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Animal Care and Use Committee Ver.2, Effective: August 9, 2012

Phone: 304-293-9368 Phone: 304-293-1050

Fax: 304-293-3098 http://oric.research.wvu.edu

Chestnut Ridge Research Building 886 Chestnut Ridge Road PO Box 6845 Morgantown, WV 26506-6845

Equal Opportunity/Affirmative Action Institution

WVU ACUC POLICY

Anesthesia, Surgery and Post-Operative Care of Rats and Mice

Table of Contents I. Introduction II. Monitoring & Documentation III. Aseptic Technique in Survival Surgery IV. Postoperative Monitoring & Care V. Surgeries on Multiple Animals VI. Clean Technique in Non-Survival Surgery I. INTRODUCTION A. Purpose This document describes appropriate procedures for anesthesia, aseptic surgery, and post-operative care of laboratory rats and mice at WVU. Other rodents and other warm-blooded mammals regulated by the United States Department of Agriculture (USDA) are covered under the USDA Species Policy for Anesthesia and Surgery. According to the Guide for the Care and Use of Laboratory Animals, 8th edition (2011; henceforth the Guide), the following areas must be given attention when performing surgery on laboratory animals: pre-surgical planning, aseptic and surgical technique, assessment of animal well-being during and after the procedure, appropriate use of analgesics, and each animal’s physiologic status during all phases of a protocol involving surgery and postoperative care. In addition, the Guide states that survival surgery on rodents must be performed in facilities intended for that purpose, using aseptic procedures to prevent clinical infections. The OLAR veterinary staff is always available to provide assistance with anesthetic or surgical issues, as well as with surgical complications, and challenging or prolonged anesthetic recoveries. For additional information, OLAR can be contacted by email at [email protected] or [email protected] or by telephone at 304-293-2721 for other information. OLAR is located in Room 186G in the Health Sciences Center North. B. Preparing Animal Care and Use Protocols This document describes the expectations of the WVU Animal Care and Use Committee (ACUC) for (a) anesthesia; (b) aseptic surgical technique; (c) appropriate pre-, intra-, and post-operative care; and (d) documentation for rats and mice undergoing anesthesia or anesthesia and surgery procedures. Investigators who adhere to the requirements of this document should provide a brief description of

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WVU ACUC Policy, Anesthesia, Surgery and Post-Operative Care of Rats and Mice, ver.2, eff. 8/9/2012

the surgical approach, intraoperative actions, and closure technique in their Animal Care as Use protocol (henceforth, protocol), and then go on to state that this policy document will be followed (i.e., the details listed here need not be repeated if this policy is referenced in the protocol). Deviations from the procedures described herein are subject to the approval of the ACUC. Any deviations must be described and scientifically justified in the protocol. C. General Issues Investigators should be familiar with the following issues that may apply to their research. 1. Acclimation period: A minimum 48-hour acclimation period is required for laboratory mice and rats prior to experimental use; in other words, animals cannot be used until the third calendar day after their arrival. See the ACUC Policy on Acclimation of New Arrivals for detailed information and exceptions. 2. Anesthesia without surgery: Animals undergoing anesthesia must be observed and monitored until fully ambulatory as described for survival surgery below, including those anesthetized for brief time periods and/or for minor or non-invasive procedures. During the anesthetic recovery period, animals must have appropriate monitoring (e.g. temperature), and active animal surveillance such as contact and stimulation by the observer to aid in arousal and anesthetic recovery is preferred. Animals can never be left unattended unless the animal has fully regained consciousness. Criteria used to define complete consciousness include: (a) a body temperature within 1-2 degrees of normal (normal is ~99 degrees F) and (b) ability of the animal to right itself (i.e. stand on all 4 limbs) from being placed in full dorsal recumbency (i.e. placed on their back). 3. Analgesic considerations: Please consult with a veterinarian from the WVU Office of Laboratory Animal Resources (OLAR) for guidance on appropriate surgery-specific analgesic options, including doses, timing and administration routes when preparing surgical animal protocols. In general, analgesics are given preemptively to avoid pain “wind-up.” However, the potential for an adverse effect of analgesic selection on the stability of the anesthetic procedure may affect the plan for analgesic use in some cases. 4. Antibiotic use: Postoperative antibiotics may be indicated in conjunction with aseptic technique. Antibiotic administration prior to surgery is more effective, because the blood supply is still uncompromised to the surgical area at this time. When needed, post-operative antibiotic administration can be used, but requires an extended treatment period. Contact an OLAR veterinarian to discuss antibiotic use as it applies to your specific needs. 5. Fluid administration: All animals undergoing anesthesia and/or surgery > 20 minutes duration benefit from warm intraperitoneal and/or subcutaneous fluids to insure adequate hydration post-operatively and to assist in a faster recovery following injectable anesthetics, such as ketamine, that are cleared by the kidneys. Contact an OLAR veterinarian for information on appropriate fluid volumes for your application. A safe rule of thumb for routine use is to deliver a balanced electrolyte solution volume of 10-12 ml to an adult rat, and 1-2 ml to an adult mouse, either subcutaneously or intraperitoneally, or divided by both routes. 6. Eye lubricant use: For must general anesthetic procedures lasting more than 20 minutes, eye lubricant (i. e., Lacrilube®, opthalmic antibiotic ointment) must be placed in each eye to avoid corneal desiccation.



Both fluids and eye lubricant may be applied immediately after anesthetic induction for ease of administration. II. MONITORING & DOCUMENTATION To comply with applicable laws and regulations, surgical records must be maintained for all surgeries performed and must include all anesthetics and analgesics (drug, dose, frequency) administered, and surgical complications encountered. The OLAR-supplied pink surgery and anesthesia cards (“pink cards” found in vivarium animal rooms, or obtained from OLAR), if completely filled out and placed on rodent cages, may suffice for this purpose. All surgical records must be accessible and readily available upon request to OLAR, the ACUC, the federal Office of Laboratory Animal Welfare (OLAW) or – in the case of certain rodents – the USDA (see the USDA Species Anesthesia and Surgery Policy). Investigators may submit copies of surgical records to OLAR, which will retain them for inspectors. Sample anesthetic monitoring and surgery record forms for use by investigators can be found in the Appendix below and on the OLAR website (see contact information above). Although use of these particular forms is not required, the type of information contained on the sample forms is required. A. Pre-Operative Records Initially, visual assessment of animal health must be made. For example, is the animal normally hydrated, or does it require additional fluids? If injectable premedications (e.g. anticholinergics, antibiotics), or anesthetics or analgesics are used, the animal’s body weight or an equivalent standard should be used to calculate dosages. It is important to note and address any abnormalities that might affect the animal’s procedural outcome. B. Intra-Operative Records Surgery records must contain the following information: PI name, surgeon name, ACUC protocol number, animal or cage ID, species/strain, and surgery date. In addition, for surgeries lasting more than 15 minutes, a meaningful assemblage of signs must be monitored and recorded: anesthetic depth (required-response to toe pinch); mucous membrane color (eye color in albinos); body temperature; respiratory rate (and respiratory depth when relevant); in some cases, monitoring of heart rate is also indicated. For short procedures, and in smaller animals, it may be acceptable to monitor only certain parameters, or to note only changes in these parameters. More complicated procedures and those of longer duration require more detailed monitoring. All anesthetics, medications, fluids, and/or test substances administered during surgery must be recorded. Completed intraoperative records or cage pink cards may be submitted to OLAR for safe-keeping or they may be maintained in the laboratory provided they are readily available for inspection, (see below; the back of the card can be used to record added details). C. Post-Operative Records Following surgery, all cages must be clearly identified (e.g. completely filled out “pink card”) during the postsurgical monitoring period within the housing room. Analgesic doses must be recorded on pink cards (i.e. including drug-circled or written in, dose, frequency, and initials of the person administering the drug). If the sutures are non-absorbable, an anticipated suture removal date


should be listed. If relevant, additional postoperative follow-up information relevant to the animal’s recovery must also be recorded and maintained in the animal room throughout the post-operative period, including all animal observations, and medications and/or experimental substances (cells, drugs, biologicals, etc.) administered spanning the surgical and postoperative periods (see the Post-Operative Monitoring and Care section below). III. ASEPTIC TECHNIQUE IN SURVIVAL SURGERY The following standards must be applied to all survival surgeries. As noted in the Introduction, any deviation must be approved, in advance, by the ACUC. A. General Considerations Surgery may only be performed at a site dedicated to that activity at that time (time or space separated), preferably with a separate area for patient preparation, and must take place on a clean, uncluttered surface (e.g. no air ducts impinging on the site or overhead shelved materials likely to fall on the sterile field). Those entering the space during surgery must abide by aseptic practices such as donning masks and/or personal protective equipment. Traffic in the area during surgery should be minimized by using appropriate signage. In addition, the cleaned surface must be covered with an appropriately cleaned heated surface, barrier cloth, or drape. All drugs, suture, and sterile gloves used for survival surgery must be used before their expiration date. B. Instrument Preparation The sterilization method used must be attuned to the durability of the materials to be sterilized, and sterilization indicators must be used to validate sterilization completeness (outside and preferably inside sterile packs). Appropriate techniques include: gas (H202 or ethylene oxide); steam (autoclave); and liquid, chemical sterilants with proper contact time (e.g. 2% glutaraldehyde x 10 hr, or 8% formaldehyde with 70% alcohol x 18 hr). When liquid sterilants are used, instruments must be rinsed in sterile saline before use. When gas sterilization is used, proper degassing times (e.g. 12 hr for ethylene oxide) should be observed. The technique of “tip sterilization” using bead or hot dry sterilizers may also be used with care, as this requires careful attention to maintenance of aseptic technique. Also, excessive heating of instruments can damage tissue upon contact. All in-house sterilized items must be used within 6 months of the sterilization date (sterilization dates are inscribed on each sterilization pack). Seventy percent (70%) alcohol is NOT a sterilant or a high-level disinfectant and, by itself, is not acceptable for surgical instrument sterilization. Novasan® (chlorhexidine gluconate) Solution (not scrub) may be used for this purpose. Contact OLAR for information on sterilization services that are available for delicate instruments or equipment that cannot be autoclaved or tip sterilized. Other items used in surgery such as gauze, suture material, irrigation fluid, syringes, needles, intravascular catheters, etc. must also be sterile. Only rust-free instruments may be used for rodent surgery. To preserve the life of instruments, “Surgical milk” can be used prior to autoclaving. Contact OLAR for additional information on surgical instrument maintenance.

C. Animal Preparation Use of a protocol approved anesthetic approach, appropriate for the species and procedure is required. Animals must be maintained at a surgical plane of anesthesia (documented as unresponsive to toe pinch, or without physiologic evidence of pain response) and and the anesthetist


must assure this state throughout the surgical procedure. Thermal support via an external heat source (e.g. circulating warm water blanket) is recommended when the duration of anesthesia or surgical conditions indicate a significant drop in body temperature of more than 3 degrees is likely, (e.g. any procedure where a body cavity is opened); if in doubt you should supply external heat. Frequency of temperature monitoring should be sufficient to avoid large fluctuations in physiological parameters that need rapid correction, (e.g. an animal’s body temperature should be monitored at least every 10–15 minutes during the procedure). When using external heat sources, minimize insulation between the heat source and the animal to get the best heating effect. If electric pads are used, a layer between the animal and the heat source may be appropriate, and the use of pads that develop hot spots must be avoided. When autoclavable pads or hot water-filled gloves are used, care must be taken to monitor and rewarm them regularly so as not to draw heat from the animal as they cool. Sterile ophthalmic ointment (e.g. Puralube®) must be applied to the eyes when animals are unconscious for more than 15 minutes, and re-applied at least every 1.5 hour (or more frequently if needed), until the animal has recovered. This treatment will keep the eyes lubricated and prevent corneal injury. Hair removal from the surgical site, including a sufficient margin, can be achieved using electric clippers, disposable razors, depilatory creams (e.g. Nair®- must be completely rinsed from the skin) or by manual plucking under anesthesia. When using depilatories, contact times may have to be modified with the species and conditions of use. Soiled skin may require cleaning before surgical skin disinfection can start. Skin disinfection of the operative site must include three alternating wipes of an approved soap disinfectant scrub (e.g. chlorhexidine [Novasan® Scrub] or iodine [Betadine Scrub®]) with wipes of 70 % alcohol or saline (i.e. for Betasept® or Hibiclens® Scrub) followed by a final application of an antiseptic solution (e.g. iodine solution without detergent). Do not expose the eyes to the scrub, alcohol or disinfectant solutions. Excessive animal wetting will speed cooling. Before or after disinfection, secure the animal at the surgery site and cover with a sterile drape, leaving only the disinfected skin exposed. In rats and mice, use only light drapes to prevent breathing difficulty in the anesthetized animal.

D. Surgeon/Support Personnel Preparation Hands and arms should be washed thoroughly using an acceptable surgical scrub/brush (e.g. chlorhexidine [Novasan® Scrub, Betasept®, or Hibiclens® Scrub] or iodine [Betadine® Scrub]) in the correct manner (contact OLAR for assistance if this is unfamiliar to you). Surgeons and assistants must don appropriate surgical attire including a sterile or clean gown, scrub shirt, or lab coat, a surgical mask, appropriate hair covering, and sterile surgical gloves. Surgical or anesthetic assistants may wear a clean covering, mask, cap and clean examination gloves, rather than sterile gloves, if they will not be touching the sterile field or instruments. E. Operative Techniques Sterile suture material must be selected specifically for the intended purpose at hand (absorbable vs. non-absorbable, braided vs. monofilament, size and needle type). For example, skin closure usually requires a monofilament non-absorbable suture such as nylon. Because rodents tend to chew out sutures, another selection such a metal staples or stainless steel suture may be more appropriate in some cases


It is critical to maintain a sterile surgical field at all times. If a non-sterile object comes in contact with the surgeon’s gloves, a new pair of sterile surgical gloves must be donned. A non-sterile surgical assistant should be present when needed to assist in maintaining the surgeon’s aseptic technique (e.g. adjust anesthesia machine, manipulate non-sterile instrument surfaces, provide supplies, etc.). Contaminated instruments (generally those touching anything outside the sterile field) are discarded from use. When surgery involves the opening of a body cavity, at least a two-layer closure is required whereby the body wall is closed separately using absorbable suture, and the skin closed with some combination of wound clips, staples, surgical adhesive, stainless steel suture or another non-absorbable monofilament suture in a simple interrupted pattern. A subcuticular pattern may be used for skin closure in larger rodents, such as rats and guinea pigs. Consult an OLAR veterinarian for additional information. Never use silk for skin closure. Silk may be used internally, but this is discouraged. Wound clips or non-absorbable skin sutures must be removed when the wound is healed sufficiently, typically by 14 days postoperative. Failure to remove sutures by postoperative day 21 is considered noncompliance unless the ACUC-approved protocol calls for euthanasia of the animal before then (in which case “n/a” should be entered for the suture removal date on the pink card. IV. POST-OPERATIVE MONITORING & CARE Oversight of anesthetic recovery monitoring, record keeping and postoperative care is the responsibility of the investigator and their staff doing the procedure. Post-anesthetic recovery services may be contracted out to the OLAR veterinary technicians for a fee (contact [email protected] for additional information). A. Anesthetic Recovery Period The recovery cage should be free of particulate bedding and lined with paper towels, “isopads”, etc. to prevent pica (ingestion of nonfood items) and gastrointestinal or tracheal obstructions during anesthetic recovery. This is especially important when using buprenorphine. To avoid cannibalism and suture chewing by cage mates in the recovery cage, rodents must be housed individually or with no more than two other individuals who have had the same procedure and are convalescing. Group-housed animals must be separated when recovered to avoid suture chewing until the time of suture removal. Only when animals are fully ambulatory can they be returned to the vivarium. Supervised heat support must be provided with an external heat source for any rodent with a depressed body temperature (e.g. 3 or more degrees below normal) or one that is slow to recover, and should remain in place until the animal is fully ambulatory. NOTE: when not in direct attendance at the recovery cage, always ensure that approx. 1/3 to 1/2 of the cage is unheated so the animal can move away from the heat source if necessary to avoid hyperthermia. If heating bulbs or elements are used, these should never be closer than 18” from the animal. Animals must be directly observed while in the recovery cage at least every 5-10 minutes until ambulatory. These observations include body temperature measurement via rectal probe or thermometer and contact stimulation of the recovering animals so they generate their own heat. Additional monitoring includes respiratory rate/quality, mucous membrane color, activity/anesthetic depth, and incisional integrity, including any leakage or bleeding.



Analgesic agents to be given at the time of surgery are preferably administered prior to anesthetic recovery, unless scientifically justified in the approved ACUC protocol, or under direct veterinary instruction. Only when animals are able to freely move around the cage can they be returned to the animal housing room and left unattended. Provision of moistened rodent food and/or dry food on the cage floor is strongly recommended for post-operative animals at least 24 hours following surgery, especially when any of the following is present: postoperative disorientation: hindleg, caudal body or abdominal surgery incisions; or overall debility. Typical situations for extended supplementation include surgeries that limit mobility or activity, such as hind limb or abdominal surgery. B. Postoperative Observations Following surgery, the investigative staff must examine postoperative animals at least once daily until the animal is stabilized. Daily examination includes at least observation of the incision for heat, swelling, drainage, or dehiscence (opening), assessment of overall animal activity, and any signs of pain or distress (described in detail in the ACUC policy entitled Recognition of Pain and Distress, Humane Endpoints). Observations are noted on the pink card on the animal’s cage. All postsurgical examinations must be recorded, including any complications encountered. If signs of pain or distress are observed, a staff member from OLAR must be notified so that appropriate and timely veterinary care can be provided. Anesthesia and post-operative monitoring records (including analgesic administration and suture removal information) must be maintained in the animal room throughout the postoperative period as defined above. V. SURGERIES ON MULTIPLE ANIMALS When performing surgeries on more than one animal consecutively, the following additional considerations apply: A. Instruments You must begin with a sterile instrument pack. Between animals, the instruments can be wiped clean and/or rinsed with sterile saline then placed in a chemical disinfectant such as chlorhexidine (Novasan®) solution, then preferably re-rinsed with sterile water or saline. When possible, instrument tips can be re-sterilized in a glass bead sterilizer, after any gross tissue contamination has been removed. A new sterile pack must be used after each 10 surgeries or three hours (whichever comes first), or any time aseptic technique is breached. The chemical disinfectant must be replaced when discolored or contaminated with blood or other body fluids. B. Surgical Gloves The surgeon may wipe his/her gloves for at least 30 seconds with sterile gauze pads soaked in chlorhexidine or iodine solution between animals that have already been scrubbed for surgery; otherwise, a new sterile (or clean for nonsurvival surgeries) pair must be donned between animals. If the surgeon is also performing animal surgical preparation (scrub) between survival surgeries, s/he must don new sterile gloves before the next animal.




C. Monitoring Multiple Anesthetized Animals If multiple animals will be anesthetized at one time, attention to anesthetic monitoring and supportive care (e. g. temperature support) is required for each animal, and extra personnel may be necessary to provide appropriate oversight. VI. CLEAN TECHNIQUE IN NON-SURVIVAL SURGERY The following considerations apply to surgeries in which animals remain under anesthesia until they are euthanized. Whereas aseptic technique is encouraged for all experimental animal surgeries, the following minimum standards must be applied to non-survival (acute) procedures. A. General Considerations Non-survival rodent surgeries must be performed on a clean, uncluttered surface in a room or portion of a room that is not used for any other purpose during the time of surgical preparation and the surgical procedure itself. All anesthetic, sedative and analgesic drugs must be used before their expiration date. Steps to minimize personnel exposure to rodent allergens should be taken. Other expired drugs, instruments or suture can be used if they are identified as such (see below) and their use does not lead to an adverse outcome. B. Instrument Preparation Surgical instruments must be clean, or sterilized according to the needs of the study. See Section III.B, Instrument Preparation, for additional information on care of surgical instruments. C. Animal Preparation Use of a protocol approved anesthetic agent is required, and animals must be maintained at a surgical plane of anesthesia (unresponsive to toe pinch, etc.) at all times. The same surgical monitoring and pre-operative steps are required as for survival surgery, including the continued verification of anesthetic depth. Site preparation should include hair removal, but a single cleaning with a disinfectant scrub is sufficient. D. Surgeon Preparation Protective gear is in large measure to protect the animal handler and surgeon rather than the animal, and can consist of standard personal protective equipment as is typically worn in the vivarium, or surgical attire as described under survival surgery above. E. Operative Technique Clean but not necessarily sterile instruments and supplies can be used, but anesthetics, euthanasia agents, analgesics or sedative must be used before their expiration date. The use of non-pharmaceutical drugs must follow the ACUC Policy on the Use of Non-pharmaceutical Drugs. Drugs, gloves, suture, and fluids used for non-survival surgeries may be expired if labeled “for acute use only” or “for non-survival use only.” F. Multiple Non-Survival Surgeries



When surgery is performed on consecutive animals, the procedures are largely the same as for other non-survival procedures. Surgical areas, instruments, and attire, including the surgeon’s gloves, must be kept clean of accumulated debris by the prudent use of glove changes or cleaning. If more than one animal is anesthetized, careful attention to monitoring and support of multiple individuals is crucial, and may require additional personnel. Reference National Research Council. 2011. Guide for the Care and Use of Laboratory Animals, 8th Ed. National Academies Press, Washington DC. pp. 115-121.


APPENDIX: Cage card option for surgery record, followed by full-page option

Post-surgical records must be completed and kept in the animal housing room during the post-op period, then returned to OLAR as part of the animal’s medical record, unless they remain on file for inspectors with the investigator’s study records.

NOTE: Individual cages must be marked with an appropriately completed “pink anesthesia/surgery card” even if a full-page record is used (and referenced thereon). This will alert the animal care and veterinary staffs of the anesthetic history of the animal(s).

Surgery cards may be obtained from the OLAR staff.

FRONT:

BACK:

WVU RODENT ANESTHESIA/ SURGERY RECORD

Principal Investigator: Date:

Surgeon: ACUC Protocol No:

Species/Strain: Animal/Cage ID:

Surgical Procedure:

Pre-Op animal condition: Suture/Clip removal date:

Preanesthetic Drug(s) Administered Drug Dose/Route

Anesthetic Drug(s) Administered Drug Dose/Route/Rate

Analgesic(s) Drug(s) Dose / Route Time given

Other medication(s) Drug(s) Dose / Route Time given

All medications and procedures above are as stated in the approved animal use protocol

WVU RODENT POSTOPERATIVE RECORD

Date/Time Surgical Site Animal

Condition* Substance(s) given (drug, dose, route)

Initials

*Animal condition: 1 = Active 2 = Inactive 3 = Moribund 4 = Found Dead

WVU RODENT POSTOPERATIVE RECORD

Date/Time Surgical Site Animal

Condition* Substance(s) given (drug, dose, route)

Initials

*Animal condition: 1 = Active 2 = Inactive 3 = Moribund 4 = Found Dead


MULTI-RODENT SURGICAL RECORD

Please ensure that all medications and procedures listed below are as stated in the approved animal use protocol.

GENERAL PROCEDURE INFORMATION

Principal Investigator: Date:

Surgeon: ACUC Protocol No.:

Species: Animal/Cage ID:

Pre-Surgical Evaluation

Animal Condition:

Preanesthetic Drug(s) Administered

Drug Dose/Route

Anesthetic Drug(s) Administered

Drug Dose/Route

Analgesic(s): Drug Dose (mg)/ Route

Other Medications: Drug Dose (mg)/ Route

INTRAOPERATIVE AND ANESTHETIC RECOVERY INFORMATION

Animal ID: Animal Weight: Anesthesia start (time):

TIME ANIMAL TEMP RESP RATE ANESTHETIC

DEPTH OTHER INITIALS

Eye lubricant applied ❑❑❑❑ Righting time: Time returned to home cage:

POSTOPERATIVE INFORMATION

DATE / TIME SURGICAL SITE (Norm/ Abnormal)

OTHER OBSERVATIONS SUBSTANCE(S) GIVEN INITIALS



Animal ID: AnimalWt: Anesthesia start (time):








Animal ID: AnimalWt: Anesthesia start (time):







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