2.2.2. Behavioral Tests to Assess Pain in OA Animal Models

There are many approaches for assessing the behaviors associated with OA pain in animal models [48]. The most common behavioral assays are the von Frey, weight bearing, hot plate, rotarod, and spontaneous pain-related behavior tests. The von Frey test is used to evaluate mechanical allodynia in mice and rats [49]. Briefly, the rodents are placed individually on a mesh grid and calibrated von Frey filaments are applied to the hind paw in ascending order of size, until the fiber bends by force. The animal response, including withdrawal and shaking or licking the paw, is considered a positive pain response [50]. Static weight bearing is assessed using an incapacitance meter, and the average weight bearing applied to the hind paw between the injured limb and contralateral limb is measured [50]. A recent study showed that the positioning of the animal in the incapacitance test may affect its weight distribution, so care should be taken to minimize the influence of positioning [51]. Catwalk automated gait analyses measure changes in spontaneous gait due to joint pain. Animals pass an illuminated glass platform while gait is recorded with a video camera connected to a computer, and the changes in gait pattern, such as swing speed and stride length, are analyzed [52]. Thermal hyperalgesia is assessed using the hot plate test, where the rodent is placed on a metal surface maintained at a constant temperature. The response time (latency) is measured until licking or shaking of the ipsilateral hind paw and jumping on a hot plate occur [53]. Testing latency should be performed without incurring tissue damage and each trial should be separated by an interval of at least 10 min [54]. The rotarod test is used to evaluate changes in motor skill. The rodent is placed on a rotarod machine with automatic timers and falling sensors, and the rod speed is accelerated gradually from 0 to 40 rpm/min [55]. The moving time until falling off is measured. The number of trials in hot plate and rotarod tests should be minimized to exclude learning effects [48,55]. Finally, the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS) test measures the spontaneous pain-related behavior of rodents, including ambulation, and exploratory behavior. Animals are housed in the LABORAS cage and their behaviors are recorded for 2–12 h [56,57]. For all pain measurement tests, the influence of stress, such as laboratory environment and testing duration, on the pain response should be taken into consideration.

It is notable that, until recently, the most commonly used induction methods to study OA pain were distinct from those most often used to investigate the pathophysiology and regulation of structural joint damage [36]. DMM and MIA models are the most commonly used models in pain research and show mechanical, thermal, and spontaneous pain behavior occurring at the onset of OA [58,59]. In general, chemically induced models exhibit pain responses evoked with von Frey, incapacitance, and hot plate tests more quickly than surgically induced models. Therefore, pain responses in the MIA model appear rapidly within 1 week post-injection [50,60–64], while more insidious responses are observed in the DMM model. In one study, STR/ort mice, which develop spontaneous OA, did not show any signs of pain, such as mechanical allodynia, cold sensitivity, or joint compression-related vocalization, with the development of OA [65]. This is reminiscent of human OA, in which pain does not develop, despite advanced joint damage. On the other hand, mechanical joint loading-induced OA animals develop a robust pain phenotype, except for thermal hyperalgesia [44]. Table 1 briefly summarizes the pathology and pain behaviors reported in OA models [38,65–90].


 **1.** Common OA models and reported pain behaviors.

**Table**

a disintegrin and

of interferon regulatory factor 4; NGF, nerve growth factor.

metalloproteinase

 with

thrombospondin

 motifs 5; RANKL, receptor activator of nuclear factor kappa-B ligand; CCL-17, chemokine (c-c motif) ligand 17; IRF-4,
