Standard Procedures for Rodent Surgery

Recommendations for the performance of rodent surgery are based on the 1996 edition of the NIH Guide for the Care and Use of Laboratory Animals and 9 CFR, the Animal Welfare Act (AWA). Part 2 of the AWA states that major surgical procedures on rodents "must be performed using aseptic procedures". Adequate procedures include the use of sterile instruments, sterile surgical gloves, and aseptic preparation of the surgical site in order to prevent postoperative infections. A separate facility for rodent surgery is not necessary. A rodent surgical area can be a room or portion of a room that is easily sanitized and not used for any other purpose during the time of the surgery.

Rodents include hamsters, gerbils and guinea pigs, as well as rats and mice. Guinea pigs and hamsters are USDA covered species, meaning that they are not exempt from USDA regulations and the provisions of the AWA. Rodent surgery can be classified as minor or major in nature.

Anesthesia and anesthetic agents of rodents will not be discussed here. One should not overlook the utility of local anesthetics. Please contact the Campus Veterinarian for more information concerning the use of various anesthetics, analgesics, and tranquilizers.

Paralytic agents may not be used without anesthesia. To prevent corneal desiccation, place ophthalmic ointment in both eyes of rodents undergoing anesthetic procedures. The administration of antibiotics and analgesics prior to commencing a procedure can make them more effective.

Minor Surgery
"Minor survival surgery is defined as any procedure which does not expose a body cavity and causes little or no physical impairment" (Guide for the Care and Use of Laboratory Animals. National Research Council. 1996.) and includes injections, venipuncture, and subcutaneous implants. When conducted with proper care, these techniques present few difficulties. "Minor procedures are often performed under less stringent conditions than major procedures but still require aseptic technique and instruments and appropriate anesthesia." (Guide for the Care and Use of Laboratory Animals. National Research Council. 1996.) Be aware that much rodent research is performed within human medical centers and that implants or instruments can contaminate rodents with human pathogens if improper technique is used.

Major Surgery
Major surgery includes invasion of the cranial, abdominal, or thoracic cavities. Any procedure that might leave the rodent with a permanent handicap, whether physical or physiological, would also be considered major surgery. The use of aseptic technique is mandatory in these surgeries to minimize the possibility of postsurgical infection. Consultation with one of the Campus veterinarian is recommended if you have questions regarding techniques appropriate for these situations.

Chronic Implants
Chronic implants, such as chronic intravenous catheters and head caps, are intermediate in nature, but are techniques presenting the most severe postsurgical infections. Surgical technique needs to be meticulous, as for major surgery. Postsurgically, use sterile technique when accessing the catheter (s). The most critical requirement is to inject only sterile solutions into the catheter. Solutions should be freshly prepared or stored under refrigeration if prepared in advance. The top of the vial or mouth of the container containing solutions for injection must be kept clean and wiped with alcohol or flamed before drawing up the solution. Inoculation of even a few organisms into an intravenous catheter may result in death of the animal due to sepsis.

Surgical instruments must be sterile. Heat sterilization is ideal. Agents such as chlorine dioxide or gluteraldehydes can be used for cold sterilization. Chlorine dioxide is not documented as being toxic to animal tissue but will corrode stainless steel instruments. Gluteraldehyde must be thoroughly rinsed off of instruments with sterile saline or water before use of delicate items, such as drills and burrs. Disinfectants should be replaced when contaminated with blood or other body fluids. Catheters and implants can be sterilized using ionizing radiation or ethylene oxide (see Appendix 2).

Performing pre-surgical evaluations help insure your prospective patients are not overtly ill. Is the animal alert with a smooth coat and clear eyes? Withholding food is not necessary in rodents unless specifically mandated by the protocol or surgical procedure. Water should NOT be withheld unless required by the protocol. Withholding food for more than six hours should be discussed with a veterinarian.

Preparation of the animal should include clipping or shaving the surgical site with a generous border (at least 1 cm) to keep hair from contaminating the incision (hair removal should be performed in a location remote from the surgical area). The surgical site should be scrubbed with a germicidal scrub (see Appendix, Table 3), being careful to scrub from the center of the site toward the periphery. The site can then be rinsed with a 70% alcohol, sterile water, or sterile saline. Three alternating preps of germicidal scrub and rinse are considered adequate. Note that alcohol will also contribute to hypothermia if liberally used. Finally, the area should be draped with sterile drapes, which not only helps prevent stray hair from entering the surgical field, but also provides a sterile area on which to lay sterile instruments during surgery.

The surgeon must thoroughly scrub his or her hands with a bactericidal scrub (see Appendix 3). The use of sterile surgical gloves is necessary. A surgical mask should be worn for major surgeries. Wearing a clean lab coat is mandatory. A sterile gown is preferable for major surgeries.

Surgical instruments, gloves and other paraphernalia may be used on more than one animal. Any item used on multiple animals must be carefully cleaned and disinfected between animals (see Appendix 4). Alternating two or more sets of instruments is one way to allow time for instruments to sit in a disinfectant or sterilant solution for more than just a few minutes.

Animal evaluation during surgery is critical. Monitoring of anesthetic depth is usually of first importance. Unfortunately, techniques for monitoring anesthetic depth vary somewhat with the agent used. A quiet animal that does not move when a painful stimulus is applied is the most certain indicator of adequate anesthesia, however, the zone between quiet and too quiet is very narrow in rodents.

Maintaining body temperature is next in importance, as anesthetics induce hypothermia either directly or indirectly. It is easier to keep animals warm than warm them up. Warm water blankets or bottles provide supplementary warmth without being too hot. Bubble wrap helps small rodents maintain body temperature. During surgeries, warm sterile fluids (saline or lactated Ringers solution) should be provided. These can be administered subcutaneously, intravenously or intraperitoneally. Any tissues exposed for long periods during surgery should be kept moist with these same warmed solutions. Some anesthetic agents, such as xylazine, will predispose an animal to volume depletion.

Observation during postsurgical recovery is important. The animal, in or out of its cage, must be kept warm. Warm water pads, bubble wrap, blankets, or the blue "diaper" pads work well. The use of electric heat pads or heat lamps may overheat the animal; their use is discouraged. If electric heat pads or heat lamps must be used, provision must be made to make frequent observations and turning of a somnolent animal so that the animal will not be overheated, with preventing burns being of the utmost importance. Provision must also be made so that an awake animal can escape the heat source when it becomes too warm. Warmed fluids can be administered subcutaneously, intravenously, or intraperitoneally if there is any suspicion the animal may be dehydrated. Over hydration is not generally a problem in animals with normal kidney function. A recovering animal should be watched continuously until in sternal recumbency, and able to move around without plugging its nostrils with bedding. Some rodents left overnight on pads or paper bedding will eat that bedding. To prevent cannibalism, house rodents individually until they are ambulatory.

Sutures and/or staples need to be removed 7-10 days following surgery, if the rodent has not already done so (see Appendix 5 for data on suture types and uses). Any foreign substance left in the incision for a long period of time serves as a nidus of irritation and infection. A veterinarian should examine incisions that do not appear to be healing. Postsurgical observations include a minimum daily observation of the condition of the animal and the surgical site. A sample "Postoperative Evaluation Record" is provided (see Appendix 6).

Please identify cages with postoperative animals to:

• explain the condition of the animals to animal care staff (e.g. sedated animals thought to be ill)
• assure animal care staff, veterinary staff, inspectors, and others that proper care is being given to the animals,
• inform animal care and veterinary staff how recently the investigator has seen the animal to avoid contacting the investigator to inform them of the animal's condition.

The following is a list of recommendations involving techniques that may be difficult to perfect.

• Touch only "prepped" areas with sterile instruments and gloved hands.
• Keep operating fields draped.
• Do not let catheters or implants become contaminated.
• Use sterile solutions.
• Disinfect the tops of containers of solutions.
• Use sterile technique to access implanted catheters.

Not only are the above recommendations more humane to our animal charges, but following these recommendations will improve one's research by providing a less stressed animal and thereby decreasing the number of variables in a research protocol. The rat has always been considered "hardy" and not subject to postsurgical infections, but published research has documented that postsurgical infections in rats are subtle. The rat appears to eat and act normally, but will not respond appropriately to research stimuli. As with all new and improved techniques, patience and practice are required to harvest full benefits from the use of aseptic surgical techniques in rodents.

There is ample literature available supporting the recommendations presented in this document. Please contact IACUC and/or the Campus Veterinarian for assistance or to provide referrals to other researchers with applicable knowledge or skills.