Subjects and study design

Twenty-one subjects were enrolled: 12 subjects (age: 26 ± 9 years, weight: 71 ± 10 kg, height: 180 ± 1 cm; BMI: 22.6 ± 2.7 kg m2) in the IPC group, and 9 subjects (age: 25 ± 11 years, weight: 75 ± 9 kg, height: 181 ± 1 cm; BMI: 23.0 ± 2.8 kg m2) in the SHAM group (3 × 5/5 min pseudo ischemia/reperfusion; 20 mmHg). Two subjects took part to both groups. Participants were eligible for the study if they met the following criteria: healthy individuals with no prior history of medical conditions, non-smokers and those free from any medications that might affect the outcome measures. Participants were excluded if they had any neuromuscular impairments that would prevent them from completing the study procedures. The study was conducted in agreement with the principles of the Declaration of Helsinki (2000) and under the approval of the Ethics Committee of the University of Torino. The subjects gave written informed consent.

Experimental setup

As can be observed in Fig. 1A, an arm rest was designed for implementing isometric stimulated contractions of the thumb adductor of the right hand. Note that the wrist is blocked by a belt, the leftward movement of the index finger is impeded by a wooden bar, the thumb is rigidly and perpendicularly connected to a load cell and the third, fourth and fifth fingers may freely move downward, so that their flexion, elicited by electrical stimulation of the ulnar nerve, does not displace other parts of the hand and interfere with the thumb adduction. The subject sat, with back and arms rested, elbows flexed at about 120 degrees and the shoulder adducted and neutrally rotated.

Fig. 1

A Experimental setup. The right hand rests over a horizontal surface, blocked by a wrist band. A rigid band (a) connects the thumb to a load cell (not visible); one of the sEMG electrodes (b) is located on the medial end of the abductor pollicis, and a near-infrared spectroscopy probe (c) over its lateral end; a temperature probe (d) is also located on the hand dorsum. B Schematic representation of the experimental protocol. A sequence of electrical stimulations of the ulnar nerve (test) is delivered before, during and after the ischemic pre-conditioning (IPC) or placebo (SHAM) treatment. C Electrical stimulation sequence. The stimulation includes four single pulses (twitches), 1 s lasting trains of stimuli at 5, 8, 10 and 12 Hz, and, four bursts consisting of four pulses at 25 Hz, all separated by 5-s intervals

Electrical stimulation protocol

A scheme of the experimental protocol is given in Fig. 1B. The subject was familiarized with the setup and invited to relax during the electrical stimulation protocol. To find the supramaximal intensity of stimulation, the subject received single pulses (1-ms duration) at increasing stimulation intensity, with steps of 5 mA, every 10 s, until both the force and the sEMG signal ceased to increase. That intensity level was further increased by 5 mA to set the supramaximal stimulation level (SSL), which was used for all subsequent stimulations. The stimulation sequence included: four single pulses (twitches), four 1-s lasting trains of stimuli at 5, 8, 10 and 12 Hz and four bursts, each consisting of five pulses at 25 Hz, all separated by 5-s intervals (Fig. 2). This stimulation sequence was delivered before, during the IPC/SHAM treatment (in-between pneumatic compressions, after 3 min of each deflation), and 15 min (POST-15) and 30 min (POST-30) after the treatment. The sequence and timing of stimuli were maintained absolutely identical and not randomized, so that any order effect should be similarly reproduced in all conditions and have no impact in the comparisons. Care was taken to maintain the arm at a constant temperature, with the help of an infrared lamp. After 2 min from the last electrical stimulation sequence, the maximum voluntary contraction (MVC) of the AP was measured as the maximum developed force (averaged over 0.5 s) in three attempts separated by 2-min rest intervals. The MVC measurement was performed at the end rather than at the beginning of the session to avoid the possible confounding effects of post-tetanic potentiation (Baudry and Duchateau 2007).

Fig. 2

Force and sEMG signals. Force and electromyography (EMG) recordings from a representative subject of contractions evoked by the different patterns of stimulation: single twitch, burst and short train stimuli at different frequencies (5, 8, 10 and 12 Hz). The black horizontal bar in each plot represents a duration of 100 ms

IPC/SHAM treatment

The IPC treatment was delivered to the right arm using a pneumatic cuff (The Occlusion Cuff® 8 × 75 cm, The Occlusion Cuff LTD, Somerset, UK). The brachial artery was occluded by rapidly inflating the cuff to 250 mmHg or to 20 mmHg according to the IPC or SHAM treatment for 5 min. This stimulus was repeated two other times, separated by 5-min rest intervals. Rapid cuff inflation and deflation was achieved by a custom system based on a PC-driven proportional valve (ITV1010, RTI Srl, Torino, Italy) (Ferraresi et al. 2019; Messere et al. 2018).

Temperature, blood pressure and hemodynamic variables

During the protocol, the cutaneous temperature (TEMP) was continuously monitored from the back of the hand (TERZMI-I, Terzano & C Srl, Milano, Italy). Arterial blood pressure (ABP; diastolic ABPd and systolic ABPs) was measured before, 5 min and 30 min after the treatment through a digital sphygmomanometer (CNSystems, Medizintechnik GmbH, Graz, Austria). The non-invasive monitoring of muscle oxygenation was performed using near-infrared spectroscopy (NIRS) (NIRO-200X, Hamamatsu Photonics K.K, Shizuoka, Japan). Two NIRS probes, with an inter-optode distance of 3 cm, were positioned over the dorsal side of the AP muscle (see Fig. 1A) and over the anterior side of the forearm. The device measures the tissue oxygenation index (TOI), which represents the oxygen-saturated (hemoglobin + myoglobin) level in percentage, and the tissue hemoglobin index (THI), which represents the total (hemoglobin + myoglobin) concentration in arbitrary units. These parameters are based on the spatially resolved methodology, which focuses on the measurement in depth (muscle tissue) and, thus, it is little affected by hemodynamic changes occurring in the superficial cutaneous tissue layer (Messere and Roatta 2013, 2015).

Electrical stimulation and EMG recording

Contraction of the AP was obtained by bipolar electrical stimulation of the ulnar nerve (DS7A, Digitimer Ltd in Welwyn Garden City, England) using two electrodes (Euro Ecg Eletrodes, Fiab Srl, Florence, Italy) located 5 and 7 cm proximal to the wrist. The other two electrodes were cut following the round shape (1 cm) and placed at a distance at 2 cm, located about 5 cm proximal to the wrist. Surface electromyographic activity of the AP was bipolarly recorded (QUATTRO, OT Bioelettronica Srl, Turin, Italy) with one electrode placed on the dorsal side of the AP, close to the NIRS probe and the other two placed over the second metacarpal bone. The skin was previously cleaned using an abrasive prepping gel.

Force recording

The force developed by the electrically stimulated contractions of the AP was recorded by a load cell (DYLY-103-5kg, CaltSensor Ltd, Shanghai, China) and a dedicated amplifier (Forza-B, OT Bioelettronica Srl, Turin, Italy). The load cell was connected to the thumb by a rigid strap. The length of the strap was adjusted to develop a slight tension, of about 2 N, in the passive muscle.

All signals were digitally sampled (1401micro, CED, Cambridge, UK) at a sampling frequency of 1024 Hz, except for the surface electromyographic signal (sEMG), sampled at 2 kHz, and stored on PC for off-line analysis (Spike2, CED, Cambridge, UK). Data acquisition was continuously performed throughout the whole experimental session.