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Evaluation of anxiety in the elevated plus maze of young rats submitted to noxious stimulation in the neonatal period
P36
The Journal of Pain
(240) Evaluation of anxiety in the elevated plus maze of young rats submitted to noxious stimulation in the neonatal period N Machado, L Sanada, E Carmo, G Silva, and V Fazan; School of Medicine of Ribeir~ ao Preto , S~ ao Paulo, Brazil Although often painful experiences in the neonatal period cannot be consciously remembered, there is evidence suggesting long-lasting psychological effects. Previous studies showed that preterm infants exposed to many painful procedures had an increased prevalence of cognitive impairment and memory loss, adult psychopathology and increased anxiety and defensive behavior. The aim of the present study was to analyze if the pain on neonate period were capable to increase anxiety in young rats. Twelve male Wistar rats with 90 days of life were separated in 2 groups: pain group (N=6) and control group (N=6). The pain group was stimulated with a needle on the right paw, twice a day for 15 days. The control group was stimulated with a swab on the right paw, twice a day for 15 days. Before evaluation, the animals remained in the experiment room for 15 minutes to adapt, then were placed for 5 minutes and filmed in the elevated plus maze (EPM). At the beginning of the experiment the animals were placed individually on the central platform of the maze, with the head turned to one of the arms. We analyzed the time spent in open and closed arms (an entry was recorded when the animal placed all four feet within one arm). Each animal was exposed to the maze only once. Neurovegetative reactions were also recorded as manure and urine of these animals during the EPM exposure. Specific static tests were applied, and p value was considered significant when p value was less than 0,05. There was a significantly difference in staying in the closed arms among the animals subjected to noxious stimulation compared to controls. Our results suggest that the painful experience in the neonatal period may exert a direct influence on behavior, triggered long-lasting anxiogenic effects. Financial Support: CNPq, Capes, Fapesp and FAEPA.
Abstracts (242) A dynamic temporal summation procedure to detect individual changes in nociceptive processing E Sohlberg, S Mackey, D Schwarz, K Johnson, and J Kong, MD; Stanford Systems Neuroscience and Pain Lab, Stanford, CA Temporal Summation (TS) is an experimental approach to study pain amplification in animals and humans. Pulses of noxious stimuli, given at a frequency typically above 0.3 to 0.5 Hz, result in a ramp up of perceived pain following each successive pulse. The mechanism of TS is thought to involve the windup of spinal secondary neurons which receive inputs from slow C-fibers, thus representing a primarily central process. There is considerable individual variability in TS, and these individual differences are valuable in characterizing chronic pain conditions. However, such variability poses a challenge to measuring within-individual changes in TS. Using fixed TS parameters, we find that some individuals are unable to tolerate a single pain pulse, while other individuals do not report any TS at all. Thus, a fixed approach may lack sensitivity to detect individual changes due to ceiling or floor effects. We have been piloting an adjustable TS protocol to more fully measure within-individual changes in TS. The protocol involves pulses of thermode heat pain lasting for 0.5 s, separated by a baseline interval of 1.5 s. We manipulate both the peak pulse temperature and the baseline temperature to find the temperature settings that induce TS for an individual, defined as the highest settings that all pulses can be tolerated. Trains of 5-10 pulses are run at one temperature setting level before adjusting to the next. This protocol is run continuously to minimize experimental time needed. The dynamic approach has reliably induced TS in healthy individuals and chronic pain patients, despite large individual variability. In several pilot studies, we have found that this TS approach is useful for detecting changes in responses to various interventions. Dynamic temporal summation procedures may have utility in measuring individual changes in nociceptive processing, across a broad range of participants.
E08 Central Sensitization (241) Supraspinal descending facilitation of central sensitization depends on peroxynitrite regulation of opioid signaling
(243) Central sensitization in the human spinal cord as measured with functional magnetic resonance imaging
J Little, Z Chen, T Doyle, F Porreca, and D Salvemini; Saint Louis University School of Medicine, Saint Louis, MO
Central sensitization, which involves an increased gain in the nociceptive system, has received growing support as a possible mechanism underlying chronic pain conditions. The heat-capsaicin model has been shown to reliably evoke symptoms of chronic pain such as secondary mechanical hyperalgesia (SMH). In experimental animals, SMH has been shown to result from central sensitization within the dorsal horn of the spinal cord, however the difficulties associated with imaging the spinal cord has mean that this is yet to be confirmed in humans. The aims of this study are to determine the activation patterns in the spinal cord due to mechanical stimuli in an experimentally induced model of central sensitization. Following the establishment of heat-capsaicin model on the volar forearm, a continuous series of fMRI brain images (212 spiral in out, gradient echo volumes, voxel size=1.25x1.25x4mm, TR=2.5seconds) were collected over a 9 minute period. The scan began with a 40 second baseline followed by 30 seconds of mechanical stimulation the site of SMH, this was repeated 7 times. The contralateral-untreated side was stimulated identically in the same location to serve as a control. Images were converted and retrospectively corrected for heart rate and respiratory motion using RETROICor software. Additional motion correction was made using Art_Repair software. Images were realigned, normalized and smoothed (3mm full width half maximum (FWHM)) using SPM8. Changes in signal intensity were determined using a box-car model convolved with a haemodynamic response function (random-effects procedure; uncorrected) Results revealed an increase in spinal BOLD activation on the side ipsilateral to painful allodynic brushing. Furthermore this was found to be anatomically appropriate to the dermatome stimulated. These results demonstrate our ability to image the effects SMH as generated by the heat-capsaicin model. This indicates the possibility of imaging central sensitization in the spinal cord in pathological pain conditions.
Over the past decade, our laboratory has demonstrated that peroxynitrite (PN) signaling in the periphery and spinal cord is critical to the development of pain of several etiologies. More recent studies have established that supraspinal regions of the central nervous system also contribute to the development and maintenance of pathologic pain. The rostral ventromedial medulla (RVM) is a supraspinal nociceptive modulating center known for its role in endogenous opioid analgesia that also drives central sensitization through descending facilitation of spinal nociception. We hypothesized that PN contributes to RVM descending facilitation of nociception through interactions with the endogenous opioid system. The studies presented herein are the first to demonstrate that PN contributes to RVM facilitation of nociception during central sensitization and that it does so through regulation of opioid signaling. Sprague Dawley rats that were cannulated for RVM microinjections received a hindpaw injection of carrageenan to induce thermal hyperalgesia. Carrageenan-induced hyperalgesia was dose-dependently reversed by therapeutic (at +3h, time of peak hyperalgesia) intra-RVM administration of a PN decomposition catalyst, FeTMPyP (0.25mg-1mg, n=4 in 0.5mL). Microinjections of a similar volume (0.5mL) of vehicle in hyperalgesic rats or FeTMPyP (1mg) in non-hyperalgesic control rats had no effect. Interestingly, the anti-hyperalgesic effects of FeTMPyP observed at 1h after injection were rapidly (within 15 min) reversed by intra-RVM administration of naloxone (5mg; n=3), a competitive antagonist of opioid receptors. When given alone, naloxone had no effect on hyperalgesia. These results suggest that formation of PN in the RVM contributes to the development and maintenance of inflammatory hyperalgesia through a local interaction with the endogenous opiate system. Experiments are underway in our lab to elucidate the mechanisms of this interaction. These findings further support our concept that targeting PN is a viable therapeutic approach in pain management.
P Nash, J Brown, and S Mackey; Stanford University, Palo Alto, CA
The Journal of Pain
(240) Evaluation of anxiety in the elevated plus maze of young rats submitted to noxious stimulation in the neonatal period N Machado, L Sanada, E Carmo, G Silva, and V Fazan; School of Medicine of Ribeir~ ao Preto , S~ ao Paulo, Brazil Although often painful experiences in the neonatal period cannot be consciously remembered, there is evidence suggesting long-lasting psychological effects. Previous studies showed that preterm infants exposed to many painful procedures had an increased prevalence of cognitive impairment and memory loss, adult psychopathology and increased anxiety and defensive behavior. The aim of the present study was to analyze if the pain on neonate period were capable to increase anxiety in young rats. Twelve male Wistar rats with 90 days of life were separated in 2 groups: pain group (N=6) and control group (N=6). The pain group was stimulated with a needle on the right paw, twice a day for 15 days. The control group was stimulated with a swab on the right paw, twice a day for 15 days. Before evaluation, the animals remained in the experiment room for 15 minutes to adapt, then were placed for 5 minutes and filmed in the elevated plus maze (EPM). At the beginning of the experiment the animals were placed individually on the central platform of the maze, with the head turned to one of the arms. We analyzed the time spent in open and closed arms (an entry was recorded when the animal placed all four feet within one arm). Each animal was exposed to the maze only once. Neurovegetative reactions were also recorded as manure and urine of these animals during the EPM exposure. Specific static tests were applied, and p value was considered significant when p value was less than 0,05. There was a significantly difference in staying in the closed arms among the animals subjected to noxious stimulation compared to controls. Our results suggest that the painful experience in the neonatal period may exert a direct influence on behavior, triggered long-lasting anxiogenic effects. Financial Support: CNPq, Capes, Fapesp and FAEPA.
Abstracts (242) A dynamic temporal summation procedure to detect individual changes in nociceptive processing E Sohlberg, S Mackey, D Schwarz, K Johnson, and J Kong, MD; Stanford Systems Neuroscience and Pain Lab, Stanford, CA Temporal Summation (TS) is an experimental approach to study pain amplification in animals and humans. Pulses of noxious stimuli, given at a frequency typically above 0.3 to 0.5 Hz, result in a ramp up of perceived pain following each successive pulse. The mechanism of TS is thought to involve the windup of spinal secondary neurons which receive inputs from slow C-fibers, thus representing a primarily central process. There is considerable individual variability in TS, and these individual differences are valuable in characterizing chronic pain conditions. However, such variability poses a challenge to measuring within-individual changes in TS. Using fixed TS parameters, we find that some individuals are unable to tolerate a single pain pulse, while other individuals do not report any TS at all. Thus, a fixed approach may lack sensitivity to detect individual changes due to ceiling or floor effects. We have been piloting an adjustable TS protocol to more fully measure within-individual changes in TS. The protocol involves pulses of thermode heat pain lasting for 0.5 s, separated by a baseline interval of 1.5 s. We manipulate both the peak pulse temperature and the baseline temperature to find the temperature settings that induce TS for an individual, defined as the highest settings that all pulses can be tolerated. Trains of 5-10 pulses are run at one temperature setting level before adjusting to the next. This protocol is run continuously to minimize experimental time needed. The dynamic approach has reliably induced TS in healthy individuals and chronic pain patients, despite large individual variability. In several pilot studies, we have found that this TS approach is useful for detecting changes in responses to various interventions. Dynamic temporal summation procedures may have utility in measuring individual changes in nociceptive processing, across a broad range of participants.
E08 Central Sensitization (241) Supraspinal descending facilitation of central sensitization depends on peroxynitrite regulation of opioid signaling
(243) Central sensitization in the human spinal cord as measured with functional magnetic resonance imaging
J Little, Z Chen, T Doyle, F Porreca, and D Salvemini; Saint Louis University School of Medicine, Saint Louis, MO
Central sensitization, which involves an increased gain in the nociceptive system, has received growing support as a possible mechanism underlying chronic pain conditions. The heat-capsaicin model has been shown to reliably evoke symptoms of chronic pain such as secondary mechanical hyperalgesia (SMH). In experimental animals, SMH has been shown to result from central sensitization within the dorsal horn of the spinal cord, however the difficulties associated with imaging the spinal cord has mean that this is yet to be confirmed in humans. The aims of this study are to determine the activation patterns in the spinal cord due to mechanical stimuli in an experimentally induced model of central sensitization. Following the establishment of heat-capsaicin model on the volar forearm, a continuous series of fMRI brain images (212 spiral in out, gradient echo volumes, voxel size=1.25x1.25x4mm, TR=2.5seconds) were collected over a 9 minute period. The scan began with a 40 second baseline followed by 30 seconds of mechanical stimulation the site of SMH, this was repeated 7 times. The contralateral-untreated side was stimulated identically in the same location to serve as a control. Images were converted and retrospectively corrected for heart rate and respiratory motion using RETROICor software. Additional motion correction was made using Art_Repair software. Images were realigned, normalized and smoothed (3mm full width half maximum (FWHM)) using SPM8. Changes in signal intensity were determined using a box-car model convolved with a haemodynamic response function (random-effects procedure; uncorrected) Results revealed an increase in spinal BOLD activation on the side ipsilateral to painful allodynic brushing. Furthermore this was found to be anatomically appropriate to the dermatome stimulated. These results demonstrate our ability to image the effects SMH as generated by the heat-capsaicin model. This indicates the possibility of imaging central sensitization in the spinal cord in pathological pain conditions.
Over the past decade, our laboratory has demonstrated that peroxynitrite (PN) signaling in the periphery and spinal cord is critical to the development of pain of several etiologies. More recent studies have established that supraspinal regions of the central nervous system also contribute to the development and maintenance of pathologic pain. The rostral ventromedial medulla (RVM) is a supraspinal nociceptive modulating center known for its role in endogenous opioid analgesia that also drives central sensitization through descending facilitation of spinal nociception. We hypothesized that PN contributes to RVM descending facilitation of nociception through interactions with the endogenous opioid system. The studies presented herein are the first to demonstrate that PN contributes to RVM facilitation of nociception during central sensitization and that it does so through regulation of opioid signaling. Sprague Dawley rats that were cannulated for RVM microinjections received a hindpaw injection of carrageenan to induce thermal hyperalgesia. Carrageenan-induced hyperalgesia was dose-dependently reversed by therapeutic (at +3h, time of peak hyperalgesia) intra-RVM administration of a PN decomposition catalyst, FeTMPyP (0.25mg-1mg, n=4 in 0.5mL). Microinjections of a similar volume (0.5mL) of vehicle in hyperalgesic rats or FeTMPyP (1mg) in non-hyperalgesic control rats had no effect. Interestingly, the anti-hyperalgesic effects of FeTMPyP observed at 1h after injection were rapidly (within 15 min) reversed by intra-RVM administration of naloxone (5mg; n=3), a competitive antagonist of opioid receptors. When given alone, naloxone had no effect on hyperalgesia. These results suggest that formation of PN in the RVM contributes to the development and maintenance of inflammatory hyperalgesia through a local interaction with the endogenous opiate system. Experiments are underway in our lab to elucidate the mechanisms of this interaction. These findings further support our concept that targeting PN is a viable therapeutic approach in pain management.
P Nash, J Brown, and S Mackey; Stanford University, Palo Alto, CA