Letter to the Editor Comment on (Comparison of Electroacupuncture in Restrained and Unrestrained Rat Models) Authors' Contribution

Abstract

Although acupuncture researchers have conducted studies using rats for decades, there is no established method for controlling the animals while performing acupuncture. Some choose to use general anesthesia Thirty-two male Sprague-Dawley rats were randomly divided into an anesthetized (AN) group and a restrained (RS) group, which were further divided into four subgroups each ( = 4 for all subgroups) consisting of (1) the sham subgroup, which had their chest opened and the heart exposed but were sutured without further intervention; (2) the IR subgroup; (3) the IR + EA subgroup (EA on PC6, ST36 for 30 min); and (4) the IR + placebo subgroup (EA on nonacupoints without conduction of electricity). On the first day, IR injury was induced in all rats (except the sham subgroups) by placing a slipknot (5-0 silk) around the left anterior descending coronary artery and releasing it after 40 min of ischemia. During the whole process, rats were anesthetized with ketamine (75 mg/kg) and xylazine (2 mg/kg) and mechanically ventilated (Harvard Apparatus, MA, USA). On the first day, the IR + EA subgroups in both AN and RS groups were anesthetized prior to surgery and preconditioned with EA on the left PC6 and ST36 starting 20 min 2 Evidence-Based Complementary and Alternative Medicine after ischemia was induced to 10 min after the snare was loosened. The IR + placebo subgroups were treated with EA on nonacupoints without conduction of electricity. From the second day to the fifth day, the IR + EA subgroup in the AN group received EA for 30 min every day under anesthesia, with additional anesthesia administered intramuscularly if needed (but to a minimum degree); the IR + EA subgroup in the RN group received the same treatment immobilized in the restrainers. Similarly, placebo EA was conducted in the IR + placebo subgroups in both the AN and RS groups. All EA stimulation was administered with acupuncture needles (0.20 × 30 mm, Dongbang Acuneedle Co, Gyeonggi-Do, Korea) with a frequency of 2 Hz for 30 min. To evaluate LV function, rats were anesthetized on the last day, and cardiac function was measured using twodimensional transthoracic echocardiography (Vivid Q; GE Medical Systems, Milwaukee, WI, USA) with a 12 MHz probe. M-mode echocardiography of the LV was performed at the papillary muscle level, guided by two-dimensional short-axis images. LV cavity size was measured during at least three beats in each projection and averaged. The m-mode images yielded systolic and diastolic wall thicknesses (anterior and posterior), and LV end-systolic and end-diastolic diameters. LV fractional shortening was calculated as (LVEDD − LVESD)/LVEDD * 100. All values are expressed as mean ± SE. Results were analyzed using the Kruskal-Wallis nonparametric test for multiple comparisons, and values less than 0.05 were considered statistically significant. In our findings, improvement in cardiac function was similar in the IR + EA subgroups of both AN and RS groups, but there were different results in the placebo subgroups. While the LVEF of the AN placebo subgroup did not improve (IR, 60.30 ± 5.49; IR + EA, 70.56 ± 4.42; IR + placebo, 54.88 ± 2.12, < 0.05), the LVEF in the RS placebo subgroup improved so that the results did not differ significantly compared to the treatment group (IR, 58.83 ± 5.80; IR + EA, 67.03 ± 3.40; IR + placebo, 60.30 ± 5.49, > 0.05). Among many possible factors that could have produced the additional effects, we believe frequent reinsertion of acupuncture needles was a significant factor. In the restrained group, the rats frequently flexed their muscles to try to escape from the restrainers, which made the acupuncture needles fall out. Although the needles at ST36 mostly stayed intact, the ones at PC6 were a problem, as the needles were inserted to a shallower depth of approximately 5 mm. Every time the needles fell out, the researchers had to stick them back in, which increased stress and produced additional somatosensory stimulation. Unlike the RS placebo subgroup, the AN placebo subgroup (which did not require reinsertions) showed no significant improvement in LVEF, and in a confirmatory trial that limited the number of reinsertions per session to less than three, the LVEF of the RS placebo subgroup did not improve. Our observation is noteworthy because in the study by Zhang et al. Much more experiments to standardize and optimize research methods in acupuncture-related preclinical studies should be conducted. Novel methods to control rats must be continuously explored and researchers should know that differences in handling techniques can unwittingly expose well-designed studies to confounding factors, reproducibility failures, and limitations. Both of the chosen methods in our study do have their limitations, sedating animals prior to every acupuncture session is not feasible, and restraining rats also induces high levels of stres