University Policy on Performing Rodent Survival Surgery

Revision 2, Approved by the Rutgers
Animal Care and Facilities Committee, April 4, 1997
Web Posting, June 4, 1999

CONTENTS

I. Objective

Experimental surgery is a powerful tool that enables researchers to obtain data that cannot be obtained otherwise. Animal welfare regulations and guidelines place great emphasis on the proper performance of surgery, as does the Animal Care and Facilities Committee (the "Committee") in its review of animal use protocols. Surgery should be done in accordance with the principles set forth in the NIH Guide for the Care and Use of Laboratory Animals (the "Guide"). Rodent surgery can be done following accepted practices of veterinary surgery, while making allowances for the unique needs of researchers. Properly performed surgery promotes animal welfare and good science. Students who learn good rodent surgical technique will be able to apply their knowledge of specific procedures to other procedures in other species. Surgery performed without proper anesthesia, monitoring and post-operative care can result in poor research data, unnecessary use of animals, and animal suffering. Animal surgery can be expensive. The Committee recognizes the concerns of researchers about the costs of implementing animal use regulations. The Committee feels the following guidelines allow reasonable accommodations for cost while meeting the spirit of animal welfare regulations and promoting good science.

These guidelines have been adopted by the Rutgers Animal Care and Facilities Committee to inform investigators of performance standards for rodent surgery at Rutgers. Principal investigators approved to do survival rodent surgery must follow these guidelines unless exceptions are granted by the Committee. Investigators are responsible for training and monitoring the surgery of all personnel who work in their laboratory.

Additional information on surgery, anesthesia and University policy is found in Care and Use of Laboratory Animals at Rutgers, provided to each investigator by Laboratory Animal Services (LAS).

II. Requirements

A number of things are required by those conducting rodent survival surgery. Each requirement will be discussed briefly.

A. Training and supervision

"It is important that persons have had appropriate training to ensure that good surgical technique is practiced,... The PHS Policy and the [Animal Welfare Act regulations] place responsibility with the IACUC [the ACFC at Rutgers] for determining that personnel performing surgical procedures are appropriately qualified and trained in the procedures to be performed." [p. 61, Guide, 1996]

Persons performing rodent surgery must have appropriate training and supervision. Qualifications will be reviewed during the protocol review process. All individuals conducting animal work as part of the research project must be listed in the protocol. Graduate students, undergraduates, and technicians doing surgery must work under close supervision until the principal investigator is confident that the surgical and postoperative care can be conducted in accordance with generally accepted practices. Laboratory Animal Services veterinarians or other qualified persons may provide training.

The Committee may require demonstration of surgical competence and compliance with these guidelines by requiring that surgery be observed by an LAS veterinarian.

B. Anesthetics, analgesics and tranquilizers

"An integral component of veterinary medical care is prevention or alleviation of pain associated with procedural and surgical protocols." [p. 64, Guide, 1996]

The use of anesthetics, analgesics and tranquilizers is required. An appropriate method of preventing and/or alleviating pain must be used. Agents should be selected in consultation with an experienced veterinarian. This is part of the protocol review process. Agents, routes, and doses must be listed in the protocol and other agents cannot be used without approval.

A good general anesthetic for rodents is a combination of ketamine and xylazine given by intraperitoneal injection. A poor anesthetic in rats is chloral hydrate which can lead to a fatal condition known as adynamic ileus. Information on rodent anesthesia is available in the manual.

C. Physical facilities

"The design of a surgical facility should accommodate the species to be operated on and the complexity of the procedures to be performed. For most rodent surgery, a facility may be small and simple, such as a dedicated space in a lab appropriately managed to minimize contamination from other activities in the room during surgery." [p. 78, Guide, 1996]

Physical facilities must be clean and otherwise prepared for aseptic surgery. Surgery must be performed in a room or area of a room that is easily sanitized and that is not used for any other purpose during the time of surgery. This means that an area of the laboratory must be cleaned and cleared of all extraneous items prior to surgery and that no other activities can be conducted in the area during the surgical procedure. Surgery may be done in an investigator’s laboratory or LAS can provide surgery facilities in many of the animal facilities.

D. Aseptic surgery techniques

"Aseptic technique is used to reduce microbial contamination to the lowest possible practical level. No procedure, piece of equipment, or germicide alone can achieve that objective. Aseptic technique requires the input and cooperation of everyone who enters the operating suite. The contribution and importance of each practice varies with the procedure. Aseptic technique includes preparation of the patient, such as hair removal and disinfection of the operative site; preparation of the surgeon, such as the provision of decontaminated surgical attire, surgical scrub and sterile surgical gloves; sterilization of instruments, supplies and implanted materials; and the use of operative techniques to reduce the likelihood of infection." [p. 62, Guide, 1996]

"Asepsis" is literally an absence of living organisms. Aseptic surgical technique is an approach to performing surgery with the goal of limiting the introduction of microorganisms to the surgical site. Performed properly, routine surgery will not require post-operative antibiotics.

Aseptic technique involves preparation of the facility, the animal, the operator and instruments and supplies in such a manner that they are sterile to start with and so they can be used in a manner which keeps them sterile. In theory, aseptic technique for rodent surgery is no different than that for any other species, including man. In practice, rodent surgery is approached differently for a number of reasons.

• Up until 1985, accepted guidelines for rodent surgery required only "clean" technique. "Older" investigators were used to doing rodent surgery under less rigorous standards.
• Rodents are perceived to be "resistant" to infection. Rodents are, of course, susceptible to infection. Rodent surgical procedures are often done quickly, through small incisions. This minimizes the chance of infection, but lack of clinically apparent signs does not mean that infection is not occurring.
• Cost is a factor in rodent surgery. Rodent surgery involves less cost in the value of the individual animal, and time spent on each procedure, so the "loss" of an animal due to surgical complications is of less consequence. However, animal welfare and quality research dictate that such losses be prevented.
• Rodent surgery is often done on groups of animals by a single individual in one session. If the same equipment and supplies are used on multiple animals, special procedures are needed to maintain aseptic technique from animal to animal.

1. Instruments

For initial sterilization, steam autoclaving or ethylene oxide gas sterilization are recommended. Both methods provide dry instruments at the time of surgery. Instruments should be wrapped in such a way that they can be introduced to surgical field in a sterile manner. Larger surgical packs can be wrapped with fabric wraps, or paper wraps. Smaller packs and individual items can be sterilized in see-through, peel-apart envelopes. Laboratory Animal Services (LAS) can help you obtain these supplies. LAS has steam autoclaves. (Ethylene oxide is not readily available at Rutgers).

2. Animal Preparation

The animal’s skin is a weak link in aseptic technique. The incision site cannot be "sterilized". Proper preparation can minimize contamination of the surgical field with skin microorganisms.

Hair around the surgical site should be removed with a surgical clipper using a #40 blade (clip "against the grain"). Shaving with a razor or use of a depilatory cream is generally not necessary. A common mistake is to not clip a sufficient area, only to find yourself looking at unsterile hair poking through your drapes during the surgery.

Scrub the surgical site with a surgical soap. Scrub in a spiral pattern starting over the intended incision site and moving outward. A typical scrub would involve use of a povidone-iodine (sudsing) soap (e.g. Betadine®), a rinse with alcohol, and final application of povidone-iodine (non-sudsing) solution.

3. Sterile Field

Based on observations of many "rodent surgeons", the two steps that would most improve their aseptic technique are 1) a surgical drape on the animal , and 2) the provision of a sterile surface to put their instruments on between use.

A surgical drape is not required by the Guide, but is strongly recommended.

Paper drapes are convenient because you can customize the hole to fit the surgical site. Any material can be used. Disposable surgical drape material is resistant to tearing when wet, and the blue/green color helps reduce glare from surgery lights.

A common mistake is to make the drape too small. The larger the sterile field you create, the easier you will find it to avoid "breaks" in sterile technique.

It is insufficient to sterilize surgical instruments only to place them on a lab bench between use.

A sterile surface for instruments between use can be the edge of the animal drape, a tray or pan used to sterilize the instruments, the inside of the instrument pack wrapper, the inside of a glove wrapper, or a separate piece of cloth or paper. This subject is discussed in more detail under the "NO-TOUCH" technique.

4. Surgeon

Sterile gloves are required for standard sterile technique. Sterile "exam" gloves are acceptable, but "surgeon’s gloves" provide a better fit which is important when doing fine work.

Cap, gown and mask are not required. They may be advisable when working with immunodeficient rodents.

5. Multiple Surgeries at one Session

One of the greatest challenges in rodent surgery is adapting sterile technique when performing multiple surgeries in one session. If an assistant is available to perform anesthesia, animal prep, and post-op care, the "surgeon" can use the same gloves and instruments on multiple animals. This is of course not acceptable technique in human or clinical veterinary surgery, as it may transmit disease (e.g. AIDS) between patients For simple surgeries, one could anesthetize and prep several animals at once. Even with this technique, a single operator will have to reglove after "breaking" sterility. Suggestions for re-use of instruments are given below.

It is always helpful to have a "non-sterile" assistant, because it is difficult to anticipate everything you will need during a surgery session. LAS can provide technical assistance on an hourly basis.

6. NO-TOUCH Technique

"The species of animal influences the components and intensity of the surgical program. The relative susceptibility of rodents to surgical infection has been debated; available data suggest that subclinical infections can cause adverse physiologic and behavioral responses that can affect both surgical success and research results. Some characteristics of common laboratory-rodent surgery--such as smaller incision sites, fewer personnel in the surgical team, manipulation of multiple animals at one sitting, and briefer procedures--as opposed to surgery in larger species, can make modifications in standard aseptic techniques necessary or desirable. Useful suggestions for dealing with some of the unique challenges of rodent surgery have been published." [p. 61, Guide, 1996]

The "NO-TOUCH" Technique is a modified approach to rodent surgery that is especially useful for multiple-surgery sessions, but one that may also be used for single surgeries such as stereotaxic procedures. The "NO-TOUCH" technique assumes that if you do all of the surgical manipulations with the working ends of your instruments, without touching the animal with your fingers directly, then it is only necessary that the tips of the instruments be sterile. The "NO-TOUCH" technique does not strictly meet the Guide’s requirements for sterile gloves, but the Committee feels that it is acceptable when certain guidelines are followed.

"NO-TOUCH" technique is not acceptable for all procedures. It should not be an excuse to circumvent accepted standards of sterile technique. In fact, you will find it requires meticulous attention to details.

Advantages of the "NO-TOUCH" technique are that sterile gloves are not required. The one surgeon is free to prep animals, introduce a pack of suture material into the sterile field, or make adjustments to a stereotaxic apparatus.

The following are guidelines for acceptable use of the "NO-TOUCH" technique in rodent surgery. Many of these techniques will also be useful in the standard aseptic technique.

1. Declare your intent to use the "NO-TOUCH" technique in your animal use protocol. The Committee will determine the suitability of the technique for the proposed procedure.
2. Sterilize all instruments and supplies in advance.
3. Prepare a sterile field on which to place instruments. Delineate a line between the area for sterile instrument tips and non-sterile handles. For example, prior to autoclaving, draw a line on a paper drape with a marker. Label each side "sterile" and "non-sterile". Or, create pockets for instrument tips by stapling a folded drape.
4. Handle instruments only by the handles.
5. Do not touch sterile tips with your hands. Do not allow tips to touch non-sterile surfaces.
6. Handle sutures, catheters and other material only with instrument tips.
7. Handle tissues only with instrument tips. Do not touch tissues with your hands.
8. After use, place instruments on the line on the drape with tips on the sterile side.
9. Assign instruments to a particular task. For example, use a heavy scissors to cut skin, then use another finer scissors for cutting internal tissues.
10. Between surgical procedures, clean instruments of blood with sterile saline and a sterile gauze or "Q-tip". Saline can be kept in a syringe or sterile cup.
11. Use two sets of instruments and alternate sets between animals. (optional)
12. Sterilize instrument tips between surgeries. An effective method to do this is with a glass bead sterilizer. Instruments placed in a cup of hot (500 F) glass beads are sterile in 15 seconds. Allow tips to cool before use. Clean instruments before heating.

E. Post operative care

Rodents must receive appropriate post operative care. Post-surgical care should include observing the animal to ensure uneventful recovery from anesthesia and surgery; administering supportive fluids, analgesics, and other drugs as required; providing adequate care for surgical incisions; and maintaining appropriate medical records. Records can be part of the regular research notebook but must be available for review by Laboratory Animal Services.

Records for rodent surgery do not require a great deal of detail. However, certain information is required to inform Laboratory Animal Services' personnel that surgery has been performed and who to contact should a problem be noted during routine observations. Each cage should be marked with a small 3 x 5 card which indicates, as a minimum, the protocol number, date and time of surgery, responsible individual with home and work phone.

Rodents should generally not be returned to their normal cage until they have recovered from anesthesia and are able to right themselves. Wire-bottom caging may allow feet to become caught, and does not allow animals to conserve body heat. Such cages are not appropriate in the immediate post-operative period. Animals should be singly housed on paper towels or soft bedding. After the initial recovery they should be observed at least daily with special attention to the appearance of the surgical site, attitude, alimentation and elimination, hydration, and weight loss. Attitude and weight loss are the two most important indicators of health in rodents. Observations should be recorded on a daily basis. Professional advice should be requested if indicated. Animals not expected to survive must be euthanatized before becoming moribund.

Properly performed aseptic surgery should not require the routine use of post-operative antibiotics. If post-operative infections become a problem, the first step should be to evaluate the aseptic technique of the operator. If antibiotics are used prophylactically, they should be started at the time of surgery and be continued for at least three days. No antibiotic is known to maintain appropriate blood levels for more than 8 - 12 hours with a single injection. An example of an acceptable drug for some rodents is oxytetracycline injection (Liquamycin, LA-200). At least in rats, therapeutic blood levels can be maintained by giving 60 mg/kg, SQ every 8 - 12 hours. Please note that some antibiotics are extremely toxic to certain species or strains of rodents such as hamsters and guinea pigs. Clinical infections should always be reported to the Laboratory Animal Services campus manager so that specific, individual guidance may be obtained.

F. Assessment of Surgical Outcomes

"A continuing and thorough assessment of surgical outcomes should be performed to ensure that appropriate procedures are followed and timely corrective changes instituted. Modification of standard techniques might be desirable or even required (for instance, in rodent or field surgery), but it should not compromise the well-being of the animals. In the event of modification, assessment of outcomes should be even more intense and might have to incorporate criteria other than obvious clinical morbidity and mortality." [p. 61, Guide, 1996]

When conducted according to these guidelines, rodent surgical procedures should result in a high success rate with few complications. Factors which might increase the incidence of problems include the implementation of new procedures, training of new people, and especially difficult surgical procedures. The ACFC and the LAS veterinary staff are obliged to assess the adequacy of current practices, and to implement changes where necessary. In order to do this in as unobtrusive a manner as possible, we have adopted the use of a "Surgical Outcome Log".  A log should be maintained for each procedure approved for each animal use protocol. The log should be maintained in your lab in a location where it will be available for inspection by LAS personnel and members of the ACFC, on request.

Surgical Outcome Log

III. Preparation for a Lab Inspection

The Animal Care and Facilities Committee inspects all animal use areas every six months. This includes investigator labs where surgery or other animal procedures are performed. The American Association for the Accreditation of Laboratory Animal Care (AAALAC) also visits investigator labs during its accreditation site visits, which occur every three years. Inspections may be unannounced.

Whether or not surgery is taking place at the time of the inspection, site visitors can determine much about surgery practices by asking appropriate questions of faculty and their laboratory staff. Here are some things you can do to demonstrate compliance. These are suggestions only.

1. Perform only surgeries for which you have an active, approved animal use protocol.
2. Train your staff in proper surgical technique. Familiarize them with these guidelines.
3. Post these guidelines prominently in your lab.
4. Have written standard operating procedures for each surgical procedure, and for instrument preparation, animal prep, etc.
5. Maintain a list of people trained to perform each procedure. These should be included on your animal use protocol.
6. Keep a surgery log. Document surgical complications, or better, the lack of them. Have post-up records available for review.
7. Convey to the site visitor that you know what acceptable procedures are, that all surgeons are trained in acceptable procedures, and that you follow them.
8. Emphasize those procedures you use to insure sterility in difficult situations.
9. Prepare for inspections with a mock inspection. A common question will be "Take me through a typical surgery."

IV. Deviations from this policy

Deviations from this policy must be approved by the Animal Care and Facilities Committee as part of an approved animal use protocol. Changes requested after approval must be requested as an amendment to the protocol.

V. Questions, comments and feedback

Investigators are encouraged to contact the Animal Care and Facilities Committee (Sponsored Programs Administrator, 445-2883) or Laboratory Animal Services (445-4168) with questions or comments about this policy.

VI. References (copies available from LAS)

1. Bradfield, JF, et al., Behavioral and physiological effects of inapparent wound infection in rats. Lab Anim Sci, 42(6): 572-578, 1992.
2. Brown, MJ. Aseptic surgery in rodents. SCAW Newsletter, 16(3): 4-12,
3. Cunliffe-Beamer, TL. Applying principles of aseptic surgery to rodents, AWIC Newsletter, 4(2): 3-6, 1993
4. Gentry, SJ. The use of aseptic surgery on rodents used in research. Contemporary Topics (AALAS), 33(1): 61-63, 1994.
5. Hicks, NJ. Reuse of biomedical implants in animals: a method for safe and effective chemical sterilization. Lab Animal, Feb., 40-43, 1995.
6. Lovaglio, J and Lawson, PT, A draping method for improving aseptic technique in rodent surgery, Lab Animal 24(10): 28-31, 1995.

VII. Equipment vendors

Both of these vendors offer a complete line of veterinary products. They provide rapid shipment of supplies. Schein offers a good catalog. Mr. Mirenna lives locally and can be of great assistance in selecting surgery equipment. Other vendors are equally as good, but these are vendors LAS has worked with in the past. LAS maintains an extensive file of vendor catalogs. Contact LAS at 908 445-4168 if you need assistance.

The Laboratory Animal Services Home Cage has links to vendors and documents such as the Guide.

1. Henry Schein, Inc (800) 772-4346

2. A. J. Buck and Son, Inc. (800) 638-8672

• Local contact: Mr. John Mirenna (908) 244-7703

This page last updated June 4, 1999

Manual Contents / LAS Homecage