The paws of mice and rats are very sensitive to heat even at temperatures which do not damage the skin. They respond by jumping, withdrawal of paws, and licking of paws. The time until these responses occur can be prolonged after administration of centrally acting analgesics, whereas peripheral analgesics of the acetyl salicylic acid or phenyl acetic acid type do not generally affect these responses.
SCREENING METHODS FOR ANALGESIC AGENTS
Centrally Acting Analgesics
Hot
plate method
The paws of mice and rats are very sensitive to heat even at
temperatures which do not damage the skin. They respond by jumping, withdrawal
of paws, and licking of paws. The time until these responses occur can be
prolonged after administration of centrally acting analgesics, whereas
peripheral analgesics of the acetyl salicylic acid or phenyl acetic acid type
do not generally affect these responses.
The hot plate consists of an electrically heated surface. The temperature is controlled for 55°–56°C. Adult albino rats are used for the test. The animals are placed on the hot plate, and the time until either licking or jumping occurs is recorded by a stopwatch. The delay in response is recorded after administration of the standard or the test compound.
In this method, the raised tail phenomena in mice are observed. Six mice per group are used. A clip is applied to the base of the tail of mice, and the reaction time is noted. The test compounds are administered orally to fasted animals. The animal quickly responds to the stimuli by biting the clip or the tail near the location of the clip. The time between stimulation onset and response is measured by a stopwatch.
This method is based on the observation that morphine-like
drugs are selectively capable of prolonging the reaction time of the typical
tail-withdrawal reflex in rats induced by immersing the end of the tail in warm
water of 55°C.
Adult albino rats are used for the test. They are placed in
individual cages leaving the tail hanging out freely. The animals are allowed
to adapt to the cages for 30 min. before testing. The lower 5 cm portion of the
tail is marked. This part of the tail is immersed in a cup of freshly filled
water of exactly 55°C. Within a few seconds, the rat reacts by withdrawing the
tail. A stopwatch records the reaction time. The reaction time is determined
before and periodically after either oral or subcutaneous administration of the
test substance. A withdrawal time of more than 6 s is regarded as a positive
response.
Radiant
heat method
This test is useful for quantitative measurements of pain
threshold against thermal radiation in man and for evaluation of analgesic
activity. It is very useful for discriminating between centrally acting morphine-like
analgesics and nonopiate analgesics.
The animal is put into a small cage with an opening for the
tail at the rear wall. The investigator holds the tail gently. By the opening
of a shutter, a light beam exerting radiant heat is directed at the end of the
tail. For about 6 s, the reaction of the animal is observed. The mouse tries to
pull the tail away and turns the head. The shutter is closed with a switch as
soon as the investigator notices this reaction. Mice with a reaction time of
more than 6 s are not used in the test. The escape reaction is the end point of
this test. Before administration of the test compound or the standard, the
normal reaction time is determined. The test compounds and the standard are
administered either orally or subcutaneously. The analgesiometer can also be
used to measure analgesic activities.
Formalin
test in rats
Rats weighing 180–300 g are administered 0.05 ml of 10%
formalin into the lower surface of the front paw. The test drug is administered
simultaneously either subcutaneously or orally. Each individual rat is placed
in a clear plastic cage for observation. Readings are taken and scored
according to a pain scale. Pain responses are indicated by elevation of the paw
or excessive licking and biting of the paw. Analgesic response or protection is
indicated if both paws are resting on the floor with no elevation of the
injected paw.
Tooth
pulp stimulation
This test is based on the fact that stimulation of the tooth
pulp induces characteristic reactions, such as licking, biting, chewing, and
head flick which can be observed easily.
Adult healthy rabbits are used for the test. Rabbits are
anaesthetized and their pulp chambers exposed with a high-speed dental drill.
On the day of the experiment, clamping electrodes are placed into the drilled
holes. After an accommodation period of 30 min, stimulation is started to
determine the threshold value. The stimulus is applied with a frequency of 50
Hz and duration of 1 s. The electrical current is started at 0.2 mA and increased
until the phenomenon of licking occurs. The test substance is either injected
intravenously or given orally. The animals serve as their own controls.
Grid
shock test
This test measures the analgesic properties by the ‘flinch-jump’ procedure in rats. The floor of the box used is wired with stainless steel wire, spaced about 1 mm apart. The stimulus is given in the form of an electric current, 30 cycles per second with duration of 2 ms per pulse. With increasing shock intensities, the mice flinch, exhibit a startling reaction, increase locomotion, or attempt to jump. The behaviour is accurately reflected on the oscilloscope by marked fluctuations of the pulse and defined as the pain threshold response. The current as measured in milliamperes is recorded for each animal before and after administration of the drug.
This method is based on the fact that since the tail of mice
is known to be sensitive to any stimulus, the stimulus can be varied either by
the duration of the electric shock or by an increase in the electric current to
check the efficacy of the analgesic agent.
Male mice weighing 20 g are placed in special cages. A pair
of clips is attached to the tail, and the positive electrode is placed at the
end of the tail. Electric current at an intensity of 40–50 V is applied. The
frequency of the stimulation is 1 shock/second, and the pulse duration is 2.5
ms. The normal response time range of the stimuli is 3–4 sec. Following
administration of the drug, the response time is registered at 15 min intervals
until the reaction time returns to control levels.
Peripherally Acting Analgesics
This method is based on the principle that inflammation
increases the peripheral analgesic sensitivity to pain. Inflammation decreases
the pain reaction threshold, but the threshold is readily elevated by
nonnarcotic analgesics of the salicylate-amidopyrine type as well as by the
narcotic analgesics.
Groups of healthy albino rats (130–175 g) are used. The
animals are starved 18 to 24 h before administration. To induce inflammation,
0.1 ml of a 20% suspension of Brewer’s yeast in distilled water is injected
subcutaneously into the plantar surface of the left hind paw of the rat. After
three hours, pressure is applied through a tip to the plantar surface of the
rat’s foot at a constant rate to the point when the animal struggles, squeals,
or attempts to bite. Each animal is tested for its control pain threshold. Any
animal with a control pain threshold greater than 80 g is eliminated and
replaced. The mean applied force is determined for each time interval.
Writhing
test
Pain is induced by injection of irritants into the
peritoneal cavity of mice. The animals react with a characteristic stretching
behaviour which is called writhing. The test is suitable to detect analgesic
activity. An irritating agent such as phenyl quinone or acetic acid is injected
intraperitoneally to mice, and the stretching reaction is evaluated.
Mice of either sex of weight 20–25 g are used. Phenyl
quinone in a concentration of 0.02% is suspended in a 1% suspension of carboxy
methyl cellulose. About 0.25 ml of this suspension is injected
intraperitoneally. The mice are placed individually into glass beakers and are
observed for a period often one minute. The number of writhes is recorded for
each animal. A writhe is indicated by a stretching of the abdomen with
simultaneous stretching of at least one hind limb.
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