Research Methodology

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Research Methodology

• LAT Chapter 13

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Toxicology

• 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.

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Dosing a Rat By Gavage
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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

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Blood Sampling - Saphenous Vein
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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 gt1 rabbits indicates the
  presence of a pyrogen.
• Limulus amebocyte lysate (LAL) test - horseshoe
  crab blood will clump together in the presence of
  a pyrogen.

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Toxicology Tests
The developing fetus
The Horseshoe Crab
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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.

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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

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Induced Immunodeficiency

• Surgery thymus gland can be removed surgically
• Chemical agents used in research suppress
  immunity
• drugs toxic, mortality rates gt20 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.

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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
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Tolerance

• 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.

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Tolerance - Neonatal
Day 2
Day 1
Day 3
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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.

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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 recipients 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.

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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, gt boosters.
• Antibody can be stored in a freezer for years.
• Exceptional response gt animal kept for a long
  period.
• It receives booster immunizations, and periodic
  blood collections.

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Polyclonal Antibodies
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Adjuvants

• 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 gt signs of
  illness.
• Unacceptable to place adjuvants IP, in foot pads,
  or gt 0.5 ml
• Complete Freunds Adjuvant (CFA), Incomplete
  Freunds 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.

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Hybridomas

• 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 gt hybridoma.

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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.

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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).
Cellmax
http//www.spectrapor.com/cell/cellmax.html
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Induced Cancer Models

• Animals exposed to cigarette by-products and
  smoke carefully compared with control group gt
  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.

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Spontaneous Cancer Models

• Some strains of inbred rodents have high
  incidence of naturally occurring cancers.
• gt 80 of AKR mice develop leukemia lt 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

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Cannulation Implants

• 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.

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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.

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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.

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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 gt 1X daily to see it is properly placed and
  patent.

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Catheter Insertion
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Catheter Insertion
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Catheter Insertion
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Catheter Insertion
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Catheter Insertion
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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.

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Catheter Maintenance/ Implants
Vascular Access Ports
Vascular Access Portin use
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Implants

• 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.

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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.

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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.

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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.

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Feeding

• 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.

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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 animals 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.

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Metabolism Cages
Mouse Metabolism Cage
Water
Food
Collection
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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.