In This Section      

Pain-related Quantitative Behavioral Core

The laboratory consists of a designated, climate-controlled rat and mouse behavior test room (400 square feet, including a 150 sq. ft. soundproof chamber) and two separate rooms (approximately 200 sq. ft. each) dedicated to surgery and mouse behavioral testing.

Major Testing Paradigms Related to Pain Behavior

Sensory Discrimination

Tests to assess the location, modality, quality and intensity of painful sensations

Mechanical Sensitivity

  1. Up-down method. For the assessment of mechanical allodynia in rats. Mechanical paw-withdrawal thresholds are determined by using an ascending series of six von Frey filaments that deliver approximately logarithmic incremental pressures (0.42–15.8 g, Stoelting Co., Wood Dale, IL, USA, rat).
     
  2. Paw withdrawal frequency method. For the assessment of mechanical allodynia in mice. Calibrated von Frey monofilaments (0.07 and 0.45 g; Stoelting Co., Wood Dale, IL, USA, mouse) are applied to the hind paw for approximately 1 sec. Each stimulation is repeated 10 times to both hind paws.
     
  3. Pin prick. Mechanical hyperalgesia is determined by pressing the plantar surface of the hind paw with a custom-designed pinprick stimulator, which produces a quick reflex withdrawal response in normal animals but is insufficient to damage the skin.
     
  4. Randall–Selitto test. A Randall–Selitto paw pressure device (IITC, Woodland Hills, USA) is used to quantify the nociceptive flexion reflex. A linearly increasing mechanical force (in g) is applied to the dorsum of the rat’s hind paw. Nociceptive threshold, expressed in grams, is applied by increasing pressure to the hind paw until a squeak (vocalization threshold) or paw withdrawal (paw withdrawal threshold) is elicited.

Heat Sensitivity

  1. Radiant heat test. Paw-withdrawal latencies to radiant heat stimuli are measured with a plantar stimulator analgesia meter (IITC model 390 radiant paw heating apparatus w/ electronic timer). Paw-withdrawal latencies measured up to tenths of a second are used for statistical analysis, and the median latency of three trials is used for analysis.
     
  2. Hot plate test. The surface of a hot plate is heated to a temperature of 55 ± 1°C. The mouse is placed on the heated plate, and the latency for it to show a nociceptive response with paw lick, paw flick, or a jump is measured with the timer. The mouse is immediately removed when this response is observed. If the mouse does not display a response within 30 sec, the mouse is removed from the heated plate to prevent any tissue damage.
     
  3. Tail-flick analgesia meters (IITC model 390 with electronic timers). In the tail flick assay, a mouse or rat is placed within a restraining tube with its tail protruding.  The tail is placed on a level surface, and radiant heat is applied at a particular intensity. The latency of the mouse to remove its tail from the heat is recorded. A cutoff time of 20 sec is maintained to prevent potential tissue damage.
     
  4. Tail immersion test. The tail of a mouse or rat is immersed in a water bath maintained at 42°C (a temperature that is normally innocuous in naive animals). The tail is kept submerged until tail withdrawal or signs of struggle are observed (cutoff time: 20 sec). The reaction time of the animal is measured three times with an inter-trial interval of at least 15 min. The tail of the animal is dried immediately with soft cellulose paper to avoid tail cooling between two measures. A shortened duration of immersion indicates allodynia.
     
  5. Two-texture and two-temperature preference assays. The floor of an 8 inch x 12 inch rectangular Plexiglas testing chamber consists of four thermally isolated aluminum blocks (for testing temperature preference; typically 32°C and 38°C) or four thin aluminum blocks, each covered by a piece of sandpaper (60 grain or 400 grain). Each diagonally opposed pair of aluminum blocks is maintained at the same temperature or covered by the same texture. The configuration of temperature or texture assignments is reversed after testing two to three mice. Mouse position is monitored by using infrared photo beams and OptoMax software (Columbus Instruments, Columbus Ohio).  The whiskers are trimmed before the texture preference assay. Mice are acclimated in a Plexiglas cylinder for 1 h before testing. The time that the mouse spends on each block is measured during a 1-h test period.

Cold Sensitivity

  1. Cold-plate test. Paw withdrawal latency in response to noxious cold (0°C) is measured with a cold plate, the temperature of which is monitored continuously. Each animal is placed in a Plexiglas chamber on a cold plate, which is set at 0°C. The length of time between the placement of the hind paw on the plate and the animal jumping, with or without paw licking and flinching, is defined as the paw withdrawal latency. Each trial is repeated three times at 10-min intervals, and a cutoff time of 60 sec is used to prevent tissue damage.
     
  2. Acetone test. Animals are placed in a transparent plastic cage with small holes on the bottom. They are habituated to the test chamber for at least 30 min before the measurements. Acetone (100 μL) is gently sprayed onto the plantar surface of the hindpaw with an Eppendorf multi-stepper pipette connected to a blunt rubber tube. A brisk foot withdrawal response after the acetone spray is considered a positive response, and the responses are graded on a 4-point scale: 0, no response; 1, brisk withdrawal or flick of the paw; 2, repeated flicking of the paw; 3, repeated flicking of the hindpaw and licking of the paw. The acetone spray is applied five times with an interval of 5 min between each application.

Cognitive Evaluation

These tests assess an animal’s ongoing perception and appraisal of the meaning of what is happening, or what might happen, in relation to a sensation.

Spontaneous/Ongoing Pain

  1. Conditioned place preference test. Custom-built, automated, three-chamber Plexiglas boxes for conditioned place preference (CPP) testing. SMART 3 video-computerized tracking system (Panlab SL, Barcelona, Spain) with two high-resolution video cameras and computer for automated recording, data acquisition and evaluation of animal behavior in a 100-square-foot soundproof chamber.
     
  2. Place escape avoidance test (to be acquired). Like CPP, this test also incorporates the motivational and affective aspects of pain relief, its central readout being based on motivational escape from a noxious stimuli to avoid pain. Animals are placed on a mesh in a Plexiglas chamber where one half of the chamber is transparent (light area) and one half is black-walled (dark area). Animals are allowed unrestricted movement throughout the chamber over a 30-min test period. Immediately after the animals are put in the test chamber they are stimulated with plantar von Frey hair application at a nociceptive intensity every 15 sec. The plantar stimulus is delivered either on the afflicted paw in the dark area or the contralateral, unaffected paw in the lit area. Over time, this conditioning leads to the avoidance of the location in the test box where the stimulus was applied to the more aversive paw.
     
  3. Formalin test. For rats, 50 µL of 1% formalin solution is injected into the dorsal surface of the paw by placing the needle above the toes and below the ankle and inserting it beneath the surface of the skin. For mice, 20 µL of 1% formalin solution is injected under the skin in the middle of the paw on the plantar side. Immediately after injection, animals are placed inside Plexiglas chambers and are video recorded for 1 h for the assessment of paw flinching, paw licking, and paw holding behavior.

Affective-Motivational

These tests assess mood and its relationship to the desire to avoid harm or an expectation of harm.

Anxiety

  1. Elevated plus maze (EPM). This test consists of a plus-shaped apparatus with two open and two enclosed arms, each with an open roof, elevated 40–70 cm from the floor. The model is based on rodents' aversion to open spaces. This aversion leads to the behavior termed thigmotaxis, which involves avoidance of open areas by confining movements to enclosed spaces or to the edges of a bounded space. In EPM, this behavior causes the mice to restrict their movement to the enclosed arms. Anxiety reduction in the plus-maze is indicated by an increase in the proportion of time spent in the open arms (time in open arms/total time in open or closed arms), and an increase in the proportion of entries into the open arms (entries into open arms/total entries into open or closed arms). The behavior testing room is soundproof and light controlled. The animal is placed in the center area of the maze with its head toward the enclosed arm and allowed to move freely for 10 min. The whole test is recorded by a video camera attached to a computer. The number of entries (an entry is defined as the center of mass of the mouse entering the arm) into each arm and the time spent in the open arms are calculated by using SMART 3 software.
     
  2. Open field test. See locomotor function heading below for details.

Depression

  1. Porsolt forced swim test (to be acquired). The Porsolt swim test (PST) was developed as a rodent screening test for potential (human) antidepressant drugs. It is based on the assumption that an animal will try to escape an aversive (stressful) stimulus. If escape is impossible, the animal eventually stops trying and gives up. In the PST, the animal is placed in a cylindrical container of water (24–30°C) from which it cannot escape. Most animals will attempt to escape by actively swimming. When the animal stops swimming and floats on the surface of the water it is considered to have “given up”.  An animal that gives up relatively quickly is thought to be displaying characteristics similar to human depression. The water must be deep enough so the animal cannot touch the bottom with its tail or feet. A depth of 30 cm is commonly recommended, although less depth may be adequate for mice. Animals are subjected to two trials. The first trial lasts 15 min. Then, after 24 h, a second trial is performed that lasts 5 min. The time that the test animal spends in the second trial without making any movements beyond those required to keep its head above water (immobility time) is measured. Animals are allowed to dry in a warm environment after removal from the water. Absorbent towel(s) may be placed in the holding cage to collect water dripping off the animal, and a heating source directed over or underneath the cage may provide warmth.
     
  2. Sucrose preference test (to be acquired). This test can measure the affective state and motivation of subject rodents. It is a reward-based test, as rodents naturally like sweet foods or solutions. Sucrose preference testing is carried out in the animal's home cage. Before the testing period, animals are habituated to the presence of two drinking bottles (one containing 2% sucrose and the other plain water) for 3 days. After this acclimation, animals have the free choice of drinking either the 2% sucrose solution or plain water for a period of 4 days. Water and sucrose solution intake is measured daily, and the positions of the two bottles are switched daily to reduce any confound produced by a side bias. Sucrose preference is calculated as a percentage of the volume of sucrose intake over the total volume of fluid intake and averaged over the 4 days of testing. A bias toward the sweetened drink is typical. Lack of bias toward the sweetened water is indicative of anhedonia/depression.

 

Locomotor Function

  1. Rota-rod testing apparatus. The rota-rod performance test involves forced motor activity by rodents on a rotating rod with accelerating speed. Rodents are trained for 2–3 days on a rota-rod at varying speeds before the final test is conducted. The test measures parameters such as riding time/fall time (seconds), and endurance. The test can be used to evaluate balance, grip strength, and motor coordination of the subjects. In the test, a rodent is placed on a horizontally oriented, rotating cylinder (rod) suspended above a cage floor. The rod is low enough that the animal will not be injured if it falls but high enough to induce avoidance of fall. The length of time that a given animal stays on the rotating rod is a measure of their balance, coordination, physical condition, and motor-planning.
     
  2.  Open field test. The open field test is used to assess behavior of the animals in a novel environment; it also represents a measure of anxiety-like behavior. The apparatus consists of a rectangular box (60 x 40 x 50 cm) inside a soundproof chamber. After the animal is placed in the box, a video camera records its activity for 10 min. Change in open field behavior, demonstrated as an increase or decrease in the number of rears and distance traveled is calculated by SMART 3 software. The advantage of the open field system is that a variety of behavioral parameters related to movement as well as exploration and anxiety are measurable within a short period. It does not require habituation or restraint, is free of reflexive or evoked behaviors, and relies entirely on objective behaviors.
     
  3. Voluntary wheel running activity. Changes in voluntary wheel running may constitute an indicator for daily wellbeing of the animal, which is an important component in the overall picture of chronic pain. Voluntary activity of the mice and rats can be measured inside their cages by using equipment from Bioseb that has an LCD display and attaches to a computer for data generation and analysis. Measurements include the Distance run both ways, the number of wheel turns, average/min/max speed, acceleration, and total time in the wheel.
     
  4. Basso, Beattie, and Bresnahan (BBB) score. Post-injury motor behavior is assessed with the BBB locomotor scale method. The scale (0–21) represents sequential recovery stages and categorizes combinations of rat joint movement, hind limb movements, stepping, forelimb and hind limb coordination, trunk position and stability, paw placement, and tail position. Each rat is assigned to one of three categories, depending on their score:
    1. Early stage (score of 0–7): Composed of isolated joint movements with little or no hind limb movement
    2. Intermediate stage (score of 8–13): Intervals of uncoordinated stepping
    3. Late stage (score of 14–21): Forelimb and hind limb coordination

Because they have natural reflex recovery, rats regain their ability to walk within the days or weeks after injury. The hind limbs are able to generate a spinal reflex arc that allows hind limb stepping without brain involvement.

Pruritic Assay: Assessing Itch Behavior in Rodents

Scratching assay. After the animals are acclimatized, pruritic compounds are injected subcutaneously into the right cheek. Scratching behavior is observed for 30 min. A bout of scratching is defined as continuous scratching movements with hind paws directed to the site of injection.

 

Major Surgical Equipment

  • Surgical microscopes (2, Fisher) and adapted surgical microscope with a multidirectional arm system
  • Small-animal miniVent (type 845, Hugo Sachs Elektronik), and a dual model ventilator (TOPO, Kent Scientific)
  • Micropositioner (Model 660, KOPF), and small-animal spinal unit (Model 980, KOPF)
  • Dual ultra-precise small-animal stereotaxic instrument with mouse and rat adapters (Model 962, 921, KOPF)
  • Isoflurane vaporizer and accessories
  • Bead sterilizer
  • Water blanket (Model 210, PolyScience)
  • MM33B Micromanipulator (F.S.T.)
  • Surgical tools

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