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Metadata of the article that will be visualized in OnlineFirst ArticleTitle Depressing effect of electroacupuncture on the spinal non-painful sensory input of the rat Article Sub-Title Article CopyRight Springer-Verlag Berlin Heidelberg (This will be the copyright line in the final PDF) Journal Name Experimental Brain Research Corresponding Author Family Name Quiroz-Gonzlez Particle Given Name Salvador Suffix Division Departamento de Acupuntura y Rehabilitacin Organization Universidad Estatal del Valle de Ecatepec Address Av. Central s/n, Esq. Leona Vicario, Col. Valle de Anhuac, Secc. A, Ecatepec, Estado de Mxico, CP 55210, Mexico Email sqg20@yahoo.com.mx Author Family Name Segura-Alegra Particle Given Name Bertha Suffix Division Facultad de Estudios Superiores, FES Iztacala Organization UNAM Address Mexico, CP 54090, Mexico Email bsegura@campus.iztacala.unam.mx Author Family Name Jimnez-Estrada Particle Given Name Ismael Suffix Division Departamento de Fisiologa, Biofsica y Neurociencias Organization Centro de Investigacin y, Estudios Avanzados del IPN Address Av. Instituto Politcnico Nacional 2508, Col. San Pedro Zacatenco, AP. 14-740, Mexico, DF, CP 07000, Mexico Email ijimenez@fisio.cinvestav.mx Schedule Received 13 February 2014 Revised Accepted 12 April 2014 Abstract The aim of this study was to explore the effect of electroacupuncture (EA) applied in the Zusanli (ST36) and Sanyinjiao (SP6) points on the N1 component of the cord dorsum potential (CDP) evoked by electrical stimulation of the sural nerve (SU) in the rat. The experiments were performed in 44 Wistar rats (250300 g) anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg). A bilateral laminectomy was performed to expose the L3 to S2 segments of the spinal cord. The SU nerve was exposed and placed on pairs of hook electrodes for electrical stimulation. The N1-CDPs were recorded with three silver-ball electrodes located on the dorsal surface of the L5 to S1 segments. Ipsilateral high and low EA stimulation (100, 2 Hz, 6 mA, 30 min) induced a considerable reduction in the amplitude (45 5.6, 41 6.2 %) of the N1-CDP recorded at the L6 segmental level. Recovery of the N1-CDP amplitude occurred approximately 13 s after EA. Sectioning of the saphenous and superficial peroneal nerves reduced the depressing effect provoked by the EA stimulation (18.7 1.3, 27 3.8 %). Similarly, sectioning of the posterior and anterior tibial, deep peroneal and gastrocnemius nerves partially reduced the effect provoked by EA (11 1.5, 9.8 1.1, 12.6 1.9 %). Intravenous picrotoxin (1 mg/kg) also reduced the action of low and high EA (23 4.8, 27 5.2 %). It is suggested that EA stimulation depresses non-painful sensory pathways through the activation of specific inhibitory pathways that receive modulatory actions from other sensory and muscle afferent inputs in the rat spinal cord. Keywords (separated by '-') Electroacupuncture - Cord dorsum potentials - Sural nerve - Spinal cord Footnote Information U N C O R R E C T E D
P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK 1 3 Exp Brain Res DOI 10.1007/s00221-014-3965-2 RESEARCH ARTICLE Depressing effect of electroacupuncture on the spinal non-painful sensory input of the rat Salvador Quiroz-Gonzlez Bertha Segura-Alegra Ismael Jimnez-Estrada Received: 13 February 2014 / Accepted: 12 April 2014 Springer-Verlag Berlin Heidelberg 2014 stimulation (18.7 1.3, 27 3.8 %). Similarly, sectioning of the posterior and anterior tibial, deep peroneal and gas- trocnemius nerves partially reduced the effect provoked by EA (11 1.5, 9.8 1.1, 12.6 1.9 %). Intravenous picro- toxin (1 mg/kg) also reduced the action of low and high EA (23 4.8, 27 5.2 %). It is suggested that EA stimulation depresses non-painful sensory pathways through the activa- tion of specic inhibitory pathways that receive modulatory actions from other sensory and muscle afferent inputs in the rat spinal cord. Keywords Electroacupuncture Cord dorsum potentials Sural nerve Spinal cord Introduction Acupuncture is a therapeutic modality that emerged from Traditional Chinese Medicine. The World Health Organi- zation recommends the use of acupuncture for the treat- ment of a wide variety of diseases (Zhang et al. 2014; Yin et al. 2010; Barnes et al. 2008). A novel contemporary form of acupuncture, electrical stimulation of acupuncture points (APs), also known as electroacupuncture (EA), has been widely used in both clinical and experimental stud- ies (Vickers et al. 2012; Zhao 2008). Several studies have demonstrated that EA exerts an analgesic effect on neuro- pathic pain in rat models (Lau et al. 2008; Kim et al. 2004; Huang et al. 2004; Hwang et al. 2002) and relieves acute or chronic inammatory pain (Kim et al. 2006; Zhang et al. 2004). There has been particular interest in determining the mechanisms involved in the antinociceptive effect of EA (Zhao 2008; Leung 2012). Acupuncture analgesia is a clear manifestation of modulatory processes occurring at differ- ent levels of the central nervous system (Zhang et al. 2014; Abstract The aim of this study was to explore the effect of electroacupuncture (EA) applied in the Zusanli (ST36) and Sanyinjiao (SP6) points on the N1 component of the cord dorsum potential (CDP) evoked by electrical stimu- lation of the sural nerve (SU) in the rat. The experiments were performed in 44 Wistar rats (250300 g) anesthe- tized with ketamine (100 mg/kg) and xylazine (2 mg/kg). A bilateral laminectomy was performed to expose the L3 to S2 segments of the spinal cord. The SU nerve was exposed and placed on pairs of hook electrodes for elec- trical stimulation. The N1-CDPs were recorded with three silver-ball electrodes located on the dorsal surface of the L5 to S1 segments. Ipsilateral high and low EA stimu- lation (100, 2 Hz, 6 mA, 30 min) induced a considerable reduction in the amplitude (45 5.6, 41 6.2 %) of the N1-CDP recorded at the L6 segmental level. Recovery of the N1-CDP amplitude occurred approximately 13 s after EA. Sectioning of the saphenous and supercial peroneal nerves reduced the depressing effect provoked by the EA S. Quiroz-Gonzlez (*) Departamento de Acupuntura y Rehabilitacin, Universidad Estatal del Valle de Ecatepec, Av. Central s/n, Esq. Leona Vicario, Col. Valle de Anhuac, Secc. A, CP 55210 Ecatepec, Estado de Mxico, Mexico e-mail: sqg20@yahoo.com.mx B. Segura-Alegra Facultad de Estudios Superiores, FES Iztacala, UNAM, CP 54090 Mexico, Mexico e-mail: bsegura@campus.iztacala.unam.mx I. Jimnez-Estrada Departamento de Fisiologa, Biofsica y Neurociencias, Centro de Investigacin y, Estudios Avanzados del IPN, Av. Instituto Politcnico Nacional 2508, Col. San Pedro Zacatenco, AP. 14-740, CP 07000 Mexico, DF, Mexico e-mail: ijimenez@sio.cinvestav.mx AQ1 AQ2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 Quiroz-Gonzlez et al. 2014; Zhao 2008). The rst conver- gence of impulses originating from pain sites and acupoints occurs in the spinal dorsal horn, where the neuronal noci- ceptive responses appear to be depressed by both pre- and post-synaptic inhibition during EA stimulation (Zhao 2008; Li et al. 1993). Although the effectiveness of EA for pain control has been demonstrated experimentally in animal models, little is known about the possible effects exerted by EA on the cutaneous non-painful sensory input in the spi- nal cord. Electrical stimulation of cutaneous nerves induces the activation of sensory neurons located in the dorsal horn of the spinal cord (Bernhard 1953; Willis et al. 1973; Coombs et al. 1956), which produces cord dorsum poten- tials (CDPs) consisting of clearly dened components. The rst observed component in the CDPs is the afferent volley (AV), which is caused by the electrical activation of low- threshold afferent bers (Coombs et al. 1956). This is fol- lowed by one negative component (named N1-CDP), which is generated by the monosynaptic activation of the dorsal horn neurons via the A afferents or non-painful bers in the cutaneous nerves (Bernhard 1953; Willis et al. 1973; Coombs et al. 1956). Subsequently, a long-lasting positive component occurs, named the P wave, which is ascribed to the current ows generated during the presynaptic depolari- zation of afferent bers (PAD) and presynaptic inhibition (Rudomin and Schmidt 1999). The present study aimed to analyze the effect of EA stimulation on the non-painful sensory neurons at the level of the spinal cord. To achieve this goal, we recorded the N1-CDP evoked by cutaneous SU nerve stimulation and during EA stimulation. In a sec- ond series of experiments, we evaluated how this effect is modied by sectioning cutaneous and muscular nerves. Because it is amply recognize that GABAergic mechanism is involved in the modulation of primary afferent depolari- zation (PAD) of cutaneous and muscular nerves, we ana- lyzed the possible effect of picrotoxin (PTX) on the actions of the EA. Some of these observations have been published previously in abstract form (Quiroz-Gonzalez et al. 2013). Methods Animals Male Wistar rats (n = 44) weighing 250300 g (8 10 weeks old) that were obtained from the animal house of our institution were used. All animals had free access to water and were housed under identical environmental con- ditions of light and dark cycles (12:12 h) and temperature (2224 C). All experiments were carried out in accordance with the guidelines of the Mexican Ofcial Norm (NOM- 062-ZOO-1999) and in accordance with the National Insti- tutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised in 1978). The study protocol was approved by the institutional bioethical committee for Care and Handling of Laboratory Animals (Protocol 0267-05, CINVESTAV). Surgical procedures Initially, the animals were anesthetized with an intraperito- neal injection of a mixed solution of ketamine (100 mg/kg) and xylazine (2 mg/kg). This injection was supplemented every hour by additional doses of ketamine (50 mg/kg), applied in the same low abdominal region to ensure that an adequate level of anesthesia, dened as the absence of with- drawal reexes. We used ketamine as an analgesic because it has been demonstrated that it did not signicantly reduce the response of spinal dorsal horn neurons to low-threshold afferent inputs in the intact animal, but in contrast suppress noxiously evoked activity of wide dynamic range neurons (Collins 1986). Subsequently, the femoral vein was exposed and cannulated for the administration of a solution of pic- rotoxin (PTX, 1 mg/kg of body weight; Quirz-Gonzalez et al. 2012). A bilateral laminectomy was performed in the lumbosacral enlargement (from the L4 to S1 segments) of the spinal cord. Several nerves of the right hind leg were carefully exposed and prepared for stimulation and/or sectioning: the main branch of the sural (SU), supercial peroneus (SP), tibial (TA), saphenous (SA) deep peroneus (DP) and gastrocnemius (GS) nerves and the rats were then secured in a stereotaxic frame. The surgical procedure for sural nerve dissection on the hind leg was carefully made to avoid the possible damage of the Zusanli (ST36) and Sanyinjiao (SP6) acupoints. The animals body temperature was monitored using a thermal probe located in the back muscles and connected to an automatic feedback control unit and a heating blanket to maintain the animals body temperature at 37 C. The skin aps and muscle around the exposed tissues were raised and tied to a metal stereotaxic frame to form a pool, which was lled with warm mineral oil to prevent the tissues from drying. SU nerve stimulation The central end of the sural (SU) nerve in the left hind limb was mounted on a pair of stimulating silver hooks connected to an isolated-current-pulse generator (Isoex D 4030), and the CDPs were produced in all experiments by single square-current pulses (0.05 ms duration; at 1 Hz) of graded intensity. The pulses were monitored by record- ing the voltage drops across a resistor (1,000 ) that was placed in the current return path. The electrical threshold (1 T) was established as the minimum stimulus strength (usually between 0.1 and 0.13 mA) necessary to cause a discernible CDP response on the surface of the spinal cord. AQ3 AQ4 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 Electroacupuncture stimulation Pairs of stainless-steel acupuncture needles (0.25 mm in diameter and 5 mm in length) were inserted perpendicu- larly at a depth of 3 mm at the Zusanli (ST36, 5 mm lateral to the anterior tubercle of the tibia) and Sanyinjiao acu- points (SP6, 3 mm proximal to the medial malleolus, at the posterior border of the tibia). These acupoints are tradition- ally used in acupuncture-induced analgesia for the treat- ment of several pain syndromes (Zhang et al. 2003; Huang et al. 2002). To disclose the region-specic effect of EA, we stimulate two non-acupoints (NAP) nearby to ST36 and SP6, the rst located 2 mm lateral to ST36 and the second 3 mm posterior to SP6. The transpositional method (Yin et al. 2008) for rats was used to determine the acupoint locations. The needles were connected to an isolated-cur- rent- pulse generator (Isoex D 4030), and square trains of high or low frequency (100 or 2 Hz, respectively) and vari- able strength current pulses (16 mA, 0.1 ms) were applied in the acupoints located both contralateral and ipsilateral of the sural nerve stimulation for a total of 30 min. CDP recording Chlorinated silver-ball electrodes were placed on the dorsal surface of several segments in the lumbosacral spinal cord (L3S2) to record the CDPs (Fig. 1), and the corresponding reference silver electrodes were inserted into the adjacent paravertebral musculature. Each recording pair of elec- trodes was connected to an individual low-noise, high-gain differential amplier (Grass, model P511; band-pass lters were set at 0.310 kHz). The resulting recordings were dig- itized, averaged (n = 40 samples at 1 Hz), and stored in a digital computer using a specially designed software (built in the Lab-VIEW environment). The peak amplitude of the N1-CDPs was measured and subsequently analyzed. Data analysis All statistical analyses were performed using the Graph- Pad Prism (version 4) software. Data were expressed as the mean standard deviation. A two-way analysis of variance test for multiple comparisons followed by a Bonferroni cor- rection was used to determine the differences in the ampli- tudes of the N1-CDPs produced by SU nerve stimulation and those produced by EA stimulation on ST36 and SP6. Repeated measure ANOVA followed by Newman-Keuls posthoc test was used to determine the differences between the amplitude of control SU N1-CDPS and the amplitude of the conditioned SU nerve responses evoked during EA stimulation. The differences were considered signicant at p < 0.05. Results CDPs provoked by SU nerve and EA stimulation Single electrical pulses applied to the SU nerve (34 T, at 1 Hz) produced N1-CDPs of a relatively large amplitude (Fig. 1a). The largest N1-CDP occurred at the L6-segmen- tal level and gradually decreased in amplitude in the adja- cent rostro-caudal spinal segments (L5, L4 and S1, respec- tively, in Figs. 1a, 2a, 3a). Similar recordings were obtained in 6 other experiments. This rostro-caudal distribution of the CDPs agreed well with previous reports (Gonzlez et al. 2011). Single electrical current pulses applied to the ST36 and SP6 acupoints (34 T, at 1 Hz) produced N1-CDPs with largest amplitude at the L5 and L6 segmental levels, decreasing in amplitude in L4 and S1 spinal segments. The N1-CDPs produced by SU nerve stimulation showed a sim- ilar rostro-caudal distribution than those produced by EA applied to ST36 and SP6, but they are smaller in amplitude (p < 0.05; Fig. 1a, b). Fig. 1 Experimental arrangement for the N1-CDP recording: a aver- age N1-CDP produced by SU nerve stimulation and EA stimulation recorded at L4 to S1 spinal cord segments. b Graphs illustrating the longitudinal distribution on the spinal segments of the N1-CDP aver- ages amplitudes (n = 7) produced by SU nerve and EA stimulation at ST36 and SP6. (*p < 0.05) 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 Effect of high frequency EA stimulation on the amplitude of the N1-CDP The N1-CDP recorded on the surface of the L5 to S1 seg- ments gradually decreased in amplitude when the high frequency (100 Hz) EA stimulation strength was progres- sively increased (from 1 to 6 mA; Fig. 2a, b). The maximal depression of the N1-CDP (45 5.6 %) was observed at the L6 segment (Figs. 2a, b, 3a, b) and occurred immedi- ately upon and during EA stimulation (30 min). In most Fig. 2 Inhibition of the N1-CDP by high (100 Hz) EA stimulation on ST36 and SP6: a average N1-CDP produced by SU nerve stimulation and recorded in several spinal segments (L5S1) before EA (control) and during ipsilateral or contralateral EA stimulation at different stimulus intensities (16 and 6 mA, respectively). b Graphs illustrating the mean reduction (n = 11) in the N1-CDP component recorded in the S1L5 spinal segments during a 100 Hz ipsilateral EA stimulation. Asterisks indicate signicant differences between N1-CDP responses produced by SU nerve stimulation before EA and during high EA stimulation (*p < 0.05 and **p < 0.01) Fig. 3 Time course of inhibi- tory effect of EA on N1-CDP component: a CDP recording showing that after EA, there was a recovery of the N1-CDP component produced by SU nerve stimulation in several spinal segments (S1L5). b Graphs illustrating the changes in the percent ampli- tude of the N1-CDPs produced before (control), during and after EA. Asterisks indicate signicant differences between N1-CDP responses produced by SU nerve stimulation before EA and during posthigh EA stimulation (*p < 0.05 and **p < 0.01) 221 222 223 224 225 226 227 228 229 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 cases, the increment in intensity of EA stimulation (above 6 mA) did not increase the magnitude of the depression at the different segments analyzed. Contralateral EA stimu- lation (6 mA) and NAP stimulation had no effect on the N1-CDP (Fig. 2a, b). After removal of the EA stimulus, recovery of depressive actions on the N1-CDP occurred within approximately 12 s (Fig. 3a, b). Effect of low frequency EA stimulation on the amplitude of the N1-CDP 30 min of low frequency EA stimulation (2 Hz) induce the reduction in amplitude of the N1-CDP (41 6.2 %), but only when the N1-CDP caused by EA stimulation occurred at 100 ms before the N1-CDP produced by SU nerve stimu- lation (Fig. 4B, C, D). No statistical differences were found when the N1-CDP caused by EA stimulation occurred after N1-CDP produced by SU nerve stimulation (Fig 4E, F). Effect of nerve sectioning on the depressive action of EA Sectioning the sensory saphenous and supercial pero- neal nerves (Fig. 5a, b) induced a signicant reduc- tion in the depressing effect provoked by EA stimulation on the SU-evoked N1-CDP (18.7 1.3 %, n = 7 and 27 3.8 %, n = 7, respectively). Meanwhile, section- ing of the tibial, deep peroneal and gastrocnemius nerves (Fig. 5a, b) reduced the depressing effect provoked by EA on the N1-CDPs but to a lesser extent (11 1.5 %, n = 7; 9.8 1.1 %, n = 7; and 12.6 1.9 %, n = 7, respectively). The effect of PTX on the depressive action of EA In order to analyze the possible GABAergic mechanism on the depressive actions of EA on the SU-evoked N1-CDP, systemic injection of a GABA A antagonist, picrotoxin (PTX, 1 mg/kg) was delivered. As shown in Fig. 6D, F, the application of PTX reduces the depressive actions of low frequency EA (23 4.8 %, n = 7), as compared with con- trol recording (Fig. 6A, F). Similar reductions were found (27 5.2 %, n = 7) on the depressive actions of high fre- quency EA stimulation (Fig. 6E, F). Intravenous saline vehicle does not produced any effect on the EA conditioned depression of the SU-evoked N1-CDP. Discussion It is well established that the N1-CDP generated by the electrical stimulation of cutaneous nerves is produced by the monosynaptic activation of dorsal horn neurons located in the Rexeds laminae III to VI via A afferent bers or low-threshold cutaneous afferent bers (Bernhard 1953; Willis et al. 1973; Coombs et al. 1956). In our study, stimu- lation of the sensory SU nerve and EA at the ST36 and SP6 acupoints provoked N1-CDPs that were simultaneously Fig. 4 Inhibition of the SU N1-CDP by low (2 Hz) EA stimula- tion applied on the ST36 and SP6 acupoints: a averaged N1-CDP (n = 16 recordings) produced by SU nerve stimulation and recorded in L6 spinal segment before EA stimulation, b during EA (EA-CDP) applied 70 ms previously to the stimulus of the SU nerve, c 40 ms, d 20 ms, and e 30 ms after SU-evoked N1-CDP (ASU-CDP). f Graphs illustrate averaged (SD) percent reduction values of the N1-CDP component recorded on the L6 spinal segment in nine animals, during a 2 Hz ipsilateral EA stimulation. Asterisks indicate signicant differ- ences between N1-CDP responses produced by SU nerve stimulation before EA and during low EA stimulation (*p < 0.05 and **p < 0.01) 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 Fig. 5 Reduction in the effect of EA at 100 Hz on the N1-CDP component by section- ing nerves: a CDPs recording showing the effect of nerve sec- tioning on the depressive action evoked in N1-CDP by high EA stimulation (100 Hz). b Graphs illustrating the changes in the percent ampli- tude of the N1-CDPs produced by EA and abolished by nerve sectioning (n = 7 animals per experimental procedure). SP supercial peroneous; SA saphe- nous; TA tibial; GS gastrocne- mius soleus nerves. Asterisks indicate signicant differences between control N1-CDP responses evoked by SU nerve stimulation and during EA stimulation before and after the sectioning of cutaneous and/or muscular nerves (*p < 0.05 and **p < 0.01) Fig. 6 Effect of picrotoxin (PTX; 1 mg/kg) on the depressive effect of EA on the N1-CDP: a averaged N1-CDP produced by SU nerve stimulation and recorded in L6 spinal segment before EA stimula- tion, b during low frequency EA (2 Hz) applied 40 ms previously to the stimulus of the SU nerve, c with high frequency EA (100 Hz), d The effect of intravenous injection of PTX on the low frequency EA conditioned depression of the SU-evoked N1-CDP, e PTX under high frequency EA conditioned depression, f, g low or high frequency EA+ vehicle intravenous administration, h graphs illustrate averaged (SD) percent reduction values of the N1-CDP component recorded on the L6 spinal segment, during a 2 Hz EA stimulation (7 animals), 100 Hz EA stimulation (7 animals) intravenous PTX (7 animals), and saline vehicle control (3 animals). Asterisks indicate signi- cant differences between N1-CDP responses produced by SU nerve stimulation before EA, during EA stimulation and intravenous PTX (*p < 0.05 and **p < 0.01) A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 recorded on several segments of the lumbosacral enlarge- ment (L4 to S1). The SU nerve evoked N1-CDP with the largest was recorded on the L6 segment, and it is consist- ent with those reported in other studies (Willis et al. 1973; Gonzlez et al. 2011). Meanwhile, the largest N1-CDPs evoked by EA were recorded at the L5L6 spinal segments. These observations may suggest that the sensory inputs activated by sural nerve and EA acupoint stimulation prob- ably share spinal pathways which probably interact synap- tically at the spinal cord level. According to the later, we also found that conditioning low and high frequency EA stimulation depressed the N1-CDPs produced by SU nerve stimulation. In contrast, EA stimulation on non-acupoint sites does not evoke signicant changes in the SU nerve evoked N1-CDP, suggesting a specic acupoint effect of EA. It is proposed that EA reduces the activation of dorsal horn neurons provoked by low-threshold cutaneous afferent bers by the activation of specic sensory pathways in the spinal dorsal horn of the rat. Transmission of non-nociceptive and nociceptive infor- mation via primary afferents is modulated at the rst spinal relay by highly complex processes (Rudomin and Schmidt 1999; Le Bars 2002; Besson and Chaouch 1987). It has been accepted that electrical stimulation of primary affer- ent bers effectively modulates the synaptic efcacy of the same and/or other afferent bers in the spinal cord (De LaTorre et al. 2009; Rudomin and Hernandez 2008). Elec- trophysiological evidences have shown that electrical stim- ulation of A bers may depress nociceptive activation of spinal dorsal horn neurons for short periods of time (Chung et al. 1984a, b). Moreover, both brief electrical stimulation of afferent C-bers and prolonged high frequency burst stimulation of the sciatic nerve at A ber strength produce long-term depression (LTD) of C-ber-evoked eld poten- tials (Liu et al. 1998). In addition, LTD of synaptic trans- mission in substantia gelatinosa neurons can be induced by low frequency stimulation of primary A-afferent bers (Sandkuhler et al. 1997). Electroacupuncture stimulation also provokes consider- able changes in the neuronal activity evoked by peripheral nerve stimulation. Kim et al. (2011) showed that EA pro- duced a signicant reversal of enhanced evoked responses of the deep dorsal horn (lamina IVVII) neurons as well as after discharges developed in ankle-sprained rats. The EA- induced inhibition lasted for at least 30 min after the ter- mination of EA. In other study, it was found that 2 Hz EA induce LTD in the C-ber-evoked eld potentials recorded within the spinal dorsal horn of rats with neuropathic pain. In contrast, 100 Hz EA-induced long-term potentiation (LTP) but LTD in control rats (Xing et al. 2007). In the present study, we found a signicant reduction in the amplitude of the N1-CDPs evoked by SU nerve stimu- lation during ipsilateral EA stimulation (46 mA) in several spinal segments, particularly at the L6 segment of the rat spinal cord. The ST36 acupoint receives sensory innerva- tion from saphenous, supercial peroneal, and lateral sural cutaneous nerves and motor innervation from the deep per- oneal and anterior tibialis nerves (Zhou et al. 2010), while the SP6 acupoint receives sensory innervation from the saphenous, sural and medial crural nerves and motor inner- vation from the tibial nerve (Zhou et al. 2010). The spinal projection of these nerves showed a considerable overlap, particularly at the L5L6 segmental level, even though they innervate different hind limb areas (Maslany et al. 1992; Panneton et al. 2005). It thus seems reasonable to expect that during EA stimulation, the sensory and motor inner- vation of the acupoints are activated and that the highest effect of EA is observed at the L6 segment. It is known that the acupuncture effect may occur in a gradual manner and last for a long period of time (Zhao 2008; Leung 2012). Several lines of evidence suggest that neurotransmitters and endogenous opioids are involved in the depressive effect of EA on spinal nociceptive neurons and that they participate in the analgesic effect of acupunc- ture (Zhao 2008; Leung 2012). In the present study, the depressive effect of EA on the N1-CDP component showed a fast onset (<1 min) and continued during EA stimulation and after the removal of the stimulation for a relative short period of time (12 s). According to the latter, it could be proposed that the effect of EA on nociceptive and non- nociceptive pathways may be mediated by different spinal mechanisms. It is well established that the N1-CDP could be depressed by the conditioning stimulation of sensory peripheral nerves and such effect is mainly attributed to PAD and presynaptic inhibition through GABAergic mechanism (Lidierth 2006; Quirz-Gonzalez et al. 2012). This kind of inhibition had its highest effect at 2030 ms and last until 100 ms (Rudomin and Schmidt 1999; Lidi- erth 2006; Quirz-Gonzalez et al. 2012). We found in this study that the depressive effect of low frequency EA occurred when the N1-CDP produced by SU nerve stim- ulation appear between 5 and 90 ms after the occurrence of the N1-CDP evoked by EA stimulation, with the high- est effect between 20 and 40 ms, this interval of depression and the time of the highest effect are quite similar to the time course of the presynaptic depolarization of afferent bers and presynaptic inhibition. This observation is rein- forced by the antagonist effect of Picrotoxin (PTX) on the depression exerted by EA on cutaneous spinal responses. In concordance with this several evidences, we proposed that presynaptic mechanism could participate in the depres- sive actions of acupuncture. It is also shown that PTX not abolish the effect of EA, the remaining PTX-resistant depression of the N1-CDP may be ascribe to the activation of non-GABAergic mechanism, most likely glycinergic AQ5 AQ6 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 A u t h o r
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P R O O F Journal : Large 221 Dispatch : 19-4-2014 Pages : 9 Article No : 3965 LE TYPESET MS Code : EBR-14-0106 CP DISK Exp Brain Res 1 3 pathways (Rudomin and Schmidt 1999) or including to the accumulation of potassium ions in the spinal cord (Kremer and Lev-Tov 1998). We also found that the depressive effects evoked by EA were partially abolished by the sectioning of cutaneous and muscle nerves that innervated the ST36 and SP6 acupoints. The changes produced by sectioning of cutaneous nerves were larger than those produced by the section of muscle nerves. According to this evidence, it could be suggested that specic heterosynaptic inhibitory pathways receiving sensory and muscular inputs could be involved in the effect of EA on low-threshold sensory pathways. It is well known that the N1-CDP is produced by a groups of sensory neurons that receive cutaneous large A, afferent bers (Bernhard 1953). These spinal neurons located in the laminae III-VI are responsible to transmit ne touch, vibration, propiocepcion to supraspinal cent- ers (Willis et al. 1973). It may be suggest that EA at low or high frequency affects the transmission of these differ- ent sensory modalities at the spinal cord level and could have some implications in the process of the information in the somatosensory cortex. Several lines of evidence have hypothesized that large diameter sensory bers play a major role in the pathogenesis of some types of neuropathic pain (Devor 2009; Campero et al. 1998). Devor (2009) has showed that dorsal root ganglion A afferents, which normally signals touch and vibration, change their electri- cal and neurotransmitter characteristics when they are sec- tioned (axotomized). Such condition seems to switch the sensory input of A afferents from non-painful to painful signals (phenotypic switching), triggering, and maintain- ing central sensitization. Since it has been shown that EA stimulation exerts anal- gesic and antinociceptive effects by modulating the activ- ity of spinal dorsal horn neurons and the experimental evidence obtained in this study indicates that high and low frequency EA stimulation also affect low-threshold non- painful sensory pathways at the spinal cord level in the rat, it could be proposed that the depression of low-threshold cutaneous pathways is involved in the reduction in neuro- pathic pain produced by EA stimulation. However, further studies are necessary to analyze this possibility by analyz- ing the effect of EA on low-threshold sensory pathways in an animal model of neuropathic pain. In conclusion, the present study showed that EA stim- ulation depressed non-painful sensory spinal pathways through the activation of specic inhibitory pathways that receive modulatory actions from sensory and muscle affer- ent inputs in the rat spinal cord. 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P r o o f Journal: 221 Article: 3965 1 3I Author Query Form Please ensure you ll out your response to the queries raised below and return this form along with your corrections Dear Author During the process of typesetting your article, the following queries have arisen. Please check your typeset proof carefully against the queries listed below and mark the necessary changes either directly on the proof/online grid or in the Authors response area provided below Query Details Required Authors Response AQ1 Please check and conrm the author names and initials are correct. Also, kindly conrm the details in the metadata are correct. AQ2 Please check whether the mail ID "quiroz@sio.cinvestav.mx" should appear in the publication. AQ3 Please check and conrm the clarity of the sentence This injectionreexes AQ4 Collins et al. (1986) has been changed to Collins (1986) so that this citation matches the list. AQ5 Reference Kim et al. (2011) is cited in text but not provided in the reference list. Please provide reference in the list or delete these citations. AQ6 Quiroz et al. (2012) has been changed to Quirz-Gonzalez et al. (2012) so that this citation matches the list. AQ7 The term transmition has been changed to transmission in the article. Please check and approve. AQ8 References Baron and Saguer (1993), Eccles et al. (1963), Jimnez et al. (1987), Kerr et al. (1978), Ochoa (1994), Rudomin (2007) are given in list but not cited in text. Please cite in text or delete from list. AQ9 Please provide volume number for reference Quiroz-Gonzalez et al. (2013). A u t h o r