The Effect of Pre-Electroacupuncture on Nociceptive Discharges of Spinal Wide Dynamic Range Neurons in Rat

Introduction

The spinal dorsal horn (SDH) is the first station that relays somatosensory afferents. Spinal wide dynamic range (WDR) neurons in the deep laminae (IV-VI) play a critical role in nociception. WDR neurons respond to a wide range of stimulation which transmits innocuous and noxious information through Aβ-, Aδ - and C-fibers.¹ Previous studies showed that the WDR neurons exhibited progressive discharges in a graded manner depending on stimulus intensity, which manifested in an increase of discharge frequency and expansion of receptive fields (RFs).^2–4 Accordingly, the WDR neurons significantly contribute to the encoding of spatial and qualitative aspects of pain and integration of various afferent inputs.^5,6

Acupuncture analgesia (AA) is closely dependent on the stimulating intensities and locations of acupoints,^7–9 where different types of afferent fibers (Aβ, Aδ and C) were activated. Electro-acupuncture (EA) with low intensity at local acupoint could activate non-nociceptive Aβ-fiber to elicit segmental analgesia, while systemic analgesia produced by heterotopic EA intervention relies on sufficient activation of noxious Aδ- and C-fibers.^10,11 Therefore, distinct intensities of EA may exert analgesic effect through different mechanisms. Meanwhile, the preventive analgesic effects of pretreatment of EA (pre-EA) have also been reported in numerous clinical trials and experimental studies.^12,13 However, the activity of WDR neurons during pre-EA with different intensities remain elusive.

As a group of convergent neurons encoding different stimulus, WDR neurons may integrate acupuncture and pain signals in SDH. Indeed, previous studies suggested that the activities of WDR neurons were greatly relevant to AA and EA intervention was able to inhibit nociceptive discharges of WDR neurons in various pain models.^14,15 A recent study also showed that EA inhibited spontaneous firing of WDR neurons by activating A-fibers in muscular inflammatory pain in rats.¹⁶ Moreover, EA intervention at homotopic or heterotopic acupoints produced ameliorated effects on visceral pain via suppressing nociceptive discharges of WDR neurons in an intensity-dependent manner.^17–19 These studies indicated that the inhibitory effects of EA intervention on WDR neurons were closely related to the activation of different afferent fibers. However, there is a lack of consensus about WDR neurons integrating the pre-EA intervention and somatic nociception.

The aim of the present study was to explore whether pre-EA regulates the nociceptive activities of WDR neurons according to different intensities and the locations of intervention. We first identified the intensity thresholds of Aβ (T_Aβ), Aδ (T_Aδ) and C (T_C)-components of WDR neurons elicited by EA intervention. Then pre-EA with distinct intensities (T_Aβ, T_Aδ, T_C or 2T_C) was administered at local RFs, adjacent or contralateral non-RFs, respectively. Following the pre-EA intervention, nociceptive discharges of WDR neurons evoked by injection of hypertonic saline (HS) were observed. At last, the effects of pre-EA with different intensities and locations on discharges of WDR neurons were estimated.

Materials and Methods

Animals

Male Sprague-Dawley rats (200 ± 20 g) were provided by the SPF (Beijing) Biotechnology Co., Ltd. [experimental animal license number: SCXK(JING)2019−0010]. All animals were kept at a constant temperature of 24 ± 2°C with a 12-h light 12-h dark cycle and with free access to food and water. After a week of adjustable feeding, animals were randomly allocated to the normal, control, model and pre-EA group. The experimental procedures were approved by the Ethics Committee of the Institutional Animal Welfare and Use Committee of Acupuncture and Moxibustion Institute of China Academy of Chinese Medical Sciences. All manipulations were performed in accordance with the recommendations of the Guideline on the Humane Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China in 2006.

Microinjection of Neural Tracer

Under respiratory anesthesia with 2% isoflurane (0.5 L/min, RWD Life Science, People’s Republic of China), 5 μL 0.1% cholera toxin subunit B conjugates of Alexa Fluor-488 (AF488-CTB, C34775, Thermo, Germany) was slowly injected into the left gastrocnemius muscle of rats at a depth of 3–7 mm, right at the medial muscle belly. To prevent leakage, the microsyringe remained in place for 1 min after injection, and was then removed.²⁰

Perfusion and Tissue Processing

Three days after injection of neural tracer, the rats were deeply anesthetized intraperitoneally with urethane (1.5 mg/kg) and then transcardially perfused with 250 mL of 0.9% NaCl, followed by 250 mL of cold 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered solution (PB, pH 7.4). The lumbar (L)1–6 and sacral (S)1 of dorsal root ganglia (DRG) were collected and post-fixed in 4% PFA for 2 h and then cryoprotected in 25% sucrose at 4°C for 24 h. Using a cryostat (Thermo, Microm International FSE, Germany), DRGs were sliced transversely into 40 μm thick sections and mounted on silane-coated glass slides. After being coverslipped with 50% glycerin, all the labeled neurons in sections were observed and recorded with fluorescent microscope equipped with a digital camera (BX53, Olympus, Japan).²¹

Surgical Exposure of Spinal Cord

After rats were deeply anesthetized intraperitoneally with urethane (1.5 mg/kg), laminectomy was performed at the thoracic (T)12 to L2 to expose the lumbar enlargement (L4-6) of the spinal cord. Carefully removed dura mater of lumbosacral segments of spinal and tightly fixed the corresponding vertebrae in a rigid frame with clamps. Exposed area of spinal cord was covered with warm (37°C) artificial cerebrospinal fluid (CZ0516, Leagene, People’s Republic of China) during surgery and recording. A feedback-controlled heating blanket (ALC-HTP, Shanghai Alcott Biotech Co., People’s Republic of China) was used to monitor and maintain core body temperature at 37.0 ± 0.5°C.²²

Extracellular Recording of the SDH Neurons

Extracellular recording of SDH neurons was performed at L4-6 segments through a 5 MΩ parylene-coated tungsten microelectrode²³ (575,500, A-M Systems, Sequim, WA, United States) or a microelectrode array²⁴ (ASSY, Lotus Biochips, United States) (Figure 1A and B). After surgery, recording electrodes were inserted perpendicularly into the SDH at a depth of approximately 600–1400 μm from the dorsal surface and 0.3 mm lateral to the central vessel by a micromanipulator (DMA-1510, Narishige, Japan). The reference electrode was placed in the nearby muscle. Signals from parylene-coated tungsten microelectrode were amplified by a preamplifier (AM-1800, AM Systems, Sequim, WA, United States) with a bandwidth of 300 Hz-5 kHz and captured online using the CED 1401-plus data acquisition system and analyzed offline by Spike 2 package software (Cambridge Electronic Devices, Cambridge, United Kingdom). Microelectrode array was attached to the headstage using a custom connector and signals were amplified by a preamplifier (LB-0164-1, Blackrock Microsystems, United States) with a bandwidth of 250 Hz–5 kHz and captured and amplified online by a data acquisition system (Cerebus-128, Blackrock Microsystems, United States).

Figure 1 Recording of neurons’ activity in the spinal dorsal horn (SDH) by a microelectrode array and experimental procedure. Notes: (A) Illustration of a microelectrode array with 4 electrodes (750-μm depth, 32 channels). (B) Schematic diagram of recording with microelectrode array and position of microelectrode across Lamine II-VI noted with dotted lines. (C) Experimental timeline. Following 20s recording of background (BG) discharge, distinct intensities (TAβ,TAδ,TC or 2 TC) of EA intervention were applied at ipsilateral RFs, adjacent or contralateral non-RFs for 1 min. Immediately after EA intervention, HS was injected at left gastrocnemius muscle (identified as muscular RFs) of rats. Then, the neuronal discharge was recorded for 1 min.

Identification of WDR Neurons and Receptive Fields

WDR neurons were identified by a mechanical press stimulator (ALMEMO2450, Ahlborn, Germany) equipped with a blunt rubber tip 0.5 cm in diameter. The mechanical threshold for escape behavior was measured at gastrocnemius muscle in awakened rats, which was 149.1 ± 13.2 g (3 rats). Therefore, 60 and 200 g were determined as innocuous and noxious stimulation, respectively.²⁵ During extracellular recording, mechanical stimulation was applied at the muscular RFs. Neurons responding to both innocuous and noxious stimulation were identified as WDR neurons. Additionally, to discriminate the responses of WDR neurons between the skin and muscle, local cutaneous afferents were blocked by hypodermic injection of 50 µL lidocaine hydrochloride (Haerbin sanmashouyaoye Co., People’s Republic of China) above the RFs.²⁶ The present study focused on WDR neurons that responded only to pressure of the muscle but not to pinch of the skin.

Identification of Intensity Thresholds Activating Aβ-, Aδ- and C-Components of WDR Neurons

Identification of thresholds was performed at WDR neurons with RFs located at the anterior tibial muscle or the gastrocnemius muscle. Electrical stimulation was delivered through an electrical stimulator (PowerlabFE180, AD Instruments, Australia) at the RFs during extracellular single-unit recording. Intensity of electrical stimulation increased gradually from 0.1 mA (0.5 ms pulse width) until 3 components with corresponding latencies (Aβ, 0–20 ms, Aδ, 20–90 ms, C, 90–300 ms) appeared.²³ Intensity that evoked discharge more than 3 times (6 times in total, 5 s intervals) was identified as threshold of corresponding component (T_Aβ, T_Aδ or T_C).^27,28

EA Intervention

EA intervention was applied at ipsilateral RFs, adjacent or contralateral non-RFs. A pair of stainless steel acupuncture needles (0.18 mm diameter, 13 mm length; Beijing Zhongyan Taihe Medicine Co., People’s Republic of China) were inserted into the muscle at a depth of 5 mm. EA intervention was applied with intensity of T_Aβ, T_Aδ, T_C or 2T_C (0.5 ms pulse width) at a frequency of 2 Hz for 1 min (Figure 1C), which was delivered by an electrical stimulator.

Administration of Saline

6% hypertonic saline (HS) was prepared as noxious stimulus. Immediately after EA intervention was finished, 200 µL HS was administered through the placement of a 10 cm long PE-10 tube at left gastrocnemius muscle of rats in advance (Figure 1C). Injection was administered at depth of 5–7 mm within 10s. Rats in control group were injected with the same volume of 0.9% NaCl.

Statistical Analysis

All data were expressed as means ± standard error of the means (SEM). Statistical analysis was performed with SPSS 23 software. The Shapiro–Wilk test was used to evaluate whether these data fit normal distributions. Normally and non-normally distributed data were analyzed via parametric or non-parametric tests, respectively. Differences among multiple groups were analyzed with one-way analysis of variance (ANOVA) test followed by the LSD, SNK, or Dunnett’s T3 post hoc test. Differences with P < 0.05 were considered significant.

Results

Identification of WDR Neurons with Muscular Receptive Fields

To locate the exact segments of electrophysiological recording, the retrograde neural tracer was injected at left gastrocnemius muscle to identify the relevant sensory innervation. As shown in Figure 2A and B, the sensory neurons labeled by AF488-CTB were distributed from L3 to sacral (S)1 and concentrated at L5-6 DRGs, indicating that the sensory inputs of gastrocnemius muscle mainly projected to L5-6 SDH. Therefore, recording of WDR neurons was performed at L5-6 segments of the SDH. WDR neurons were identified by mechanical stimulation, including innocuous 60 g and noxious 200 g pressure as well as innocuous brushing at left gastrocnemius muscle and anterior tibial muscle. Neurons responded to both innocuous and noxious pressure but not brushing were identified as WDR neurons with muscular RFs (Figure 2C).^16,25 A total of 106 WDR neurons were recorded from 70 rats. Most WDR neurons were distributed at a depth of 650–1300 μm below the dorsal surface of the spinal cord, corresponding to laminae IV-VI of the SDH (Figure 2D).

Figure 2 General properties of WDR neurons with receptive fields (RFs) at left gastrocnemius muscle. Notes: (A) Representative images of retrogradely labeled primary sensory neurons (white arrows) in lumbar (L)4 and L5 dorsal root ganglia (DRG) associated with left gastrocnemius muscle. (B) The number of labeled sensory neurons in L3-6 and sacral (S)1 DRGs (n=3). (C) Representative discharge mode of WDR neurons evoked by 60 and 200 g pressure on muscular RFs and brush on local skin. (D) Percentages of WDR neurons distributed among different depths of SDH (106 neurons from 70 rats). All data were presented as means ± SEM.

Thresholds of Aβ-, Aδ- and C-Components of WDR Neurons

Typical discharge of WDR neurons to multiple types of primary sensory inputs can be separated into Aβ-, Aδ- and C-components by the corresponding latencies (0–20, 20–90 and 90–300 ms, respectively).²³ To determine the activation thresholds of 3 components of WDR neurons, increasing intensities of electrical stimulus (0.1–3 mA, 0.5 ms pulse width) was applied at the muscular RFs during extracellular recording (Figure 3A). Conduction velocity (CV) of different afferent fibers was calculated through dividing the distance between stimulus and recording site (about 0.12 m) by the latency, without regard to the synaptic delays occurring within the SDH. Elicited discharges of WDR neurons were shown in Figure 3B and Table 1. Aβ- was the fastest component (CV, 12.24 ± 8.86 m/s) with lowest threshold (T_Aβ, 0.53 ± 0.06 mA) and shortest latency (12.60 ± 4.97 ms). Aδ- was the slower component (CV, 3.36 ± 1.57 m/s) with higher threshold (T_Aδ, 0.90 ± 0.26 mA) and longer latency (44.26 ± 23.24 ms). C- was the slowest component (CV, 1.07 ± 0.28 m/s) with highest threshold (T_C, 2.06 ± 0.13 mA) and longest latency (117.88 ± 35.61 ms). Accordingly, intensity of T_Aβ, T_Aδ, T_C and 2T_C was determined as 0.5 mA, 1 mA, 2 mA and 4 mA in later experiments, respectively.

Table 1 Identification of the Threshold, Latency, and CV of Aβ-, Aδ- and C-Components of WDR Neurons (9 Neurons from 7 Rats) by Electrical Stimulation

Figure 3 Intensity thresholds of electrical stimulation activating Aβ, Aδ and C-components of WDR neurons. Notes: (A) Schematic diagram of experimental setup. Electrical stimulation (0.1–3 mA, 0.5 ms) was applied at muscular RFs of WDR neurons and action potentials of WDR neurons were recorded by a microelectrode inserted in the enlargement of spinal cord. (B) Representative discharges of Aβ (0–20 ms latency), Aδ (20–90 ms latency) and C (90–300 ms latency) components of WDR neurons evoked by electrical stimulation with intensity of 0.5, 1 and 2 mA, respectively (9 neurons from 7 rats).

Nociceptive Discharges of WDR Neurons Elicited by HS

After identification of the general properties of WDR neurons, the nociceptive discharges were elicited by injection of HS at left gastrocnemius muscle of rats (Figure 4A). As expected, 11 WDR neurons from 9 rats exhibited robust discharges immediately after HS injection, while no firing was evoked by 0.9% NaCl (Figure 4B and C). Since the discharges of WDR neurons induced by HS exhibited short duration and decreased rapidly, the firing rates during the 1st 1 min after injection were further analyzed. The discharge frequency gradually decreased over time. During the 1st 20s it was 28.3 ± 11.6 Hz, but it decreased sharply to 16.2 ± 10.5 Hz in the 3rd 20s (P < 0.05, Figure 4C). Further analysis of firing rates of WDR neurons in the 1st 20s course showed that a majority of WDR neurons (55%) fired at a rate of 20–30 Hz, 18% WDR neurons fired at 10–20 Hz, and 27% WDR neurons fired at 30–60 Hz (Figure 4D). Therefore, the activities of WDR neurons within the 1st 20s after HS were selected to be observed.

Figure 4 Injection of hypertonic saline (HS) at RFs evoked robust discharges of WDR neurons. Notes: (A) Schematic diagram of experimental setup. 200 μL HS was injected at gastrocnemius muscle and action potential of WDR neurons was recorded by a microelectrode array inserted in L5-6 segment of spinal cord. (B) Representative traces of discharges of WDR neurons in the 3 groups. (C) Discharge frequency of WDR neurons in the 3 groups. Frequency of model group was further analyzed during 0–20, 20–40 and 40–60 s post-injection of HS (5 neurons from 3 rats in normal group, 6 neurons from 3 rats in control group, 11 neurons from 9 rats in model group). (D) Percentages of WDR neurons with different discharge frequency after injection of HS within 20s (11 neurons from 9 rats). All data were presented as means ± SEM. *, compared with normal, *P < 0.05, **P < 0.01, ***P < 0.001. #, compared with control, #P < 0.05, ##P < 0.01, ###P < 0.001. &Compared with 0–20s, &&P < 0.05.

Effects of Pre-EA at RFs on Nociceptive Discharges of WDR Neurons Evoked by HS

There were quite a number of WDR neurons with RFs located at both gastrocnemius muscle and anterior tibial muscle. Pre-EA of T_Aβ (0.5 mA), T_Aδ (1 mA), T_C (2 mA) or 2T_C (4 mA) was administered at left anterior tibial muscle. Immediately after pre-EA intervention, HS was applied to the ipsilateral gastrocnemius muscle. Nociceptive activities of WDR neurons were compared to evaluate the effects of pre-EA with distinct intensities at RFs. Figure 5A displayed representative firing traces of WDR neurons in 4 EA groups. Compared with the model group (28.3 ± 11.6 Hz), pre-EA of T_Aδ (8.92 ± 4.26 Hz, P < 0.05), T_C (10.20 ± 6.50 Hz, P < 0.05) and 2T_C (6.62 ± 3.04 Hz, P < 0.01) significantly reduced discharge frequencies of WDR neurons (Figure 5B), with suppressive rates of 68.48%, 63.96% and 76.61%, respectively. However, pre-EA of T_Aβ had no inhibitory effect on WDR neurons. These results suggested that pre-EA administered at the RFs required Aδ- or C- afferents to inhibit nociceptive responses of WDR neurons.

Figure 5 Changes of noxious firing of WDR neurons after pre-EA at ipsilateral RFs or non-RFs with different intensities. Notes: (A, B) Representative discharge traces (A) and frequency (B) of WDR neurons after pre-EA at ipsilateral RFs (6 neurons from 3 rats in TAβ group, 6 neurons from 4 rats in TAδ group, 8 neurons from 3 rats in TC group, 5 neurons from 4 rats n=5 in 2TC group). (C, D) Representative discharge traces (C) and frequency (D) of WDR neurons after pre-EA at ipsilateral non-RFs (8 neurons from 8 rats in TAβ group, 5 neurons from 4 rats in TAδ group, 6 neurons from 3 rats in TC group, 5 neurons from 3 rats in 2TC group). All data were presented as means ± SEM. *, compared with normal, *P < 0.05, **P < 0.01, *** P < 0.001. #Compared with control, #P < 0.05, ##P < 0.01, ###P < 0.001. &Compared with model, &P < 0.05, &&P < 0.01.

Effects of Pre-EA at Adjacent Non-RFs on Nociceptive Discharges of WDR Neurons

WDR neurons that responded to mechanical stimulation only on the gastrocnemius muscle but not the anterior tibial muscle were observed in this part. Likewise, pre-EA of 4 intensities was administered at left anterior tibial muscle (identified as adjacent non-RFs) and HS was applied to the ipsilateral gastrocnemius muscle. As shown in Figure 5C and D, discharge frequency of WDR neurons in T_Aβ (11.9±5.37 Hz, P < 0.05), T_Aδ, (9.32 ± 7.66 Hz, P < 0.05), T_C (9.43±2.85 Hz, P < 0.05) and 2T_C (4.32 ± 4.46 Hz, P < 0.01) groups decreased significantly compared with that of the model (Figure 5D). The inhibition rate of 4 groups was 57.95%, 67.06%, 66.68% and 84.73%, respectively. These results indicated that pre-EA at adjacent non-RFs could effectively inhibit the nociceptive activities of WDR neurons, especially pre-EA of T_Aβ. The other intensities of pre-EA at adjacent non-RFs produced the same effects as that in the RFs.

Effects of Pre-EA at Contralateral Non-RFs on Nociceptive Discharges of WDR Neurons

It has been reported that heterotopic EA intervention can elicit extrasegmental analgesia.^7,29 Meanwhile, the activities of the WDR neurons could be inhibited by heterotopic stimulation with high intensity in different pain models of rats.^30,31 Here, we examined the effective intensity of pre-EA at right anterior tibial muscle (contralateral non-RFs) which suppressed nociceptive firing of WDR neurons in an acute muscle pain model. Interestingly, pre-EA of T_C and 2T_C remarkably reduced HS-evoked WDR neuronal activities to 7.49 ± 4.70 Hz and 6.25 ± 6.74 Hz, and the inhibiting rate was 73.53% and 77.92%, respectively (Figure 6, P < 0.01). However, no obvious alleviation was observed for pre-EA with the intensity of T_Aδ (13.58±5.07 Hz, Figure 6, P > 0.05) and T_Aβ (15.74 ± 5.36 Hz, Figure 6, P > 0.05) on firing frequency of WDR neurons. Overall, these results indicated that intensity of heterotopic pre-EA intervention should reach at least T_C to suppress nociceptive discharges of WDR neurons.

Figure 6 Changes of activity of WDR neurons after contralateral pre-EA with different intensities. Notes: (A) Representative traces of discharge of WDR neurons in the 4 groups. (B) Discharge frequency of WDR neurons in the 7 groups (8 neurons from 6 rats in TAβ group, 5 neurons from 4 rats in TAδ group, 8 neurons from 3 rats in TC group, 5 neurons from 3 rats in 2TC group). All data were presented as means ± SEM. *, compared with normal, **P < 0.01, ***P < 0.001. #Compared with control, ##P < 0.01, ###P < 0.001. &Compared with model, &&P < 0.01.

Discussion

In the present study, the inhibitory effects of pre-EA with distinct intensities and locations on nociceptive discharges of WDR neurons were compared. Specifically, pre-EA of T_Aβ, T_Aδ, T_C or 2T_C at ipsilateral adjacent non-RFs could inhibit nociceptive discharges of WDR neurons, while in local RFs, only T_Aδ, T_C or 2T_C of pre-EA could exert inhibitory effects. Meanwhile, at contralateral non-RFs, intensity of pre-EA should reach at least T_C to suppress discharges of WDR neurons. These results suggested that pre-EA could inhibit nociceptive activities of WDR neurons and the effective intensities varied depending on the locations of stimulation.

Intramuscular injection of HS is commonly used to evoke acute muscle pain.^32–34 Application of HS at gastrocnemius muscle induced mechanical hyperalgesia^35,36 and c-Fos expression in deep (IV-VI) lamina of SDH of L4-6 segments.^37,38 In this study, WDR neurons in deep lamina IV-VI of SDH exhibited obvious discharges, indicating that WDR neurons were activated following intramuscular injection of HS. Interestingly, pretreatment of EA effectively inhibits nociceptive activities of WDR neurons and the effects were dependent on intensities and locations of intervention, showing the preventive analgesic effects of pre-EA.

Behavior and electrophysiological evidence have demonstrated that activation of low-threshold mechanoreceptors could alleviate pain symptoms and inhibit the spontaneous discharges of dorsal horn neurons.^39–42 This analgesic effect has been generally explained by the gate control theory, which suggests that the nociceptive (Aδ/C) inputs are gated by feed-forward activation of non-nociceptive (Aβ) afferents in SDH. The spinal WDR neurons receive inputs from both non-nociceptive (Aβ) and noxious (Aδ and C) afferents.⁴³ Activation of A-fibers inhibited the nociceptive discharges of WDR neurons evoked by C-fiber.⁴⁴ In addition, the underlying mechanisms of the segmental analgesia of acupuncture have been suggested to be associated with the gate control theory.^10,16,45 In this study, pre-EA with low intensity T_Aβ inhibited nociceptive discharges of WDR neurons, indicating that anti-nociceptive effects of pre-EA may also be related to the gate control theory. However, the phenomena only occurred when pre-EA with T_Aβ was administered at adjacent non-RFs. As we know, there are also inhibitory fields (adjacent non-RFs) around the excitatory fields (known as RFs) of WDR neurons.⁴⁶ It was demonstrated that tactile stimulus of adjacent non-RFs could reduce the activities of WDR neurons to nociceptive stimulation.^47–49 That is why pre-EA of T_Aβ at non-RFs was effective rather than at RFs. Besides, pre-EA of T_Aδ at both RFs and non-RFs was effective on the nociceptive firing of WDR neurons.

Apart from local acupoints, contralateral or distal acupoints are also frequently used to relieve pain.^7,50,51 Different from segmental analgesia induced by homotopic acupoint, systemic analgesia by heterotopic acupoints requires relatively higher intensity which is enough to activate myelinated fibers (thinly Aδ- and/or C-fibers).^9,52,53 Mechanisms underlying systemic AA involve the diffuse noxious inhibitory controls (DNICs),^54–57 which refer to the strong inhibitory effects on spinal WDR neurons by a nociceptive stimulus applied to any part of the body distinct from their RFs.⁵⁸ In the present study, heterotopic nociceptive afferent stimulation by pre-EA inhibited nociceptive discharges of WDR neurons, indicating the information was convergent and integrated by WDR neurons. Specifically, ipsilateral pre-EA with high intensity (T_Aδ, T_C or 2T_C) significantly decreased discharge frequency of WDR neurons. While at contralateral non-RFs, only intensity higher than T_C could generate inhibitory effects on WDR neurons. This was consistent with a previous study which reported that threshold of Aδ-fiber activation showed less effective when administered at contralateral acupoints.¹¹ Consequently, these results implied that inhibitory effects of pre-EA with high intensity on nociceptive discharges of WDR neurons may be through DNIC pathway, and the extent to which DNIC was triggered varied with distinct intensities.

Activation of different somatic afferents play important roles in AA. Previous studies determined the intensity of EA by recordings of nociceptive reflexes (RIII reflex).^10,11,16 As the C-fiber reflex was a kind of nociceptive response,^59,60 it was difficult to find out the threshold of Aβ-fiber. Other studies employed single-unit recording of primary afferent fibers or sensory neurons to determine the intensity.^25,45,61 In the present study, to better investigate the inhibitory effects of pre-EA on spinal WDR neurons, it was more reliable to determine the intensities of EA by thresholds of the 3 components of WDR neurons. Moreover, this measurement may reveal the integration of pre-EA and nociceptive signaling in the dorsal horn.

However, the present study has some limitations. We only analyzed discharges of WDR neurons by in vivo electrophysiological recording, further study is needed to evaluate whether pre-EA could relieve acute pain behavior. Additionally, we merely examined the effects of pre-EA at spinal level. Supraspinal components may also participate in this complex loop, especially in systemic analgesia of heterotopic pre-EA. Further studies should be performed to elucidate the supraspinal mechanisms of antinociceptive effects of pre-EA with distinct intensities.

Conclusion

In summary, different afferent stimulation of pre-EA and nociceptive information were convergent and integrated by WDR. Pre-EA with distinct intensities inhibits noxious discharges of WDR neurons depending on the locations of stimulation. Our studies suggest novel therapeutic strategies for parameters’ selection of pre-EA for acute pain.