High frequency transcutaneous electrical nerve stimulation
attenuates post-surgical pain and inhibits excess substance P
in rat DRG Neurons
Yu-Wen Chen1,2, Ph.D., Jann-Inn Tzeng3,4, M.S., M.D., Min-Fei Lin5, M.S.,
Ching-Hsia Hung5,*, Ph.D., Pei-Ling Hsieh5, M.S., Jhi-Joung Wang2, M.D., Ph.D.
1
Department of Physical Therapy, China Medical University, Taichung, Taiwan
2
Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan
3
Department of Food Sciences and Technology, Chia Nan University of Pharmacy
and Science, Jen-Te, Tainan City, Taiwan
4
Department of Anesthesiology, Chi-Mei Medical Center, Yong Kang, Tainan City,
Taiwan
5
Institute & Department of Physical Therapy, National Cheng Kung University,
Tainan, Taiwan
Institution:
This work was done in National Cheng Kung University, Tainan, Taiwan.
1
Running Head (< 45 characters):
TENS diminishes prolonged SMIR-evoked pain
Funding: The financial support provided for this study by grants NSC
100-2314-B-039-017-MY3 and NSC 101-2314-B-006-037-MY3 from the National
Science Council, Taiwan.
*Corresponding Author:
Ching-Hsia Hung, Ph.D.
National Cheng Kung University,
Institute & Department of Physical Therapy,
No.1 Ta-Hsueh Road, Tainan, Taiwan
Phone: 886-6-2353535 ext 5939
FAX: 886-6-2370411
Email: chhung@mail.ncku.edu.tw
Conflict of Interest:
The authors declare no conflict of interest.
2
ABSTRACT
1
2
Background: Transcutaneous electrical nerve stimulator (TENS) is a common
3
therapeutic modality for pain management but its effectiveness in skin–muscle
4
incision retraction (SMIR)-evoked pain is unknown. We aim to examine the effects of
5
TENS on postoperative pain and the levels of substance P, N-methyl-D-aspartate
6
receptor subunit 1 (NR1), and interleukin-1β (IL-1β) in dorsal root ganglion (DRG).
7
Methods: High frequency (100Hz) TENS was administered daily on postoperative
8
day 5 (POD5). Mechanical sensitivity to von Frey stimuli (6g and 15g), the levels of
9
NR1, substance P, and IL-1β in DRG were assessed in the sham-operated,
10
SMIR-operated, TENS after SMIR surgery, and placebo-TENS after SMIR surgery
11
groups.
12
Results: SMIR rats exhibited a significant hypersensitivity to von Frey stimuli on
13
POD5. In contrast with SMIR rats, SMIR-operated rats received TENS therapy
14
demonstrated a rapid recovery of mechanical hypersensitivity. SMIR-operated rats
15
showed an up-regulation of NR1, substance P, and IL-1β in DRG on POD32, whereas
16
SMIR-operated rats after TENS administration inhibited it. By contrast, the
17
placebo-TENS after SMIR operation did not alter post-surgical pain and the levels of
18
NR1, substance P, and IL-1β.
19
Conclusions: Our data reveal TENS intervention reduces persistent postoperative
3
20
pain caused by SMIR operation. Up-regulation of NR1, substance P, and IL-1β in
21
DRG, activated after SMIR surgery, is important in the development of prolonged
22
postincisional pain. TENS pain relief may be relating to the suppression of NR1,
23
substance P, and IL-1β levels in DRG of SMIR rats.
24
25
Key words: Transcutaneous electrical nerve stimulator, Postoperative pain,
26
N-methyl-D- aspartate receptor 1, Substance P, Interleukin-1β
27
4
INTRODUCTION
28
29
Persistent post-surgical pain remains a major challenge to therapy after surgical
30
procedures 1 and is a major factor in delaying hospital discharge and increasing the
31
utilization of health services.2,3 Pain management methods for postoperative pain
32
often require administration of adequate doses of parenteral opioids, which have a
33
multitude of side effects including nausea, respiratory depression, addiction and
34
tolerance.1,4 Furthermore, the laboratory data from studies have demonstrated that the
35
application of opioids may increase the risk for developing chronic pain as they may
36
worsen hyperalgesia in some patients.4,5 Transcutaneous electrical nerve stimulation
37
(TENS) is a safe analgesic therapy provided to alleviate pain of patients. For instance,
38
the application of exclusively TENS was able to attenuate activity related pain in
39
spinal surgery patients 6 and increase pain relief postoperatively after sterilization
40
surgery.7 The pathogenic mechanism underlying persistent postoperative pain is under
41
debate.
42
Substance P (SP), a neuromodulator or neurotransmitter, is a pain-related
43
neuropeptide contained in small size DRG neurons.8 Whether SP release in DRG was
44
regulated through TENS application was examined in this present experiment. It has
45
been well known that the DRG can be a target of pain relief for physicians and
46
regulate pain perception before they enter the spinal cord and travel to the brain.
5
47
Increasing evidence suggests that pro-inflammatory cytokines induce pain,9,10
48
whereas treatments with inhibitors of pro-inflammatory cytokines or
49
anti-inflammatory cytokines reduce pain.11-13 Additionally, recent evidence has
50
suggested that interleukin-1β (IL-1β) content was markedly increased in damaged
51
sciatic nerve.14,15 In this present study, we employed a skin/muscle incision and
52
retraction (SMIR) model 16 and assessed the release of IL-1β in DRG.
53
The varieties of mechanisms trigger the perception of pain following nerve and
54
tissue injuries, including spontaneous ectopic firing in peripheral nociceptive neurons,
55
sensitization of pain receptors, and alterations in gene expression of receptors and ion
56
channels within neurons and nociceptors.16-19 Hyperalgesia to heat after plantar
57
incision was reversed via the non-N-methyl-D- aspartate (NMDA) receptors
58
antagonists, and inflammatory hyperalgesia was mediated through NMDA
59
receptors.20 NMDA receptors include NMDA receptor 1 (NR1), NR3, and NR2A-D
60
subunits in rodents,21,22 and the NR1 subunit is necessary to form functional NMDA
61
receptors predominantly.21,23 In the previous study, we found a significant increase in
62
NR1 levels in the spinal cord of SMIR rats.24
63
The use of high-frequency, but not low-frequency TENS daily reduced
64
mechanical allodynia in the hind paw compared to untreated chronic constriction
65
injury animals.25 To date, few experiments have evaluated the effects of
6
66
high-frequency TENS on postoperative pain and expression of NR1, substance P, and
67
IL-1β in DRG of the SMIR rat. It is well established that the SMIR model does
68
accurately reflect the clinical scenarios of postincisional pain, i.e. prolonged tissue
69
retraction resulting in persistent pain.16 The aim of this study was to examine the
70
effect of TENS on mechanical sensitivity and the levels of NR1, substance P, and
71
IL-1β in DRG of rats after SMIR surgery.
72
73
7
MATERIALS AND METHODS
74
75
Animals
76
This experimental procedure was approved by the Institutional Animal Care and
77
Use Committee of National Cheng Kung University (Tainan, Taiwan) and conducted
78
according to IASP ethical guidelines.26 Male Sprague-Dawley rats (200 to 250 g)
79
were purchased from the Laboratory Animal Center of National Cheng Kung
80
University and kept in the animal housing facilities at National Cheng Kung
81
University, with controlled humidity (approximately 50% relative humidity), room
82
temperature (22C), and a 12-hour (6:00 AM to 6:00 PM) light/dark cycle.
83
TENS preparation
84
TENS was applied daily and started postoperative day 5 (POD5) while animals
85
were under isoflurane anesthesia for high-frequency (100 Hz) TENS. Evidences from
86
studies showed that the use of high-frequency daily attenuated mechanical/tactile
87
allodynia in the hind paw when compared with untreated chronic constriction injury
88
rats.25 In the following days, the TENS was applied to rats while they were lightly
89
anesthetized with isoflurane (0.5―1.0%). Animals were treated using an TENS
90
application (Trio 300, Ito Co., Tokyo, Japan) through the self-adhesive surface
91
electrodes, and the stimulator was used to run continuously through no
92
preprogrammed options. The medial thigh of SMIR-treated leg was shaved, and 2 small
8
93
pre-gelled adhesive electrodes with gel were applied on the proximal part close to the hip
94
joint and proximal close to the knee joint. The mode of TENS was set at 80% of that
95
needed to elicit visible muscle contractions. The pulse duration was kept at 100 μs
96
and the treatments were lasted for 20 min.27
97
SMIR operation
98
SMIR procedures were performed on rats as previously described.16,24 In brief,
99
rats were anesthetized with pentobarbital sodium (45mg/kg, i.p.), and a 15 – 20 mm
100
incision was made in the skin of the medial thigh approximately 4 mm medial to the
101
saphenous vein to expose the muscle of the thigh. Then, a 7 – 10 mm incision was
102
made in the gracilis muscle layer of the thigh, approximately 4 mm medial to the
103
saphenous nerve. The prongs of retractor (Cat. No. 13-1090, Biomedical Research
104
Instruments Inc, USA) were subsequently inserted into the gracilis muscle, to position
105
all prongs underneath the superficial layer of thigh muscle. The skin and superficial
106
muscle of the thigh were then retracted by 2 cm, exposing the fascia of the underlying
107
adductor muscles and the retraction time was maintained for 1 hour with covering the
108
incision site with cotton swabs. Following the SMIR procedure, the tissues in the
109
surgical site were closed with 4.0 Vicryl® sutures. Sham-operated rats underwent the
110
same procedure without the skin/muscle retraction. The SMIR surgery evoked
111
significant mechanical hypersensitivity in the ipsilateral hindpaw was seen by POD3
9
112
and increased to maximal levels from POD5, as previously described.16
113
Mechanical sensitivity
114
For consistency, an experienced investigator, who was blinded to the groups, was
115
responsible for handling all the animals and behavioral measurements. All behavioral
116
assessment was performed between 9:00 a.m. and 11:00 a.m., and rats were evaluated
117
for mechanical hypersensitivity after a period of at least three days of habituation to
118
the testing environment and experimenters. In brief, rats were placed individually in a
119
clear plexiglass chamber (23 cm [length] x 17 cm [width] x 14 cm [height]) and
120
supported by a wire mesh floor (40 cm [width] x 50 cm [length]). Unless otherwise
121
specified, behavioral tests were conducted on the day of SMIR operation and on
122
PODs 5, 11, 18, 25, and 32. Mechanical sensitivity was evaluated by two von Frey
123
filaments with bending forces of 6g and 15g (Linton Instruments, UK).16 In ascending
124
order of force, each filament was applied 10 times vertically, to the mid-plantar area
125
of the hind paw. Care was taken to avoid stimulating the same spot repeatedly within
126
this region and to avoid stimulating the tori/footpads themselves. Withdrawal
127
responses caused by mechanical stimulation was determined including foot lifting,
128
shaking, licking and squeaking. Paw movements associated with weight shifting or
129
locomotion were not counted.
130
NR-1 assay
10
131
Certain animals were anesthetized with pentobarbital sodium (200mg/kg, i.p.)
132
and then killed on POD 32. Under aseptic conditions, L3-L5 DRG neurons were
133
removed. The nerve specimen was homogenized in 150μl RIPA buffer and 10 μl
134
protease inhibitor (P3840, Sigma, St. Louis, MO, USA) by a glass homogenizer. After
135
incubating on ice for 1 hour, the lysates were centrifuged at 12000 rpm for 10 min at
136
4℃ with High Speed Micro Refrigerated Centrifuge (Model 3740, KUBOTA Corp.,
137
Tokyo, Japan). The supernatant was collected and determined protein concentration
138
using a protein assay. We added 25μl with Laemmli Sample Buffer (Bio-Rad,
139
Hercules, CA) into lysates, and heated at 100℃ for 5 minutes. An ELISA reader was
140
used to assay protein with bovine serum albumin (BSA) as standard at 620nm. Protein
141
samples (30 μg/lane) were loaded to separate by 12% SDS polyacrylamide gel
142
electrophoresis (SDS-PAGE) at a voltage of 75 V, and then were transferred to a
143
polyvinylidene difluoride membrane with a 0.45 μm pore size (Millipore, Bedford,
144
MA) through a transfer apparatus (Bio-Rad, Hercules, CA, USA). Then this
145
polyvinylidene difluoride membrane was blocked in TBS (20 mM Tris, 500 mM NaCl,
146
and 0.1% Tween 20, pH 7.5) containing 5% skim milk (Difco, Detroit, MI) for an
147
hour. The primary antibody of anti-NR1 against the intracellular C terminus (1: 1000,
148
Millipore) was diluted to 1:1,000 in antibody binding buffer overnight at 4ºC. The
149
membrane was then washed 3 times with TBS (10 minutes per wash) and incubated
11
150
for 1 hour with goat-anti-mouse IgG-HRP (Santa-Cruz, Santa Cruz, CA) and diluted
151
5,000-fold in TBS buffer at 4°C. This membrane was washed in TBS buffer for 10
152
minutes 3 times again. Immunodetection for NR1 was detected by the enhanced
153
chemiluminescence ECL Western blotting luminal reagent (Santa Cruz Biotechnology)
154
and then the membrane was quantified through a Gel-Pro Analyzer (version 4.0;
155
Media Cybernetics, USA). Actin was used as the internal control.
156
Substance P assay
157
The L3-L5 DRG neurons were homogenized in 200μl RIPA buffer and 10 μl
158
protease inhibitor (P3840, Sigma, St. Louis, MO, USA) using a glass homogenizer.
159
After incubating on ice for 1 hour, the lysates were centrifuged at 12000 rpm for 10
160
min at 4℃ with High Speed Micro Refrigerated Centrifuge (Model 3740, KUBOTA
161
Corp., Tokyo, Japan). The supernatant was collected and determined protein
162
concentration using a protein assay. We added 25μl with Laemmli Sample Buffer
163
(Bio-Rad, Hercules, CA) into lysates, and heated at 100℃ for 5 minutes. An ELISA
164
reader was used to assay protein with bovine serum albumin as standard at 620nm.
165
Protein samples (30 μg/lane) were separated by 12% SDS polyacrylamide gel
166
electrophoresis (SDS-PAGE) at a constant voltage of 75 V. These electrophoresed
167
proteins were transferred to a polyvinylidene difluoride (PVDF) membrane with a
168
0.45 μm pore size (Millipore, Bedford, MA) by a transfer apparatus (Bio-Rad,
12
169
Hercules, CA, USA). Then the PVDF membrane was blocked in TBS (20 mM Tris,
170
500 mM NaCl, and 0.1% Tween 20, pH 7.5) containing 5% fat-free milk (Difco,
171
Detroit, MI) for 1 hour. The primary antibody of substance P (Millipore, Billerica,
172
MA, USA) and the primary antibody of actin were diluted to 1:2,000 in antibody
173
binding buffer overnight at 4ºC. The membrane was then washed 3 times with TBS
174
(10 minutes per wash) and incubated for 1 hour with goat-anti-mouse IgG-HRP
175
(Santa-Cruz, Santa Cruz, CA) and diluted 5,000-fold in TBS buffer at 4°C. The
176
membrane was washed in TBS buffer for 10 minutes 3 times. Immunodetection for
177
substance P was performed by the enhanced chemiluminescence ECL Western
178
blotting luminal reagent (Santa Cruz Biotechnology) and then the membrane was
179
quantified by a Gel-Pro Analyzer (version 4.0; Media Cybernetics, USA). Actin was
180
used as the internal control.
181
Cytokine (IL-1β) analysis
182
Some rats were anesthetized with pentobarbital sodium (200mg/kg, i.p.) and
183
sacrificed on POD32. Skin was cut to expose the L3-L5 segments of rat DRGs was
184
removed. The nerve specimen was immediately stored at −80℃ for the protein assay.
185
Ice cold (4ºC) homogenization buffer was freshly prepared by adding protease
186
inhibitor (P 8340 cocktail, Sigma-Aldrich, St. Louis, MO) to T-PER™ Tissue Protein
187
Extraction Reagent (Pierce Chemical Co., Rockford, IL) prior to tissue lysis. After
13
188
adding the buffer (300 μl/each spinal nerve), a homogenization probe (Tissue Tearor,
189
Polytron; Biospec Products, Inc., Bartlesville, OK, USA) was applied for 20 seconds
190
on ice at 21,000 rpm. Then the homogenized samples were centrifuged for 40 minutes
191
at a speed of 13,000 rpm at 4°C, stored at −80°C and used subsequently for protein
192
quantification. The protein concentration in the supernatant was quantified using the
193
Lowry protein assay. Samples were pipetted as duplicates (1 μl/50 μl/well) in a
194
96-well microtiter plate (Costar). Each plate was inserted into a plate reader
195
(Molecular Device Spec 383, Sunnyvale, CA, USA) to read the optical density of
196
each well at an absorbance of 750 nm. Data were analyzed using Ascent Software
197
(London, UK) for iEMS Reader. The concentrations of IL-1β in the supernatants were
198
determined by the DuoSet® ELISA Development Kit (R&D Systems, Minneapolis,
199
MN).15,28 All experimental procedures were practiced in accordance with the
200
manufacturer’s recommended protocols. Plates were individually inserted into the
201
plate reader for reading optical density by a 450-nm filter. Data were then analyzed
202
using Ascent Software for iEMS Reader and a four-parameter logistics curve-fit. Data
203
were expressed in pg/mg protein of duplicate samples.
204
Groups and design
205
All the rats used in this study were randomly divided into four groups. The group
206
ONE, sham rats received sham-operated. The group TWO, SMIR rats received SMIR
14
207
operation. The group THREE, SMIR-TENS rats received high-frequency TENS
208
through stimulating electrodes positioned on skin overlying the thigh musculature
209
(ipsilateral to injury) after SMIR surgery, and the group FOUR, SMIR-Placebo-TENS
210
rats was treated exactly like the SMIR rats that received TENS, including isoflurane
211
administration, except that no TENS was administered. Some rats were considered for
212
the overall behavioral analysis (n = 8, 8, 8, 8 for Sham, SMIR, SMIR-TENS, and
213
SMIR-Placebo-TENS, respectively), while certain part of rats were killed for tissue
214
IL-1β analysis on POD32 (n = 6, 6, 6, 6 for Sham, SMIR, SMIR-TENS, and
215
SMIR-Placebo-TENS, respectively), and other rats were killed for substance P (or
216
NR1) analysis on POD32 (n = 4, 4, 4, 4 for Sham, SMIR, SMIR-TENS, and
217
SMIR-Placebo-TENS, respectively).
218
Statistical analysis
219
The resulting data are presented as the mean ± S.E.M. of N observations unless
220
noted otherwise. Statistical significance between multiple experimental groups was
221
determined by one-way or two-way ANOVA with a Bonferroni multiple comparison
222
post-hoc analysis. A statistical software, SPSS for Windows (version 17.0; SPSS, Inc.,
223
Chicago, IL, USA) was used for all statistical analyses. In each case, statistical
224
significance was set at P < 0.05.
225
15
226
RESULTS
227
TENS constrained the development of SMIR-evoked mechanical hypersensitivity
228
The sham-operated rats showed a similar sensitivity to von Frey hair stimulus
229
over the course of the study (Fig. 1A and 1B). By comparison, those rats received
230
SMIR operation manifested an incremental sensitivity (4.8 ± 0.3, n = 8, Fig. 1A; 7.0 ±
231
0.3, n = 8, Fig. 1B) to innocuous von Frey hair test on POD5, and incessant
232
mechanical hypersensitivity remained over the four-week course of this experiment
233
(Fig. 1), which was consistent with the original study, prominent mechanical
234
allodynia in rats raised 3 days after animals had been received SMIR surgery and
235
maintained for up to 22 days.16 Both SMIR-Placebo-TENS and SMIR rats exhibited
236
similarly to mechanical stimulations on PODs 5, 11, 18, 25 and 32, suggesting that
237
this Placebo-TENS procedure applied in this study does not alter tactile/mechanical
238
sensitivity (P > 0.05; two-way repeated measures ANOVA). SMIR rats displayed a
239
persistent significant mechanical hypersensitivity which was markedly in the SMIR
240
ipsilateral hind paw response to the von Frey hair test (6 and 15g) when compared to
241
the pre-surgery baseline data (Fig. 1 A and B, P < 0.05, two-way repeated measures
242
ANOVA, Bonferroni’s post-hoc). By contrast, SMIR rats after 3 week underwent
243
TENS treatment had the average numbers of paw withdraws of 2.4 ± 0.3 (n = 8),
244
lower than these (3.6 ± 0.4, n = 8) of SMIR-operated rats (Fig. 1A). Furthermore,
16
245
TENS markedly suppressed mechanical hypersensitivity (P < 0.05; two-way repeated
246
measures ANOVA) in SMIR-operated rats after 2-4 weeks TENS intervention (Fig.
247
1B) and significantly attenuated mechanical/tactile allodynia (P < 0.05; two-way
248
repeated measures ANOVA) in SMIR-operated rats on PODs 25 and 32 (Fig. 1A).
249
TENS prevents the up-regulation of NR1 in DRG following SMIR operation
250
Rat DRG tissues obtained on POD32 are shown in Fig. 2 that assayed the
251
expression of NR1 in sham, SMIR, SMIR-TENS, and SMIR-Placebo-TENS groups.
252
The NR1 expression in DRG was not markedly different between SMIR and
253
SMIR-Placebo-TENS rats (Fig. 2), suggesting that Placebo-TENS procedure used in
254
this study does not affect NR1 expression. The NR1 level in DRG was prominently
255
increased in the SMIR (P < 0.05) and SMIR-Placebo-TENS (P < 0.01) groups on
256
POD 32, respectively, when compared with the sham group (Fig. 2). Furthermore, the
257
SMIR-operated rats underwent 4-week TENS program (Fig. 2) displayed lower NR1
258
expression (P < 0.05) than those in SMIR-operated rats without TENS intervention.
259
TENS decreases substance P release in DRG after SMIR operation
260
Figure 3 depicts substance P level in DRG on POD32 in four different groups
261
including sham, SMIR, SMIR-TENS, and SMIR-Placebo-TENS. The data manifested
262
that the substance P release in DRG were significantly increased in SMIR-operated (P
263
< 0.05) rats on POD32 when compared to the sham-operated rats (Fig. 3). The
17
264
SMIR-operated rats underwent a 4-week TENS program exhibited the substance
265
P content, lower than that in SMIR-operated rats without TENS intervention (P < 0.05,
266
Fig. 3). The level of substance P in DRG was not markedly different between the
267
SMIR and SMIR-Placebo-TENS groups (Fig. 3), suggesting that the Placebo-TENS
268
regimen in this experiment does not alter substance P release.
269
TENS suppresses excess IL-1β release in DRG following SMIR surgery
270
Figure 4 reveals the level of IL-1β in DRG of sham, SMIR, SMIR-TENS, and
271
SMIR-Placebo-TENS rats on POD32. The IL-1β release in DRG on POD32 was
272
markedly increased in the SMIR (P < 0.01) and SMIR-Placebo-TENS (P < 0.01)
273
group, respectively, compared with the sham group as shown in Fig. 4. The
274
SMIR-Placebo-TENS and SMIR rats exhibited similar cytokine levels in DRG,
275
suggesting that this Placebo-TENS procedure in this study does not regulate IL-1β
276
release. By comparison, the SMIR-operated rats undergoing TENS intervention
277
demonstrated significantly lower IL-1β (P < 0.05, Fig. 4) than those in
278
SMIR-operated rats without TENS intervention.
279
18
DISCUSSION
280
281
In this study we reported for the first time that high-frequency TENS attenuates
282
SMIR-evoked pain and inhibits excess IL-1β release in DRG of the SMIR rat. Our
283
resulting data are in resemblance to the previous study that exercise diminishes
284
post-surgical pain and decreases cytokine level in SMIR rats.24 Another finding is that
285
SMIR rats following high-frequency TENS treatment prevents the up-regulation of
286
NR1 and substance P expression induced by SMIR surgery in DRG. Overall, our
287
results presume TENS suppresses the development of persistent postoperative pain, in
288
part, possibly relating to inhibit the up-regulation of NR 1, substance P, and IL-1β
289
levels in DRG.
290
High-frequency TENS suppresses the progression of SMIR-evoked pain
291
It is still unclear whether the onset for the pain syndrome is nociceptive from the
292
skin incision, muscle injury, neuropathic from surgical injury to peripheral nerves,
293
inflammatory responses, or a combination of all the possible etiologies. However, it
294
has been known that inflammation and nerve damage give rise to changes in sensory
295
processing at central and peripheral layers with a resultant sensitization.29,30 In this
296
present study we showed SMIR-operated rats on POD5 developed a markedly plantar
297
responsiveness to mechanical stimulus (Fig. 1). Our findings are in agreement with
298
the report by Flatters S.J. 16 who reported rats received SMIR operation exhibited at
19
299
least three weeks of hypersensitivity to mechanical stimulation of the plantar hind
300
paw.
301
Increasing evidence suggests that TENS can be extensively used for effective
302
treatment of phantom pain,31 chronic inflammatory hyperalgesia,32 and certain
303
neurological disorders.33 The absence of adverse effects and complications of TENS
304
compared with conventional nonopioid and opioids analgesics makes TENS a reliable
305
and safe therapeutic technique.7 Our present study showed that high-frequency TENS
306
diminished postincisional pain evoked by SMIR surgery in rats. Although TENS is
307
extensively used by physiotherapists and pain clinics, the ambiguous conclusions of
308
several experiments suggest that the location of the electrodes, the mode of TENS
309
application and the individuals disease stage may variously influence the therapeutic
310
results of this procedure on pain.34-37 The theoretical principle of TENS was well
311
established on the gate control theory reported by Melzack and Wall in 1965,38 which
312
suggests that the nociceptive information transmitted through small diameter fibers is
313
inhibited by large diameter fibers stimulation, and in this rule this painful stimulation
314
does not reach that superspinal centers.
315
It is interesting to observe that mechanical hypersensitivity was reduced after
316
high-frequency TENS intervention from POD18 to POD32 (Fig. 1B), and in the
317
meanwhile TENS also significantly suppressed tactile allodynia on POD25 to POD32
20
318
(Fig. 1A). This result suggested that one-week results remained unsatisfactory and
319
further intervention was necessary, whereas 2- to 4-week clinical outcome could be
320
maintained by TENS therapy. Further, it appeared that approximately 2-week of
321
TENS intervention was required before differences in pain behavior occur. The pain
322
behaviors altered by TENS may have been dependent on the stimuli (6g vs 15 g). Or
323
it perhaps related to the healing postoperative phase (acute inflammation vs.
324
proliferation) in that TENS may be more beneficial later in recovery than earlier.
325
We noticed that the SMIR-operated rats received TENS therapy did not exhibit
326
normal sensitivity to mechanical stimulation (Fig. 1). In marked contrast, the degree
327
of reduction (< 50%) in postoperative mechanical/tactile hypersensitivity by TENS
328
(Fig. 1) is quite small and indicates the relevance of the findings in relation to
329
post-surgical pain that is still present. Interestingly, the protective effect of TENS was
330
found to be not so strong, with SMIR rats ultimately recovery.
331
High-frequency TENS prevents the up-regulation of NR1 expression caused by
332
SMIR surgery
333
It has been proved that central sensitization contributes to some formations of
334
painful feelings after surgeries.39 In addition, the progression leading to central
335
neuronal sensitization is also related to the process underling long-term potentiation
336
and involves the contract of NMDA receptors as well.40-42 On the other hand,
21
337
treatment of NMDA receptor antagonists had potential both for the prevention and
338
management of pain.30,43 In our current study, we observed a predominant increase in
339
NR1 expression in DRG of SMIR rats.
340
Evidences from studies manifested that central sensitization of dorsal horn
341
neurons caused by peripheral nerve injury and/or inflammation underlying
342
pathological, painful, chronic states sustains a long period, and may be associated
343
with changes in gene expression of NR1, and ultimately, morphological modifications
344
in NR1 expression.44,45 In addition, NMDA receptors antagonist pre-treatment was an
345
effective analgesic therapy for thermal aspects of neuropathic pain and suggested that
346
the NMDA receptors may markedly play an important role in the onset and
347
development of thermal hyperalgesia than of mechanical allodynia.46 Here, we found
348
TENS suppressed the up-regulation of NR1 subunits induced by SMIR surgery. Those
349
experimental results and our laboratory data suggested that TENS may suppress
350
central sensitization caused by inflammation or nerve (tissue) damage by inhibiting
351
the increased expression of NR1 subunits.
352
High-frequency TENS inhibits excess substance P release in DRG in SMIR rats
353
Substance P has been implicated in regulating relatively high intensity
354
nociceptive transmission occurring with the administration of strong chemical,
355
mechanical and thermal stimuli. Additionally, substance P appears to be involved in
22
356
the mechanisms of hyper-excitability of dorsal horn neurons through potentiation of
357
the excitatory effects of glutamate or through the direct action on the postsynaptic
358
cells in the spinal cord and DRG.8,47,48 In this present experiment, we manifested that
359
SMIR rats showed a significant increase in substance P levels in DRG and
360
accompanied to develop an aggrandized plantar responsiveness to mechanical
361
stimulus. Our data are consistent with the report that substance P creates nociceptive
362
sensitization after mouse paw incision.49
363
Increasing evidence suggested that TENS can produce a significant suppression
364
of chronic hyperalgesia,32 formalin-induced pain,50 and neuropathic pain caused by
365
partial nerve injury 8 accompanying with a reduction of the substance P level in DRG
366
and the spinal cord. To investigate the mechanisms of therapeutic effects of TENS
367
intervention on postincisional pain, we explored the possible role played by substance
368
P in DRG in SMIR-operated rats. We found that TENS prevented the increased
369
release of substance P following SMIR surgery. It is possible that lack of this
370
substance attenuates the intensity of the inflammatory reaction surrounding incisions
371
thereby providing a second mechanism for the reduced thermal and mechanical
372
sensitization.49 These results suggest that TENS may inhibit central sensitization
373
induced by nerve (tissue) damage and inflammation through decreasing the
374
up-regulation of substance P release.
23
375
High-frequency TENS decreases SMIR-induced increased levels of IL-1β in DRG
376
Results from animal studies gave definite evidence for the crucial role of
377
cytokines in the induction and development of pain.29,51,52 Interestingly, we showed
378
that IL-1β in DRG increased significantly in SMIR-operated rats on POD32 when
379
compared to sham-operated rats (Fig. 4). Our experimental results are in agreement
380
with the report by Loram et al. 53 who showed that the inflammatory cytokine
381
secretion at the site of incision in an animal model of postoperative hyperalgesia. The
382
tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 levels increased during 2 h and 2
383
days after rat tail incision, and that cytokine concentration markedly elevated for 4
384
(IL-6 and IL-1β) to 8 days (TNF-α) after a 20 mm long incision made by the skin and
385
fascia of the tails. One mechanism not addressed in our study involves SP-induced
386
activation of glial cells and subsequent cytokines release as has been implicated in the
387
genesis of neuropathic pain and inflammatory states.54-58
388
In TENS analgesic mechanism, neuropharmacologic studies support the role of
389
spinal and superspinal neurotransmitters 59 and endogenous opioids released by
390
central nervous system are involved.60 Other experimental reports presume that spinal
391
opioids and serotoninergic receptors may mediate TENS antihyperalgesic effect,38,61,62
392
whereas spinal muscarinic receptors are activated after low- or high-frequency TENS
393
treatment.61 Here our observations are apparent that high-frequency TENS did inhibit
24
394
excess IL-1β level in DRG of rats after SMIR operation.
395
We conclude high-frequency TENS decreases the progression of prolonged
396
postoperative/post-surgical pain caused by SMIR surgery. Elucidating the method by
397
which kind of TENS treatment reduces excess NR1, substance P, and IL-1β levels in
398
DRG may demonstrate the TENS therapeutic mechanisms to manage persistent
399
postincisional pain. This treatment strategy by TENS that suppresses the development
400
of postoperative pain in DRG may be possible in the future.
401
25
402
ACKNOWLEDGMENTS
403
We gratefully acknowledge the financial support provided by grants NSC
404
100-2314-B-039-017-MY3 and NSC 101-2314-B-006-037-MY3 from the National
405
Science Council, Taiwan.
406
26
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36
No. withdraw from stimulus
A. von Frey 6g
Sham
SMIR
SMIR-TENS
SMIR-Placebo-TENS
10
8
b,c,d
6
b,c,d
b,c,d
4
c,d
c,d
a,b
2
a
0
0
5
11
18
25
32
No. withdraw from stimulus
Days after SMIR surgery
B. von Frey 15g
Sham
SMIR
SMIR-TENS
SMIR-Placebo-TENS
10
8
b,c,d
b,c,d
c,d
6
c,d
4
a
a,b
c,d
a
2
0
0
5
11
18
25
32
Days after SMIR surgery
Fig. 1.
37
Fig. 2.
38
Fig. 3.
39
la
c
-P
IR
40
o-
S
*
TE
N
S
IR
-T
EN
IR
SM
**
eb
SM
250
SM
am
Sh
IL-1 (pg/mg)
**
**
200
150
100
50
0
Fig. 4.
FIGURE LEGENDS
Fig. 1. The behavioral time courses of no. withdraw from mechanical stimulation. The
graphs show mean ± S.E.M. of the number of rat hindpaw withdrawals out of 10
stimuli with (A) von Frey 6g and (B) von Frey 15g. For all time points n = 8 rats per
group, including sham-operated (sham), skin–muscle incision retraction
(SMIR)-operated (SMIR), transcutaneous electrical nerve stimulation (TENS) after
SMIR surgery (SMIR-TENS), and placebo-TENS after SMIR surgery
(SMIR-Placebo-TENS) groups. The Placebo-TENS group of SMIR rats was received
exactly like the rats that treated TENS, including halothane application, except that no
TENS was applied. The symbol (a) indicates P < 0.05 when the SMIR-TENS group
was compared with the SMIR group; the symbols (b, c, d) indicate P < 0.05 when the
SMIR-TENS, SMIR, and SMIR-Placebo-TENS group were compared with the sham
group, respectively (2-way ANOVA of repeated measures followed by post hoc
Bonferroni’s test).
Fig. 2. The expression of N-methyl-D-aspartate receptor 1 (NR1) in DRG on
postoperative days 32 in different groups of rats: sham, SMIR, SMIR-TENS, and
SMIR-Placebo-TENS (sham = sham-operated; skin–muscle incision retraction (SMIR)
= SMIR-operated; SMIR-TENS = transcutaneous electrical nerve stimulation (TENS)
after SMIR surgery; SMIR-Placebo-TENS = placebo-TENS after SMIR surgery). The
41
Placebo-TENS group of SMIR rats was treated exactly like the rats that received
TENS, including halothane application, except that no TENS was administered. The
values are presented as mean ± S.E.M. for 4 rats per group. The asterisk (*, **)
indicates P < 0.05 and P < 0.01, respectively, when compared with the SMIR-TENS
or sham group (1-way ANOVA followed by post hoc Bonferroni’s test).
Fig. 3. Substance P release in DRG on postoperative day 32 after skin–muscle
incision retraction (SMIR) operation was quantified by the western blotting method in
4 different groups of rats: sham, SMIR, SMIR-TENS, and SMIR-Placebo-TENS
(sham = sham operated; SMIR = SMIR-operated; SMIR-TENS = transcutaneous
electrical nerve stimulation (TENS) after SMIR surgery; SMIR-Placebo-TENS =
placebo-TENS after SMIR surgery). The Placebo-TENS group of SMIR rats was
received exactly like those rats that treated TENS, including halothane application,
except that no TENS was administered. The values are presented as mean ± S.E.M.
for 4 rats per group. Compared with the sham group and SMIR-TENS group, the
SMIR group showed a significant increase in SP level in DRG (P<0.05). The asterisk
(*, **, ***) indicates P < 0.05, P < 0.01 and P < 0.001, respectively, when compared
with the SMIR-TENS or sham group (1-way ANOVA followed by post hoc
Bonferroni’s test).
Fig. 4. The level of interleukin-1β (IL-1β) on postoperative day 32 in DRG in sham,
42
SMIR, SMIR-TENS, and SMIR-TENS-Control rats, where sham = sham operated;
SMIR = skin–muscle incision retraction (SMIR)-operated; SMIR-TENS =
transcutaneous electrical nerve stimulation (TENS) after SMIR surgery;
SMIR-Placebo-TENS = placebo-TENS after SMIR surgery. The Placebo-TENS
group of SMIR rats was treated exactly like those rats that received TENS, including
halothane application, except that no TENS was administered. The values are
presented as mean ± S.E.M. for 6 rats per group. The asterisk (*, **) indicates P <
0.05 and P < 0.01, respectively, when compared with the SMIR-TENS or sham group
(1-way ANOVA followed by post hoc Bonferroni’s test).
43