Screening of Local Anaesthetics

Dr. Advaitha M.V

KSHEMA, Mangalore

Types of local Anaesthesia in Preclinical models

1. Conduction Anaesthesia

2. Infiltration Anaesthesia

3. Surface Anaesthesia

4. Epidural Anaesthesia

5. Spinal Anaesthesia

Conduction Anaesthesia in the Sciatic Nerve of the Rat

Definition:

• It is a type of regional anesthesia.

• local anesthetic solution is injected to nerves to inhibit nerve transmission.

• It includes

Spinal, epidural,

Nerve block, and field block anesthesia, but not topical anesthesia.

• But for all practical Purposes, Here it only means Nerve Block

• Wistar or Sprague Dawley rats

• Weight : 125 to 175 g

Procedure: –

• Grasp the base of the tail and thoracic cage.

• The animal is suspended in a prone position.

• A hind limb is extended to its full length.

• Site of injection : area under the skin at the junction of the biceps femoris and the gluteus maximus muscles.

• The sciatic nerve is blocked in the midthighregion with 0.2 ml of the drug solution.

(use a 24- to 25-gauge needle attached to a 0.25 ml tuberculin syringe).

• The other leg is used for the control drug.

• Immediately after the injection,

repeated check of the digit of the foot,

the walking behavior.

is observed.

• In the normal foot, the digits are wide apart.

• In the blocked leg the digits of the foot are close together.

• The successful block is evidenced by

Dragging of the leg

Inability of the animal to use the leg in walking up the inclined wire mesh cover of the cage.

Lastly,

• The time of recovery :

After the time of block for each leg is noted, each animal is examined every 5 to 10 min.

Similar Experiments of conduction Anaesthesia :

Conduction Anaesthesia in Sciatic nerve of Frog.

Conduction anaesthesia on Mouse Tail.

Infiltration Anaesthesia in Guinea Pig’s Wheals

BASED ON

works of

Mc Intyre & sievers(1937)

Bulbring & wajda (1945)

This method is one of the standard operating procedure for testing local anesthesia

• Adult Guniea Pigs

• Weight: 250 – 300 g

• Procedure: –

• The sensitivity of the skin is greatest in the midline.

• Also, It is slightly more in the front than in the back area.

• So, each concentration of a local anesthetic must be tested in both areas.

• One day preceding the experiment two areas of 4–5 cm diameter are shaved.

• This produces certain amount of sensitivity which disappears overnight

• The doses of local anesthetics are injected intracutaneously in 0.1 ml saline.

• The size of the wheal is marked with ink.

• One side is used for the test preparation, the other side for the standard

(e.g 1%butanilicaine) .

triple response,a triad of phenomena that occur in sequence after the intradermal injection of histamine. First, a red spot develops, spreading outward fora few millimeters, reaching its maximal size within 1 minute and then turning bluish. Next, a brighter red flush of color spreads slowly in anirregular flare around the originalred spot. Finally, a wheal filled with fuid forms over the original spot. Also call

ed triple response ofLewis.

• The reaction to pin prick is tested 5 min after injection in the following way--

• Firstly, normal reaction to a prick applied outside the wheal is seen.

• Then six pricks are applied inside the wheal.

• The pricks are applied at intervals of about 3– 5 s.

• Six pricks are applied every 5 min for 30 min.

• Now, the number of pricks to which the guinea pig fails to react inside the whealduring 30 min noted.

• This gives an indication of degree of anesthesia.

Surface Anaesthesia on the Cornea of Rabbits

• Animals: Albino Rabbits

• Weight: 2.5 – 3 kg

Procedure: –

• The upper and lower eyelashes are carefully clipped.

• The conjunctival sac of one eye is held open, forming a pocket.

• Apply 0.5 ml of a solution of the anesthetic into the conjunctival sac for 30 s.

(use, 1 ml syringe with a 22-gauge needle)

• 1 ml of the standard is applied to the other eye ( 0.1% solution of tetracaninehydrochloride).

• Effective local anesthetics abolish the corneal reflex (blinking) elicited by any touch of the cornea.

• The test is started 5 min after application of the drug.

• It is repeated every 5 min until effect subsides and blinking occurs again.

• The time between disappearance and reappearance of the corneal reflex is noted.

Method of irritating cornea

• For quantitative purpose the irritation, “von frey hairs” has been widely recommended.

• An equine(HORSE) hair bending at a load of 230 mg is attached perpendicularily to a glass rod.

• Within 25 sec, the cornea is touched 100 times.

• The summation of many stimuli applied this way gives better result than a single touch with the glass rod (Ther and Mugge1953)

Von Frey hairs. Through the use of a camel hair, various levels of pressure are applied to the skin and pressure is calculated. The hair is mounted inside a tube. Calculations are made respective

to how much the hair is bent during the examination

Today, nylon fibers are used to perform the same task.

Similar experiments:

Abolition of Sneezing Reflex in Rabbits

• New Zealand Albino rabbits weighing 3kg are used.

• The test solution is applied to the mucous membrane of one nostril and standard to other.

• After 2-5 min mucosa is stimulated with a lead pencil.

• Loss of sneezing reflex is considered as the sign of complete local anaesthesia.

• Test is repeated at 3,5,10 and 15min and continued every 5 min until reflex reappears.

Epidural Anesthesia in Rabbits

• Animals: Adult Rabbit

• Weight: 2.5 – 3 kg

• Procedure: –

• The rabbit is carefully restrained by an assistant.

• Palpate the midline at L7 spine and the depression over the lumbosacral fossa. –

The Rabbit Vertebrae column consists of 7 Cervical Vertebrae (As in all mammals), 12-13 Thoracic

Vertebrae, 7 Lumbar, 4 Sacral, and a variable amount of caudal vertebrae (usually 16)

• At this point, through a short bevel 1.5 cm, 20 gauge spinal needle to be introduced.

• This is at right angles to the skin in the midline with the bevel aligned longitudinally

• After passage through the skin,

only minor resistance would be felt until the ligamentum flavum is reached.

When passing through the ligament, a definite “pop” is felt and resistance to advancement of the needle is lost.

The stylet would be then withdrawn and the hub inspected for the presence of blood or cerebrospinal fluid.

If absent, the needle should be rotated through 90° to direct the bevel caudally.

A 1.0 ml syringe to be attached and 0.1 ml of air injected.

A syringe containing the desired dose of the local anesthetic is injected over a 5– 10 s period.

• The effectiveness is assessed by

(1) Sensory loss,

(2) loss of weight-bearing ability, and

(3) flaccid paresis.

Spinal Anaesthesia in Rats

• Animals: Sprague-Dawley rats

• Weight: 75- 100 g

Procedure: –

• A 30-gauge needle is attached to a 25µl Hamilton syringe.

• This is introduced on one side of the L5 or L6 spinous process at an angle of about 20°.

• The needle is advanced to the groove between the spinous and transverse processes.

• Then moved forward to intervertebral space at an angle of about 10°.

• Correct placement of the needle is indicated by an arching of the tail.

• Drugs are dissolved in saline or water and administered in a volume of 5 µl.

• Antinociception is determined by tail flickassay.

(by placing the tail under a focused radiant heat source on an analgesiometer.)

• The reaction time is noted in each case.

References:

• Drug Discovery and Evaluation: Pharmacological Assays, 3rd Edition; by H. Gerhard Vogel.

Thank You