This experimental protocol was approved by the Institutional Animal Care and Use Committee of Anhui Medical University. A total of 40 male skeleton mature New Zealand white rabbits were used in this study (Anhui Medical University Experimental Animal Center, Hefei, China, age 3-4 months, weight 2-2.5 kg). The rabbit which were maintained in 60 cm × 50 cm × 40 cm cages, were exposed to a 12-h light–dark cycle at an ambient temperature of 24 °C, then allowed free activities in cages and free access to water and food. All of them were anesthetized by ear intravenous administration of 30mg/kg sodium pentobarbital. Then 35 rabbits underwent unilateral immobilization of a knee joint at full extension using a plaster cast from groin to proximal toes as the previous research (Figure 1A), 11 and 5 rabbits were used for control group corresponding to C.
After 8 weeks immobilization, the plaster cast of the left knee joint was removed. Then 35 rabbits were randomly divided by random number table method into 7 groups, 5 rabbits in each group, corresponding to I-8, R-1, R-2, R-4, T-1, T-2, and T-4.
In Group C, the rabbits did not undergo immobilization, and the rabbits only underwent 8 weeks immobilization in Group I-8. In addition, there were two intervention methods about self-recovery and ultrashort wave treatment. Specifically, Group R-1, R-2 and R-4 only underwent self-recovery for 1, 2, 4 weeks respectively. Relatively, Group T-1, T-2 and T-4 not only experienced self-recovery, but also experienced ultrashort wave treatment. For instance, each animal that needed to receive ultrashort wave treatment was placed on the floor and the experimental knee joint was exposed to the electrodes of ultrashort wave treatment equipment. After the equipment was preheated, the mode of the ultrashort wave treatment equipment was transferred to micro heat and the time of treatment was set to 15 mins, once a day (Figure 1B).
Tissue sampling and H&E
At the end of each time point, the rabbits were euthanized with an excess of sodium pentobarbital. The left hind limb was dislocated at the left hip joint and completely removed. Then ROM of the left knee joint was measured by the joint motion measuring instrument as the previous experiment. Later, two muscle tissues of 1 cm × 1 cm × 0.5 cm in size were cut in the middle of the rectus femoris. One of them was used for H&E staining, and the other was frozen in liquid nitrogen at -80 °C until histological analysis.
Rectus femoris were stained with Hematoxylin and Eosin (H&E), and the CSA of individual myofibers was photographed using Nikon TE2000-U inverted microscope (Nikon Corporation, Tokyo, Japan) and measured using Image-Pro Plus (IPP) 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, USA). Six randomly selected fields of view were analyzed in each group.
Calculation of the total and the myogenic contracture
Like our previous study, ROM of the left knee joint was measured by the joint motion measuring instrument. 11,12 According to the method described by Trudel G,13 we evaluated myogenic contracture caused by the muscular structures including their tendons and fascia, and arthrogenic contracture caused by the articular structures including bone, cartilage, synovium, capsule, and ligaments (Figure 2). The formulas used were as follows:
- Decrement in ROM as a result of total contracture = ROM before myotomy (of the control knee) − ROM before myotomy (of the contractured knee).
- Decrement in ROM as a result of arthrogenic contracture = ROM after myotomy (of the control knee) - ROM after myotomy (of the contractured knee).
- Decrement in ROM as a result of myogenic contracture = Decrement in ROM as a result of total contracture − Decrement in ROM as a result of arthrogenic contracture.
Protein Extraction and Western Blot
The skeletal muscle samples were ground into powder with liquid nitrogen using a grinder and homogenized in RIPA buffer (Beyotime, China) containing protease inhibitors at 4 ℃. Homogenates were centrifuged at 12,000×g for 30 min three times at 4 ℃, and the resulting supernatants were collected. The protein concentrations were determined using the bicinchoninic acid method. Protein lysates were separated on a 10 % sodium dodecyl sulfate-poly-acrylamide electrophoresis gel and transferred onto polyvinylidene fluoride membranes (Millipore, USA). After being blocked with 5 % non-fat dry milk in Tris-buffered salineTween-20 at RT for 2 h, the membranes were incubated with mouse anti-MyoD mAb (dilution 1:600; BF0314, Affinity Biosciences, USA) at 4 ℃ overnight. On the second day, after being washed in TBST solution three times per 10 mins, membranes were incubated with peroxidase conjugated affinipure goat anti-mouse IgG-HRP (dilution 1:3000; S0002, Affinity Biosciences, USA) as the secondary antibody for 2 hours at room temperature. After being washed three times with TBST per 10 mins, the membranes were then detected with the enhanced chemiluminescence system according to the manufacturer's instructions. The densities of bands were quantified using Image J software. The relative protein levels were calculated by comparison with the amount of beta-Tubulin (T0023, Affinity Biosciences, USA) as a loading control.
All data are expressed as the mean ± standard error of the mean (S.E.M.). The assumptions of normality of data and homogeneity of variances between the groups was analyzed by SPSS 21.0 (Chicago, IL, USA). Differences in the total contracture, the myogenic contracture, the CSA, and the average protein levels for MyoD between the Group R and Group T at each recovery time point were assessed using Student’s t-test. The significant difference between the Group R and Group T at the same time point was measured at 95% CI not overlapping zero. One-way analysis of variance (ANOVA) and the Tukey-Kramer test were performed to examine differences in the total contracture, the myogenic contracture, the CSA, and the average protein levels for MyoD among the time points.