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Research Methodology
LAT Chapter 13
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Chapter 13
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Toxicology
Chapter 13
• Science of poisons and their harmful or noxious effects
on living organisms
• Toxicologist assesses the toxicity of materials
 pharmacology, biochemistry, pharmacodynamics, physiology,
inorganic and organic chemistry, and cellular and molecular
biology.
• Acute Toxicity Tests - a single dose of a test substance
 most often a rodent or rabbit
 LD5O - dose of that kills 50 percent of the animals tested
 Draize skin and eye assays
 criticized, if used to evaluate non-pharmaceuticals
 definitive means of maintaining public safety.
Dosing a Rat By Gavage
Chapter 13
Subchronic & Chronic Testing
Chapter 13
• Subchronic - 13 to 26 weeks in duration.
 Daily dose by the same route substance administered normally
 Observed for toxicity, changes in weight or food consumption
 Evaluate clinical chemistry and hematology values
 4 groups, each 15 to 20 rats or four dogs of each gender
 Euthanized and evaluated for histopathologic toxicity
• Chronic Testing - longer-term toxic and carcinogenic
 Mice and rats, 4 to 5 groups of 60 to 100 per sex per group
 As subchronic, but observation period is longer (~ two years)
 Animals are palpated to detect tumor formation.
 Postmortem for histopathologic toxicity and carcinogenicity
Blood Sampling - Saphenous Vein
Chapter 13
Repro., Teratology, Pyrogens
Chapter 13
• Reproductive - usually conducted on rats and rabbits
 to detect changes in reproductive cycle and toxic effects on
fertility, organogenesis and behavior
• Teratology = exposure of developing litters to chemicals
 Teratogens = substances that damage the developing fetus.
 Changes in normal fetal anatomy, litter size, or fetus weight may
indicate that the test substance is a teratogen.
• Pyrogen = substance which produces a fever
 To detect bacterial toxins in products administered by injection
 A rise in temp. in >1 rabbits indicates the presence of a pyrogen.
 Limulus amebocyte lysate (LAL) test - horseshoe crab blood will
clump together in the presence of a pyrogen.
Toxicology Tests
Chapter 13
The developing fetus
The Horseshoe Crab
Immunodeficiency Models
Chapter 13
• Immunodeficient = defect in normal immune system
 By studying animals that lack one or more parts of the normal
immune system, it is possible to gain information about how the
total system functions.
• Good models of spontaneous or infectious diseases,
such as AIDS in humans
• Means of studying immune system vs. neoplasia
 Method of keeping various tumor cell lines alive
• Nude mice lack immune mechanism responsible for
transplant rejection.
 As a result, they act as living (in vivo) culture vehicles for certain
tumor cell lines which will not grow properly in vitro.
Spontaneous Immunodeficiency
Chapter 13
• Genetic manipulation primary method to induce.
• Low lymphocytes, macrophages, or hematologic factors
• Nude mice most widely used
 Hairlessness and lack of thymus gland
 No thymus = no T-cells, attack viruses and
tumor cells and helps other cells make antibodies.
 Makes them more susceptible to infections.
 Usually maintained as pathogen-free.
• Athymic Rats and Hamsters: T-cell deficient
• Other Immunodeficient Animal Models:
 B-cell hereditary defects = CBA/N and Xid mice
 SCID mice lack B-cells and T-cells
 Beige mice lack “natural killer” (NK) cell
Induced Immunodeficiency
Chapter 13
• Surgery: thymus gland can be removed surgically
• Chemical: agents used in research suppress immunity
 drugs toxic, mortality rates >20 % anticipated
• Irradiation faster & easily measured.
 High energy radiation inhibits protein synthesis.
 Source of radiation cobalt or cesium gamma irradiators
1. Single exposure: Total body radiation easiest method. Small
animals are confined in plastic tubes or aluminum boxes for
exposure, dogs and larger animals anesthetized. suppresses
immune response to foreign cells.
2. Low level protracted semicontinuous radiation
3. Partial body radiation: uses lead shields to protect certain parts.
Irradiation Induced Immunodeficiency
Chapter 13
For rodent irradiation, animals
are placed in a specially
designed holder which is inserted
into a ventilated chamber for a
brief period of exposure.
Holding chamber
Tolerance
Chapter 13
• Ability to differentiate between self and foreign
substances
 Ability is acquired early in life.
 Exposure to substances at the appropriate time during
development = tolerance substances.
 Subsequent exposure does not result in an immune response.
 May be short or long in duration.
 Differs from immunodeficiency in that immune system fully
functional and capable of responding to other antigens normally.
• Neonatal are easiest for induction of tolerances, but adult
animals can also be used.
 Less developed the immune system of a species at birth, more
easily tolerance induced.
Tolerance - Neonatal
Chapter 13
Day 1
Day 3
Day 2
Immunocompromised Care
Chapter 13
• Structural and procedural barriers to transmission of
infectious agents = positive pressure rooms or MicroIsolator™ cages.
• Experimental subjects frequently require extra attention
 Poor appetite and difficulty eating and drinking
 Acidified (pH 2.4-2.8) or chlorinated water used to suppress
bacterial growth
 Food, bedding, and cages are sterilized before use, and filter
bonnets or isolator cages frequently used.
 Recovery period from wounds or ailments
likely to be much longer than normal animal.
 Be alert to differences and provide the food
and medication specified by the protocol.
Antibody Production
Chapter 13
• Classic Vaccination: When a person or animal receives a
vaccination to protect against a specific disease, the
protection is in the form of antibodies.
• The vaccine is composed of the disease organism, or
some part of it, and when administered to the recipient
results in production of antibodies by the recipient’s
lymphocytes (B-cells).
• The immunity conferred by the vaccination can last for as
short as a few months to as long as many years,
depending on the organism.
 For example, it is recommended that dogs be vaccinated against
canine parvovirus annually, whereas humans vaccinated against
tetanus only require re-vaccination every ten years.
Polyclonal Antibodies
Chapter 13
• Bacteria, viruses, plant pollen and toxins = antigens
 Antigen X stimulates production of anti-X antibody; anti-X
antibody reacts only with antigen X. Antigens have multiple,
unique areas on their surface which stimulate the production of
different antibodies.
• Rabbits, sheep and goats for size and ease of collection
•
~ 3 wks after series of injections, blood is collected and
serum is evaluated for presence of antibody.
• If antibody titer is not adequate, => boosters.
• Antibody can be stored in a freezer for years.
• Exceptional response => animal kept for a long period.
 It receives booster immunizations, and periodic blood collections.
Polyclonal Antibodies
Chapter 13
Adjuvants
Chapter 13
• Some antigens are poor stimulators of antibody.
• Adjuvants enhance the antibody response by:
1) directly stimulating immune cells to produce antibodies
2) prolong absorption of antigen from injection site
• Locally irritating can cause ulceration at injection site.
• Immunize with antigen/adjuvant => signs of illness.
 Unacceptable to place adjuvants IP, in foot pads, or > 0.5 ml
• Complete Freund’s Adjuvant (CFA), Incomplete Freund’s
Adjuvant (IFA), Titer-Max and RIBI.
 CFA produces ab most consistently, + most frequent side effects.
 Part of formulation of CFA includes killed Mycobacteria.
• IFA does not contain Mycobacteria.
 Usual immunization involves first injection w/ CFA, and then IFA.
Hybridomas
Chapter 13
• To get monoclonal Antibodies (MAb):
 Immunized spleen lymphocytes isolated in individual chambers.
• Cancer cells reproduce easily both in vitro and in vivo.
• When a cancer cell is combined with antibody-producing
lymphocyte, a hybrid results.
• Hybridoma has the properties of both parent cells = good
growth and production of desired antibody.
• Techniques available to researchers unite these two
types of cells in vitro => hybridoma.
Hybridoma Culture
Chapter 13
• In vitro = involves growing
hybridoma cells in a special medium.
 As these cells grow, they secrete MAb into the medium.
 MAb are purified from the medium.
 Not all MAb can be produced in sufficient quantities.
• In vivo = hybridoma cells IP into mice, producing MAb in
the fluids that accumulate in the abdominal cavity.
 Ascites fluid, collected by inserting needle into abdomen and
allowing fluid to drip into tube, or aspirating w/ syringe.
 Monitor animals frequently once fluid accumulation begins.
 Aspirate fluid before distention interferes with respiration.
• Since the hybridoma is a type of cancer, animals may
become sick as the disease spreads.
Hybridoma Cell Culture
Chapter 13
Cellmax®
Cross section of the artificial capillary
system. All cells grow on and between
hollow fibers. Fiber number is designed to
optimize diffusion of oxygen CO2 and
nutrients to the cells. Low molecular weight
inhibitory factors (TGFß, TNF ) diffuse away.
High molecular weight product (MAB)
secreted proteins are concentrated in small
volumes (ECS).
http://www.spectrapor.com/cell/cellmax.html
Induced Cancer Models
Chapter 13
• Animals exposed to cigarette by-products and smoke
carefully compared with control group => lung cancer.
• Causes under investigation = toxins, viruses, pollutants.
• Course, diagnosis, or treatment = induced in a group.
 Method variable, depending on the type of cancer being studied.
 Injection of cancer cells, topical application of some cancercausing chemical directly on the skin or mucous membranes.
 Be aware of potential dangers in handling cancer cells or
carcinogenic chemicals.
• Follow proper safety techniques to avoid contamination of workplace.
 Spontaneous tumors can be transplanted from one inbred animal
to another of same strain.
• Cancer reproduces without immunologic rejection of tumor that would
occur in outbred animals.
Spontaneous Cancer Models
Chapter 13
• Some strains of inbred rodents have high
incidence of naturally occurring cancers.
 > 80 % of AKR mice develop leukemia < 1 yr.old
 for evaluating diagnostic and preventive
treatments in susceptible population
 group of animals left untreated acts as a control for the study
• Size of the tumor or metastasis of cancer is evaluated.
• Rarely is it required that disease in test animals be
allowed to progress to the point that undue suffering or
death occurs.
 Test animals are euthanized when clinically ill (listless, loss of
appetite, loss of weight) or tumors become large or ulcerated
Cannulation & Implants
Chapter 13
Catheterization
• Cannula = catheter
 Hollow tube through which body fluids [out] or test substances [in]
 Place aseptically, chronic catheters require careful monitoring.
 Flexible tube with tapered end, stainless steel tube or blunt needle.
 Measure length that is to be inserted, apply sterile lubricating jelly to
tip, advance gently through urethral orifice.
• The possibility of introducing bacteria into the bladder exists.
• Females more difficult than males.
 Visualize the orifice with vaginal speculum.
• Cystocentesis for sterile sample.
 Pass hypodermic needle through surgically prepared abdomen into
bladder, tranquilize animal, skin site numbed.
Cannulating Teats
Chapter 13
• Technicians handling cows and
goats may be required to cannulate
the teats of these animals.
 Reusable metal and disposable plastic
 Only use sterile cannulae.
 Carefully clean and disinfect ends of teats before insertion of
cannula, clean and disinfect again after removal .
 Removable caps permit cannula to be left in place for long
periods of time. The cap is removed periodically to drain milk
from the gland.
Types of Catheters
Chapter 13
• 3 types of IV catheters: through-the-needle, over-theneedle, and butterfly types
 Needle portion of the over-the-needle and through-the-needle
catheter is removed following penetration of the blood vessel,
leaving only the flexible plastic catheter inside the vessel. The
butterfly needle, however, remains in the vessel.
• Over-the-needle: short, easiest, admin. of fluids
• Through the needle: difficult, hub prevents needle
removal, any length, pass far into interior areas of body
• Butterfly: Needle has two plastic flaps that attach to the
hub a means of securing to skin.
 Insertion site immobilized to prevent dislodging or laceration.
Catheter Insertion
Chapter 13
• Aseptic technique 
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Clip and prep insertion site, just as it would be for surgery.
Restrain animal adequately.
Assemble necessary equipment, remove from wrapping.
Occlude vein by manual pressure or by tourniquet.
Puncture as for a routine intravenous injection.
Once blood appears, pass needle into vessel.
Stop occlusion, advanced catheter, remove needle.
Place antibiotic ointment at site, cover with sterile gauze.
• Intravenous catheters may be secured with tape and
routinely left in place for up to 72 hours.
 Longer possible if no infection present.
 If sign of infection is present, remove catheter .
 Check > 1X daily to see it is properly placed and patent.
Catheter Insertion
Chapter 13
Catheter Insertion
Chapter 13
Catheter Insertion
Chapter 13
Catheter Insertion
Chapter 13
Catheter Insertion
Chapter 13
Catheter Maintenance
Chapter 13
• “Cut-down” - insert by directly visualizing a blood vessel.
 Incision over vein, vein isolated by blunt dissection.
 Needle inserted directly into the vein, or vein nicked with scissors
and a catheter inserted.
 Routine use of indwelling intravenous catheters in all surgical
patients eliminates most of the indications for emergency cutdowns before they occur.
• Arterial cannulation - directly measure blood pressure.
 “Cut-down” is more often used as a means to directly visualize
proper insertion of the cannula.
 General anesthesia required, trained dogs repeatedly
catheterized can accept femoral artery puncture through skin.
Catheter Maintenance/ Implants
Chapter 13
Vascular Access Ports
Vascular Access Port
in use
Implants
Chapter 13
• Implantable injection ports and osmotic pumps provide
other ways of administering substances.
• Injection ports connected to vessels or body chambers
 Subcutaneous implantation accessible for injections.
• Osmotic pumps capable of continuous delivery for weeks.
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


Capacity of pumps varies from 200 ul to 2 ml.
The pumps are cylindrical with rounded ends.
Influx of body fluids forces contents of pump to be slowly injected.
Subcutaneous and intraperitoneal implantation
• Aseptic incision made through the skin.
• SubQ pocket formed with hemostats to receive the pump.
• Incision closed w/ wound clips or suture.
 Intraperitoneal placement requires incision through muscular and
peritoneal layers of abdomen as well as through the skin.
Behavioral Motivation
Chapter 13
• Rat most frequently used.
 Pigeons, Columba sp., also used.
 Cats well mapped neuroanatomy,
for neurophysiological brain electrode implant experiments.
 Few primates are used & frequency declining.
• Rhesus monkeys, Macaca mulatta, most commonly used Old World
monkeys. Of New World spp, squirrel monkey, Saimiri sciureus.
• Conceptual problem approach - phenomenon essentially
same in wide variety of species.
• Substitute-for-human approach - animals as proxies when
complexity of study behavior closely parallels humans.
• Evolutionary-comparative approach - how behavior in that
species compares with behavior in other species.
Behavioral Motivation Terms
Chapter 13
• Learning can be defined as modification of a behavioral
tendency by experience.
 Basic requirements for learning experiment are motivation,
perception, response, and reward.
• Motivation means that an animal needs or desires something.
• Perception involves an awareness of the environment.
• What animal does following perception is considered response.
• The reward is something an animal gets as a result of its behavior.
• + reinforcement = reward reinforces the behavior.
• - reinforcement = learn to avoid aversive treatment.
• Deprivation of food or water for motivation.
• It should be remembered an animal learns at all times,
not just when under observation by an investigator.
Special Dietary Studies
Chapter 13
• The choice of species depends on the
purpose of the experiment and the resources available.
 Use the species for which the information is desired, in order to
avoid the problem of cross-species interpretation.
• Some dietary studies are performed to gain knowledge
applicable to many species - rat most popular.
 Long growth period after weaning and
rapid weight gain during that period.
• To study a particular nutrient, it must be required.
 Rats do not require vitamin C in their diet.
 For vitamin C studies, guinea pigs and
nonhuman primates must be used.
Feeding
Chapter 13
• Laboratory animal technicians should be aware of the
types of feeding procedures used in dietary studies.
 In a typical experiment using ad libitum feeding, two groups of
animals are fed diets that differ only by the single factor being
studied or the test substance is introduced into the water.
• Ad libitum feeding and watering may not be satisfactory for
some studies.
 If test diet has a bad flavor, test animals may eat less of it than the
control animals. Substances added to drinking water may cause
animals to drink more or less water than controls
 Pair-feeding is a method of assuring that the control group and the
experimental group receive the same amount of food.
 Simplest to start control group on the study one day later than test
group. Amount of food provided to control animals is determined by
how much was consumed by study group on previous day.
Metabolism Cages
Chapter 13
•
•
•
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Measurement of food, water intake & feces, urine output
More sophisticated measure gases.
Practice taking it apart and reassembling it.
Food is placed in a recessed barred container so animal
cannot readily remove and waste large quantities.
• Water bottle recessed so drops of water will be caught in
a special trough rather than mixing with animal’s urine.
• Bottom of cage is wire mesh to allow feces to fall to floor.
• A double inverted funnel separates urine from feces so
each collects in a separate receptacle.
Stereotaxic Equipment
 Used for rigid stabilization of head and precise measurement of
dimensions for correct placement of items during a surgery.
Metabolism Cages
Chapter 13
Mouse Metabolism Cage
Water
Food
Collection
Additional Reading
Chapter 13
1.Kirk, R.W. and Bistner, S.I. Handbook of Veterinary Procedures and
Emergency Treatment, 6th Ed. W.B. Saunders, Philadelphia, PA,
1995.
2.Waynforth, H.B., Experimental and Surgical Technique in the Rat. 2nd.
Edition. Academic Press, San Diego, CA, 1994.
3.Crow, S.E. and Walshaw, S.O., Manual of Clinical Procedures in the
Dog, Cat and Rabbit, 2nd. Edition, Lippincott-Raven, Philadelphia,
PA, 1997.