4.1 Basic Principles of Working with Laboratory Animals

To begin training, one must first become familiar with the rules of the laboratory and with handling laboratory animals. One’s attitude toward laboratory animals must be as humane and respectful as it is to humans in acknowledgment of the sacrifice made.

In order to work with laboratory animals, an application for the work should be approved by an ethics committee at the institution. In many countries, there is a formal body (e.g., an institutional animal care and use committee) that supervises such work and ensures that it adheres to international standards. The researcher takes responsibility to ensure that the care of the animals is exemplary and, if necessary, may be obligated to take care of the animals personally, including feeding, watering, anaesthetizing during pain or distress, and promptly performing euthanasia, if needed.

Considering the necessity of information about handling laboratory animals and the relatively rare literature on this topic, we decided to provide essential principles that everyone who undergoes microsurgical training should know.

4.2 The Three “R” Principles

In most countries, researchers are guided by the three “R” principles for humane treatment of research animals that were proposed by William Moy Stratton Russell and Rex Leonard Burch in 1959. ¹ These principles are referred to as reduction, refinement, and replacement. Reduction refers to methods that enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals; refinement refers to methods that alleviate or minimize potential pain, suffering, or distress and enhance welfare for the animals used; and replacement is the practice of preferring to use nonanimal methods over animal methods whenever it is possible to achieve the same scientific aim.

4.3 Symptoms of Pain and Distress in Laboratory Animals

When working with laboratory animals, it is important to be able to recognize the signs of pain and distress that they may exhibit. Pain is defined as an unpleasant sensory and emotional experience associated with potential or actual tissue damage, or described in terms of such damage (from the International Association for the Study of Pain). Distress is defined as the biological responses that an animal exhibits in an attempt to cope with a threat to its homeostasis. ² Signs of pain and distress in laboratory animals are presented in Table 4.1 and Fig. 4.1.

4.4 Anesthesia

Appropriate pain relief and sedation, as well as proper care of laboratory animals, are the responsibility of the researcher. There are two primary ways to induce anesthesia: injection and inhalation. Injection can be performed via intravenous, intraperitoneal, or intramuscular (Fig. 4.2) routes.

The mode of anesthesia is usually chosen based on the availability of a certain drug and the peculiarities of the study being undertaken. A variety of injectable pharmaceuticals useful for the narcosis of laboratory rats is presented in Table 4.2. ^{3 ,​ 4 ,​ 5 ,​ 6 ,​ 7 ,​ 8 ,​ 9} Metabolism is higher in rodents than in humans, and drugs are therefore metabolized and excreted faster. Thus, dosages for anesthetics differ significantly from those for humans. Many substances are narcotics and require special permission and conditions for their storage and use.

For rat anesthesia during microsurgical training, we prefer using a xylazine/ketamine cocktail. The recommended combination is 8 mL of 100 mg/mL ketamine + 1 mL of 100 mg/mL xylazine + 1 mL of sterile isotonic saline, resulting in 10 mL total cocktail. The dosage of 0.1 mL per 100 g intramuscular injection delivers 10 mg/kg xylazine and 80 mg/kg ketamine.

Laboratory animals are small in size, so the volume of injected solution is small (Table 4.3). For aqueous solutions, intramuscular injection sites should be rotated. For nonaqueous solutions, not more than two intramuscular injection sites and not more than three subcutaneous injection sites should be used per day. ¹⁰ Intraperitoneal injections should be infrequent in survival experiments due to the risk of peritonitis.