The purpose of this study was to investigate whether opposing electroacupuncture (EA) could produce similar analgesic effects as operated side EA after knee surgery in rats. Sprague Dawley rats were randomly divided into the sham surgery group, and three surgery groups: opposing EA, operated side EA, and model. After surgery, compared with the sham surgery group, three kinds of pain behavior test methods (mechanical withdrawal threshold (MWT), cumulative pain score [CPS], and mechanical hypersensitivity of knee) were used to assess the pain behavior of the rats in the surgery groups. After knee surgery, the three surgery groups were intervened for three consecutive days: EA on the nonoperated side in the opposing EA group, EA on the operated side in the operated side EA group, and no intervention in the model group. It was shown that MWT was higher and CPS was lower in the two EA groups than in the model group on the first and second days after surgery. On the third day after surgery, MWT in the two EA groups was the highest among the 3 days, CPS was the lowest among the 3 days, and the number of nonvocalizations in rats also increased compared with the model group. Moreover, the MWT of the nonoperated side increased more in the opposing EA group than in the model and operated side EA groups. This indicated that both opposing EA and operated side EA methods can be used to relieve pain after knee joint surgery.
Total knee arthroplasty (TKA) is a common orthopedic surgical procedure [
In previous studies, the most commonly used animal experimental model for postoperative acute pain was the hyperalgesia induced by the plantar skin incision model, which is useful for evaluating the phenotype of postoperative pain [
Acupuncture has always been a good supplement and alternative therapy for pain [
Forty-nine healthy adult Sprague Dawley rats, weighing 220–260 g, of both sexes, were included. They were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in a 12 h/12 h light/dark cycle environment with five rats in each cage in the laboratory animal center of Shanghai University of Traditional Chinese Medicine. They were quiet, at 24 ± 1°C and humidity of 60 ± 5%. The experiment was approved by the Animal Room Ethics Committee of Shanghai University of Traditional Chinese Medicine (No. PZSHUTCM190830007). We strictly referred to and implemented the ethical guidelines of the International Association for the Study of Pain [
All Sprague Dawley rats were numbered according to their weight and then randomly divided into four groups as follows: the sham surgery group (12 rats), and three surgery groups (the opposing EA (12 rats), operated side EA [13 rats], and model [12 rats] groups).
This study used the knee surgery rat model developed by Dr. Buvanendran et al. We slowly poured 1.5% isoflurane into the air anesthesia machine, the pipe switch was adjusted to ensure that the induction concentration of anesthesia was 4%, and the concentration was maintained at 2%. From the supine position, the rats were bent 90 degrees after depilation around the right knee joint and firmly supported on both sides by the blunt end of the stereotactic ear rod (Figure
Photographs of the procedure of KSM.
The anesthetic method remained the same; the sham surgery animals only had a skin incision on the knee.
After the recovering from anesthesia, the rats were placed in the test cage to adapt for 20 min. Pain was measured, and the mechanical withdrawal threshold (MWT) was measured. The pain protective behavior appeared, and the decrease in pain threshold indicated that the model was successful.
Rats were intervened continuously for 3 days, once a day, for 30 min in each intervention (Figure
Experimental procedures, acupoints, and intervention. (a) Abbreviations: MWT, mechanical withdrawal threshold; CPS, cumulative pain score; MHK, mechanical hypersensitivity of knee. (b) SD rats received KSM on the right knee joint, EA on the the contralateral (left) side in opposing EA group, while EA on the operated (right) side in the operated side EA group. (c) The acupoints futu (ST32), housanli (ST36), and yangling quan (GB34) are located on lateral lower leg, yingling quan (SP9) on the medial lower leg, all are around the knee joint of rats.
On the first day after surgery, the rats were bound and fixed on the acupuncture fixator, and then aseptic acupuncture (0.25 mm in diameter, 13 mm in length, Hwato Brand, Suzhou Medical Appliance Factory, China) was inserted to four points of the nonoperated side of the rats (Figure
On the first day after surgery, the rats were bound and fixed on the acupuncture fixator, and then aseptic acupuncture (0.25 mm in diameter, 13 mm in length, Hwato Brand, Suzhou Medical Appliance Factory, China) was inserted to four points of the operated side of the rats, namely, Futu (ST32), Housanli (ST36), Yanglingquan (GB34), and yinlingquan (SP9), with a 2 mm depth. The two connecting wires of the EA instrument were, respectively, connected at two pairs of acupoint needle handles (GB34 to ST32, ST36 to SP9), with a continuous wave of 2 Hz and a strength of 1 mA.
On the first day after surgery, the rats were bound and fixed on the acupuncture fixator without intervention.
On the first day after surgery, the rats were bound and fixed on the acupuncture fixator without intervention.
The MWT of 50% was calculated by the “up-and-down” method with von Frey hair [
The test time was 2 h and 1–3 days after surgery.
The cumulative pain scoring method of the model was as follows: the rats were placed in plastic cages on the pain test bracket, and the method of adapting to the test environment was the same as described in the abovementioned section. The position of the right hind paws of the rats was closely observed within 1 min and repeated every 5 min. The observation procedure lasted 1 h (12 recordings). According to the position of most right hind paws during this 1 min observation period, 0, 1, and 2 scores were given, where (1) score = 0: it can completely contact the grid floor, and it is completely load-bearing; (2) score = 1: the rear paws are partially or just in contact with the grid floor and are partially load-bearing; and (3) score = 2: the hind paws are completely separated from the grid floor or suspended on the tail and cannot bear the load [
The test time was 2 h and 1–3 days after surgery.
The hyperalgesia reaction was evaluated by squeezing the knee joint as follows: the knee joint was held with the thumb and forefinger and squeezed firmly (constant pressure kept for 3 s) to determine the hyperalgesia response of the rats. The hyperalgesia reaction during five trials was recorded, and each operation was recorded as Yes/No. The data were analyzed as the number of times that nonvocalizations were made [
The test time was 2 h and the 3rd day after surgery.
The figures were drawn using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). SPSS statistical software (SPSS version 26.0, IBM Corp, New York) was used for statistical analyses of the experimental data. Univariate analysis of variance (ANOVA) was used for the measurement data with normal distribution and homogeneity of variance among the groups. The Bonferroni method was used when the variance was homogeneous, whereas the Tamhane method was used when the variance was not uniform. The Kruskal-Wallis nonparametric test was used for non-normally distributed data. The pain behavior data collected belong to repeated measurement data and the normal distribution as means ± standard deviations (
A total of 49 Sprague Dawley rats were purchased. Of those, 37 rats underwent surgery, of which 2 died due to excessive anesthesia. There were 12 rats in the sham surgery group, and 1 died because of excessive anesthesia. Therefore, this study effective study was 46 rats. After the surgery or sham surgery, the wound healed well, the fur was smooth and tidy, the appetite was good, and the activity was slow because of pain in the right lower limb. There were no significant differences in the weight of the rats before and after intervention among the four groups (
As shown in Figure
Mechanical withdrawal threshold.
MWT of KSM rats (median (Q1, Q3), g).
Group | 2H | 1D | 2D | 3D | ||
---|---|---|---|---|---|---|
Operated side | Sham surgery | 11 | 29.35 (23.66, 54.59) | 23.66 (23.66, 29.35) | 34.07 (23.66, 60.00) | 34.07 (23.66, 60.00) |
Model | 11 | 12.39 (12.39, 14.21) | 12.39 (12.39, 12.81) | 12.39 (9.16, 12.39) | 12.39 (11.30, 17.82) | |
Opposing EA | 12 | 12.39 (9.44, 13.47) | 23.66 (17.77, 23.66) | 23.66 (17.87, 60.00) | 57.30 (23.66, 60.00) | |
Operated side EA | 12 | 12.39 (9.79, 18.41) | 23.66 (15.13, 23.66) | 28.87 (18.59, 58.65) | 23.66 (23.66, 28.79) | |
Nonoperated side | Sham surgery | 11 | 60.00 (60.00, 60.00) | 60.00 (60.00, 60.00) | 60.00 (60.00, 60.00) | 60.00 (60.00, 60.00) |
Model | 11 | 60.00 (22.98, 60.00) | 60.00 (23.66, 60.00) | 26.13 (12.39, 60.00) | 23.66 (20.75, 60.00) | |
Opposing EA | 12 | 60.00 (40.55, 60.00) | 60.00 (36.54, 60.00) | 60.00 (55.94, 60.00) | 60.00 (60.00, 60.00) | |
Operated side EA | 12 | 60.00 (23.15, 60.00) | 60.00 (24.28, 60.00) | 60.00 (40.55, 60.00) | 26.51 (23.15, 60.00) |
As shown in Figure
As shown in Figure
CPS and number of nonvocalizations.
CPS and number of nonvocalizations of KSM rats (mean ± SD).
Group | 2H | 1D | 2D | 3D | ||
---|---|---|---|---|---|---|
CPS | Sham surgery | 11 | 2.82 ± 1.92 | 2.09 ± 1.45 | 1.64 ± 1.12 | 1.55 ± 1.04 |
Model | 11 | 10.09 ± 2.12 | 10.00 ± 3.41 | 9.00 ± 3.74 | 8.36 ± 3.14 | |
Opposing EA | 12 | 9.33 ± 2.19 | 5.75 ± 3.08 | 5.08 ± 3.00 | 3.50 ± 2.58 | |
Operated side EA | 12 | 9.67 ± 2.74 | 5.42 ± 4.21 | 5.25 ± 3.17 | 4.25 ± 2.05 | |
Number of nonvocalizations | Sham surgery | 11 | 4.36 ± 0.67 | — | — | 4.55 ± 0.69 |
Model | 11 | 1.55 ± 1.04 | — | — | 1.36 ± 1.12 | |
Opposing EA | 12 | 1.75 ± 1.14 | — | — | 3.58 ± 1.38 | |
Operated side EA | 12 | 1.92 ± 1.24 | — | — | 3.50 ± 1.00 |
There was no significant difference in CPS on the first (one intervention) and second days (two cumulative interventions) (
As shown in Figure
As a kind of subjective discomfort, pain is often manifested in the behavioral representation of pain in animals, including lifting, withdrawing, limping, licking, and so on. In this study, two methods were used to calculate the MWT and CPS. During the study, the von Frey fair with corresponding g number was used to stimulate the same plantar position of the operated and the nonoperated sides. The rats' foot retraction reaction was observed, and the corresponding “X” and “O” marks were recorded. Finally, the 50% foot retraction threshold was calculated using the recorded data for statistical analyses. This method is widely used in the detection of mechanical pain threshold of animals [
In the present study, it was found that both opposing EA and operated side EA could improve the mechanical pain threshold of the operated side of KSM rats, unlike those in the model group. There were a total of three interventions, and the effect was the best at the time of three cumulative interventions. The mechanical pain threshold of opposing EA group was similar to that of the operated side EA group.
Peripheral sensitization is characterized by (primary) hyperalgesia in the operated area, which in turn is characterized by a stronger response of sensitized nociceptors to suprathreshold stimuli and a lower activation threshold. Central sensitization is responsible for spreading pain and hyperalgesia to uninjured tissues (secondary hyperalgesia) and participates in the pain transmitting neurons of the central nervous system, usually located in the dorsal horn of the spinal cord [
The CPS method was similar as the MWT test method. In this method, the behavioral performance of the lower limbs of rats with weight-bearing can be directly observed and recorded, and pain scores can be accumulated to evaluate the pain of rats after surgery or intervention. The method is simple and intuitive. In this experiment, the CPSs of the two EA groups were lower than those of the model group. The pain score of the two EA groups were the lowest at the time with three cumulative interventions, and then there was no significant difference between the two EA groups.
The method of knee mechanical hypersensitivity was referred to original paper of KSM [
Through the measurement of the above three pain behaviors, it was verified that the KSM is also suitable for the study of acupuncture analgesia. The analgesic effect of the opposing needling method is equivalent to the analgesic effect of acupuncture on the operated side, which is similar to the results in many other research researches [
We explored the regulation of pain after knee surgery in rats through opposing EA and operated side EA and found a similar analgesic effect of the two EA methods from the animal behavior characterization, which suggests the clinical application of opposing needling is an effective way of pain management after TKA surgery. However, the mechanism of acupuncture analgesia by opposing needling is unclear. Acupuncture signals are transmitted from the periphery, and the spinal cord is the first station to process pain and acupuncture information [
Opposing EA can effectively improve the plantar mechanical pain threshold of operated side and contralateral side of rats after knee joint surgery, reduce the cumulative pain scores of rats, and reduce the mechanical hypersensitivity of the knee. Further, the analgesic effect is similar to that of the operated side EA method. This experiment provides an effective supplementary and alternative treatment for postoperative analgesia of TKA. However, the principle of its analgesic mechanism needs further study.
The data used to support the findings of this study are available upon request from the corresponding author.
Hai Huang and Xiu-ling Song should be considered co-first authors.
The authors declare that they have no conflicts of interest and no financial interests regarding the publication of this article.
Hai Huang and Xiu-ling Song contributed equally to this work.
The facilities and the examination of the Animal Ethics Committee for this study were provided by the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine. We thank Dr. AnFeng Xiang for the technical guidance.