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By Kalani M PhD candidate in Immunology

What kind of animals? – Rattus Norvegicus – Rabbit (Oryctolagus cuniculus) – Hamster (syrian) – Guinea pig – Mus domesticus domesticus (musculus) 3

1.Abundance of literature published 2. Ease of handling 3.High fertility rate 4.Short gestation period 5.Low maintenance 6.Disease model for various human disorders NOD, Nude, EAE, CIA, NZB/NZW Why animals? 4

Mice Restraining 5

Mechanical Restrainers 6

Injections  Intramuscular (IM) Intramuscular (IM)  Subcutaneous (SC) Subcutaneous (SC)  Intraperitoneal (IP) Intraperitoneal (IP)  Intravenous (IV) Intravenous (IV) 7

Blood Collection  Tail Tail  Orbital sinus Orbital sinus  Saphenous vein Saphenous vein  Heart Heart 8

Intramuscular Injections 9

Intramuscular Injections cont. 10

Subcutaneous Injections 11

Pick up a nude mouse and spin it’s tail to put it in a faint condition Subcutaneous Injections cont. 12

Grasp the loose skin on the back of the mouse from ears along the legs and restrain the legs with your ring finger and little finger Subcutaneous Injections cont. 13

After disinfect the surface area, insert the needle in the lateral side of the abdominal wall and push upwards to the armpit of the mouse Subcutaneous Injections cont. 14

Inject the substance slowly Subcutaneous Injections cont. 15

A lump of injection substance can be seen through the skin after injection Subcutaneous Injections cont. 16

Intraperitoneal Injections 17

Place a mouse on a cage lid and grasping the loose skin behind the ears with your thumb and forefinger Intraperitoneal Injections cont. 18

As soon as the mouse’s head is restrained, the mouse can be picked up and the tail secured within your ring finger and little finger Intraperitoneal Injections cont. 19

The injection site should be in the lower left quadrant of the abdomen because vital organs are absent from this area. Only the tip of the needle should penetrate the abdominal wall to prevent injection into the intestine. Intraperitoneal Injections cont. 20

Intravenous Injections Warm the mouse under a heat lamp or other heating device being sure not to OVERHEAT the animal. 21

Placing a mouse on a cage lid and grasping the loose skin behind the ears by the thumb and forefinger 22 Blood collection from Tail

Push the mouse into the restrainer 23 Blood collection from Tail cont.

Leave the tail of the mouse outside the cover of the restrainer 24 Blood collection from Tail cont.

Amputate the tip of the mouse tail by scissors 25 Blood collection from Tail cont.

Massage the tail and collect blood by sampler 26 Blood collection from Tail cont.

Lateral Tail Veins 27 Blood collection from Tail cont.

Blood Collection From Orbital Sinus in Mouse Should apply anesthetic before blood withdraw A convenience and easy apply method for blood collection in mouse Collect amount up to 0.5 ml 28

29 Blood Collection From Orbital Sinus cont.

Anesthetize a mouse by intraperitoneal injection of Hypnorm 30 Blood Collection From Orbital Sinus cont.

Use a sharp end glass capillary tube to penetrate the orbital conjunctiva and rupture the orbital sinus 31 Blood Collection From Orbital Sinus cont.

Collect blood with a vial 32 Blood Collection From Orbital Sinus cont.

Tools for blood collection from Saphenous vein in mice 75% alcohol cotton ball for surface disinfection 50 ml syringe tube with small holes at the end as restrainer a scalpel and shaver for remove of hair 24 G “ needle for release of blood tips and pipetteman for blood collection 33

Placing a mouse on a cage lid and grasping the loose skin behind the ears with your thumb and forefinger 34 Blood collection from Saphenous vein cont.

Place the mouse in the restainer 35 Blood collection from Saphenous vein cont.

Pull out the leg and removed the hair by a assistant 36 Blood collection from Saphenous vein cont.

Hair can also be shaved by using a small scalpel 37 Blood collection from Saphenous vein cont.

The saphenous vein is seen on the surface of the thigh 38 Blood collection from Saphenous vein cont.

Apply vaseline after disinfect the surface area to reduce clotting and coagulation during blood collection. 39 Blood collection from Saphenous vein cont.

Use a 24 G 1” needle to puncture the vein and release blood from the saphenous vein 40 Blood collection from Saphenous vein cont.

Use a Microvette or a pipetteman with tip to collect blood from the saphenous vein 41 Blood collection from Saphenous vein cont.

Approximate 100 microliters can be collected 42 Blood collection from Saphenous vein cont.

Flex the foot of the mouse to reduce the flow of blood back to the puncture site 43 Blood collection from Saphenous vein cont.

A cotton ball is applied to the puncture site to stop further bleeding 44 Blood collection from Saphenous vein cont.

Blood Collection From Cardiac Puncture in Mouse For collect up to 1 ml of blood within a short period of time Must be performed under general anesthetic 45

Tools for Cardiac puncture in Mouse 75% alcohol cotton ball for surface disinfection Hypnorm used as anesthetic 27G needle with 1 ml syringe for injection 24G needle with 3 ml syringe for blood withdraw 46

47 Blood Collection From Cardiac Puncture cont.

Anesthetize a mouse by intraperitoneal injection of Hypnorm 48 Blood Collection From Cardiac Puncture cont.

Disinfect the thorax area with 75% alcohol cotton ball 49 Blood Collection From Cardiac Puncture cont.

Search for the maximum heart palpitation with your finger 50 Blood Collection From Cardiac Puncture cont.

Insert a 24G 1” needle through the thoracic wall at the point of maximum heart palpitation 51 Blood Collection From Cardiac Puncture cont.

Withdraw blood slowly by your right hand 52 Blood Collection From Cardiac Puncture cont.

Administration volumes considered good practice (and possible maximal dose volumes) Orals.c.i.p.i.m. i.v. (bolus) i.v. (slow) Mouse10 (50)10 (40)20 (80)0.5b (0.1)b5(25) Rat10 (40)5 (10)10 (20)0.1b (0.2)b5(20) Rabbit10 (15)1 (2)5 (20)0.25 (0.5)2(10) Species route and volumes (mL kg-1) Journal of Applied Toxicology 53

SpeciesInjection Site SCIMIPIV FelineScruff, back, 21 – 23G Quadriceps, 23G21 – 23GCephalic vein, 21 – 25G CanineScruff, back, 21 – 23G Quadriceps, G 21 – 23GCephalic vein, 21 – 25G FerretScruff, 21 – 23G Quadriceps, G 21 – 23GCephalic vein, 21 – 25G Guinea pigScruff, back, 23 – 25 Quadriceps, 25G23 – 25GEar vein, Saphenous vein, 25 – 27G HamsterScruff, 25G Quadriceps, 25G23 – 25GFemoral or jugular vein, 25 – 27G MouseScruff, back, 25G Quadriceps, 27G25 – 27GLateral tail vein, 26 – 28G Primate (small) Scruff, back, 23 – 25G Quadriceps, G 21 – 25GLateral tail vein, 21 – 25G Primate (large) Scruff, 21 – 25G Quadriceps, triceps, G 21 – 23GCephalic vein, recurrent tarsal vein, jugular vein 21 – 25G RabbitScruff, flank, 21 – 25G Quadriceps, lumbar muscles, 25G 21 – 23GMarginal ear vein, 23 – 25G RatScruff, 25G Quadriceps, 25G23 – 25GLateral tail vein, 21 – 23G BirdPectoral, interscapular or inguinal fold 1 – 3% BW bid or tid, <21 G Pectoral/per site 0.2 ml/ 500g BW <25G Not applicableCutaneous ulnar vein, <25G, short bevel Needle Sizes and Recommended Injection Sites; Adapted from: Formulary for Laboratory Animals, 3rd Ed., Hawk, Leary, Morris,

Identification methods 55

Euthanasia 56

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