Page 1 of 43
1 An Intensive Locomotor Training Paradigm Improves Neuropathic Pain following Spinal Cord Compression Injury in Rats Dugan, Elizabeth A., PhD, and Sagen, Jacqueline, PhD,
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
University of Miami, Miller School of Medicine
Running Title: Intensive locomotor training for SCI pain
Corresponding Author: Elizabeth A. Dugan, PhD University of Miami, Miller School of Medicine The Miami Project to Cure Paralysis 1095 NW 14th Terrace Lois Pope LIFE Center Miami, Fl 33136 USA p. (305) 243-6038 f. (305) 243-3923 [email protected]
Page 2 of 43
2 Jacqueline Sagen, PhD University of Miami, Miller School of Medicine The Miami Project to Cure Paralysis 1095 NW 14th Terrace
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Lois Pope LIFE Center Miami, Fl 33136 USA p. (305) 243-6038 f. (305) 243-3923 [email protected]
Page 3 of 43
3 Abstract Spinal cord injury (SCI) is often associated with both locomotor deficits and sensory dysfunction including debilitating neuropathic pain. Unfortunately, current conventional
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
pharmacological, physiological, or psychological treatments provided only marginal relief for more 2/3rds of patients highlighting the need for improved treatment options. Locomotor training is often prescribed as an adjunct therapy for peripheral neuropathic pain but is rarely used to treat central neuropathic pain. The goal of this study was to evaluate the potential antinociceptive benefits of intensive locomotor training (ILT) on neuropathic pain consequent to traumatic SCI. Using a rodent SCI model for central neuropathic pain, ILT was initiated either 5 days after injury prior to development of neuropathic pain symptoms (the “prevention” group), or delayed until pain symptoms fully developed (~3 weeks post injury, the “reversal” group). The training protocol consisted of 5 days/week of a ramping protocol that started with 11m/min for 5 minutes and increased in speed (+1m/min/wk) and time (1-4 minutes/wk) to a maximum of two 20 minute sessions/day at 15m/min by the 4th week of training. ILT prevented and reversed the development of heat hyperalgesia and cold allodynia as well as reversed developed tactile allodynia suggesting analgesic benefits not seen with moderate levels of locomotor training. Further, the analgesic benefits of ILT persisted for several weeks once training had been stopped. The unique ability of an ILT protocol to produce robust and sustained antinociceptive effects, as assessed by 3 distinct outcome measures for below-level SCI neuropathic pain, suggests that this adjunct therapeutic approach has great promise in a comprehensive treatment strategy for SCI pain.
Key words: Central neuropathic pain, locomotor training, spinal cord injury, rat, analgesia
Page 4 of 43
4
Introduction Spinal cord injury (SCI) is often associated with both locomotor deficits and sensory dysfunction including debilitating neuropathic pain.1-3 These sensory abnormalities can manifest Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
as spontaneous or evoked dysesthesias including allodynia (painful response to non-noxious stimuli) and/or hyperalgesia (increased pain response to noxious stimuli).4 The emergence of neuropathic pain following SCI contributes to and may even be the primary impediment to participation in normal daily activities, productivity, and overall quality of life. Conventional pharmacological, physiological, or psychological treatments provide only marginal relief.5-9 The long-term use of analgesic medications for treatment of chronic SCI pain can be limited by untoward side effects and abuse potential owing to high doses needed to achieve even modest benefit. In addition the presence of persistent neuropathic pain can discourage participation of SCI patients in locomotor rehabilitation training, leading to poorer long-term functional outcomes. Locomotor training, which relies on intrinsic and automatic control of movement by lower neural circuits, is frequently used to assist in functional recovery in SCI patients.
10-16
Training such as passive cycling and treadmill training prevent muscle atrophy in the affected limbs and may promote locomotor recovery.17-22 Locomotor training is utilized by many incomplete SCI patients and often uses a treadmill with bodyweight support and manual assistance to appropriately cue the different phases of gait.23 This improves self-selected walking velocity in chronic hemiplegia24 and has been reported to raise patients to a functional locomotion category after both acute and chronic incomplete SCI.25,
26
Mechanistically,
locomotor training normalized the overexpression of GABAA receptors in sensory and motor
Page 5 of 43
5 neurons 27 and stimulated neurotrophic factor secretion such as brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 and 4 (NT-3/4) in the spinal cord and muscle.28, 29 These changes suggest that locomotor training can promote spinal plasticity and spinal learning.30-35
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Treadmill training may also provide some improvement in sensory function and has been used as adjunct therapy for peripheral neuropathic pain syndromes.35-41 For example, in diabetic humans and rats with peripheral neuropathy, routine exercise reduces neuropathic pain symptoms42-44. While mechanisms of these analgesic benefits of exercise remain unclear, it has been associated with increases in endogenous opioids
45-49
, neurotrophic factors
50-52
, decreased
expression of pro-inflammatory cytokines 53-56 and the serotonin system.57, 58 Recently, our colleagues at the Miami Project to Cure Paralysis have shown that Overground Bionic Ambulation training not only provides improved mobility but also greatly reduces chronic, SCI associated neuropathic pain symptoms.59 Although not currently prescribed for the management of central neuropathic pain
60-63
, a few studies have examined the analgesic
benefits of locomotor training for SCI neuropathic pain in animal models.51,
64
In a thoracic
contusion model, moderate treadmill training ameliorated mechanical allodynia.51 Recently, moderate locomotor training (wheel-running) was reported to prevent the development of mechanical allodynia through normalization of glial cell-line derived neurotrophic factor and artemin in a cervical contusion animal model.64 Dugan et al.,
65
extended these findings in an
excitotoxic dorsal horn spinal cord injury model to incorporate an intensive locomotor training (ILT) protocol that also provided analgesic benefits for heat hyperalgesia and spontaneous pain (over-grooming behavior). ILT (as defined by measuring the increase and maintenance of a 20% increase in heart rate) both prevented and reversed the development of heat hyperalgesia suggesting analgesic benefits not seen with moderate levels of locomotor training.65 The goal of
Page 6 of 43
6 this study was to evaluate the potential antinociceptive benefits of ILT on neuropathic pain consequent to traumatic SCI. Materials and methods
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Animals Male Sprague-Dawley rats (150-200g) were used for the clip-compression spinal cord injury (n=72). Half of the animals (n=36) were used for the initial behavioral studies. Based on the behavioral findings a second set of animals (n=36) were utilized to examine prolonged effects of the ILT. Animals were housed two per cage with free access to food and water in a 12h light/dark cycle. Animals were handled daily and were taken to the training area regardless of the experimental group they were assigned. Animals were given enrichment tools in their home cages such as tubes and nestlets throughout the study. Experimental procedures were reviewed and approved by the University of Miami Animal Care and Use Committee and followed the recommendations of the “Guide for the Care and Use of Laboratory Animals” (National Research Council). Surgical Procedures The method to induce SCI via compression was adapted from previous reports,66 and has been used successfully by our laboratory for pharmacologic antinociceptive evaluations over the past several years.67-70 Male Sprague-Dawley rats (Harlan, IN) were anesthetized with 4-5% isoflurane in O2 and maintained on 2-3% isoflurane/O2. The back of the rats, from lumbar to cervical vertebrae were shaved and the skin swabbed with an iodine solution. Following incision of the skin of the back, 2-3 thoracic vertebrae were cleared and a laminectomy was performed to expose spinal cord segments T6-T8. An aneurism clip 1 mm wide (20 g compression force; Harvard Apparatus) was oriented in a vertical position and a spinal segment in the area between
Page 7 of 43
7 T6/T7 was compressed. The dura and spinal nerve roots were not disturbed and the clip was left in place for 60 sec. The clip was then removed and the wounds closed. Rats were allowed to recover in their home cages and allowed free access to food and water. Following spinal
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
compression, the bladder was expressed twice daily for 7-10 days, or until voiding is regained. Rats were tested up to 4 months after spinal compression. We have included a representative histological image of the injury site to show the extent of damage at 20 wks post injury. Due to planned molecular analysis (mRNA), we only had fresh non-fixed tissue to examine, thus images of the lesion epicenter were not intact. However, we have imaged sections from the non-fixed tissue immediately adjacent to the epicenter which show pathology consistent with SCI damage at T7/8 (Fig. S1). Exercise Protocol Treadmill training was initiated either 5 days after injury prior to development of neuropathic pain symptoms (the “prevention” group), or delayed until pain symptoms fully developed (~3 weeks post injury, the “reversal” group). ILT training was done daily for 5 days (Monday – Friday), followed by 2 days off on weekends. The training protocol consisted of 5 days/week of a ramping protocol that started with 11m/min for 5 minutes and increased in speed (+1m/min/wk) and time (1-4 minutes/wk) to a maximum of two 20 minute sessions/day at 15m/min by the 4th week. A 10-minute break was given between the two daily sessions where the animals were given access to water. This protocol was designed by examining normal animals for increased heart rate while treadmill training. Briefly, animals were monitored at varying speeds and times to assess the protocol that provided a 20% increase in heart rate similar that seen in intensive and endurance trained humans. An 8 degree incline of the treadmill was
Page 8 of 43
8 used requiring more load to be placed on the hind limbs during exercise which is beneficial for locomotor improvement following SCI.71-74 Behavioral Analysis
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
To determine changes in neuropathic pain symptoms as a result of treatment we used a battery of sensory tests. Sensory behavioral tests routinely employed for evaluation of neuropathic pain syndromes in our laboratory and others included tests for tactile allodynia, heat hyperalgesia, and cold allodynia. Cutaneous mechanical sensitivity has been examined in rats with spinal cord compression; however, SCI patients report pain that is also evoked by cold and heat stimulus.75 Therefore, a more comprehensive set of behavioral testing is likely to yield the most promising and relevant information. The compression SCI model chosen has the ability to generate stable and long-term behaviorally measurable pain symptoms allowing for chronic testing (at least 12 weeks). Behavioral testing was conducted for mechanical allodynia (Von Frey hairs) and thermal hyperalgesia (Hargreaves test) twice per week (prior to ILT training on Mondays and following ILT training on Fridays). Behavioral testing for cold allodynia (acetone drop) and locomotor recovery (BBB) was conducted once a week on Mondays prior to ILT training (see weekly training schedule below). For a positive response to be recorded during the Von Frey and acetone testing session, animals had to display a higher center behavior as well as the reflexive paw withdrawal. Higher center behaviors included vocalization, head orientation towards the stimulus, and licking or grooming of the testing paw. Sedentary control animals with clip compression SCI received no treadmill training. All behavioral evaluations were carried out by individuals blinded to the treatment groups. Monday Behavioral testing ILT training
Tuesday ILT training
Wednesday ILT training
Thursday ILT training
Friday ILT training Behavioral testing
Saturday No training
Sunday No training
Page 9 of 43
9
Heat Hyperalgesia To assess response to a noxious heat stimulus, the Hargreaves test was used.76 Rats were
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
placed beneath an inverted clear plastic cage on an elevated glass floor and a radiant heat source (52º C) beneath the glass aimed at the plantar hind paw which activated a timer. Withdrawal latencies were the length of time between the activation of the heat source and the hind paw withdrawal from the glass (normal baseline ~10 sec). To avoid tissue damage in the absence of a withdrawal, the cutoff is set at 20 sec. Testing is alternated on both hind paws for 3 trials at least 30 sec apart, the average values used for statistical analysis. Mechanical Allodynia For assessment of mechanical allodynia, the threshold level to an mechanical stimulus was measured with calibrated von Frey hairs ranging from 0.4 to 15 g. To determine von Frey threshold, animals were placed beneath an inverted clear plastic cage (17.5 x 28.0 x 12.5 cm) on an elevated wire mesh floor. Using the Dixon up-down method, calibrated von Frey filaments were applied to the plantar skin of the hind paw.77 The filaments were applied sequentially with increasing force until withdrawal. Once the rat responded, the lower force filament was used until no response was observed at which time the filament force was again increased sequentially. This process was repeated until a pattern of six responses were collected and used to calculate the 50% withdrawal threshold (g). Generally, uninjured rats do not respond to a force of less than 15 g. An upper limit of 15 g was used since a greater force may lift the hind paw itself. To insure cognition of pain responses, only reflexive responses that also include a higher center response was counted. This required the animal to vocalize or turn their head in the direction of the filament.
Page 10 of 43
10 Cold Allodynia For assessment of cold allodynia, we evaluate the effect of the hind paw skin cooled with an acetone droplet.78 Previous studies have examine the use of acetone for cold allodynia in
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
peripheral and central neuropathic pain animal models and suggest that is similar to the response seen in human neuropathic pain patients that suffer from cold and pressure allodynia.78-81The frequency of responses was calculated from the number of times the rat responds to application of acetone to the hind paw skin. The rats are placed in individual chambers and allowed to acclimate for at least 20 minutes. For testing, acetone is applied to the rat hind paw. 100 µl of acetone is slowly ejected from a blunt syringe to the plantar skin of the rat’s hind paw. The fluid is ejected from the syringe over a period of 2-3 seconds. After application of acetone to the hind paw, rats quickly lift and vigorously shake the paw. Both the left and right hind paws are tested with at least 5 minutes between each application of acetone. Response frequency (%) is calculated by: Response frequency % = (number of response / 5 acetone trials) x 100. To insure cognition of pain responses, only reflexive responses that also include a higher center response were counted. This required the animal to vocalize or turn their head in the direction of the tested paw. BBB for functional recovery assessment The open field locomotor score (Basso, Beattie, Bresnahan Locomotor Rating Scale; BBB test)82 measures hindlimb function while the animals freely move in a 2.5 x 3-ft diameter enclosure. It is a widely used and a replicable method of evaluating loss and recovery of overground hindlimb motor function after spinal cord injury. Rats were placed in an enclosure and scored by 2 individuals who were blinded to the experimental conditions. This test is particularly valuable in detecting recovery in hindlimb function after contusion, and has been adapted for
Page 11 of 43
11 other injury models including thoracic transaction.82, 83 The BBB scores range from 0 to 21. A score from 0 to 8 indicates the animal exhibits no, slight, or extensive hindlimb locomotor-like movements (LM) up to coordinated hindlimb sweeping. A score of 9 or above indicates some
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
degree of hindquarter weight support with weight-supported stepping.82 Statistical analyses Data for behavioral tests over time approximate normal distributions and were analyzed by two-way ANOVA between groups and time post injury, with time taken as a repeated measure. However, post hoc analysis between groups at individual time points remained nonparametric and analyzed where appropriate using the Mann Whitney test. Results Intensive locomotor training prevented and reversed heat hyperalgesia Intensive locomotor training prevented the development of thermal hyperalgesia to a noxious heat stimulus (Hargreaves) when training was begun 5-days following SCI (prevention group: Fig. 1a; **P
1 An Intensive Locomotor Training Paradigm Improves Neuropathic Pain following Spinal Cord Compression Injury in Rats Dugan, Elizabeth A., PhD, and Sagen, Jacqueline, PhD,
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
University of Miami, Miller School of Medicine
Running Title: Intensive locomotor training for SCI pain
Corresponding Author: Elizabeth A. Dugan, PhD University of Miami, Miller School of Medicine The Miami Project to Cure Paralysis 1095 NW 14th Terrace Lois Pope LIFE Center Miami, Fl 33136 USA p. (305) 243-6038 f. (305) 243-3923 [email protected]
Page 2 of 43
2 Jacqueline Sagen, PhD University of Miami, Miller School of Medicine The Miami Project to Cure Paralysis 1095 NW 14th Terrace
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Lois Pope LIFE Center Miami, Fl 33136 USA p. (305) 243-6038 f. (305) 243-3923 [email protected]
Page 3 of 43
3 Abstract Spinal cord injury (SCI) is often associated with both locomotor deficits and sensory dysfunction including debilitating neuropathic pain. Unfortunately, current conventional
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
pharmacological, physiological, or psychological treatments provided only marginal relief for more 2/3rds of patients highlighting the need for improved treatment options. Locomotor training is often prescribed as an adjunct therapy for peripheral neuropathic pain but is rarely used to treat central neuropathic pain. The goal of this study was to evaluate the potential antinociceptive benefits of intensive locomotor training (ILT) on neuropathic pain consequent to traumatic SCI. Using a rodent SCI model for central neuropathic pain, ILT was initiated either 5 days after injury prior to development of neuropathic pain symptoms (the “prevention” group), or delayed until pain symptoms fully developed (~3 weeks post injury, the “reversal” group). The training protocol consisted of 5 days/week of a ramping protocol that started with 11m/min for 5 minutes and increased in speed (+1m/min/wk) and time (1-4 minutes/wk) to a maximum of two 20 minute sessions/day at 15m/min by the 4th week of training. ILT prevented and reversed the development of heat hyperalgesia and cold allodynia as well as reversed developed tactile allodynia suggesting analgesic benefits not seen with moderate levels of locomotor training. Further, the analgesic benefits of ILT persisted for several weeks once training had been stopped. The unique ability of an ILT protocol to produce robust and sustained antinociceptive effects, as assessed by 3 distinct outcome measures for below-level SCI neuropathic pain, suggests that this adjunct therapeutic approach has great promise in a comprehensive treatment strategy for SCI pain.
Key words: Central neuropathic pain, locomotor training, spinal cord injury, rat, analgesia
Page 4 of 43
4
Introduction Spinal cord injury (SCI) is often associated with both locomotor deficits and sensory dysfunction including debilitating neuropathic pain.1-3 These sensory abnormalities can manifest Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
as spontaneous or evoked dysesthesias including allodynia (painful response to non-noxious stimuli) and/or hyperalgesia (increased pain response to noxious stimuli).4 The emergence of neuropathic pain following SCI contributes to and may even be the primary impediment to participation in normal daily activities, productivity, and overall quality of life. Conventional pharmacological, physiological, or psychological treatments provide only marginal relief.5-9 The long-term use of analgesic medications for treatment of chronic SCI pain can be limited by untoward side effects and abuse potential owing to high doses needed to achieve even modest benefit. In addition the presence of persistent neuropathic pain can discourage participation of SCI patients in locomotor rehabilitation training, leading to poorer long-term functional outcomes. Locomotor training, which relies on intrinsic and automatic control of movement by lower neural circuits, is frequently used to assist in functional recovery in SCI patients.
10-16
Training such as passive cycling and treadmill training prevent muscle atrophy in the affected limbs and may promote locomotor recovery.17-22 Locomotor training is utilized by many incomplete SCI patients and often uses a treadmill with bodyweight support and manual assistance to appropriately cue the different phases of gait.23 This improves self-selected walking velocity in chronic hemiplegia24 and has been reported to raise patients to a functional locomotion category after both acute and chronic incomplete SCI.25,
26
Mechanistically,
locomotor training normalized the overexpression of GABAA receptors in sensory and motor
Page 5 of 43
5 neurons 27 and stimulated neurotrophic factor secretion such as brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 and 4 (NT-3/4) in the spinal cord and muscle.28, 29 These changes suggest that locomotor training can promote spinal plasticity and spinal learning.30-35
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Treadmill training may also provide some improvement in sensory function and has been used as adjunct therapy for peripheral neuropathic pain syndromes.35-41 For example, in diabetic humans and rats with peripheral neuropathy, routine exercise reduces neuropathic pain symptoms42-44. While mechanisms of these analgesic benefits of exercise remain unclear, it has been associated with increases in endogenous opioids
45-49
, neurotrophic factors
50-52
, decreased
expression of pro-inflammatory cytokines 53-56 and the serotonin system.57, 58 Recently, our colleagues at the Miami Project to Cure Paralysis have shown that Overground Bionic Ambulation training not only provides improved mobility but also greatly reduces chronic, SCI associated neuropathic pain symptoms.59 Although not currently prescribed for the management of central neuropathic pain
60-63
, a few studies have examined the analgesic
benefits of locomotor training for SCI neuropathic pain in animal models.51,
64
In a thoracic
contusion model, moderate treadmill training ameliorated mechanical allodynia.51 Recently, moderate locomotor training (wheel-running) was reported to prevent the development of mechanical allodynia through normalization of glial cell-line derived neurotrophic factor and artemin in a cervical contusion animal model.64 Dugan et al.,
65
extended these findings in an
excitotoxic dorsal horn spinal cord injury model to incorporate an intensive locomotor training (ILT) protocol that also provided analgesic benefits for heat hyperalgesia and spontaneous pain (over-grooming behavior). ILT (as defined by measuring the increase and maintenance of a 20% increase in heart rate) both prevented and reversed the development of heat hyperalgesia suggesting analgesic benefits not seen with moderate levels of locomotor training.65 The goal of
Page 6 of 43
6 this study was to evaluate the potential antinociceptive benefits of ILT on neuropathic pain consequent to traumatic SCI. Materials and methods
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
Animals Male Sprague-Dawley rats (150-200g) were used for the clip-compression spinal cord injury (n=72). Half of the animals (n=36) were used for the initial behavioral studies. Based on the behavioral findings a second set of animals (n=36) were utilized to examine prolonged effects of the ILT. Animals were housed two per cage with free access to food and water in a 12h light/dark cycle. Animals were handled daily and were taken to the training area regardless of the experimental group they were assigned. Animals were given enrichment tools in their home cages such as tubes and nestlets throughout the study. Experimental procedures were reviewed and approved by the University of Miami Animal Care and Use Committee and followed the recommendations of the “Guide for the Care and Use of Laboratory Animals” (National Research Council). Surgical Procedures The method to induce SCI via compression was adapted from previous reports,66 and has been used successfully by our laboratory for pharmacologic antinociceptive evaluations over the past several years.67-70 Male Sprague-Dawley rats (Harlan, IN) were anesthetized with 4-5% isoflurane in O2 and maintained on 2-3% isoflurane/O2. The back of the rats, from lumbar to cervical vertebrae were shaved and the skin swabbed with an iodine solution. Following incision of the skin of the back, 2-3 thoracic vertebrae were cleared and a laminectomy was performed to expose spinal cord segments T6-T8. An aneurism clip 1 mm wide (20 g compression force; Harvard Apparatus) was oriented in a vertical position and a spinal segment in the area between
Page 7 of 43
7 T6/T7 was compressed. The dura and spinal nerve roots were not disturbed and the clip was left in place for 60 sec. The clip was then removed and the wounds closed. Rats were allowed to recover in their home cages and allowed free access to food and water. Following spinal
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
compression, the bladder was expressed twice daily for 7-10 days, or until voiding is regained. Rats were tested up to 4 months after spinal compression. We have included a representative histological image of the injury site to show the extent of damage at 20 wks post injury. Due to planned molecular analysis (mRNA), we only had fresh non-fixed tissue to examine, thus images of the lesion epicenter were not intact. However, we have imaged sections from the non-fixed tissue immediately adjacent to the epicenter which show pathology consistent with SCI damage at T7/8 (Fig. S1). Exercise Protocol Treadmill training was initiated either 5 days after injury prior to development of neuropathic pain symptoms (the “prevention” group), or delayed until pain symptoms fully developed (~3 weeks post injury, the “reversal” group). ILT training was done daily for 5 days (Monday – Friday), followed by 2 days off on weekends. The training protocol consisted of 5 days/week of a ramping protocol that started with 11m/min for 5 minutes and increased in speed (+1m/min/wk) and time (1-4 minutes/wk) to a maximum of two 20 minute sessions/day at 15m/min by the 4th week. A 10-minute break was given between the two daily sessions where the animals were given access to water. This protocol was designed by examining normal animals for increased heart rate while treadmill training. Briefly, animals were monitored at varying speeds and times to assess the protocol that provided a 20% increase in heart rate similar that seen in intensive and endurance trained humans. An 8 degree incline of the treadmill was
Page 8 of 43
8 used requiring more load to be placed on the hind limbs during exercise which is beneficial for locomotor improvement following SCI.71-74 Behavioral Analysis
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
To determine changes in neuropathic pain symptoms as a result of treatment we used a battery of sensory tests. Sensory behavioral tests routinely employed for evaluation of neuropathic pain syndromes in our laboratory and others included tests for tactile allodynia, heat hyperalgesia, and cold allodynia. Cutaneous mechanical sensitivity has been examined in rats with spinal cord compression; however, SCI patients report pain that is also evoked by cold and heat stimulus.75 Therefore, a more comprehensive set of behavioral testing is likely to yield the most promising and relevant information. The compression SCI model chosen has the ability to generate stable and long-term behaviorally measurable pain symptoms allowing for chronic testing (at least 12 weeks). Behavioral testing was conducted for mechanical allodynia (Von Frey hairs) and thermal hyperalgesia (Hargreaves test) twice per week (prior to ILT training on Mondays and following ILT training on Fridays). Behavioral testing for cold allodynia (acetone drop) and locomotor recovery (BBB) was conducted once a week on Mondays prior to ILT training (see weekly training schedule below). For a positive response to be recorded during the Von Frey and acetone testing session, animals had to display a higher center behavior as well as the reflexive paw withdrawal. Higher center behaviors included vocalization, head orientation towards the stimulus, and licking or grooming of the testing paw. Sedentary control animals with clip compression SCI received no treadmill training. All behavioral evaluations were carried out by individuals blinded to the treatment groups. Monday Behavioral testing ILT training
Tuesday ILT training
Wednesday ILT training
Thursday ILT training
Friday ILT training Behavioral testing
Saturday No training
Sunday No training
Page 9 of 43
9
Heat Hyperalgesia To assess response to a noxious heat stimulus, the Hargreaves test was used.76 Rats were
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
placed beneath an inverted clear plastic cage on an elevated glass floor and a radiant heat source (52º C) beneath the glass aimed at the plantar hind paw which activated a timer. Withdrawal latencies were the length of time between the activation of the heat source and the hind paw withdrawal from the glass (normal baseline ~10 sec). To avoid tissue damage in the absence of a withdrawal, the cutoff is set at 20 sec. Testing is alternated on both hind paws for 3 trials at least 30 sec apart, the average values used for statistical analysis. Mechanical Allodynia For assessment of mechanical allodynia, the threshold level to an mechanical stimulus was measured with calibrated von Frey hairs ranging from 0.4 to 15 g. To determine von Frey threshold, animals were placed beneath an inverted clear plastic cage (17.5 x 28.0 x 12.5 cm) on an elevated wire mesh floor. Using the Dixon up-down method, calibrated von Frey filaments were applied to the plantar skin of the hind paw.77 The filaments were applied sequentially with increasing force until withdrawal. Once the rat responded, the lower force filament was used until no response was observed at which time the filament force was again increased sequentially. This process was repeated until a pattern of six responses were collected and used to calculate the 50% withdrawal threshold (g). Generally, uninjured rats do not respond to a force of less than 15 g. An upper limit of 15 g was used since a greater force may lift the hind paw itself. To insure cognition of pain responses, only reflexive responses that also include a higher center response was counted. This required the animal to vocalize or turn their head in the direction of the filament.
Page 10 of 43
10 Cold Allodynia For assessment of cold allodynia, we evaluate the effect of the hind paw skin cooled with an acetone droplet.78 Previous studies have examine the use of acetone for cold allodynia in
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
peripheral and central neuropathic pain animal models and suggest that is similar to the response seen in human neuropathic pain patients that suffer from cold and pressure allodynia.78-81The frequency of responses was calculated from the number of times the rat responds to application of acetone to the hind paw skin. The rats are placed in individual chambers and allowed to acclimate for at least 20 minutes. For testing, acetone is applied to the rat hind paw. 100 µl of acetone is slowly ejected from a blunt syringe to the plantar skin of the rat’s hind paw. The fluid is ejected from the syringe over a period of 2-3 seconds. After application of acetone to the hind paw, rats quickly lift and vigorously shake the paw. Both the left and right hind paws are tested with at least 5 minutes between each application of acetone. Response frequency (%) is calculated by: Response frequency % = (number of response / 5 acetone trials) x 100. To insure cognition of pain responses, only reflexive responses that also include a higher center response were counted. This required the animal to vocalize or turn their head in the direction of the tested paw. BBB for functional recovery assessment The open field locomotor score (Basso, Beattie, Bresnahan Locomotor Rating Scale; BBB test)82 measures hindlimb function while the animals freely move in a 2.5 x 3-ft diameter enclosure. It is a widely used and a replicable method of evaluating loss and recovery of overground hindlimb motor function after spinal cord injury. Rats were placed in an enclosure and scored by 2 individuals who were blinded to the experimental conditions. This test is particularly valuable in detecting recovery in hindlimb function after contusion, and has been adapted for
Page 11 of 43
11 other injury models including thoracic transaction.82, 83 The BBB scores range from 0 to 21. A score from 0 to 8 indicates the animal exhibits no, slight, or extensive hindlimb locomotor-like movements (LM) up to coordinated hindlimb sweeping. A score of 9 or above indicates some
Journal of Neurotrauma Downloaded from online.liebertpub.com by Univ Of Pennsylvania on 01/11/15. For personal use only.
degree of hindquarter weight support with weight-supported stepping.82 Statistical analyses Data for behavioral tests over time approximate normal distributions and were analyzed by two-way ANOVA between groups and time post injury, with time taken as a repeated measure. However, post hoc analysis between groups at individual time points remained nonparametric and analyzed where appropriate using the Mann Whitney test. Results Intensive locomotor training prevented and reversed heat hyperalgesia Intensive locomotor training prevented the development of thermal hyperalgesia to a noxious heat stimulus (Hargreaves) when training was begun 5-days following SCI (prevention group: Fig. 1a; **P
