Hargreaves apparatus

Animals were habituated to the testing environment daily for at least two days before baseline testing. To assess mechanical allodynia after intrathecal injections of inhibitor or siRNA, animals were put in individual transparent plastic chambers on an elevated wire mesh floor. After 30 min habituation, von Frey filaments (Stoelting, Wood Dale, IL) were applied perpendicular to the plantar surface of each hind paw, starting with the 0.6 g filament. The response to the filament was considered positive if immediate flinching, licking/biting, or rapid withdrawal of the stimulated paw was observed. The 50% paw withdrawal threshold was determined by Dixon’s up-down method [39 (link)]. Heat hyperalgesia was measured by radiant heat using the Hargreaves apparatus (IITC Life Science Inc.), which measures withdrawal latency of the paw from a noxious radiant heat source with baseline latencies of 10–14 s and a cutoff time of 18 s to prevent tissue injury.

Animals were habituated to the testing environment daily for at least two days before baseline testing. The room temperature and humidity remained stable for all experiments. For testing mechanical sensitivity, animals were put in boxes on an elevated metal mesh floor and allowed 30 min for habituation before examination. The plantar surface of each hindpaw was stimulated with a series of von Frey hairs with logarithmically incrementing stiffness (0.02–2.56 g, Stoelting, Wood Dale, IL), presented perpendicular to the plantar surface (2–3 s for each hair, 3 min interval between the tests). The 50% paw withdrawal threshold was determined using Dixon’s up-down method63 (link). For testing heat sensitivity, animals were put in plastic boxes and allowed 30 min for habituation before examination. Heat sensitivity was tested by radiant heat using Hargreaves apparatus (IITC Life Science Inc., Woodland Hills, CA) and expressed as paw withdrawal latency. The test was repeated at least three times/animal allowing at least 5 min in between each test. The radiant heat intensity was adjusted so that basal PWL is between 10 and 14 s, with a cut-off of 18 s to prevent tissue damage8 (link).

To test the mechanical sensitivity of the hind paw ipsilateral plantar surface to the SNL, animals were put in boxes on an elevated metal mesh floor and allowed 30 min for habituation before the examination. The plantar surface of each hind paw was stimulated with a series of von Frey hairs with logarithmically incrementing stiffness (0.02-2.56 g, Stoelting, Wood Dale, IL), presented perpendicular to the plantar surface (2 to 3s for each hair). Dixon’s up-down method determined the 50% paw withdrawal threshold (22 (link)). For testing heat sensitivity, animals were put in plastic boxes and allowed 30 min for habituation. Heat sensitivity was tested by radiant heat using the Hargreaves apparatus (IITC Life Science Inc., Woodland Hills, CA) and expressed as paw withdrawal latency (PWL). The radiant heat intensity was adjusted to 30% of the maximal so that basal PWL is between 10 and 12s, with a cut-off of 20 s to prevent tissue damage (23 (link)).

Before testing, animals were habituated to the Hargreaves apparatus (IITC Life Science Inc., Woodland Hills, CA, USA) [50 (link)] and the experimenter for at least 2 days. The baseline of paw withdrawal latency (PWL) to noxious heat stimulation was tested 1 day before HDAC injection. After 30 min of acclimation on a temperature-controlled glass platform in a clear plastic enclosure, a radiant light was directed towards the plantar surface of the left hind paw. A 20 s cutoff was applied to prevent tissue damage. Three measurements were done for each animal per test session separated by 90 s. The PWL value for baseline was tested the day before CFA injection. After creating the inflammatory lesion, PWL was tested 30, 40, and 50 min, and 1, 2, 3, and 4 h after injection of HDACIs or vehicle for at least 3 days. One additional day for HDACI injection and testing was conducted for some compounds without significant attenuation of thermal hyperalgesia between HDACI and vehicle during the first 3 days. Then, a once daily PWL test was accomplished until inhibition of hyperalgesia was abrogated (Figure 1). The mean value of PWL at several time points each day during the first 3 or 4 days after CFA injection was used to assess the daily change in thermal sensitivity thresholds.