Pain and Analgesic Mechanisms Section Editors: Martin Angst/Jianren Mao
Spinal Cord Stimulation Reduces Mechanical Hyperalgesia and Glial Cell Activation in Animals with Neuropathic Pain Karina L. Sato, PT, PhD,* Lisa M. Johanek, PhD,† Luciana S. Sanada, PT,* and Kathleen A. Sluka, PhD, PT* BACKGROUND: Spinal cord stimulation (SCS) is commonly used for neuropathic pain; the optimal variables and mechanisms of action are unclear. We tested whether modulation of SCS variables improved analgesia in animals with neuropathic pain by comparing 6-hour vs 30-minute duration and 50%, 75%, or 90% motor threshold (MT) intensity (amplitude). Furthermore, we examined whether maximally effective SCS reduced glial activation in the spinal cord in neuropathic animals. METHODS: Sprague-Dawley rats received the spared nerve injury model and were implanted with an epidural SCS lead. Animals were tested for mechanical withdrawal threshold of the paw before and 2 weeks after spared nerve injury, before and after SCS daily for 4 days, and 1, 4, and 9 days after SCS. Spinal cords were examined for the effects of SCS on glial cell activation. RESULTS: The mechanical withdrawal threshold decreased, and glial immunoreactivity increased 2 weeks after spared nerve injury. For duration, 6-hour SCS significantly increased the mechanical withdrawal threshold when compared with 30-minute SCS or sham SCS; 30-minute SCS was greater than sham SCS. For intensity (amplitude), 90% MT SCS significantly increased the withdrawal threshold when compared with 75% MT SCS, 50% MT SCS, and sham SCS. Both 4 and 60 Hz SCS decreased glial activation (GFAP, MCP-1, and OX-42) in the spinal cord dorsal horn when compared with sham. CONCLUSIONS: Six-hour duration SCS with 90% MT showed the largest increase in mechanical withdrawal threshold, suggesting that the variables of stimulation are important for clinical effectiveness. One potential mechanism for SCS may be to reduce glial activation at the level of the spinal cord. (Anesth Analg 2014;118:464–72)
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europathic pain is defined as pain resulting from damage or dysfunction of peripheral nerves and more broadly, as a result of injury or disease of the somatosensory system. There are a number of diseases associated with neuropathic pain. Examples include autoimmune diseases (e.g., multiple sclerosis), metabolic diseases (e.g., diabetic neuropathy), infection (e.g., shingles and the sequel, postherpetic neuralgia), vascular diseases (stroke), trauma, and cancer. Neuropathic pain in experimental animals can be induced using multiple models that injure peripheral nerves: partial sciatic ligation, chronic constrictive injury, spinal nerve ligation, and spared nerve injury.1,2 Specific mechanisms for the pain associated with peripheral nerve injury are complex and involve changes at the site of injury and in the central nervous system. Pain hypersensitivity was originally thought to result exclusively from From the *Department of Physical Therapy, University of Iowa, Iowa City, Iowa; and †Medtronic, Minneapolis, Minnesota. Accepted for publication November 11, 2013. Funding: Funded by Medtronic, Inc. and NIH AR052316 (KAS). Conflict of Interest: See Disclosures at the end of the article. Reprints will not be available from the authors. Address correspondence to Kathleen A. Sluka, PhD, PT, Department of Physical Therapy, University of Iowa Carver College of Medicine, 500 Newton Rd., 1–248 Medical Education Building, Iowa City, IA 52242. Address e-mail to [email protected]. Copyright © 2013 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000047
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altered neuronal activity in primary sensory and spinal cord neurons. It is now clear that glial cells also play a significant role in the pathogenesis of neuropathic pain.3–7 In fact, there is a time-dependent activation of both astrocytes and microglia after spared nerve injury in the spinal cord.8–10 Furthermore, inhibition of glial activation reduces mechanical allodynia in animals with nerve injury.11–14 Thus, nerve injury is associated with activation of glia that can alter mechanical allodynia in animals. Spinal cord stimulation (SCS) has been successful in providing analgesia, improving function, and enhancing the quality of life for patients suffering from chronic pain.15 In animals with nerve injury, short-duration (10–60 minutes) SCS at high frequencies (50–60 Hz) reduces mechanical allodynia that outlasts the stimulation time.16–19 Our previous work also shows that 4 Hz SCS (30 minutes) reduces mechanical allodynia.19 Furthermore, high-frequency SCS (60 Hz) activates δ-opioid receptors spinally, and low-frequency SCS (4 Hz) activates μ-opioid receptors spinally, showing that different frequencies of stimulation use different mechanisms.20 Clinically, SCS is typically applied at higher frequencies (i.e., 60 Hz), two-thirds motor threshold intensity (amplitude), and for longer durations. Previous studies using peripheral stimulation techniques, such as transcutaneous electrical nerve stimulation (TENS) and electroacupuncture, show that modulation of stimulation parameters can affect outcomes.21–25 Therefore, we tested different parameters of SCS: duration (30 minutes vs 6 hours); February 2014 • Volume 118 • Number 2
and intensity (amplitude) (50%, 75%, 90% motor threshold). We further examined whether the most effective parameters of SCS would reduce glial cell activation in the spinal cord as a potential underlying mechanism.
METHODS Experimental Procedures
Experiments were performed on adult Sprague-Dawley rats, weighing 250 to 350 g and housed in transparent plastic cages with free access to food and water, in a 12-hour light–dark cycle. All the experimental procedures were approved by the Animal Care and Use Committee at the University of Iowa.
Nerve Injury Model
All rats were anesthetized with 2% to 3% isoflurane. The tibial and common peroneal nerves on 1 limb were tightly ligated with 4-0 silk, and the sural nerve was kept intact, as previously described.26 The overlying muscle was sutured with 4-0 silk, and the skin was sutured closed with 3-0 silk.
Implantation of Electrode
After nerve injury, a small laminectomy was performed at the level of T13 vertebra, and the SCS lead was inserted epidurally in the rostral direction. The lead was fixed with sutures to the muscle, the wound was sutured in layers, and the lead was tunneled to exit the skin at the base of the neck.19 We implanted a spinal cord lead designed for use in rats (Medtronic, Minneapolis, MN) that is similar to that used in humans. The proximal end of the lead was tunneled outside the animal for later connection to an external neurostimulator (model # 37021) and programmer (model: #8840) (Medtronic Inc., Minneapolis, MN).
Behavior Tests
Rats were acclimated to the behavior room for 30 minutes followed by acclimation to transparent plastic cubicles on elevated wire mesh floor for 15 minutes. To test for mechanical withdrawal thresholds of the paw, calibrated von Frey filaments (1–402 mN) were applied to the lateral surface bilaterally in the area innervated by the sural nerve. Each filament was applied for approximately 1 second with enough force to bend the filament. Each filament was applied twice, and a positive response was 1 withdrawal. Once a positive response was found, the filament above and below the filament that caused a positive response was tested. Confirmation of mechanical withdrawal threshold was established if there was a positive withdrawal from the filament above and no withdrawal from the filament below. The lowest force that produced a withdrawal was recorded as the mechanical withdrawal threshold. A decrease in mechanical withdrawal threshold is interpreted as cutaneous allodynia in this study.
Immunohistochemistry
We tested for glial cell activation in the spinal cord using immunohistochemistry of astrocyte and microglial markers. Rats were anesthetized with 100 mg/kg sodium pentobarbital and transcardially perfused with heparinized saline (10 U/mL) followed by 4% paraformaldehyde with
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15% picric acid. The spinal cords were removed, post fixed in 4% paraformaldehyde for 1 hour, transferred to 30% sucrose solution for 24 hours, and then frozen on dry ice with Tissue Tek OCT (Sakura Finetek, Torrance, CA). Twenty-micrometer cryostat sections were stained onto slides, and all sections in a group were stained simultaneously. All sections were first blocked with 3% normal goat serum for 30 minutes, followed by Avidin-Biotin block (15 minutes/blocker). For sections used for astrocyte staining, sections were incubated overnight with monoclonal antimouse anti-GFAP (Millipore, Billerica, MA, 1:5000, Cat.#MAB360). On the second day, sections were incubated with biotinylated goat antimouse IgG (Invitrogen, Carlsbad, CA, 1:1000, 1 hour), followed by Strep-568 (Invitrogen, Carlsbad, CA, 1:1000, 1 hour). Sections were then incubated overnight in goat antirabbit MCP-1 (Millipore, Billerica, MA, 1:500, Cat.#1834P). On the last day, sections were incubated in biotinylated goat antirabbit IgG (Invitrogen, Carlsbad, CA, 1:1000, 1 hour), followed by Strep-488 (Invitrogen, 1:1000, 1 hour). Slides were covered with Vectashield. For microglia staining, we used immunohistochemistry staining for OX-42, changing the first primary antibody to OX-42 (AbD Serotec, Raleigh, NC, 1:2500, Cat.#MCA275G). Five spinal cord sections (L4-L5) were randomly chosen from each rat and imaged with an Olympus BX-51 fluorescence microscope (Olympus, Center Valley, PA). Images were analyzed for the density of the staining with Image J (National Institutes of Health, Bethesda, MD version 1.24) as we previously described.27,28 Specifically, each tissue section was first converted to 8-bit gray scale and then calibrated independently using the “uncalibrated OD” function with pixel values ranging from 0 to 255. A background reading taken from the white matter of the dorsal column in the same section was subtracted from the density reading taken from the gray matter of the same tissue section to control for differences in nonspecific staining.29 The superficial laminas (I-II) and intermediate and deep dorsal horn (III–VI) were analyzed separately.
Experimental Protocol Experiment 1 Experiment 1 examined different durations of SCS either 30 minutes or 6 hours. SCS was applied at 4 or 60 Hz frequency and 90% motor threshold intensity (amplitude) (0.35 V) for 4 days. Mechanical withdrawal thresholds were tested before and 2 weeks after spared nerve injury, before and after daily application of SCS for 4 days, and 1, 3, and 9 days after stopping SCS. SCS was applied 2 weeks after spared nerve injury. The following experimental groups were compared: (1) 4 Hz, 30-minute SCS (N = 8), (2) 4 Hz, 6-hour SCS (N = 6), (3) 60 Hz, 30-minute SCS (N =8), (4) 60 Hz, 6-hour SCS (N =6), (5) Sham, 30-minute SCS (N =4), and (6) Sham and 6-hour SCS (N =6). Sham animals were implanted with the neurostimulation hardware and were tethered to the neurostimulator system but did not receive SCS. Experiment 2 Experiment 2 tested different intensities (amplitude) of SCS: 50%, 75%, or 90% motor threshold. SCS was applied at either 4 or 60 Hz frequency, for 6-hour duration daily for 4 days. Mechanical withdrawal thresholds were tested before
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Experiment 3 Experiment 3 tested the effects of SCS on glial cell activation in the spinal cord using immunohistochemistry of astrocyte and microglial markers. SCS was delivered for 4 days beginning 2 weeks after spared nerve injury as follows: 90% motor threshold intensity (amplitude), 6-hour duration, either 4 or 60 Hz frequency. Animals were divided as follows: (1) naive control for OX-42, or GFAP and MCP-1 (N = 5/marker), (2) 60 Hz SCS for OX-42, or GFAP and MCP-1 (N = 5/marker), (3) 4 Hz SCS for OX-42, or GFAP, and MCP-1 (N = 5/marker).
Statistical Analysis
Analysis of the data was performed using SPSS 13.1 (Statistical Package for the Social Sciences, IBM, Armonk, NY). The effect of nerve injury on withdrawal thresholds was tested with a simple paired t test and is in paragraph 1 of the results. Withdrawal threshold data were normalized using a difference score between thresholds obtained after SCS compared with those before SCS that therefore led to 1 number for each day of treatment (days 1–4). Repeatedmeasures analysis of variance (ANOVA) assessed for differences across time for days 1 to 4 with a post hoc Tukey test to examine for differences among groups. Data were also transformed to an area under the curve for the first 4 days of measures after SCS and analyzed with a 1-way ANOVA with a post hoc Tukey test to examine for differences among groups. The first hypothesis examined whether longer-duration SCS was more effective than shorter-duration SCS. We used repeated-measures ANOVA to examine difference scores immediately after SCS over the 4 days (days 1–4) using duration (0 [sham], 30 minutes, 6 hours) and frequency (0 [sham], 4 Hz, 60 Hz) as between-subject factors. Post hoc testing with a Tukey test examined for differences among groups. The second hypothesis tested whether higher amplitude SCS produced greater analgesia. We used repeated-measures ANOVA to examine difference scores immediately after SCS over the 4 days using amplitude (intensity) (days 1–4) (0 [sham], 50%, 75%, 90% motor threshold) and frequency (0 [sham], 4 Hz, 60 Hz) as between-subject factors. Post hoc testing with a Tukey test examined for differences among groups. The third hypothesis tested whether there was a carryover effect of TENS examining difference scores between values 24 hours, 3, and 9 days after stopping SCS to those before SCS on day 1. Differences with 4 and 60 Hz SCS were compared with differences from sham SCS with a 1-way ANOVA followed by a Tukey test at each time period.
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Post hoc testing between different groups was performed with a Tukey test. The fourth hypothesis examined for differences in the responders and nonresponders. Responders and nonresponders were established based on the mechanical withdrawal threshold response after SCS. Responders were defined as those with an increase in mechanical withdrawal threshold by 2 filaments or more during SCS.19 A Pearson χ2 test compared differences between responders and nonresponders by frequency (60 vs 4 Hz) and duration (30 minutes vs 6 hours). The fifth hypothesis examined whether SCS reduced glial cell activation in the spinal cord. The density of immunostaining was analyzed using a 1-way ANOVA followed by post hoc testing with a Tukey test. Data are presented as the mean ± SEM. A P ≤ 0.05 was considered significant.
RESULTS Spared Nerve Injury Model Induces Hyperalgesia
Before spared nerve injury, baseline mechanical withdrawal threshold averaged 295 ± 18 mN. Two weeks after spared nerve injury, all groups showed a significant decrease in their mechanical withdrawal threshold ipsilaterally, averaging 20 ± 3.2 mN. The contralateral side showed a decrease that averaged 89 ± 4.6 mN, but this was not statistically significant from baseline (Fig. 1).
Longer-Duration SCS Produces Greater Analgesia
After SCS, between-subject comparisons show there was a significant effect for the duration of SCS (F1,31 = 15.3, P = 0.0001) and frequency of SCS (F1,31 = 8.1, P = 0.008). There was no interaction between frequency and duration (F1,31 = 1.6, P = 0.21). Six-hour SCS showed significantly greater analgesia when compared with 30-minute SCS (P = 0.0001) or sham (P = 0.0001); 30-minute SCS was significantly greater than sham SCS (P = 0.0001) (Fig. 2A). SCS produced a significant increase in the mechanical withdrawal threshold ipsilaterally after 60 Hz SCS (P = 0.0001) or 4 Hz SCS (P = 0.0001), when compared with sham SCS. The greatest increase in the mechanical withdrawal threshold occurred with 60 Hz SCS delivered for 6 hours at 90% motor threshold. 1000
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and 2 weeks after spared nerve injury, before and after daily application of SCS for 4 days, and 1 day after stopping SCS. SCS began 2 weeks after spared nerve injury. The following experimental groups were compared: (1) 4 Hz, 50% motor threshold (N =8), (2) 4 Hz, 75% motor threshold (N = 8), (3) 60 Hz, 50% motor threshold (N = 8), (4) 60 Hz, 75% motor threshold (N = 8), (5) Sham, 6-hour SCS (N = 8). These data were compared with the groups from Experiment 1, which received 90% motor threshold intensity (amplitude) for 6 hours. Sham animals were implanted with the neurostimulation hardware and were tethered to the neurostimulator system but did not receive SCS.
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Figure 1. Average withdrawal thresholds of the paw before (baseline) and 2 weeks after spared nerve injury for the ipsilateral and contralateral sides. Black column is naive, and white column is spared nerve injury. Data are mean ± SEM. *P < 0.05.
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Figure 2. A, Time course for changes in paw withdrawal thresholds after spared nerve injury and after spinal cord stimulation (SCS) for up to 14 days. SCS significantly increased the mechanical withdrawal threshold bilaterally when compared with sham SCS. Greater analgesia was observed with 6-hour SCS (60 Hz) compared with 30-minute SCS (60 Hz). The arrow shows the time of SCS treatment. B, Average area under the curve for the changes in withdrawal thresholds during SCS compared with before SCS for all groups averaged over the first 4 days of spared nerve injury. Data are mean difference scores between after SCS compared with before SCS with SEM. *P < 0.05, different from sham group.
There was a significant increase in withdrawal thresholds after stopping SCS 24 and 3 days after SCS that was reversed by 9 days after SCS. This effect was significant for the group that received 6-hour SCS (P = 0.003) but not 30-minute SCS (P = 0.07). Figure 2B shows the area under the curve for the groups on days 1 to 4. On day 1, the number of responders (animals that had an increase in their withdrawal thresholds by 2 filaments or more)19 in the 60 Hz 6-hour SCS group was 100%, compared with 62% in the 60 Hz 30-minute SCS group, 67% in the 4 Hz 6-hour SCS group, and 13% in the 4 Hz 30-minute SCS group. An overall difference was observed among groups (χ2 = 11.3, P = 0.01).After SCS, there were significantly more responders in the groups receiving 60 Hz (79%) compared with those receiving 4 Hz (36%) (χ2 = 5.2, P =0.02). Furthermore, there were more responders in the group receiving 6-hour (83%) SCS when compared with 30-minute SCS (37%) (χ2 = 5.9, P = 0.01).
Greater Intensity (Amplitude) of SCS Produces Greater Analgesia
After SCS, between-subjects comparisons show there was a significant effect for amplitude (intensity) of SCS (F2,45 = 30.0, P = 0.0001) but not frequency of SCS (F1,45 = 2.3, P = 0.14) (mean with 95% confidence intervals: sham −2.9 [−11.2 to 5.3]; 4 Hz 17.3 [12.3–22.3]; 60 Hz 19.7 [14.8–24.7]). However, there was a significant interaction between
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Figure 3. A, Time course for changes in paw withdrawal thresholds after spared nerve injury and after delivery of different intensities of spinal cord stimulation (SCS). SCS significantly increased the mechanical withdrawal threshold bilaterally when compared with sham SCS when delivered at 75% and 90% motor threshold intensity (amplitude). Fifty percent motor threshold intensity (amplitude) had no analgesic effect and was similar to sham. Data are mean (± SEM) difference scores between after SCS and before SCS. B, The area under the curve for the first 4 days after SCS for changes in withdrawal threshold for increasing intensity (amplitude) of SCS compared with sham SCS. MT = motor threshold, *P < 0.05, different from sham group.
frequency and amplitude (F2,45 = 9.7, P = 0.0001), and post hoc tests show that sham SCS is significantly different from 4 and 60 Hz SCS (P = 0.0001). SCS delivered at 90% motor threshold amplitude produced the largest increase in withdrawal thresholds and was significantly different from 75% (P = 0.0001) and 50% (P = 0.0001) motor threshold SCS and sham SCS (P = 0.0001). Seventy-five percent motor threshold SCS significantly increased withdrawal thresholds when compared with sham SCS (P = 0.0001) and 50% motor threshold SCS (P = 0.0001). Fifty percent motor threshold SCS was similar to sham SCS (P =0.91). There was a significant difference when comparing the 3 intensities as follows: 90% motor threshold >75% motor threshold >50% motor threshold = sham SCS (Fig. 3).
SCS Suppressed Spinal Glial Activation in Neuropathic Pain
After SCS, immunohistochemistry was performed to determine whether SCS suppressed glial activation in the spinal dorsal horn in animals with neuropathic pain. Both microglial (OX-42 positive) (Fig. 4A) and astrocytic markers (GFAP positive and MCP-1 positive) (Fig. 5, A and B) were significantly increased bilaterally 2 weeks after spared nerve injury. Four days of SCS (4 or 60 Hz, 6 hours, 90% motor threshold) significantly decreased OX-42, GFAP, and MCP-1
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Figure 4. A, Representative tissue sections for OX-42 immunostaining in the dorsal horn of naive control rats, spared nerve injury rats, 60 Hz SCS, and 4 Hz SCS. Bar = 100 μm. B, There is a significant increase in the density of OX-42 staining bilaterally in the dorsal horn 2 weeks after spared nerve injury. Sixty hertz spinal cord stimulation (SCS) and 4 Hz SCS show significantly less OX-42 immunostaining density. (*) compared with the naive controls, (#) compared with the spared nerve injury, *P ≤ 0.05, data represented by mean ± SEM.
immunoreactivity bilaterally when compared with sham SCS (Figs. 4A, 5, A and B). The density of the microglia marker OX-42 was increased bilaterally in laminae I-II 2 weeks after spared nerve injury when compared with naive controls (ipsilateral P = 0.01; contralateral P = 0.008). Both 60 and 4 Hz SCS significantly decreased OX-42 staining in the superficial laminae bilaterally when compared with sham SCS: 60 Hz (ipsilateral P = 0.0001; contralateral P = 0.001); 4 Hz (ipsilateral P = 0.0001; contralateral P = 0.003). In laminae III-V, the density of immunoreactivity for OX-42 was also increased after spared nerve injury both ipsilaterally (P = 0.001) and contralaterally (P = 0.001) when compared with naive controls. SCS significantly decreased the OX-42 staining in the deep dorsal horn when compared with sham SCS: 60 Hz (ipsilateral P = 0.005; contralateral P = 0.004); 4 Hz group (ipsilateral P = 0.005; contralateral P = 0.004) (Fig. 4B). For astrocyte markers, we examined GFAP and MCP-1. In animals with spared nerve injury, significant increases in the density of immunoreactivity for GFAP and MCP-1 occurred in laminae I-II for both the ipsilateral (GFAP P = 0.0001; MCP-1 P = 0.007) and the contralateral (GFAP P = 0.001; MCP-1 P = 0.03) sides when compared with naive animals. SCS reduced the spared nerve injury-induced increased immunoreactivity for GFAP both ipsilaterally (60 Hz P = 0.0001;
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4 Hz P = 0.0001) and contralaterally (60 Hz P = 0.002; 4 Hz P = 0.0001) in the superficial laminae. Similarly, statistically significant increases in the density of GFAP and MCP-1 in laminae I-II and III-V occurred after spared nerve injury, and these increases were reduced by both 60 and 4 Hz SCS laminae I-II ipsilaterally (GFAP: control vs spared nerve injury P = 0.0001, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.002; MCP-1: control vs spared nerve injury P = 0.007, spared nerve injury vs 60 Hz P = 0.002, spared nerve injury vs 4 Hz P = 0.843) and laminae I-II contralaterally (GFAP: control vs spared nerve injury P = 0.001, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.123; MCP-1: control vs spared nerve injury P = 0.003, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.269) (Fig. 5, C and D).
DISCUSSION
In the present study, we demonstrated that longer-duration SCS (6 hours) produces greater analgesia than shorterduration SCS (30 minutes); that 90% motor threshold intensity (amplitude) is better than 75% motor threshold, and both are better than 50% motor threshold or sham SCS. Furthermore, both 60 and 4 Hz SCS decrease activation of glial cells (microglia and astrocytes) in the spinal cord, suggesting that SCS reduces central excitability.
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Figure 5. A and B. Representative tissue sections for GFAP and MCP-1 immunostaining in the dorsal horn of control rats, spared nerve injury, 60 Hz spinal cord stimulation (SCS), and 4 Hz SCS. Sketches delineating boundaries of different laminae are superimposed over the spinal sections of control rats. Bar =100 μm. C, There is a significant increase in the density of GFAP and MCP-1 staining bilaterally in the dorsal horn 2 weeks after spared nerve injury. Both 60 Hz SCS and 4 Hz SCS show significantly less staining density when compared with animals with spared nerve injury. (*) compared with naive controls, (#) compared with the spared nerve injury, (&) compared with 60 Hz SCS. *P ≤ 0.05, data represented by mean ± SEM.
Effectiveness of SCS Depends on Intensity (Amplitude)
SCS is generally delivered both clinically and in experimental studies below motor threshold. Most of these use intensities (amplitudes) approximately two-thirds motor threshold to produce analgesia.19,30–34 The current study showed an intensity-dependent analgesia with the greatest analgesia at 90% motor threshold and no analgesia at 50% motor threshold.
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Similarly, intensity-dependent reductions in inflammationinduced hyperalgesia or pain thresholds are observed with TENS in both animal and human subjects, respectively.35,36 Higher intensities (amplitudes) of SCS and TENS produce a longer and greater magnitude of analgesia with greater amplitudes,18,24,25,35 and greater intensities (amplitudes) of SCS correlate with longer duration and magnitude of pain relief in rats.18,20,30 Thus, intensity-dependent analgesia occurs
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for both SCS and TENS, suggesting that intensity (amplitude) is a key factor in stimulation-produced analgesia.
SCS Is More Effective with Longer-Duration Stimulation
We showed a small but significantly larger reduction in hypersensitivity with 6 hours of stimulation, particularly with 60 Hz SCS. Previous studies clearly show reductions with stimulation durations as little as 5 minutes with the most of studies showing good reductions in hypersensitivity with 30 minutes to 1 hour of SCS.19,36–42 Clinically, however, the duration of SCS is variable. For example, the duration of SCS has been reported as 6 to 8 sessions per day for 10 to 60 minutes each session and for longer periods between 5 and 12 hours in a single session.43–45 The current study confirms that longer-duration SCS produces greater analgesia than shorter-duration SCS.
SCS Analgesia Can Persist After Stimulation
Previous work in a rat model of neuropathic pain (ligature of the sciatic nerve) shows 10 minutes of SCS attenuated allodynia for up to 40 minutes after cessation of the stimulation.33 SCS also inhibited nociceptive discharges of dorsal horn neurons for approximately 30 to 40 minutes after the cessation of SCS.46,47 The current study extends these previous findings and shows a small yet significant reduction in mechanical hypersensitivity with 60 Hz SCS for up to 3 days after cessation of stimulation. Some previous studies also show that repeated SCS produces a cumulative analgesic effect.19,36,48–50 In humans, how long the pain relief remains after SCS and which factors influence the duration of postSCS pain relief is unknown.43 Some authors suggest that the carryover and cumulative analgesic effect may involve a complex set of plastic changes and remodeling in spinal and supraspinal pain-processing structures. However, in the current and in our previous study, we were unable to show this cumulative analgesic effect with repeated stimulation.43
SCS Reduces Glial Cell Activation in the Spinal Cord
The current study showed for the first time that both 4 and 60 Hz SCS reduced microglia and astrocyte activation in the spinal cord in animals with neuropathic pain. Similarly, other therapeutic treatments that used our endogenous analgesia system also reduced glial activation, including electroacupuncture, peripheral nerve stimulation, and joint mobilization.51–54 SCS increases release of inhibitory neurotransmitters γ-aminobutyric acid, serotonin, acetylcholine, and opioids in the spinal dorsal horn, which could directly inhibit activation of glial cells.16,18,31,41,55,56 In fact, both astrocytes and microglia express inhibitory neurotransmitter receptors including GABAergic, serotonergic, and opioidergic receptors.57–59 Alternatively, inhibitory neurotransmitters could indirectly reduce glial cell activity by reducing neuronal release of excitatory neurotransmitters.60 It is well known that neuronal release of excitatory neurotransmitters such as glutamate are increased in response to nociceptive stimuli and that excitatory neurotransmitters can directly activate glial cells to perpetuate the nociceptive response.61–64 Therefore, further studies
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examining the underlying mechanisms of the reduced glial cell activity are required. Glial cells have been implicated in producing hypersensitivity after nerve injury with both spinal microglia and astrocytes activated after nerve injury.29,65–70 In the current study, we showed activation of both microglia and astrocytes after nerve injury with an early increase in phosphorylation of mitogen activated kinase p38, similar to previous studies.29,71,72 However, our results showed that another microglial surface marker, CD11b (OX-42), continued to show enhanced immunoreactivity and that microglial cells appeared to remain activated 10 days to 2 weeks after nerve injury.73,74 The activation of astrocytes in the later phases also agrees with previous studies implicating these cells in the maintenance of mechanical allodynia in neuropathic pain.75,76
CONCLUSION
In the present study, we examined stimulation variables and showed that the most effective analgesia was produced by 60 Hz SCS for 6-hour duration at 90% motor threshold. We further showed that SCS, both 4 and 60 Hz, reduces glial cell activation in the dorsal horn of the spinal cord. We suggest that SCS has the ability to modulate nociceptive input at the spinal cord using multiple inhibitory neurotransmitters that subsequently reduce glial cell activation. E DISCLOSURES
Name: Karina L. Sato, PT, PhD. Contribution: This author helped design and conduct the study, collect data, analyze data, and prepare the manuscript. Attestation: Karina Sato attests to having approved the final manuscript and reviewed the original study data and data analysis. Karina Sato attests to the integrity of the original data and the analysis reported in this manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author has no conflicts of interest to declare. Name: Lisa M. Johanek, PhD. Contribution: This author helped design the study and prepare the manuscript. Attestation: Lisa Johanek attests to having approved the final manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author is an employee of Medtronic, Inc. Name: Luciana S. Sanada, PT. Contribution: This author helped design and conduct the study, collect data, and prepare the manuscript. Attestation: Luciana Sanada attests to having approved the final manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author has no conflicts of interest to declare. Name: Kathleen A. Sluka, PhD, PT. Contribution: This author helped design and conduct the study, analyze data, and prepare the manuscript. Attestation: Kathleen Sluka attests to having approved the final manuscript and reviewed the original study data and data analysis. Kathleen Sluka attests to the integrity of the original data and the analysis reported in this manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: Dr. Sluka receives research funding for this study from Medtronic, Inc. This manuscript was handled by: Jianren Mao, MD, PhD.
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REFERENCES 1. Campbell JN, Meyer RA. Mechanisms of neuropathic pain. Neuron 2006;52:77–92 2. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355–63 3. Meller ST, Dykstra C, Grzybycki D, Murphy S, Gebhart GF. The possible role of glia in nociceptive processing and hyperalgesia in the spinal cord of the rat. Neuropharmacology 1994;33:1471–8 4. Watkins LR, Martin D, Ulrich P, Tracey KJ, Maier SF. Evidence for the involvement of spinal cord glia in subcutaneous formalin induced hyperalgesia in the rat. Pain 1997;71:225–35 5. Watkins LR, Milligan ED, Maier SF. Glial activation: a driving force for pathological pain. Trends Neurosci 2001;24:450–5 6. Watkins LR, Milligan ED, Maier SF. Spinal cord glia: new players in pain. Pain 2001;93:201–5 7. DeLeo JA, Yezierski RP. The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 2001;90:1–6 8. Obata H, Sakurazawa S, Kimura M, Saito S. Activation of astrocytes in the spinal cord contributes to the development of bilateral allodynia after peripheral nerve injury in rats. Brain Res 2010;1363:72–80 9. Okubo M, Yamanaka H, Kobayashi K, Kanda H, Dai Y, Noguchi K. Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury. Mol Pain 2012;8:8 10. Giordano C, Siniscalco D, Melisi D, Luongo L, Curcio A, Soukupova M, Palazzo E, Marabese I, De Chiaro M, Rimoli MG, Rossi F, Maione S, de Novellis V. The galactosylation of N(ω)-nitro-L-arginine enhances its anti-nocifensive or anti-allodynic effects by targeting glia in healthy and neuropathic mice. Eur J Pharmacol 2011;656:52–62 11. Sweitzer SM, Schubert P, DeLeo JA. Propentofylline, a glial modulating agent, exhibits antiallodynic properties in a rat model of neuropathic pain. J Pharmacol Exp Ther 2001;297:1210–7 12. Mika J, Osikowicz M, Rojewska E, Korostynski M, WawrzczakBargiela A, Przewlocki R, Przewlocka B. Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain. Eur J Pharmacol 2009;623:65–72 13. Tawfik VL, Nutile-McMenemy N, Lacroix-Fralish ML, Deleo JA. Efficacy of propentofylline, a glial modulating agent, on existing mechanical allodynia following peripheral nerve injury. Brain Behav Immun 2007;21:238–46 14. Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, Decosterd I, Ji RR. A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. J Neurosci 2006;26:3551–60 15. Jeon Y, Huh BK. Spinal cord stimulation for chronic pain. Ann Acad Med Singapore 2009;38:998–1003 16. Cui JG, O’Connor WT, Ungerstedt U, Linderoth B, Meyerson BA. Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via a GABAergic mechanism. Pain 1997;73:87–95 17. Li D, Yang H, Meyerson BA, Linderoth B. Response to spinal cord stimulation in variants of the spared nerve injury pain model. Neurosci Lett 2006;400:115–20 18. Meyerson BA, Ren B, Herregodts P, Linderoth B. Spinal cord stimulation in animal models of mononeuropathy: effects on the withdrawal response and the flexor reflex. Pain 1995;61: 229–43 19. Maeda Y, Wacnik PW, Sluka KA. Low frequencies, but not high frequencies of bi-polar spinal cord stimulation reduce cutaneous and muscle hyperalgesia induced by nerve injury. Pain 2008;138:143–52 20. Sato KL, King EW, Johanek LM, Sluka KA. Spinal cord stimulation reduces hypersensitivity through activation of opioid receptors in a frequency-dependent manner. Eur J Pain 2013;17:551–61 21. Le K, Yang HY, Jiang J, Chen H. [Comparison of analgesic effects of electroacupuncture of multi-factor quantitative parameters on inflammatory pain in rats]. Zhongguo Zhen Jiu 2008;28:829–32
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22. Taguchi T, Taguchi R. Effect of varying frequency and duration of electroacupuncture stimulation on carrageenan-induced hyperalgesia. Acupunct Med 2007;25:80–6 23. Gobbo M, Gaffurini P, Bissolotti L, Esposito F, Orizio C. Transcutaneous neuromuscular electrical stimulation: influence of electrode positioning and stimulus amplitude settings on muscle response. Eur J Appl Physiol 2011;111:2451–9 24. Sato KL, Sanada LS, Rakel BA, Sluka KA. Increasing intensity of TENS prevents analgesic tolerance in rats. J Pain 2012;13:884–90 25. Rakel B, Cooper N, Adams HJ, Messer BR, Frey Law LA, Dannen DR, Miller CA, Polehna AC, Ruggle RC, Vance CG, Walsh DM, Sluka KA. A new transient sham TENS device allows for investigator blinding while delivering a true placebo treatment. J Pain 2010;11:230–8 26. Decosterd I, Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000;87:149–58 27. Guen S Le, Catheline G, Besson JM. Development of tolerance to the antinociceptive effect of systemic morphine at the lumbar spinal cord level: a c-Fos study in the rat. Brain Res 1998;813:128–38. 28. Wei LC, Shi M, Chen LW, Cao R, Zhang P, Chan YS. Nestincontaining cells express glial fibrillary acidic protein in the proliferative regions of central nervous system of postnatal developing and adult mice. Brain Res Dev Brain Res 2002;139:9–17 29. Hoeger-Bement MK, Sluka KA. Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections. J Neurosci 2003;23:5437–45 30. Meyerson BA, Linderoth B. Mode of action of spinal cord stimulation in neuropathic pain. J Pain Symptom Manage 2006;31:S6–12 31. Song Z, Ultenius C, Meyerson BA, Linderoth B. Pain relief by spinal cord stimulation involves serotonergic mechanisms: an experimental study in a rat model of mononeuropathy. Pain 2009;147:241–8 32. Truin M, Janssen SP, van Kleef M, Joosten EA. Successful pain relief in non-responders to spinal cord stimulation: the combined use of ketamine and spinal cord stimulation. Eur J Pain 2011;15:1049.e1–9 33. Yakhnitsa V, Linderoth B, Meyerson BA. Spinal cord stimulation attenuates dorsal horn neuronal hyperexcitability in a rat model of mononeuropathy. Pain 1999;79:223–33 34. Linderoth B, Gherardini G, Ren B, Lundeberg T. Preemptive spinal cord stimulation reduces ischemia in an animal model of vasospasm. Neurosurgery 1995;37:266–71 35. Moran F, Leonard T, Hawthorne S, Hughes CM, McCrum Gardner E, Johnson MI, Rakel BA, Sluka KA, Walsh DM. Hypoalgesia in response to transcutaneous electrical nerve stimulation (TENS) depends on stimulation intensity. J Pain 2011;12:929–35 36. Yang F, Carteret AF, Wacnik PW, Chung CY, Xing L, Dong X, Meyer RA, Raja SN, Guan Y. Bipolar spinal cord stimulation attenuates mechanical hypersensitivity at an intensity that activates a small portion of A-fiber afferents in spinal nerve-injured rats. Neuroscience 2011;199:470–80 37. Gherardini G, Lundeberg T, Cui JG, Eriksson SV, Trubek S, Linderoth B. Spinal cord stimulation improves survival in ischemic skin flaps: an experimental study of the possible mediation by calcitonin gene-related peptide. Plast Reconstr Surg 1999;103:1221–8 38. Qiu Y, Li T, Li H, Zuo Y. Continuous spinal cord stimulation reduced cardiac ischaemia/reperfusion injury in a rat model. Heart Lung Circ 2012;21:564–71 39. Barchini J, Tchachaghian S, Shamaa F, Jabbur SJ, Meyerson BA, Song Z, Linderoth B, Saadé NE. Spinal segmental and supraspinal mechanisms underlying the pain-relieving effects of spinal cord stimulation: an experimental study in a rat model of neuropathy. Neuroscience 2012;215:196–208 40. Smits H, van Kleef M, Holsheimer J, Joosten EA. Experimental spinal cord stimulation and neuropathic pain: mechanism of action, technical aspects, and effectiveness. Pain Pract 2013;13:154–68
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41. Song Z, Meyerson BA, Linderoth B. Spinal 5-HT receptors that contribute to the pain-relieving effects of spinal cord stimulation in a rat model of neuropathy. Pain 2011;152:1666–73 42. Guan Y, Wacnik PW, Yang F, Carteret AF, Chung CY, Meyer RA, Raja SN. Spinal cord stimulation-induced analgesia: electrical stimulation of dorsal column and dorsal roots attenuates dorsal horn neuronal excitability in neuropathic rats. Anesthesiology 2010;113:1392–405 43. Wolter T, Kieselbach K. Spinal cord stimulation for Raynaud’s syndrome: long-term alleviation of bilateral pain with a single cervical lead. Neuromodulation 2011;14:229–33 44. Koeze TH, Williams AC, Reiman S. Spinal cord stimulation and the relief of chronic pain. J Neurol Neurosurg Psychiatry 1987;50:1424–9 45. Daousi C, Benbow SJ, MacFarlane IA. Electrical spinal cord stimulation in the long-term treatment of chronic painful diabetic neuropathy. Diabet Med 2005;22:393–8 46. Lindblom U, Tapper DN, Wiesenfeld Z. The effect of dorsal column stimulation on the nociceptive response of dorsal horn cells and its relevance for pain suppression. Pain 1977;4:133–44 47. Rees H, Roberts MH. Antinociceptive effects of dorsal column stimulation in the rat: involvement of the anterior pretectal nucleus. J Physiol 1989;417:375–88 48. Stiller CO, Linderoth B, O’Connor WT, Franck J, Falkenberg T, Ungerstedt U, Brodin E. Repeated spinal cord stimulation decreases the extracellular level of gamma-aminobutyric acid in the periaqueductal gray matter of freely moving rats. Brain Res 1995;699:231–41 49. Ren B, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on the flexor reflex and involvement of supraspinal mechanisms: an experimental study in mononeuropathic rats. J Neurosurg 1996;84:244–9 50. El-Khoury C, Hawwa N, Baliki M, Atweh SF, Jabbur SJ, Saadé NE. Attenuation of neuropathic pain by segmental and supraspinal activation of the dorsal column system in awake rats. Neuroscience 2002;112:541–53 51. Gim GT, Lee JH, Park E, Sung YH, Kim CJ, Hwang WW, Chu JP, Min BI. Electroacupuncture attenuates mechanical and warm allodynia through suppression of spinal glial activation in a rat model of neuropathic pain. Brain Res Bull 2011;86:403–11 52. Sun S, Cao H, Han M, Li TT, Zhao ZQ, Zhang YQ. Evidence for suppression of electroacupuncture on spinal glial activation and behavioral hypersensitivity in a rat model of monoarthritis. Brain Res Bull 2008;75:83–93 53. Martins DF, Mazzardo-Martins L, Gadotti VM, Nascimento FP, Lima DA, Speckhann B, Favretto GA, Bobinski F, CargninFerreira E, Bressan E, Dutra RC, Calixto JB, Santos AR. Ankle joint mobilization reduces axonotmesis-induced neuropathic pain and glial activation in the spinal cord and enhances nerve regeneration in rats. Pain 2011;152:2653–61 54. Fu H, Bartz JD, Stephens RL Jr, McCarty DM. Peripheral nervous system neuropathology and progressive sensory impairments in a mouse model of Mucopolysaccharidosis IIIB. PLoS One 2012;7:e45992 55. Cui JG, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on touch-evoked allodynia involve GABAergic mechanisms. An experimental study in the mononeuropathic rat. Pain 1996;66:287–95 56. Stiller CO, Cui JG, O’Connor WT, Brodin E, Meyerson BA, Linderoth B. Release of gamma-aminobutyric acid in the dorsal horn and suppression of tactile allodynia by spinal cord stimulation in mononeuropathic rats. Neurosurgery 1996;39:367–74 57. Raghavendra V, Rutkowski MD, DeLeo JA. The role of spinal neuroimmune activation in morphine tolerance/hyperalgesia in neuropathic and sham-operated rats. J Neurosci 2002;22:9980–9 58. Song P, Zhao ZQ. The involvement of glial cells in the development of morphine tolerance. Neurosci Res 2001;39:281–6
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59. Mika J, Osikowicz M, Makuch W, Przewlocka B. Minocycline and pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain. Eur J Pharmacol 2007;560:142–9 60. Chen T, Willoughby KA, Ellis EF. Group I metabotropic receptor antagonism blocks depletion of calcium stores and reduces potentiated capacitative calcium entry in strain-injured neurons and astrocytes. J Neurotrauma 2004;21:271–81 61. Li W, Neugebauer V. Block of NMDA and non-NMDA receptor activation results in reduced background and evoked activity of central amygdala neurons in a model of arthritic pain. Pain 2004;110:112–22 62. You HJ, Mørch CD, Arendt-Nielsen L. Electrophysiological characterization of facilitated spinal withdrawal reflex to repetitive electrical stimuli and its modulation by central glutamate receptor in spinal anesthetized rats. Brain Res 2004;1009:110–9 63. Casamenti F, Prosperi C, Scali C, Giovannelli L, Colivicchi MA, Faussone-Pellegrini MS, Pepeu G. Interleukin-1beta activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer’s disease. Neuroscience 1999;91:831–42 64. Chen T, Koga K, Li XY, Zhuo M. Spinal microglial motility is independent of neuronal activity and plasticity in adult mice. Mol Pain 2010;6:19 65. Scholz J, Woolf CJ. The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 2007;10:1361–8 66. Ren K, Dubner R. Neuron-glia crosstalk gets serious: role in pain hypersensitivity. Curr Opin Anaesthesiol 2008;21:570–9 67. Milligan ED, Watkins LR. Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 2009;10:23–36 68. Piao ZG, Cho IH, Park CK, Hong JP, Choi SY, Lee SJ, Lee S, Park K, Kim JS, Oh SB. Activation of glia and microglial p38 MAPK in medullary dorsal horn contributes to tactile hypersensitivity following trigeminal sensory nerve injury. Pain 2006;121:219–31 69. Terayama R, Omura S, Fujisawa N, Yamaai T, Ichikawa H, Sugimoto T. Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury. Neuroscience 2008;153:1245–55 70. Tsuda M, Mizokoshi A, Shigemoto-Mogami Y, Koizumi S, Inoue K. Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 2004;45:89–95 71. Wen YR, Suter MR, Kawasaki Y, Huang J, Pertin M, Kohno T, Berde CB, Decosterd I, Ji RR. Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model. Anesthesiology 2007;107:312–21 72. Colburn RW, DeLeo JA, Rickman AJ, Yeager MP, Kwon P, Hickey WF. Dissociation of microglial activation and neuropathic pain behaviors following peripheral nerve injury in the rat. J Neuroimmunol 1997;79:163–75 73. Winkelstein BA, DeLeo JA. Nerve root injury severity differentially modulates spinal glial activation in a rat lumbar radiculopathy model: considerations for persistent pain. Brain Res 2002;956:294–301 74. Ji RR, Kawasaki Y, Zhuang ZY, Wen YR, Decosterd I. Possible role of spinal astrocytes in maintaining chronic pain sensitization: review of current evidence with focus on bFGF/JNK pathway. Neuron Glia Biol 2006;2:259–69 75. Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS, Lim S. Acupuncture inhibits microglial activation and inflammatory events in the MPTP-induced mouse model. Brain Res 2007;1131:211–9 76. Svensson CI, Brodin E. Spinal astrocytes in pain process ing: non-neuronal cells as therapeutic targets. Mol Interv 2010;10:25–38
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Spinal Cord Stimulation Reduces Mechanical Hyperalgesia and Glial Cell Activation in Animals with Neuropathic Pain Karina L. Sato, PT, PhD,* Lisa M. Johanek, PhD,† Luciana S. Sanada, PT,* and Kathleen A. Sluka, PhD, PT* BACKGROUND: Spinal cord stimulation (SCS) is commonly used for neuropathic pain; the optimal variables and mechanisms of action are unclear. We tested whether modulation of SCS variables improved analgesia in animals with neuropathic pain by comparing 6-hour vs 30-minute duration and 50%, 75%, or 90% motor threshold (MT) intensity (amplitude). Furthermore, we examined whether maximally effective SCS reduced glial activation in the spinal cord in neuropathic animals. METHODS: Sprague-Dawley rats received the spared nerve injury model and were implanted with an epidural SCS lead. Animals were tested for mechanical withdrawal threshold of the paw before and 2 weeks after spared nerve injury, before and after SCS daily for 4 days, and 1, 4, and 9 days after SCS. Spinal cords were examined for the effects of SCS on glial cell activation. RESULTS: The mechanical withdrawal threshold decreased, and glial immunoreactivity increased 2 weeks after spared nerve injury. For duration, 6-hour SCS significantly increased the mechanical withdrawal threshold when compared with 30-minute SCS or sham SCS; 30-minute SCS was greater than sham SCS. For intensity (amplitude), 90% MT SCS significantly increased the withdrawal threshold when compared with 75% MT SCS, 50% MT SCS, and sham SCS. Both 4 and 60 Hz SCS decreased glial activation (GFAP, MCP-1, and OX-42) in the spinal cord dorsal horn when compared with sham. CONCLUSIONS: Six-hour duration SCS with 90% MT showed the largest increase in mechanical withdrawal threshold, suggesting that the variables of stimulation are important for clinical effectiveness. One potential mechanism for SCS may be to reduce glial activation at the level of the spinal cord. (Anesth Analg 2014;118:464–72)
N
europathic pain is defined as pain resulting from damage or dysfunction of peripheral nerves and more broadly, as a result of injury or disease of the somatosensory system. There are a number of diseases associated with neuropathic pain. Examples include autoimmune diseases (e.g., multiple sclerosis), metabolic diseases (e.g., diabetic neuropathy), infection (e.g., shingles and the sequel, postherpetic neuralgia), vascular diseases (stroke), trauma, and cancer. Neuropathic pain in experimental animals can be induced using multiple models that injure peripheral nerves: partial sciatic ligation, chronic constrictive injury, spinal nerve ligation, and spared nerve injury.1,2 Specific mechanisms for the pain associated with peripheral nerve injury are complex and involve changes at the site of injury and in the central nervous system. Pain hypersensitivity was originally thought to result exclusively from From the *Department of Physical Therapy, University of Iowa, Iowa City, Iowa; and †Medtronic, Minneapolis, Minnesota. Accepted for publication November 11, 2013. Funding: Funded by Medtronic, Inc. and NIH AR052316 (KAS). Conflict of Interest: See Disclosures at the end of the article. Reprints will not be available from the authors. Address correspondence to Kathleen A. Sluka, PhD, PT, Department of Physical Therapy, University of Iowa Carver College of Medicine, 500 Newton Rd., 1–248 Medical Education Building, Iowa City, IA 52242. Address e-mail to [email protected]. Copyright © 2013 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000047
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altered neuronal activity in primary sensory and spinal cord neurons. It is now clear that glial cells also play a significant role in the pathogenesis of neuropathic pain.3–7 In fact, there is a time-dependent activation of both astrocytes and microglia after spared nerve injury in the spinal cord.8–10 Furthermore, inhibition of glial activation reduces mechanical allodynia in animals with nerve injury.11–14 Thus, nerve injury is associated with activation of glia that can alter mechanical allodynia in animals. Spinal cord stimulation (SCS) has been successful in providing analgesia, improving function, and enhancing the quality of life for patients suffering from chronic pain.15 In animals with nerve injury, short-duration (10–60 minutes) SCS at high frequencies (50–60 Hz) reduces mechanical allodynia that outlasts the stimulation time.16–19 Our previous work also shows that 4 Hz SCS (30 minutes) reduces mechanical allodynia.19 Furthermore, high-frequency SCS (60 Hz) activates δ-opioid receptors spinally, and low-frequency SCS (4 Hz) activates μ-opioid receptors spinally, showing that different frequencies of stimulation use different mechanisms.20 Clinically, SCS is typically applied at higher frequencies (i.e., 60 Hz), two-thirds motor threshold intensity (amplitude), and for longer durations. Previous studies using peripheral stimulation techniques, such as transcutaneous electrical nerve stimulation (TENS) and electroacupuncture, show that modulation of stimulation parameters can affect outcomes.21–25 Therefore, we tested different parameters of SCS: duration (30 minutes vs 6 hours); February 2014 • Volume 118 • Number 2
and intensity (amplitude) (50%, 75%, 90% motor threshold). We further examined whether the most effective parameters of SCS would reduce glial cell activation in the spinal cord as a potential underlying mechanism.
METHODS Experimental Procedures
Experiments were performed on adult Sprague-Dawley rats, weighing 250 to 350 g and housed in transparent plastic cages with free access to food and water, in a 12-hour light–dark cycle. All the experimental procedures were approved by the Animal Care and Use Committee at the University of Iowa.
Nerve Injury Model
All rats were anesthetized with 2% to 3% isoflurane. The tibial and common peroneal nerves on 1 limb were tightly ligated with 4-0 silk, and the sural nerve was kept intact, as previously described.26 The overlying muscle was sutured with 4-0 silk, and the skin was sutured closed with 3-0 silk.
Implantation of Electrode
After nerve injury, a small laminectomy was performed at the level of T13 vertebra, and the SCS lead was inserted epidurally in the rostral direction. The lead was fixed with sutures to the muscle, the wound was sutured in layers, and the lead was tunneled to exit the skin at the base of the neck.19 We implanted a spinal cord lead designed for use in rats (Medtronic, Minneapolis, MN) that is similar to that used in humans. The proximal end of the lead was tunneled outside the animal for later connection to an external neurostimulator (model # 37021) and programmer (model: #8840) (Medtronic Inc., Minneapolis, MN).
Behavior Tests
Rats were acclimated to the behavior room for 30 minutes followed by acclimation to transparent plastic cubicles on elevated wire mesh floor for 15 minutes. To test for mechanical withdrawal thresholds of the paw, calibrated von Frey filaments (1–402 mN) were applied to the lateral surface bilaterally in the area innervated by the sural nerve. Each filament was applied for approximately 1 second with enough force to bend the filament. Each filament was applied twice, and a positive response was 1 withdrawal. Once a positive response was found, the filament above and below the filament that caused a positive response was tested. Confirmation of mechanical withdrawal threshold was established if there was a positive withdrawal from the filament above and no withdrawal from the filament below. The lowest force that produced a withdrawal was recorded as the mechanical withdrawal threshold. A decrease in mechanical withdrawal threshold is interpreted as cutaneous allodynia in this study.
Immunohistochemistry
We tested for glial cell activation in the spinal cord using immunohistochemistry of astrocyte and microglial markers. Rats were anesthetized with 100 mg/kg sodium pentobarbital and transcardially perfused with heparinized saline (10 U/mL) followed by 4% paraformaldehyde with
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15% picric acid. The spinal cords were removed, post fixed in 4% paraformaldehyde for 1 hour, transferred to 30% sucrose solution for 24 hours, and then frozen on dry ice with Tissue Tek OCT (Sakura Finetek, Torrance, CA). Twenty-micrometer cryostat sections were stained onto slides, and all sections in a group were stained simultaneously. All sections were first blocked with 3% normal goat serum for 30 minutes, followed by Avidin-Biotin block (15 minutes/blocker). For sections used for astrocyte staining, sections were incubated overnight with monoclonal antimouse anti-GFAP (Millipore, Billerica, MA, 1:5000, Cat.#MAB360). On the second day, sections were incubated with biotinylated goat antimouse IgG (Invitrogen, Carlsbad, CA, 1:1000, 1 hour), followed by Strep-568 (Invitrogen, Carlsbad, CA, 1:1000, 1 hour). Sections were then incubated overnight in goat antirabbit MCP-1 (Millipore, Billerica, MA, 1:500, Cat.#1834P). On the last day, sections were incubated in biotinylated goat antirabbit IgG (Invitrogen, Carlsbad, CA, 1:1000, 1 hour), followed by Strep-488 (Invitrogen, 1:1000, 1 hour). Slides were covered with Vectashield. For microglia staining, we used immunohistochemistry staining for OX-42, changing the first primary antibody to OX-42 (AbD Serotec, Raleigh, NC, 1:2500, Cat.#MCA275G). Five spinal cord sections (L4-L5) were randomly chosen from each rat and imaged with an Olympus BX-51 fluorescence microscope (Olympus, Center Valley, PA). Images were analyzed for the density of the staining with Image J (National Institutes of Health, Bethesda, MD version 1.24) as we previously described.27,28 Specifically, each tissue section was first converted to 8-bit gray scale and then calibrated independently using the “uncalibrated OD” function with pixel values ranging from 0 to 255. A background reading taken from the white matter of the dorsal column in the same section was subtracted from the density reading taken from the gray matter of the same tissue section to control for differences in nonspecific staining.29 The superficial laminas (I-II) and intermediate and deep dorsal horn (III–VI) were analyzed separately.
Experimental Protocol Experiment 1 Experiment 1 examined different durations of SCS either 30 minutes or 6 hours. SCS was applied at 4 or 60 Hz frequency and 90% motor threshold intensity (amplitude) (0.35 V) for 4 days. Mechanical withdrawal thresholds were tested before and 2 weeks after spared nerve injury, before and after daily application of SCS for 4 days, and 1, 3, and 9 days after stopping SCS. SCS was applied 2 weeks after spared nerve injury. The following experimental groups were compared: (1) 4 Hz, 30-minute SCS (N = 8), (2) 4 Hz, 6-hour SCS (N = 6), (3) 60 Hz, 30-minute SCS (N =8), (4) 60 Hz, 6-hour SCS (N =6), (5) Sham, 30-minute SCS (N =4), and (6) Sham and 6-hour SCS (N =6). Sham animals were implanted with the neurostimulation hardware and were tethered to the neurostimulator system but did not receive SCS. Experiment 2 Experiment 2 tested different intensities (amplitude) of SCS: 50%, 75%, or 90% motor threshold. SCS was applied at either 4 or 60 Hz frequency, for 6-hour duration daily for 4 days. Mechanical withdrawal thresholds were tested before
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Experiment 3 Experiment 3 tested the effects of SCS on glial cell activation in the spinal cord using immunohistochemistry of astrocyte and microglial markers. SCS was delivered for 4 days beginning 2 weeks after spared nerve injury as follows: 90% motor threshold intensity (amplitude), 6-hour duration, either 4 or 60 Hz frequency. Animals were divided as follows: (1) naive control for OX-42, or GFAP and MCP-1 (N = 5/marker), (2) 60 Hz SCS for OX-42, or GFAP and MCP-1 (N = 5/marker), (3) 4 Hz SCS for OX-42, or GFAP, and MCP-1 (N = 5/marker).
Statistical Analysis
Analysis of the data was performed using SPSS 13.1 (Statistical Package for the Social Sciences, IBM, Armonk, NY). The effect of nerve injury on withdrawal thresholds was tested with a simple paired t test and is in paragraph 1 of the results. Withdrawal threshold data were normalized using a difference score between thresholds obtained after SCS compared with those before SCS that therefore led to 1 number for each day of treatment (days 1–4). Repeatedmeasures analysis of variance (ANOVA) assessed for differences across time for days 1 to 4 with a post hoc Tukey test to examine for differences among groups. Data were also transformed to an area under the curve for the first 4 days of measures after SCS and analyzed with a 1-way ANOVA with a post hoc Tukey test to examine for differences among groups. The first hypothesis examined whether longer-duration SCS was more effective than shorter-duration SCS. We used repeated-measures ANOVA to examine difference scores immediately after SCS over the 4 days (days 1–4) using duration (0 [sham], 30 minutes, 6 hours) and frequency (0 [sham], 4 Hz, 60 Hz) as between-subject factors. Post hoc testing with a Tukey test examined for differences among groups. The second hypothesis tested whether higher amplitude SCS produced greater analgesia. We used repeated-measures ANOVA to examine difference scores immediately after SCS over the 4 days using amplitude (intensity) (days 1–4) (0 [sham], 50%, 75%, 90% motor threshold) and frequency (0 [sham], 4 Hz, 60 Hz) as between-subject factors. Post hoc testing with a Tukey test examined for differences among groups. The third hypothesis tested whether there was a carryover effect of TENS examining difference scores between values 24 hours, 3, and 9 days after stopping SCS to those before SCS on day 1. Differences with 4 and 60 Hz SCS were compared with differences from sham SCS with a 1-way ANOVA followed by a Tukey test at each time period.
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Post hoc testing between different groups was performed with a Tukey test. The fourth hypothesis examined for differences in the responders and nonresponders. Responders and nonresponders were established based on the mechanical withdrawal threshold response after SCS. Responders were defined as those with an increase in mechanical withdrawal threshold by 2 filaments or more during SCS.19 A Pearson χ2 test compared differences between responders and nonresponders by frequency (60 vs 4 Hz) and duration (30 minutes vs 6 hours). The fifth hypothesis examined whether SCS reduced glial cell activation in the spinal cord. The density of immunostaining was analyzed using a 1-way ANOVA followed by post hoc testing with a Tukey test. Data are presented as the mean ± SEM. A P ≤ 0.05 was considered significant.
RESULTS Spared Nerve Injury Model Induces Hyperalgesia
Before spared nerve injury, baseline mechanical withdrawal threshold averaged 295 ± 18 mN. Two weeks after spared nerve injury, all groups showed a significant decrease in their mechanical withdrawal threshold ipsilaterally, averaging 20 ± 3.2 mN. The contralateral side showed a decrease that averaged 89 ± 4.6 mN, but this was not statistically significant from baseline (Fig. 1).
Longer-Duration SCS Produces Greater Analgesia
After SCS, between-subject comparisons show there was a significant effect for the duration of SCS (F1,31 = 15.3, P = 0.0001) and frequency of SCS (F1,31 = 8.1, P = 0.008). There was no interaction between frequency and duration (F1,31 = 1.6, P = 0.21). Six-hour SCS showed significantly greater analgesia when compared with 30-minute SCS (P = 0.0001) or sham (P = 0.0001); 30-minute SCS was significantly greater than sham SCS (P = 0.0001) (Fig. 2A). SCS produced a significant increase in the mechanical withdrawal threshold ipsilaterally after 60 Hz SCS (P = 0.0001) or 4 Hz SCS (P = 0.0001), when compared with sham SCS. The greatest increase in the mechanical withdrawal threshold occurred with 60 Hz SCS delivered for 6 hours at 90% motor threshold. 1000
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and 2 weeks after spared nerve injury, before and after daily application of SCS for 4 days, and 1 day after stopping SCS. SCS began 2 weeks after spared nerve injury. The following experimental groups were compared: (1) 4 Hz, 50% motor threshold (N =8), (2) 4 Hz, 75% motor threshold (N = 8), (3) 60 Hz, 50% motor threshold (N = 8), (4) 60 Hz, 75% motor threshold (N = 8), (5) Sham, 6-hour SCS (N = 8). These data were compared with the groups from Experiment 1, which received 90% motor threshold intensity (amplitude) for 6 hours. Sham animals were implanted with the neurostimulation hardware and were tethered to the neurostimulator system but did not receive SCS.
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Figure 1. Average withdrawal thresholds of the paw before (baseline) and 2 weeks after spared nerve injury for the ipsilateral and contralateral sides. Black column is naive, and white column is spared nerve injury. Data are mean ± SEM. *P < 0.05.
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Figure 2. A, Time course for changes in paw withdrawal thresholds after spared nerve injury and after spinal cord stimulation (SCS) for up to 14 days. SCS significantly increased the mechanical withdrawal threshold bilaterally when compared with sham SCS. Greater analgesia was observed with 6-hour SCS (60 Hz) compared with 30-minute SCS (60 Hz). The arrow shows the time of SCS treatment. B, Average area under the curve for the changes in withdrawal thresholds during SCS compared with before SCS for all groups averaged over the first 4 days of spared nerve injury. Data are mean difference scores between after SCS compared with before SCS with SEM. *P < 0.05, different from sham group.
There was a significant increase in withdrawal thresholds after stopping SCS 24 and 3 days after SCS that was reversed by 9 days after SCS. This effect was significant for the group that received 6-hour SCS (P = 0.003) but not 30-minute SCS (P = 0.07). Figure 2B shows the area under the curve for the groups on days 1 to 4. On day 1, the number of responders (animals that had an increase in their withdrawal thresholds by 2 filaments or more)19 in the 60 Hz 6-hour SCS group was 100%, compared with 62% in the 60 Hz 30-minute SCS group, 67% in the 4 Hz 6-hour SCS group, and 13% in the 4 Hz 30-minute SCS group. An overall difference was observed among groups (χ2 = 11.3, P = 0.01).After SCS, there were significantly more responders in the groups receiving 60 Hz (79%) compared with those receiving 4 Hz (36%) (χ2 = 5.2, P =0.02). Furthermore, there were more responders in the group receiving 6-hour (83%) SCS when compared with 30-minute SCS (37%) (χ2 = 5.9, P = 0.01).
Greater Intensity (Amplitude) of SCS Produces Greater Analgesia
After SCS, between-subjects comparisons show there was a significant effect for amplitude (intensity) of SCS (F2,45 = 30.0, P = 0.0001) but not frequency of SCS (F1,45 = 2.3, P = 0.14) (mean with 95% confidence intervals: sham −2.9 [−11.2 to 5.3]; 4 Hz 17.3 [12.3–22.3]; 60 Hz 19.7 [14.8–24.7]). However, there was a significant interaction between
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Figure 3. A, Time course for changes in paw withdrawal thresholds after spared nerve injury and after delivery of different intensities of spinal cord stimulation (SCS). SCS significantly increased the mechanical withdrawal threshold bilaterally when compared with sham SCS when delivered at 75% and 90% motor threshold intensity (amplitude). Fifty percent motor threshold intensity (amplitude) had no analgesic effect and was similar to sham. Data are mean (± SEM) difference scores between after SCS and before SCS. B, The area under the curve for the first 4 days after SCS for changes in withdrawal threshold for increasing intensity (amplitude) of SCS compared with sham SCS. MT = motor threshold, *P < 0.05, different from sham group.
frequency and amplitude (F2,45 = 9.7, P = 0.0001), and post hoc tests show that sham SCS is significantly different from 4 and 60 Hz SCS (P = 0.0001). SCS delivered at 90% motor threshold amplitude produced the largest increase in withdrawal thresholds and was significantly different from 75% (P = 0.0001) and 50% (P = 0.0001) motor threshold SCS and sham SCS (P = 0.0001). Seventy-five percent motor threshold SCS significantly increased withdrawal thresholds when compared with sham SCS (P = 0.0001) and 50% motor threshold SCS (P = 0.0001). Fifty percent motor threshold SCS was similar to sham SCS (P =0.91). There was a significant difference when comparing the 3 intensities as follows: 90% motor threshold >75% motor threshold >50% motor threshold = sham SCS (Fig. 3).
SCS Suppressed Spinal Glial Activation in Neuropathic Pain
After SCS, immunohistochemistry was performed to determine whether SCS suppressed glial activation in the spinal dorsal horn in animals with neuropathic pain. Both microglial (OX-42 positive) (Fig. 4A) and astrocytic markers (GFAP positive and MCP-1 positive) (Fig. 5, A and B) were significantly increased bilaterally 2 weeks after spared nerve injury. Four days of SCS (4 or 60 Hz, 6 hours, 90% motor threshold) significantly decreased OX-42, GFAP, and MCP-1
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Figure 4. A, Representative tissue sections for OX-42 immunostaining in the dorsal horn of naive control rats, spared nerve injury rats, 60 Hz SCS, and 4 Hz SCS. Bar = 100 μm. B, There is a significant increase in the density of OX-42 staining bilaterally in the dorsal horn 2 weeks after spared nerve injury. Sixty hertz spinal cord stimulation (SCS) and 4 Hz SCS show significantly less OX-42 immunostaining density. (*) compared with the naive controls, (#) compared with the spared nerve injury, *P ≤ 0.05, data represented by mean ± SEM.
immunoreactivity bilaterally when compared with sham SCS (Figs. 4A, 5, A and B). The density of the microglia marker OX-42 was increased bilaterally in laminae I-II 2 weeks after spared nerve injury when compared with naive controls (ipsilateral P = 0.01; contralateral P = 0.008). Both 60 and 4 Hz SCS significantly decreased OX-42 staining in the superficial laminae bilaterally when compared with sham SCS: 60 Hz (ipsilateral P = 0.0001; contralateral P = 0.001); 4 Hz (ipsilateral P = 0.0001; contralateral P = 0.003). In laminae III-V, the density of immunoreactivity for OX-42 was also increased after spared nerve injury both ipsilaterally (P = 0.001) and contralaterally (P = 0.001) when compared with naive controls. SCS significantly decreased the OX-42 staining in the deep dorsal horn when compared with sham SCS: 60 Hz (ipsilateral P = 0.005; contralateral P = 0.004); 4 Hz group (ipsilateral P = 0.005; contralateral P = 0.004) (Fig. 4B). For astrocyte markers, we examined GFAP and MCP-1. In animals with spared nerve injury, significant increases in the density of immunoreactivity for GFAP and MCP-1 occurred in laminae I-II for both the ipsilateral (GFAP P = 0.0001; MCP-1 P = 0.007) and the contralateral (GFAP P = 0.001; MCP-1 P = 0.03) sides when compared with naive animals. SCS reduced the spared nerve injury-induced increased immunoreactivity for GFAP both ipsilaterally (60 Hz P = 0.0001;
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4 Hz P = 0.0001) and contralaterally (60 Hz P = 0.002; 4 Hz P = 0.0001) in the superficial laminae. Similarly, statistically significant increases in the density of GFAP and MCP-1 in laminae I-II and III-V occurred after spared nerve injury, and these increases were reduced by both 60 and 4 Hz SCS laminae I-II ipsilaterally (GFAP: control vs spared nerve injury P = 0.0001, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.002; MCP-1: control vs spared nerve injury P = 0.007, spared nerve injury vs 60 Hz P = 0.002, spared nerve injury vs 4 Hz P = 0.843) and laminae I-II contralaterally (GFAP: control vs spared nerve injury P = 0.001, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.123; MCP-1: control vs spared nerve injury P = 0.003, spared nerve injury vs 60 Hz P = 0.001, spared nerve injury vs 4 Hz P = 0.269) (Fig. 5, C and D).
DISCUSSION
In the present study, we demonstrated that longer-duration SCS (6 hours) produces greater analgesia than shorterduration SCS (30 minutes); that 90% motor threshold intensity (amplitude) is better than 75% motor threshold, and both are better than 50% motor threshold or sham SCS. Furthermore, both 60 and 4 Hz SCS decrease activation of glial cells (microglia and astrocytes) in the spinal cord, suggesting that SCS reduces central excitability.
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Figure 5. A and B. Representative tissue sections for GFAP and MCP-1 immunostaining in the dorsal horn of control rats, spared nerve injury, 60 Hz spinal cord stimulation (SCS), and 4 Hz SCS. Sketches delineating boundaries of different laminae are superimposed over the spinal sections of control rats. Bar =100 μm. C, There is a significant increase in the density of GFAP and MCP-1 staining bilaterally in the dorsal horn 2 weeks after spared nerve injury. Both 60 Hz SCS and 4 Hz SCS show significantly less staining density when compared with animals with spared nerve injury. (*) compared with naive controls, (#) compared with the spared nerve injury, (&) compared with 60 Hz SCS. *P ≤ 0.05, data represented by mean ± SEM.
Effectiveness of SCS Depends on Intensity (Amplitude)
SCS is generally delivered both clinically and in experimental studies below motor threshold. Most of these use intensities (amplitudes) approximately two-thirds motor threshold to produce analgesia.19,30–34 The current study showed an intensity-dependent analgesia with the greatest analgesia at 90% motor threshold and no analgesia at 50% motor threshold.
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Similarly, intensity-dependent reductions in inflammationinduced hyperalgesia or pain thresholds are observed with TENS in both animal and human subjects, respectively.35,36 Higher intensities (amplitudes) of SCS and TENS produce a longer and greater magnitude of analgesia with greater amplitudes,18,24,25,35 and greater intensities (amplitudes) of SCS correlate with longer duration and magnitude of pain relief in rats.18,20,30 Thus, intensity-dependent analgesia occurs
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for both SCS and TENS, suggesting that intensity (amplitude) is a key factor in stimulation-produced analgesia.
SCS Is More Effective with Longer-Duration Stimulation
We showed a small but significantly larger reduction in hypersensitivity with 6 hours of stimulation, particularly with 60 Hz SCS. Previous studies clearly show reductions with stimulation durations as little as 5 minutes with the most of studies showing good reductions in hypersensitivity with 30 minutes to 1 hour of SCS.19,36–42 Clinically, however, the duration of SCS is variable. For example, the duration of SCS has been reported as 6 to 8 sessions per day for 10 to 60 minutes each session and for longer periods between 5 and 12 hours in a single session.43–45 The current study confirms that longer-duration SCS produces greater analgesia than shorter-duration SCS.
SCS Analgesia Can Persist After Stimulation
Previous work in a rat model of neuropathic pain (ligature of the sciatic nerve) shows 10 minutes of SCS attenuated allodynia for up to 40 minutes after cessation of the stimulation.33 SCS also inhibited nociceptive discharges of dorsal horn neurons for approximately 30 to 40 minutes after the cessation of SCS.46,47 The current study extends these previous findings and shows a small yet significant reduction in mechanical hypersensitivity with 60 Hz SCS for up to 3 days after cessation of stimulation. Some previous studies also show that repeated SCS produces a cumulative analgesic effect.19,36,48–50 In humans, how long the pain relief remains after SCS and which factors influence the duration of postSCS pain relief is unknown.43 Some authors suggest that the carryover and cumulative analgesic effect may involve a complex set of plastic changes and remodeling in spinal and supraspinal pain-processing structures. However, in the current and in our previous study, we were unable to show this cumulative analgesic effect with repeated stimulation.43
SCS Reduces Glial Cell Activation in the Spinal Cord
The current study showed for the first time that both 4 and 60 Hz SCS reduced microglia and astrocyte activation in the spinal cord in animals with neuropathic pain. Similarly, other therapeutic treatments that used our endogenous analgesia system also reduced glial activation, including electroacupuncture, peripheral nerve stimulation, and joint mobilization.51–54 SCS increases release of inhibitory neurotransmitters γ-aminobutyric acid, serotonin, acetylcholine, and opioids in the spinal dorsal horn, which could directly inhibit activation of glial cells.16,18,31,41,55,56 In fact, both astrocytes and microglia express inhibitory neurotransmitter receptors including GABAergic, serotonergic, and opioidergic receptors.57–59 Alternatively, inhibitory neurotransmitters could indirectly reduce glial cell activity by reducing neuronal release of excitatory neurotransmitters.60 It is well known that neuronal release of excitatory neurotransmitters such as glutamate are increased in response to nociceptive stimuli and that excitatory neurotransmitters can directly activate glial cells to perpetuate the nociceptive response.61–64 Therefore, further studies
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examining the underlying mechanisms of the reduced glial cell activity are required. Glial cells have been implicated in producing hypersensitivity after nerve injury with both spinal microglia and astrocytes activated after nerve injury.29,65–70 In the current study, we showed activation of both microglia and astrocytes after nerve injury with an early increase in phosphorylation of mitogen activated kinase p38, similar to previous studies.29,71,72 However, our results showed that another microglial surface marker, CD11b (OX-42), continued to show enhanced immunoreactivity and that microglial cells appeared to remain activated 10 days to 2 weeks after nerve injury.73,74 The activation of astrocytes in the later phases also agrees with previous studies implicating these cells in the maintenance of mechanical allodynia in neuropathic pain.75,76
CONCLUSION
In the present study, we examined stimulation variables and showed that the most effective analgesia was produced by 60 Hz SCS for 6-hour duration at 90% motor threshold. We further showed that SCS, both 4 and 60 Hz, reduces glial cell activation in the dorsal horn of the spinal cord. We suggest that SCS has the ability to modulate nociceptive input at the spinal cord using multiple inhibitory neurotransmitters that subsequently reduce glial cell activation. E DISCLOSURES
Name: Karina L. Sato, PT, PhD. Contribution: This author helped design and conduct the study, collect data, analyze data, and prepare the manuscript. Attestation: Karina Sato attests to having approved the final manuscript and reviewed the original study data and data analysis. Karina Sato attests to the integrity of the original data and the analysis reported in this manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author has no conflicts of interest to declare. Name: Lisa M. Johanek, PhD. Contribution: This author helped design the study and prepare the manuscript. Attestation: Lisa Johanek attests to having approved the final manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author is an employee of Medtronic, Inc. Name: Luciana S. Sanada, PT. Contribution: This author helped design and conduct the study, collect data, and prepare the manuscript. Attestation: Luciana Sanada attests to having approved the final manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: The author has no conflicts of interest to declare. Name: Kathleen A. Sluka, PhD, PT. Contribution: This author helped design and conduct the study, analyze data, and prepare the manuscript. Attestation: Kathleen Sluka attests to having approved the final manuscript and reviewed the original study data and data analysis. Kathleen Sluka attests to the integrity of the original data and the analysis reported in this manuscript. Kathleen Sluka is designated as the archival author. Conflicts of Interest: Dr. Sluka receives research funding for this study from Medtronic, Inc. This manuscript was handled by: Jianren Mao, MD, PhD.
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