Behavioral Assessments in Preclinical Pain Models

1. Stimulus-Evoked Pain Assessments

Von Frey Test: The Von Frey test is one of the most widely used methods for assessing mechanical allodynia, a condition in which normally non-painful stimuli elicit pain. In this test, researchers use mechanical stimuli from hair-like filaments to apply pressure to the animal’s paw. The animal’s withdrawal response is recorded. This test is particularly useful for evaluating pain sensitivity in models of peripheral nerve injury and other neuropathic pain conditions. 

Tail Flick Test: The tail flick test is a common method for assessing heat-induced pain in rodents. The tail flick test involves exposing the tail to a heat stimulus, and the time it takes for the animal to flick or twitch its tail is measured. The latency time measures the nociceptive sensitivity and can be prolonged using analgesics.

Hot Plate Test: The hot plate test involves placing the animal on a heated surface and measuring the time it takes for the animal to withdraw its paw in response to the heat. While this test provides useful data on pain response to heat, one challenge is variability due to learned behavioral responses from the rodents. 

Cold Pain Testing: Cold-induced pain is a critical factor in pain research, particularly in neuropathic pain conditions. Several methods exist to measure responses to cold stimuli, each with its own advantages and limitations.

• Dry Ice Test: In the dry ice test, rats are placed on a glass surface with dry ice applied near their hind paws (without direct contact) to induce a cold stimulus. Researchers then measure the latency of the rat’s response, such as lifting, licking, or shaking the paw. This method is known for its robustness and reproducibility, as well as its ability to reliably induce a clear response without causing harm to the animal. 
• Acetone Test: The acetone test involves applying a drop of acetone to the animal's paw to evoke a cold sensation. The animal’s response, which may include licking or shaking the paw, is recorded. Although this test is relatively easy to perform, it has a subjective element since the interpretation of the animal's behavior can vary.
• Cold Plate Test: In this test, animals are placed on a cold metal plate and the latency time for paw withdrawal is measured. While effective, the cold plate method can be less reliable due to the potential for uncontrolled hind limb touches, leading to ambiguity in the results.

Video: Dry Ice Test in Rodents

2. Spontaneous Pain Tests

Weight-Bearing / Gait Analysis Tests: Weight-bearing tests are commonly used to measure spontaneous pain in animals. The test measures the distribution of weight across the hind paws where the pressure is measured using independent pressure sensors. Unequal distribution between the ipsilateral and contralateral paws is recorded and used as an indication of the nociception experienced. However, static weight-bearing tests have limitations. The test requires the animals to assume a specific stance for the test to be accurate, and the test can only be used in models with unilateral pain. 

Figure 1: Body load distribution of the symptomatic neuroma pig model. The pig carries more weight on the intact hind leg than on the operated hind leg two months after surgery.

Dynamic Weight-Bearing (DWB) Test: The dynamic weight-bearing (DWB) test can assess weight distribution across all four paws in freely moving animals. By calculating the weight ratio and paw surface area, researchers can determine the extent of pain-related changes in posture and movement. This method is particularly useful in preclinical models of chronic pain, as it offers improved translatability to human pain conditions, where altered weight-bearing is a hallmark of many chronic pain syndromes.

(1) Prevalence of chronic pain among adults in the United States. (2021) Journal for the International Study of Pain

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