Guidelines for Rodent Survival Surgery

(Material adapted from the University of Iowa Animal Care Unit and Stanford University)

• Introduction
• Monitoring Anesthesia
• Preoperative Considerations and Care
  • Preoperative Withholding of food
  • Animal Health Selection
  • Presurgical Medication
  • Pre- and Postoperative Antibiotics
• Postsurgical Care
• Considerations when Performing serial Surgeries
• Sterilization of Instruments and Supplies for Aseptic Surgical Procedures
• References

Rodents include the following species: mice, rats, Guinea pigs, hamster, and gerbils. Rabbits and most other species, except amphibians and fish, must have major survival surgery performed in an approved aseptic surgery suite. If you have questions, concerning this policy, please call the Office of Campus Veterinarian, 909-787-6332.

Introduction:

Post-operative infections in rodents can and do occur. The misconception that rodents have an innate resistance to bacterial infection has not been scientifically substantiated. Such infections which may not be apparent on casual observation, will cause loss of vessel cannulations ²⁶, and numerous changes in physiologic parameters ²⁷. In accordance with good scientific practice and standards set forth in the Public Health Service Guide for the Care and use of Laboratory Animals and the Federal Animal Welfare Act, aseptic surgical procedures must be used.

A separate room used primarily for aseptic procedures is desirable; however, it is appropriate to perform survival rodent surgical procedures in a conventional laboratory setting using aseptic technique. The following standards for aseptic procedures should be adopted by the investigator.

1. A clean, uncluttered work area and a sanitized work surface should be utilized for the surgery area. The work area should be located to minimize laboratory traffic not related to the surgical procedure and dedicated exclusively for surgery, when in use. Considerations should also be given to locate the surgery away from potential sources of contamination such as open windows, fans, or fume hoods which can blow dust into the area and increase desiccation of exposed tissues. The surgery surface should be disinfected (70% alcohol or a quaternary ammonium compound) before use. It often simplifies the maintenance of asepsis if a sterile drape is then applied over the surgery surface.
2. Animal preparation includes preparation of the surgical site by clipping of the hair using a #40 clipper blade and disinfection of the skin. Plucking of the hair may be appropriate for mice and similarly sized rodents. Application and removal of an adhesive pad will often remove any extraneous loose hair from the surgical site. Disinfection can be achieved by using surgical iodine solution. Sterile cotton pledgets or swabs dipped in disinfectant should be applied to the incision site and applied (if possible) in a circular fashion proceeding from the incision site to the edge of the clipped area. The used pledget or swab should then be discarded. After reaching the hair the pledget should not be reapplied to the incision site since it has been contaminated. This process should be performed a minimum of three times and the disinfectant should be in contact with the skin for a minimum of three minutes before the initial incision. Do not saturate other areas of the body with disinfectant since this enhances hypothermia which is a common postoperative complication in rodents.
3. The use of sterile instruments, supplies, and wound closure materials (suture, wound clamps)is required. Instruments used in pediatric or ophthalmic surgery are sized appropriately for rodent surgery ²⁸. These tend to be delicate instruments and the user should examine them prior to each sterilization to insure their integrity.
4. Draping the animal with sterile drapes to avoid contamination of the incision, instruments and supplies is strongly recommended. Opaque and non-opaque materials have been utilized. Clear materials have the advantage of allowing the investigator to monitor respirations and perfusion through the drape. Autoclavable plastic and sterile adhesive dressings are available for use.
5. Surgeon preparation: Surgical scrub of the surgeon hands utilizing the appropriate disinfectant; use of sterile gloves and wearing a surgical mask by the surgeon and any assistants working in the immediate area.

Monitoring Anesthesia:

The small size of rodents precludes the use of several methods to evaluate anesthesia commonly used in larger species. However, periodic observation of respiration, color of mucous membranes and loss of reflected eye color (in albino animals) will provide the surgeon with a good assessment of the animal's status. Except for Guinea pigs, the absence of the pedal reflex is a good indication that a surgical plane of anesthesia has been attained in rodents. The absence of the pinna reflex is a good indicator in Guinea pigs.

Preoperative Considerations and Care:

• Animal Health Selection:

A healthy rodent is a prerequisite to a successful surgery. Rodents undergoing clinical or subclinical disease often experience anesthetic complications and are not good candidates for a successful procedure. It is recommended that animals purchased for surgery be barrier housed prior to surgery to insure the absence of rodent diseases. Also, in general, a minimum of 48 hours is required for an animal to recover from the stress of shipping; therefore, surgery should not be performed immediately upon arrival.

• Preoperative Withholding of Food:

While it is common in larger specie to withhold food prior to surgery to prevent the possibility of aspiration pneumonia after regurgitation this practice is not necessary in rodents. Fasting for four hours before surgery, however, my promote the absorption of intraperitoneally administered anesthetics ²⁹. Water should never be withheld.

• Presurgical Medication

To decrease tracheobronchial secretions, which may cause obstruction of the trachea, atropine or glycopyrrolate should be considered. Also, the investigator should be prepared to aspirate secretions from the trachea if necessary.

• Pre- and Postoperative Antibiotics:

If proper aseptic technique is utilized antibiotics should not be necessary. In fact, antibiotics are contraindicated in hamsters and Guinea pigs due to the frequent development of fatal Clostridial enteritis. If the interior of the intestinal tract is exposed, however, antibiotics are commonly administered. To have the desired effect, antibiotics should be administered prior to surgery to provide adequate blood/tissue levels at the time of surgery.

Postsurgical Care:

After surgery the animal should be placed back in a cage that is lined with an absorbent pad. Animal bedding should not be present since the unconscious animal may aspirate bedding into the nares thereby compromising respiration.

The most common complication that occurs during and after surgery is the development of hypothermia. This is often exacerbated by performing surgery directly on a heat conducting surface (stainless steel). This can be avoided by using sterile pads under the animal or utilizing a circulating water pad. Electrical heating pads cannot be appropriately regulated and should never be utilized as a heat source. Many procedures entail the loss of body fluids either through bleeding or drying during surgery. In those cases the administration of warmed sterile saline either subcutaneously or intraperitoneally will hasten the animal's recovery.

The surgical site should be monitored daily to insure that the surgical wound is healing properly and that stitch abscesses, dehiscence or other complications have not occurred. Usually in seven to ten days the sutures can be removed from a properly healed incision.

Analgesics should be administered during surgery or immediately postoperatively. For minor procedures it may be appropriate to administer only one dose of an analgesic. For major procedures narcotic analgesics should be administered for the first 24 hours postoperatively and continued if necessary. A table of anesthetic and analgesic doses is available for review.

Considerations when Performing Serial Surgeries:

It is often necessary to surgically prepare several different animals during one session using one sterile pack. This is appropriate, providing care is taken to maintain sterility of the instruments. The following considerations should be made:

1. It may be appropriate to segregate instruments based on potential for contamination. For example, the instruments used to incise the skin could be dedicated solely for that purpose and separate instruments utilized to manipulate exposed tissues and organs.
2. Manipulate the tissues with only the tips of the instruments and avoid handling the tissues directly with your hands, which tend to be more easily contaminated. Using a dry bead sterilizer to resterilize the tips of instruments between surgeries will further insure adequate aseptic technique if precautions are taken to allow cooling of the instruments before reuse.
3. Consider using a separate sterile pack for no more than four animals.

Sterilization of Instruments and Supplies for Aseptic Surgical Procedures

Survival surgical procedures on all mammalian species must be conducted using aseptic technique which requires the use of sterile instruments and supplies. Many supplies such as gloves, surgical blades, and suture materials are commercially available as sterile packs. However, it is frequently necessary to sterilize, in house, items such as surgical instruments, drapes, gowns, etc.

In considering methods for sterilization procedures, it is important to differentiate between sterilization and disinfection. Sterilization kills all viable microorganisms while disinfection only reduces the number of viable microorganisms. High level disinfection will not kill the more resistant bacterial spores. Commonly used disinfectants such as alcohol, iodophors, quaternary ammonium and phenolic compounds are not effective sterilants and, therefore, are not acceptable for use on items intended to be used in survival surgical procedures.

The preferred methods of sterilization are high pressure/temperature (in autoclaves) and dry heat for items that can withstand high temperature and ethylene oxide gas for items that cannot withstand high temperature. However, cold chemical sterilants may be used effectively for many items.

The following are approved sterilization procedures:

1. High pressure/temperature steam sterilization using an autoclave and appropriate monitoring systems to assure sterility.
2. High temperature dry heat systems. Since it is difficult to drape instruments prepared in this fashion they cannot be stored for future use. Typically instruments are sterilized and allowed to cool immediately prior to use by the surgeon.
3. Gas sterilization with ethylene oxide using an appropriate gas sterilizer and appropriate monitoring systems to assure sterility and personnel safety.
4. Cold (chemical) sterilization:

Effective and proper use of cold sterilization is dependent on many factors including:

1. The use of chemicals classified as "sterilants". Those classified only as disinfectants (70% alcohol) are not adequate.
2. The physical properties of the items being sterilized: instruments must be relatively smooth, impervious to moisture, and be of a shape that permits all surfaces to be exposed to the sterilant. Instruments tend to degrade when exposed to sterilants requiring that their integrity be assured prior to use.
3. Exposure:

   All surfaces, both interior and exterior, must be exposed to the sterilant. Tubing must be completely filled and the materials to be sterilized must be clean and arranged in the sterilant to assure total immersion.

   The items being sterilized must be exposed to the sterilant for the prescribed period of time.

4. Use of fresh solutions. The sterilant solution must be clean and fresh. Most sterilants come in solutions consisting of two parts that when added together form what is referred to as an "activated" solution. The shelf life of activated solutions is indicated on the instructions for commercial products.
5. Rinsing chemically sterilized items. Instruments, implants, and tubing (both inside and out) should be rinsed with sterile saline or sterile water prior to use to avoid tissue damage.

There are several acceptable commercial sterilants available. Only products classified as sterilants are to be used for sterilizing instruments and implants for surgery and they must be used according to the manufacturer's recommendations for sterilization.

Following are examples of commercial products:

Discretion is required in using these agents to assure that they are used with appropriate safety precautions and that they are compatible with the items being sterilized. The use of sterilization procedures other than those listed must be approved by the IACUC.