Study aim, design, and setting
Mice
This study adheres to the applicable Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. The experiments were approved by the Institutional Animal Experiment Committee of Jichi Medical University (17217-01). Tables 1 and 2 show the groups (total, 34) and numbers of mice used in this study and the treatment provided to each group. Figure 1 shows the experiment time course. Six- to seven-week-old male wild-type C57BL/6J, C57BL/6-Tg (CAG-EGFP; SLC, Hamamatsu, Japan), and LysM Cre tandem TOMATO (CLEA, Tokyo, Japan) mice (19–23 g) were maintained in individual cages with a 12-h light/dark cycle at constant temperature and provided with water and food ad libitum. No mice had any abnormal health condition before the experiments. Invasive procedures were performed under general anesthesia.
Table 1. Group allocation and testing for C57BL/6J mice
C57BL/6J Mice
|
Control
|
CCI Surgery
|
|
|
No Tx
|
Clod
|
Dex
|
Preg
|
Loxo
|
Neuro
|
Amitryp
|
Muscle weight
|
8
|
8
|
8
|
8
|
8
|
8
|
−
|
−
|
Total bone density
|
8
|
8
|
8
|
8
|
8
|
8
|
−
|
−
|
Immunohisto-chemistry
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
Flow cytometry
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
8
|
RT-PCR
|
8
|
8
|
−
|
−
|
−
|
−
|
−
|
−
|
A total of 30 × 8 = 240 mice were tested. Control, surgery without chronic constriction injury (CCI); No Tx, no treatment; Clod, Clodronate liposome; Dex, Dexamethasone; Preg, Pregabalin; Loxo, Loxoprofen; Neuro, Neurotropin; Amitryp, Amitriptyline.
Table 2. Intravital microscopy group allocation and testing
Test/Drug
|
Control
|
CCI-No Tx
|
C57BL/6-Tg (CAG-EGFP)
|
8
|
8
|
LysMCre tandem TOMATO
|
8
|
8
|
A total of 4 × 8 = 32 mice were tested. The C57BL/6-Tg (CAG-EGFP) mice were tested immediately after CCI (chronic constriction injury), whereas the LysM Cre tandem TOMATO were tested one week after CCI. No Tx = no treatment.
Animal Models
Chronic constriction injury (CCI) was performed as described previously [18]. The mice were anesthetized with 1.5% isoflurane; the right common sciatic nerve was exposed by blunt dissection through the biceps femoris muscle. Four ligatures (5-0 Monocryl monofilament), 1 mm apart, were tied loosely around the nerve. The muscle and skin layers were closed using 5-0 silk thread. In control mice, the right sciatic nerve was exposed, but not ligated.
To investigate macrophages’ influence on muscle/bone atrophy after CCI, they were depleted by a single injection of clodronate liposomes (Xigieia Bioscience, Tokyo, Japan) into the tail veins (100 μg/mouse) immediately after CCI.
We also tried to prevent muscle/bone atrophy due to CCI using anti-inflammatory and neuropathic pain drugs. Immediately after CCI, dexamethasone (2 mg/kg subcutaneously; Fuji Pharma Co., Ltd., Toyama, Japan), pregabalin (30 mg/kg oral; Pfizer Japan, Inc., Tokyo, Japan), loxoprofen (3 mg/kg oral; Daiichi Sankyo, Ltd., Tokyo, Japan), neurotropin (60 NU/kg intraperitoneal; Nippon Zoki Pharmaceutical Co., Ltd., Osaka, Japan), and amitriptyline (10 mg/kg oral; Nichi-Iko Pharmaceutical Co., Ltd., Toyama, Japan) were administrated/day for 1 week. Mice were randomly assigned to each group.
Muscle Weight Measurement
To evaluate muscle atrophy, 1 week after CCI, C57BL/6J mice were sacrificed via cervical dislocation. The right biceps femoris and gastrocnemius muscles were collected; muscle weights were measured.
Total Bone Density
To evaluate bone atrophy, total hind limb bone density was measured by computed tomography (CT) in C57BL/6J mice anesthetized with 1.5% isoflurane (Latheta Laboratory CT; Hitachi, Tokyo, Japan). CCI was performed; CT of the hind limb were obtained/week for 5 weeks. Images were analyzed using the CT scanner software. Total bone density of the femur and tibia were measured with 0.5-mm slices; their average values were compared.
Immunohistochemistry
Inflammation cells and osteoclasts in the femoral bone were analyzed by immunohistochemistry. One week after CCI, C57BL/6J mice were sacrificed via cervical dislocation; the right femurs were collected. Pathologic specimen preparation and immunostaining [tartrate-resistant acid phosphatase (TRAP) staining] were outsourced (Kyodo Byori, Kobe, Japan). Images were captured on a FSX100 microscope (Olympus, Tokyo, Japan) using a ×4 (N.A. 0.1) objective and analyzed with Fiji win64 software (Rasband, W.S., ImageJ; National Institutes of Health, Bethesda, MD, USA). With a 1.5 × 2 mm view, osteoclasts were separated in a single color according to difference in staining; the osteoclast numbers were compared.
Intravital Microscopy
To visualize inflammation cell dynamics in the nerve and surrounding tissues, we used in vivo multiphoton microscopy [19]. One week after CCI, LysM Cre tandem TOMATO mice were anesthetized by urethane injection (1.5 g/kg); the hind limb skin was removed. The hind limb tissue was visualized with an inverted microscope (Eclipse Ti; Nikon). Fluorescein isothiocyanate dextran (5 mg/mouse; Merck KGaA, Darmstadt, Germany) and Hoechst 33342 (3 mg/mouse; Thermo Fisher Scientific, Waltham, MA, USA) were injected into the tail vein to visualize cell dynamics and blood flow. The tissues were excited at 920 nm, using a Ti:sapphire laser (Vision II; Coherent, Inc., Santa Clara, CA, USA), and images were captured as XY images using an A1R-MP system (Nikon). A ×40 (N.A. 1.15) water immersion objective lens (Nikon) was used. The dwelling time for one pixel was 0.1 µs. To observe the inflammatory response in the acute phase, CAG-EGFP mice were anesthetized immediately after CCI; Rhodamine B dextran (5 mg/mouse, Thermo Fisher Scientific), Hoechst 33342 (3 mg/mouse), and F4/80 Alx647 (25 μg/mouse; BioLegend, San Diego, CA, USA) were injected into the tail veins. We continuously observed the sciatic nerve and surrounding tissue for 3 h. Collected data were analyzed via an automatic algorithm in NIS-Elements and Fiji win64 software. With a 500 × 500 μm view, macrophages were separated in a single color based on the difference in fluorescence color, and their numbers were compared.
Flow Cytometry
Cell infiltrations in muscles were analyzed by flow cytometry. One week after CCI, C57BL/6J mice were sacrificed via cervical dislocation; right biceps femoris and gastrocnemius muscles were collected, minced, and treated with collagenase. Cells were isolated, washed twice with phosphate-buffered saline, incubated for 8.5 min in erythrocyte-lysing buffer, and finally suspended in phosphate-buffered saline. Isolated cells were then incubated with FcBlock antibody (BD Biosciences, Bedford, MA, USA) for 15 min on ice, labeled with dye-conjugated antibodies (CD45-FITC, 25 μg/mL; Ly6G-BV605, 10 μg/mL; F4/80-PE, 10 μg/mL; CD11b-BV711, 2.5 μg/mL; CD301-Alx647, 2.5 μg/mL; Ly6C-BV421, 2.5 μg/mL; BioLegend), and analyzed by flow cytometry using BD LSR Fortessa (BD Biosciences) and FlowJo V10 (Tomy Digital Biology, Tokyo, Japan) software. DRAQ7 (BioLegend) was used to exclude dead cells.
RNA Isolation and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
To investigate cytokine changes in the muscles after CCI, RT-PCR was performed. One week after CCI, C57BL/6J mice were sacrificed, the right biceps femoris and gastrocnemius muscles were collected; RNA was extracted from the muscle tissues using Trizol (Thermo Fisher Scientific). cDNA was synthesized using primescript reverse transcriptase (Takara Bio, Kusatsu, Japan). Taqman probes of TNFα, IL1-β, CCL2, and CCR2 (Thermo Fisher Scientific) were used in RT-PCR, performed with 10 ng cDNA with a Vii A real-time PCR system (Thermo Fisher Scientific). Processing of raw data and normalizing the relative quantities were performed using the ΔΔ-Ct-method. mRNA expression levels are expressed relative to the control group.
Statistics
Statistical analysis was performed using Graphpad Prism7 software (GraphPad Software, Inc., San Diego, CA, USA). All data were of equal variance in the F test; however, in the D'Agostino and Pearson normality test, except RT-PCR data, all data were not normally distributed. Taking into account that n = 8 is the least number of samples detectable by the D'Agostino and Pearson test, a nonparametric test was chosen, except for RT-PCR. The Mann–Whitney U test was used to compare the groups. The Kruskal–Wallis test was used to compare ≥3 groups. The Friedman test was used for repeated measurements of the same sample. The Dunn's multiple comparison test was used for post hoc tests. The results are expressed as median and interquartile range. Since RT-PCR data were normally distributed, Student’s t-test was chosen as the parametric test. Results were expressed as means ± SD. P < 0.05 was considered significant.
Sample sizes were calculated using GPower 3.1 software (Heinrich-Heine-University, Düsseldorf, Germany) for primary outcomes [muscle weight (control vs. CCI groups, 1 week later) and total bone density (CCI group, 0–5 weeks)] based on the pilot studies of five mice. To detect a 50 mg decrease in muscle based on a standard deviation (SD) of 30 mg, n = 8 in each group was considered appropriate (α = 0.05; 1 − β = 0.8). To detect a 60 mg/cm3 decrease in total bone density based on a SD of 50 mg/cm3, n = 8 in each group was considered appropriate (α = 0.05; 1 − β = 0.8). Therefore, n = 8 was chosen for each group (total groups = 34, total n = 272).