Animals and experimental design
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 was used for euthanasia. The mice were separated into four groups:
(1) Control group – saline injection, no exercise
(2) Treadmill group – saline injection, treadmill exercise
(3) BoNT-A control (BC) group – BoNT-A injection, no exercise
(4) BoNT-A treadmill (BT) group – BoNT-A injection, treadmill exercise
For the control and saline groups, 10 μL of saline was injected into the right gastrocnemius muscle of each mouse, while 0.5 unit 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 conformed 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 each) for 5 min of running at 50 m/min on a 45-cm treadmill belt to ensure that all mice performed similar treadmill work before BoNT-A administration. A JD-A-09 treadmill manufactured by JEUNGDO Bio & Plant Co., Ltd. (Seoul, Korea) was used in this study. The 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. The treadmill exercise was performed for 20 min at a speed of 15 m/min and temperature of 10°C five times a week over a 6-week period.
Nerve conduction study (NCS): Electrophysiology
The Dantec™ Keypoint® Focus (Natus Neurology, Middleton, WI, USA) instrument was used to record nerve conduction of the gastrocnemius muscle (5 mice per group); 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 received supramaximal electric stimulation at the same position each time. The compound muscle action potential 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 individual nerve conduction study (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 sciatic functional index (SFI). A 10 cm × 10 cm × 24 cm box was made and a piece of paper of the corresponding length and width 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. The box was then tapped and 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 the damaged (E) and normal (N) foot: (1) print length, the distance from the heel to the third toe; (2) toe spread, the distance from the first to the fifth toe; and (3) intermediary toe spread (IT), the distance from the second to the fourth toe.
SFI was measured using the Bain-Mackinnon-Hunter SFI formula: SFI = -38.3*(EPL - NPL)/NPL + 109.5*(ETS - NTS)/NTS + 13.3*(EITS - NITS)/NITS - 8.8. An SFI = 0 is normal, while an SFI = -100 indicates serious nerve damage. The SFI values of four mice per group were measured in weeks 1–5, while those of three mice per group were measured in the final week.
Muscle mass and volume
The mice (3 mice per group) were euthanized every week for 6 weeks; after their skin was removed, changes in calf muscle volume reduction were evaluated using 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 to analyze 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 (three mice per group) was homogenized using a homogenizer (TissueLyser Ⅱ; QIAGEN, Tokyo, Japan) in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 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 supernatants were collected. The protein concentration was subsequently quantified using the 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 blotting 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 the 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 (three mice per group) were collected, immediately fixed in 10% paraformaldehyde in phosphate buffered saline (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 the 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, Rocklin, CA, USA). The sections were treated with 3% H2O2 solution for 30 min at room temperature to halt any endogenous peroxidase activity. After nonspecific proteins were blocked in 10% normal serum with 1% bovine serum albumin in PBS with 0.1% Tween-20 (PBST), 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 being washed in PBST, the slides were incubated with fluorescein isothiocyanate (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 using mounting solution. All stained sections were then examined using light microscopy (DM750; Leica, Wetzlar, Germany) to assess histological changes. Data were analyzed using ImageJ 1.38 software (NIH, Bethesda, 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, the 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. The slides were washed with 1× Tris-buffered saline and mounted in fluorescent mounting medium with DAPI (Golden Bridge International Inc., Mukilteo, WA, USA). Fluorescent images were acquired using a confocal microscope (DMI400; Leica).
All quantitative data are presented as mean ± standard deviation (SD) for three independent experiments. Statistical analyses were performed using SPSS software (SPSS Inc., Chicago, IL, USA). For multiple comparisons, one-way analysis of variance was used with post hoc Bonferroni test. We considered P values<0.05 statistically significant. Significance levels are designated as follows: *p<0.05; **p<0.01.