Origin v6

Mechanical and thermal pain thresholds were measured in a quiet room between 09:00 and 11:00. Rats were allowed to acclimatize to surroundings for 2 h in a set of Plexiglas^TM cages with wire mesh floor. Von Frey filaments (North Coast Medical, Gilroy, CA, United States) of increasing stiffness were applied to six sites distributed across the plantar surface of the rat hindpaw until the filaments bent slightly. A positive withdrawal was scored if rat showed a brisk withdrawal. Eight von Frey monofilaments (0.5, 1, 2, 4, 6, 8, 10, and 12 g) were used in testing. Pain threshold was defined as the force corresponding to a 50% withdrawal, and was determined by a Hill equation linear fitting [Origin v6.0 (MicroCal, Studio City, CA, United States)] (Xiang et al., 2008 (link)). Thermal threshold was undertaken in individual cages with a transparent acrylic floor. After acclimatization, the heating device was placed under the hindpaw at certain heat intensity and a cut-off time of 30 s to avoid tissue damage. The withdrawal latency of the hindpaw was detected thrice for each rat at an interval of 5 min, and the mean value was taken as the thermal threshold.

Latency (ms), peak-to-peak amplitude (mV; not reported), total area (mV•ms) and area of the terminal phase (mV•ms) of the recorded M_max130 were extracted for analysis. Latency was measured between the stimulus event and the beginning of the first positive phase of M_max130 (Fig. 3b). Total M_max130 area was calculated from the start of the first positive phase of M_max130 to the end of the terminal phase in the action potential (Fig. 3b). The area of the terminal phase (M_max130TP) was calculated in order to infer possible intervention-induced changes in action potential propagation along the muscle fibre membrane [31 (link)].
AH excitability was also quantified by calculating the intensity (mA) required to elicit an M_wave peak-to-peak amplitude equivalent to 50% of the M_max (AH_MT@50) by way of a sigmoidal fit function (Boltzman plot, Origin v6.0, Microcal Software Inc., USA) applied to the M_wave recruitment curve at each testing period in the protocol (Fig. 3c).