The aim of the present study was to find the most safe and appropriate intraperitoneal injection dose of Ketamine-Xylazine cocktail for short to medium-duration surgical procedure (ultrasound guided liver biopsy) in rats suffering chronic liver injury. Four anesthetic doses of Ketamine-Xylazine combination were compared for their safety and efficacy (death rate and surgical tolerance), using observations and reflex tests. Anesthesia evaluated during ultrasound guided liver biopsy procedure. The reactions of physiological parameters to surgical stimuli were used to determine anesthesia depth and were correlated with reflex test results. Full dose of Ketamine-Xylazine (87 mg/kg-13 mg/ kg) rapidly induced deep state of anesthesia that lasted for about 70 minutes followed by complete cessation of respiration and death. Three quarters dose of the cocktail also, rapidly induced deep state of anesthesia that lasted 45 minutes. Anesthesia was adequate to perform the procedure. Recovery was long. No postoperative complications detected. Half dose of the Ketamine-Xylazine cocktail was short acting. Very good analgesia and muscle relaxation were recorded. Anesthesia lasted for about 30 minutes that was adequate for performing the procedure. Physiological parameters decreased followed by rapid stabilization. Smooth recovery noted. No postoperative complication recorded. Quarter dose produced a state of sedation. Analgesia and muscle relaxation were poor. Animals showed pain during manipulation. Procedure could not be achieved. The optimal intraperitoneal dose of Ketamine-Xylazine cocktail for balanced anesthesia in lab rats suffering chronic liver injury is (43.5 mg/kg and 6.5 mg/kg) respectively.

Keywords: anesthesia, intraperitoneal, rats, ketamine, xylazine

Mice and rats are served as the preferable lab animal for biomedical research model due to their small size, ease of handling, short life span and similarity in anatomical, physiological and genetic disceplens to humans. They have been used for in vivo studying of mechanisms of liver fibrosis and its possible treatments. Carbon tetrachloride induced liver fibrosis resembles human liver fibrosis and serves as an attractive model for chronic liver intoxication on the molecular levels.^1,2 Completion of certain research may require keeping all participated animals alive, despite of frustrated procedures of examination or samples collection. These procedures are performed in anesthetized mice in order to facilitate the procedure and enable the operator to perform safely and accurately. General anesthesia has been described in lab mice. Ether and isoflurane inhalant agents have been reported for inducing general anesthesia in mice.³ Several injectable protocols have been described. Xylazine 10mg/kg-Ketamine 100mg/kg, Xylazine 10mg/kg-Ketamine 100mg/kg-Acetyl promazine 3mg/kg and Diazepam 5mg/kg -Ketamine 200 mg/kg mixtures have been designated for survival procedures in mice.⁴⁻⁷ The combination of ketamine and xylazine is still the most widely used ketamine combination in mice, providing good immobilization with some degree of analgesia. Several different dosage combinations of the ketamine/xylazine mixture have been reported for mice in the medical literature, varying from 65/4 to 100/13 mg/kg. The large variability of the recommended dosages depends on differences related to strain, sex, age, and type of experimental procedure.⁸⁻¹² Injectable anesthetics may be associated with cardiovascular and respiratory depression, prolonged recoveries, lower margin of safety and hard to control the depth of anesthesia. Both xylazine and ketamine are metabolized by liver enzymes.¹³ Animals with end stage liver disease are at significant risk of mortality during and after anesthesia and surgery.¹⁴ In the text below, we studied four doses of Ketamine-Xylazine mixture, and we report our own experience in selection of a safe and reliable anesthetic protocol.

Wistar rats used in this study were selected from the reservoir rats dedicated to a research designed for studying the effect of mesenchymal stem cells on chronic liver injury. Rats were of both sexes and weighting 180-200gm. They were all treated with carbon tetrachloride (CCL4), eight doses (2ml/kg/dose), two weeks apart. Acute liver injury was confirmed and rats were left for chronic liver disease progression. Rats were divided into four equal groups (A, B, C and D) with five for each (ƞ=5). All mice were prepared for short to medium-duration surgical procedure (ultrasound guided liver biopsy) under general anesthesia. At the time of surgery, group A received freshly prepared Ketamine-Xylazine (87mg/kg-13mg/kg) mixture (dose A), the dose was according to,^15,7 group B received, K 66 mg/kg- X 10 mg/kg (Dose B), group C received; K 43.5 mg/kg- X 6.5 mg/kg (Dose C) and group D administered; K 22mg/kg- X 4mg/kg (Dose D). The four doses were presented as full dose (A), threequarters dose (B), half dose (C) and quarte dose (D). The anesthetic mixture was injected intraperitoneal IP. Criteria of assessment were; induction time, analgesia, anesthesia, duration of action, recovery time, and post anesthetic complications. Criteria and assessment are tabulated in Table 1. Ultrasound guided liver biopsy was performed to all rats. Reliability of the anesthetic protocol was also evaluated for its suitability to short/ medium-duration surgical intervention.

Rats of group (A) that received the full dose of (K 87 mg/ kg- X13 mg/ kg) have shown rapid induction of deep state of anesthesia. Profound analgesia and muscle relaxation were induced with sever bradycardia and shallow very slow respiration. Rectal temperature decreased and respiratory rate remained shallow and slow. Bradycardia also persists. No reflexes recorded during anesthesia. All rats of group A died few hours after the procedure. Rats of group (B) that received threequarters dose (ketamine 66 mg/kg and xylazine 10mg/kg) have also shown rapid induction. Analgesia was profound, good muscle relaxation were predicted with long recovery period. Anesthesia remained 44±1 minutes. The physiological parameters decreased along with anesthesia and begin to increase at the recovery. Rats of group (C) that received half dose (ketamine 43.5 mg/kg and xylazine 6.5 mg/kg) have shown short induction period. Analgesia was good, no pain detected during the procedure. Stabilization of physiological parameters and prober analgesia in combination with good muscle relaxation indicates balanced anesthesia. Recovery period was short. All Rats of this group regained normal status in short period. No post anesthetic complications detected. Rats of group (D) that received quarter dose (ketamine 22 mg/kg and xylazine 4 mg/kg) have good sedative effect. Analgesia and muscle relaxation were poor. General anesthesia was not induced in any animal of this group. Physiological parameters decreased firstly then raised after stimulation (Table 2).

To the authors' knowledge, this study represents the first clinical evaluation and optimization of intraperitoneal ketamine-xylazine anesthetic dose in rats suffering chronic liver injury. The goal of the present study was to provide the reliable, safe and adjustable anesthetic dose of Ketamine-Xylazine combination suitable for short to medium-duration surgical procedures in liver disease rat models, a dose that is reliable to adapt the needs of a wide range of researchers. Ketamine alone in high doses was used as a sole anesthetic agent,¹⁶ it produced only sedation without analgesia that altered induction and recovery behaviors. The results of this study support the use of anesthetic combination. Results of the present study are supportive to the former studies demonstrating the reliable effect of Ketamine-Xylazine mixture as general anesthetic for short to medium-duration surgical procedures. Ketamine-Xylazine combination has been used with various doses in almost all species of animals and birds.¹⁷ Regarding the findings of previous literature, the reviewed doses have narrow therapeutic range and poor safety margin.¹⁸⁻²⁰ To obtain reliable anesthetic status and increase the safety and surgical tolerance of anesthesia, variable doses of the combination were examined. At a parallel line, Arras et al.²⁰ reported that addition of some sedatives to the combination in combination with decreasing the doses resulted in more reliable and safe anesthesia. Lack of some sedatives may limit the capability for obtaining similar results

A study conducted by Suliburk et al.²¹ showed that ketamine has a liver protective effect over isoflurane. This result plus other technical facts supports the use of ketamine rather than inhalation anesthesia. Despite these supportive results, both Ketamine and Xylazine are bio-transformed in liver²² with high hepatic clearance ratio,²³ the initial dose is to be reduced.²⁴ Drug information service released a bulletin on (2013) recommended decreasing the dose of high hepatic clearance drugs such as ketamine and xylazine by 50%. Animals with liver dysfunction are considered at greater risk for complications associated with general anesthesia as reported by Weil.²⁵ Several studies were conducted to optimize Ketamine-Xylazine anesthetic dose for preclinical imaging²⁶ and medium-duration surgical procedures,²⁰ establishment of anesthesia optimization study in rats suffering chronic liver disease will help researchers in their biomedical daily tasks.

The most appropriate and highest safety margin was associated with the three quarters dose for medium-duration and half dose for short-duration procedures, doses that are not widely known. Wide range dosage reported indicates that strain, administration rout and health status of the lab rat are principle rules in dose adaptation. The three quarters and half dose regimens were adapted for induction of surgical plane of anesthesia. Those protocols provided surgical tolerance for 45 minutes that is adequate for medium-duration surgical procedures and 35 minutes suitable for short-duration surgical procedures as transcutaneous liver biopsy performed here.

The appropriate intraperitoneal dose of Ketamine-Xylazine combination to obtain balanced anesthesia in lab rats suffering chronic liver injury is (43.5 mg/kg and 6.5 mg/kg).

None.

Author declares that there is no conflicts of interest.

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