Seventy-two 6-week-old male ICR mice (25 ~ 30 g) were purchased from Orient Bio (Seongnam, Korea). The animals were fed standard solid feed (antibiotic‑free) and water ad libitum and housed in sawdust-lined cages in an air-conditioned environment with a 12-hour light/dark cycle. The mice were anesthetized with Zoletil 50 (50 mg/kg) and xylazine (10 mg/kg). CO2 administration is used for euthanasia. 72 mice were separated into four groups: control, treadmill (saline), BoNT-A control (BC), and BoNT-A treadmill (BT) group. For control and saline groups, 10 µl of saline was injected into the right gastrocnemius muscle of each mouse, while 0.5 units of BoNT-A (Botox®; Allergan, Inc., Irvine, CA, USA) was diluted with 10 µl of saline and injected into the right gastrocnemius muscle of each mouse in the BC and BT groups. All animal procedures were approved by the Institutional Animal Care and Use Committee of Chung-Ang University (201700028) and confirmed to all applicable National Institutes of Health guidelines.
One week before starting the treadmill exercise, 72 mice were randomly assigned to four groups (18 mice per group) for 5 min of running at 50 m/min on a 45-cm treadmill belt to ensure that all mice performed similarly in treadmill work before BoNT-A administration. The treadmill used in these studies was a JD-A-09 treadmill manufactured by JEUNGDO Bio & Plant Co., Ltd. (Seoul, Korea). Mice of the two exercise groups (saline plus exercise and BoNT-A plus exercise) were run at the same time in the six-lane treadmill. Treadmill exercise was carried out for 20 min at a speed of 15 m/min at a temperature of 10 °C, five times a week over a 6-week period.
Nerve Conduction Study (ncs): Electrophysiology
A Dantec™ Keypoint® Focus (Natus Neurology, Middleton, WI, USA) instrument was used for nerve conduction recording of the gastrocnemius muscle (5 mice per group); the data were automatically analyzed and averaged. Three surface disc electrodes (recording anode, cathode, and ground electrode) were used. An incision was made from the gluteus muscles to the popliteal region to expose the sciatic nerve with standard settings (electric potential = 5.8 mA; stimulus duration = 0.1 ms). The proximal side of the nerve was stimulated with a supramaximal electric stimulation at the same position each time. The compound muscle action potential (CMAP) amplitude (mV) (peak to peak), distal latency (ms), and area (mm2) were recorded each time. The protocol was repeated three times for each mouse during all individual NCS experiments. Amplitude was chosen as the principal variable in the data analysis because it was the best predictor of the physiologic changes at the muscle motor unit level.
Sciatic Functional Index (sfi) And Walking Track
After injection of BoNT-A and saline, the walking track of each group (6 mice per group) was observed weekly for 6 weeks and analyzed to calculate the SFI. A 10 cm × 10 cm × 24 cm box was made and a piece of paper of the same length and width as the box was placed under it. The hind feet of the mice were painted with ink, and the mice were placed at the right end of the box. Then, by tapping the box, the mice were forced to move to the left end of the box. The footprints of the mice were marked on the paper. The following three indicators were measured for both the damaged foot (E) and the normal foot (N): (1) print length (PL), which is the distance from the heel to the third toe; (2) the distance of the toe spread (TS), which is the distance from the first to the fifth toe; and (3) intermediary toe spread (IT), which is the distance from the second to the fourth toe.
SFI was measured using the Bain-Mackinnon-Hunter (BMH) sciatic functional index formula: SFI = -38.3*(EPL-NPL)/NPL + 109.5*(ETS-NTS)/NTS + 13.3*(EITS-NITS)/NITS − 8.8. An SFI = 0 shows normalcy, while SFI = -100 indicates serious nerve damage. The SFIs of four mice per group were measured from week 1 to 5 and three mice per group in the final week.
Muscle Mass And Volume
The mice (6 mice per group) were sacrificed every week for 6 weeks, and after peeling their skin, the changes in calf muscle volume reduction were evaluated by stereoscopic microscopy (OLYMPUS, SZ2-LGB, Tokyo, Japan) and PRIMOSLITE (GFMesstechnik GmbH, Berlin, Germany). The volume measurement result refers to the following: PRIMOSLITE software (PRIMOSLITE version 5.8E) was used for analysis of the degree to which parallel projection stripes transmitted on the gastrocnemius mass was changed by the height difference of the gastrocnemius mass. The value refers to the volume. The average of three volume measurements taken from one mouse was used.
Protein Extraction And Western Blot Analysis
The total protein content of the gastrocnemius muscle tissue samples (3 mice per group) was homogenized using a homogenizer (TissueLyser Ⅱ, QIAGEN, Tokyo, Japan) in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, Triton X-100, and protease inhibitors). Tissue homogenates were incubated on ice for 15 min, centrifuged (GYROZEN, 1730MR, Korea) at 18,000 g for 20 min at 4 °C and the supernatants were collected. The protein concentration was subsequently quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty milligrams of protein from each sample was separated by 12% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Immobilon, Millipore, Bedford, MA, USA). The membrane was saturated with 5% skim milk in Tris-buffered saline containing 0.5% Tween 20. Western blot analysis was performed by first incubating the membrane in antibodies against transforming growth factor (TGF)-β1 (ab2486, Abcam, Cambridge, UK), SNAP-25 (sc-7539, Santa Cruz Biotechnology, Santa Cruz, CA, USA), brain-derived neurotrophic factor (BDNF) (sc-546, Santa Cruz Biotechnology), and β-actin (sc-1616, Santa Cruz Biotechnology) at 4℃ for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Vector Labs, Inc., Burlingame, CA, USA) at room temperature for 1 h. Bound antibodies were detected using a SuperSignal™ West Pico Chemiluminescent Substrate (PIERCE Biotechnology Inc., Rockford, IL, USA) and assessed using a ChemiDoc™ XRS + System (Bio-RAD, Hercules, CA, USA).
Muscle tissue biopsy specimens (3 mice per group) were collected, immediately fixed with 10% paraformaldehyde in PBS and incubated overnight at 4℃. The samples were dehydrated, embedded in paraffin wax, and cleaved with a microtome into 5-µm serial transverse sections. The sections were then transferred to treated slides (Thermo Fisher Scientific, Pittsburgh, PA, USA), deparaffinized, and stained with hematoxylin and eosin (DAKO, Carpinteria, CA, USA) or Trichrome Stain Kit (Modified Masson’s; ScyTek Laboratories, Inc., Logan, UT, USA). For immunohistochemical analysis, paraffin-embedded tissues were deparaffinized, rehydrated, and subjected to antigen retrieval using Trilogy (1:20, 920P-06-RUO, CELL MARQUE, California, USA). The sections were treated with 3% H2O2 solution for 30 min at room temperature to halt any endogenous peroxidase activity. After blocking nonspecific proteins in 10% normal serum with 1% bovine serum albumin (BSA) in PBST (0.1% Tween-20), the slides were incubated with antibodies against α-smooth muscle actin (SMA) (1:500, ab5694, Abcam), TGF-β1 (1:500, ab2486, Abcam), or CD34 (1:500, BD553731, BD Biosciences, Heidelberg, Germany). After PBST washing, the slides were incubated with FITC-conjugated goat-anti-rabbit IgG (1:1000, sc-2012, Santa Cruz Biotechnology). The slides were washed, incubated with the biotinylated secondary antibody at room temperature, and stained with diaminobenzidine (DAB Plus Substrate System Kit, Thermo Scientific, Fremont, CA, USA). After counterstaining with Harris hematoxylin counterstain (Sigma, St. Louis, MO, USA), the sections were dehydrated and mounted on slides with mounting solution. All stained sections were then examined by light microscopy (DM750, Leica, Wetzlar, Germany) to assess the histological changes. Data were analyzed using ImageJ 1.38 software (NIH, MD, USA). For the immunofluorescence assay, the same process used for immunohistochemical analysis was executed up until the step of incubation with the primary antibody. Briefly, gastrocnemius muscle tissue was incubated with primary antibodies to BDNF (1:200, sc-546, Santa Cruz Biotechnology) overnight at 4℃, followed by further incubation with anti-FITC-IgG at 37℃ for 1 h. Slides were washed with 1X TBS buffer and mounted in fluorescent mounting medium with DAPI (Golden Bridge International Inc., Mukilteo, WA, USA). Fluorescent images were acquired using a confocal microscope (Leica DMI400; Leica, Wetzlar, Germany).
All quantitative data are presented as the mean ± standard deviation (SD) for three independent experiments. Statistical analyses were performed using SPSS software (SPSS Inc., Chicago, IL, USA) program. Analysis of variance (ANOVA) was used for multiple comparisons. The significance of differences between two groups was evaluated by a paired t-test. Significant values were *p < 0.05 and **p < 0.01.