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Part III:
Biomedical Research Animal Use

Minimizing pain and distress in laboratory animals

The University of Minnesota categorizes the amount of pain and distress that an animal experiences according to these classifications:

Class A

Animals do not experience anything more than momentary or slight pain and distress and no pain relieving measures are used. For instance, animals that are euthanized (according to guidelines) and their tissues harvested for study are considered Class A animals. Animals that are receiving minor injections or non-invasive behavioral tests are considered Class A animals also.
USDA AWA, 2.36(b)(5): [studies]...involving no pain, distress, or use of pain-relieving drugs. Routine procedures (e.g. injections, tattooing, blood sampling) should be reported with this group;

Class B

Animals are undergoing procedures with the potential to cause pain and distress but appropriate pain relieving medications, including analgesics, anesthetics or tranquilizers, are being used. Any animal that undergoes a surgical procedure or general anesthesia, even a non- survival surgery, would be considered Class B.
USDA AWA 2.36(b)(6): [studies]...involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs were used;

Class C

Animals are undergoing pain and distress which is not relieved by appropriate anesthetics, analgesics, tranquilizers or other treatments. For instance, some pain studies require that animals not receive pain medication. If relief of pain or distress will interfere with the objectives of the experiment, the scientific justification for this must be clearly demonstrated. Other methods for minimizing discomfort that will not interfere with experimental objectives or early end points should also be considered.
USDA AWA 2.36(b)(7): [studies]...involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs would have adversely affected the...results;

Investigators using animals that are considered Class B or Class C must demonstrate that a search for alternatives to procedures which cause pain and distress was conducted. This search must include addressing the three "R"s and a search of the current literature for alternatives to painful and distressful procedures. This literature search should be conducted using more than one database.

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Relief of pain and distress

The relief of pain and distress in laboratory animals is an important issue and one that is addressed in detail in many laws and regulations. When completing the Animal Usage Form, investigators must address the relief of pain and distress as it relates to their experimental design. For further details on any of these topics or assistance with the Animal Usage Form contact Research Animal Resources, the Institutional Animal Care and Use Committee or the Office of Regulatory Affairs.

Anesthesia

Anesthesia is defined as creation of a state of analgesia, immobilization and amnesia. Anesthesia must be used for all surgical procedures, and may be used in cases where only restraint or immobilization is needed. There are several key issues to understand when using anesthesia. There is great variability in the effects of anesthetics depending on the species, strain, individual, route of administration, type of procedures, physiologic status, etc. Therefore, it is important to:

• Give to effect. There is no one dose of anesthetic that will work under all circumstances. Normally a dose range is calculated and a low to mid range if the dose is given. With injectable anesthetics, more can be given if needed, but an overdose of medication must be avoided.
• Monitor the depth of anesthesia at all times during the procedure and during recovery. This includes monitoring body temperature, hydration, capillary refill time, mucous membrane color, respiratory rate, heart/pulse rate, muscular tone and reflexes.
• Maintenance of normal physiology is essential, as anesthetics have profound effects on body temperature, fluid balance, and cardiovascular and respiratory function. This underscores the necessity for monitoring the depth of anesthesia. In addition, appropriate supportive care, such as fluids or a heating pad, may be needed.

Anesthetic Drugs

Many of these drugs are controlled substances and must be used according to University policy.

Sedatives

such as acepromazine, xylazine, medetomidine, opiates, diazepam, midazolam and others can be used by themselves for performing minor procedures. Usually sedatives can not be used for painful procedures, although xylazine, medetomidine, and the opiates do provide pain relief. These sedatives are also useful adjuncts to general anesthesia. They can be used to smooth induction and recovery, and to decrease the dosages of other drugs being used.

Local anesthesia drugs

such as lidocaine or bupivicaine can be used for localized anesthesia. Typically, some type of sedative or physical restraint is needed to allow procedures to be performed. Several methods of local anesthesia can be used including topical, regional, spinal or nerve blocks.

General anesthesia

these drugs cause generalized depression of the central nervous system, resulting in loss of consciousness, loss of pain perception, immobilization, amnesia, as well as side effects such as cardiopulmonary depression and loss of thermoregulation. Different drugs have differential effects in each of these areas.

Inhalant Anesthetics

such as halothane or isoflurane can be used as sole agents for surgery. Inhalant anesthetics must be used with a scavenging system or fume hood to prevent hazards to personnel.

Barbiturates

such as pentobarbital, thiopental or methohexital can be used as sole agents for surgery.

Dissociatives

such as ketamine and tiletamine do not provide adequate analgesia or muscle relaxation for many procedures, and must be combined with other drugs. Their use as sole agents is limited to restraint for non painful procedures, or in some species such as cats and monkeys, for minor skin procedures.

Neuroleptanalgesia

refers to the use of several drugs to provide a trance like state with analgesia. Typical combinations of drugs include dissociatives or opiates with an analgesic and/or sedative added. Examples include Telazol/xylazine, ketamine/xylazine, ketamine/diazepam, ketamine/acepromazine/butorphanol, or fentanyl/acepromazine.

Paralytics

such as succinylcholine or gallamine require concurrent use of an anesthetic, as they provide no analgesia or loss of consciousness. Monitoring anesthetic depth must focus on physiological parameters, such as heart rate and blood pressure, as normal reflexes may not be used.

Analgesia

Analgesics, or pain relievers, are drugs that can be used to prevent or relieve pain. RAR veterinarians may be consulted on analgesic options.

Types of pain:
(the intensity and character of pain differs depending on the location and source of the pain stimulus)

• Superficial - refers to pain transmitted by somatic efferent nerves from skin and muscles. Generally less intense than deep pain.
• Deep - often referred to as visceral pain. Transmitted by visceral efferent nerves from viscera, the pleura and peritoneum, and the periosteum. Generally the most intense pain.
• Nociceptive - the type of pain detected by receptors sensitive to pressure, heat or electrical stimulus
• Inflammation - the type of pain stimulated by release of intercellular messaging molecules such as substance P, prostaglandin's and leukotrienes
• Chronic - some types of pain tend to last longer than the inciting stimulus. This includes neuropathic pain, chronic inflammation, and others

Analgesic Drugs used to relieve pain

Methods of administration

Parenteral

Dosing by injection is the most reliable method, however, in certain species restraint can be problematic, and there are practical difficulties when treating large numbers of animals.

Enteral

Oral administration may be effective for many analgesics. Bolus dosing is the most reliable method, but suffers from the same disadvantages as does parenteral dosing. Dosing in the animals food or water can be used in some situations.

 

For more information on the use of anesthetics and analgesics see: http://www.ahc.umn.edu/rar/anesthesia.html

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Surgery

Surgery in a research setting has potential for causing pain or distress to animals if not performed properly. Surgery can result in pain, damage to tissue and post-operative infections. Therefore stringent guidelines for training, management of surgical facilities, anesthesia, analgesia, asepsis, and post-operative monitoring have been established.

Persons performing surgical procedures should be trained in the specific surgery and in the specific animal model that is being used. The training and/or experience of the surgeon must be documented in the Animal Usage Form. The use of post-operative analgesia is required after almost all survival surgeries. Pre-emptive analgesia (given before the surgical procedure begins) can also be considered for some surgeries. Post-operative records should be kept on all animals. Dogs, cats, farm animals and non-human primates must be enrolled in RAR's post-op program.

There are different requirements for:

• survival surgeries versus non-survival surgeries,
• major surgeries versus minor surgeries, and
• rodent surgeries versus non-rodent surgeries.

Major surgical procedures are defined as those that enter a body cavity (thorax, abdomen, calvarium), or that have the capacity for having significant complications. Included would be orthopedic procedures and extensive cannulation procedures. Minor procedures include peripheral vessel cannulations and skin incisions.

Rodents are defined by the USDA as laboratory strains of mice and rats only. Rabbits and rodents such as guinea pigs or hamsters are classified as non-rodents with regard to surgical requirements.

Non-rodent survival surgery requirements

• Asepsis. This includes using sterilized instruments (autoclaved or a high level disinfectant, not just alcohol), clipping the surgical site, scrubbing with a disinfectant such as betadine or chlorhexidine, draping of the surgical site, and use of sterile gloves. Sterile gowns, caps and surgical masks must be used for major surgeries. Requirements for asepsis are similar for major or minor procedures in non-rodent animals.
• An IACUC approved surgical area is required for non-rodent surgery. For major procedures, there must be separation of the operating room from animal preparation and instrument cleaning areas, and an adjacent, but separate area for surgeon preparation. The area must be inspected by the IACUC and approved prior to starting any animal procedures. All surgical areas are subject to post-approval inspections at any time.
• Post-operative care. This includes support of body temperature, cardiovascular and respiratory function, monitoring, analgesics and record keeping must by followed.

Rodent surgery requirements

Incision size is smaller, reducing the potential for contamination.

• Asepsis. This includes using sterilized instruments (autoclaved or a high-level disinfectant, not just alcohol), clipping the surgical site, scrubbing with a disinfectant such as betadine or chlorhexidine, draping for major procedures, and use of sterile gloves. A clean lab coat, hair covering and mask are highly recommended, especially for major surgeries.
• A dedicated work area is required. This is a room or bench top that is out of major traffic and from which all materials are removed at the time of the surgery. The area must be disinfected before starting the procedure.
• Post-operative care. This includes support of body temperature, cardiovascular and respiratory function, monitoring, analgesics and record keeping.

Non-survival surgery requirements

• A dedicated work area, similar to that used for rodents may be used. For short non-survival procedures, dedicated surgical facilities and aseptic techniques are not needed.
• Asepsis is still required for longer non-survival surgeries, as infections can begin affecting the animals within several hours.
• Anesthetic requirements are identical between survival and non-survival procedures.
• Post-operative analgesia, monitoring and care is obviously not a requirement for non-survival procedures.

For more information on surgery see: http://www.ahc.umn.edu/rar/surgery.html

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Euthanasia

Euthanasia is defined as a pain free or stress free death. All personnel should understand the criteria and the approved methods for euthanasia. RAR offers euthanasia as a free service. This is arranged by contacting the business office at 612-624-6169.

Criteria for Euthanasia

Experimental Endpoint

Most animals are euthanized at the end of the experiment. It is important that this endpoint be clearly defined in the Animal Usage Form.
Examples include: a specific time point following manipulation, specific size of a lesion, a measurable physiological parameter such as a serum chemistry level, blood pressure, etc., detection of a gene product, or some other experiment-specific measurement.
If an animal is experiencing unrelieved pain or distress before the end of the experiment, euthanasia may need to occur prior to the defined experimental endpoint.

Unrelieved pain or distress

Any pain or distress that cannot be relieved by analgesics, anesthetics, or sedatives is a condition for euthanasia, unless there is specific written approval from the IACUC and it is scientifically justified that doing so would interfere with the goals of the experiment. This would include general debilitation, infection that is non-responsive to treatment or severe organ dysfunction. Appendix N of the Animal Usage Form lists the criteria defined by the IACUC that would indicate euthanasia.

Approved methods

1. CO2 inhalation in small animals. The source of carbon dioxide must be compressed carbon dioxide gas in a cylinder. Dry ice is not an acceptable source of carbon dioxide.
2. Anesthetic overdose. Certain anesthetics, such as sodium pentobarbital are appropriate to use in high doses (i.e. four times the anesthetic dose) as a means of euthanasia.
3. KCL, cervical dislocation or decapitation may be used as methods of euthanasia when the animals are UNDER ANESTHESIA. This applies to all species. Approval by the IACUC for cervical dislocation or decapitation on awake animals may be attained, with scientific justification, if necessary.
4. Rodents for Raptors. Euthanized rodents may be donated to the Raptor Center at the University of Minnesota. See the RAR web site for more details on this program.

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Procedures

Certain procedures have the potential to cause pain and distress. The IACUC and RAR have developed guidelines for some of these procedures in order to minimize the pain and distress experienced by an animal. Deviation from these guidelines requires IACUC approval and scientific justification. Specific experiment guidelines listed for each of these procedures can be found on the RAR web site.

Contact an RAR veterinarian for assistance with these or other procedures not described below.

Immunization

inflammation from immunization with adjuvants can result in pain or distress. Complete Freunds Adjuvant may be used only for the first (priming) dose. Subsequent immunizations should be with incomplete Freund's or another adjuvant.

Tumor induction

tumors can result in pain or distress when they reach an advanced stage. Unless necessary for the experiment, animals should be euthanized before the tumor begins having adverse effects, such as anorexia, depression, weight loss, ulceration of the skin, internal bleeding or interference with the animal's locomotion.

Blood collection

Blood collection can result in anemia or hypovolemic shock. Certain methods, such as orbital sinus collection or intracardiac blood collection, can be painful and normally require use of anesthesia

Food or water restriction

Food and water restriction can cause stress in animals. Restriction should be modest and should be based on something measurable (e.g. a percentage of ad lib consumption or ad lib body weight). It is critical to monitor animal well-being, as well as physiological parameters, during times of food or water restriction.

Restraint and handling

Proper handling procedures can minimize stress to both the handler and the animal. If restraint devices are used, it is important that animals be adapted to the device (place them in the device several days in a row for gradually increasing intervals). If prolonged restraint is to be performed (over 1 hour), it may be necessary to provide food, water, and the opportunity for the animal to exercise during the restraint period.

Monoclonal antibody production using the mouse acites method

must be conducted according to guidelines and it must be demonstrated that the investigator searched for in-vitro methods.

Tail snip guidelines

The use of tail snips in rodents as a means of genetic monitoring is also subject to guidelines developed by the IACUC.

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Husbandry and Housing

The environment an animal lives in and the care it receives can have significant impacts on its health and well-being. Guidelines for husbandry and housing must be followed at all times, unless specific approval is given by the IACUC for exceptions.

These guidelines include the amount of cage space needed for animals; temperature, humidity, and ventilation requirements; guidelines for the quality of, and the frequency of providing feed and water; frequency of and methods for sanitation; and the need for environmental enrichment and social behavior.

At the University of Minnesota, Research Animal Resources (RAR) provides a centralized husbandry and housing program for all laboratory animals. All animals must be housed within centrally managed housing areas, unless there is a special need for the investigator to provide housing. This need is usually based on a housing or experimental requirement that cannot be provided within the central housing areas. IACUC approval is needed to house animals outside of RAR managed areas for more than 12 hours.

In the cases where investigators house animals in their laboratory, a written description of the facilities and the husbandry procedures must be given and approved by the IACUC. RAR will visit the areas on a regular basis to ensure the well being and veterinary care of the animals that are not housed within the central housing areas.

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Safety and Security

The animal housing areas and some of the laboratory areas are locked and access is limited to those with a legitimate need. Do not prop open locked doors, even for a short period of time. Be alert for persons who do not belong in the area and report any suspicious behavior. Wear your ID badge at all times when on campus.

Use 911 from any campus phone for all emergencies, including fire, natural disasters, medical emergencies, threats or security breaches. (Do NOT dial 8 for an outside line)

For chemical/biohazard/radiation spills Call DEHS 612-626-6002
For animal emergencies Call RAR 612-624-9100
For building problems Call Facilities Management 612-624-5765

More tips for keeping research areas secure have been developed by The University Police Department. If you have questions about security, or to arrange for a presentation covering safety tips, call the University Police at (612) 624-2575.

If you are the manager or supervisor of an animal area, we recommend you print off and read the Animal Care Program Emergency Guide.

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