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 midthigh
region 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 o
f histamine. First, a red spot develops, spreading outward fora few millimeters
, reaching its maximal size within 1 minute and then turning bluish. Next, a bri
ghter red flush of color spreads slowly in anirregular flare around the original
red 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 wheal
during 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 tetracanine
hydrochloride).
• 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 flick
assay.
(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