Adult male Wistar Han rats (n = 58) weighing 250 g ± 4g were randomly divided into two groups: the naïve group (n = 10) and the CCI group (n = 48). At day 0, sciatic nerve ligation was performed on the right hindlimb of all the rats belonging to the CCI group. The left (contralateral) hindlimb was operated but without ligation of the sciatic nerve.
One day before the surgery, baseline body weight and body composition were assessed. The same parameters were assessed once per week till the end of the study.
Magnetic resonance imaging (MRI) was performed at day zero before ligation and at day 28 before sacrifice. The day of sacrifice, animals were anesthetized and euthanized, and muscles were rapidly removed, weighed, and immediately frozen in liquid nitrogen or fixed in 10% formalin for further analysis. After the sacrifice a Micro-computed tomography on tibias was performed.
All the experimental procedures conduced on animals were performed according to protocols approved by the Animal Care of University Magna Graecia of Catanzaro and were carried out in compliance with the ARRIVE guidelines. All experiments were performed in accordance with the European Commission guidelines (Directive 2010/63/EU) for the animals used for scientific purposes.
Chronic Constriction Injury (CCI)
The surgery was performed under anesthesia, induced with 5% isoflurane in oxygen and then maintained with 2% isoflurane in oxygen. Anesthetized animals were monitored constantly during the surgery. On the right hindlimbs (ipsilateral), the sciatic nerve was exposed by making a skin incision, and cutting through the connective tissue, 3–4 mm below the femur, between the gluteus superficialis and biceps femoral muscle. Four loosely constrictive ligatures of 4.0 chromic gut were tied around the right sciatic nerve with a double knot, 1 mm apart, proximal to the trifurcation of the sciatic nerve, to occlude, but not arrest epineural blood flow. An identical surgery, without sciatic nerve damage, was performed in the left hindlimb (contralateral). Staples were used to suture (Autoclip, 9 mm) and the wounds were disinfected using an iodine solution (Riodine). After the surgery, the rats were housed individually in cages .
Body composition analysis
Total body fat, lean mass, and body fluids were measured using a nuclear magnetic resonance spectroscopy device EchoMRI-700TM (Echo Medical System, Houston, TX, USA), as previously described .
Magnetic resonance imaging
Before magnetic resonance imaging, rats were anaesthetized with 4% isoflurane (Forane, Abbott) vaporized in oxygen (flow: 2 l/min) and then kept during the acquisition between 2 and 3% to maintain the breathing rhythm between 40 and 60 breaths per minute. All the vital parameters were monitored, through a multiparametric monitor. The body temperature was maintained at 37°C. MRI images were acquired with a Bruker Pharmascan 70/16 US 7 Tesla bore MR scanner (Bruker Biospin MRI GmbH, Ettlingen, Germany), equipped with a total body transmitter receiver coil. Axial T2_turboRARE weighted images with fat suppression were acquired with the following parameters: relaxation time (TR) 800.000 ms, echo time (TE) 25.00 ms, averages 8, slices 9, thickness of 1 mm, repetitions 1, field of view 58x58 mm2.
Manual segmentation was performed using OsiriX imaging software (v. 12.0.2, Pixmeo SARL, Switzerland). The margins of individual muscle in Axial T2_turboRARE weighted images were manually traced through the "Draw tool" as defined in Greene’s Rat Anatomy Atlas , and in a more recently experiment performed by Zhang et al. . The slice used is the same for all animals and it represents the exact distance from epiphysis and diaphysis. To improve the viewing of individual muscle margins, a Default WL / WW, “Vr Muscles-Bones Clut" and a Logarithmic opacity table were used.
Protein extraction and western blot
50mg of GC muscle were homogenized in 500µL ice-cold lysis buffer (20mM Tris–HCl pH 7.5; 150mM NaCl; 1mM EDTA; 1mM EGTA; 1% 43 Triton X-100; 2,5mM Na4P2O7; 20mM NaF; 1mM dithiothreitol; 1mM Na3VO4; 1mM β-glycerophosphate; and 10 µL/mL freshly added protease and phosphatase inhibitor cocktails), centrifuged at 14000×g for 20min at 4°C and supernatant was collected. A total of 20µL of the supernatant was used to determine the total protein concentration with a Bradford assay (Bio-Rad, Hercules, California, USA) using bovine serum albumin as a standard. Proteins were heat denatured for 5min at 95°C in sample loading buffer (500mM Tris/HCl, pH 6.8; 30% Glycerol; 10% sodium dodecyl sulfate; 5% β-mercaptoethanol; and 0,024% bromophenol blue), and 30µg of protein lysate was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare,10600001). After incubation in a blocking solution (5% dry non-fat milk, Sigma-Aldrich, St. Louis, MO), membranes were incubated overnight at 4°C shaking with the following primary antibodies: Beclin-1 (3738, Cell Signaling Technology, Boston, USA), LC3-II/LC3-I (PM-036, MBL, Woburn, USA), p62 (M162-3, MBL, Woburn, USA), TRAF-6 (Ab 33915, Abcam, Cambridge, UK), Atrogin-1 (ab168372, Abcam, Cambridge, UK), Pax-7 (MABD20 Merk Millipore, Darmstadt Germania) and GAPDH (ab181602, Abcam, Cambridge, UK); all antibodies were used at 1:1000 concentration. Membranes were then washed in TBS (pH 7.6) with 0.1% Tween-20 and incubated with a secondary antibody for 1h at RT with shaking .
GC muscles of rats were harvested and fixed in 10% formalin for 24 hours and paraffin embedded for morphological analysis of cross-sectional area of muscle fibers. For the assessment of tissue morphology, 5-µm-thick sections of muscles were stained H&E and visualized at room temperature on a microscope (Olympus bx53, U-LH100HG) using a digital camera (Nikon) at 20X magnification, and Olympus cellSens Dimension 1.1 Software. The images, stored as JPEG files, were equally adjusted using Photoshop CS2 software (Adobe). For each muscle, the distribution of fiber CSA was calculated by analyzing 400 myofibers.
After the sacrifice, the tibias were explanted from the animal, and stored in alcohol at a temperature of 4°C. Right and left tibias were scanned using a SkyScan 1176 (SkyScan, Kontich, Belgium). The explanted bones were removed from alcohol storage and dried superficially on paper tissue, before being wrapped in plastic “cling-film” or in parafilm, to prevent drying during scanning (and associated movement artefacts). Each plastic-wrapped bone was placed in a plastic/polystyrene foam tube which was mounted horizontally in the 1176 scanner sample chamber, for micro-CT imaging. Reconstruction was carried out using the Skyscan Nrecon2 software which facilitates network distributed reconstruction carried out on four pcs running simultaneously. The time needed for the reconstruction of the dataset scan is usually much less than the scan duration.
Trabecular bone was assessed in 400 slices of proximal tibia (immediately distal to the epiphyseal plate). Settings were Source Voltage 65 KVp, Source Current 380 µA. Trabecular bone was manually segmented. Parameters are reported according to published guidelines. Trabecular parameters included bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), separation (Tb.Sp), structure model index (SMI), connectivity density (Conn.D) .
Bone calcium content
Right and left tibias were isolated and cleaned of soft tissue. The bones were dried at 110°C for 6 hours and weighed, then were ashed at 800°C for 4 hours, weighed again and dissolved in 1mL 6N HCl. Calcium content was determined by colorimetric determination with Quantichrom calcium Assay kit (BioAssay Systems, Hayward, CA).
Reverse Transcription and Quantitative Real Time PCR (qRT-PCR) performed on GC muscle tissue
Total RNA from GC muscle was extracted with Trizol reagent (Gibco, Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. The RNA quantity and quality were assessed through NanoDrop® ND-2000 Spectrophotometer (Waltham, MA, USA). To evaluate transcript changes, 1000 ng of total RNA was reverse-transcribed to cDNA using the “High Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA, USA).
The following TaqMan gene expression assays (Applied Biosystems, Carlsbad, CA, USA) were used to detect and quantify genes using the Viia7 DX real time PCR instrument (Life Technologies, Waltham, MA, USA): TRAF-6 (Rn00590197_m1), Atrogin-1 (fbx32) (Rn00591730_m1), and GAPDH (Rn01462661_g1).
Reverse Transcription and Quantitative Real Time PCR (qRT-PCR) performed on femurs bone
The rat femurs were homogenized using gentleMACS Dissociators (Miltenyi). Total RNA from femurs were extracted with Trizol reagent (Gibco, Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions.
The RNA quantity and quality were assessed through NanoDrop® ND-2000 Spectrophotometer (Waltham, MA, USA). To evaluate transcript changes, 1000 ng of total RNA was reverse transcribed to cDNA using the “High-Capacity cDNA Reverse Transcription Kit” (Applied Biosystems, Carlsbad, CA, USA). mRNA expression of TRAF-6, alkaline phosphatase (ALP), RANKL, osteoprotegerin (OPG), RUNX2 and β-ACTIN were quantified by real time-PCR using SYBR® Green dye (SYBR® Green PCR Master Mix, Applied Biosystems, Foster City, CA, USA) (see Table 1).
Table 1. Primers of genes used for real-time PCR on femurs bone.
Real-time PCR data analysis
To carry out the analysis of data obtained by real-time PCR, the arithmetic mean of the Ct (Threshold Cycle) in triplicate values was performed. ΔCt was calculated as the difference between Ct of the refence gene and Ct of the target gene. ΔΔCt was calculated as the difference between the mean of the ipsilateral ΔCt and the mean of the contralateral ΔCt. The statistical significance was calculated on the ΔCt values .
The data were analyzed with GraphPad PRISM 9.1.2 (Graph Pad Software, Inc., La Jolla, CA, USA). All data were expressed as mean ± S.E.M. Normally distributed data of body weight and body composition were analyzed by one way ANOVA, followed by the Tukey’s test, while data not normally distributed were analyzed using Kruskal–Wallis analysis of variance, followed by Dunn's tests. The Unpaired Two-tailed Student’s t-test was used for statistical analysis of data of muscle weight, Cross sectional area of fibers, western blotting analysis, and bone parameters. PCR data was tested by a Student's t-test and 95% confidence interval (CI). Values with p < 0.05 were considered statistically significant.