Irrespective of the technique used, all anesthetic procedures aim to promote reversible depression of the central nervous system and have three important pillars: analgesia, hypnosis, and muscle relaxation [1]. However, the anesthetic recovery process directly depends on the techniques and anesthetic drugs used. Other factors that directly interfere with the time and quality of recovery are the presence or absence of pre-anesthetic medication and surgical time [2,3].

The dissociative anesthesia technique has characteristics that meet the objectives of general anesthesia, and the association of drugs used promotes analgesia, hypnosis, and muscle relaxation. It is a safe, practical, and cost-effective technique compared to other techniques available for general anesthesia in small animals. Dissociative anesthesia has become the most used technique in the routine castration campaigns and in small veterinary clinics. Safe and low-cost anesthetic protocols and its beneficial and deleterious effects are dose dependent, maintaining a direct relationship with postoperative quality [4].

The quality and maintenance of thoracic expansion and pulmonary ventilation are influenced during anesthesia, usually by reducing the volume or increasing the pressure gradient, resulting in decreased compliance and thoracic expansion and decreasing the efficiency of gas exchange in the lungs [2]. Cardiorespiratory depression caused by anesthetics can be explained by the interference of the anesthetic drugs in the central nervous system. Seisdedos et al. [5] demonstrated this interference through the analysis of hypoxia biomarkers, such as lactate, in the cerebrospinal fluid of healthy dogs, and showed that its effects are related to the drugs used and the duration of the anesthesia.

The study by Ferreira et al. [6] points out that anesthetic recovery after dissociative anesthesia associated with diazepam and ketamine lasts an average of 19 minutes. This period might be extended according to the response of each individual, and in cases of exacerbated cardiorespiratory depression, hypothermia, and dehydration. Therefore, it is important to study techniques to reduce the period of anesthetic recovery or combat the deleterious anesthetic effects.

Acupuncture is a Traditional Chinese Medicine (TCM) technique, with holistic bases aimed at energy restructuring through the application of needles that promote acupuncture stimulation [7], restoring altered functional states until rebalancing. The energy channels have the purpose of taking the Qi (energy) and the Xue (blood) to the spaces between the main energy channels, promoting the connections between the Zang Fu (organs and viscera) and the energy channels [8]. The Governing Vessel 26 (GV26 or Ren Zhong) is one of the most used acupoints in emergency cases for both human and animal, such as in the aid of brain-cardiopulmonary resuscitation to promote improvement of central nervous system depression and recovery of spontaneous breathing movements [9].

The aim of the present study was to evaluate the effectiveness of acupuncture GV26 (Ren Zhong) in the recovery of postoperative cardiorespiratory depression in dogs submitted to dissociative anesthesia, by monitoring the respiratory rate, amplitude, respiratory movement, capillary filling time, temperature, presence or absence of laryngotracheal reflex, and presence or absence of interdigital reflexes and to evaluate the effects of the GV26 point on the anesthetic recovery time.

The study was approved by the Animal Experimentation Ethics Committee (CEUA-UFAL) with protocol number 45/2017, and used 20 healthy, non-neutered, mixed-breed bitches, with a mean age of 3 ± 2 years, from tutors in the city of Viçosa and Maceió, Alagoas (AL), which were brought to the ambulatory of the Federal University of Alagoas.

The bitches were selected after a pre-surgical consultation, where the animal’s history was evaluated through anamnesis and physiological parameters such as heart rate, respiratory rate, mucosa coloration, capillary filling time, abdominal palpation, lymph node palpation, and hematological exams, all being within normal limits.

After the pre-surgical consultation, the animals were brought to the outpatient clinic on a scheduled date after fasting from solid food (12 hours) and water (4 hours). The hematological exams and baseline physiological parameters were then evaluated and the animals within the normal range for the species were sent to the pre-surgical room.

In the non-sterile, pre-surgical room, pre-anesthetic medication (PAM) comprising 0.2% acepromazine (0.1 mg/kg) and tramadol hydrochloride (2 mg/kg) was administered intramuscularly. After 15 minutes, abdominal trichotomy was performed, and venous access of the cephalic vein was conducted by administering lactated Ringer’s solution (10 ml/kg/h).

Anesthetic induction was performed using midazolam (0.5 mg/kg) and ketamine (10 mg/kg) associated in the same syringe, administered intravenously, and the animals were transported to a sterile operating room. Antiseptic conditions were ensured, and the surgical technique of ovariohysterectomy (OH) was performed by the same surgeon for all the cases. Only bitches submitted to a single application of midazolam/ketamine with no need for reapplication until the end of the surgery were considered in the study. The reapplication of anesthetics was considered as an exclusion criterion.

The animals were randomly assigned into two groups of 10 animals each using a simple randomization method. The groups included the control group (C), which did not receive acupuncture, and the acupuncture group (AP), which received acupuncture. Needling was performed with a 25 × 08 needle at the GV26 acupoint (Ren Zhong) located in the midline of the nasolabial philtrum 20 minutes after anesthetic induction, at a depth of 0.2 to 0.5 cm depending on the size of the patient, and the manual stimulation of the point was held for 5 minutes.

Respiratory rate (RR), amplitude (superficial, normal or deep) based on the inspiratory expansion of the chest cage, type of respiratory movement (abdominal, abdominocostal or thoracoabdominal based on the region where the inspiratory effort occurs (mainly in the abdomen or chest or both), heart rate (HR), capillary filling time (CFT), and temperature by means of the standard semiological technique were measured. Additionally, the presence or absence of laryngotracheal reflex assessed using a laryngoscope for direct palpation, and the presence or absence of interdigital reflexes using tweezers were also performed immediately before the application of PAM or (T0), and 2 minutes (T1), 5 minutes (T2), 10 minutes (T3), 15 minutes (T4), 20 minutes (T5), 25 minutes (T6) and 30 minutes (T7) after treatment.

Anesthetic recovery time was also evaluated, considering the moment when the patient lifts her head, recognizes the environment, and remains in sternal recumbency without assistance.

In the postoperative period after the evaluations, a single dose of benzathine penicillin (30,000 IU/kg/IM), ketoprofen (2 mg/kg/IV) and tramadol hydrochloride (2 mg/kg/IM) was administered. The animals were observed for 8 hours after the procedure, and then forwarded to their guardians with a post-surgical prescription and a return appointment scheduled for removal of the stitches in 10 days. No adverse effects occurred after the protocols and no animal was excluded from the experiment after pre-surgical selection.

In the analysis of the parametric data, the Kolmogorov-Smirnov test was performed to verify the normality in the distribution of HR, RR, CFT, temperature, and recovery time. These parameters were then compared between the control group and the AP group using the unpaired t-test. The possible association between the parameters amplitude of the chest cage (superficial, normal, or deep), type of respiratory movement (abdominal, abdominocostal, or thoracoabdominal), mucosa coloration, laryngotracheal reflex and interdigital reflex and the treatment was analyzed using the chi-square test.

All analyses were performed using GraphPad Prism, Version 5.0 (Trial), 2007 while assuming a 5% probability of error [10].

The 20 surgeries were performed within a mean time of 20 ± 15 min, allowing experimentation with the proposed anesthetic protocol and maintenance of patients with a single application of midazolam/ketamine through induction. The parametric data for HR, RR, CFT and temperature are shown in Table 1.

HR = heart rate; RR = respiratory rate; CFT = capillary filling time..

^a,bIndicate statistical differences between each other..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

Effects of acupuncture on heart rate, respiratory rate, capillary filling time and temperature in bitches.

Times (min)	HR			RR			CFT			Temperature
	Control	AP		Control	AP		Control	AP		Control	AP
2	105.1 ± 15.4a	155.5 ± 34.4b		28.9 ± 10.6	23.3 ± 5.6		1.9 ± 0.4	1.6 ± 0.5		36.6 ± 1.0	37.2 ± 0.6
5	128.3 ± 49.6	160.5 ± 27.3		26.9 ± 8.9	27.2 ± 7.7		1.7 ± 0.5	1.7 ± 0.5		36.7 ± 0.8	37.1 ± 0.9
10	142.0 ± 47.1	156.4 ± 26.7		26.9 ± 7.0	27.1 ± 9.3		1.8 ± 0.4	1.7 ± 0.5		36.9 ± 0.7	36.9 ± 1.0
15	140.4 ± 46.2	170.8 ± 32.4		28.9 ± 12.3	28.9 ± 9.3		1.8 ± 0.4	1.7 ± 0.5		36.8 ± 0.6	36.9 ± 0.9
20	140.0 ± 40.2	171.6 ± 31.5		29.3 ± 10.8	31.8 ± 8.2		1.8 ± 0.4	1.8 ± 0.4		36.8 ± 0.5	36.9 ± 0.9
25	154.2 ± 32.9	177.2 ± 34.3		33.8 ± 12.0	32.0 ± 8.0		1.8 ± 0.4	1.7 ± 0.5		36.7 ± 0.4	36.9 ± 0.9
30	148.5 ± 43.0	170.2 ± 28.9		32.0 ± 8.0	32.0 ± 11.0		1.8 ± 0.4	1.8 ± 0.4		36.6 ± 0.5	36.6 ± 1.0

HR = heart rate; RR = respiratory rate; CFT = capillary filling time..

^a,bIndicate statistical differences between each other..

No significant difference was observed when respiratory rate, capillary filling time and temperature were correlated in the evaluated data. When comparing the control group with the treated group, the mean heart rate of the treated group (155.5 ± 34.4 bpm) was significantly higher than that of the control group (105.1 ± 15.4 bpm) at T1 (2 minutes), representing an immediate positive result of the chronotropic action of acupuncture, and remaining numerically superior during all evaluation moments, but without statistical significance.

Regarding the information on the amplitude of the chest cage, type of respiratory movement, mucosa coloration, laryngotracheal reflex, and digital reflex, it was observed that among all the evaluation results, only the amplitude of the chest cage was associated with the treatment; the AP group showed a direct relationship with the thoracoabdominal amplitude of the animals at all times, as shown in Table 2.

T1 = 2 minutes; T2 = 5 minutes; T3 = 10 minutes; T4 = 15 minutes; T5 = 20 minutes; T6 = 25 minutes; T7 = 30 minutes after treatment..

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

Analysis and comparison between control group (C) and acupuncture (AP) between times from non-parametric data: amplitude of the chest cage, type of respiratory movement, mucosa coloration, laryngotracheal reflex and, interdigital reflex.

Non parametric data		Assessment times between groups																					Total		p-value
		T1			T2			T3			T4			T5			T6			T7
		C	AP		C	AP		C	AP		C	AP		C	AP		C	AP		C	AP		C	AP
Amplitude of the chest cage	Superficial	5	0		5	1		5	1		5	0		5	1		2	1		0	0		27	4	< 0.0001
	Normal	1	3		2	6		4	7		4	6		4	5		7	7		9	7		31	41
	Deep	1	6		1	2		0	1		0	2		0	2		0	1		0	1		2	15
Type of respiratory movement	Thoracoabdominal	2	6		2	5		4	5		5	4		6	4		6	3		7	2		32	29	0.6194
	Abdominocostal	1	0		2	2		3	2		3	3		2	2		3	6		2	6		16	21
	Abdominal	4	3		4	3		2	2		1	2		1	3		0	1		0	1		12	15
Mucosa coloration	Hipo	1	2		1	4		2	4		2	3		2	3		2	3		2	3		12	22	0.1029
	Normo	6	7		7	6		7	6		7	7		7	6		7	7		7	6		48	45
Laringotraqueal reflex	Yes	0	0		2	0		1	0		1	1		1	2		2	2		4	2		11	7	0.9590
	No	5	3		3	4		5	4		5	3		5	1		4	3		2	1		29	19
Interdigital reflex	Yes	0	0		0	2		1	3		1	3		3	4		7	6		7	7		19	25	0.5044
	No	7	9		8	8		8	7		8	7		6	5		2	4		2	2		41	42

T1 = 2 minutes; T2 = 5 minutes; T3 = 10 minutes; T4 = 15 minutes; T5 = 20 minutes; T6 = 25 minutes; T7 = 30 minutes after treatment..

Recovery time was significantly shorter in the AP group (54.1 ± 14.9 min) as compared to the control group (79.9 ± 17.9 min), as shown in Fig. 1.

Figure 1. Effects of acupuncture (GV26) on the recovery from ketamine-midazolam anesthesia in bitches.

Cominato Boer [11] states that moderate sympathetic stimulation favors systolic release and contributes to the production of positive inotropic effect. Furthermore, this stimulation of adrenergic receptors by catecholamines acts as one of the most important regulatory mechanisms of cardiovascular performance, modulating the frequency and the force of contraction by changing the functional properties of ion channels and excitation-contraction coupling components.

Corroborating the results of the present study, Alvarenga et al. [12] state that acupuncture stimulation is responsible for the release of catecholamines and other neuromodulators into the bloodstream, which stimulate the cardiac receptors in the myocardium. As HR is one of the main determinants of cardiac output and oxygen consumption, its increase is expected [13], culminating in an increase in blood pressure and improvement in blood perfusion. Another study demonstrated that the placement of needles in GV26 increased tissue oxygenation in rat brains and that electroacupuncture in GV26 and GV20 sites was able to reduce cerebral ischemic lesions [14].

A study with 69 cases of apnea or respiratory depression during anesthetic procedures demonstrated a 100% success after 10 to 30 seconds of stimulation of the GV26 point with acupuncture needles. However, in cases of apnea followed by cardiac arrest, the success rate was only 43% 4 to 10 minutes of stimulation [9]. This percentage of success can be extrapolated for patients with respiratory alterations caused by sedatives, anesthetics, lung and pleural cavity diseases. and airway obstructions [15].

The decrease in recovery time can be justified by the increase in heart rate with a consequent increase in blood volume and improvements in blood perfusion leading to greater cerebral oxygenation. According to Hall [16], even small changes in blood volume or the capacity of the circulatory system caused by the varying levels of sympathetic activity can have large effects on mean circulatory filling pressure. Adequate average pressure guarantees tissue oxygenation, thus increasing cerebral circulation of oxygen [17,18]. Recovery is related to adequate GV26 stimulation and in dogs it increases ejection volume, heart rate, and cardiac output and causes changes in pulse pressure [19-21] compatible with changes induced by epinephrine injections [19], such as what has been observed in experimental animals. In the clinical case, the effectiveness of GV26 was demonstrated in restoring the sinus rhythm in a dog with cardiorespiratory arrest. According to the study, the dog only responded to continuous stimulation of the point. Previously, cardiopulmonary resuscitation with epinephrine, sodium bicarbonate, and fluid therapy had been tried, but without satisfactory results [19-21].

The study suggests that the GV26 point was effective in maintaining better respiratory amplitude, immediate positive chronotropic action and accelerating anesthetic recovery. It is concluded that it can be indicated in cases where there is a need for a better breathing pattern and faster recovery in bitches under dissociative anesthesia.

Other studies can be conducted using a larger number of patients and other acupoints to complement the technique using GV26.

Conceptualization: Carolina, Jackellyne; Data curation: Carolina; Amanda; Jackellyne; Formal analysis: Carolina; Funding acquisition: Jackellyne; Pierre; Investigation: Carolina; Marcia; Methodology: Pierre; Marcia; Carolina; Project administration: Pierre; Marcia; Resources: Pierre; Supervision: Pierre; Marcia; Carolina Validation: Marcia; Visualization: Pierre; Amanda; Writing - original draft: Carolina; Writing - review & editing: Amanda.

The authors declare no conflict of interest.