Laboratory Animal Handling Technique and routes of drug administration Dr.Raz Mohammed Lab. 1

Objective To comply with the Animal Welfare Ordinance and avoid mishandling of animal in research To provide basic concepts of animal handling technique to the new animal user. While offering our concept and techniques to our animal user, we also encourage comments from experienced animal users. By doing so, we would enrich our knowledge in the field of laboratory animal research on both sides and further benefit animal welfare as well as the credibility of research result in our university.

Laboratory Animal Handling Technique - Rat A.Intraperitoneal injection B.Blood collection from tail vein C.Blood collection from cardiac puncture D. Oral feeding E. Sexing

Intraperitoneal Injection in Rat (IP) The most common method of administering drugs to rats, due to the ease of administration compared with other parenteral methods.

Intraperitoneal injection This is a common method to introduce drugs to animals. The drug is injected into the peritoneal cavity where absorption is rapid.

Tools for intraperitoneal injection in rat 75% alcohol, cotton ball for surface disinfection medium size towel for restraining the rat 25G 5/8” needle with 1ml syringe for injection

Let the rat relax on the top of the lid.

Stretch the body of the rat by pulling up it’s tail and then cover the rat with a towel by your left hand

Fold the skirts of towel under the rat from all directions

Grasp up the left hind limb of the rat to expose the abdomen

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 intestines.

Collection of blood from tail vein in rat General anesthesia needed small volume: 0.1-1 ml

Tools for collection of blood from tail vein 75% alcohol cotton ball for surface disinfection 27G1/2” needle with 1 ml syringe for blood withdrawal a vial for blood collection

Optimal site of blood withdrawal is around the distal one-third of the tail since this part of tail gives better visualization of the veins

Disinfect the tail with 75% alcoholic cotton ball

When the needle penetrates the epithelium of the tail, pull back the plugger a bit to create negative pressure inside the syringe, then push the needle in the vein slowly until blood get into the dead space of the needle head

Pull back the plugger by the ring finger to withdraw blood from the tail vein

Using a scalpel to make a small wound on the tail is also an option for collecting blood from tail vein

Blood can be collected b using a pipetteman after then

Collection of Blood from Cardiac puncture in Rat General anesthesia needed Large volume: up to 3% of body weight

Tools for collection of blood from cardiac puncture 75% alcohol cotton ball for surface disinfection 24G needle with 10cc syringe for blood withdraw 15 cc centrifuge tube for blood collection

Disinfect the left thorax with 75% alcoholic cotton ball

Search for the point of maximum heart beat

Insert the needle straight on the selected point and withdraw blood by your left hand

Oral Feeding in Rat Feeding amount limited to 1% of body weight

Tools for oral feeding in rat 16 G ball-tipped feeding needle with syring Leather glove

Restrain the shoulders of the rat by your thumb and index finger, then support the lower limbs with your right hand

Restrain the tail of the rat in between your ring finger and little finger

Let the rat lie on your left palm and introduce the ball-tipped feeding needle from the pharynx in to the esophagus when the rat is in the act of swallowing

Sexing Rat- the distance between the anal and genital orifices is greater in the male (left) compared to the female (right)

Routes of drug administration to rats Oral administration Subcutaneous injection Intravenous injection Intraperitoneal injection Intramuscular injection Rectal administration Inhalation

Oral administration (Gavage tube) Rodents have several unique characteristics One of most important characteristic is the lack of an emetic response. The lack of this response allows for a higher dose of a potential emetic compound to be administered and evaluated. There are two types of gavage tube: Straight and curved

Advantages The gavage method can be used when the test compound is not stable in the diet or might not be palatable to the animals. With the gavage method of dosing, a more precise amount of test compound can be delivered and this might reduce the amount of test compound required to complete the study.

Disadvantage Handling of the rat for each dosing. Handling of the rat has been shown to increase corticosteroids levels and could affect study results. Daily intubation might lead to death due to esophageal puncture or aspiration pneumonia.

Drug Excretion Dr. Raz Mohammed 18.4.016

The major organ for the excretion of drugs is the kidney. Renal elimination of a drug 1. Glomerular filtration 2. Proximal tubular secretion 3. Distal tubular reabsoption

1. Glomerular filtration Most drugs are readily filtered from blood into the glomerulus, except drugs that are extensively bound to plasma proteins. But the overall renal excretion is controlled by what happens in the tubules. More than 90% of the filtrate is reabsorbed into circulation.

2. Proximal tubular secretion Secretion occurs by energy-requiring active transport (carrier-requiring) system. 3- Distal tubular reabsoption As a drug moves toward distal convoluted tubule, its concentration increases & exceeds that of the perivascular space. Uncharged drug may diffuse out of the lumen, back into the systemic circulation. Changing the pH of the urine to increase the ionized form of the drug in the lumen may be used to minimize the amount of back-diffusion, and increase the clearance of an undesirable drug.

Weak acids can be eliminated by alkalinization of urine Weak bases may be eliminated by acidification of urine. The excretion of weak acidic drugs (patient who has taken overdose of aspirin) can be increased by taking sodium bicarbonate .

Weak acids can be eliminated by alkalinization of urine Weak bases may be eliminated by acidification of urine. This process is called ion trapping EX- a patient with phenobarbital (weak acid) overdose can be given bicarbonate, which alkalinizes the urine and keeps the drug ionized, thereby decreasing its reabsorption. -If overdose is with a weak base like cocaine, acidification of the urine with NH4Cl, increases its clearance.

Drug-induced coloration of urine Discoloration could be due to: Serious adverse effects of drugs such as hemolysis. Harmless discoloration of urine is induced by some drugs.

A patient taking drugs like rifampicin, phenazopyridine, metronidazole, levodopa..etc Should be informed in advance, other wise, he might alarmed by the changes of his/ her urine color.

Drug Color Rifampicin Orange-red Metronidazole Dark brown Tetracycline Yellow Phenazopyridine Levodopa Phenolphthalin Red Salicylates Pink to brown Nitrofurantoin Yellow to brown Cascara Red (in alkaline urine)

Procedure: One student (volunteer) in each group after emptying his bladder and preserving a sample of blank urine, will receive two capsules of rifampicin (300mg) and the other will receive nitrofurantoin. Urine is then collected after 45 minutes and the color is compared to the blank sample.

Notes Color changes may interfere with laboratory tests. Food and food additives might also change the color of urine (beet root) Drugs might interfere with urine tests, Drug, in addition, could discolor stool (iron, charcoal, and red wine)

Rifampicin t1/2 = 3 hrs Metronidazole t1/2= 8 hrs Tetracycline t1/2= 16 hrs Nitrofurantoin t1/2= 30 min.