AALAS INTRODUCTION TO RATS
Which statement is TRUE regarding venipuncture procedures in rats?
Cardiac puncture is most often allowed only as a terminal procedure.
Barrier facilities should only contain shoebox cages contained in ventilated racks.
False
General anesthesia has no or little effect on thermoregulation so warming pads are generally not needed.
False
Rats should never be housed on wire flooring, because the Guide forbids it.
False
Dissociative agent combinations
Lethal doses of dissociative agents such as ketamine are commonly used in laboratory settings. In some species, ketamine alone can result in stimulatory activity prior to sedation and loss of consciousness. In conscious rodents, ketamine and similar dissociative agents should be used in combination with an a2-adrenergic receptor agonist such as xylazine or benzodiazepines such as diazepam.
Buprenorphine is a common and effective opioid analgesic used in rats.
True
Tribromoethanol is acceptable with conditions
as a method for euthanasia when prepared, stored, and administered at the appropriate dosage.
Hantavirus is a
bunyavirus which can be transmitted by rodent species. The virus is transmitted to man by excretions and aerosols from the lungs, saliva, and urine of infected animals.
Rats have
incisors that are open-rooted, meaning that these teeth grow continuously throughout adult life. A diet of soft foods, i.e., in liquid or powder form, or a developmental jaw malformation will cause tooth overgrowth. Staff must be alert to detect any signs of this condition and to provide appropriate treatment. Teeth can be trimmed with small, sharp scissors or nail-trimmer type cutters. Ask your veterinary staff to help or to perform the task for you.
The antigen preparation should be free of
extraneous microbial contamination and byproducts such as polyacrylamide gel. The protocol should describe how the antigen-adjuvant emulsion will be prepared.
Humans infected with Salmonella may have inapparent clinical signs (and be carriers) or may have a
febrile enterocolitis, septicemia, and focal infections in diverse tissues. Increased severity of the disease occurs due to reduced immunocompetence, e.g., in persons with AIDS, neoplasia, immunosupression therapy, etc., and due to treatment with antibiotics.
Sporothrix schenckii is
fungus associated with multiple species, including rats. Humans acquire Sporothrix schenckii via bites or direct contact. Disease manifests as a nodule on the hand or arm, which may progress with additional nodules forming along the path of lymphatic vessels. Deep visceral infections may also occur.
Unlike mice, rats do not have
gallbladder. Bile passes from the liver through a bile duct directly to the duodenum.
Outbred stocks are
genetically dissimilar animals (males and females that are not related are mated). Outbred stock tend to be more healthy and produce more young (also called outbred vigor). Using these rats is sometimes desirable in scientific studies, when genetic heterogeneity (differences) is not a concern. Example: Wistar rats, Hsd:WI
Inbred strains are
genetically similar rats that have continued to be inbred (brother-sister or father-daughter matings) and are used for genetic engineering and finely controlled studies. Inbred strains with characteristics of human diseases or physiological conditions are generally preferred models for biomedical research. Example: Lewis rats, LEW/CrlBR
If non-survival surgery will be performed (when the animal is euthanized before regaining consciousness),
gloves should be worn by the surgeon, the area and instruments should be clean, and the surgery site should be clipped.
If the use of Freund's complete adjuvant (FCA) is proposed, your IACUC may require justification of this choice of adjuvant. FCA has been associated with
granulomatous inflammation, focal necrosis, ulceration of skin, fistulous tracts, muscle atrophy, self-induced trauma, hypersensitivity reactions, and weight loss. The USDA states that the injection of FCA may cause more than momentary or slight pain. This means that FCA injections might put an animal into USDA pain category D (painful/stressful but relieved), requiring the use of post-injection analgesics. Check with your IACUC to determine your institution's policy.
Due to the interaction of metabolic factors and drug effects that can cause animal
mortality, rats should receive good supportive care and frequent monitoring during anesthesia, whether or not the procedure involves surgery.
Aseptic technique should be used when performing
survival surgery on rats.
In common booster schedules, the initial and subsequent immunizations are spaced at intervals of
two to three weeks (minimum). Booster immunizations may be delayed if significant inflammatory reactions are still present from the initial immunization.
A chronic state of pain or distress may be more
ubtle and difficult to detect. A good knowledge of the animal's normal appearance and behavior is especially important to recognize chronic pain or distress.
Rats do not vomit because
they lack the neurophysiological mechanisms for emesis. Therefore, presurgical fasting is not necessary in rats, as it is for nonrodent species.
Check all that apply. Which of the statements below are TRUE regarding the assessment of pain or distress in rats?
- A rat with a mild to moderately painful condition may appear and behave normally. - Pain or distress in rats is commonly expressed by subtle changes in activity level and behavior.
Monitoring during anesthesia:
- Analgesia - toe pinch - Respiration - gross changes in rate, character of breathing - Color of mucous membrane and skin - blue (poor oxygenation), pale (poor blood perfusion) The photo shows a rat with the trachea intubated, ready to be connected to a ventilator.
Monitoring post procedure:
- Assess appearance, activity, and behavior as indications of pain and discomfort (see lesson on Detecting Pain and Distress). - Assess food and water intake. - Provide floor-level access of food and water post procedure if stretching overhead (up to the wire cage lid) for these items may be painful. - Assess wound healing.
When CO2 is introduced to an uncrowded chamber from a compressed gas cylinder slowly, it mixes with the room air and anesthetizes the animals before they are asphyxiated. This prevents the following situations, which should be avoided:
- Climbing or jumping to avoid exposure to CO2 - Severe irritation to the mucous membranes - Prolonged distress due to fluctuating levels of CO2 in the chamber After animals are anesthetized and asphyxiated, gas flow should be maintained for at least 1 minute after apparent clinical death. It is important to verify that an animal is dead before removing it from the chamber. If an animal is not dead another method of euthanasia should be used.
Keep in mind that:
- General anesthesia causes disturbances of thermoregulation and other physiological functions. Maintaining body temperature, e.g., via insulating materials and heating pads, is an important objective of supportive care because rats may be unable to properly thermoregulate for some time after some procedures, including anesthesia and surgery. - During surgery, an animal may experience pain if anesthesia is inadequate at any time during the procedure. - Postoperatively, the animal may experience pain, discomfort, and distress unless treated with analgesics and appropriate supportive care measures. It is recommended that the first analgesic dose be administered during surgery, or before the animal wakes up from anesthesia. Follow your approved IACUC protocol, and keep documentation on when all analgesic treatments are given.
Supportive care aims to:
- Maintain the animal's physiological status as near normal as possible - Minimize animal pain and distress
Supportive care:
- Provide a source of warmth to rats from the onset of anesthesia to the end of anesthetic recovery. - Inject sterile physiological fluid (warmed) to compensate for blood loss during a procedure and depressed fluid intake post-procedure. This should be done in concert with input from the veterinary staff.
An investigator's responsibilities towards the use of laboratory animals include assuring that staff is properly trained for which of the following?
- Provide the necessary supportive care to the animals during a procedure. - Provide the necessary supportive care to the animals following a procedure. - Perform the animal procedures humanely.
This periprocedural care requirement covers:
- properly preparing the animal to humanely undergo the procedure; - supporting the animal's physiological functions during the procedure; and - providing additional supportive care to aid the animal in recovering from the procedure.
Alternative procedures are those which may:
- replace animals with nonanimal methods, - reduce the number of animals used, or - refine the methodology to minimize animal pain or distress.
Recommendations for FCA from the Institute of Laboratory Research are as follows
1. FCA should be used only once, usually for the initial immunization. 2. Formulations of FCA should not exceed 0.1 mg dry mycobacterial cell mass/ml. 3. Less inflammatory alternatives to Freund's adjuvant are available and should be considered.
The assurance often takes the form of a written narrative that describes what sources were used to determine that alternatives were not available. Typically, you may be asked to provide the results of a database search including information on:
1. The databases searched 2. The date the search was performed 3. The years of citations covered by database searches 4. The key words and/or search strategy used when searching a database It is strongly recommended that this information be sought during development of a protocol.
Recommended needle sizes are
23 to 27 gauge. Larger needles may be necessary for injecting large volumes or viscous materials.
Typically, acclimation periods range from
4 days to 1 week.
What is a procedure?
A procedure is any activity carried out on the animal, such as a controlled behavioral task (e.g., use of a maze), food or water restriction, blood collection, breeding, or surgery.
Which statement is TRUE regarding anesthesia in rats?
A stable plane of anesthesia for long-term surgery is best achieved by gas anesthesia using a vaporizer.
Severe pain and distress causes overt clinical signs in rats. Laboratory staff working with rats should be trained to recognize abnormalities in:
Activity level: - Hypoactivity - Hyperactivity - Lethargy - Restlessness Behavior: - Vocalization - Self-trauma - Isolation from cage mates - Aggressiveness - Ataxia Appearance: - Unkempt greasy fur - Porphyrin staining around eyes and nostrils - Hunched posture - Cyanosis - Pale mucous membranes - Soiled anogenital area Vital signs: Respiratory distress Body condition: - Weight loss - Emaciation - Dehydration Intake: Reduced intake of food and water
Methods are classified as acceptable or conditionally acceptable, as set by the
American Veterinary Medical Association's document, the AVMA Guidelines for the Euthanasia of Animals. The inclusion of conditionally acceptable methods in your protocol may require scientific justification and IACUC approval.
Injection Site Selection and Preparation
Anatomic sites used for grasping, handling, or restraint....should be avoided when possible. Extension of granulomatous inflammation into the spinal cord following inadvertent injection of a FCA-antigen mixture into the paraspinal musculature has been associated with posterior paresis in guinea pigs (Kleinman et al., 1993). Care therefore should be taken when making injections near the dorsal spinal column. Granulomas can also be noted in other organs after injections with FCA (Schiefer and Stunzi, 1979).
Analgesics:
Available in two drug types - the opioids and the nonsteroidal anti-inflammatory drugs (NSAIDs). The rapid clearance of many of these drugs in rats results in the need for an increased frequency of administration.
Acceptable:
Barbiturate overdose Dissociative agent combinations
Anesthetics:
Because rats metabolize drugs so rapidly, many anesthetic agents have brief durations of effect. An anesthetic regimen should be chosen to match the duration of drug effects with the length of the procedure. In particular, short-acting agents (and regimens) should be not be used for long procedures because repeat drug administrations, necessary to prolong anesthesia, will produce uneven blood concentrations and therefore an uneven plane of anesthesia. For long procedures, gaseous anesthesia is often the most practical method to sustain uniformly adequate levels of anesthesia.
All of the following are appropriate components of an aseptic survival surgery EXCEPT:
Beginning the surgery with instruments that have been cleaned with soap and water
Carbon monoxide
Carbon monoxide used for individual animal or mass euthanasia is acceptable for dogs, cats, and other small mammals, provided that commercially compressed CO is used and the following precautions are taken: 1. Personnel using CO must be instructed thoroughly in its use and must understand its hazards and limitations; 2. The CO chamber must be of the highest quality construction and should allow for separation of individual animals 3. The CO source and chamber must be located in a well-ventilated environment, preferably out of doors; 4. The chamber must be well lit and have view ports that allow personnel direct observation of animals; 5. The CO flow rate should be adequate to rapidly achieve a uniform CO concentration of at least 6% after animals are placed in the chamber, although some species (e.g., neonatal pigs) are less likely to become agitated with a gradual rise in CO concentration; and 6. If the chamber is inside a room, CO monitors must be placed in the room to warn personnel of hazardous concentrations. It is essential that CO use be in compliance with state and federal occupational health and safety regulations.
According to the AVMA, which statement is TRUE for the use of CO2 as a euthanasia agent of rats?
Compressed CO2 gas in cylinders is the only acceptable source of carbon dioxide.
Microwave irradiation
Focused beam microwave irradiation is a humane method for euthanizing small laboratory rodents if instruments that induce rapid loss of consciousness are used. Only instruments that are designed for this use and have appropriate power and microwave distribution can be used. Microwave ovens designed for domestic and institutional kitchens are unacceptable for euthanasia.
The term euthanasia is derived from
Greek and means "good death." Animals should be euthanized when killed for any purpose, including research. To euthanize a rat, you must be trained in the concepts of euthanasia, the method to be used, and the proper handling of rats.
viruses
Hantavirus
parasites:
Hymenolepsis nana is a common type of tapeworm, and is transmissible to humans. Depending on the parasite burden, humans may have no apparent clinical signs or may have nausea, anorexia, vomiting, diarrhea, and central nervous system signs (agitation).
The following are volume recommendations for intravenous fluid administration and blood collection in adult rats (250-500 g):
IV fluid volume (mL) max. acute admin - 0..5-1.0 mL Total blood volume - 64 mL/kg Safe bleed volume - 5.5 mL/kg Bleedout volume - 7.5-15 mL
Acceptable with Conditions:
Inhalant anesthetic overdose Carbon dioxide (compressed tanks only) Carbon monoxide Cervical dislocation Decapitation Microwave irradiation Tribromoethanol
Barbiturate overdose
Injectable barbiturates act quickly and smoothly to render rodents unconscious...Pentobarbital is the most commonly used barbiturate for laboratory rodents because of its long shelf life and rapidity of action.
Which of the following choices is NOT a commonly used nonvascular type of injection in rats?
Intracardiac
Bacteria:
Leptospira spp. Salmonella spp. Streptobacillus moniliformis and Spirillum minus
Cervical dislocation
Manual cervical dislocation is acceptable with conditions for euthanasia of small birds, poultry, mice, rats weighing < 200 g, and rabbits when performed by individuals with a demonstrated high degree of technical proficiency. In lieu of demonstrated technical competency, animals must be unconscious or anesthetized prior to cervical dislocation. For heavy rats and rabbits, the large muscle mass in the cervical region makes manual cervical dislocation physically more difficult ... Those responsible for the use of this technique must ensure that personnel performing cervical dislocation techniques have been properly trained and consistently apply it humanely and effectively.
What would be a good choice to help you determine if a rat is in pain?
Observe normal healthy rats and compare their behavior and physical appearance to the rat you are trying to assess.
Rats and mice ocular
Rats and mice may develop red staining around the eyes and nostrils when they are distressed, (e.g., by disease, trauma, etc.) or in response to some types of drugs. This staining is due to the accumulation of porphyrins produced by the Harderian gland, a lacrimal gland. Though a normal constituent of tears in rodents, lacrimal porphyrin is produced in limited amounts and rodents keep themselves clean of debris through frequent grooming. Porphyrin staining in distressed animals occurs because stress stimulates porphyrin production in tears, and distressed animals groom themselves less often.
Typical Rat Housing
Rats are generally housed in shoebox cages with a solid floor covered with bedding material that absorbs liquid wastes. Most institutions use microisolation cages, which are made of hard plastic and use a filter top to contain allergens and protect animals from potential pathogens in the environment.
Why do rats require thermal support during anesthesia?
Rats lose body heat rapidly under anesthesia.
Leptospira spp.
Rats may be a reservoir for Leptospira spp. bacteria, which are shed in the urine. Transmission occurs by contact with urine and tissues, or inhalation or ingestion of aerosol droplets. Humans with leptospirosis may have influenza-like symptoms, orchitis, rash, skin and mucosal hemorrhage, hemolytic anemia, hepatorenal failure, jaundice, encephalitis, and pneumonia.
Salmonella spp.
Rats may carry Salmonella spp., which are ubiquitous in nature. These bacteria are transmitted via the fecal-oral route.
Streptobacillus moniliformis and Spirillum minus
Rats may carry Streptobacillus moniliformis and Spirillum minus as commensal organisms living in the nasopharynx. Transmission to humans occurs by rat bite. Symptoms may include wound inflammation, lymphadenopathy, fever, headache, malaise, myalgia, joint pain, and arthritis.
Monitoring post anesthesia:
Rats must be monitored until fully recovered from anesthesia as indicated by the ability to ambulate and maintain core body temperature.
What occupational health and safety concerns are NOT relevant to performing research on rats?
Risks for zoonotic diseases when working with healthy specific-pathogen free rats
Sedatives:
Sedatives are used to calm animals and facilitate handling, but have no effect on consciousness, the perception of pain or other sensations. When combined with general anesthetics, the result is a balanced anesthesia where muscle relaxation, unconsciousness, and analgesia are enhanced.
Pentobarbital combinations
Several euthanasia products combine a barbituric acid derivative (usually sodium pentobarbital) with local anesthetic agents, other CNS depressants (e.g., phenytoin, ethanol), or agents that metabolize to pentobarbital. Although some of the additives are slowly cardiotoxic, euthanasia makes this pharmacologic effect inconsequential. These combination products are listed by the DEA as schedule III drugs, making them somewhat simpler to obtain, store, and administer than schedule II drugs such as sodium pentobarbital. The pharmacologic properties and recommended use of euthanasia products that combine sodium pentobarbital with agents such as lidocaine or phenytoin are interchangeable with those of pure barbituric acid derivatives. Mixing of pentobarbital with a neuromuscular blocking agent in the same injection apparatus is not an acceptable approach to euthanasia because of the potential for the neuromuscular blocking agent to induce paralysis prior to onset of unconsciousness.
Sedatives + Analgesia:
Some sedatives also have analgesic effects. When combined with general anesthetics, a balanced anesthesia is attained, and these sedatives enhance analgesia through specific effects.
Fungi:
Sporothrix schenckii Trichophyton spp.
Most rats used in research are albinos, such as the
Sprague Dawley stock. Albinism in rats is an inherited disorder of melanin metabolism caused by the lack of the enzyme tyrosinase, which has an impact both on melanocytes and neurons. Neuronal morphological abnormalities and functional impairments involve the following sites: medial vestibular nucleus, cochlear nuclei, and retina. Studies comparing albino and pigmented animals have shown differences even in pharmacotoxic kinetics in these tissue areas.
Which statement is true regarding the conduct of surgery in rats?
The Guide requires the use of aseptic technique for survival surgery in rats.
What information should be provided with the results of a database search for alternatives?
The names of the databases used, the date of the search, the years of citations accessed, and the key words and/or search strategy
Which concept is relevant to the proper conduct of euthanasia?
The selection of an appropriate euthanasia method should address considerations of humaneness to the animal, and safety and esthetics for the staff.
Carbon dioxide (compressed tanks only)
The use of CO2 to euthanize rats is acceptable with conditions; precautions must be taken to ensure the least amount of stress to the animals prior to death. The only source of CO2 that is acceptable is from a compressed gas cylinder (some institutions have CO2 piped in through the plumbing, which is acceptable since it originates from a compressed gas tank that is regulated). The tank must have a pressure-reducing regulator and flow meter. An optimal flow rate for CO2 euthanasia systems should displace 10% to 30% of the chamber or cage volume/min.
What is one result of rats' high rate of metabolism?
They eliminate drugs rapidly from their bodies.
Which of the following does NOT oversee research with laboratory strains of rats and mice?
USDA
Which of the following descriptions is NOT true about shoebox caging for rats?
Urine and feces can fall through perforations in the bottom of the cage.
Which statement regarding preparation of a rat for surgery is TRUE?
You should firmly pinch the rat's foot or toe to check for a pain reflex before beginning surgery.
Post-injection Observations
Your animal protocol should describe how animals will be monitored for post-injection lesions and how affected animals will be treated.
Animals should be exsanguinated only under
anesthesia
In rats, herbaceous foodstuffs are broken down by microbial action in the cecum, which is a large organ in the rat. To assimilate the microbial byproducts of digestion, the rat regularly eats its own feces, a habit known as
coprophagy. Stomach digestion and intestinal absorption of this fecal material yields nutrients that are essential to the rat.
Tricophytum are
dermatophytic fungi that grow in the skin and hair follicles and cause a condition of reddened skin and patchy hair loss known as ringworm. The symptoms are the same in animals and humans. Infection may be inapparent in individual animals. Dermatophytes are spread by direct contact. Fungal spores are long-lived and may become widely dispersed in the environment.
Some microisolation-type cages are housed on
egular racks and are not individually ventilated. Because of the filter tops, ammonia from the urine can build up to toxic levels if the cage is not changed on an appropriate schedule. Usually microisolation-type cages in a ventilated rack can be changed at longer intervals than non-ventilated cages.
Rats have a large body surface area (relative to body volume) plus many hairless body parts (tail, ears, feet). As a result, rats are vulnerable to a profound
hypothermia under conditions of sedation and anesthesia. Sedation and anesthesia induce hypothermia due to drug effects on the hypothalamus and a rapid drop in core body temperature. If surgery is being performed, additional heat is lost by convection from an open incision during surgery.
Removing greater quantities of blood (exceeding 10% of total blood volume) can produce
hypovolemic shock. Repeated collections of smaller amounts of blood will have the same effect. In such procedures, animals should receive warmed, physiological fluids to replace the volume of blood collected. In addition, monitor the animal's hematocrit for anemia
Animal Biosafety Levels (ABSLs).
maintains rats in studies involving pathogens may need to be housed in biocontainment facilities
The rat's high rate of
metabolism produces a rapid clearance of drugs from the body. Drugs administered at dose rates used in larger species (with lower metabolic rates) would reach lower blood concentrations and exert less pharmacological effect in the rat. As a result, rats should receive drug doses that have been scaled to the rat's metabolism. Through a discipline known as allometry, mathematical formulas have been developed to adjust dose rates between species of disparate size.
Inhalant anesthetic overdose
nhaled anesthetics are acceptable with conditions for euthanasia of small animals (< 7 kg) where the following contingencies can be met: (1) In those species where aversion or overt escape behaviors have not been noted, exposure to high concentrations resulting in rapid loss of consciousness is preferred. Otherwise, gradual fill methods can be used, keeping in mind the effect that chamber volume, flow rate, and anesthetic concentration will have on the time constant and rate of rise of anesthetic concentration. Inhaled anesthetics can be administered as the sole euthanasia agent or as part of a 2-step process, where animals are first rendered unconscious through inhaled anesthetic agent exposure and then subsequently killed by a secondary method. (2) Order of preference is isoflurane, halothane, sevoflurane, enflurane, methoxyflurane, and desflurane, with or without N2O. Nitrous oxide should not be used alone. Methoxyflurane is acceptable with conditions only if other agents or methods are not available. Ether is not acceptable for euthanasia.
[Decapitation] is acceptable with conditions if
performed correctly, and it may be used in research settings when its use is required by the experimental design and approved by the IACUC. Decapitation is justified for studies where undamaged and uncontaminated brain tissue is required. The equipment used to perform decapitation must be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. The use of plastic cones to restrain animals appears to reduce distress from handling, minimizes the chance of injury to personnel, and improves positioning of the animal. Those responsible for the use of this technique must ensure that personnel who perform decapitation have been properly trained to do so and are monitored for competence.
During quarantine, diagnostic tests are
performed on the animals.
Many laboratory animal programs house rats in a "barrier" facility, where appropriate precautions are taken to
prevent disease from entering and spreading through the animal populations.
The purpose of quarantine is to help
prevent transmission of diseases between new animals and established colonies.
Because rats have a high rate of metabolism, drugs are
rapidly eliminated from their bodies. Dose rates appropriate for larger species produce ineffective drug concentrations in the bloodstream when used in rats.
Fasting of rats before surgery is not necessary because:
rats have no risk for respiratory aspiration of vomitus under anesthesia because they cannot vomit.
nonvascular routes of injection that are commonly used in rats and volume recommendations
safe administration of fluids acutely in adults (average 200 g). Subcutaneous (SQ or SC) - 25 ml/kg Intraperitoneal (IP) - 25 ml/kg Oral (PO) - 10 ml Intradermal (ID) - 0.05 ml/site Intramuscular (IM) - 0.1 ml per site
Assessing pain and distress in rats is difficult at times. This is because rats, like many other species, commonly conceal outward signs of moderate pain and distress
the behavioral changes that reveal a rat's pain and distress may be subtle and detectable only with specialized behavioral methods.
Supportive care includes
the monitoring of both physiological parameters and analgesia during anesthetic and surgical procedures. Monitoring of vital signs and potential signs of pain should be conducted throughout the procedure and the recovery period.
The investigator has the responsibility to see that staff working with the animals are properly trained to not only perform
the procedure humanely but also to provide the necessary supportive care to the animals.
Rats should have a source of warmth throughout a procedure that lowers their body temperature (e.g., anesthesia, surgery) and afterwards until they recover the ability
to thermoregulate themselves.
