Animal Care Services

BASIS:
Recommendations for the performance of survival surgery on mice, rats, and birds are based on the 1996 edition of the NIH Guide for the Care and Use of Laboratory Animals (the Guide, pp. 60-65, 78-79) and the USDA Animal Welfare Act Regulations (AWARs) §2.31(d)(1)(ix) and §2.33(a)(5).

Note: For the purpose of these guidelines, the term “rodent” refers to mice of the genus Mus and rats of the genus Rattus. For information on survival surgery in warm-blooded species other than rats, mice, or birds, please refer to the ACS Guidelines on Survival Surgery in USDA-Covered Species.

DEFINITIONS:

Aseptic Technique:
Aseptic technique reduces microbial contamination to the lowest possible practical level and includes preparation of the animal, 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.

Survival Surgery:
Any surgical procedure from which an animal regains consciousness for any period of time.

Major Survival Surgery:
Any survival surgical procedure penetrating and exposing a body cavity or producing substantial impairment of physical or physiologic functions. Examples of major surgery include laparotomy, thoracotomy, craniotomy, joint replacement, spinal transection, and limb amputation.

Multiple Survival Surgeries:
Multiple survival surgical procedures are not permitted on animals unless scientifically justified. For additional information, please refer to the ACS Guidelines on Multiple Survival Surgeries.

GENERAL GUIDELINES:

Location of Surgery

A surgical area for rodents and birds can be a room or portion of a room that is easily sanitized and not used for any other purpose during the time of surgery. However, because dedicated surgical facilities provide advantages with regard to reduced investigator regulatory burden, consideration must be given for using ACS facilities (Communicore Basement, Brain Institute, etc.), whenever possible. There is no charge to use the facilities or the gas anesthetic machines (when used within the facilities). An investigator’s laboratory may be used as a survival surgery area provided the investigator provides scientific rationale for such use and the location is inspected and approved by the IACUC. In selecting a surgical location, the investigator should bear in mind that “the number of personnel and their level of activity have been shown to be directly related to the level of bacterial contamination and the incidence of post-operative wound infection” (the Guide, p. 78-79). Thus, every attempt should be made to sufficiently physically separate the surgical area from other areas in the room to minimize unnecessary traffic and decrease the potential for contamination of the wound. Further, the location should be designed to include the following three areas.

(1)   An area should be designated for preparation of the animal, including weighing, hair or feather removal, and initial skin disinfection. The prep area should be sufficiently separate from the surgery table to minimize the potential for contamination of the surgery area by aerosols generated during animal preparation.   Again, physical separation of the prep and surgical areas is expected.

(2)   A separate area should be set aside for conducting surgical procedures (i.e., from skin incision to wound closure). The surgical table and immediate surrounding areas must be constructed of material that can be cleaned (washed with soap and water) and then disinfected using appropriate agents (see Table 1 below). The immediate surgical area should be disinfected prior to and between surgeries to decrease dust-borne contamination and may not be used for other purposes during the time of surgery. 

(3)   Finally, a separate recovery area should be established. This should be a quiet, undisturbed location where the animals can be observed. 

Table 1. Recommended Hard Surface Disinfectants
(e.g., table tops, equipment) Always follow manufacturers’ instructions.

 

Agents	Examples	Comments
Alcohols	70% alcohol	Minimum contact time required is 15 minutes.  Contaminated surfaces take longer to disinfect.  Remove gross contamination before using.  Inexpensive.  Flammable.
Chlorine	Sodium hypochlorite (Clorox® 10% solution) Chlorine dioxide (Clidox®, Alcide®)	Corrosive.  Presence of organic matter reduces activity.  Chlorine dioxide must be fresh (<14 days old).  Kills vegetative organisms within 3 minutes of contact.
Aldehydes	Gluteraldehyde (Cidex®, Cide Wipes ®)	Rapidly disinfects surfaces.  Toxic.  Exposure limits have been set by OSHA.

Surgical Instruments

Surgical instruments must be sterile. A list of acceptable methods for instrument sterilization is included in Table 2 below.

Table 2. Recommended Methods of Instrument Sterilization
Always follow manufacturers’ instructions.

Agents	Examples	Comments
Physical: Steam sterilization  (moist heat)	Autoclave	Effectiveness dependent upon temperature, pressure and time (e.g., 121.6°C for 15 min. vs. 131°C for 3 min.).
Chemical: Gas sterilization	Ethylene Oxide	Requires 30% or greater relative humidity for effectiveness against spores.  Gas is irritating to tissue; all materials require safe airing time.  Carcinogenic.  Suitable for catheters and implants.
Chlorine Dioxide	Clidox®, Alcide®	A minimum of 6 hours required for sterilization.  Corrosive.  Presence of organic matter reduces activity.  Must be freshly prepared (<14 days).  Must be thoroughly rinsed from instruments using sterile distilled water before use.
Aldehydes	Gluteraldehyde	Many hours required for sterilization.  Corrosive and irritating.  Consult Biosafety Officer on proper use.  Must be thoroughly rinsed from instruments using sterile distilled water before use.

Surgical instruments may be used on more than one animal; however, any item used on multiple animals must be carefully cleaned and disinfected between animals (see Table 3 below). Hot bead sterilizers are the preferred method, although prolonged soaking in disinfectant is also acceptable. Because the effectiveness of disinfection is directly dependent upon the contact time with the disinfectant, the surgeon is expected to anticipate the number of surgical instruments required to guarantee uninterrupted conduct of the procedures while affording ample disinfectant contact time. Replace disinfectants when contaminated with body fluids or tissues.

Table 3. Recommended Instrument Disinfectants
Always follow manufacturers’ instructions.

 

Agents	Examples	Comments
Dry heat	Hot bead sterilizer	Fast.  Instruments must be cooled before contacting tissue.
Alcohols	70% alcohol	Minimum contact time required is 15 minutes.  Contaminated surfaces take longer to disinfect.  Remove gross contamination before using.  Inexpensive.  Flammable.
Chlorine	Sodium hypochlorite (Clorox® 10% solution) Chlorine dioxide (Clidox®, Alcide®)	Corrosive.  Presence of organic matter reduces activity.  Chlorine dioxide must be fresh (<14 days old).  Kills vegetative organisms within 3 min.  Must be thoroughly rinsed from instruments using sterile distilled water before use.
Peracetic Acid/ Hydrogen Peroxide	Spor-Klenz®	Corrosive to instrument surfaces.  Must be thoroughly rinsed from instruments using sterile distilled water before use.
Aldehydes	Gluteraldehyde	Minimum contact time required is 15 min.  Corrosive and irritating.  Consult Biosafety Officer on proper use.  Must be thoroughly rinsed from instruments using sterile distilled water before use.

 

Pre-Surgical Evaluation & Treatment
Pre-existing health conditions may negatively affect the immediate or long-term success of the surgical procedures and the experiment. Performing pre-surgical evaluations helps ensure the animals are not overtly ill. This includes visual inspection of the animal and an assessment of their behavioral status. The animal should be alert and behaving normally, and should have a smooth coat and clear eyes. Bring animals with physical or behavioral abnormalities to the attention of the ACS veterinary staff.

Withholding food or water is generally not necessary in rodents or birds unless specifically mandated by the protocol or surgical procedure (e.g., gastrointestinal surgery). Withholding food or water for more than six hours should be discussed with an ACS veterinarian.

In some cases, it may be preferable to initiate antibiotic or analgesic treatment prior to surgery. All antibiotic or analgesic treatment regimens must be discussed with an ACS veterinarian.

Surgical Preparation
Animal preparation includes hair/feather removal from the surgical site with a generous border (at least 1 cm) to avoid contaminating the incision site. Perform the hair/feather removal in a location separate from the surgical area. When removing the hair/feather with a vacuum cleaner, it must be HEPA filtered. Scrub the surgical site with a germicidal scrub, being careful to scrub from the center of the surgical site toward the periphery. Recommended germicidal scrub and rinse combinations include povidone-iodine scrub followed by a 70% isopropyl alcohol rinse, or a 4% chlorhexidine scrub followed by a sterile saline rinse. At least three alternating preparations of germicidal scrub and rinse are considered adequate. When using chlorhexidine and sterile saline in combination, the sterile saline can be warmed up to help minimize heat loss. If povidone-iodine scrub is used, the third 70% isopropyl alcohol rinse should be followed by an application of povidone-iodine solution to the area. Finally, drape the area with sterile drapes to prevent contaminants from entering the surgical field and provide a sterile area on which to lay sterile instruments during surgery.

The surgeon must thoroughly wash his or her hands with a bactericidal scrub. The use of sterile surgical gloves is required. Gloves dipped in bleach are not acceptable for this purpose. A surgical mask must be worn for major surgeries and implanted devices, but is also recommended for minor procedures. Wearing a clean lab coat is mandatory; however, a sterile gown is preferable, especially for major surgeries or surgeries where materials are implanted.

Anesthesia
The anesthetic regimen for any surgical procedure must be determined in consultation with an ACS veterinarian and must be described in the IACUC approved research protocol. Generally, gas anesthesia (e.g., isoflurane or halothane) is recommended. In any case, the animal must be fully anesthetized prior to initiating the procedure and maintained in a consistent anesthesia plane throughout the surgery. Anesthetic depth may be monitored in a number of ways (e.g., respiration rate, corneal reflex, positive toe pinch) and may vary depending upon the species and the anesthetic agent used. For rodents and birds, it is generally not necessary or feasible to monitor the heart rate.

For guidance in selection and use of anesthetics, please contact an ACS veterinarian (x22978).

Surgical Procedures
All aspects of the surgical procedures must be conducted as described in the IACUC approved protocol. Animal evaluation during surgery is critical. In addition to monitoring anesthetic depth as described above, maintaining normal body temperature is of particular importance, as anesthetics can directly or indirectly induce hypothermia. Water-circulating heat pads are recommended for this purpose. Using electric heating pads may overheat or burn the animal; if these are used, the pad must be set on low, and a light cloth covering or bubble wrap should be placed between the animal and the pad, and the animal must be observed frequently for signs of hyperthermia. Even the use of rectal thermostat heating blankets can result in burned and overheated animals. Because heat lamps may cause severe hyperthermia or other thermal injury, their use is prohibited.

To prevent corneal desiccation, bland ophthalmic ointment must be placed in the eyes immediately following anesthetic induction. If the animals are undergoing survival stereotaxic surgery, blunt ear bars must be used to prevent tympanic membrane damage.

Paralytic agents must not be used without anesthesia. If a neuromuscular blocking agent is required for the surgical procedures, please refer to the ACS Guidelines on Neuromuscular Blocking Agents.

Suture Selection
Use an absorbable suture material for body wall closure or other internal wound closures, and a nonabsorbable monofilament suture material for skin closure. Subcuticular suture placement, although more technically challenging, is acceptable for skin closure and may be performed using absorbable materials. The smallest gauge suture material should be used as practicable; typically 3-0 or 4-0 material is acceptable. A list of acceptable suture materials is included in Table 4 below.

Table 4. Acceptable Suture Materials

Suture	Characteristics and Frequent Uses
Vicryl®, Dexon®	Absorbable; 60-90 days.  Suitable for internal wound closure.
PDS®, Maxon®	Absorbable; 6 months.  Suitable for internal wound closure where extended wound support is desirable.
Prolene®	Nonabsorbable.  Suitable for skin closure.
Nylon	Nonabsorbable.  Suitable for skin closure.
Stainless Steel Wound Clips, Staples	Nonabsorbable.  Suitable for skin closure.  Requires instrument for removal from skin.

Because silk and chromic gut may cause tissue inflammation, these materials are not acceptable for wound closure.

Sutures, staples, or wound clips must be removed 7-14 days following surgery. Suture removal prior to euthanasia is not necessary for those animals euthanized within 14 days of surgery. Foreign substances (e.g.: suture material) remaining in the incision for an extended period serves as a nidus of irritation and infection. Please contact an ACS veterinarian to examine any incisions that do not appear to be healing.

For guidance in suture selection and use, please contact an ACS veterinarian (x22978).

Post-Operative Recovery
Observation during post-surgical recovery is imperative. The animal, whether recovering in or out of its ‘home’ cage, must be kept warm. Water-circulating heating pads are recommended for this purpose. Using electric heating pads may overheat or burn the animal; if these are used, the pad must be set on low, and a light cloth covering should be placed between the animal and the pad, and the animal must be observed frequently for signs of hyperthermia. Turn somnolent animals periodically to prevent burns or other thermal injury. Provisions must also be made for a conscious animal to escape the heat source when it becomes too warm. Because heat lamps may cause severe hyperthermia or other thermal injury, their use is prohibited.

A recovering animal must be watched continuously until in sternal recumbency and showing a degree of purposeful movement. Unconscious animals must never be unattended. To prevent undue risk, rodents must be housed individually following surgery until they are ambulatory.

Post-Operative Analgesic and Antibiotic Treatment
As described in the ACS Guidelines on Post-Operative Analgesia, analgesia must be provided to all animals following survival surgery, unless scientific justification for withholding such agents is approved by the IACUC as part of the investigator’s research protocol, or if a veterinarian examines the animal and determines that analgesic administration is no longer necessary.

A list of commonly used analgesics is included in Table 5 below.

Table 5. Frequently Used Analgesics

 

Drug	Species	Dose	Route	Frequency
Opiate drugs:
Buprenorphine	Mice	.05-.1 mg/kg	SQ	Every 12 hours
	Rats	.01-.05 mg/kg	SQ	Every 12 hours
	Birds	.01-.05 mg/kg	IM
Non-steroidal drugs (NSAIDs):
Carprofen	Mice & Rats	5 mg/kg	SQ	Once daily
Ketoprofen	Mice & Rats	5 mg/kg	SQ	Once daily
Flunixin meglumine	Mice & Rats	1.1-2.5 mg/kg	SQ	Every 12 hours

 

The use of local pain-relieving drugs such as Marcaine® (bupivacaine), in addition to systemic analgesia, may be indicated for some procedures resulting in significant disruption of the skin (e.g., Alzet® pump placement, catheter exteriorization), as these drugs may help to block the onset of the pain cascade due to disruption of the dermal nerve cells. Local analgesics are not intended for use in lieu of systemic analgesics, unless the withholding of systemic analgesia is scientifically justified.

Post-operative antibiotic treatment should be discussed with an ACS veterinarian to determine whether routine antibiotic administration is necessary. In general, post-operative antibiotics should be provided if the animal will survive long enough to develop severe infection, but may also depend upon other factors such as the invasiveness of the procedure and the animal’s immune status. Administering antibiotics prior to commencing a procedure can further minimize post-operative infections.

Long-Term Recovery and Monitoring
Post-surgical observations include a minimum daily observation, including weekends and holidays, of the animal’s condition and the surgical site. Animals should be observed for continued recovery, which may include state of arousal; indices of pain or discomfort; condition of the surgical wound; appetite; hydration status; capillary refill time; mucous membrane color; or fecal and urine production.

Some surgical manipulations may require an extended period of post-operative monitoring. The ACS veterinary staff, in consultation with the investigator, can determine the appropriate duration and extent of monitoring. Some situations constituting prolonged monitoring periods include animals with chronic debilitating disease states (e.g., diabetes mellitus), animals undergoing organ transplantation or immunosuppressive therapy, and animals with chronically implanted instruments or catheters.

The on-call veterinarian pager number ((352-413-0810) should be kept readily available in the event that post-surgical complications are observed during after hours, weekends, and holidays.

Record-keeping Requirements
In accord with recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, surgical records are required for rodents and birds. These records should include the administration of anesthetics, fluids, and any drugs; procedure details, including intra-operative monitoring; daily post-operative recovery observations and treatment, including administration of analgesics and antibiotics; monitoring of incision healing, including suture/staple removal if applicable; and the initials of the individual performing these tasks. Record all medications, including the name, dose, route, and time of administration. Additionally, any adverse outcomes should be noted. A sample Procedure Record for Rats, Mice, and Birds and a Post-Operative Evaluation Record for Mice, Rats, and Birds are available at the ACS website.

To facilitate the veterinary staff’s evaluation of post-operative healing and to ensure sutures are appropriately removed, each cage card must be clearly marked with the date of surgery. If animals will be provided antibiotics in the drinking water, special treatment cards (purple Special Care by PI cards) should be placed on each cage to communicate this special care to the veterinary and husbandry staff.

All records relating to surgical procedures and post-operative care may be subject to review during IACUC inspection or audit.

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