Pl3516

Handgrip force was sampled at 1,000 Hz using a grip force transducer (MLT004/ST, AD Instruments) and a data acquisition device (PL3516, AD Instruments). Sampled data were smoothed by an online low-pass filter with a cut-off frequency of 20 Hz using LabChart software (LabChart 8, AD Instruments). Maximal handgrip force was determined as the highest value. Furthermore, the maximal handgrip force in Test 1 and 2 were averaged and the BLD was calculated using the following equation.
Surface electromyography (EMG) signals were recorded from the flexor digitorum superficialis using a wireless EMG sensor (pico, cometa). On the basis of a previous study (Kong et al., 2010 (link)), disposable Ag/AgCl electrodes were placed slightly ulnarly on the line between the oblique line of radius and the second middle phalanx at 1/4 from the oblique line of the radius. The signals were sampled at 1,000 Hz using a data acquisition device (PL3516, AD Instruments) and filtered with a band-pass filter (10–500 Hz). Recorded data were rectified and smoothed with a fourth order zero-lag Butterworth low-pass filter with a cut-off frequency of 10 Hz. Integrated EMG (IEMG) was calculated as 125 ms around the maximal handgrip force.

The temperature in three regions (rectum, dorsal skin, and tail skin) and the blood flow (BF) in two regions (dorsal skin and tail skin) were measured in real time using fiber-optic thermometers (FL-2400, Anritsu Meter Co., Ltd., Japan) and Doppler blood flow meters (FLO-C1 TWIN, Omegawave, Tokyo, Japan), respectively. The animals were anesthetized with isoflurane (3%) in 100% oxygen (Narcobit-E, Natsume Seisakusho Co., Ltd., Japan) for placement in an acrylic holder (Rev1, Kyoto Jushi-Seiko, Co., Ltd., Japan) and to set up the following probes: one thermometer probe was inserted into the rectum (2 cm from the anus), and the other two thermometer probes and BF-meter probes were placed in contact with the dorsal skin (below the antenna) and the base of the tail. All probes were made of non-metallic materials. Analog data acquired from the fiber-optic thermometers and Doppler BF meter were digitized using an A/D converter (PL3516, ADInstruments Ltd., New Zealand) at a sampling rate of 200 Hz (20 (link)). Because the Doppler BF meter was unable to measure the absolute flow values, the BF was revealed as a value relative to the reference response. This is displayed as the amount of change, with the value at the beginning of the exposure as 100%. BF data were analyzed after removing the upper 0.01 Hz with a low-pass filter.

As in the previous surgical procedure (Wen et al., 2018 (link)), the skin was cut along the medial border of the scapula. The brachial plexus was exposed after the muscle tissue was isolated. Phrenic nerve was found deep in the brachial plexus and dissociated with glass minute needles. Two Bipolar protective stimulation electrodes for transmitting stimulation signals were hung on the phrenic nerves on both sides and fixed with medical tape. The positive and negative output ends of an electrode wire were connected to the stimulator. Outputs of two stimulators were recorded by the physiological recorder (PL3516, PowerLab 16/35, ADInstruments, Sydney, Australia) to verify the effectiveness of stimulation signals sent to the phrenic nerve (Figure 1).