2.1 Animals and experimental materials
Our procedures on rabbits obeyed the guidelines for humane treatment, which were set by Animal Ethics Committee of Anhui Medical University (LLSC20190761). A total number of 48 male skeletally mature New Zealand white rabbits (aged 3-4 months; weight 2-2.5 kg) were purchased from the Experimental Animal Center of Anhui Medical University. The rabbits were individually reared in a cage of 60×50×40 cm3, with an ambient temperature of 24°C, and a light-dark cycle for 12 hours. Rabbits had unlimited activities in cages with enough food and water. All rabbits were fed with a standard rabbit diet for two weeks before the experiment.
Electronic Acupuncture Treatment Instrument (SDZ-Ⅳ type) was purchased from Hwato Co (Suzhou, China). The joint range of motion measuring instrument (ZL201720251124.6) with utility model patent was designed by our research group. Antibodies against Atg7(ab133528), LC3B-I/II (ab243506) and p62 (ab56416) were bought from Abcam (Cambridge, MA). p-mTOR (5536S) was bought from Cell Signaling Technology (Beverley, MA). GAPDH (F2612) antibody was bought from Santa Cruz Biotechnologies. Thiazolyl Blue Tetrazolium Bromide (MTT, M8180) was purchased from Solarbio (Beijing, China). Chemiluminescence (ECL) detection kit was from Thermo Scientific (Massachusetts, US).
2.2 Grouping and intervention measures
The whole experiment was divided into two parts. In the first part of the experiment, to explore the effects of immobilization on skeletal muscle autophagy, disuse muscle atrophy and joint contracture, 24 rabbits were randomly divided into control 1 group (Ctrl1 group), immobilization for 2 weeks group (I-2 group), immobilization for 4 weeks group (I-4 group) and immobilization for 6 weeks group (I-6 group), each group has 6 animals. Three groups of rabbits in need of immobilization were anesthetized by injection of 30 mg/kg sodium pentobarbital through ear vein and the left knee joints were fixed in extension. In the Ctrl1 group, the rabbits moved freely for 6 weeks; in the I-2, I-4, and I-6 groups, plaster casts were used to immobilize the rabbit knee joint from the groin to the proximal interphalangeal joint at full extension posture , removing the tubular plaster at the end of each fixed time point.
In the second part of the experiment, in order to study the therapeutic effect and mechanism of low-frequency electrical stimulation on disuse muscle atrophy and joint function, 24 rabbits were randomly divided into control 2 group (Ctrl2 group), natural recovery group (NR group), electrical stimulation treatment group (EST group) and pure electrical stimulation group (ES group), with 6 rabbits in each group. (A table with group characteristics is in the Supplementary Table S1 section.) In the Ctrl2 group, the rabbits were free to move for 7 weeks; in the ES group, the rabbits were free to move for 4 weeks, followed by 10 Hz low-frequency electrical stimulation treatment for 3 weeks; in the NR group, the rabbits’ left knee joint were fixed as described above, the plaster was removed after 4 weeks of immobilization, followed by natural recovery for 3 weeks; in the EST group, the left knee joint of the rabbit was fixed for 4 weeks and then the plaster was removed, followed by low-frequency electrical stimulation at 10 Hz for 20 min a day for 3 weeks.
2.3 Low frequency electrical stimulation treatment
Each rabbit in the ES and EST groups received 3 weeks of 10Hz low-frequency electrical stimulation once a day for 20 minutes with an Electronic Acupuncture Treatment Instrument (SDZ-Ⅳ). The intervention site of electrical stimulation was the quadriceps femoris of the left hind limb of the rabbit. First, the hair of the left hind leg was shaved off, and then two 3 × 3 cm² non-woven silica gel electrode sheets were attached to the skin on the front side of the left hind leg. The distance between the two electrodes was 0.5 cm. The output current of electronic acupuncture instrument was less than 10mA. We adjust the size of the output current to cause the quadriceps muscle contraction, but not to cause the rabbit's strong resistance. A current of 5 mA could cause obvious muscle contraction without the rabbit’s excessive struggling in our preliminary experiment, so the current was set to 5 mA in our former experiment. The electronic acupuncture instrument worked in an intermittent wave mode with a pulse duration of 15 sec and a pause time of 5 sec.
2.4 Tissue preparation and joint ROM measurement
Each rabbit’s left hind limb was dislocated at the left hip joint after euthanasia with an overdose of sodium pentobarbital via an auricular vein, this method of euthanasia was approved by Animal Ethics Committee of Anhui Medical University (LLSC20190761). The starting point of the thigh muscles at the hip joint was cut off, and the left hind limb was completely detached from the torso. As in previous experiment, a joint range of motion measuring instrument was used to measure the range of motion of the left knee joint . The proximal end of femur, the proximal end and the distal end of tibia were fixed on the arthrometer with a metal clamp. All knee joints started at 0° flexion position before applying the force. The driving wheel rotated to drive the dial to rotate, and the tibia rotated indirectly, and the femur remained motionless. Since the radius of the dial is fixed, the torque applied can be calculated by multiplying the force with the dial constant radius. In our previous experiment, by measuring the range of motion of the normal rabbits, it was found that the torque of 0.077 N.m could pull the knee joint of rabbits to about 140 degrees of buckling. After this, even though the torque continued to increase the bending angle of knee joint was difficult to increase. Therefore, we used 0.077 N.m as the standard torque to measure the knee joint ROM. The grouping information of rabbits was kept secret from the surveyors. The ROM measurements were made by 2 surveyors and repeated 3 times for each rabbit. The surveyors kept their measurements secret from each other and the buckling angle of each rabbit was the average of six measurements. (A table with contracture angle is in the Supplementary Table S2 section.)
Subsequently, three muscle tissue specimens were removed from the middle of the separated rectus femoris muscle, about 1×1×0.5 cm3 in size, two samples were used for hematoxylin-eosin and immunofluorescence staining, and the other sample was stored in a refrigerator at -80°C for the detection of skeletal muscle autophagy proteins.
2.5 Hematoxylin and eosin (H&E) staining
Rabbit rectus femoris tissues were fixed with 4% paraformaldehyde (PFA) and then embedded in paraffin. The rectus femoris sections were stained using hematoxylin and eosin (H&E) staining. In the case of magnification of 400 times, the cross section of the rectus femoris muscle was photographed with Nikon TE2000-U microscope (Nikon Corporation, Tokyo, Japan), and 4 fields were randomly selected for each HE staining section. Image-Pro Plus 6.0 software was used to count the number of muscle fibers and the total area of muscle fibers in each field. The average muscle fiber area under each field was statistically analyzed with SPSS 23.0. (A table with total number of myofiber cells is in the Supplementary Table S2 section.)
2.6 Western blotting
Weighed the rectus femoris muscle approximately 70-80 mg and added 600µl lysis buffer to each sample which was a 100:1 proportion of RIPA and PMSF. The homogenized tissue sample was transferred to a 1.5 ml EP tube, and the EP tube was put into a centrifuge (4℃). The tube was centrifuged at 12,000 g of centrifugal force for 15min. After centrifugation, 300µl of the intermediate layer clear liquid was absorbed to prepare for the subsequent protein quantification of BCA method. The antibody diluent we used was 5% skimmed milk (5g powdered milk plus 100ml TBST). The loading volume of the sample is determined according to the expression intensity of the protein. For the reference protein with relatively stable expression, such as GAPDH, the loading volume of 8µg is sufficient; however, for the target protein with relatively weak expression, such as LC3B, the loading volume usually needs to reach 75µg. For other target proteins, the loading volume of the sample is determined according to the expression intensity of the protein. Total lysate was separated with 12.5% SDS-PAGE electrophoresis buffer and then transferred onto PVDF membranes. The membranes were first sealed with milk for 1.5h, and then incubated with primary antibodies for 1-2h. GAPDH was applied as a loading control. After being washed, the membranes were incubated with secondary antibodies which were conjugated to HRP for 90 minutes. After being washed, the enhanced chemiluminescence reagent was used for development. The signal was then detected with a digital imaging equipment.
Thin sections (10 µm) of rectus femoris were fixed for 1 h with 4% PFA. Nonspecific binding sites in the slides were blocked using 10% normal goat serum. The slides were incubated for 2 hours with LC3B (1:200) at 37℃. The slides were incubated by the Alexa Fluor 488 conjugated secondary antibody (711-545-152, Jackson Immuno Research) for 90 min after PBS washing. The sections were stained with DAPI (C1002, Beyotime) for 5 min. All sections were then mounted and observed using a fluorescence microscope (BX53F, Olympus) under a 400x magnification field. Four fields were randomly selected and photographed for each slice. LC3B positive points in each visual field were counted out for statistical analysis. The mean value of green fluorescence points in individual muscle fiber under each field of vision was calculated.
2.8 Statistical analysis
Mean ± SD was used to present the quantified data. All data were entered and analyzed in SPSS (version 23.0). Mean differences among groups of rabbits in (1) range of motion, (2) cross-sectional area of the RF, and (3) expression of p-mTOR, Atg7, LC3B-I/II and p62 proteins associated with muscle autophagy, were assessed using one-way analysis of variance. When analysis of variance detected differences, Bonferroni or Tamhane’s T2 tests were used to assess multiple comparisons between groups. To examine all pairwise comparisons among groups of rabbits. P <0.05 indicated that the difference was statistically significant.