ALAT: EXPERIMENTAL DESIGN AND METHODOLOGY LAT:...

ALAT: EXPERIMENTAL DESIGN AND METHODOLOGYAND METHODOLOGY

LAT: RESEARCH METHODOLOGY

Anusak Kijtawornrat, DVM, PhD

February 25, 2009

EXPERIMENTAL DESIGNEXPERIMENTAL DESIGN

Influences the outcome of a research studyy

First step, identifying and defining the questionObjective: realistic, achievable

Next step, choose the research model that is best suited for the experiment

In vitro model : procedure outside the living organism

In vivo model : an experiment on a living subject

IN VITRO VS IN VIVOIN VITRO VS IN VIVO

In vitroIn vivo

THE RESEARCH PROJECTTHE RESEARCH PROJECT

Experimental plan:p p1. A literature search

2 The model to be used2. The model to be used

3. The hypothesis

4 A concise step by step schedule of 4. A concise step-by-step schedule of experimental manipulation

5 A description of the methods for data 5. A description of the methods for data collection and assessment

THE RESEARCH PROJECTTHE RESEARCH PROJECT

Experimental plan (cont.):p p ( )6. adverse reactions or potential personnel hazards expected from the manipulationp p

7. housing methods and nursing care for animals.

8. A budget

9. the qualifications of the investigatorsq gIf animal are to be used, protocol -->IACUC to review

CONTROL AND EXPERIMENTAL GROUPSEXPERIMENTAL GROUPS≥ 1 control groupg p

Not subjected to experimental treatment

≥ 1 test group≥ 1 test group

ElectrocardiogramssalineGroup 1

Control gr.Control gr.

Saline+Drug A

Group 2

Test gr.Test gr.

CONTROL AND EXPERIMENTAL GROUPSEXPERIMENTAL GROUPS

Any factor or condition that can change = y gvariable

Research manipulations,

Animal strain

Age and gender

E i l di iEnvironmental conditions

Avoid introducing unplanned variablesSOP d t l h l t id th l d SOP and protocol help to avoid the unplanned variables

Utilize highly homogenous populationsUtilize highly homogenous populationsBreed, strain, sex, age, body weight, etc.

CONTROL AND EXPERIMENTAL GROUPSEXPERIMENTAL GROUPS

Unintentionally influencing experimental variables1. Mixing or exchanging strains of animals

2. Improper animal identification

3. Errors in weighing or medicating animals

4. incorrect recording of data

5 Variations in room temperature lighting diet 5. Variations in room temperature, lighting, diet, bedding or cage type

6. increasing the noise level by careless handling of cages and equipment

7. Changing or supplementing a standard diet

8 Showing favoritism toward individual animals8. Showing favoritism toward individual animals

ANIMAL MODELSANIMAL MODELS

Animal models: studying diseases, normal functionBenefit to animals and humans

Category of animal models1. Natural models: disease or entity occurs spontaneously

A h l i i h S i l kAtherosclerosis in the Squirrel monkey

Epilepsy in Mongolian gerbils

Diabetes in mice

2. Induced animal models: disease or condition must be artificially produced

Injection of tumor cells in to animals

IMMUNODEFICIENCY MODELS

Immunodeficient animals = animals that have some defect in their normal immune system

Used in immunology and cancer research

Make good models of spontaneous or infectious diseases, i.e., AIDS in humans

Types of immunodeficient animals1 S t I d fi i1. Spontaneous Immunodeficiency

2. Induced Immunodeficiency


Spontaneous Immunodeficiencyp yNude mice: hairless and no thymus

Lack T-cellLack T cell

Susceptible to infections

Maintained as pathogen free animals in Maintained as pathogen-free animals in barrier facilities

Athymic Rats and hamstersAthymic Rats and hamsters


Induced immunodeficiencyySurgery: the thymus gland can be removed surgically from newborn mice, rats, and cats

Chemical: agents can be used in research to gsuppress immunity, i.e., 6-mercaptopurine, etc

Act by interfere with DNA or RNA synthesis

Suppress antibody synthesis and immune function

Toxic, mortality rate up to 20% can be anticipated


Irradiation: faster and more easily measured

Common sources of radiation: gamma irradiators that utilize cobalt or cesium isotopes

T f di ti Types of radiation exposure

1. Single exposure: total body radiation easiest method

Small animals are confined in plastic tubes or aluminum boxes for exposure

dogs and larger animals anesthetizeddogs and larger animals anesthetized

2. Low level protracted semicontinuous radiation

3. Partial body radiation: using lead shields

RODENT IRRADIATIONRODENT IRRADIATION

Holding chamber

CARING FOR IMMUNOCOMPROMISED ANIMALS

Experimental subjects require extra attentionp j qPoor appetite and difficulty eating and drinking

Acidified (pH 2.4-2.8) or chlorinated water should be used to suppress bacterial growth

Food, bedding, and cages should be sterilized before use

Recovery period from wounds or ailments likely to be much longer than normal animallikely to be much longer than normal animal

CANCER MODELSCANCER MODELS

Induced cancer modelsVariable method, depend on type of cancer

Injection of tumor cells

Spontaneous cancer modelsUseful for evaluating Dx and preventive Tx in a l l i k b ibl large population known to be susceptible to cancer

80% AKR mice: leukemia before 1 yr

Observed closely euthanized when Observed closely, euthanized when they become clinically ill or tumor become large or ulceratedg

TOXICOLOGYTOXICOLOGY

Toxicology: the science of poisons and the harmful or noxious effects of these substances have on living organisms

Toxicologist: an individual who is responsible for Toxicologist: an individual who is responsible for predicting the toxic or harmful nature of a substance

Principal toxicological experimentsAcute toxicity test (Short-term)

Subchronic toxicity testing

Chronic testingChronic testing

Reproductive testing

Pyrogen testing

ACUTE TOXICITY TESTACUTE TOXICITY TEST

Acute Toxicity Tests: single dose of test substanceLD50 (Lethal dose 50) = the dose of a substance that kills 50 percents of the animal tested

The Draize Test: assess the toxicity of products placed in contact y p pwith animal tissue to evaluate local tissue irritation

ACUTE TOXICITY TESTACUTE TOXICITY TEST

Criticized, if used to evaluate non-pharmaceuticalsCriticized, if used to evaluate non pharmaceuticals

SUBCHRONIC TOXICITYSUBCHRONIC TOXICITY

Subchronic toxicity testing: 13-26 wks in durationy gDaily dose by the same route substance administered normally

Observed for toxicity, changes in weight or food consumption

Evaluate clinical chemistry and hematology valuesEvaluate 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 TOXICITYCHRONIC TOXICITY

Chronic Testing: longer term toxic, carcinogenicg g gMice and rats, 4 to 5 groups of 60 to 100 per sex per group

As subchronic, but observation period is longer (~t )( two years)

Animals are palpated to detect tumor formation

Postmortem for histopathologic toxicity and Postmortem for histopathologic toxicity and carcinogenicity

TOXICOLOGYTOXICOLOGY

Reproductive - usually conducted on rats and p d y d d d

rabbits

to detect changes in reproductive cycle and toxic to detect changes in reproductive cycle and toxic

effects on fertility, organ development, and behavior

Teratology = exposure of developing litters to Teratology = exposure of developing litters to

chemicals

T t b t th t d th d l i Teratogens = substances that damage the developing

fetus.

Changes in normal fetal anatomy litter size or fetus Changes in normal fetal anatomy, litter size, or fetus

weight may indicate that the test substance is a teratogen

PYROGEN TESTINGPYROGEN TESTING

Pyrogen: a substance that produces a fever in an y g panimal

To detect bacterial toxins in medical product administered by injection

A rise in body temp in one or more rabbits indicates the presence of a pyrogen in the sample

LIMULUS AMEBOCYTE LYSATE TEST (LAL)( )

RESEARCH METHODOLOGYRESEARCH METHODOLOGY

Implants and CannulationspImplant: a device placed into living tissue

Permanent:

Temporary:

Cannulation: insertion of a small tube into a body i d lcavity, duct, or vessel

May be inserted through a natural opening

May be inserted into artery or veinMay be inserted into artery or vein

Inherent danger: introducing undesirable bacteria

Possible problemspExcessive discharge around the implant, kinked tubing, dislocation of the cannula

URETHRA CATHETERIZATION

Simple in male but difficult in femalep

Vaginal speculum should be used in female

Male cannulaFlexible tube with a tapered end

Stainless steel tube or blunt needle

Sterile lubricating jelly should be used

Aseptic technique should be used

CystocentesisPassing a hypodermic needle through the surgically

d bd d i t th bl ddprepared abdomen and into the bladder

CANNULATIONS OF THE TEATS

Technicians handling cows and goats may be required to g g y q

cannulate the teats of these animals.

Reusable metal and disposable plasticReusable metal and disposable plastic

Only use sterile cannulae

C f ll l d di i f t d f t t b f Carefully clean and disinfect ends of teats before

insertion of cannula, clean and disinfect again after

removalremoval

CATHETERSCATHETERS

3 Types of IV cathetersypThrough the needle

Catheter can be any length, difficult to y g ,insert, hubs prevent needle to remove

Over the needleShort, easy to insert, widely used

Butterfly Two plastic flaps attach to the hub to secure to the skin

Needle remain in the vessel !!Needle remain in the vessel !!

OVER THE NEEDLE CATHETEROVER THE NEEDLE CATHETER

CATHETER INSERTIONCATHETER INSERTION

Requires Aseptic techniqueq p qRestrained animal adequately

Clipped and prepped insertion site

Assemble equipment, remove from wrapping

Occluded the vein

P h lPuncture the vessel

Once blood appears, pass needle into the vessels

Stop occlusion advance catheter and remove needleStop occlusion, advance catheter, and remove needle

Cap the catheter and wrap around

IV catheter may be secured with tape and left in place for y p pup to 72 hours, longer is possible

Remove catheter if sign of infection is present

CATHETER INSERTIONCATHETER INSERTION

IMPLANTSIMPLANTS

Implantable injection ports and osmotic pumps p j p p pprovide other ways of administering substances

Injection ports connected to vessels or body chambers

IMPLANTIMPLANT

Osmotic pumps capable of continuous delivery p p p yup to 4 weeks

The pumps are cylindrical with rounded ends

Capacity of pumps varies from 100 ul to 2 ml

Influx of body fluids forces contents of pump to be slowly injectedslowly injected

BEHAVIORAL MOTIVATION EXPERIMENTS

Behavior: the response of an individual, group or p g pspecies to its environment

Psychology: the science of mind and behavior that pertains exclusively to human beings

Ethology: the scientific study of animal behavior

Physiological psychology: the structures and functions of the body responsible for behavior

BEHAVIORAL MOTIVATION EXPERIMENTS

Rat most frequently used.

Pigeons and cat are also used.

Few primates are used & frequency declining.

OWM: Rhesus monkeys, Macaca mulatta

NWM: Squirrel monkey, Saimiri sciureus

LEARNINGLEARNING

Learning: the modification of a behavioral gtendency by experience

Basic requirements for a learning experimentMotivation: an animal needs or desires something; hunger, thirst

Perception: an awareness of the environment being manipulated

Response: anything that an animal does following the perceptionResponse: anything that an animal does following the perception

Reward: something an animal gets as a result of its behavior; food, water

P i i i f i l h Positive reinforcement: animal repeats the behavior to acquire the reward

N ti i f t l t id i Negative reinforcement: learn to avoid aversive treatment

SPECIAL DIETARY STUDIESSPECIAL DIETARY STUDIES

Choice of species: the purpose of the p p pexperiment, the resources available

Rat: the most popular subject animal for dietary studies

Long growth period after weaning and

Rapid weight gain during that period

Rats do not require vitamin C in their diet

Guinea Pig and some NHP require vitamin C in their diet

FEEDINGFEEDING

Lab animal technicians should be aware of the types of feeding procedures used in dietary studies

Ad libitum feeding

Ad libitum feeding and watering may not be i f f di satisfactory for some studies. If test diet has a bad flavor, test animals may eat less of it than the control animalsof it than the control animals

Pair-feeding is a method of assuring that the control group and the experimental group receive the same amount of food

METABOLISM CAGESMETABOLISM CAGES

Metabolic cage: an apparatus used in dietary g pp ystudy to measure the amount of food and water intake and feces and urine output

LATG: EXPERIMENTAL DESIGN & DATA HANDLING

Anusak Kijtawornrat, DVM, PhD

February 25, 2009

EXPERIMENTAL DESIGN & DATA HANDLING

Scientific research begins with a QUESTION.g

The research question is usually expressed as a HYPOTHESIS.

Sources of research questionObservation

Literature review

Result from previous study

RESEARCH QUESTIONR R Q

Anecdotal reports: “Garlic has been lowering p gblood pressure in people taking garlic powder as a supplement”

Question: “Will a diet containing garlic powder lowers BP in SHR rats?”

THE HYPOTHESIS

Hypothesis: a prediction of the results of a study,yp p y

It is a statement of the answer the researcher expects to find to the research question.

“During the study, the SHR that received garlic g y gpowder in their diet will have lower BP than SHR that does not receiving garlic powder”

RESEARCH TERMS AND CONCEPTSRESEARCH TERMS AND CONCEPTS

Variables: anything that can potentially affect the y g p yresults of the experiment

Independent variable: the variable that is manipulated by the investigator

What you think will cause the change that you d ib i h h i > li ddescribe in your hypothesis--> garlic powder

Dependent variable: the variable that is measured during the studyduring the study

What you measure in order to determine if a change has taken place --> Blood pressurep p

SAMPLE SIZE & SELECTIONSAMPLE SIZE & SELECTION

Sample size: how many animals are to be p yincluded in the study

3Rs principle & others considerations: the PI must determine the minimum number of animals needed to obtain results that one can h fidhave confidence

Randomization: a statistical method that helps t l i bl b li i ti l bi control variables by eliminating personal bias

from the study

ACCURACY OF RESULTSACCURACY OF RESULTS

Confidence level: an index of certainty, any difference found between experimental and control data is due to experimental treatment not variation

Expressed as a decimal i.e., 0.95

p- value: the probability that the difference observed in the data happened by chance and is observed in the data happened by chance and is due to normal distribution rather than to the experimental tx i.e., 0.05p ,

NORMAL DISTRIBUTIONNORMAL DISTRIBUTION

Normal distribution: statistical distributions that symmetric and have bell-shaped density curves with single peak

STANDARD DEVIATIONSTANDARD DEVIATION

Standard deviation: a measure of the dispersion pof the data measurements.

Mean ± 1 SD, a 68% of the measurement fall between the means plus or minus 1 SD.

CALCULATION OF MEAN AND SDCALCULATION OF MEAN AND SD

Control group BP (mmHg): g p gMean±SD = 179±3.8

Experimental group BP (mmHg): Mean±SD = 150±4.3

Are these two means really different from each other h fid l l f 95 ?at the confidence level of 95%?

There is significant difference = what is that mean?

There is a 95% or greater chance that the BP There is a 95% or greater chance that the BP difference observed is due to the garlic powder.

There is a 5% or less chance (p≤0.05) that the “We found that there was a significant different difference between the BP is due to normal BP variation within the SHR population.between the BP of the SHR that received the garlic powder and the BP of the control SHR (p≤0.05)”

ANY QUESTIONS ?ANY QUESTIONS ?

PRACTICE QUESTIONSPRACTICE QUESTIONS

1. What does “ in vivo” mean?

E i ti thi li ia. Experimenting on something non-living

b. Experimenting on cells in culture

c. Experimenting on the whole animal

d. Experimenting on something livingp g g g


2. What group must approve every g p pp yexperiment using animals?

a. USDA

b Cb. AAALAC

c. GLP

d. IACUC


3. How is a control group used in an experiment?g p p

a. It is used to measure the difference between having or not having the experimental variable

b. It is used to monitor the health status of the experimental group

c. It is maintained under the same conditions as the experimental to replace any animals that die

d. It is the experimental group that has variables controlled


4. What is “variable” ?

Th f t diti b i t di da. The factor or condition being studied

b. Any factor or condition that can change

c. The factor or conditions out of one’s control

d. Any difference between the control and experimental groupsp g p


5. What is the device that is placed under pthe skin to deliver a continuous flow of medication?

V l ta. Vascular port

b. Electrode

c. Osmotic pump

d. Pacemaker


6. What is a teratogen?g

A b t th t d d l i a. A substance that damages a developing fetus

b d i bb. A mutated gene in an embryo

c. A substance that causes cancer

d. An animal that developed abnormally in utero


7. What is a pyrogen?py g

A fl bl b ta. A flammable substance

b. A substance that produces a fever in an i lanimal

c. A substance that causes blistering when applied to the skin

d. A substance that causes cancer

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