Principles of therapy and care of laboratory animals after chronic administration into Xylasine-Zoletyl® Anesthesia

The use of a combination of dissociative anesthetics and alpha-2-agonists for chronic experiments in laboratory animals is advisable in the case of studying the functions of the limbic system. This requires the development of a protocol for withdrawing animals from this anesthesia, since frequent anesthesia leads to damage to organs and tissues, the need to increase the dose with frequent use of anesthesia, and distortion of the research results. This article provides recommendations for the elimination of chronic xylazine-Zoletil® anesthesia, including pharmacological therapy and additional care measures for experimental animals (based on a comparative analysis of changes in respiration on a pneumogram, the nature of dysfunctions during the period of anesthesia and the mortality rate of animals that were or did not use measures to withdraw from anesthesia). Research Article Principles of therapy and care of laboratory animals after chronic administration into Xylasine-Zoletyl® Anesthesia Vovk AN, Karantysh GV*, Kosenko PO and Medvedev DS Research Technology Center Neurotechnologies, Southern Federal University, Rostov-on-Don, Russia Received: 18 August, 2020 Accepted: 05 September, 2020 Published: 07 September, 2020 *Corresponding author: Karantysh GV, Leading Researcher, Research Technology Center Neurotechnologies, Southern Federal University, Rostov-on-Don, Russia, Tel: Tel: +7-928-138-88-57; E-mail:


Introduction
The progress of medicine at the present time is closely related to the need to conduct experimental research on laboratory animals [1]. For humane treatment of animals in laboratory conditions, anesthesia and sedation are used, the combination of which, as well as the technique of their application on animals, are widely described in the literature [2][3][4][5]. However, the technology providing the required level of safety of chronic use of anesthesia and sedatives for the health of laboratory animals has not been fully developed. This often leads to damage to organs and tissues and, as a result, distortion of research results and even death of animals. Frequent anesthesia leads to a number of negative consequences at the stage of recovery from anesthesia: severe salivation, bronchial hypersecretion, the development of apnea, as well as tolerance to anesthesia drugs and, as a result, the need to increase the dose of drug to achieve the desired effect [6].
To neutralize the negative phenomena caused by anesthesia, there are a number of measures, including pharmacological therapy and additional care measures for experimental animals after anesthesia procedures. These measures are described for drugs such as acetaminophen, acetylsalicylic acid, butorphanol, codeine, fentanyl + medetomidine, ibuprofen, ketamine + xylazine, mepiridine, methadone, morphine, nalbufen, nalorphine, oxymorphine, phenobarbital / zolotophazine, tylepaxol and a number of others [6]. For a combination of drugs Zoletil ® (Zoletil20, 50 or 100) and XilaVet (Xila (xylazine)), measures that prevent the development of negative effects on the animal's body are insuffi ciently developed and described in the literature [7,8], although the action of a combination of dissociative anesthetics and alpha-2-agonists is detailed investigated in laboratory animals [9][10][11]. Xilazine is an alpha-2-adrenergic agonist, is a muscle relaxant and antihypertensive agent. It is use as a treatment of chronic drug addiction is considered justifi ed [12]. Simultaneously, according to the ASA (American Society of Anesthesiologists), the use of Zoletil ® , a complex injection anesthetic for veterinary medicine, has a wide range of adverse effects. Thus, it is inappropriate for use in the surgical stage of anesthesia. The reason for this is the fact that Zoletil® can cause serious functional disorders of the cerebral cortex. Nevertheless, the use of Zoletil ® to study functions controlled by the centers of the limbic system is promising, since this drug induces dissociative anesthesia: it inhibits only certain areas of the brain, such as the thalamus and cortex, while other areas, in particular the limbic system remain active [13]. The purpose of this work is to develop a protocol for the withdrawal of a laboratory animal from xylazine-Zoletil ® anesthesia and additional / concomitant drug therapy, as well as measures for caring for the animal in order to reduce negative symptoms caused by regular anesthesia with these drugs. Feeding was carried out with complete ration feed PK 120-2_35 at the rate of 35-45 grams per day per head; water without restrictions on consumption.
The animals were divided into groups: control (n = 43) and experimental (n = 50). The control group was treated according to the standard scheme [13]. The following changes have been made to the rules for the care and maintenance of this group of animals. The animals were on a starvation diet 12 hours before anesthesia. During the recovery from anesthesia, the animal was placed in a cage with sides insulated from the outside, located on a heating pad. The temperature of the pad was adjusted manually at the control unit to maintain body temperature of 37.5°C. After coming out of anesthesia (after about 5.5 hours), the animal was placed in a cage with normal conditions.

Research results
One of the most controllable indicators of the effect of anesthesia on the animal organism is monitoring the frequency and depth of breathing [15]. The study of respiration during experimental sessions accompanied by xylazine-Zoletil ® anesthesia showed that cases with a favorable outcome of anesthesia with the use of these drugs are characterized by the following changes in the pneumogram of rats: compared with the state of wakefulness (Figure 1), after entering the deep stage of anesthesia, the frequency respiration is signifi cantly reduced, while the nature of the teeth of the pneumogram, as a rule, changes during this stage of anesthesia (Figure 2A-B). Recovery from anesthesia is characterized by an increase in the respiratory rate ( Figure 3). A negative reaction to xylazine-Zoletil® anesthesia was characterized by the formation of irregular breathing with a signifi cant change in the amplitude of the teeth on the pneumogram up to respiratory arrest ( Figure 4).
During anesthesia, in animals with a negative reaction to xylazine-Zoletil ® anesthesia, in addition to disturbances in the rhythm and frequency of respiratory movements, hypersalivation and apnea were noted. With each subsequent drug addiction, negative symptoms intensifi ed. At the stage of recovering from anesthesia, the animals showed a perversion of appetite and choking with vomit were often observed.
Long-term use of xylazine-Zoletil ® anesthesia led to the development of tolerance to Zoletil ® and the need to increase its dose (up to double) to achieve the desired effect (Figure5). In these cases, the cause of death was polyetiological in nature (myocardial infarction, pulmonary edema and cerebral edema, in some cases -oncology).
According to the analysis, the percentage of death of animals in the control group was higher than in the experimental group throughout the experiment ( Figure 6).  Table 1.

Conclusion
The use of xylazine-Zoletil ® anesthesia in a chronic experiment has features. The advantage of this type of anesthesia is the ability to conduct studies of body functions controlled by the structures of the limbic system. However, the presence of serious side effects of this type of anesthesia is the basis for limiting its use during chronic anesthesia [6].
The results of the study prove the possibility of using xylazine-Zoletil ® anesthesia for this purpose as well. The condition for the success of its chronic use is the observance of the measures described in this work for the conduct of therapy and the observance of special measures for the care of animals after each anesthesia.
The use of therapeutic measures and changes in the conditions of keeping the animals of the experimental group made it possible to reduce the mortality rate, as well as to increase their resistance to chronic drug addiction. Thus, the use of atropine sulfate as a premedication made it possible to minimize of salivation and salivation during and after anesthesia, which reduced the cases of fl ooding with saliva and minimized the effect of salivation on the results of the study of the olfactory system function. The introduction of Cordiamine after each session of the experiment helped to reduce the phenomenon of apnea at the stage of recovery from anesthesia. So, if in the control group, after 3 months of the experiment during anesthesia, the development of apnea was observed in all animals, in the experimental group this phenomenon was characteristic for 1 / 4 of the animals at the 3rd month of the experiment, at 4-5 months -for 1 / 3 , on the 6th month of the experiment -for 1 / 2 rats (out of the number of survivors). The introduction of Antisedan ® provided a decrease in the time of recovery from the state of anesthesia by about half.
Transferring the animal to a starvation diet 12 hours before     These recommendations can form the basis for the formation of a protocol for animal management when using chronic xylazine-Zoletil ® anesthesia.