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1、BASIC PROTOCOL 1: MEASUREMENT OF ACUTE PAIN USING THE HOT-PLATE TESTThis protocol describes the use of a hot plate to measure the potential antinociceptive effects of test compounds to an acute thermal stimulus. Animals are injected with test compounds or vehicle (controls) and then placed on the ho
2、t plate one at a time. Latency to respond to the heat stimulus is measured by the amount of time it takes for the animal to lick one of its paws.MaterialsAnimal: 30- to 35-g male CD mice, group-housed (e.g., Charles River Labs) or 200- to 250-g male Sprague-Dawley ratsTest compounds in saline or oth
3、er appropriate vehicleReference analgesic compound (e.g., morphine, to be given 10 mg/kg, i.p.)1-ml syringes with 27-G needlesHot-plate apparatus or metal plate in a controlled temperature water bathStopwatch or timer1. Bring animals to test room and record their body weights. Allow animals to accli
4、mate to test room for 15 to 30 min.Separate groups of animals are used for each treatment.2. Set hot plate to 55C for mice or 52.5C for rats.Morphine treatment may be used to determine the optimal hot-plate temperature. Doses of 8 to 10 mg/kg morphine (i.p.) typically provide a near-maximal to maxim
5、al antinociceptive response in acute pain assays. The apparatus is set to the temperature at which this type of antinociceptive response is observed with these doses of morphine.3. Prepare doses of test compounds, vehicle (control), and reference agent such as morphine (if desired).4. Inject animals
6、 with test compound, vehicle, or reference agent. Inject animals in a staggered fashion to allow a consistent time to pass between treatment and testing for each animal. Randomize the order in which different treatments are administered such that each condition is distributed throughout the duration
7、 of the entire experimental procedure.For example, administer vehicle, then dose 1, then dose 2, and repeat until the desired number of animals for each group (e.g., n = 8) is reached. The time interval used to stagger the injections will depend on the time it takes to test each animal. Testing time
8、 is generally based on the cutoff timethe preselected time at which the test will be terminated if the animal shows no nociceptive response. Adhering to preselected cutoff times helps minimize the tissue damage that can occur with prolonged exposure to a heated surface. For the purposes of staggerin
9、g treatments, if the cutoff time is set to 30 sec, the experimenter may want to leave 45 to 60 sec between the injection of animals. The number of animals that can be treated at one time depends on the time between treatment and testing. For example, if testing is to be performed 30 min after treatm
10、ent, a single experimenter, treating animals at a rate of one per minute, should treat only 30 animals. At 30 min, it will be time to test the first treated animal.5. When post-treatment time (e.g., 30 min) has elapsed, begin testing animals. Place a single animal on the hot plate and immediately st
11、art a stopwatch or timer. Observe the animal until it shows a nociceptive response (e.g., licks its paw) or until the cutoff time is reached. Remove the animal from the hot plate. Record its latency to respond. For animals that do not respond prior to the cutoff time, record the cutoff time (e.g., 3
12、0 sec). Repeat for all animals in the order in which they were treated.Mice or rats placed on a heated surface will often lick their paws, typically the front paws first. For scoring, the latency to respond is measured as the amount of time that elapses between when the animal is placed on the hot p
13、late and when it first licks one of its rear paws. Front paw licking is a common grooming response and may have no relation to discomfort. Therefore, rear paw licking is a more reliable measure of discomfort.The duration of action of a compound may also be determined by measuring a single animals re
14、sponse over time. For example, measurements may be taken 15, 30, and 60 min after treatment with a test compound. Of course, similar measurements must be made for vehicle-treated animals (controls).6. Perform appropriate statistical analysis on the data. With multiple groups, use an ANOVA followed b
15、y post-hoc analysis (if appropriate) to compare the treatment groups to the controls (vehicle treatment).Data from hot-plate tests are often expressed as percent maximum possible effect (%MPE). %MPE = (test - baseline)/(cutoff - baseline) 100, where test is the latency to respond after treatment; baseline is the latency to respond prior to treatment; and cutoff is the preset time at which the test will be ended in the absence of a response. To determine a compounds %MPE, a baseline measure must be obtained for each animal prior to treatment with vehicle or test compound.
