Anesthesia protocol for ear surgery in Wistar rats (animal research)

Abstract Objective To formulate an anesthesia protocol for safe and satisfactory anesthesia for ear surgery in rats. Methods The rats were anesthetized with xylazine (10 mg/kg body weight) and ketamine at doses of 80, 50, 40, and 30 mg/kg body weight or with isoflurane anesthesia (2%–3.5% in 100% oxygen; maintenance dose 1.5%–3.5%). The anesthesia induction, surgery, and recovery time were recorded. Results In total, 17 rats were induced by varying doses of ketamine‐xylazine and 28 rats with isoflurane. Mean induction time with ketamine‐xylazine was 6 ± 2.9 min compared with 3.8 ± 1.1 min with isoflurane. Mean recovery time with ketamine‐xylazine was 142.6 ± 49.3 min compared with 4.1 ± 1.2 min with isoflurane. A mortality of 4 animals after developing dyspnea was recorded with ketamine‐xylazine. Conclusion Isoflurane anesthesia offers appropriate induction and recovery times and low mortality rates for the surgeries performed. Isoflurane anesthesia offers reliable results for ear surgery in rats. However, more equipment and technical skills are needed.


| INTRODUC TI ON
Anesthetic agents that are safe, repeatable, inexpensive, and minimally traumatic are ideal for invasive animal experiments. Ketamine hydrochloride is one of the most commonly used injectable anesthetic agents, and its usefulness as a rapidly acting general anesthetic has been confirmed. Xylazine hydrochloride is an effective sedative and commonly used as an adjunct to anesthesia in animals.
A combination of ketamine and xylazine is commonly used to anesthetize rats, mice, and other rodents. 1 This combination is known to have a number of advantages as opposed to using either drug alone.
Xylazine supplements ketamine with its analgesic properties, muscle relaxation, and sedation, which are beneficial in reducing side effects of ketamine such as tremor and muscle rigidity. Moreover, the combination of ketamine and xylazine provides an optimal anesthesia without the need for specialized equipment. 2 Among the volatile anesthetic agents, isoflurane is a commonly used inhalation anesthetic in animal research, known for its rapid induction and recovery, and control over depth of anesthesia. It has been reported to undergo less biotransformation than other agents, thus making it a suitable choice for in vivo studies. It is commonly used for short-and moderate-duration procedures. Inhalation anesthesia, however, requires substantial physical infrastructure and trained manpower. [2][3][4] Ear surgeries in rats for otological research are usually shortto-moderate-duration procedures performed using an operating microscope. Such procedures present unique challenges for the anesthetist. The surgical field is cramped due to close proximity to the other sense organs and to the airway that needs to be accessible to the anesthetist. Such unique challenges will dictate the choice of the anesthesia technique and agents. Though extensive studies exist on the merits and demerits of the various anesthesia techniques and agents used in the current study, they have not been studied in the context of the otology procedures. The present work describes the experience of the authors while establishing the protocols for injectable and inhalational anesthesia in adult Wistar rats for nonmutilating, short-to-moderate-duration survival ear surgeries by post-aural approach. All the animals (Saha Enterprises, Kolkata, India) were maintained in polycarbonate cage (BIK Industries, India) with corn cob bedding (qCob, Altromin, Germany) under laboratory conditions of temperature 22 ± 2°C, relative humidity 45 ± 5%, and 12 h light cycle and were allowed food (standard cereal-based pellet diet) (Altromin, Germany) and water ad libitum.

| Experimental design
This study was conducted as a part of an ongoing project that involved ear surgery in rats, as described later. Only clinically healthy animals were selected for the study. The animals had been housed in the Animal House for at least 2 months before they were operated.
Rats that had been operated previously for any other disease were excluded.
The rats procured for the study were shortlisted and numbered sequentially. The rats for each procedure were identified 1 day prior in no particular order after considering the general clinical health of the rats. The observations were recorded with the veterinary anesthetist blinded to the outcomes. The veterinary anesthetist (P.P.S.) and surgeon (A.B.) were blinded to the numbering sequence, identification of rats for surgery, and observations. The data were analyzed by A.B. The results were noted with reference to otological surgeries conducted in this study.

| Ketamine/xylazine (injectable anesthesia)
Xylazine (10 mg/kg) followed by ketamine in variable doses (80, 50, 40, and 30 mg/kg) was injected intramuscularly into the femoral musculature of the hind leg. After loss of the reflexes, the rat was placed in prone position on the heating pad on an operating table.
Following the surgical procedure and wound dressing, the rat was kept under observation till the righting reflex returned. Thereafter, the animal was placed back in its cage and kept in the animal housing room under controlled environmental conditions.

| Isoflurane
The rat was placed in an induction chamber and subjected to induction dose varying from 2% to 3.5% isoflurane in 100% oxygen at a rate of 1.5 L/min till loss of the reflexes. The rat was then placed in prone position on the heating pad, and then the dose of isoflurane was individually regulated at a maintenance dose also varying from 1.5% to 3.5% administered through a sterile nose cone (NCON-D; Orchid Scientific & Innovative India Pvt. Ltd, Nasik, India). A gaseous anesthesia system (Hospital Devices, India) was used for this purpose. Depth of anesthesia was monitored regularly. After surgery and wound dressing, isoflurane administration was ceased and the rat was administered oxygen and monitored till its righting reflex returned.
The animals were administered a single dose each of ceftriaxone

| Statistical analysis
All statistical analyses were performed using GNU PSPP version 1.2.0-g0fb4db, software. The differences in the anesthesia induction time and recovery time between ketamine-xylazine and isoflurane anesthetized groups were analyzed statistically using independent samples t-test. A p value less than .05 was considered statistically significant.

| RE SULTS
A total of 45 animals (27 male; 17 female) with gender of one rat unrecorded, 4-18 months old (mean 8.7 ± 3.8 months) and weighing 100-570 g (mean 252.9 ± 107.1 g) were included in the surgery. Seventeen rats also underwent middle ear exploration, prior to implant insertion.
Surgery time on such animals was longer than on the rest of the animals.

| Injectable anesthesia
Initially, 4 rats (4 ears) were administered 80 mg/kg ketamine and 10 mg xylazine. The anesthesia induction time was in the range of 4-6 min (mean 5 ± 1 min), and the recovery time was in the range of 125-165 min (mean 146.7 ± 20.2 min) against a mean surgery time of 20 ± 20 min (Table 1). One animal developed dyspnea during recovery from anesthesia and died. The recovery time from this animal was, therefore, not recorded.
Due to the long recovery period with 80 mg/kg ketamine, the dose of ketamine was reduced to 50 mg/kg. Nine animals were anes-

| Gaseous anesthesia
Twenty-eight animals were anesthetized with a mean induction dose of isoflurane of 2.9 ± 3% isoflurane and 1.5 L/min oxygen in the induction chamber. The mean maintenance dose of isoflurane was 2.5 ± 0.5%. During the surgical procedure, 10 animals displayed dyspnea; however, this complication was managed uneventfully by reducing the maintenance dose of isoflurane to 1.5% and increasing the oxygen volume to 2-2.5 L/min. The time taken to induce anesthesia was 2-8 min (mean 3.8 ± 1.1 min). The mean time taken to complete the surgical procedure was 29.8 ± 28.9 min, while the time taken by the animals to recover from anesthesia was 1-6 min (mean 4.1 ± 1.2 min) ( Table 1) The procedure for administration of inhalational anesthesia was observed to be technically demanding, with use of substantial equipment. The anesthesia was administered through a nose cone, which significantly hindered access to the surgical field. The manipulation of the head and body was also cumbersome. However, it was also observed that with experience, and a short learning curve, the issues of access to the surgical field and convenience of manipulation were circumvented.

| DISCUSS ION
Though the induction time with isoflurane anesthesia was significantly less than that with ketamine-xylazine, it was of an acceptable duration with both agents. However, the recovery time was unac- ceptably prolonged with all the tested doses of ketamine-xylazine with respect to the surgery time. Further, a mortality of 5 animals was also recorded, following dyspnea in animals anesthetized with ketamine-xylazine. The recovery time with isoflurane was of an acceptable duration with respect to surgery time and significantly less than that with ketamine-xylazine. Although complications such as dyspnea were encountered intraoperatively in 3 animals, recovery was uneventful because of greater control over anesthesia depth.
There were no cases of mortality or sequelae recorded in isofluraneanesthetized animals.
Ketamine is an N-methyld-aspartate receptor antagonist, classified as a dissociative anesthetic that produces analgesia and immobility. 1 Xylazine is an α2 adrenoreceptor agonist. 1 It acts as a sedative, analgesic, and muscle relaxant. The properties of ketamine and xylazine complement each other. The sedative and musclerelaxant properties of xylazine are beneficial in reducing side effects of ketamine such as tremor and muscle rigidity. Ketamine-xylazine is a commonly used and cost-effective injectable anesthetic combination for rodents, particularly for long-duration procedures. 1,5 Though ketamine-xylazine can be administered by different routes, the intramuscular route is less stressful for the animals, and also avoids inadvertent injection into the gastrointestinal tract. 6 In rats, the half-lives of ketamine and xylazine are approximately 2 and 1 h, respectively, and both drugs are chiefly metabolized in the liver. 7 For anesthesia duration of 60-80 min, the doses of ketamine and xylazine recommended by various laboratories in United States range from 40 to 100 mg/kg and 5 to 13 mg/kg body weight, respectively. 8,9 Ketamine and xylazine at the recommended doses have been reported in various studies to have an induction time ranging from 5 to 10 min and recovery period ranging from 60 to 120 min.
The long recovery time might be attributed to its slow metabolism. [10][11][12][13] The induction time was similar to that in the current study, but the recovery time was shorter, though it was significantly longer than the surgery time required for ear surgeries conducted in the current study at all recommended doses. Depth of anesthesia was satisfactory for the surgical procedures performed, and the surgical field was unhindered with the combination. Repeated manipulation of the head required for the surgical procedures on the ear was unhindered as well.
Although the use of ketamine-xylazine offers numerous advantages, the side effects associated with their use have also been reported to include respiratory depression, which might even be fatal.
Hypothermia is a major risk in anesthetized animals, hence the need to maintain body temperature during the procedure. Muscle necrosis has frequently been observed following intramuscular administration of the combination. Cardiac depression has also been commonly observed. 2,14 In the current study, 5 rats developed dyspnea and could not recover from anesthesia. No abnormality was noted at the injection site in any of the animals after i.m. injection. In view of the safety concerns, injectable anesthetics, primarily ketaminexylazine, are preferred anesthetic agents in settings where infrastructure for inhalational anesthesia is not available. 2,14 Inhalation anesthesia using isoflurane is the most widely used method of general anesthesia in animal research as it produces rapid induction and recovery from anesthesia and undergoes less biotransformation. 15 Other volatile inhalational anesthetics such as ether, halothane, methoxyflurane, isoflurane, sevoflurane, desflurane, and enflurane are also commonly used in different species. Isoflurane has been successfully used to anesthetize rats at a concentration of 2.5%-5% with a maintenance dose of 1%-3%. 16,17 The use of isoflurane in the current study enabled regulation of depth of anesthesia in real time.
However, isoflurane has been reported to depress cardiovascular function in a dose-dependent manner. 6,18 Isoflurane administration in the current study necessitated the use of a nose cone, which limited the manipulation of the head during the surgery, and also interfered with the surgical access. This shortcoming was overcome with experience. The nose cone was sterilized before use. Since inhalation anesthesia provided greater safety, appropriate induction and recovery times and real-time regulation of depth of anesthesia, greater numbers of animals were operated using the technique, and injectable anesthesia was not used thereafter.
Ear surgery in rats is routinely conducted for numerous oto-

| CON CLUS IONS
Isoflurane anesthesia provided superior outcomes with respect to morbidity and mortality of the animals. It proved to be more reliable in terms of the ability to regulate the anesthesia duration for the required surgical period, and it induced a short recovery time with no mortality and minimal morbidity recorded. However, it entails more equipment and, hence, a substantially higher cost. The inhalation anesthesia also involves technical expertise.

ACK N OWLED G M ENTS
The study was a part of the research project funded by Department

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

AUTH O R CO NTR I B UTI O N S
Dr Abhijeet Bhatia: Made substantial contributions to the con-