Behavioral Measures of Pain Yhresholds

Behavioral Measures of Pain ThresholdsMichael S. Minett,1,2 Kathryn Quick,1 and John N. Wood1,2

1Molecular Nociception Group, Wolfson Institute for Biomedical Research, UniversityCollege London, London, United Kingdom2London Pain Consortium, Kings College London, London, United Kingdom

ABSTRACT

Pain afflicts a fifth of the population, and animal models have proven useful in targetvalidation and analgesic drug development. Thresholds to pain are tested by applying asensory stimulus, such as heat or pressure, and observing the resulting withdrawal be-havior. Sensitized pain models involve provoking an inflammatory response or damagingthe nerves themselves, and testing the changes in pain threshold. In this article, mousemodels of acute mechanical and thermal pain and inflammatory, visceral, and neuropathicpain are discussed. These behavioral measures can be used to phenotype transgenic micefor target validation and mechanistic studies, as well as to screen potential analgesiccompounds. Curr. Protoc. Mouse Biol. 1:383-412 C© 2011 by John Wiley & Sons, Inc.

Keywords: nociception � reflex � pain � neuropathic � inflammatory � visceral

INTRODUCTION

Animal models of pain have been critical for our current understanding of the under-lying mechanisms of pain and the development of pharmacological interventions. Allhuman analgesic drugs are active in rodent models of pain, although the converse isnot necessarily true—for example, NK1 antagonists are analgesic in rodents but notman. Nonetheless, animal models have proven important in both target validation andunderstanding mechanisms underlying pain sensation.

These models explore various aspects of pain mainly through the interpretation of phys-iological responses, such as withdrawal reflexes or more complex escape behaviors. Thebehavioral responses occur in reaction to thermal, mechanical, and chemical stimuli.Sensory stimuli have a dynamic range—for example, mechanical sensation ranges fromlight touch to noxious pressure, and thermal sensation ranges from noxious cold throughwarm temperatures up to noxious heat. The thresholds to these different types of acutestimuli can be tested using the protocols described here.

Acute pain is a vitally important signaling system that warns us of imminent and/oractual tissue damage. However, chronic pain has no biologically relevant purposeand dramatically reduces quality of life and productivity. The cost of pain is es-timated at over £13 billion per year in the UK (British Pain Society, http://www.britishpainsociety.org/bps nl sum 2009.pdf) and over $100 billion per year in the U.S.(Mayday Fund, http://www.maydaypainreport.org/). There are a number of chronic painmodels outlined in this unit that model some of the different types of pain experiencedin humans—inflammatory and neuropathic. When inflammation or injury is inducedusing inflammatory, visceral, or neuropathic pain models, the thresholds to sensorystimuli change—producing hyperalgesia, an enhanced response to a normally painfulstimulus—and allodynia, a painful response to a normally innocuous stimulus.

The most commonly used animal in pain research has been the laboratory rat (Mogilet al., 2001), but more recently the application of these pain models to mice with targetedgene mutations has proved to be a powerful tool. However, the behavioral responses ofrats and mice can differ; as Mogil et al. (2001) succinctly explain, “mice are not small

Current Protocols in Mouse Biology 1: 383-412, September 2011Published online September 2011 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/9780470942390.mo110116Copyright C© 2011 John Wiley & Sons, Inc.

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rats.” It is important to bear this in mind if applying an established rat model to mice forthe first time.

We describe here the steps required to characterize sensory thresholds of mice andmodel inflammatory, neuropathic, and visceral pain states. The first section containsprotocols for establishing acute pain thresholds to thermal stimuli, using tail flick (seeBasic Protocol 1 and Alternate Protocols 1 and 2), hot and cold plate test (see BasicProtocols 2 and 3), Hargreaves’ apparatus (see Basic Protocol 4 and Support Protocol 1),and acetone evaporation (see Basic Protocol 5); the second section provides protocolsfor testing threshods to mechanical stimuli using von Frey hairs (see Basic Protocol 6,Support Protocol 2, and Alternate Protocol 3) and the Randall-Selitto apparatus (seeBasic Protocol 7 and Alternate Protocol 4). Additional sections outline animal modelsof pain split into three types—inflammatory pain (see Basic Protocol 8 and AlternateProtocol 4), visceral pain (see Basic Protocol 9 and Support Protocol 3), and neuropathicpain (Basic Protocol 10 and Alternate Protocols 5 and 6).

STRATEGIC PLANNING

While planning an experiment, it is important to consider the following: issues.

Ethical considerationsBefore beginning any work with mice, proper training and protocol approval must beobtained according to the European Directive 86/609/EEC, Animals (Scientific Proce-dures) Act 1986, or equivalent legislation in other parts of the world, and the study mustconform to government regulations. Also, it is advisable to consult the InternationalAssociation for the Study of Pain ethical guidelines (http://www.iasp-pain.org) for moredetails and advice. Also see the relevant article in Current Protocols in Mouse Biology,Baertschi and Gyger (2011).

Cut-off timeThe nature of nociceptive testing means that it is important to limit the risk of tissuedamage by limiting unnecessary exposure to noxious stimuli. This is especially truewhen working with transgenic mice and analgesics, where nociceptive thresholds maybe altered. It is recommended to conduct a literature search for previous findings foryour specific mouse strain. If this is unavailable, a cut-off of three times the averagewithdrawal response of naıve mice is desirable.

Motor functionIt is critical to demonstrate that the motor function and coordination of any mice under-going nociceptive testing are intact. A loss in motor function and coordination may alterwithdrawal latencies or other nociceptive behaviors.

Operator blindingThe test operator should be blinded to the test groups (e.g., genotype, treatment etc). Thisensures unbiased results.

Pain behaviorsThroughout this chapter, “pain behaviors” or “nocifensive behaviors” are referred tofrequently as outcome measures of many of the tests.

Common pain behaviors are:

FlinchingLickingShaking


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BitingJumpingStretching or squashing the abdomenGuarding of hindpawChange in posture.

It is essential to become practiced in the “normal” behavior of a mouse, so that anydifferences in behavior during a nociceptive test can be noted. It is also important to noteif anything may be affecting pain behavior, such as grooming, sleeping, defecating, ordistraction by changes in noise or light levels.

ControlsAs with all scientific experiments, proper controls are essential. Always test a groupof control mice (e.g., wild-type or non-treated) at the same time as the test group.When administering drugs or compounds, it is good practice to use an appropriatevehicle control. Similarly, with surgical models, it is important to perform sham surgeryon littermates alongside any real surgery. This can indicate if any other factors havecontributed to any changes in nociceptive behavior,

Baseline pain measuresBefore inducing any of the inflammatory, visceral, or neuropathic pain models, determinewhich pain measures are to be investigated and obtain consistent baseline measurements.It is recommended that at least two baseline studies be performed.

TESTING THRESHOLDS TO THERMAL STIMULI

Heating the skin to temperatures above 45◦C is generally considered noxious, and isthe most commonly used method for assessing nociception. When using a noxious heatstimulus, it is critical to limit exposure time in order to avoid tissue damage.

BASICPROTOCOL 1

Radiant Heat Tail-Flick

This protocol describes the Radiant heat tail-flick test developed 70 years ago byD’Amour and Smith (1941). The tail-flick was one of the first, and subsequently mostcommonly used, nociceptive tests. The D’Amour and Smith technique involves applica-tion of focused beam of radiant heat and measuring the flexor withdrawal reflex latency.It is a robust thermal nociception assay that is stable during repeated tests.

Materials

Mice of optimal age (6 to 8 weeks)Mouse restrainer (consisting of base plate, body tube, and head cover)Infrared heat source with built-in timer and motion detector. (e.g., Tail Flick

Analgesia Meter #33; IITC Life Science, http://www.iitcinc.com/)Infrared heat-flux radiometer (Ugo Basile, cat. no. 37300), for calibrating and

maintaining apparatus

Habituation1. Clean the mouse restrainer.

2. Place a single subject mouse into the restrainer (Fig. 1).

It is recommended to use a restrainer with a removable base plate. Remove the base plateand cover the tube of the restrainer with tissue to make an ideal hiding place for a mouse.Next, place the test subject mouse next to the tube opening and gently tug on its tail totrigger escape behavior, encouraging the mouse to enter the restrainer tube. If the mouse


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Figure 1 Mouse in a restrainer.

repeatedly places its head within the tube only to withdraw it again, this may indicatethat the bore is too narrow and a larger restrainer is required.

3. Adjust head cover for body length of mouse.

Adjust the head cover so that the mouse’s movements are minimized without causingunnecessary distress.

4. Check tail for evidence of injury (e.g., from fighting).

If the tail is significantly injured, consider using another mouse or testing the injuredmouse at a later time.

5. Place restrainer on top of the tail-flick apparatus and leave mouse until settled(average 10 min).

A cover can be placed over the restrainer to help calm the animal, but this must beremoved before the test so the animal can be seen fully.

Testing6. While the radiant heat light source is in the “idle” state, guide the tail over the test

head so the stimulus spot is midway along the tail.

Location of the stimulus spot used on the tail may vary; the most important point is to keepthe stimulus site constant within your experiment. If tail color is not constant over thewhole tail or not the same for all subjects, it is suggested to use the hot water immersionprotocol (Alternate Protocol 1), since tail color has been shown to alter the responselatency of this test (Vetulani et al., 1988).

7. Switch the radiant heat light source to the high-intensity setting and leave in placeuntil nociceptive reflex/withdrawal behavior (e.g., a sudden twitch of the tail) isobserved.

The user interface, controls, and feature of the tail-flick apparatus vary between manu-facturers and models. Please consult user manual for exact details

8. Once a response time has been recorded, repeat the test twice more.

The mouse should be allowed 5 min to recover each time, and the next stimulus spotshould be 10 mm to either side of the original stimulus spot.

Threshold calculation9. Use the multiple test results to calculate an average response time for each mouse.


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ALTERNATEPROTOCOL 1

Hot Water Immersion Tail-Flick

Ben-Bassat et al. (1959) describe an alternative version of the above protocol using ahot-water bath (typically 46◦ to 52◦C) instead of an infrared radiant heat source. Thisprotocol minimizes the effects of tail color or stimulation site, which can alter heat-transfer properties. Also, this version of the tail flick test is particularly useful if a labhas no specialist in analgesia assay equipment.

Additional Materials (also see Basic Protocol 1)

Mice of optimal age (6 to 8 weeks)Water bathThermometer (accurate to 0.5◦C)Timer

Habituation1. Habituate the mouse to the apparatus as described in Basic Protocol 1, steps 1 to 5.

Testing2. Heat water bath to 46◦ to 52◦C. Make sure temperature is stable.

Start with the lower end of the temperature range, and adjust it as necessary so thatwithdrawal latency occurs within a reasonable amount of time for the majority of the mice.

3. Gently guide the distal half of the tail into the water bath and time the withdrawallatency.

This is where it can be useful to have an assistant to handle the mouse while the observeroperates the timer. It is suggested that the experimenter with the most mouse husbandryexperience restrain and handle the mice.

4. Allow the mouse 60 sec to recover before repeating test. Each mouse can be testedat least three times per session.

Threshold calculation5. Use the multiple test results to calculate an average response time for each mouse.

ALTERNATEPROTOCOL 2

Cold Water Immersion Tail-Flick

Pizziketti et al. (1985) describe an alternative version of Alternate Protocol 1 using a cold-water bath (0◦ to 5◦C) instead of a hot water bath. The protocol is identical to the radiantheat tail-flick with the exception that the distal half of the tail is immersed in cool water.It is possible to test subzero (as low as −18◦C) temperatures by using other fluids such asethanol. However, this increases risk of tissue damage, and, therefore, cut-off times shouldbe reduced accordingly. The needed materials are as tabulated for Alternate Protocol 1.Habituation, testing, and threshold calculation are as described for Alternate Protocol 1.

BASICPROTOCOL 2

Hot Plate: Nociceptive Response to Noxious Heat

This protocol describes the steps required to assess behavioral responses to temperaturesup to 55◦C. The most commonly used version of the hot plate test was originally describedby Woolfe and Macdonald (Macdonald et al., 1946) and later modified by Eddy andLeimbach (1953). In contrast to the tail flick test (Basic Protocol 1), which is a spinalresponse, the hot plate test seems to represent a supraspinal thermal assay (discussed inmore detail in the Commentary).

Materials

Mice of optimal age (6 to 8 weeks)Hot plate apparatus with enclosure (e.g., Hot/Cold Plate; Ugo Basile, cat.

no. 35100)Surface thermometer, for calibrating and maintaining apparatus (optional)


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Habituation1. Set plate temperature to match that of the test room and clean test plate surface.

2. Place a single subject mouse into the test chamber and leave to explore undisturbedfor 15 min.

Exposure to novel environments may mask or alter withdrawal threshold

3. Remove mouse and clean any urine or feces from the plate surface.

Urine or fecal matter on the hindpaws may alter the heat transfer properties of the hotplate.

4. Set the hot plate to the test temperature.

This is typically between 50o and 56◦C.

Testing5. Place the habituated mouse onto the hot plate, once temperature has been reached.

6. Measure the time latency to first nocifensive behavior (e.g., hindpaw lift or jumping).

Using forepaw behaviors is NOT recommended, since the forepaws are commonly involvedin grooming and exploratory behaviors. Similarly, a simple hindpaw lift may be due togrooming and exploration.

7. Quickly remove mouse from test surface to avoid unnecessary suffering and/or tissuedamage.

Repeat testing within one session is NOT recommended. Repeated exposure to the hot-plate test at noxious or even innocuous temperatures can decrease behavioral responselatencies (Mogil et al., 2001).

Preparation of next mouse8. Place tested mouse into a separate cage from untested mice.

Mice can communicate via a variety of olfactory and ultrasound systems, which can be asource of anxiety and fear (Gray, 1978).

9. Return the test plate temperature to that of the room and clean surface in preparation.

Multiple temperatures10. If testing same mice at multiple temperatures, then separate the tests by at least 24

hr to avoid the effects of stress and repeated testing on latency times (detail above).

BASICPROTOCOL 3

Cold Plate: Noxious Cold

This protocol describes the steps to assess behavioral responses to noxious cold. Anumber of different versions of the cold plate test have been described, such as Leeet al. (1999), Zimmermann et al. (2007), and Abrahamsen et al. (2008). However, allversions involve observing hindpaw nociceptive behaviors of mice placed onto a surfacemaintained at a temperature between 0◦ to 4◦C. Unlike nocioceptive responses to noxiousheat, noxious cold stimuli do not consistently induce nocioceptive responses within wellestablished time frames. Therefore, it is useful to record the number of nocioceptiveresponses within a fixed period, as well as the latency to the first response.

Materials

Mice of optimal age (6 to 8 weeks)Cold plate apparatus with enclosure (e.g. Hot/Cold Plate; Ugo Basile, cat. no.

35100)Surface thermometer, for calibrating and maintaining apparatus (optional)


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Habituation1. Set plate temperature to match that of the test room, and clean test plate surface.

2. Place a single subject mouse into the test chamber and leave to explore undisturbedfor 15 min.

Exposure to novel environments may mask or alter behavioral response.

3. Remove mouse and clean any urine or feces from the plate surface.

Urine or fecal matter on the hindpaw may alter the heat transfer properties of the coldplate.

4. Set the cold plate to the test temperature.

Testing5. Place the habituated mouse onto the cold plate, once temperature has been reached.

6. Measure the time latency to first nocifensive behavior (e.g., hindpaw flutter-ing/licking).

Using forepaw behaviors is NOT recommended since the forepaws are commonly involvedin grooming and exploratory behaviors. Similarly, a simple hindpaw lift may be due togrooming and exploration

7. Measure the amount of time the mouse spends displaying nociceptive behaviors for5 min.

8. Quickly remove mouse from the test surface to avoid unnecessary suffering and/ortissue damage.

Preparation of next mouse9. Place tested mouse into a separate cage from untested mice.

Mice can communicate via a variety of olfactory and ultrasound systems, which can be asource of anxiety and fear (Gray, 1978).

10. Return the test plate temperature to that of the room and clean surface in preparation.

BASICPROTOCOL 4

Hargreaves’ Test: Withdrawal Threshold to Noxious Heat

This protocol describes the steps required to assess noxious heat thresholds to radiantheat. The Hargreaves test was originally described as a method for measuring thermalnociception in cutaneous hyperalgesia, in response to carrageenan-induced inflammation(Hargreaves et al., 1988). However, it can also be used for comparative phenotyping oftransgenic mice, measuring analgesic drug efficacy etc.

Materials

Mice of optimal age (6 to 8 weeks)Hargreaves’ apparatus, infrared or light (IITC Life Science, cat. no. 390;

http://www.iitcinc.com/; Ugo Basile, cat. no. 37370)Raised glass pane with clear plastic enclosuresOptional: Infrared heat-flux radiometer (Ugo Basile, cat. no. 37300), for calibrating

and maintaining apparatus

Habituation1. Design an identification key, assigning each subject mouse to a specific test com-

partment.

If the test group (e.g., genotype, dose, control etc.) of subject mice is known, the abovetask cannot be performed by the test operator, as it may lead to test bias.


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2. Place the subject mice into the assigned test compartments, with a piece of tissue toabsorb any urine that may collect within the test compartment.

3. Leave subject mice to explore test compartments, undisturbed for at least 1 hr.

Exposure to novel environments may mask or alter withdrawal thresholds

4. Check that exploratory behavior has stopped or decreased to a minimum. If not,leave undisturbed for an additional 30 min before rechecking.

It may be necessary to repeat this more than once.

5. Remove any tissue fragments from the test compartments.

6. Use a fresh tissue to remove as much urine and feces as possible from the glass floorof each test compartment. Slow and gentle movements will minimize any disturbanceto the subject mice.

If the mouse’s paw is submerged in substantial pool of urine, it may alter the Hargreavesmachine’s ability to constantly heat the plantar surface of the paw. Any fecal mattersmeared on the glass floor may also have a similar effect.

7. Leave the mice to resettle for any additional 5 to 10 min following cleaning.

The test operator should remain in the room during this period. Remaining in the roomduring this period familiarizes the subject mice with the test operator’s presence, meaningthe test operator is less of a novel event during the test.

Testing8. While the radiant heat light source is in the “idle” state, use the guide mirror on

top of the test head to position the stimulus spot over the plantar surface of the lefthindpaw.

The majority of studies use the hindpaw. Choosing either left or right is arbitrary; however,it is advisable to consistently use the same hindpaw for all animals and repeats during atest.

9. Switch the radiant heat light source to the high intensity setting and leave in placeuntil a nociceptive reflex/withdrawal behavior is seen.

The user interface, controls, and features of the Hargreaves apparatus vary betweenmanufacturers and models.

10. If the behavioral response is ambiguous (e.g., the mouse engages in grooming be-havior or the movement is triggered by a sudden noise), then disregard the reading.Otherwise, record reading and move on to the next mouse. Allow the mouse torecover by ensuring at least 2 min between each stimulus on the same mouse.

It is important that the mouse be still but not asleep when testing. Similarly, the miceshould not be grooming, as this may influence the response (Callahan et al., 2008).

11. Once a response time has been recorded for each mouse, return to the first mouseand repeat previous step. Several response times (three is common practice) can berecorded from each mouse within one session without causing harm and therebyaltering the response.

12. Upon completion of test, use the identification key to double check that the responselatency time corresponds to the correct mouse.

Threshold calculation13. Use the multiple test results to calculate an average response for each mouse.


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SUPPORTPROTOCOL 1

Alternative Measurement for Hargreaves’ Apparatus

Paw withdrawal latency can also be supplemented by additional behavioral outcomemeasures:

Velocity of paw withdrawal (ordinal score of 0 if movement complete within 1 sec, or ascore of 1 if persisted beyond 1 sec).

Time spent licking affected paw following withdrawal.

Duration of hindpaw withdrawal/lift until affected paw is returned to glass surface.

These additional behavioral measures can be seen long after the paw withdrawal latencyhas return to baseline, following induced hyperalgesia (Hargreaves et al., 1988).

BASICPROTOCOL 5

Acetone Evaporation Test: Innocuous Cooling and Cold Allodynia

This protocol describes the steps required to assess cold sensitivity in the 10◦ to 15◦Crange, which is usually considered innocuous. The application of acetone causes a rapiddecrease in temperature (of ∼10◦C). This can be used to examine sensitivity to cooling, ormore commonly as a measure of cold allodynia following an inflammatory or neuropathicpain model (see relevant sections below).

Materials

Mice of optimal age (6 to 8 weeks)Acetone

Test compartments with a mesh floor (e.g., Mesh Stand; IITC Life Science, cat. no.410; http://www.iitcinc.com)

Acetone applicator (blunting two 33-G needles and inserting them in either end ofa 10-cm length of thin tubing, and using a 5-ml syringe, have approvedsuccessful previously; Fig. 2)

Two timers

Figure 2 Acetone applicator.


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Habituation1. Design an identification key, assigning each subject mice to a specific test compart-

ment.

If the test group (e.g., genotype, dose, control etc.) of subject mice is known, the abovetask cannot be performed by the test operator, as it may lead to test bias.

2. Place the subject mice into the assigned test compartments.

3. Leave subject mice to explore test compartments, undisturbed for 1 hr.

Exposure to novel environments may mask or alter withdrawal thresholds

4. Check exploratory behavior has stopped or decreased to a minimum. If not, leaveundisturbed for an additional 30 min before rechecking.


5. Once settled, have the test operator sit in the room for 5 min before starting the test.

Remaining in the room during this period familiarizes the subject mice with the testoperator’s presence, meaning that the test operator is less of a novel event during thetest.

Testing6. Gently apply pressure to the syringe to convex the acetone meniscus at the open end

of the needle (see Fig. 2).

7. Apply the acetone to the center of the hindpaw and immediately start your first timer.

It is important that the mouse be still but not asleep when testing. Similarly, the miceshould not be grooming, as this may influence the response (Callahan et al., 2008).

8. Use your second timer to measure the amount of time mice displays nocifensivebehavior (e.g., hindpaw fluttering/licking).

It is recommended that two people perform this test, one person to operate the timers anda second applying the acetone.

9. Repeat this at least three times with approximately 1 min between each application.

Threshold calculation10. Use the multiple test results to calculate an average response for each mouse.

TESTING THRESHOLDS TO MECHANICAL STIMULI

Pain can result from a range of noxious stimulus modalities. Mounting evidence showsthat these pain modalities diverge at the primary sensory neurons as well as at thespinal and supraspinal levels. Therefore it is critical to apply a full range of tests whencharacterizing nociceptive responses.

BASICPROTOCOL 6

Von Frey Test: Light Touch Perception Threshold

This protocol describes the steps required to assess touch thresholds in mice. Touchthresholds in humans and animals have been determined using von Frey hairs since the1890s. The calibrated monofilaments are most commonly made of nylon and apply aconsistent accurate force when used correctly. The up-down method for obtaining the50% threshold using von Frey hairs was described by Chaplan et al. (Chaplan et al.,1994). This method makes use of the statistical formula described by Dixon in 1980 todetermine the LD50 (Dixon, 1980).


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Materials

Mice of optimal age (6 to 8 weeks)Test compartments with a mesh floor (e.g., Mesh Stand; IITC Life Science, cat. no.

410; http://www.iitcinc.com)Calibrated set of von Frey hairs (e.g., Stoelting Touch Test Sensory Evaluator)

Habituation1. Design an identification key, assigning each subject mice to a specific test compart-

ment.

If the test group (e.g., genotype, dose, control etc.) of subject mice is known, the abovetask cannot be performed by test operator as it may lead to test bias.

2. Place the subject mice into the assigned test compartments.

3. Leave subject mice to explore test compartments, undisturbed for at least 1 hr.

Exposure to novel environments may mask or alter withdrawal thresholds.

4. Check exploratory behavior has stopped or decreased to a minimum. If not, leaveundisturbed for an additional 30 min before rechecking.


5. Once settled the test operator should sit in the room for 5 min before starting the test.

Remaining in the room during this period familiarizes the subject mice with the testoperator’s presence, meaning the test operator is less of a novel event during the test.

Figure 3 Mouse paw. Circle indicates the area of the plantar surface of the paw to be stimulated(e.g., with von Frey hair), as well as the site for intraplantar injection. Scale bar = 1 mm.


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Testing6. Apply the first von Frey hair, perpendicular to the paw (Fig. 3), until it buckles

slightly, and hold for 3 sec. If a response (pain behavior) is seen, mark the firstcolumn of the scoring grid with an X. If no response is seen mark with an O.

It is important that the mouse should be still but not asleep when testing. Similarly themice should not be grooming as this may influence the response (Callahan et al., 2008).

7. If the last hair produced a response, select the next lower weight hair. Conversely, iflast hair failed to produce a response, select the next higher weight hair.

Ensure that the application of the hair was correct before considering the response or noresponse. Leave approximately 1 min between each application. If may be time efficientto serially time each mouse, once you return to the first mouse a sufficient amount of timeshould have passed.

8. Continue applying increasing or decreasing weighted hairs and marking the resultsin the first column until a change in response occurs—e.g., XXXO or simply OX.

9. Once this perception threshold has been crossed, continue applying increasing (no re-sponse) or decreasing (response) weighted hairs, and record the next five applications.

Threshold calculation10. Use the reference table (Table 1) to look up the corresponding κ value for each

response series.

Note that the κ value is inverted if the series begins with X (or multiple Xs).

Table 1 von Frey Kappa Value Reference Tablea

K for test series whose first part is:

O OO OOO OOOO

XOOOO −0.547 −0.547 −0.547 −0.547 OXXXX

XOOOX −1.250 −1.247 −1.246 −1.246 OXXXO

XOOXO 0.372 0.380 0.381 0.381 OXXOX

XOOXX −0.169 −0.144 0.142 −0.142 OXXOO

XOXOO 0.022 0.039 0.040 0.040 OXOXX

XOXOX −0.5 −0.458 −0.453 −0.453 OXOXO

XOXXO 1.169 1.237 1.247 1.248 OXOOX

XOXXX 0.611 0.732 0.756 0.758 OXOOO

XXOOO −0.296 −0.266 −0.263 −0.263 OOXXX

XXOOX −0.831 −0.763 −0.753 −0.752 OOXXO

XXOXO 0.831 0.935 0.952 0.954 OOXOX

XXOXX 0.296 0.463 0.500 0.504 OOXOO

XXXOO 0.500 0.648 0.678 0.681 OOOXX

XXXOX −0.043 0.187 0.244 0.252 OOOXO

XXXXO 1.603 1.917 2.000 2.014 OOOOX

XXXXX 0.893 1.329 1.465 1.496 OOOOO

X XX XXX XXXX

−K for test series whose first part is:aThe number of responses before the response threshold is crossed determines the column. The followingpattern of responses determines the row. Note that the κ value is inverted if the response series prior to

threshold crossing with X.


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11. Use the formula below to calculate the 50% threshold (grams).

50% threshold = (10[χ+κδ])/10,000)χ = log of the final von Frey hair usedκ = tabular valueδ = log of mean difference between stimuli (typically 0.224 for most von Frey

sets).

SUPPORTPROTOCOL 2

Repeated Measures Von Frey Test

This alternative to the up-down method for the von Frey test (Basic Protocol 6) is used toassess light touch. In contrast to the up-down method, the repeated measures test enablestesting of both sub- and supra-threshold stimuli.

For materials, see Basic Protocol 6. For habituation procedures, see Basic Protocol 6.

Testing1. Apply the lightest von Frey hair, perpendicular to the paw (Fig. 3), until it buckles

slightly, and hold for 3 sec.

2. Repeat application of each filament an additional four times at 60 second intervals.

3. Record number of responses and repeat steps 1 and 2 using the next hair in order offorce.

The upper cut-off should be 6 g. von Frey hairs above this weight will simply lift thehindpaw without buckling.

Threshold calculation4. Determine the threshold as the von Frey hair that elicited a withdrawal response in

40% (two out of five) or more of applications.

ALTERNATEPROTOCOL 3

Automatic Von Frey Test

Digital von Frey instruments are also available. The mice are habituated in the same way,but the automatic von Frey instrument applies the hair perpendicular to the paw when thebutton is pressed, and returns a threshold value. The speed of acceleration and maximumforce applied can be altered. This protocol measures the absolute threshold and not acalculated 50% threshold, as with the manual von Frey hairs.

Materials

Mice of optimal age (6 to 8 weeks)Test compartments with a mesh floor (e.g., Mesh Stand; IITC Life Science, cat. no.

410; http://www.iitcinc.com)Automatic von Frey apparatus (e.g., Electronic von Frey IITC Life Science, cat. no.

2390; http://www.iitcinc.com/)

Additional reagents and equipment for habituation of mice to von Frey apparatus(Basic Protocol 6)

Habituation1. Habituate mice as described in Basic Protocol 6.

Testing2. Using the mirror, position the automatic von Frey probe directly underneath the

stimulus area of the hindpaw.

It is important that the mouse should be still but not asleep when testing. Similarly, themice should not be grooming as this may influence the response (Callahan et al., 2008).


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3. Extend the probe at the chosen acceleration rate by pressing start button.

Ensure that the probe is hitting the paw at the correct point (Fig. 3) and not the wiremesh. The probe will automatically retract and display the threshold value, following awithdrawal response.

4. Record a maximum of six responses.

Leave ∼1 min between each application of the probe to the same mouse.

Threshold calculation5. Average the threshold values obtained for each mouse.

BASICPROTOCOL 7

Randall-Selitto Test: Noxious Mechanical Pressure

This protocol outlines the steps to determine the noxious pain threshold of mouse usinga modified version of the Randall-Selitto test that applies pressure to the tail via a 3-mm2 blunt probe (Takesue et al., 1969). Randall and Selitto originally described theapplication of uniformly increasing pressure to the rat paw as a measure of inflammation,but measuring the tail in the mouse is more reliable (Randall and Selitto, 1957).

Materials

Mice of optimal age (6 to 8 weeks)Mouse restrainer (consisting of base plate, body tube, and head cover)Randall-Selitto apparatus (e.g., Analgesy-Meter; Ugo Basile, cat. no. 37215)

Habituation1. Clean the mouse restrainer.

2. Place a single subject mouse into a restrainer (Fig. 1).

It is recommended to use a restrainer with a removable base plate since it facilitates gettingthe mouse into the restrainer. Remove the base plate and cover the tube of the restrainerwith tissue to make an ideal hiding place for a mouse. Next, place the test subject mousenext to the tube opening and gently tug on its tail to trigger escape behavior, encouragingthe mouse to enter the restrainer tube. If the mouse repeatedly places its head withintube only to withdraw again, this may indicate that the bore is too narrow and a largerrestrainer is required.

3. Adjust head cover for body length of mouse.

Adjust the head cover so that the mouse’s movements are minimized without causingunnecessary distress.

4. Check tail for evidence of injury (e.g., from fighting).

If tail is significantly injured, consider using another mouse or testing the injured mouseat a later stage.

5. Habituate the mouse for 5 to 10 min, until breathing is normal and mouse is notagitated.

A cover can be placed over the restrainer to help calm the animal, but this must beremoved before the test so that the animal can be seen fully.

Testing6. Place tail onto the pedestal of the Randall-Selitto apparatus and rest the blunt cone

on top of the tail.

Choose a point on the tail approximately one quarter of the way down from the base ofthe tail, so that the animal is able to withdraw the tail easily.

7. Apply pressure to the foot pedal to increase the weight exerted onto the tail.


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8. Observe the mouse closely. Release the foot pedal and lift the blunt cone at the firstsign of struggling, vocalization, or withdrawal of the tail.

The animal may not always be physically able to move the tail, so other pain behaviorsshould always be considered when deciding an endpoint.

9. Record the number reached on the scale and multiply by the weights to obtain thefinal force exerted on the tail.

If the number is very close to the beginning or the end of the scale, consider adding orsubtracting a weight to minimize errors from user reaction time or “maxing out.”

10. Repeat a maximum of three times in one session with at least 60 sec between stimuli.Be careful not to choose the exact same point on the tail each time.

ALTERNATEPROTOCOL 4

Digital Paw Pressure Test

A hand-held digital paw pressure analgesia instrument is also available. The protocol isthe same as above (Basic Protocol 7), but the force is automatically calculated and thereis no need to add or subtract additional weights.

Materials

Mice of optimal age (6 to 8 weeks)Mouse restrainerDigital Paw Pressure Analgesia Instrument (IITC Life Science, cat. no. 2500)

Habituation1. Place mouse into restrainer so that the head of the mouse is snug against the nose

cone and the tail is extending out of the end plate.

Choose the right size of restrainer—the mouse should be unable to turn around in therestrainer but not be uncomfortable. Time spent in the restrainer should be kept to <30min, to minimize restraint-induced stress.

2. Habituate the mouse for 5 to 10 min until breathing is normal and mouse is notagitated.

A cover can be placed over the restrainer to help calm the animal, but this must beremoved before the test so the animal can be seen fully.

Testing3. Open the pressure applicator and place the probe underneath the tail.

Choose a point on the tail approximately one quarter of the way down from the base ofthe tail, so that the animal is able to withdraw the tail easily.

4. Close the pressure applicator of the digital paw pressure test instrument.

Be careful to keep the pressure applicator horizontal, to maintain a consistent force.

5. Observe the mouse closely and release the pressure applicator at the first sign ofstruggling, vocalization, or withdrawal of the tail.

The animal may not always be physically able to move the tail so other pain behaviorsshould always be considered when deciding an endpoint. A cut-off limit of 500 g shouldalso be used to prevent tissue damage.

The electronic unit will capture and store the peak force applied.

6. Repeat a maximum of three times in each session with at least 60 sec between stimuli.Be careful not to choose the exact same point on the tail each time.


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INFLAMMATORY PAIN

BASICPROTOCOL 8

Mechanical and/or Thermal Hyperalgesia in the Hindpaw

This protocol describes the method for inducing inflammation in the hindpaw of a mouse.A number of agents can be used to induce inflammation of varying durations and severity,some of which are listed in Table 2. The method for injection remains the same for all ofthese agents. See Table 2 for specific information about the inflammatory agent and thesubsequent behavioral measures and experiment time course, as these are not the samefor all inflammatory agents.

Materials

Mice of optimal age (6 to 8 weeks)Hamilton syringe and 29-G needle, or 0.5-ml disposable insulin syringeInflammatory agent (see Table 2 for concentrations and dosage)

Additional reagent and equipment for determining mechanical and thermal painthresholds (see protocols above)

Hindpaw injection1. Restrain mouse gently by covering with tissue so the mouse is cupped under your

hand securely.

You should be able to access the paw and hold it firmly with the plantar surface facingupwards.

2. Insert the needle into the center of the paw at a shallow angle and inject the requireddose of the inflammatory agent.

This is an intraplantar injection—some agents may require intradermal injections, whichare in the same location on the paw, but by inserting the needle almost parallel to the pawit is possible to inject just under the surface of the skin.

The needle does not need to be inserted deeply, and the mouse should not bleed duringor after injection.

Table 2 Examples of Inflammatory Agents and Suggested Doses

Inflammatory agent Concentration Dose Time course of inflammationType ofinjection

Type ofhyperalgesia

Complete Freund’sadjuvant

100% 20 μl More than 2 weeks Intraplantar Mechanicaland thermal

Carrageenan 2% 20 μl 30 min to up to 6 days Intraplantar Mechanicaland thermal

PGE2 100 ng 2.5 μl Intradermal Mechanicaland thermal

Zymosan 0.2 mg/ml 20 μl 30 min to up to 6 days Intraplantar Mechanicaland thermal

Formalin 0.5%-5% 20 μl Acute phase: 0-10 minInflammatory phase:10-60 min

Intraplantar Spontaneous

Bradykinin 1-10 nM 10-20 μl 0-60 min Intraplantar Spontaneous


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Testing3. After injection, record the mechanical and/or thermal thresholds of the inflamed paw

using Hargreaves and/or von Frey protocols described in Basic Protocols 4 and/or 6at the desired time points

If desired, both paws can be injected; if not, the uninflamed paw can be used as anipsilateral control.

After the measurements are taken, the mice should be culled.

ALTERNATEPROTOCOL 5

Spontaneous Inflammatory Pain

Inflammation can also cause spontaneous pain in the form of flinching/licking biting ofthe affected area, usually the hindpaw. See Strategic Planning for more descriptions ofpain behaviors.

Materials

Mice of optimal age (6 to 8 weeks)Inflammatory agent (see Table 2 for concentrations and dosage)

Clear, lidded Perspex box, ∼15 cm × 15 cmMirrorStopwatchesHamilton syringe and 29-G needle or 0.5-ml disposable insulin syringe

Habituation1. Habituate mouse to the Perspex box for 30 to 60 min or until exploratory behavior

has ceased.

It is useful to have two boxes available so that one mouse can be habituating while thefirst mouse is being observed.

Hindpaw injection2. Remove the mouse from the box for injection. Restrain mouse gently by covering it

with tissue so the mouse is cupped under your hand securely.

You should be able to access the paw and hold it firmly with the plantar surface facingupwards.

3. Insert the needle into the center of the paw at a shallow angle and inject the requireddose.

This is an intraplantar injection—some agents may require intradermal injections, whichare in the same location on the paw, but by inserting the needle almost parallel to thepaw, it is possible to inject just under the surface of the skin.

The needle does not need to be inserted deeply and the mouse should not bleed during orafter injection.

Observation4. After injection place the mouse into the Perspex box and use the stopwatch to time

the amount of time spent conducting pain behaviors for the desired amount of time.

A time course of the pain can be graphed if the pain behaviors are put into 5-min bins.

The mirror should be used to ensure all views of the mouse can be seen and no behaviorsare missed.

VISCERAL PAIN

This protocol describes the steps required to produce viscera-specific behaviors in mice;this model causes both spontaneous pain and hyperalgesia. Common chemical irritants


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used are mustard oil and capsaicin, but this method would also be suitable to test morespecific agonists/antagonists if required.

BASICPROTOCOL 9

Intracolonic Administration of Irritants

Materials

Mice of optimal age (6 to 8 weeks)Anesthetic (e.g., isoflurane)IsofluraneOxygenIrritant (mustard oil or capsaicin)

Catheter (0.6 mm diameter, ∼4 cm long; e.g., Harvard Apparatus, cat. no. 732851)1-ml syringeTest compartment with mesh floor (e.g., IITC Life Science, cat. no. 410;

http://www.iitcinc.com/)

Habituation1. Habituate mouse to the enclosure for 30 to 60 min or until exploratory behavior has

ceased.

It is useful to have two enclosures available so one mouse can be habituating while thefirst mouse is being observed.

Intracolonic administration2. Remove the mouse from the enclosure, and, under brief anesthesia (using ∼3%

isoflurane/oxygen mix), insert the catheter approximately 2.5 cm into the rectum.

This ensures that the catheter has reached the colon; it can be useful to mark the catheterbefore insertion to make sure sufficient depth is achieved. Vaseline can also aid insertionand prevent topical contact of the perineal area with the irritant chemicals.

3. Administer 50 to 100 μl of irritant such as mustard oil (0.25 to 1%) or capsaicin(0.03% to 0.1%).

Observation4. Replace mouse into box and observe behaviors (licking, stretching, and contractions

of abdomen and squashing abdomen against the floor) for 20 min.

The latency for appearance of first behavior can be recorded along with the numbers andtypes of behaviors. After the measurements have been taken, the mouse should be culled.

SUPPORTPROTOCOL 3

Von Frey Hair

Mechanical hypersensitivity after intracolonic administration of irritants can also betested. The following protocol describes a method to test mechanical sensitivity of theabdomen using von Frey hairs. Referred hyperalgesia on other parts of the body suchas the hindpaws may also be present in this model and can be tested using the protocoldescribed in Basic Protocol 6.


Calibrated set of von Frey hairs (e.g., Stoelting Touch Test Sensory Evaluator)

Before intracolonic administration1. Apply a range of von Frey hairs 10 times each in ascending order of force and note

the number and intensity of responses.

Each hair should be applied for 1 to 2 sec with an inter-stimulus interval of 5 to 10 sec.Do not stimulate the same point twice in succession, and avoid the external genitaliaregion.


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2. Observe withdrawal response.

A positive withdrawal response is either a sharp retraction of the abdomen, immediatelicking or scratching of site of application of the von Frey hair, or jumping.

20 min after intracolonic administration3. Repeat application of von Frey hairs as before.

NEUROPATHIC PAIN: HYPERALGESIC ALLODYNIA MEDIATED BYPERIPHERAL NERVE DAMAGE

BASICPROTOCOL 10

Chronic Constriction Injury (CCI): Loose Ligation of the Sciatic Nerve

Originally developed by Bennett and Xie in rats, CCI has also been shown to sensitizethe affected limb to mechanical and thermal (both hot and cold) stimuli (Bennett andXie, 1988; Bennett, 1994). The CCI model seems to involve a large immune-mediatedcomponent. The model involves tying sutures around the sciatic nerve (Fig. 4).

Figure 4 CCI and Seltzer diagram.


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Materials

Mice of optimal age (6 to 8 weeks)IsofluraneOxygenIodine antiseptic solution

Apparatus for administering isoflurane anesthesia, including muzzle for mouseHeat matSurgical swabsTwo size-5 forcepsSize-15 scalpel3-0 non-absorbable sutureSuture needles and needle holders3-0 absorbable sutureWound clipsWound clip remover

Anesthetize mouse1. Place mouse in induction chamber. Fill chamber with ∼3% isoflurane/oxygen mix.

Exact isoflurane concentration will depend on exact equipment being used. It is recom-mended that you consult the manual and optimize the isoflurane/oxygen mix beforehand.

2. Once mouse is unconscious, transfer to anesthetic muzzle with∼1% isoflu-rane/oxygen mix. Place the mouse on a heat mat to maintain body temperatureduring surgery.

Again, isoflurane concentration will depend on exact equipment being used. It is recom-mended that you consult the manual and optimize the isoflurane/oxygen mix beforehand.

3. Check the mouse’s withdrawal reflex by pinching the hindpaw with forceps. If mousewithdraws paw, wait 30 sec and check again. If after three attempts, the mouse stillwithdraws, adjust isoflurane concentration.

If the isoflurane oxygen mix is too high this can be lethal. So any adjustments should bemade gradually.

4. Once withdrawal reflex is no longer seen, proceed with surgery.

However, anesthetic depth should be monitored throughout surgery. This can be achievedthrough monitoring breathing rate and depth.

Surgery5. Clean the shaved skin over the dorsal side designated for surgery with an iodine

antiseptic solution (or equivalent).

This area should extend from the midline to the knee, with a width of at least 15 mm.

6. Make a 0.15-mm incision in the skin along the length of the femur.

7. Break through the biceps femoris using blunt dissection to expose the sciatic nerve.

The sciatic nerve lies just beneath the biceps femoris. Using two sharp size-5 forceps toexpose and gently separate the muscle avoids the risk of nicking the sciatic nerve.

IMPORTANT NOTE: For sham surgery, proceed directly to step 12. DO NOT performsteps 8 to 11.

8. Carefully separate a 6-mm section of the sciatic nerve of from the surrounding tissueusing blunt dissection.

9. Gently slide a closed forceps under the sciatic nerve and lift enough to pass the sutureneedle under the nerve.


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10. Pull the non-absorbable suture through, leaving 10 mm, protecting the sciatic nerveby lifting with forceps to avoid friction damage.

11. Loosely tie the non-absorbable suture around the nerve, trimming any excess sutures.Repeat previous step three times, leaving 1 mm between each ligature.

Bennett and Xie (1988) state that ligatures should be tied such that the diameter of thenerve should just barely be constricted when viewed with 40× magnification.

Wound closure12. Close the biceps femoris with a 3-0 absorbable suture, if necessary.

13. Bring together the two sides of the incision, making sure both sides are aligned.Close with three surgical wound clips, or the minimum number of clips possible.

Avoid putting the wound clips too close to the edge of the incision as these may rip out.Concomitantly, avoid putting the clips too ‘deep’ and bunching up the skin, as this maycause irritation and distress to the mice.

Recovery14. Remove the anesthetic muzzle and transfer to a recovery cage.

It is important that mice be allowed to recover in isolation before being returned to theiroriginal littermates in their original cage.

15. Monitor mice until they regain consciousness.

It is recommended that a second person monitor the recovery of the previous mouse whilethe first person continues performing surgery with the remaining mice.

Post-operative care

24 hr after surgery

16. Check that none of the wound clips have fallen out and that the incision has notreopened. Apply additional wound clips where necessary.

17. Inspect the mice for any signs of infection, lameness, or other unexpected adverseside effects. Treat the mice if possible; otherwise it may be necessary to cull micewith severe adverse side effects.

7 days after surgery

18. Remove any remaining wound clips and inspect for signs of infection. Treat the miceif possible.

Pain tests19. Allow mice 48 hr to recover before undergoing any behavioral measures of nocicep-

tion.

The time taken for allodynia and hyperalgesia-like responses to develop can vary. There-fore, it may be useful to test the mice on the 3rd, 5th, and 7th day following surgery.Following this, less frequent measures are necessary, i.e., once or twice per week. Theeffects of surgery can be detected up to 60 days (or more) following surgery.

ALTERNATEPROTOCOL 6

Seltzer: Partial Ligation of the Sciatic Nerve

Originally developed by Seltzer using rats, partial ligation of the sciatic nerve has alsobeen shown to sensitize the affected limb to mechanical and thermal (both hot and cold)stimuli (Seltzer et al., 1990). Of the listed models included in this unit, it is practicallythe most simple to perform. The model involves tying a suture through half the sciaticnerve (Fig. 4).


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Dissection microscope

Anesthesia1. Anesthetize mouse as described in steps 1 to 4 of Basic Protocol 10.


antiseptic solution (or equivalent).

This area should extend from midline to the knee, with a width of at least 15 mm.

3. Make a15-mm incision in the skin along the length of the femur.

4. Break through the biceps femoris using blunt dissection to expose the sciatic nerve.

The sciatic nerve lies just beneath the biceps femoris. Using two sharp size-5 forceps toexpose gently separate the muscle avoids the risk of nicking the sciatic nerve.

IMPORTANT NOTE: For sham surgery, proceed directly to step 9. DO NOT performsteps 5 to 8.

5. Carefully separate an ∼3 mm section of the sciatic nerve from surrounding tissueusing blunt dissection.

6. Gently slide a closed forceps under the sciatic nerve and lift enough to pass the sutureneedle under the nerve.

7. Use a dissection microscope to insert the needle one-third to one-half way throughthe thickness of the sciatic nerve.

8. Loosely tie the suture around the nerve, trimming any excess suture.

Be careful not to pull the suture too tight, thereby cutting the nerve.

Wound closure9. Close wounds with wound clips as described in steps 12 to 13 of Basic Protocol 10.

Recovery10. Allow animals to recover as described in steps 14 to 15 of Basic Protocol 10.

Post-operative care11. Care for mice post-operatively as in steps 16 to 18 of Basic Protocol 10.


tion.

The time taken for allodynia- and hyperalgesia-like responses to develop can vary. There-fore, it may be useful to test the mice on the 3rd, 5th and 7th day following surgery.Following this, less frequent measures are necessary—i.e., once or twice per week. Theeffects of surgery can be detected up to 60 days (or more) following surgery.

ALTERNATEPROTOCOL 7

Spinal Nerve Ligation (SNL)

For almost 20 years, the Kim and Chung spinal nerve ligation model has been widely usedin both rats and mice. SNL has been shown to sensitize the affected limb to mechanicaland thermal (both hot and cold) stimuli (Kim and Chung, 1992). However, its widespreaduse has led to a number of slight variations, such as tight ligation of L5 or L5 and L6, aswell as axotomy of one or both of L5 and L6. These differences are likely to account forsome phenotypic difference and should be taken into account when comparing results topublished findings. The protocol below specifically describes L5 axotomy (Fig. 5).


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Figure 5 SNL diagram.


Surgical swabsRetractor, glass hook: surgical hooks are commercially available; however these

has proven awkward to use and can result in damage to the other sciatic nerveroots—using a Bunsen burner to melt and draw out glass Pasteur pipets into finehooks has proven a more useful tool (Fig. 6)

Dissection microscopeSize-7 forcepsSize-11 scalpelMicro-scissors

Anesthetic1. Anesthetize mouse as described in steps 1 to 4 of Basic Protocol 10.


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Figure 6 Glass hook for surgery. Scale bar = 1 mm.


antiseptic solution (or equivalent) and place in the prone position.

This area should extend along the midline from second lumbar vertebrae (L2) and secondsacral vertebra (S1), with a width of at least 15 mm.

3. Make a 15-mm longitudinal incision in the skin along the spinal column, from L3 toS1.

4. Use the forefinger and thumb to gently locate the iliac crests. Starting ∼2 mm belowthe iliac crests make a 7-mm incision in the paraspinal muscle ∼2 mm from thecenter of the spinal column, ∼3 mm deep. Be careful not to extend the incision pastL4, as this can increase the risk of the incision rapidly filling with blood, therebyobstructing the view of the spinal nerves. If the incision causes a bleed, insert a swabinto the incision and gently apply pressure in the direction of the bleed source (itmay be necessary to hold this in place for up to 60 sec).

When the incision is in the correct location, you should be able to feel slight ‘bumps’ asthe blade runs along the transverse processes.

IMPORTANT NOTE: Sham surgery: move directly to step 12; DO NOT perform steps 5to 11.

5. Carefully insert the retractor into the incision and gently expand the opening.

Be careful not to insert the retractor between the iliac crest and the spinal column, sinceopening the retractor in this case may fracture the hip.


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6. Using a dissection microscope, gently feel for the L5 transverse processes usingclosed forceps. Once located, use two size-5 forceps to separate the muscle to revealthe bone.

7. Once the L5 transverse process is exposed, use a size-11 scalpel to gently score theprocesses as close to the base as possible. Then, using the original cut as a guide,repeatedly score the bone while gently applying pressure until the bone becomesdetached.

It may be necessary to remove bone splinters, using size-7 forceps, from the stump of theL5 transverse process.

8. Firmly grip the ligand attached to the tip of the L5 transverse process, and cut withmicro-scissors, allowing the transverse process to be removed.

The L5 branch of the sciatic nerve should be immediately below the recently removed L5transverse process.

9. Use the glass hook of the retractor to separate the nerve from the surrounding tissue.

10. Slide the glass hook under the L5 nerve and gently lift away from the surroundingtissue. Use the micro-scissors to cut the nerve.

11. Grip the distal section of the cut nerve with the size-5 forceps and cut off a 1- to2-mm section.

Wound closure12. Close the paraspinal muscle with two 3-0 absorbable sutures.

13. Bring together the two sides of the incision, making sure both sides are aligned.Close with four surgical wound clips (or the minimum number of possible).

Recovery14. Allow mice to recover as described in steps 14 to 15 of Basic Protocol 10.

Post-operative care

24 hr after surgery

15. Check that none of the wound clips have fallen out and that the incision has notreopened. Apply additional wound clips where necessary.

16. Inspect the mice for any signs of infection, lameness, or other unexpected adverseside effects. Treat the mice if possible; otherwise, it may be necessary to cull micewith severe adverse side effects.

Dragging of the operated side is indicative of damage to the L4 spinal nerve, since itinnervates a number of the hind-limb muscles. These mice should be removed since thenociceptive measures require paw withdrawal as a cutoff point.

7 days after surgery

17. Remove any remaining wound clips and inspect for signs of infection. Treat the miceif possible.


tion.

The time taken for allodynia and hyperalgesia-like responses to develop can vary. There-fore it may be useful to test the mice on the 3rd, 5th, and 7th day following surgery.Following this, less frequent measures are necessary, i.e., once or twice per week. Theeffects of surgery can be detected up to 60 days (or more) following surgery.


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COMMENTARY

Background Information

Thermal thresholds

HeatThe Hargreaves test is a recent addition

to the arsenal of thermal nociception mea-sures. The test paradigm has some advantagesover other commonly used thermal nocicep-tion measures, namely the tail flick (D’Amourand Smith, 1941) and the hot plate (Macdon-ald et al., 1946). The main strengths of the testparadigm are: (1) the mice are unrestrained,minimizing stress-related analgesia; (2) a clear(in some cases automated) and quantifiableoutcome measure; (3) repeated testing withina single session is not associated with the in-duction of hyperalgesia; (4) laterality, enablingwithin-subject controls. However, there seemsto be some disparity between the thermalstimuli in the various tests described above.Chapman et al. (1985) among others suggestthat the tail-flick represents a spinal reflex,while the hot plate involves supraspinal pro-cessing. Therefore, experimental conditionsthat affect one and not the other can be consid-ered spinal or supraspinal, respectively. How-ever, it seems a number of other factors mayalso contribute to differences between the twotests.

ColdAbrahamsen et al. (2008) demonstrate that

noxious heat and cold are very separate painmodalities. Ablating peripheral neurons ex-pressing SCN10A leads to loss of nociceptivebehavior to cold (0◦C), while nociceptive be-havior to heat remains intact (both at 50◦ and55◦C). Single-fiber recordings further high-light that the sensory neuron sodium channelNav1.8 is essential for nociception in the coldand therefore cold pain (Zimmermann et al.,2007). Therefore it is critical to use separateassays for both noxious heat and cold to fullycharacterize thermal nociception. The temper-ature border for noxious cold is less clear; how-ever, it can generally be considered that <0◦Cis noxious. By this definition, the acetone testdoes not induce cold pain when applied to anaıve mouse, since it only causes a decrease of∼5◦C. However, it is listed, since it can detectcold allodynia associated with some neuro-pathic pain models.

Mechanical thresholds

von FreyTouch thresholds in humans and animals

have been determined using von Frey hairs

since the 1890s. The calibrated monofilamentsare most commonly made of nylon and applya consistent, accurate force when used cor-rectly. The up-down method for obtaining the50% threshold using von Frey hairs was de-scribed by Chaplan et al. (1994). This methodmakes use of the statistical formula describedby Dixon (1980) to determine the LD50. Theadvantage of the up-down method is reducedapplications of von Frey hairs while maintain-ing the same level of accuracy.

Randall-SelittoThe Randall-Selitto test was developed by

L.O. Randall and J.J. Selitto in 1957 as away of measuring efficacy of analgesics oninflamed tissue (Randall and Selitto, 1957).The test was originally applied to a rat’s pawbut has been modified for use on mice usingthe tail. It is possible to use the Randall-Selittotest on a mouse paw, but the level of restraintrequired for this increases stress levels andmakes the pain behaviors difficult to observe.Instead, mechanical sensitivity on the mousepaw should be measured using the von Freymethod

Inflammatory models

Complete Freund’s adjuvant (CFA)CFA has been used as a reliable model of

persistent inflammatory hyperalgesia since the1980s (Larson et al., 1986). The thermal andmechanical hyperalgesia observed is linked toTNF-α and cytokine release in the periphery(Cunha et al., 1992), develops approximately24 hr after injection, and remains for up to 2weeks.

CarrageenanThe injection of carrageenan into the hind-

paw was originally developed as a modelof inflammation for the screening of anti-inflammatory drugs (Crunkhorn and Meacock,1971). The edema develops over 6 hr and, aswith CFA, is linked to activation of cytokinecascades (Cunha et al., 1992).

ProstaglandinE2 (PGE2)PGE2 is synthesized from arachidonic acid

by COX-2 and PGE synthase enzymes, apathway which is the major site of ac-tion for NSAIDs. Intraplantar administrationof PGE2 causes a short-lasting mechani-cal hyperalgesia by acting directly on pri-mary afferent nociceptors causing sensitiza-tion (Taiwo et al., 1987; Southall and Vasko,2000).


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FormalinThe formalin test allows assessment of con-

tinuous pain, lasting ∼1 hr in the absence ofevoked stimuli. The behaviors occur in twodistinct phases. The first phase starts imme-diately after injection and lasts 5 to 10 min,and is due to direct chemical stimulation ofC fibers acting through TRPA1 (McNamaraet al., 2007). The second phase starts around15 to 20 min after injection and lasts for20 to 40 min. This phase involves centralchanges within the spinal cord and inflamma-tory molecules (Coderre et al., 1990). The con-centration of formalin injected can be alteredto study the two phases—a low concentration(0.5% to 1%) is sufficient to study the earlyphase, whereas concentrations of over 1% arerequired to produce the second phase of painbehaviors (Rosland et al., 1990). The concen-tration should be kept as low as possible toavoid unnecessary suffering.

Bradykinin (BK)Bradykinin is a major inflammatory media-

tor that produces severe spontaneous pain andmechanical and thermal hyperalgesia (Coutureet al., 2001). Hyperalgesia is caused by sensiti-zation of sensory ion channels such as TRPV1and TRPA1 (Chuang et al., 2001;Wang et al.,2008).

ZymosanZymosan is a glucan that forms the cell wall

of Saccharomyces cerevisiae, which producesthermal and mechanical hyperalgesia alongwith edema and spontaneous pain (Meller andGebhart, 1997).

Visceral pain modelThis intracolonic stimulation model was

developed by Laird et al. (2001) to providea way to obtain information on visceral pain,hyperalgesia, and colon inflammation from thesame animal (Laird et al., 2001). The extent ofspontaneous pain behaviors is concentrationdependent, with 1% mustard oil and 0.1% cap-saicin giving the peak behavioral responses.However, high concentrations can also pro-duce ‘freezing’ in-between the pain behaviors.Mechanical hyperalgesia may only be seen atthe higher concentrations of irritant.

Neuropathic pain modelAlthough the mechanisms underlying neu-

ropathic pain are currently not fully under-stood, a number of factors can lead to nerveinjury and, in turn, neuropathic pain—for ex-ample, diabetes, herpes zoster, nerve compres-sion, channelopathies, and autoimmune dis-

ease. Hence, there are a large variety of neuro-pathic pain models that address these differentetiologies. Similarly, neuropathic pain can alsoresult from central sensitization mechanisms.Information regarding central pain and the rel-evant animals models can be found in Bennett(1994). However, this unit focuses on threecommonly used models of surgically inducedperipheral nerve damage.

Critical Parameters

Operator influenceAs with many behavioral measures, the op-

erator’s scoring criteria can vary and result invariation with respect to the actual outcomescore. Therefore, the same operator, when con-ducting multiple repetitions on the same testgroup (e.g., before and after treatment), cancounteract this. It seems that an important fac-tor in operator influence is animal-handlingtechnique (Chesler et al., 2002), which pos-sibly exerts its effect by inducing varying de-grees of stress. On the other hand, it seems thatoperator age, sex, and experience level play aninsignificant role.

Test environmentThe influence of the test environment on

behavioral outcome measures has previouslybeen shown (Chesler et al., 2002). These fac-tors include cage density, humidity, lighting,etc., as well as non-physical factors such astime of day. Careful monitoring of these fac-tors in order to keep them consistent may min-imize their influence. However, if the test sub-ject mice fail to settle after the habituationperiod, a number of measures can be taken:

a. Reducing light levels to a minimum andensuring that it is consistent for all subjectmice.

b. Covering transparent test compartmentsso that operator is not in the direct line of sight.

c. Checking background ultrasound levels,as this may cause distress to mice but withoutbeing audible to test operator.

d. Playing low-level white noise may maskoperator and/or external noises that may alarmthe mice.

e. Using unscented cleaning products andminimizing exposure to novel or threateningsmells (e.g., operator wearing a new perfumeor animal scents, such as rat).

f. Checking room temperature and environ-ment: either too hot or cold can alter behavioralresponse or habituation. Similarly, high levelsof humidity can also be detrimental.

g. Carrying out tests at same time of day,since activity levels can vary during a working


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Table 3 Troubleshooting Summary

Problem Possible cause Solution

One mouse will not settle Various Remove mouse from experiment andcontinue with the remaining eleven

All mice will not settle Multiple possible causes, e.g., ultrasonicnoise, high intensity light, smells, time of day

The test environment may beunsuitable. Find alternative location.

All mice reach cut-off point Apparatus faulty or incorrectly calibrated Refer to apparatus manual

Table 4 Approximate Anticipated Results for Acute Pain Tests

Mouse strain 129sv BALB/c C57BL/6 DBA/2

von Frey hairs 0.8 g 0.8 g 1-2 g 1 g

Automatic von Frey 9-10 g 8-9 g 9-10 g 8-9 g

Randall-Sellitto 100-120 g 150-200 g

Hot plate (53◦C) 20-25 sec 30-35 sec 15-20 sec 25-30 sec

Cold plate (15◦C) 15-20 sec 20-25 sec 5-10 sec 10-15 sec

Hargreaves 10 sec 15 sec 10 sec 10 sec

day. Also, mice are nocturnal, and thereforeit may be necessary to house them in a roomwith an inverted dark-light cycle if specificallyinvestigating behavior during the active phase.

Alternatively, see Mogil et al. (2001) andRacz and Zimmer (2006) for further discus-sion.

Strain-specific variationAs with humans, mice display highly vari-

able nociceptive responses to identical stimuli,injuries, or pathologies. Mogil et al. (1999)systematically surveyed the relative sensitiv-ity of 11 inbred mouse strains using 12 dif-ferent behavioral measures of nociception andreported that performance in all of the strainsvaried greatly. This highlights the importanceof using appropriate littermate controls, aswell as establishing the baseline response forany novel mouse strain. When investigating anew strain or behavioral paradigm, it is advis-able to establish the baseline response for yourparticular setup, even if the strain or behavioralparadigm is commonly used. This will ensurethat your apparatus is calibrated, that your testenvironment is suitable, and that your test op-erator is confident with the test paradigm.

Inter-stimulus intervalSlugg et al. (2000) demonstrate that C fibers

undergo significant fatigue in response whenthe inter-stimulus interval between the pairedstimuli was <150 sec (Slugg et al., 2000). Onthe other hand, A fibers did not demonstratea significant fatigue until the inter-stimulus

interval was <30 sec in monkeys. This canbe used as guideline for determining inter-stimulus interval for the above tests.

Sample sizeWhen comparing two groups, it recom-

mended to start with at least six animals pergroup. However, it may be necessary to in-crease this number.

TroubleshootingA list of common issues and solutions is

provided in Table 3.If inflammation fails to develop, first check

that the agents have been stored and made upcorrectly. CFA needs to be shaken vigorouslybefore administration to emulsify. Also, prac-tice injection technique to ensure the full doseof inflammatory agent is being administered.

Anticipated ResultsTest results can be influenced by the op-

erator and environment (Chesler et al., 2002)as well as mouse strain (Mogil et al., 1999).Therefore, it is recommended to perform a lit-erature search for similar experimental condi-tions. Example results for wild-type C57BL/6,129sv and BALB/c mouse strains are listed inTable 4.

Mechanical hyperalgesiaAt the peak of inflammation, the 50%

threshold measured by von Frey hairs willoften be reduced to absolute minimum (a


411


response given at the lowest weight hair used).The threshold measured by an automatic vonFrey should drop by more than 50%. Thethresholds will slowly improve back to base-line depending on the duration of inflamma-tion.

Thermal hyperalgesiaAt the peak of inflammation, latency should

drop by more than 50% and slowly improveback to baseline depending on the duration ofinflammation.

Spontaneous painSpontaneous pain behaviors have a large

amount of subjectivity, which can make it dif-ficult to predict absolute numbers. Consistencyis more important, and in the case of the forma-lin test, the plotted data should show a biphasicresponse (>1% formalin concentrations).

Time Considerations

HabituationHabituation of the animals for 30 min to

2 hr as stated in the individual protocols is es-sential. The following estimates of time con-siderations exclude this habituation time, sothis should be added on accordingly.

Mechanical and thermal thresholddetermination

The majority of commercial enclosures forthe Hargreaves’ test, von Frey test, and ace-tone test allow habituation and testing of up to12 animals in one session. For each of thesetests, a minimum of 1.0 to 1.5 hr should be al-lotted for 12 animals. The hot plate, cold plate,tail-flick, and Randall-Selitto tests need to becarried out individually, and will take around10 min per mouse per temperature, includinghandling time.

Inflammatory and visceral pain modelsWith practice, the injection or intracolonic

administration of the chemical or irritantshould take no more than 5 min per mouse.

SurgeryIt is preferable to have two people for the

surgical procedures, one to anesthetize andperform the nerve ligation and one for woundrepair and recovery. With two people, an entireday should be allotted to perform surgery on12 to 18 animals. The time needed for eachsurgery will depend greatly on the level ofexpertise.

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Behavioral Measures of Pain Yhresholds

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