run on a treadmill with an electric shock-grid. Left iliac artery of the rats was then surgically occluded and animals were allowed to recover for 48h. At 48h, 72h, and weekly from 1wk through 8wk, heat and mechanical thresholds were measured again. Animals were then placed on the treadmill and forced to run for 5 min using mild electric shock motivation. Pain behavior was measured as 1) onset time for lifting, shaking or dragging the hind limb; 2) incapacity of the animal to run on the treadmill, indicated by the number of hits on the shock-grid; 3) reduction in heat and/or mechanical threshold after treadmill test. Our results show that the onset time for exercise-induced pain was shorter, number of hits on shock-grid was higher, and the mechanical threshold was reduced (mechanical allodynia) in ligated animals compared to sham. The pain behavior was present for 3-6 weeks with peak at 48-72h. Preliminary results show that at 72h time-point, TRPV1 antagonist capsazepine (25 mg/kg.,i.p.) increased the capacity of the ligated animals to run, but did not affect the onset of pain or allodynia, indicating a role for TRPV1 receptors in this model. Studies are underway to further characterize the model and to confirm the preliminary findings.