REGULAR EXERCISE REVERSES SENSORY HYPERSENSITIVITY IN A RAT NEUROPATHIC PAIN MODEL: ROLE OF ENDOGENOUS OPIOIDS methods
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METHODS:
After baseline measurements, the L5 and L6 spinal nerves of male Sprague-Dawley rats were tightly ligated. Animals were randomized to sedentary or 5-week treadmill exercise-trained groups. Thermal and tactile sensitivities were assessed 23 hours after exercise, using paw withdrawal thresholds to von Frey filaments and withdrawal latenciesto noxious heat.Opioid receptor antagonists were administered by subcutaneous,intrathecal, or intracerebroventricularinjection. Opioid peptides were quantified usingimmunohistochemistry with densitometry.
MATERIALS AND METHODS
Approval was obtained from the University of Arizona Animal Care and Use Committee (Tucson, Arizona). Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) were 250-380 g throughout testing. They were allowed water and food ad libitum andhoused in a climate-controlled room.They weremaintained under reverse light/dark conditions,with the room litfrom 10 pm to 10 am. They were handled twice daily in a stress-free environment and were allowed to equilibrate to the surroundings for 7 days before experimental manipulations were begun. All procedures conformed to the ethical guidelines for the care and use of laboratory animals published by the International Association for the Study of Pain (IASP) and the National Institutes of Health (NIH).
Assessment of tactile sensitivity
Tactile sensitivity was assessed by measuring the threshold for withdrawal of the hindpawfrom normally non-noxious tactile stimuli. Rats were allowed to acclimate for 30 min within plexiglass enclosures with wire mesh bottoms. Paw withdrawal thresholds were determined in response to probing of the lefthind paw with a series of calibrated von Frey filaments, in logarithmically spaced increments, applied perpendicularly to the plantar surface of the paw. A maximal cutoff of 15 g was used because larger filaments lift the paw,preventing the assessment of active paw withdrawal. Data were analyzed by the up-and-down method of Dixon, as described by Chaplan et al. Tactile sensitivity was measured at baseline, 1 week after surgery, and at weekly increments during exercise training. Each time point consisted of one day of testing. Where applicable, measurements were performed 23 hours after e...
RESULTS
There were no differences between animals assigned to the SNL and sham-operated groups in pre-operative withdrawal thresholds or latencies. By one week after surgery, SNL animals developed sensory hypersensitivity (lower thermal withdrawal latencies and tactile withdrawal thresholds) compared to presurgical values ( ), while sham- or non-operated animals did not. Statistical analysis using repeated measures two-way ANOVA (group x time) demonstrated a significant time-treatment interaction effect for both thermal withdrawal latencies (P <0.0001) and paw withdrawal thresholds (P<0.0001). Post-hoc testing was performed using Bonferroni's Method for Multiple Comparisons, specifically making the following comparisons. Spinal nerve ligation reduced thermal withdrawal latency from 17.7 (16.0 to - 19.4) to 12.1 (11.0 to 13.2) sec (P<0.0001) and tactile withdrawal thresholdfrom 14.7 (14....
Exercise effects were dependent on exercise intensity but not frequency.
Exercise training was conducted in SNL animals at speeds of either 10 m/min (lower intensity) or 16m/min (higher intensity). Walking was required at the lower intensity and running at the higher intensity. Animals could not be trained at speeds of more than 16 m/min, because animals exercised at higher speeds exhibited signs of stress. The higher intensity group had a more complete reversal of tactile hypersensitivity than did the lower intensity group ( ). Statistical analysis using repeated measures two-way ANOVA demonstrated a significant intensity-time interaction effect (P <0.0001) andintensity effect (P = 0.0064). Post-hoc testing was performed using Bonferroni's Method for Multiple Comparisons, comparing paw withdrawal thresholds between higher- and lower-intensity training at each time point. Tactile threshold was 12.3(9.3 to 15.3) g after 5 weeks of higher-intensity exe...
The onset of exercise effects was determined by the number of weeks of exercise training
The observed timing of exercise effects might be due to the number of weeks of exercise training required to reverse sensory hypersensitivity. Alternatively, the timing of exercise effects might be due to a maturation of the SNL model in which sensory hypersensitivity was not susceptible to exercise reversal until 3 weeks after surgery. To distinguish between these possibilities, in one group of animals exercise was initiated 4 weeks after SNL surgery as opposed to the 1-week recovery period used in other experiments. Reduction of sensory hypersensitivity occurred 3 weeks after initiation of exercise, regardless of the interval after surgery ( ).Statistical analysis using repeated measures two-way ANOVA (group X time) demonstrated a significant group-time interaction effect (P<0.0001) and group effect (P = 0.0009).Post-hoc testing was performed using Bonferroni's Method for Mult...
Sensory hypersensitivity returned within 1 week of cessation of exercise
Tactile hypersensitivity began to return5 days after cessation of exercise training and returned to pre-exercise levels 8 days after discontinuing exercise ( ). Statistical analysis with one-way repeated-measures ANOVA demonstrated a significant time effect(P<0.0001). Post hoc analysis was performed using Bonferroni's Method for Multiple comparisons, specifically comparing paw withdrawal threshold at each time point with paw withdrawal threshold before discontinuation of exercise training. The first statistically significant decrease in withdrawal threshold occurred on day 5 (P = 0.0006).
Opioid receptor antagonists reversed exercise effects
Subcutaneous naloxone (1 mg/kg)decreased tactile withdrawal threshold in exercise-trained SNL animals from 13.9 (12.1 to 15.7) g to 3.6 (2.8 to 4.4)g 20 min after injection ( ). Statistical analysis using one-way repeated-measures ANOVA demonstrated a significant time effect(P< 0.0001). Post-hoc testing using Bonferroni's Method for Multiple Comparisons was used to specifically compare paw withdrawal threshold at each time point to the pre-naloxone value. P<0.0001 for paw withdrawal threshold 20 min after injection vs. the pre-injection value.
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After baseline measurements, the L5 and L6 spinal nerves of male Sprague-Dawley rats were tightly ligated. Animals were randomized to sedentary or 5-week treadmill exercise-trained groups. Thermal and tactile sensitivities were assessed 23 hours after exercise, using paw withdrawal thresholds to von Frey filaments and withdrawal latenciesto noxious heat.Opioid receptor antagonists were administered by subcutaneous,intrathecal, or intracerebroventricularinjection. Opioid peptides were quantified usingimmunohistochemistry with densitometry.
Approval was obtained from the University of Arizona Animal Care and Use Committee (Tucson, Arizona). Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) were 250-380 g throughout testing. They were allowed water and food ad libitum andhoused in a climate-controlled room.They weremaintained under reverse light/dark conditions,with the room litfrom 10 pm to 10 am. They were handled twice daily in a stress-free environment and were allowed to equilibrate to the surroundings for 7 days before experimental manipulations were begun. All procedures conformed to the ethical guidelines for the care and use of laboratory animals published by the International Association for the Study of Pain (IASP) and the National Institutes of Health (NIH).
Tactile sensitivity was assessed by measuring the threshold for withdrawal of the hindpawfrom normally non-noxious tactile stimuli. Rats were allowed to acclimate for 30 min within plexiglass enclosures with wire mesh bottoms. Paw withdrawal thresholds were determined in response to probing of the lefthind paw with a series of calibrated von Frey filaments, in logarithmically spaced increments, applied perpendicularly to the plantar surface of the paw. A maximal cutoff of 15 g was used because larger filaments lift the paw,preventing the assessment of active paw withdrawal. Data were analyzed by the up-and-down method of Dixon, as described by Chaplan et al. Tactile sensitivity was measured at baseline, 1 week after surgery, and at weekly increments during exercise training. Each time point consisted of one day of testing. Where applicable, measurements were performed 23 hours after exercise. Measurements were confirmed by an observer blinded to the treatment received.
There were no differences between animals assigned to the SNL and sham-operated groups in pre-operative withdrawal thresholds or latencies. By one week after surgery, SNL animals developed sensory hypersensitivity (lower thermal withdrawal latencies and tactile withdrawal thresholds) compared to presurgical values ( ), while sham- or non-operated animals did not. Statistical analysis using repeated measures two-way ANOVA (group x time) demonstrated a significant time-treatment interaction effect for both thermal withdrawal latencies (P <0.0001) and paw withdrawal thresholds (P<0.0001). Post-hoc testing was performed using Bonferroni's Method for Multiple Comparisons, specifically making the following comparisons. Spinal nerve ligation reduced thermal withdrawal latency from 17.7 (16.0 to - 19.4) to 12.1 (11.0 to 13.2) sec (P<0.0001) and tactile withdrawal thresholdfrom 14.7 (14.0 to15.4) to 1.8 (1.6 to 2.1) sec (P<0.0001). Regular exercise training ameliorated thermal and tactile hypersensitivity within 3 weeks. Thermal withdrawal latency increased after 3 weeks of exercise compared to sedentary animals (P = 0.0064), and after 5 weeks withdrawal latency had returned to 17....
Exercise training was conducted in SNL animals at speeds of either 10 m/min (lower intensity) or 16m/min (higher intensity). Walking was required at the lower intensity and running at the higher intensity. Animals could not be trained at speeds of more than 16 m/min, because animals exercised at higher speeds exhibited signs of stress. The higher intensity group had a more complete reversal of tactile hypersensitivity than did the lower intensity group ( ). Statistical analysis using repeated measures two-way ANOVA demonstrated a significant intensity-time interaction effect (P <0.0001) andintensity effect (P = 0.0064). Post-hoc testing was performed using Bonferroni's Method for Multiple Comparisons, comparing paw withdrawal thresholds between higher- and lower-intensity training at each time point. Tactile threshold was 12.3(9.3 to 15.3) g after 5 weeks of higher-intensity exercise, compared to 4.9(1.3 to 8.5) g (P = 0.0003) after 5 weeks of lower-intensity exercise. Post hoc analysis with repeated measures one-way ANOVA also demonstrated that lower-intensity exercise did not significantly increase paw withdrawal threshold compared to pre-exercise values (P=0.068). In co...
The observed timing of exercise effects might be due to the number of weeks of exercise training required to reverse sensory hypersensitivity. Alternatively, the timing of exercise effects might be due to a maturation of the SNL model in which sensory hypersensitivity was not susceptible to exercise reversal until 3 weeks after surgery. To distinguish between these possibilities, in one group of animals exercise was initiated 4 weeks after SNL surgery as opposed to the 1-week recovery period used in other experiments. Reduction of sensory hypersensitivity occurred 3 weeks after initiation of exercise, regardless of the interval after surgery ( ).Statistical analysis using repeated measures two-way ANOVA (group X time) demonstrated a significant group-time interaction effect (P<0.0001) and group effect (P = 0.0009).Post-hoc testing was performed using Bonferroni's Method for Multiple Comparisons, specifically comparing paw withdrawal threshold at each time point after initiation of exercise to paw withdrawal latency after surgery and before exercise testing. The results of this analysis are presented in.
Tactile hypersensitivity began to return5 days after cessation of exercise training and returned to pre-exercise levels 8 days after discontinuing exercise ( ). Statistical analysis with one-way repeated-measures ANOVA demonstrated a significant time effect(P<0.0001). Post hoc analysis was performed using Bonferroni's Method for Multiple comparisons, specifically comparing paw withdrawal threshold at each time point with paw withdrawal threshold before discontinuation of exercise training. The first statistically significant decrease in withdrawal threshold occurred on day 5 (P = 0.0006).
Subcutaneous naloxone (1 mg/kg)decreased tactile withdrawal threshold in exercise-trained SNL animals from 13.9 (12.1 to 15.7) g to 3.6 (2.8 to 4.4)g 20 min after injection ( ). Statistical analysis using one-way repeated-measures ANOVA demonstrated a significant time effect(P< 0.0001). Post-hoc testing using Bonferroni's Method for Multiple Comparisons was used to specifically compare paw withdrawal threshold at each time point to the pre-naloxone value. P<0.0001 for paw withdrawal threshold 20 min after injection vs. the pre-injection value.
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