The concept of preemptive analgesia was coined at the beginning of the 20th century [1], when the association of local and inhalation anesthesia was recommended to prevent postoperative pain. There is a consensus that preemptive multimodal pharmacological analgesia, when compared with postoperative, minimizes central and peripheral sensitization of the incisional and lesional phases, better controls pain, and reduces the need for postoperative analgesia [2,3].

Non-steroidal anti-inflammatory drugs (NSAID) and opioids are the most commonly used drugs to treat perioperative pain in dogs [4], however, the adverse effects of NSAID include gastrointestinal and renal lesions and increased clotting time [4,5] and of opioids include dependence, sedation, respiratory depression, nausea, emesis, serotonin syndrome, and hyperalgesia [6-8]. Various animal pain guidelines [9,10] consider acupuncture as an effective analgesic modality to control pain in dogs. Preoperative acupuncture and its modalities provide comparable or superior postoperative analgesia to opioids and NSAIDs in dogs [11-16] and the Large intestine 4, Spleen 6, and Stomach 36 acupoints are some of the most commonly used for this [17-20]. Performing preoperative acupuncture in unmanageable animals is a major limitation of this procedure, however, this drawback could be overcome by performing acupuncture in the immediate postoperative period when the residual effect of anesthetics would provide sedation and facilitate the procedure. A gap to be filled is whether acupuncture is also effective for controlling postoperative pain if used after nociceptive stimulation, as reported in humans [21-23]. To our knowledge, there is no comparison, in any species, between the preemptive versus postoperative analgesic efficacy of acupuncture. Like for analgesic drugs, we hypothesized that postoperative acupuncture is a worse analgesic than preemptive acupuncture or meloxicam.

This study aimed to compare the preemptive or postoperative analgesic efficacy of acupuncture, in relation to the preemptive use of meloxicam in dogs submitted to ovariohysterectomy.

This is a horizontal prospective positive control, randomized, blinded study approved by the School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp) Ethical Committee for the Use of Animals in Research, under protocol 0133/2019. All owners signed a Free and Informed Consent Form.

Thirty-six dogs were randomly divided (www.randomizer.org) into three groups respectively: GA - preemptive acupuncture, GPA - postoperative acupuncture, and GM - preemptive meloxicam (0.2 mg/kg intravenous - IV). The random allocation sequence was performed by an employee of the veterinary practice where the study was carried out. ACZBF was in charge of inviting and designating participants to each specific function. Recruitment of dogs occurred from May to July 2019 and the study occurred between September and November 2019.

As inclusion criteria, the female dogs were required to be manageable, non-pregnant, and healthy according to clinical and laboratory tests (complete blood count, urea, creatinine, alkaline phosphatase, and alanine aminotransferase). Dogs that did not meet these criteria were excluded. The animals were admitted 24 hours before surgery and placed in individual kennels (120 × 80 × 60 cm) with water and food.

A single anesthetist (CC) blind to treatments was responsible for anesthesia. A single certified veterinary acupuncturist (JCR), performed the acupuncture sessions. Another veterinarian performed the blind assessment of pain (ACZBF).

After 6 hours of fasting, the evaluator measured the degree of sedation [24] and pain using the short version of the Glasgow pain scale [25]. The dogs were sedated with 0.05 mg/kg of acepromazine (Acepran 0.2%, Vetnil, Brazil) intramuscularly (IM), followed by cephalic vein cannulation (Safelet Etfe, Nipro Medical LTDA, Brazil). Benzathine penicillin (30,000 IU/kg) (Penikel LA, Ceva, Brazil) was administered IM. The GM dogs received meloxicam (0.2 mg/kg) (Flamavet 0.2%, Agener União, Brazil) IV and dogs from the GA and GPA received 0.9% IV saline. Western medical acupuncture was performed once on the floor on a rubber mat in a quiet, traffic-free room immediately afterwards in the GA (before surgery) and after surgery in the GPA. Acupuncture dry needles of 0.25 × 3 mm (Goldlife, Huan Qiu, China) were inserted bilaterally in the acupoints Large intestine 4, Spleen 6, and Stomach 36 (Fig. 1) [26]. The needles were rotated in both directions until the firm adhesion of the needle at the site, trying to simulate the Deqi sensation [27]. The needles remained in position for 20 minutes. After sedation and administration of meloxicam (GM) or saline (GPA), a “sham acupuncture session” was simulated for 20 minutes before surgery only by placing the dogs on the table but not performing any needling. In the GA and GM dogs, this procedure was performed immediately after surgery.

Figure 1. Anatomic location of Large intestine 4 (5 mm depth, located on the medial side of the thoracic limb between the first and the second metacarpal bone, at the midpoint of the 2nd metacarpal bone on the radial side - https://youtu.be/MxEQEoGTH4o), Spleen 6 (2 mm depth, located on the medial caudal region of the tibia, three tsun, proximal to the medial malleolus of the tibia. Three tsun corresponds to the width between the lateral and medial side of the distal interphalangeal joint - https://youtu.be/BsuoORX8JiU), and Stomach 36 (1 cm depth, located on the lateral region of the pelvic limb in a depression lateral to the tibial tuberosity, three tsun distal to the patella on the protrusion of the cranial tibial muscle - https://youtu.be/t9mxZ_79-Jc) acupoints.

About 5 mg/kg of propofol (Provive 10 mg/ml, Claris, Brazil) was used for induction of anesthesia until orotracheal intubation was possible. Anesthesia was maintained with isoflurane (Isoflurane 100 ml, BioChimico, Brazil) diluted in 100% oxygen using an avalvular (dogs < 5kg) or valvular (dogs > 5 kg) circuit. Lactate Ringer (2 ml/kg/h, IV) (Solução de Ringer lactato, Eurofarma, Brazil) was administered during anesthesia and fentanyl (2 µg/kg, IV) (Fentanest, Cristália, Brazil) was administered immediately before surgery. Heart rate was monitored every 5 minutes during the surgery with an electrocardiogram (ECG DL 660, Delta Life, Brazil). Oxygen saturation in hemoglobin was measured with an oximeter sensor positioned on the tongue (Oximeter Contec - CMS60D- VET, Brazil). Systolic arterial blood pressure was measured with a Doppler sensor (DV 610V Medmega, Brazil) positioned on the metacarpal artery, and rectal temperature was measured with a digital clinical thermometer (G - Tech, Brazil). Transoperative parameters were recorded at baseline and immediately, 5, 10, 15, and 20 minutes after the beginning of the surgery period.

Secondary outcome was based on pain and sedation evaluations that took place before (baseline) and 1, 2, 4, 8, 12, and 24 hours after surgery. The primary outcome was the comparison of the number of dogs treated with intervention analgesia between groups to test postoperative analgesia efficiency. Rescue analgesia was performed with 0.5 mg/kg of IM morphine (Dimorf 10 mg/ml, Cristália, Brazil) in animals with a score ≥ 6/24 on the CMPS-SF short-form Glasgow Composite Measure Pain Scale. Thirty minutes after the rescue analgesia, if the pain score remained ≥ 6 / 24, a further rescue was performed with 25 mg/kg of IM dipyrone (Sodium Dipyrone 500 mg/ml, Climax, Brazil). Thirty minutes later, ketamine 1 mg/kg IM (Ketamine 100 mg/ml, Vetnil, Brazil) was administered if a third rescue analgesic was needed. Data were maintained from dogs receiving rescue analgesia. After the final evaluation, the dogs were discharged with a prescription for oral meloxicam (0.1 mg/kg per day) for 5 days.

The sample size was calculated (http://biomath.info/power/ttest.htm) by comparing a mean and standard deviation pain score of 4 ± 1.5 in one group and 6 ± 1.5 in another group, with 80% power and 0.05 significance level for the hypothesis test, assuming that animals with scores ≥ 6 in the moments of postoperative pain would receive rescue analgesia and, therefore, the analgesia would be insufficient. This analysis estimated the sample size at 10 animals per group. Statistical analyses were performed using R software in the RStudio integrated development environment (RStudioTeam, 2016). Data distribution was analyzed using the Shapiro Wilk test. Differences between groups for cardiorespiratory data, weight, age, and duration of surgery were analyzed by one-way ANOVA, followed by Tukey’s test. For nonparametric data (propofol dose, pain and sedation scores, number of doses and number of dogs treated with rescue analgesia), the Kruskal-Wallis test was used, followed by Dunn’s test. Results are expressed as means ± standard deviation and were considered significant when p < 0.05.

All dogs were included in the study and all data reported in methods were analyzed. There were no differences between groups for weight, propofol, and time of surgery (Table 1). The transoperative parameters did not differ between groups or moments, except for the rectal temperature, which was lower in the GM (36.4 ± 1℃) than in the GA (37.1 ± 1℃) 10 minutes after the start of surgery (p = 0.021).

Table 1

Weight, age, propofol dose, and duration of surgery of dogs submitted to ovariohysterectomy after preemptive (GA = 12) or postoperative acupuncture (GPA = 12) or preemptive meloxicam (GM = 12).

	GA	GPA	GM	p-value
Weight (kg)	10.48 ± 7.97	10.87 ± 5.09	8.81 ± 7.13	0.73
Age (months)	37.17 ± 22.18	24.41 ± 15.29	36.92 ± 20.32	0.20
Propofol dose (mg/kg)	5.00 ± 0.00	5.58 ± 0.86	5.49 ± 0.92	0.05
Duration of surgery (minutes)	13.92 ± 3.99	13.50 ± 3.03	12.75 ± 5.51	0.59

Rescue analgesia was performed in 5 of the 36 female dogs (14%). Two GA and one GPA dogs received rescue analgesia once. Two GM dogs received rescue analgesia and one of those was treated again for two times. There were no significant differences in the number of dogs receiving rescue analgesia (p = 0.80) or number of rescue analgesia episodes between groups (p = 0.78; Table 2).

Differences between groups for number of doses and number of dogs treated with rescue analgesia was analyzed by the Kruskal-Wallis test, followed by Dunn’s test..

&md=tbl&idx=2' data-target="#file-modal"">Table 2

Number of doses administered and number of dogs submitted to ovariohysterectomy that received postoperative rescue analgesia, after preemptive (GA = 12) or postoperative acupuncture (GPA = 12) or preemptive meloxicam (GM = 12).

Group	Number of doses and moments of postoperative rescue				Number of dogs treated with rescue analgesia
	1 h	2 h	4 h	8 h
GA			1		2
				1
GPA	1				1
GM	1	1	1		2
	1
p-value	0.78				0.80

Differences between groups for number of doses and number of dogs treated with rescue analgesia was analyzed by the Kruskal-Wallis test, followed by Dunn’s test..

There were no differences in pain or sedation scores between groups at any time-point. In all groups significantly greater pain scores occurred at 1 and 2 hours (Fig. 2) and significantly greater sedation scores occurred at 1, 2, and 4 hours postoperatively, compared to baseline (Fig. 3).

Figure 2. Box plot of perioperative scores of short-form Glasgow Composite Measure Pain Scale in bitches submitted to ovariohysrerectomy, treated with preemptive acupuncture (GA = 12), postoperative acupuncture (GPA = 12), or preemptive meloxicam (GM = 12). Asterisks indicate significant differences in pain scores between the measurement moments in relation to the baseline (p < 0.05; Kruskal Wallis, followed by Dunn). The top and bottom box lines represent the interquartile range (25 to 75%), the line within the box represents the median, the extremes of the vertical lines represent the minimum and maximum values. Basal (preoperative) and 1, 2, 4, 8, 12, and 24 hours after surgery.

Figure 3. Box plot of perioperative sedation scores in bitches submitted to ovariohysrerectomy, treated with preemptive acupuncture (GA = 12), postoperative acupuncture (GPA = 12), or preemptive meloxicam (GM = 12). Asterisks indicate significant differences in sedation scores between the measurement moments in relation to the baseline (p < 0.05; Kruskal Wallis, followed by Dunn). The top and bottom box lines represent the interquartile range (25 to 75%), the line within the box represents the median, the extremes of the vertical lines represent the minimum and maximum values. Basal (preoperative) and 1, 2, 4, 8, 12, and 24 hours after surgery.

Preemptive analgesia is based on three fundamental pillars: i) the analgesic intervention prior to the nociceptive stimulus must be more effective than when administered after tissue injury; ii) the duration of analgesia must be longer than the expected effect of the drug or technique used [28], and iii) the intervention should reduce the postoperative analgesic requirement [29]. Our hypothesis was denied because the analgesic effect of postoperative acupuncture was as effective as preemptive acupuncture or meloxicam in dogs submitted to ovariohysterectomy. Although there is evidence regarding the preemptive analgesic action of NSAIDs and opioids, controversy remains. A systematic review reported no preemptive analgesic effect with the use of NSAIDs or opioids in humans [30]. Meanwhile, another more recent review [3] and experimental study in rats [2] reported that preemptive analgesia with these drugs reduced the nociceptive response, pain scores, and need for rescue analgesia compared to their postoperative use. Our findings are different to those observed in women undergoing gynecological surgery, where preoperative electroacupuncture (EA) reduced the trans and postoperative dose of opioids in relation to postoperative EA [21].

The mechanism of action of acupuncture has been previously detailed and is not the scope of this study [31]. The analgesic effect of preemptive acupuncture is already well described in humans. Preemptive EA reduced the consumption of morphine for 24 hours after lower abdominal surgery [32]. Like in the current study, in humans submitted to hemorrhoidopexy, preemptive acupuncture and pharmacoanalgesia were equally effective for analgesia [33]. The inclusion of EA in the trans or postoperative period increased the analgesic effect in relation to its exclusive use in the preoperative period in laparoscopies [34].

The only analgesic used in addition to the treatments tested in this study was fentanyl, to guarantee, for ethical reasons, transoperative analgesia, and this could be a limitation of the study. Although fentanyl could trigger preemptive analgesia, it is a short-acting analgesic with a rapid redistribution and short duration. In dogs even a dose five times higher than in the current study reduced the thermal nociceptive threshold to just over half after 30 minutes, returning to baseline two hours after administration [35]. Although the preemptive effect of fentanyl cannot be completely ruled out, it is unlikely that the analgesic effect of fentanyl remained postoperatively. This can be supported by the findings of a previous study under similar conditions [36], where all dogs treated with preoperative meloxicam and transoperative fentanyl and submitted to ovariohysterectomy, required rescue analgesia at 30, 60, and 120 minutes postoperatively. Another possibility is the possible synergistic opioidergic effect of fentanyl with acupuncture and meloxicam [35], but this would occur in the three groups. Several studies assessing the analgesic effect of different modalities of acupuncture used transoperative fentanyl [18,37,38].

The distribution of rescue analgesia in the three groups differed. None of the dogs treated with either pre or postoperative acupuncture required more than one analgesic intervention. In all groups rescues were mostly concentrated in the first hours after the end of the surgery, when sedation scores were high, overestimating the Glasgow pain score because dogs might be depressed, reluctant to walk, and unresponsive to stimuli. Although the residual effect of the drugs used during anesthesia may increase the pain scores assigned by inexperienced observers at the anesthetic recovery phase [39], in the current study the evaluator was experienced and trained to use the pain assessment tool.

To guarantee reliability, the present study was designed to minimize the factors that interfere with pain assessment. Dogs were admitted 24 hours before surgery to be familiarized with the environment and professionals involved. Although there are no studies of this nature in dogs, prior adaptation to the environment in cats minimizes their fear and anxiety bias, which can interfere with pain scale scores and lead to false-positive results [40]. Usually, cats require two days to adapt to stressful hospitalization situations, such as blood collection, human traffic, and noise [41]. However, except for blood collection before the beginning of the study, these situations did not occur in our conditions, as the room was exclusive and quiet, guaranteeing an appropriate environment for the adaptation and well-being of the dogs.

In addition to environmental adaptation, the personality of the dogs and the evaluator are also important when evaluating pain [42]. The same anesthetist and acupuncturist were responsible for all anesthesia and acupuncture sessions, respectively, and the same evaluator performed all blinded pain evaluations.

The choice of acupoints in this study was based on their postoperative analgesic efficacies, demonstrated in similar experimental models. The combination of Gall bladder 34, Stomach 36, and Spleen 6 [14,15] and Large intestine 4, Stomach 36, and Spleen 6 [43] are the most widely used in the literature. The stimulus was bilateral to guarantee the lowest latency and the longest duration of analgesia [20].

Meloxicam is the most widely used NSAID in research, the third most popular among dog owners [4] and the most commonly reported NSAID in studies evaluating the analgesic effect of different acupuncture techniques [38,44,45]. Our study confirmed previous findings that acupuncture and its modalities have equivalent effectiveness to NSAIDs, in this case, meloxicam, to guarantee postoperative analgesia in cats [38] and dogs submitted to ovariohysterectomy [44]. The differential of our results was that even when used in the postoperative period, acupuncture also demonstrated the same efficacy as preemptive meloxicam.

The postoperative analgesic effectiveness of acupuncture in the current study agrees with previous data in dogs submitted to ovariohysterectomy. Acupuncture applied to Stomach 36, Spleen 6 and Gall bladder 34 acupoints [12] or to the same points as in the current study combined to Liver 3, Gall bladder 34 and 39 was as effective as meloxicam [44], showing that the inclusion of these three acupoints may be redundant for the analgesic outcome. The analgesic efficacy of acupuncture occurs regardless of the technique employed. Postoperative analgesia after electroacupuncture was more effective than butorphanol [17], similar to tramadol [13] and similar [13,14] or superior [15] to morphine. Even for major surgery like mastectomy, the number of rescue analgesia was smaller in female dogs treated with preemptive electroacupuncture than morphine [11]. LASERpuncture provided a superior postoperative pain relief than meloxicam [18] and injection of ozone into acupoints was as effective as meloxicam for postoperative analgesia [45].

Some limitations of this study should be mentioned. There was no negative control group, sham group, or group treated with a preoperative opioid. Placebo treatment was not performed for ethical reasons and because ovariohysterectomy causes moderate pain and requires postoperative rescue analgesic, even when preemptive analgesia is provided [46]. No false acupuncture group was included. The analgesic effect of the needling of false points is absent or small compared to real points [11,20], and the analgesic effect of acupuncture is already well documented [9,10]. As for the non-inclusion of a group treated with preoperative opioids, the analgesic effect of preemptive morphine or electroacupuncture appears to be similar [11].

Since acupuncture does not have the adverse effects of NSAIDs, it can be used to replace NSAIDs for postoperative analgesia in dogs submitted to ovariohysterectomy in the clinical routine, when NSAIDs are contraindicated, or in combination with NSAIDs for multimodal analgesia. Given the difficulty of using acupuncture in the preoperative period in animals with unmanageable behavior, we demonstrated an equally effective analgesic alternative provided by the postoperative use of acupuncture, in a period when the residual anesthetic effect affords sedation to allow the placement of the needles.

This work was supported by São Paulo Research Foundation (FAPESP) thematic research project (2017/12815-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Experimental design, planning and writing were performed by ACZBF and SPLL. Practical work, including anesthesia, surgery and pain assessment, and proof reading were performed by ACZBF, CC and JCR. All authors contributed to the final writing process and have read and approved the manuscript.

The authors declare no conflict of interest.