A pain is a subjective symptom and it is told that other people cannot understand how painful it is. It is hard to explain our painful states objectively but we can verbally complain our pains. Some animals also vocalize but we cannot understand their words. Evaluation of pains in rodents is conducted by observation of their behaviors respond to painful stimuli (nociceptive stimuli). For example, in a typical method with a hot plate we observe behaviors in rodents respond to the heated hot plate. As nociceptive stimuli, pressure stimulation such as tail pinch, chemical nociception such as intraperitoneal injection of acetic acid, surgical nociception and so on as well as thermal nociception are used. If we evaluate pains in rodents by their responses to these stimuli as parameters, analgesics suppress such behavioral changes. It is namely one of evidences that we can evaluate pains in these methods, which clinically potent drugs suppressed the behaviors induced by nociceptive stimuli. We introduce below actual models in rodents evaluating pains.

5-1 Thermal Stimulation

5-1-1 Hot Plate

We place rodents on the hot plate keeping a high temperature and measure a time (latency) until they show responses such as behaviors lifting and licking their tails and paws to thermal stimuli. We measure the latencies until the first escape behaviors on the hot plate with a constant high temperature (e.g. 50 - 55 degree C) or gradually increase the temperature of the hot plate and measure the temperature to induce the escape responses. Mice and rats can freely move in a cage on the hot plate and we manually observe and evaluate the escape behaviors. Because responses to the thermal stimuli may be suppressed by drugs such as analgesics, it is important that we determine a maximal observation time (e.g. 1 min) and a maximal temperature of the thermal stimuli to protect tissues from heat damages by over stimulation.

5-1-2 Tail flick

Many rodents lift their tails to thermal stimuli, which is called as a tail flick. An evaluation method of pains by observation of this behavior to thermal stimulation of tails in rodents is called as a tail flick method. Some devices can automatically detect the tail flick by infrared beam sensors and record a time and a temperature (latency) until the tail flick is appeared. Because we need to immobilize the rodent on the device for stimulation and detection of the tail flick, we must consider a change of an analgesic threshold by immobilization stresses.

5-1-3 Paw flick (Hargreaves plantar test)

Rodents usually lift and lick their paws to thermal plantar stimuli, which are also added on the hot plate. A measurement method that the thermal stimulus is added to a paw from a sole by a hot beam and a paw flick behavior is automatically detected by a sensor is called as a paw flick test or a Hargreaves planter test. Although rodents are not restrained for measurement, we project the hot beam when rodents do not move after an end of exploratory behaviors to hit surely the target. Because effects of the hot beam stimulation will diminish wet paws in rodents, we must take care that the rodents' paws do not wet with urine and so on.

5-2 Pressure Stimulation

5-2-1 Tail Pinch

We evaluate pains in rodents by a score based on behaviors that they wave and lick a tail pinched with tweezers and so on. It is a simple method and does not need expensive instruments but is difficult to evaluate objectively a level of pains. We should give the same level of stimulation to each rodent using by instruments which is able to pinch with a constant pressure such as clamp, forceps, and clothespins. Because a stimulus level may different by pinching sites whether distal or proximal portions of the tail, we must pinch the constant site.

5-2-2 Randall Sellito

We hold a rat or a mouse, add a pressure on a hind paw with a constant speed and measure a threshold to show escape behaviors such as a paw flick in a Randall Sellito test. We give the pressure on a tail as well as a paw in some mice. We can continuously add the pressure in rodents with a measurement device until the escape behavior which is turned off with a foot switch and automatically measure the pressure level. We must consider a change of an analgesic threshold by immobilization stresses because we restrain the animal like the tail flick in this method.

5-2-3 von Frey

We vertically press filaments with various diameters called as von Frey filaments or von Frey hairs into a sole of a paw in rodents until the filaments bend, and observe whether they show escape behaviors (paw movements) or not. Because pressures to bend the filaments are different depend on their diameters, we can guess what pressures the rodents feel pains by determination of the filament induced escape behaviors. The pressure stimulation by the filament is usually weak not to feel pains and it is used to evaluate allodynia. We can conduct a von Frey test by manual observation with a mesh cage that we can stimulate a rat or mouse from a bottom and von Frey filaments but an automatic measuring device of a pressure threshold to show escape behaviors is available.

It is named as an electronic von Frey anesthesiometer or dynamic plantar anesthesiometer by device makers.

5-3 Chemical Nociceptive Stimuli

5-3-1 Acetic Acid Writhing Test

We administer 0.6% acetic acid intraperitoneally in mice at a volume of 0.1 mL/10 g and count writhing (behaviors pressing their abdominal site to a floor and stretching their body) by intraperitoneal acidic inflammation for c.a. 10 min. Because we manually observe the writhing, the acetic acid writhing test is simple and does not need special devices. However, it is currently few used due to animal welfare.

5-3-2 Formalin Paw Edema

We subcutaneously inject 1.5-5% formalin in a hind paw of mice or rats at a volume of 0.05-0.1 mL and observe their behaviors such as licking and biting the paw injected and flinching. Biphasic responses are observed in first phase by formalin stimulation and later in second phase by inflammation. We can observe licking and biting for c.a. 30 min immediately after an injection of 10 mL of 5% formalin in a hind paw plantar of a mouse. We count such behaviors and measure duration of these behaviors (c.a. 30-60 min). Because we can manually observe the behaviors, we do not need the measuring devices except for a stop watch.

5-3-3 Carrageenan Paw Edema

We subcutaneously inject 1% carrageenan in a hind paw of mice or rats at a volume of 0.1 mL (or 0.5mL of 0.1% carrageenan) and measure a volume of hind paws 4 hrs after the injection. We guess that inflammation induces pains and evaluate edema as inflammatory parameter but cannot observe acute pain responses. The inflammation gradually progresses for several hours and continue about 1 day. If hyperalgesia is formed by the inflammation in rodents, we can observe hypersensitivity to stimulation by von Frey filaments and so on. A volume of the edema can be measured by a plethysmometer.

5-3-4 Adjuvant Arthritis

We intradermally inject complete Freund's adjuvant (CFA: killed mycobacterium cell suspension in paraffin oil) in a hind footpad of mice or rats and induce arthritis. The inflammatory edema is evaluated by its volume with plethysmometer in the similar way as the carrageenan paw edema compared to secondary inflammation in a contralateral paw. A swelling level of the edema may be scored or measured by a caliper. CFA is may be administered in synovial cavity of knee joint and intra-articular injection in an ankle or intradermal injection in a tail is also possible. Arthritis is evaluated for from several hours to c.a. 4 weeks after the injection. Inflammation-induced hyperalgesia is measured by the von Frey test or the Hargreaves plantar test described above.

5-4 Weight Bearing Method

We evaluate surgical pains in rodents by surgical operation such as tendinous and intraperitoneal incisions under anesthesia and behavioral observation of recoveries from anesthesia. If we conduct the operation to one limb in rodents, they walk protecting that limb. If intraperitoneal incision induces pains, they press abdominal sites on a floor. We usually conduct megascopic evaluation of these behaviors and can consider that those disappear as diminishing pains. We are difficult to evaluate pain levels objectively by the manual observation but can measure the behaviors protecting one painful limb in a weight bearing method as digits. The rodents behave to relief the limb pains by their attitudes that body weights are not loaded on the painful limb. We measure a lord on each limb separately and evaluate an unbalance between the both lords as a pain parameter. Its measuring device is sometime called as an incapacitance tester, which can measure each lord on right and left hindlimbs by 2 weight sensors. A shape of the device cage is created that the rodents do not place forelimbs on the weight sensors and the lord is measured under freely moving states. We add pains on one limb in rodents using by inflammation by carrageenan or adjuvant as well as surgical operations described above.

The weight bearing method is remarkable on a point that it can measure painful states in rodents without direct stimuli such as thermal or pressure nociception.

5-5 Itch Measurement Method

We briefly introduce an itch measurement method because itches are transmitted to cortical brains via the same nerve fibers (C fibers) for pains. Since rodents show scratching behaviors by itches, we measure and evaluate the scratching behaviors as itches. The scratching behaviors are measured by manual observation, PC analysis of video tracking with a high speed-shooting camera, and changes of magnetic fields by a magnet subcutaneously implanted in a hindlimb. In the last method scratching by the hindlimb with the magnet changes magnetic fields and we can count scratching by the automatic measuring device (1).

5-6 Closing

Many methods in rodents to evaluate pains and analgesics measure responses to nociceptive stimuli and must be not enough as an animal model to evaluate pains by various human diseases. Evaluation of inflammatory pains by chemical nociception is close to a human pathological condition but an inflammatory area is limited in a limb except for the acetic acid writhing test and cannot become an animal model for headaches or cancer pains which need analgesics in humans. The animal model for pains is difficult on a point that we objectively evaluate subjective symptoms and it may be impossible because we cannot understand the animal's words. On this point of view the weight bearing method supplies a remarkable model measuring behaviors protecting a painful limb by subjective pains as objective values. When we can clarify behaviors induced by pains of various pathological models in rodents, we may get novel animal models for pains.