Course Material Introduction Welcome to the Course Material. You must complete this course if you perform surgical procedures on rodents (mice or rats) at UAB. The goal of this course is to make you aware of the policies and procedures to implement during Survival and Non-Survival Surgeries and how to perform the Aseptic Technique. If you have any questions or need further instruction, contact the UAB ARP Veterinary Staff. Objectives At the conclusion, participants will be able to do the following for rodents: 1. Recognize the importance of planning, preparation, and practicing before performing any procedure. 2. Implement the use of Aseptic Technique when performing procedures. 3. Provide monitoring before, during, and after procedures to maintain their health. 4. Understand the essential differences between Survival and Non-Survival Surgery. Pre-Planning Key Terms 1. Analgesics: A term that describes administering drugs to animals that relieve pain without losing consciousness. Sometimes analgesics are given before a procedure to prevent the animal from waking up in pain. 2. Anesthesia: A term that describes the loss of feeling or sensation and especially pain. Anesthetics may need to be re-administered if the animal regains consciousness. 3. Aseptic Technique: Refers to surgery performed using procedures that limit microbial contamination so that significant infection does not occur. This technique is a fundamental premise of proper surgical conduct for all animals. However, some modifications have been allowed in the case of rodents since there are many unique aspects to this laboratory species. Deviations require an approved amendment to the protocol before using the animal. 4. Disinfection: A term that describes killing all the vegetative forms of bacteria only. In other words, disinfection decreases the number of bacteria. Course Material Page 1 Last Updated: 08/13/2018
Course Material 5. Induction: A term that describes the administration of pre-surgical anesthesia. It begins the patient preparation process. 6. Major Surgery: Any surgical intervention that penetrates and exposes a body cavity, procedures producing substantial impairment of physical or physiological functions, or involves extensive tissue dissection or transection. Examples include (but are not limited to): Laparotomy, Joint Replacement, Implantation of an Indwelling Catheter, and Limb Amputation. 7. Minor Surgery: A surgical procedures that does not expose body cavities and causes little or no physical impairment. Examples include (but are not limited to): Tail Clipping, Subcutaneous Implants, Intracranial Injections, and Cannulation of a Peripheral Blood Vessel. 8. Non-Survival Surgery: A surgical procedure in which an animal is euthanized before recovery from general anesthesia. 9. Pre-Emptive Analgesia: A term describing a theory stating if analgesia is present before a painful procedure, it will block the pain receptors during the painful stimulus. The theory also proposes that less analgesia will be needed after the procedure when administering pre-emptive analgesics. 10. Sterilization: A term that describes killing all living microorganisms and their spores. 11. Survival Surgery: A surgical procedure from which an animal will recover from the procedure. All survival surgical procedures must be performed using Aseptic Techniques regardless of species. Personnel and Roles The Pre-Planning Stage includes identifying personnel who are a part of the surgical team. Personnel are assigned their specific roles during this time. Roles may include the following tasks: Anesthetist, Surgical Nurse, and Surgeon. Equipment and Supplies The Pre-Planning Stage includes determining the equipment and supplies required for the planned procedures. Examples include obtaining sterilized instruments, specialized equipment, or analgesics. Your IACUC Approved Protocol lists these. Course Material Page 2 Last Updated: 08/13/2018
Course Material Antibiotics If your procedure requires the use of antibiotics, they must be included in your protocol and discussed with the UAB ARP Veterinary Staff during the Pre-Planning Stage. However, should an infection occur, call the UAB ARP Veterinary Staff immediately for appropriate treatment options. A bacterial infection of the surgery site could cause severe problems for the animals and your research, and it should be treated as instructed by the veterinary staff. Out-of-Date All drugs administered to relieve pain or distress, produce euthanasia or anesthesia, and treat any emergency conditions must be in-date. Analgesics and Anesthetics used pre, during, and post-surgery may not be used past their expiration date even in Non-Survival Surgeries. Other drugs (not related to relief of pain or distress) that exceed their expiration date may be used in animals for acute non-survival procedures if the usage will not adversely affect an animal s well-being! Out-of-date materials must be labeled as expired and stored in a separate container from other materials. If you have questions, check with the UAB ARP Veterinary Staff for more information. Location The Pre-Planning Stage includes identifying the location for the procedure. Regulations allow you to perform rodent surgeries in a laboratory. However, you should have an area designated for the procedures. Examples include a desk, counter-top, or table-top. The area can be a multi-use area except during surgery. The area must be appropriately disinfected and partitioned. The area should be out of the way of traffic flow and air vents. By doing this, it prevents other contaminants from entering the surgical area or incision. The area must be uncluttered. The Preparation Area and Surgical Area must be separate to prevent contamination. Course Material Page 3 Last Updated: 08/13/2018
Course Material The IACUC Approved Protocol must list the location(s) for performing surgery. These areas are subject to inspection by the IACUC. You should implement adequate veterinary sanitation (e.g., equivalent to those used in a standard examination or treatment room). ARP Facilities A dedicated Animal Resources Program (ARP) Facility is ideal because it has room to separate the preparation area from the surgical area. It is required to schedule ARP Facilities in advance. If you are not using a dedicated ARP Surgical Space or a Biosafety Cabinet (BSC) located in the room, the surgical area you choose must be dedicated to the surgery during the procedure. Pain Management If you have questions or need assistance in determining an animal s pain level, contact the UAB ARP Veterinary Staff. Anesthesia With so many options, which one is the best choice? The answer depends on the type of surgical procedure, procedure length, and weight of the animal. If you have questions or need assistance, contact the UAB ARP Veterinary Staff. Types There are two major types of anesthesia injectable and inhalant. Injectable Anesthesia Advantages 1. No equipment needed other than a needle and syringe 2. Allows a defined period of anesthesia (by precise control of the dosage) Disadvantages 1. Requires a precise body weight for appropriate dosage 2. May require locked storage and detailed records of use 3. Accidental overdosing is non-reversible 4. Requires extensive metabolism to eliminate (The rodent must process it through the liver or kidney to remove it.) 5. Some combinations have a very narrow margin of safety Example: Ketamine or Xylazine Course Material Page 4 Last Updated: 08/13/2018
Course Material Inhalant Anesthesia Advantages 1. Easily administered 2. Easily reversed (The rodent s body has only to exhale to remove it.) 3. Wide margin of safety 4. A precision vaporizer regulates the depth of anesthesia Disadvantages 1. Can be extremely short-acting 2. Procedures lasting longer than a minute or two require a precision vaporizer 3. Requires, at least in most cases, a system to capture excess gas (a scavenging system) 4. Requires use of expensive equipment; however, this equipment is available for use in surgery areas 5. Equipment requires maintenance Example: Isoflurane Pinch Test Pinching the foot of the rodent following anesthesia induction before surgery ensures that your animal is not feeling pain. Any reaction, no matter how slight, indicates that the animal is not sufficiently anesthetized. Practice administering anesthesia with more experienced staff members or under the direction of the UAB ARP Veterinary Staff. Warming Devices When anesthetizing a rodent, a warming device should be used to prevent hypothermia before, during, and after anesthesia. After anesthesia, a rodent should never be placed back in the cage directly on the cage bedding or any surface without a warming device. Once anesthetized animals lose the ability to thermoregulate (regulate body temperature), and rodents lose body heat very quickly. Water Warming A water warming device controls the temperature of the water running through the pad preventing overheating and burns. Always check the temperature before placing the rodent on the pad. The proper way to test any warming device is to put your hand on it. If it is too warm for you, it is also too warm for the animal! Course Material Page 5 Last Updated: 08/13/2018
Course Material Slide Warmers Slide warmers can be used to hold many rodents or cages with bedding. Cages do not require pads since the cage bedding prevents overheating and burns. Placing a portion of the cage on the slide warmer allows the animals to escape the heat once they begin to recover. Hand Warmers Place a pad or paper towel between the rodent and the hand warmer to prevent overheating and burns. Break the pad open and make sure it is warm before use. Heat Lamps The use of a heat lamp is strongly discouraged; however, if using heat lamps place the cage so that only one end is under the heat lamp, and the rodents can move away from the heat when conscious. Monitor the rodents until they are fully awake. Never leave anesthetized rodents unattended before, during or after surgical procedures! They should always be awake and moving around normally before the monitoring process is complete. Your protocol should include a plan for post-procedure monitoring as well. Analgesics Knowing How to Choose Choosing the correct type of analgesia depends on the: 1. Nature of the Surgery: a relatively simple, quick procedure requires different analgesics than a complicated, time-consuming operation. 2. Rodent s Pain: watch for signs of avoidance, vocalization, shivering, inappetence, and hyperesthesia. 3. Rodent s Weight Course Material Page 6 Last Updated: 08/13/2018
Course Material The amount of analgesic necessary must be listed in your protocol. However, if you have questions, please contact the UAB ARP Veterinary Staff. Performing Surgery Pre-Surgery Checklist Prepare the Preparation Area Prepare the Surgical Area Prepare a sterile field for any instruments. To avoid contaminating instrument tips during surgery, always keep at least the tips on a sterile field (e.g., 4X4 sterile gauze sponge). Lay out your surgical instruments Check the animals Locate any necessary Personal Protective Equipment (PPE). o Put on a clean gown or surgical scrub shirt to protect your clothes. o Wear a mask to avoid contamination. o Put on a surgical cap if you have long hair. o Don the appropriate gloves. Partitioning There are a minimum of three separate areas: 1. Surgical Area 2. Preparation Area 3. Recovery or Holding Area Draping While popular, clothe, gauze, or paper cover the animal but do not allow for direct visualization. Commercially available plastic wrap is sterile coming off the roll. You do have to be very careful while laying it over the animal, so that does not cling to itself or bunch around the surgical incision site. Its use is an advantage because it allows direct visualization and monitoring of the rodent. It also helps retain body heat during the surgery. Be careful not to inhibit breathing. Course Material Page 7 Last Updated: 08/13/2018
Course Material Sterilization Anything and everything that comes in direct contact with the incision site or animal requires sterilization. Examples (not limited to): Surgical Instruments Fluids Implants Suture or Staples Surgical Drapes Sterilization Methods Physical Autoclave Dry Heat (glass bead sterilizer) Liquid Filtration (0.2 µ) Radiation Chemical Gas (ethylene oxide) Liquid cold sterilant (aldehydes, peroxides, chlorites) Plasma (hydrogen peroxide) Experimental Treatment or Drugs Sterilization Parameters for Autoclaves Time 15 minutes Some different methods accomplish sterilization. The standard practice is autoclaving. However, sterility is dependent upon other parameters as well Temperature Pressure 121.5 C 15 psi (e.g., pressure and time exposure). If you have questions, contact the UAB ARP Veterinary Staff. Glass Bead Sterilizer When using a Glass Bead Sterilizer, you should remember the following: Only the tips of the instruments achieve sterilization. The tips of delicate instruments may become damaged during immersion in the glass beads. The instruments must be allowed to cool before use. The tips of the tools become extremely hot and could burn the animal. Do not place the instruments in the sterilizer for longer than the manufacturer s recommendations (typically this is less than fifteen seconds). Rinse instruments thoroughly to remove any tissue and to prevent other tissue and particles from being baked onto the tools. Course Material Page 8 Last Updated: 08/13/2018
Course Material Pre-Sterile Packaged Items Pre-sterile packaged items are acceptable. However, these are only pre-sterilized inside the package. Make sure that you do not contaminate the instruments when you remove them from the package. Open pre-sterile packs with ungloved hands being careful not to touch the instruments inside. Immediately don sterile gloves before handling tools. If you touch the outside of the pre-sterile package with sterile gloves, you have contaminated the gloves and will have to repeat the gloving process. If you touch the instruments with contaminated gloves, you must open another sterile pack and change gloves. Disinfection For disinfection to occur, specific conditions must be met. These conditions depend on the procedure used for disinfection and following the instructions on the label correctly. Tabletops, hoods, microscopes, rodent s skin, and non-surgical instruments are items that require adequate disinfection. Types No matter which one you decide to use, take care not to soak the animal. Soaking the animal increases the risk of hypothermia and could cause anesthetic complications. PCMX Betadine Chlorhexidine Apply for two minutes of contact time with a cotton tip applicator and remove the excess after the affliction. PCMX does not require rinsing off or repeating. Apply in concentric circles from the proposed incision site moving outward. A 70% alcohol rinse going in the same direction follows the Betadine application. Repeat this sequence three times. Apply in concentric circles from the proposed incision site moving outward. A saline rinse going in the same direction follows the Chlorhexidine application. Repeat this sequence three times. Applying Betadine or Chlorhexidine in a sequence of three applications enables them to achieve the appropriate critical contact time. Otherwise, they are ineffective. Alternate the three applications of Betadine with a 70% alcohol rinse, which increases the effectiveness of Betadine alone. However, alcohol inactivates Chlorhexidine, so it must be rotated with saline instead. Course Material Page 9 Last Updated: 08/13/2018
Course Material Alcohol has no detergent activities, and thus should not be used alone to disinfect or prepare the surgical incision site. Please contact the UAB ARP Veterinary Staff for more information on any of the surgical prep options. There is an alternative surgical prep-agent that only requires one application along with two minutes of contact time on the animal. The agent is Chloroxylenol. It also has the added advantage of having some residual disinfectant effect, while Betadine and Chlorhexidine do not. Aseptic Technique Non-Survival Procedures Non-Survival Surgery is as any surgery after which the animal will not regain consciousness. It is not necessary to follow aseptic technique when performing non-survival operations in rodents. However, at a minimum, the following should be done. The animal is anesthetized. The surgical site should be clipped. The surgeon should wear gloves. The instruments should be clean. Survival Procedures For survival procedures, you must start with a completely sterile set of instruments. If you drop a sterile instrument, you may not disinfect it and reuse it for a procedure. You must have the tool re-sterilized. You must use sterile gloves. In some cases, a tips-only procedure may be performed wherein only the tips of the instruments touch the sterile field. In these cases, disinfected gloves may be used. Please be aware that in these procedures your gloves cannot contact the incision, sterile field or sterile parts of the instruments at any time! Course Material Page 10 Last Updated: 08/13/2018
Course Material Potential Negative Effects Failure to use aseptic technique can hurt your research! The problem is infection often manifests differently in rodents. The signs may be subtle but can have profound implications. Any infection becomes an uncontrolled variable in your study. Clipping and Shaving The clipping area should be in a separate location with little to no airflow to avoid contamination of the surgical and postprocedural care areas. Once anesthesia is given, and the animal feels no pain, then place the patient on a warming pad to prevent hypothermia. Hypothermia is one of the most common complications associated with rodent surgery. Clipping or shaving removes fur which may add to the loss of body heat. Incision Site Preparation 1. Clip the fur from the incision site. Remove all the fur from the site with a hair depilatory cream or scalpel blade if necessary. 2. Apply a topical disinfectant to remove bacteria from the area. Be careful not to soak the animal with disinfectant. In this image, Betadine was applied in concentric circles starting at the proposed incision site and then moving outward to cleanse the skin. Then followed by a 70% Alcohol rinse going in the same direction. Repeat this sequence three times. Course Material Page 11 Last Updated: 08/13/2018
Course Material Eye Preparation Anesthesia eliminates the blink reflex. Apply a sterile ophthalmic ointment to prevent the eyes from drying out during surgery. Do not touch the tip of the applicator or swab to the patient s eyes (this can cause an infection). Gently massage the ointment in the eyes Styrofoam Boards Styrofoam boards are suitable for use as a surgical surface, but only for nonsurvival procedures; however, discard them once soiled. Closing the Incision Suturing Internal Selecting the internal suture material is critical. It must be sterile. It must be able to reduce the risk of infection and provide minimal tissue reaction. Examples are Vicryl, PDS, Dexon, Monocryl, and Gut. External External suture material should be a non-braided, non-wicking monofilament, sterile, non-absorbable, and removed within 7-10 days. Examples are Nylon, Prolene, and PDS. External absorbable, or wicking, sutures are not used in animals because of the nature of their skin, sleeping habits, etc. Surgical glue does not require removal; however, check to make sure the rodent does not chew the glued tissue area. Course Material Page 12 Last Updated: 08/13/2018
Course Material Braided Braided suture material is never allowed to close the skin. This type of suture material tends to wick and harbor bacteria allowing entry into the incision. It allows for the potential infection of the incision Silk suture material is braided and unacceptable for skin closure. Sequential Procedures Performing sequential procedures with multiple rodents is conditional. It must be approved in your protocol first. When performing sequential procedures with many rodents, should you use numerous instrument packs or an individual set? Ideally, each surgery will have its own sterilized instruments and supplies. However, in some cases, this may not be possible. The decision to do more than one operation on multiple animals with a single set of instruments should be based upon the type of surgery and recognize that in some instances only one operation is justified. An acceptable example of using the same instruments for sequential surgeries would be Implanting tumor cells under the skin a relatively clean area Make the incision. Insert the tumor cells. Surgery completion. The instruments would be relatively clean so you could disinfect the instruments and move on to the next animal. An unacceptable example of using the same instruments for sequential surgeries would be Intestinal surgery a typically dirty area Normal contents of the intestines might contaminate the wound. You could not disinfect the instruments between surgeries due to contamination. You must have sterile instruments for each surgery. Course Material Page 13 Last Updated: 08/13/2018
Minimally between sequential surgeries you should: Course Material Clean up the surgery area Change the surgical drape Change the suture materials Wash and disinfect instruments Change gloves Multiple Packs You can create multiple packs by forming pockets within a large pack or using individual packs with standard instruments. Individual Packs Wash and disinfect specialized instruments then rinse with a sterile solution, or Use a glass bead sterilizer to sterilize the tips of the tools between surgeries. One Set of Instruments Since contamination is directly proportional to the length of time exposed to the environment, the following requirements are in place: No more than five surgeries or 2 hours of exposure per pack! You may perform five operations (starting with a fully sterile pack), but between each surgery, you must stop and disinfect the instruments. After the fifth surgery, stop and get a new sterile pack. Do not exceed two hours with an open pack because this limits the environmental exposure on any instrument. Post-Operative Monitoring Monitor any rodent that undergoes surgical procedures until they are awake and moving around normally. An anesthetized animal should never be left unattended. Course Material Page 14 Last Updated: 08/13/2018
Course Material Before being placed back in its original cage with other alert animals, the rodent should be exhibiting signs of normal behavior. If the rodent appears to be unable to defend itself or maintain its care and feeding, it should not be put back in the cage with other rodents until it can do so. During the recovery period, administer supplemental heat or warmed subcutaneous fluids. Also, check for: Evidence of pain Signs of infection Range of motion Food and water consumption Fecal and urine production After the animal recovers from anesthesia, it can be placed back in the animal room and observed for signs of infections. Infections will interfere with healthy recovery. Post-Procedure Checklist Monitor the incision and the animal after surgery. On the chart below, the green blocks are good symptoms. The white indicates there could be problems. Contact the UAB ARP Veterinary Staff for assistance. Course Material Page 15 Last Updated: 08/13/2018
Course Material The Incision Appetite Urination/Defecation Pain Desirable Is the incision clean and dry? Is the animal eating frequently? Is the animal urinating and defecating normal amounts? Is the animal acting normally with no hesitation to movements? Problematic Is the incision infected or is there pus visible between the sutures? Is there fresh unclotted blood on the incision even after several days postprocedure? Has the animal been eating irregularly since the procedure? Has the animal refused to eat since the procedure? Is there no evidence of feces since the surgery? Is there a bloody puddle in the cage that could be urine or diarrhea? Does the animal try to bite when touched? Does the animal huddle in the corner of the cage? Recordkeeping The UAB ARP Veterinary Staff must know which animals have had surgery by noting on the cage card that the animal has had surgery. You should also maintain records indicating surgical procedures performed for study groups and any post-operative care including administration of analgesia. Making notes when the animals recover, eat, drink, urinate, and defecate is recommended since it can help improve future surgeries and techniques. Appetite and weight gain are easy parameters to measure for general animal health. These are also excellent indicators of successful operation. Keeping a detailed, individual animal record of the operation may be required depending on the species used. Check with the UAB ARP Veterinary Staff for more information. Course Material Page 16 Last Updated: 08/13/2018
Course Material Euthanasia UAB requires two methods of euthanasia listed on your approved IACUC Protocol. If you have not been trained to euthanize rodents, please contact one of the ARP Veterinary Staff before attempting the procedure. Primary Methods Following are examples of acceptable primary methods of euthanasia with IACUC approval: 1. Inhalants (e.g., CO 2 and Isoflurane) 2. Injectables (e.g., Fatal Plus, Ketamine/Xylazine) 3. Physical, (e.g., rapid decapitation, microwave euthanasia) Secondary Methods To ensure death, you must follow primary euthanasia (e.g., inhalants and injectables) with a secondary method such as: Cervical Dislocation Cervical dislocation is used in unconscious rodents and involves the physical separation of the skull and vertebrae by application of force and results in severing the cervical spinal cord. This method is not appropriate in animal >200g. Thoracotomy Thoracotomy is also used in unconscious rodents and involves cutting open the thoracic (chest) cavity to induce a pneumothorax and prohibit the expansion of the lungs. This leads to irreversible oxygen deprivation and death. Exsanguination Exsanguination involves allowing an unconscious animal to bleed out. In essence, this acute loss of a large volume of blood will result in death. Course Material Page 17 Last Updated: 08/13/2018
Course Material Decapitation Decapitation involves physical removal of the head from the body. The equipment used to perform decapitation should be maintained in good working order and serviced on a regular basis. Other Techniques and Information Restraint devices are available that minimize the chance of injury to personnel and improve positioning of the animal for this procedure. Other techniques are available for rodent euthanasia and may be acceptable when scientifically justified by the user and approved by the IACUC. For more information on euthanasia techniques, please consult with the ARP Veterinary Staff, and see the IACUC SOP on Animal Euthanasia. Carcass Disposal Before placing euthanized rodents in a bag, you must make sure they are dead! Mice can stop breathing for a minute or more then can regain respiratory function and survive. Younger mice are resistant to Carbon Dioxide asphyxiation and take longer to succumb than adult mice. Locations with an Animal Morgue 1. Place the carcasses in a black plastic bag. 2. Place the black bag in the red-lined Stericycle box in the morgue. Location without an Animal Morgue Non-Hazardous Carcasses 1. Place the carcasses in a black plastic bag. 2. Place the bag in the designated refrigerator. Hazardous Carcasses 1. Place the bag inside a red bag marked with the hazard 2. Place it in the designated refrigerator or red-lined Stericycle box in the morgue. Course Material Page 18 Last Updated: 08/13/2018
Course Material Carcasses Contaminated with Radioactive Hazards Carcasses contaminated with radioactive hazards should be disposed of as indicated by the PI s Radioactive Materials License. Conclusion This section concludes the Course Material. You should take the assessment now. The passing score is 80% or higher. Other Training If you are responsible for monitoring rodents after any procedure, you should complete Post Procedure Care of Mice and Research at UAB (AU_PP). If you are working with any animal at UAB, you must complete Using Animals for Teaching, Testing, and Research at UAB (AU_UA). If you are ordering or handling scheduled substances in your research area, you must complete Using Controlled Substances in Research, Teaching, and Veterinary Care at UAB (OHS_CS245). If you are listed on an approved IACUC Protocol using mice in research at UAB, you must complete Working with Mice in Research at UAB (AU_M). If you are listed on an approved IACUC Protocol using rats in research at UAB, you must complete Working with Rats in Research at UAB (AU_R). Course Material Page 19 Last Updated: 08/13/2018