Behavioral measurements of sensory and motor function
Von Frey assay of mechanical sensitivity. An electronic Von Frey device (Bioseb, Model: BIO-EVF4) was used to assess mechanical sensitivity in rats before and after paw incision injury. Animals were habituated for 1 hour, one day prior to baseline testing. Animals were given 30 minutes to settle before testing. An average mechanical threshold was calculated using 5 measurements taken 5 minutes apart for each animal. For baseline measurements two testing sessions were performed on separate days prior to injury and averaged together. 50 µL of BW-031 or saline were administered into the plantar region of the hind paw adjacent to the incision 24 hours post injury. Animals were then tested 1, 3, 5, and 24 hours post treatment. Additional timepoints were added at 7 and 9 hours for higher concentrations of treatments.
A manual Von Frey assay was used to assess mechanical sensitivity in mice before and after UV burn, as previously described (Lee et al., 2019). After mice were habituated to the testing cage (7.5 × 7.5 × 15 cm) with a metal grid floor for 45 min for 2 days, baseline values were measured using nine von Frey filaments with different bending forces (0.04, 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2, and 4 g). The response patterns were collected and converted into corresponding 50% withdrawal thresholds using the Up-Down Reader software and associated protocol (Gonzalez-Cano et al., 2018). Based on the baseline measurement, mice were assigned to three groups so that the baseline mechanical sensitivity among the groups was similar. Each group consisted of 10 mice, based on previous experiments showing sufficient power to detect significance with 95% confidence. Twenty-four hours after UV irradiation, mice received a 10-μL bolus intraplantar injection of either 2% BW-031, 2% QX-314, or vehicle (normal saline) to the irradiated paw. The von Frey test was performed at 1, 3, 5, 7, and 24 hours after the drug injection.
Radiant heat assay of thermal sensitivity. Thermal hypersensitivity was measured using the plantar radiant heat test (Hargreaves et al., 1988) (Ugo Basile, Model code: 37370) in CFA injected rats. Rats were habituated to testing enclosures for 1 hour one day prior to baseline testing. Rats were given 30 minutes to settle before testing. An average paw withdrawal latency was calculated using 3 measurements taken 5 minutes apart. Animals were tested 1, 4, and 24 hours after injury from CFA injection.
Toe spread assay of motor function. Mouse toe movement was evaluated in the ipsilateral hind-paws as previously described (Ma et al., 2011) in order to assess the presence of motor block after peri-sciatic injection of lidocaine or charged sodium channel blockers. Briefly, 5 minutes after peri-sciatic injection, mice were lifted by the tail, uncovering the hind paws for clear observation. Under this condition, the digits spread, maximizing the space between them (the toe spreading reflex). This reflex was scored as previously described: 0, no spreading; 1, intermediate spreading with all toes; and 2, full spreading. Full toe spreading was defined as a complete, wide, and sustained (at least 2 seconds) spreading of the toes. Full toe spreading was observed in the contralateral paws for all mice tested.
Pinprick assay of sensory function. Mouse responses to pinprick were measured as previously described (Ma et al., 2011), with modifications. Mice were placed in wire mesh cages and habituated for 3 sessions prior to peri-sciatic injection. After peri-sciatic injection and measurement of motor function, mice were immediately placed in wire mesh cages and an Austerlitz insect pin (size 000) (FST, USA) was gently applied to the plantar surface of the paw without moving the paw or penetrating the skin. The pinprick was applied three times to the sole of the ipsilateral hind paw and three times to the sole of the contralateral hind paw. A response was considered positive (1) when the animal briskly removed its paw. If none of the applications elicited a positive response, the overall grade was 0.
Blinding. All behavioral measurements of sensory and motor function were performed by investigators blinded to the drug treatment; the test order was randomized with multiple groups being represented in each cage.