Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedures for testing nociception and pain sensitivity
Data
References
Workflow
Step
Procedure performed
Mice pain sensitivity to thermostatically controlled water bath is investigated
- Balance scale
- Stopwatch
- A focused, high-intensity projector lamp beam (IITC Model 336 Plantar Test and Tail-flick Analgesia Meter, Woodland Hills, CA, USA)
- 3/16th-inch thick glass floor within small (9 cm L Ã 5 cm W Ã 5 cm H) Plexiglas cubicles
- Metal surface (IITC Inc., Hotplate Analgesia Meter, Woodland Hills, CA, USA)
- Transparent Plexiglas cylinder (15 cm D; 22.5 cm H) with Plexiglas lid
- Thermostatically controlled water bath (TW47) by Boekel Scientific/Grant Optima Immersion Circulator Model GR150 (Boekel Scientific, Feasterville, PA, USA)
- Plexiglas-bound arena measuring 13.5 in L Ã 16 in W Ã 15 in H
- Alligator clips with a rubber cuffs around each jaw (exerting â600 g of force)
- Von Frey type nylon monofilament: A set of eight calibrated Von Frey fibers (Stoelting, IL, USA), ranging from 0.067 to 9.33 g of force
- Surface cleanser MB-10 (QuipLabs, Wilmington, DE, USA)
- Paper towels
Acclimation to test conditions
Mice are allowed to acclimate for 1 wk to new housing conditions.
Procedures for testing nociception and pain sensitivity
Hargreaves paw withdrawal test
a. Mice are allowed to habituate for a period of 2 hrs on a 3/16th-inch thick glass floor within small (9 cm L Ã 5 cm WÃ 5 cm H) Plexiglas cubicles.
b. Following a period of habituation, a focused, high-intensity projector lamp beam is shone from below onto the mid-plantar surface of the hindpaw (Hargreaves et al, 1988).
c. The projector lamp is set to 10% idle intensity (II10), while the light-beam is aligned to the mid-plantar surface of the left paw.
d. The lamp is then switched to 25% active intensity (AI25); the latency to respond by withdrawing the paw away from the light, or by licking of the paw, is recorded with the internal timer.
e. The lamp is turned to idle intensity after 30 s and the paw is removed from from the platform to avoid tissue damage.
f. Following an intra-trial period of at least 5 min, the above process is repeated for the right hindpaw.
g. Mice are tested for 3 - 6 trials depending on the variance observed on each paw. Only the three tightest latencies are averaged and recorded.Hot plate test
a. Mice are allowed to habituate to the testing room for 30 min.
b. Following a period of habituation, the mice are placed on the center of a hot metal surface maintained at 54 ± 0.2°C temperature (HP52) within a transparent Plexiglas cylinder (15 cm D; 22.5 cm H) equipped with a Plexiglas lid and a built-in timer that is started immediately.
c. The latency to respond with a jump, or hindpaw lick or shake/flutter is measured to the nearest 0.1 s.
d. Two trial-latencies are recorded per mouse with a 30 s intra-trial interval and a maximum trial duration of 30 s.
e. To prevent tissue injury from the hot plate, each mouse is promptly removed after 30 s.
f. The apparatus is thoroughly cleansed with MB-10 between each testing.Notes from submitting PI: "This test occurs approximately 2â3 h after the completion of the open field test. The lights in the testing area are turned off at least an hour prior to testing and animals are allowed to sit undisturbed in the darkened room. A lamp (15 W bulb) behind the hot plate (Hotplate Analgesia Meter, Model 39, IITC, Inc.) is faced away from the hot plate surface. A mirror is placed behind the hot plate so that the experimenter can observe the animal."
Tail withdrawal test
a. Mice are allowed to habituate for 30 min to the testing environment.
b. Each mouse is lightly restrained in a denim pocket while the distal half of it's tail is dipped into a thermostatically controlled recirculating waterbath at 47.0 ± 0.1°C) (TW47) temperature.
c. Latency to respond to the heat stimulus by vigorous flexion of the tail is measured with a stopwatch.
d. Each mouse is given 3-5 trials with an inter-trial interval of 10 s, and a maximum trial duration of 30 s.
e. To prevent tissue injury each mouse's tail is promptly removed from the hot water after 30 s.
f. Latency performance of the last 3 trials are recorded.Tail clip test
a. Mice are allowed to habituate for 30 min to the testing environment. The enclosure is a Plexiglas-bound arena measuring 13.5 in L Ã 16 in W Ã 15 in H.
b. The front of the arena is opened and aligned with the leading edge of the table, such that the experimenter can easily restrain and release mice quickly.
c. All mice are lightly restrained in a denim pocket, while an a rubber cuff protected alligator clip (exerting â600 g of force) is applied to the tail 1 cm from the base and vertically oriented with respect to the table.
d. The mouse is immediately removed from the holder, and the latency to lick, bite, or grab the clip or bring its head within 1 cm of the clip is measured with a stopwatch to the nearest 0.1 s, after which the clip is immediately removed.
e. Each mouse is tested only once with maximum trial duration of 60 s.
f. To prevent tissue injury the tail clip is promptly removed after 60 s.
g. The enclosure and clip are thoroughly cleansed with MB-10 between each mouse.Von Frey test
a. Mice are allowed 120 min of habituation to the same Plexiglas enclosure (9 cm L Ã 5 cm W Ã 5 cm H) on a wire-mesh floor (aquarium/vivarium top) instead of a glass floor.
b. For assessment of mechanical sensitivity thresholds, mice are tested with von Frey type nylon monofilaments.
c. A set of 8 calibrated Von Frey fibers, ranging from 0.067 to 9.33 g of force, are applied to the plantar surface of the hindpaw until they bend.• 1 of a series of 8 Von Frey fibers with logarithmically increasing stiffness (0.067 to 9.33 g) is used to stimulate the hindpaw.
• The Von Frey fiber is positioned perpendicular to the plantar surface, enough pressure is applied to cause slight bending against the paw, and held in place for approximately 6-8 s.
• Only when behavioral responses to previous stimuli have resolved that the series of stimuli are presented at several seconds intervals.
• If the paw is sharply withdrawn, a positive response is noted.
• If the mouse flinch immediately when the fiber is removed, a positive response is noted .
• If a mouse walks away, the response is considered ambiguous, and the stimulus is repeated.d. The threshold force required to elicit withdrawal of the paw (median 50% paw withdrawal) is determined using the upâdown method (Chaplan et al. 1994).
• Testing is started with the 2.0 g fiber, in the middle of the series.
• Stimuli are always presented in a consecutively, either ascending or descending.
• If there is no hindpaw withdrawal response to the first selected fiber, a stronger stimulus is tried; if a hindpaw withdrawal is elicited, the next weaker stimulus is selected.
• Optimal threshold calculation by this up-down method requires 6 withdrawal responses in the immediate vicinity of the 50% threshold.
• Series of similar responses may be generated as the threshold is approached from either up or down direction.
• Although all responses are noted, counting of the critical 6 data points does not begin until the response threshold is first crossed, at which time the 2 responses straddling the threshold are retrospectively designated as the first 2 responses of the series of 6.
• Four additional responses to the continued presentation of stimuli that were varied sequentially up or down, based on the rat's response, constituted the remainder of the series.
• Thus, the number of actual responses collected using this paradigm can vary from a minimum of 4 (in the case of paw withdrawal sequentially to the 4 hairs in the descending range 2.0-0.4 g: threshold lies below the range of actual stimuli) to a maximum of 9 (in the case of the first withdrawal occurring on the fifth ascending stimulus presentation at 15.1 g, followed by elicitation of 4 additional responses, assuming that withdrawals continue to occur at or below 15.1 g).
• In cases where continuous positive or negative responses are generated to the exhaustion of the stimulus set, values of 15.00 g and 0.25 g are assigned respectively.
• The resulting pattern of positive and negative responses is tabulated using the convention, X= withdrawal; 0 = no withdrawal, and the 50% response threshold is interpolated using the following formula:50% g threshold = (10 [Xf+κδ)/10,000
• Xf = value (in log units) of the final Von Frey fiber used
• k = tabular value (see Appendix) for the pattern of positive/negative responses
• δ = mean difference (in log units) between stimuli (here, 0.224)
e. A maximum of 9 trials are required for each paw with 4 trials performed after the 1st response cross-over.
f. Trials begin with the left hindpaw and then to the right hindpaw after all mice in the enclosure receive the stimulus.
g. An intra-trial period of 150 s is used between left and right paw.
References
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994 Jul;53(1):55-63.
PubMed 7990513 Chesler EJ, Wilson SG, Lariviere WR, Rodriguez-Zas SL, Mogil JS. Identification and ranking of genetic and laboratory environment factors influencing a behavioral trait, thermal nociception, via computational analysis of a large data archive. Neurosci Biobehav Rev. 2002 Dec;26(8):907-23.
PubMed 12667496 Chesler EJ, Wilson SG, Lariviere WR, Rodriguez-Zas SL, Mogil JS. Influences of laboratory environment on behavior. Nat Neurosci. 2002 Nov;5(11):1101-2.
PubMed 12403996 Hargreaves K, Dubner R, Brown F, Flores C, Joris J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 1988 Jan;32(1):77-88.
PubMed 3340425 Lariviere WR, Chesler EJ, Mogil JS. Transgenic studies of pain and analgesia: mutation or background genotype?. J Pharmacol Exp Ther. 2001 May;297(2):467-73.
PubMed 11303031 Mogil JS, Chesler EJ, Wilson SG, Juraska JM, Sternberg WF. Sex differences in thermal nociception and morphine antinociception in rodents depend on genotype. Neurosci Biobehav Rev. 2000 May;24(3):375-89.
PubMed 10781697 Mouse Phenotype Database Integration Consortium. Mouse Phenotype Database Integration Consortium: integration [corrected] of mouse phenome data resources. Mamm Genome. 2007 Mar;18(3):157-63. Epub 2007 Apr 10.
PubMed 17436037 Wilson SG, Bryant CD, Lariviere WR, Olsen MS, Giles BE, Chesler EJ, Mogil JS. The heritability of antinociception II: pharmacogenetic mediation of three over-the-counter analgesics in mice. J Pharmacol Exp Ther. 2003 May;305(2):755-64. Epub 2003 Feb 20.
PubMed 12606637 Wilson SG, Smith SB, Chesler EJ, Melton KA, Haas JJ, Mitton B, Strasburg K, Hubert L, Rodriguez-Zas SL, Mogil JS. The heritability of antinociception: common pharmacogenetic mediation of five neurochemically distinct analgesics. J Pharmacol Exp Ther. 2003 Feb;304(2):547-59.
PubMed 12538806
