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.