1. Lab. No.1 : Introduction to experimental pharmacology , handling of laboratory animals & drug administration

2. Introduction:
Animals are widely used experimentally in almost every laboratory where a biological study is needed
In pharmacological research, animals are being used to test the effect of drugs before their introduction to clinical trials where drugs are tested on humans
In addition, animals are used in educational institutions to demonstrate the effect of drugs for teaching purposes.

3. Experimental animals should always be:
A. Suitably bred.
B. Gently handled
C. Properly used

4. Ethical and legal responsibility
Careful handling of animals in such a manner that they do not suffer from unnecessary pain.
Providing a good care towards the health and well being of animals.
It is important that animals are aware of the handler’s presence before attempting to restrain them, particularly if the animal is initially asleep. This will reduce stress for the animal and help to avoid bite injuries.
Experimental animals should always be:
A. Suitably bred.
B. Gently handled
C. Properly used

5. ANIMAL HOUSE
Diurnal light cycles in animal holding rooms (12:12-h light:dark cycle)
Controlled temperature (22º C)
Controlled humidity (range 30%-70%)
Free access to water ‘ad libitum’ and chow diet
Adaptation period
Standard operating procedures….

6. CAGES

7. Selection of an experimental animal for a particular experiment depends on:
Nature of the experiment.
Type of drug activity to be evaluated.
Specific character of the experimental animals

8. Animals can be used in:
“In Vivo” experiments when the whole intact animal is used, or in
“Ex Vivo” experiments when only a part of the animal is used. This part could be an organ (e.g. heart, uterus, trachea.etc.), a tissue (e.g. skeletal muscle) or even some cells (e.g. blood cells).

9. Types of animals: Large (monkey, pigs, dogs and cats) OR
Small (mice, rats, rabbits and frogs)
For teaching purposes the small animals are commonly used.
Both large (monkey, pigs, dogs and cats) as well as small (mice, rats, guinea pigs, rabbits and frogs) are used as experimental animals. However, for teaching purposes the small animals are commonly used.

10. Selection of species depends on many factors:
Technical issues, e.g. availability of the species in the numbers needed, health and genetic status of the species, animal size and volume of blood required
Animal husbandry issues, e.g. availability of suitable housing, ease of handling of the animals, training and level of competence of personnel
Public concern about use of certain species
Financial considerations
Animal husbandry is the management and care of farm animals by humans for profit

11. Types of experimental animals:
1.Mouse
2. Rat
3. Guinea pig
4. Rabbit
Rodents are mammals of the order Rodentia, characterized by a single pair of continuously growing incisors in each of the upper and lower jaws. The name is derived from the Latin rodere, to gnaw.
Well known rodents include mice, rats and guinea pig
The rabbit is from another order…..lagomorpha

12. MOUSE

13. Mice The most common mammal among laboratory animals. Characteristics:
1. Mice are small laboratory animals weighing about g.
2. They are very sensitive and consume small doses of drugs.
3. They can be easily handled.
4. Highly exploring and mobile
5. Drugs are best injected intraperitoneally or intravenously into one of their superficial tail veins.

14. Mice Uses in research: 1. In cancer and genetic research.
2. In acute toxicity studies.
3. In screening of various drug activities specially central nervous system (CNS) activity.

15. Mice sexing (how to discriminate between male and female mouse)
The distance between the anal and genital orifices is greater in the male (left) compared to the female (right).

16. HANDLING & RESTRAINT OF MICE
Instructions:
Gently but firmly.
Wear disposable gloves
Wash your hands prior to and after handling
Wear a clean laboratory coat

17. HANDLING & RESTRAINT OF MICE
The animal should be grasped by the tail, and should then be placed on a rough surface such as a cage top while holding the tail firmly
The mouse is lifted from its tail (caudal end) with the right hand.
2. The animal is allowed to grip the wire mesh of the cage with its fore legs.
3. The animal is grasped from the nape of the neck between thumb and index finger.
4. The tail is then transferred from the right to the left hand in between the small and ring fingers.

18. To carry the mouse for injection , you can use one-handed or two-handed methods:
One-handed method :
Place the mouse’s tail between the last two fingers of the hand that is holding the nape.

19. Two-handed method:
The scruff can be grasped between the thumb and forefinger whilst maintaining a grip on the tail.

20. Injecting the mouse 1. Intraperitoneal injections:
can be made into the lower quadrant of the abdomen.

21. 2. Subcutaneous injection
Subcutaneous injection can be made into the scruff of the neck. Care must be taken to direct the needle into the scruff and not into the handlers finger or thumb.

22. 3. Intramuscular injections
can be made into the quadriceps muscle groups on the anterior of the thigh

23. GENETICALLY MODIFIED MICE
Knockout mouse (KO):
Is a genetically engineered mouse in which researchers have inactivated, or "knocked out", an existing gene
This causes changes in a mouse's phenotype (appearance, behavior, physical and biochemical characteristics)
Important animal models to study the role of genes that have been sequenced but whose functions have not been determined
….Causing a specific gene inactivation, and observing any differences from normal behaviour or physiology, we can infer its probable function
May also be a useful experimental pathological model to test the efficacy of new drugs
Is a genetically engineered mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA.

24. GENETICALLY MODIFIED MICE
Knockin mouse
Is a genetically engineered mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA.

25. NUDE MOUSE
Mouse from a strain with a genetic mutation that causes a deteriorated or absent thymus, resulting in an inhibited immune system due to a greatly reduced number of T cells.
The phenotype (main outward appearance) of the mouse is a lack of body hair…..nude" nickname.
Valuable in research! it can receive different types of tissue and tumor grafts (xenografts ), with no rejection response.
The genetic basis of the nude mouse mutation is a disruption of the FOXN1 gene

26. RAT
The best-known rat species are the black rat (Rattus rattus) and the brown rat (Rattus norvegicus).
Over time, breeding the rats for these contests may have produced variations in color, notably the albino and hooded varieties.
Scientists have bred many strains or "lines" of rats specifically for experimentation. Most are derived from the albino Wistar rat, which is still widely used. Other common strains are the Sprague Dawley, Fischer 344,[6] Holtzman albino strains, the Long-Evans, and Lister black hooded rats

27. RAT
Characteristics:
1. Rats have a larger weight than mice (about g).
2. They can withstand long periods of experimentation under anaesthesia.
3. They can be easily handled if treated kindly.
4. Drugs can be injected intravenously (I.V) into their tail veins, subcutaneously (S.C), intramuscularly (I.M) or intraperitonealy.
5. They can be also given drugs orally by means of stomach tube.
6. Isolated preparations can be taken from them easily. (stomach, fundus, colon and uterus are the most commonly used preparations)

28. Proper method of handling:
To initially restrain a rat, the handler should gently grasp it around the shoulders.
Depending on the manner of treatment they
receive; rats can be handled easily or with
difficulty. The following is the best way to
handle them:
1. The rat is grasped from its tail.
2. It is placed on a hard surface

29. Proper method of handling:
The handler’s thumb can then be placed under the rat’s mandible, to prevent bites, and the rat’s hind limb scan be supported with the other hand. Restraint should be firm but not too tight as this will impede the animal’s respiration.
Note: Bites from rats are uncommon and will typically only occur if the animal is stressed or in pain.

30. Intravenous administration in rat

31. Intraperitonial injection
Intraperitoneal injections may be made into caudal half of the abdomen with the needle directed along the line of the hind limb.

32. Gastric gavage

33. Use in research:
1. In acute and chronic toxicity studies.
2. In preliminary testing of some drug activities specially CNS activity. For example, rats are used for testing psychotropic activity of drugs since they can be trained to perform skilful tasks.

34. The guinea pig

35. The guinea pig Characteristics: 1. very docile and tame animals.
2. very susceptible to infections and highly sensitive to action of many drugs, e.g. histamine and penicillin.
3. cannot synthesize endogenous vitamin C and this vitamin should be supplied in their diet.
4. Some isolated muscle preparations can be taken from guinea pigs, eg. Guinea pig Vas Deferens, tracheal chain or ileum.
5. Drugs can be injected intravenously (I.V) into their tail veins, subcutaneously (S.C), intramuscularly (I.M) or intraperitonealy.
6. They can be also given drugs orally by means of stomach tube.

36. The guinea pig Use in research:
1. Screening for histaminic, antihistaminic drugs, respiratory or cardiovascular activity.
2. Testing the effect of drugs on their isolated muscle preparations.
The guinea pig was most extensively implemented in research and diagnosis of infectious diseases (because of its similarity to humans with regard to symptoms and immune response.)

37. Handling and Restraint of Guinea Pig
To initially restrain a guinea pig, the handler should be rapid and smooth, to avoid frightening the animal.
The handler’s thumb is placed beneath the jaw of the guinea pig. The hindquarters of the guinea pig are supported by the handler’s other hand

38. The rabbit

39. The rabbit Characteristics: 1. Rabbits are very docile animals.
2. They can be easily injected intravenously into their marginal ear veins.
3. They can be also given drugs orally by means of stomach tube.
4. Some isolated muscle preparations can be taken from rabbits.

40. Intravenous injection in rabbit
Rabbits may also be restrained for injection by wrapping the animal in a drape or towel. If the rabbit is securely wrapped, it will not struggle.
Intravenous injections into the rabbit’s marginal ear vein can be readily made if the rabbit is restrained with a drape.

41. Handling and restraint of the rabbit:
Rabbits are especially susceptible to the effects of stress and should always be approached in a calm and confident manner.
The handler is restraining the rabbit firmly by the scruff with the other hand ready to support the animal’s hindquarters.
Uses in research:
1. In pyrogen testing of drugs.
2. To study the effect of drugs on the eye, cardiovascular (CVS) and autonomic (ANS) and neuromuscular junction (NMJ).
3. Some isolated muscle preparations can be taken from rabbits, e.g. intestine.

42. BLOOD SAMPLING IN MICE/RATS:
from tail vein
from orbital sinus
from cardiac puncture
from saphenous vein

43. 1. Blood collection from Tail vein:
It may be used to collect a small volume of blood (Approximately 0.1 ml )
Carefully warm animal with heat lamp or disposable hand warmers.
Place animal in an appropriate restraint device.
Clean withdrawal site with alcohol.
Using a sterile scalpel blade, nick the lateral tail vein as shown in this picture.
Collect blood into an appropriate container.
Apply gentle pressure to stop bleeding.

45. 2-Blood Collection From Orbital Sinus
The Orbital Sinus method may be used to collect larger volumes of blood(up to 0.5 ml)
Appropriate anesthesia must be used for this procedure. Animals may receive topical anesthetic or general anesthesia.
Blood Collection: The peri-orbital sinus of the mouse may be used as a source of venous blood. The mouse has a large peri-orbital venous sinus that fills the bony orbit of the eye. Blood can be collected from either medial or lateral canthus approach.

46. 3-Blood Collection From Cardiac Puncture
To collect up to 1 ml of blood within a short period of time
Must be performed under general anesthesia .

47. 4-Blood Collection From Saphenous Vein
The Saphenous Vein is located along the lower portion of the hind leg. This technique is used for collecting small volumes of blood.
It can also be applied on rats and guinea-pigs

48. The frog

49. The frog Characteristics:
1. Frogs are amphibian (cold-blooded) animals and must be kept moistened with water to absorb moisture throughout their skin.
2. They are usually administered drugs subcutaneously (S.C) in their dorsal lymph sac
Use in research:
1. To preliminarily test drug effect on NMJ, CNS or CVS.
2. In pregnancy tests (more advanced methods are now available).
3. Frog isolated muscle preparations (e.g. Rectus abdominus, heart) are also used.
Proper method of handling:
1. The frog is taken by left hand and is held by grasping its trunk.
2. The head of the frog is held between the thumb and index fingers.
3. The hind limbs are placed between the forth and fifth fingers.

50. Dose calculation

51. Dose calculation
It is important that each individual involved in prescribing, dispensing and administering drug understands how to calculate drug dose.
Many errors, some fatal, could be avoided if all physician, pharmacists and nurses had the ability to calculate dose quickly and accurately.
Dr. Rana Abu Farha

52. Dose calculation
When one deals with drug solutions rather than solid preparations, the concentration defines how much drug there is in a volume of solution.
The most common way expressing concentration is mg/ml.
Dr. Rana Abu Farha

53. Dose calculation
The administered dose of a drug is defined as the amount of drug to be given and is generally expressed in terms of some physiological parameter such as body weight, surface area or volume.
Injection volume (ml) = dose (mg/kg) * body wt (kg)
Conc. of solution (mg/ml)
Dr. Rana Abu Farha

54. Dose calculation
Many errors are made when non-comparable units such as dose in mg/kg and a weight in grams are used. It is necessary to be certain that all the terms are expressed in comparable units.
Dr. Rana Abu Farha

55. Dose calculation
In the lab. it is advisable to avoid very small or large injection volumes. This can be accomplished by using a constant dosage volume ( a volume of drug solution per a given physiological parameter such as weight).
For the laboratory mice or rat a common constant dosage volume is 5 ml/kg for intraperitonial injections.
Dr. Rana Abu Farha

56. Example
You have a drug solution at a concentration of 5%, what is the injection volume for a rat (weight 100 gm) if the dose is 50 mg/kg?
1st convert gm to kg and % to mg/ml.
**100 gm = 0.1 kg
**5 % = 5 gm/ 100 ml = 5000 mg / 100ml
= 50 mg/ml
Dr. Rana Abu Farha

57. Example (cont) Injection volume = 50 mg/kg * 0.1 kg = 0.1 ml 50 mg/ml
In the above example, what is the constant dosage volume?
Constant dosage volume (ml/kg) = dose (mg/kg)
conc. mg/ml
= 50 mg/kg = 1 ml/kg.
Dr. Rana Abu Farha