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I. Purpose

To provide guidelines to ensure optimal survival surgery results in laboratory rodents to promote good science and animal welfare. The goal of aseptic surgery is to reduce microbial contamination to the lowest possible practical level. 

II. Scope

This  policy is for performing survival surgery in rodents and applies to all personnel performing survival surgery on rodents and providing post-operative care.

III. Definitions

  1. Survival Surgery: Survival surgery is defined as a surgical intervention in which it is anticipated that the animal will recover from anesthesia, regardless of the survival time.  Survival surgery is further defined as either major or minor.
  2. Major Survival Surgery: As a general guideline, major survival surgery (e.g., laparotomy, thoracotomy, joint replacement, and limb amputation) penetrates and exposes a body cavity, produces substantial impairment of physical or physiological functions, or involves extensive tissue dissection or transection.
  3. Minor Survival Surgery: As a general guideline minor survival surgery does not expose a body cavity and causes little or no physical impairment (e.g., jugular, or femoral cannulation, subcutaneous osmotic pump placement, wound suturing).
  4. Sternal Recumbency: An animal is resting upright on its sternum

IV. General Guidance

  1. The National Research Council Guidestates that aseptic technique must be used for all survival surgery, including the use of sterile gloves. Aseptic technique is used to reduce microbial contamination to the lowest possible practical level. Components of aseptic technique include preparation of the animal, preparation of the surgeon, sterilization of surgical instruments and implanted materials, and good tissue handling technique.
  2. Personnel who perform survival surgery must be appropriately trained on each procedure that they plan to perform. An individual learning a procedure may do so under the direct supervision of an individual whose surgical skills has been evaluated and approved by the MSU Attending Veterinarian. Prior to performing survival surgeries on their own, each individual must have their surgical technique evaluated by the MSU AV.
  3. Aseptic surgery must be conducted in dedicated facilities or spaces unless justified as an essential component of the research protocol in the IACUC approved protocol. Surgical facilities should be maintained and operated in a manner that ensures cleanliness, minimizes contamination from other activities conducted in the room or space at other times and minimizes unnecessary traffic. If it is necessary to use an operating room or space for other purposes, it is imperative that the room be returned to an appropriate level of hygiene before its use for major survival surgery. The number of personnel in the operating room or space, and their level of activity, have been shown to be directly related to the level of bacterial contamination and the incidence of postoperative wound infection.
  4. Pre-Operative Preparation: Preoperative preparation must include an assessment of the animal and review of the requirements for postsurgical monitoring, care, and recordkeeping. This includes assignment of personnel to attend to each of these items. In addition, all surgical equipment must be identified and prepared prior to the start of surgery to decrease the amount of time that the animal is under anesthesia.
    1. Health Status evaluation: Successful survival surgery is dependent on a healthy, acclimatized animal. The health status of the animal must be evaluated before beginning the surgical procedure.
      1. A period of acclimatization (generally 7 days) to new housing or handling procedures is also recommended before the surgical procedure to provide time for the animal to recover from shipping stress.
      2. The animal must be weighed prior to surgery and the baseline weight recorded.
      3. Preoperative anesthesia and analgesia must be provided according to the approved IACUC protocol. Anesthetics and analgesics must be clearly labeled with an expiration date and used prior to expiration. All drugs administered parenterally must be mixed fresh (within a few days) to reduce risk of contamination. Sterile saline for injection is preservative free and provides an excellent media for bacterial growth, prompting the manufacturer to label the vials for single use only. Containers of sterile saline for injection must be discarded no later than 3 days after opening. A new needle must be used for each animal.
    2. Post-operative care logistics: Personnel must not perform surgery unless arrangements have been made to perform post-operative care according to IACUC policy Post-operative Care of Rodents.
  5. Animal Monitoring: Animals undergoing survival surgery must be monitored to ensure appropriate depth of anesthesia and maintenance of vital functions. Careful monitoring and timely attention to problems increase the likelihood of a successful surgical outcome.
    1. Animals must not be left unattended while anesthetized. Failure to provide constant monitoring of the animal may result in death of the animal due to anesthetic complications or recovery of the animal during the surgical procedure, which would result in significant pain and/or distress.
    2. Depending on the species, monitoring may include observation of respiration rate, color of extremities, and response to peripheral reflex stimulation. Additional monitoring may include measurement of temperature, heart rate, end-tidal carbon dioxide, peripheral oxygen saturation, electrocardiogram, or electroencephalogram. Use of a transparent sterile drape may be helpful to facilitate observation of the animal.
  6. Facility and Equipment
    1. Surgery must be performed in ARC procedure rooms unless specified in the IACUC approved protocol. Animals removed from ARC for surgery must be returned within 24 hours.
    2. The immediate surgical area should be dedicated to surgery and related activities and should not be used for other purposes during the time of surgery.
    3. Surgery should be conducted inside a biosafety cabinet (BSC) when possible, or on a clean, uncluttered bench or table that is impervious to liquids. Procedure rooms should be reserved through the ARC. To prepare the surgical area, disinfect the worksurface with an appropriate disinfectant (e.g., accelerated hydrogen peroxide) place a clean pad or drape on the surgical surface.
    4. If using isoflurane, the rodent face mask should be disassembled and cleaned prior to use as it comes into direct contact with the animal and therefore may serve as a nidus of infection. Cleaning the mask also ensures that fur and other debris is not obstructing the waste gas removal system.
    5. Thermoregulation: The surgical table/board/pad should be equipped with a mechanism to provide controlled supplemental heat to the animal to help it maintain a normal body temperature. This will reduce hypothermia and decrease the recovery time.  A water circulating heating pad is preferred and  very effective for this purpose. Heat lamps (without feedback mechanisms) or household electric heating pads are not acceptable methods for providing supplemental heat during anesthesia/ surgery.
      1. Maintenance of normal body temperature minimizes cardiovascular and respiratory disturbances caused by anesthetic and is of particular importance in small animals, such as rodents, where the high ratio of surface area to body weight may easily lead to hypothermia. Animals must be provided with a supplemental heat source to prevent loss of body temperature during the procedure. A large variety of supplemental heat sources are available for use to 

        prevent hypothermia in small mammals and birds. Selection of the most appropriate heat source depends on several factors.

        1. Water circulating heating pads, which can be set to a desired temperature, and electric heating pads designed specifically for use in small animal surgery (i.e., have a self-regulating system via a thermocouple attached to the animal) are recommended.
        2. Chemical hand warmers may be used in cases where other supplemental heat sources are not available or cannot be used. This type of heat source may reach temperatures of up to 130ºF; therefore, caution must be used to provide a layer of material (e.g., paper towels) between the hand warmer and the animal to prevent direct contact which may result in thermal burns and over-heating the animal. Use of this method of temperature support is best coupled with use of a rectal temperature probe to closely monitor the body temperature of the animal.
        3. Electric heating pads designed for domestic use by humans must never be used for the surgical procedure. This equipment may be used only for placement under the cage (cage half-on and half-off) during animal recovery. These heating pads have been shown to cause thermal burns and can over-heat small animals. Self-regulating electric heating pads designed for use in rodents that are equipped with a thermocouple to monitor the animal may be used when performing survival surgery.
  7. Sterilization of Instruments
    1. Use of Sterile Instruments: Surgical instruments must be sterilized for use in survival rodent surgery. Several techniques (steam, dry heat, gas sterilization or chemical agents) can be used to sterilize instruments and other materials that will come in contact with the animal’s tissues. Specific sterilization methods should be selected based on the physical characteristics of the materials to be sterilized. Steam or dry heat are the preferred methods to sterilize surgical instruments.Other methods (e.g., chemical) may be used but must be documented in the IACUC approved protocol.
      1. Plastic implantables, suture, surgical clips/staples and other materials must also be sterile.  Sterilization can be achieved by several methods:

        1. Steam: 250°F, 15 psi, for 30 minutes. Sterilization indicators (e.g., sterilization chemical indicator strips or autoclave indicator tape) should be used to validate that materials have been properly sterilized. If instruments are placed inside a sealed wrapped instrument pack, a sterility indicator strip should be placed inside of the pack to ensure steam penetration and effective sterilization of the contents of the pack. Instrument packs must be labeled with the date of sterilization and must be used with one year of sterilization.
        2. Physical: A glass bead sterilizer may be used to help maintain sterility of instrument tips if re-using instruments.  The optimal method for re-sterilization of instrument tips on the day of surgery is using a glass bead sterilizer. While the first set of instruments is being re-sterilized, the second set is used. After using a set of instruments, remove all organic material, a sterile saline/water bath and gauze can assist with this. Then place the tips of the instruments in a glass bead sterilizer for 20-30 seconds (follow manufacturers guidelines). Make sure the tips of the instruments have cooled before using them on tissue. Tips may be cooled by dipping in sterile water. Note: glass bead sterilizers and tips of instruments sterilized in glass bead sterilizers can produce severe burns. Care must be exercised when using a glass bead sterilizer, and all manufacturer instructions and safety precautions must be followed to avoid injury. Sterilized instruments are to be placed on a sterile drape to reduce the chances of contamination.  It is acceptable to re-sterilize surgical instruments utilizing a glass bead sterilizer for use up to five rodents during the course of a day before they are washed and re-sterilized. One sterile instrument pack may be used on a maximum of 5 rodents.
  8. Animal Preparation
    1. Individuals performing aseptic preparation of the surgical area will wear, at the minimum, head cover, lab coat, mask, and
    2. While anesthetized at least a centimeter of hair (where possible) be removed on either side of the incision site. Hair can be removed by clipping with an appropriately sized clipper, shaving with a razor or by using a depilatory cream. The animal’s hair must be removed from the area over and adjacent to the surgical site on all sides. A large area of hair must be removed to ensure that hair is not present in the sterile field including the opening created by the surgical drape. Removal of the hair decreases the risk of inadvertent introduction of skin bacteria and/or hair into the surgical incision and therefore decreases risk of infection.
      1. Clipping: Loose hair must be removed using adhesive tape after clipping Clippers must be thoroughly cleaned after each using a disinfectant provided by the ARC  (e.g., Peroxigard) to ensure optimal operation and decrease risk of transfer of bacteria (including fecal matter) to the surgical site. Clippers must be maintained to prevent rusting and/or malfunction that can result in damage to the skin. For information on clipper purchase, please contact the Attending Veterinarian.
      2. Depilatory Cream: Use of an over the counter depilatory cream may be used for fur removal but caution must be used to prevent skin damage due to the chemical reaction. Additionally, care must be used to remove all depilatory cream from the animal to prevent ingestion of the cream or transfer of the cream to the eyes when the animal grooms.
    3. The surgical site must be disinfected with an appropriate skin antiseptic. Use of chlorhexidine scrub and solution is recommended. Povidone iodine solutions (e.g., Betadine) do not adhere to the skin and can be quickly removed by the animal after surgery during grooming. Chlorhexidine scrub and solution adhere to the skin and cannot be removed via grooming and provides residual antimicrobial for up to 24 hours after surgery. Additionally, povidone is classified as an environmental hazard (due to the presence of iodine) during disposal of expired products. Monitor expiration date of the skin antiseptics and discard appropriately when expired.
      1. Use of cotton buds to apply the skin antiseptic is recommended. This allows for more precise application and reduces the risk of wetting the animal which reduces the risk of death due to hypothermia. Three alternating applications of chlorhexidine scrub (to remove oils and gross debris) followed by chlorhexidine solution should be applied. A total of 6 applications (3 of the scrub and 3 of the solution) is recommended. Start the scrubbing process at the intended incision site and progress outward in a circular pattern away from the intended incision site. Do not return the cotton bud to the incision site once it has been in contact with fur along the margin of the aseptic field.
    4. Sterile ophthalmic ointment (e.g., Puralube) must be applied to the corneas to prevent corneal drying or other injury for all surgical procedures greater than 5 minutes duration.  Ophthalmic ointment must be used unless it interferes with the surgery being performed.  Failure to lubricate then eyes may result in corneal damage and infection of the eyes during the post-operative period.
    5. Sterile drapes are always required for surgery in which a major body cavity is exposed (thorax, abdomen). A sterile drape helps to decrease risk of inadvertent introduction of skin bacteria and/or fur into the surgical incision and decreases risk of infection. Caution must be taken to ensure that the sterile drape remains sterile once it is placed (the sleeves of a gown or instruments that have been handled with a non-sterile glove should not come in contact with the drape). If incisions are closed with sterile surgical wound clips or glue, or if the incision is not closed but the defect is filled with dental acrylic, the patient need not be covered with a sterile drape.
    6. Anesthetic depth must be assessed prior to beginning the surgical procedure.
  9. Surgeon Preparation
    1. Personnel who perform or assist with surgery must wear a clean coat, face mask, head cover, and clean or sterile gloves (see below for criteria for use of sterile gloves).
    2. Use of Sterile Gloves:
      1. For certain surgical procedures in rodents in which the surgeon’s gloved hands do not come into contact with the sterile field, the use of sterile gloves is not required by the MSU IACUC. An example of such surgeries includes cranial implants using stereotaxic equipment. If sterile gloves are not required, the surgeon must wear a clean lab coat, surgical face mask and don clean examination gloves. If uncertain regarding the use of sterile surgical gloves for certain procedures, contact the AV.
      2. Sterile gloves must be used if any part of the hands will come into contact with the surgical incision area or with sterile equipment, including suture material, or the tips of surgical instruments.
  10. Surgical Instruments and Intraoperative Technique
    1. All items that come in contact with an internal body tissue must be sterile, including surgical instruments, suture material, surgical staples, and implants.
    2. Sterile packaged equipment must be inspected for tears or damage before use. Damaged packages should not be used for survival surgery procedures.
    3. If sterile gloves will not be worn sterile instruments must be held by the handles, and only the sterilized tips of the instruments must come into contact with the operative site.
      1. If the tips of the instruments come into contact with non-sterile material, they must be re-sterilized by a method appropriate for the instrument (e.g., glass bead sterilizer, autoclaving, or chemical sterilant).
      2. During the procedure, sterility of the tips of the instruments must be maintained by placing them in a designated sterile area (e.g., under a fold of sterile drape or paper towel or in a sterile beaker).
      3. Caution must be used to ensure that instruments and suture material are not contaminated by touching non-sterile surfaces (e.g., hands, work surfaces, non-sterile paper towel on hood surface, etc.).
    4. If multiple rodents will undergo surgery in a single day, the same instruments may be used for up to 5 animals. In between animals, the instruments must be cleaned with sterile saline to remove any organic debris and then re-sterilized using a hot-bead sterilizer per manufacturer guidelines. (see G.1.b. above).
    5. If incisions are closed using suture material, the patient must be covered with a sterile drape during surgery to prevent contamination of suture material by contact with non-sterile surfaces such as the animal or its surroundings. Sutures used for survival surgery must be used prior to the expiration date. Expired suture material may be used for terminal procedures but must never be used for survival surgery. Use of a running suture to close the muscle layer followed by simple interrupted or subcutaneous sutures to close the skin must be used for abdominal surgery. Knots should have at least 4 throws to ensure security of each knot. This is especially important in animals as they tend to over-groom the incision area and may loosen knots during the grooming process. Use of 4-0 or 5-0 suture PDS (absorbable, monofilament), Vicryl or Dexon (both absorbable and braided) with a reverse cutting 3/8 curve needle is recommended for skin closure in rodents. 4-0 or 5-0 suture may be used in rats depending on age and sex. Silk and gut must never be used for skin sutures.  Nylon sutures are not absorbable and must be removed in 10-14 days. Percutaneous sutures should be tied loosely to barely appose wound edges; otherwise, postoperative swelling may cause the suture to be too tight, strangle cutaneous vessels and lead to pain, infection, and wound failure.
  11. Documentation
    1. A written survival surgery/post-operative care log must be maintained in the animal housing room for the first 7 days after surgery and be available for review by the AV. The IACUC may also request copies of the documents. See IACUC SOP "Post-operative Care of Rodents" for details. 
  12. A notation must be made on the standard cage card on the day of surgery indicating the name and contact information of the person performing surgery, the type of surgery, and the date the surgery was performed.
    1. Animal(s) must be visually assessed daily, and the surgical/post-operative care log completed daily. See IACUC  policy Post-Operative Care in Rodents.

IACUC Approval Date: 08/18/2021

Review Date: 08/18/2021

Issue Date: 08/30/2021