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Research Methodology. LAT Chapter 13. LAT Presentations Study Tips. If viewing this in PowerPoint, use the icon to run the show. Mac users go to “Slide Show > View Show” in menu bar Click on the Audio icon: when it appears on the left of the slide to hear the narration.

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research methodology

Research Methodology

LAT Chapter 13

lat presentations study tips
LAT Presentations Study Tips
  • If viewing this in PowerPoint, use the icon to run the show.
    • Mac users go to “Slide Show > View Show” in menu bar
  • Click on the Audio icon: when it appears on the left of the slide to hear the narration.
  • From “File > Print” in the menu bar, choose “notes pages”, “slides 3 per page” or “outline view” for taking notes as you listen and watch the presentation.
    • Start your own notebook with a 3 ring binder, for later study!
  • 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.
subchronic chronic testing
Subchronic & Chronic Testing
  • 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
repro teratology pyrogens
Repro., Teratology, Pyrogens
  • 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
Toxicology Tests

The developing fetus

The Horseshoe Crab

immunodeficiency models
Immunodeficiency Models
  • 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
Spontaneous Immunodeficiency
  • 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
Induced Immunodeficiency
  • 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

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

  • 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
Tolerance - Neonatal

Day 2

Day 1

Day 3

immunocompromised care
Immunocompromised Care
  • Structural and procedural barriers to transmission of infectious agents = positive pressure rooms or Micro-Isolator™ 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
Antibody Production
  • 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
Polyclonal Antibodies
  • 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.
  • 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.
  • 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
Hybridoma Culture
  • 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
Hybridoma Cell Culture

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).


induced cancer models
Induced Cancer Models
  • 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 cancer-causing 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
Spontaneous Cancer Models
  • 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
Cannulation & Implants


  • 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
Cannulating Teats
  • 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
Types of Catheters
  • 3 types of IV catheters: through-the-needle, over-the-needle, 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
Catheter Insertion
  • Aseptic technique -
    • 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 maintenance
Catheter Maintenance
  • “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 cut-downs 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
Catheter Maintenance/ Implants

Vascular Access Ports

Vascular Access Portin use

  • 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.
    • 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
Behavioral Motivation
  • 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
Behavioral Motivation Terms
  • 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
Special Dietary Studies
  • 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.
  • 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
Metabolism Cages
  • 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 cages1
Metabolism Cages

Mouse Metabolism Cage




additional reading
Additional Reading

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.