IACUC Policy: Rodent Surgery
Policy # IBT-200.00 IACUC Approval: January 27, 2014

Purpose:
This document is intended to provide guidelines for investigators who perform survival surgical procedures utilizing rodents. The importance of utilizing appropriate surgical techniques should not be underestimated. They are designed to reduce post-surgical complications (e.g., infections and wound dehiscence), improve animal survival rates, and hasten return to the basal physiological functions that were present in the animal prior to surgery. Therefore, by observing appropriate procedures for aseptic surgery, the investigator will help ensure that the post-surgical rodent will be a suitable research subject.

Scope:
This policy applies to all surgical procedures performed on rodents at Texas A&M Health Science Center in which the animals are expected to recover from anesthesia. Prior to performing any survival surgery techniques on rodents, an approved IACUC protocol must be in place with appropriately trained personnel and procedures available.

Responsibilities:
The IACUC is responsible for ensuring all protocols meet this policy’s standard as a requirement for approval.
Principal Investigators are responsible for complying with the requirements of this policy including maintaining appropriate surgical documentation and for enforcing compliance with their research staff. It is also the responsibility of the PI to consult with the Attending Veterinarian in the pre-planning portion of protocol preparation.

Definitions:
Major surgery penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic functions. Examples of a major surgery include laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation.
Minor surgery does not expose a body cavity and causes little or no physical impairment. Examples of a minor surgery include wound suturing, and peripheral-vessel cannulation.
Multiple survival surgical procedures on a single animal are discouraged. Under special circumstances, such as if the procedures are essential related components of the research project, more than one major surgical procedure may be permitted with adequate scientific justification and the approval of the IACUC.
Survival surgery -the animals are allowed to recover from anesthesia.
Non-survival surgery- the animal is euthanized prior to recovery from anesthesia.
Sterilization is process whereby all viable microorganisms are eliminated or destroyed. The criterion of sterilization is the failure of organisms to grow if a growth supporting medium is supplied.
Disinfection is chemical or physical process that involves the destruction of pathogenic organisms. All disinfectants are effective against vegetative forms of organisms, but not necessarily spores.
Asepsis – the freedom of living tissue from pathogenic organisms.
Aseptic surgery is surgery performed in an environment sufficiently free of microorganisms such that significant infection with pathogenic organisms does not occur. 

Procedure:
I. Preparation of the Environment

The Surgery Room/Area

The Guide recommends that rodent survival surgery be performed in a dedicated facilities or space, but it is not required.  The surgical area must be easily sanitized. The immediate surgical area should not be used for other purposes during the time of surgery and traffic in this area should be minimized.

Preparation of the Surgery Area

Prior to and between surgeries, clean and disinfect the surface upon which surgery will be performed. Use soap and water, rinse thoroughly, and follow with an appropriate disinfectant. Commonly used disinfectants are quaternary ammonium compounds (such as Roccal); household bleach diluted 1 part to 32 parts water, chlorine dioxide-based sterilant (Clidox), chlorhexidine (Nolvasan), or other antimicrobial agent. Disinfectants must be prepared and used according to the manufacturer's recommendations.

Preparation of Surgical Instruments

Surgical instruments must be sterilized before use in a rodent survival surgery. Several techniques (steam, dry heat, ethylene oxide, or chemical agents) can be used to sterilize instruments and other materials that will come in contact with the animal's tissues. Steam or dry heat is the preferred methods to sterilize surgical instruments.

  1. Chemicals: Chemicals used to sterilize surgical instruments must be classified as a sterilant not a disinfectant. Chemical sterilants typically require a contact time of 6-24 hours, depending on the chemical used. For example, chlorine dioxide requires a minimum of 6 hours of contact time. Chemical sterilants must be prepared and used according to the manufacturer’s recommendations. All instruments sterilized by chemicals must be rinsed in sterile water before use in tissues.

2. Multiple Surgeries: When performing surgeries on multiple animals it is recommended to have at least 2 sets of sterile instruments to allow re-sterilization of instruments between animals. Chemical sterilants typically require hours of contact time, therefore they are seldom practical for re-sterilizing instruments on the same day as surgery. It is suggested that a new sterile instrument pack be used after every 6 major surgical procedures.

3. Glass bead sterilizer: 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 and then immerse 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. It should be noted that glass bead sterilizers and tips of instruments sterilized in glass bead sterilizers are capable of producing 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.

II. Preparation of the Animal 

Preparation of a rodent for surgery requires attention to both the specific surgical site and the physiological status of the animal as a whole. It is vital that the animal’s body temperature be maintained within acceptable limits as hypothermia is a significant cause of peri-surgical mortality in rodents.
The normal body temperature of a mouse is approximately 96.4° – 99.7°F (35.8° – 37.6° C). The normal body temperature of a rat is approximately 96.6° - 99.5°F (35.9° - 37.5°C).

  1. An ophthalmic ointment (e.g., Artificial Tears®) must be applied to the eyes of any animals receiving injectable anesthetics or to those animals anesthetized with gas anesthetics for greater than 5 minutes.
  2. Hair must be removed from the area surrounding the incision site. This may be accomplished with animal clippers, a razor, or a depilatory agent such as Nair®. If a depilatory agent will be used, all of the substance must be thoroughly removed from the skin after use to prevent skin irritation. In order to avoid contaminating the area of the lab in which surgery will be performed, hair removal should be performed at a separate location such as a nonadjacent portion of the lab bench. Loose hair must then be removed from both the animal and the environment. This can be done through use of a vacuum or sticky/Scotch tape (which is a practical and effective alternative for removing small quantities of hair).
  3. It is highly recommended that pre-operative analgesics and fluids be provided per approved IACUC protocol. Tissue injury causes the release of various inflammatory and pain mediators resulting in peripheral sensitization. Pre-op analgesics stop the inflammatory cascade and pain before it starts. Consultation with the Veterinarian regarding analgesics, anesthetics and fluids is highly recommended.
  4. After hair removal, the animal should be placed on a clean, absorbent surface, such as an absorbent pad, to minimize heat loss during surgery. A heating source must be provided when the duration of anesthesia will be greater than 10 minutes or for any procedure in which a body cavity will be exposed (ie. thoracotomy, laporatomy). Use of a recirculating warm water blanket or a self-regulating heating pad specifically designed for use with rodents is highly recommended. Electric heating pads (i.e., drug store heating pads) not specifically designed for medical use are only acceptable for use when placed under the caging unit. Use of warm water bottles, microwaveable heating packs (eg. Snuggle Safe – R.C. Steele Inc), and slide warmers is acceptable. To decrease the risk of burns, the heat source should never be in direct contact with the animal. Instead, a pad must be placed between the animal and heat source. Alternate heat sources may be used only with prior approval by IACUC or the veterinary staff. Heat sources should not be so hot as to elevate the animal’s body temperature beyond its normal physiologic range. Once the animal has been stabilized in the area in which surgery will be performed, the skin must be cleaned and/or disinfected prior to any surgical manipulations. The easiest and most efficacious manner of surgical preparation is to use a one-step patient preparation such as, Techni-Care® Surgical Scrub or ChloraPrep. These one-step systems involve applying the product to the area where the incision will be made, swabbing with a sterile swab and allowing about 2 minutes of contact time. Technicare must be wiped off just prior to the incision with a sterile gauze from the surgical pack. Another option for patient sterilization can be achieved by cleaning the area with three alternating scrubs of an appropriate disinfectant and rinse solution (see below). Materials (such a gauze pads or cotton-tipped applicators) should not be used for more than one scrub. The scrub should be performed so that the proposed incision site is scrubbed first and the scrub then continued in a spiral pattern which radiates out from the proposed incision line. If dirt or debris is still visible after three alternating scrubs, additional scrubs must be performed.

Disinfectants Recommended for Use for Skin Preparation
a) iodophors (e.g., Betadine® scrub or Prepodyne® scrub). Prolonged contact with these substances may irritate the skin. Therefore, these substances should be rinsed from the skin with alcohol, between subsequent scrubs.
b) chlorhexidine scrub (e.g., Nolvasan®, Hibiclens®)
c) Alternate skin disinfectants may be used only with prior approval from the IACUC office or veterinary staff. 

Appropriate Rinse Solutions
a) Warm, sterile saline or sterile water
b) 70% ethyl alcohol 

5. After disinfection of the skin, rodents should be covered with a sterile drape containing a hole over the proposed incision site. The drape helps minimize contamination of the surgical site and also serves as a clean surface upon which instruments, sponges, etc. can be placed during surgery. If desired, clear adhesive drapes (3M™ Steri-Drape™ Incise Drape) can be used which completely cover the animal without impeding visual assessment of the animal. However, care must be taken to ensure that the drape does not obstruct the animal’s breathing.

III. Preparation of the Surgeon

To perform rodent surgeries, the surgeon must, at a minimum, perform the procedures listed below. 

Minimally Required Preparations for the Surgeon

1. Wash hands prior to surgery
2. Wear a mask
3. Wear sterile surgical gloves
4. Wear a clean gown
5. Wear a hair cover
6. A sterile gown is desirable for major procedures 

IV. Suture Materials and Wound Closure

Selection and use of appropriate suture materials is imperative for successful wound closure and healing. Sutures are either absorbable or nonabsorbable dependant upon the materials from which they are manufactured.

For routine surgical procedures in rodents, commercial suture materials with swaged (attached) needles in sterile packets are available. Materials should be selected that are the correct size and have appropriate absorption (if absorbable) and handling characteristics for the intended procedure and animal species

  • Suture material must be sterile as they are a foreign material and provide a substrate where bacteria may proliferate.
  • Silk causes tissue reactions and is generally not considered to be an appropriate material for use in animals.
  • Monofilament (non-braided), synthetic materials (Prolene, PDS, nylon, mersilene), vetbond or wound clips are appropriate for skin.
  • Synthetic absorbable materials should be used for closure or ligation of deep tissues (Vicryl, PDS, prolene).
  • Proper wound closure is essential to avoid wound dehiscence. Surgery in which a body cavity is opened requires a two-layer closure in which the body wall is closed separately from the skin.
  • Wound clips or skin sutures must be removed 7-10 days after placement unless previously approved through IACUC.

V. Required Documentation

Intra- and post-operative documentation is required. A Rodent Post-Op Monitoring template is available in the PAR and is recommended to be utilized by the IACUC. Some basic information should include, date of surgery, pre-surgical weight, anesthetic/analgesic administration, type of surgery, and documentation of 5 day post-operative monitoring.

VI. Postoperative Care

It is the investigator's responsibility to oversee the animal’s anesthetic recovery. Monitoring involves regularly checking the animal in its cage until it is fully ambulatory. During the anesthetic recovery, it is important that the animals be separated from bedding within the cage as animals have previously choked on bedding at this time. This may be accomplished by placing a recovery pad or a paper towel between the animal and bedding. Alternatively, the bedding can be removed, and an absorbent pad placed in the cage. Once the animal has recovered, the pad can be replaced with fresh bedding. Animals recovering from anesthesia should be rotated from side to side every 15-20 minutes until they are able to remain upright. After this, the animal should be examined by the laboratory staff (or PAR staff if prior arrangements are made) for postoperative complications at least once a day for 5 days or until skin sutures or wound clips are removed. A veterinarian or veterinary technician should be contacted if the animal or surgical site appears abnormal. This would include, but is not limited to, excessive swelling, redness, dehiscence (opening) or drainage of the surgical site.

Analgesics (pain-relieving medication) should be provided as approved in the animal use protocol. Aside from bleeding or another direct complication of surgery, the common complications affecting rodents recovering from anesthesia are hypothermia, dehydration, and injury from cagemates.

Hypothermia: The risk of hypothermia can be minimized by keeping the animal in a warm environment and on a bedded or padded surface. An external heat source such as a recirculating warm water blanket or a self-regulating heating pad specifically designed for use with rodents is highly recommended.

Dehydration: To help prevent dehydration warm (not hot) sterile 0.9% NaCl or LRS be administered subcutaneously or intraperitoneally before, during, or after surgery. Additional fluids may be required under certain circumstances such as if 1) the animal suffers significant blood loss, 2) the animal will be anesthetized for prolonged periods (such as greater than 2 hours), 3) the animal is prone to dehydration due to any factors not related to surgery (such as if the animal is diabetic), or 4) the animal has not recovered from anesthesia within a reasonable amount of time depending on the anesthetic.

Injury from other animals: A rodent that has been anesthetized (especially with injectable anesthetics) should not be put back in a cage with other rodents until it is fully ambulatory. This is because rodents may cannibalize nonresponsive cage mates. Even if all of the rodents in a cage were anesthetized, some will be slower to recover than others. These animals may be injured by more alert animals. Animals may be housed together following anesthesia but prior to recovery only if they are continually observed (at least once every 2-3 minutes) by a member of the research lab.

Rodents may be returned to the animal room when they are fully ambulatory. The cage card should be marked to indicate the type and date of surgery.

Skin sutures or wound clips should be removed 7-10 days after surgery unless otherwise approved by IACUC.

VII. Training
Principle Investigators (PI’s) are required to ensure all staff conducting or assisting with rodent survival surgeries are properly trained and training has been documented. Researchers conducting surgical procedures must have appropriate training to ensure that good surgical technique is practiced – that is, asepsis, gentle tissue handling, minimal dissection of tissue, appropriate use of instruments, effective hemostasis, and correct use of suture materials and patterns (Guide, 115)” PI’s and staff may receive training from other proficient researchers and members of the (PAR) Veterinary staff.  Technical hands-on training is offered by the PAR staff on an individual basis.  In addition, the following online module is available:  (http://www.jove.com/video/2586/principles-of-rodent-surgery-for-the-new-surgeon).

VIII. Non-survival rodent surgeries
In nonsurvival surgery, an animal is euthanized before recovery from anesthesia. It may not be necessary to follow all the techniques outlined in this section if nonsurvival surgery is performed but, at a minimum, the surgical site should be clipped, the surgeon should wear a lab coat and wear gloves, and the instruments and surrounding area should be clean. For nonsurvival procedures of extended duration, attention to aseptic technique may be more important in order to ensure stability of the model and a successful outcome, (Guide, 118).

IV. Rodent surgery in investigator laboratories

The specific location of the surgical area within the laboratory should promote the proper conduct of sterile technique, and to the extent possible, it should be isolated from other activities in the laboratory. The surgical area should be dedicated for that purpose while surgery is performed.

Other factors that may impact the risk of contamination include the invasiveness and complexity of the surgical procedure, duration of surgery, and the nature of other non-surgical activities conducted in the laboratory (i.e. their likelihood of increasing the risks of surgical contamination). For complex or long procedures, or if the layout of the laboratory does not permit a suitable dedicated surgical space, it may be advisable to temporarily stop other laboratory activities, thereby dedicating the laboratory to surgery in order to maximize the potential for a good surgical outcome.

For minor surgeries of short duration, conducted in a suitable area within the laboratory, it may be acceptable to allow other laboratory activities to continue if they do not jeopardize sterile technique. The investigator, IACUC and veterinarian should evaluate surgical facilities to ensure they are appropriate.

References:

  1. Animal and Plant Health Inspection Service, USDA. US Animal Welfare Act (AWA 1990) and Regulations (PL-89-544; USDA 1985) 1991. CFR Title 9, Subchapter A - Animal Welfare. U.S. Government Printing Office, Washington, D.C.
  2. National Research Council. Institute for Laboratory Animal Research. 2011. Guide for the Care and Use of Laboratory Animals. Public Health Service, Bethesda, MD.
  3. Public Health Service Policy on Humane Care and Use of Laboratory Animals http://grants.nih.gov/grants/olaw/references/phspol.htm.
  4. AAALAC Guidelines

History:
00 –Original Version – January 27, 2014