Male normotensive Sprague-Dawley rats (n = 6; 9 to 12 weeks old; 276-499g) obtained from Nihon SLC (Japan SLC, Inc. Shizuoka, Japan) were housed in individual cages in a controlled room (temperature: 24-250C; humidity: 50-60%) and a 12:12 hours light-dark cycle. The rats were allowed free access to food (CE-2, CLEA Japan, Inc., Tokyo, Japan) and water. The research protocol was approved by the Institutional Animal Care and Use Committee of The Jikei University School of Medicine (Protocol number: 2019-043C1). All experimental procedures were conducted under the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions issued by the Japanese Ministry of Education, Culture, Sports, Science and Technology .
The fastened zip-tie rat model of ACS procedure
A vascular surgeon performed the fastened zip-tie rat model of ACS. Anesthesia was induced using 3% isoflurane kept within the laboratory “Small Animal Anesthesia System” (SBN-487, Shinano, Tokyo, Japan) and titrated to maintain an acceptable standard during the experiment. This isoflurane concentration is commonly used for MR procedures without impacting the results obtained. Respiratory was monitored in real-time using a Magnetic Resonance (MR)-compatible Small Animal Monitoring & Getting System (SA Instruments, Inc.,10, NY, USA). First, the rats were placed on a heating pad in a supine position and shifted into a lateral position. Then, two plastic zip-ties (ELPA, Osaka, Japan; length: 150mm, width: 3.6mm) were held with three fixed positions on the rat’s inguinal region (3 fixed points were sewn directly around the inguinal region avoiding vascular damages) (fig.1). After the pre-ischemic scanning, the ischemic was induced by fastening two zip ties. Pre-ischemic and ischemic states were monitored continuously from time zero to 120 minutes. After releasing the zip-ties, the reperfusion state was followed for 90 minutes.
After preparation for the fastened zip-tie rat model of ACS, the rats were placed on a supine position and immobilized on the MR cradle with the hindlimb in the full-extended position. The MR cradle is connected to a small animal ventilator (SA Instruments, Inc., 10, NY, USA). MR coils were positioned on the hindlimb’s lateral side and firmly secured with tape at pre-ischemic state. Ischemic was then induced by fastening the zip tie. After 120 minutes of ischemic, perfusion was resumed immediately by cutting the zip tie. 31P-MRS spectra were acquired at rest, during muscle was compressed (120 minutes) and reperfusion (90 minutes). During the MR procedure, the concentration of pH and CPK were analyzed by blood samples obtained from the tail artery. The rat’s respiration rate was continuously monitored (SA Instruments, Stony Brook, NY, USA) and controlled 80 to 120/minutes throughout the MR scanning. Body temperature was maintained at about 37 degrees by heating bath circulator equipment (CW-05G, lab companion, Daejeon, Korea) connected to the MR cradle. All the experiment’s items are demonstrated in fig.2
1H-MRI and 31P-MRS acquisition
The measurements were performed on a preclinical MR scanner biospec 9.4 Tesla (Bruker Optik GmbH, Ettlingen, Germany) and controlled by the paravision 6.2 software package (Bruker Biospin). The rats’ knee and leg were fully extended, and a Bruker dual resonance linearly polarized coils 1H/31P (1H: 400.525 MHz & 31P: 162.056 MHz) with 20 mm inner diameter was attached underneath the hind limb to perform shimming, and the knee was centered over the surface coil. The leg was secured to the 1H/31P dual coil covered by masking tape to avoid potential motion during the experiment.
Localizer 1H-images were acquired to detect the leg’s position within the sensitive area of the 1H/31P dual coil. Subsequently, the wobble adjustment was performed that allows the radiofrequency (RF) coil (measuring its absorption spectra) for manual tuning/ matching. We selected two coils (elements 1H and 31P, respectively) with an isotropic voxel of 48 x 48 x 48 mm3 in the surface coil’s sensitive field. The wobble curve was tuned and matched until the dip reaches the center, and its minimum is close to the zero line. After that, the B0 map, used to measure a field map of the object used in a study to calculate shims, was set up. The shim was calculated based on a previously measured B0-field map to optimize the field homogeneity within the Shim volume. As a result, a proton linewidth of 120-140 Hz was obtained. When the line width at half height of the proton signal was about 0.5 ppm for one free induction decay (FID), the magnetic field homogeneity was accepted. Then, the spectrometer was turned to 31P nuclei. The entire procedure took about five minutes on average.
31P spectra were acquired, followed by T2-weighted images, leading to a total acquisition time of 10 minutes at pre-ischemic. The interleaved 31P-MRS, T2-weighted images, and blood samples were acquired on the hindlimb of rats from pre-ischemic to ischemic and followed by reperfusion phase. The 31P-MRS dynamic protocol consisted of 15 min for pre-ischemic, 120 min for ischemic, and 90 min for recovery phase, respectively, to quantify phosphate metabolite changes. Each acquisition of 31P-MRS (a non-localized spectra with a single pulse) was acquired with parameters: flip angle = 900; repetition time (TR) = 2000ms; Averages = 192. Total acquisition time was 6 minutes, 24 seconds. The parameters used for axial slice T2-weighted images (fig.4) as follows: "fast spin-echo sequence”; TR = 2000 ms; time to echo (TE) = 30.69; Refocusing angle = 143.70; Rare factor = 8; Averages = 3, matrix = 256 x 256 pixels; Field of view = 35x50mm2; Slice thickness = 1.00 mm, Slices = 18; Scanning time = 3minutes 12 seconds). The experimental procedure workflow is illustrated in fig.3.
Blood sampling procedure
Blood samples were collected for ex vivo analysis. The 22-gauge catheter for invasive blood sampling collection was inserted into the ventral tail artery. After sampling arterial blood at the pre-ischemic state, the heparinized physiological saline was flushed into the catheter. The catheter was kept until the end of the experiment. Each amount of blood sample (0.25mL) was obtained and filled into the i-STAT CG4 + cartridge (Abbott product, USA) and FUJI DRI-CHEM slide (Fujifilm Medical, Tokyo, Japan) blood test kit. Blood samples were obtained at pre-ischemic, ischemic (60 minutes & 120 minutes), and after pressure removal (5 minutes, 60 minutes & 90 minutes). It was assessed at each time point for measurement of inflammatory biomarker CPK. After the needle was withdrawn, we applied gentle pressure with a cotton ball to the puncture site until the bleeding stopped.
Data Processing, Statistical Analysis
The MRS data were first processed with TopSpin 4.0.7 software (Bruker Biospin Corp., Billerica MA). The resulting datasets were fitted in the time domain using AMARES (Advanced Method for Accurate, Robust, and Efficient Spectral Fitting) algorithm implemented in jMRUI software. The NMRSCOPE tool (jMRUI software package) created a basic set of five metabolites spectra that include Pi, PCr, and ATP. PCr and Pi peaks were fitted to Lorentzian line shapes, whereas µ-ATP, α-ATP, and β-ATP signals were fitted to Gaussian line shapes. PCr was used as an internal reference for calculating the absolute concentration of Pi and ATP[20, 21]. The PCr/(Pi+PCr) ratio, a marker of energy state’s level, was calculated from Pi and PCr areas. The intracellular pH was calculated from the chemical shift of Pi relative to PCr utilizing the following equation 
pH = 6.75 + log (δ- 3.27/ (5.69- δ),
Where δ is the chemical shift of the Pi peak in parts per million relatives to PCr.
T2-weighted images were analyzed using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA). In order to quantitively analyze the signal intensity changes, the signal intensity was measured in two compartments that correspond to the tibialis anterior and the gastrocnemius muscles (locations were indicated in fig.8). The signal intensities of these two regions of interest (ROI) were calculated as the mean values of all pixels within the ROI. The tibia bone’s signal intensity was then used as the reference value to normalize the signal intensity. The signal intensity through each different phase from the pre-ischemic phase to the recovery phase was quantified.
Blood samples analysis: Blood samples were analyzed using an automatic biochemical analyzer (FUJI DRI-CHEM 3500v, Fujifilm Medical, Tokyo, Japan) and handheld blood analyzer i-STAT 1(Abbott product, USA). Results were obtained after 2 minutes. The analysis included pH and CPK [16, 24]. We set all the results over 2000 U/L to 2000 U/L.
Results are presented as mean ± SD. A repeated-measures nonparametric Friedman test was used to compare 31P-MRS and CPK between time points. Potential relationships between T2-weighted and CPK were assessed using Pearson’s correlation analysis or the nonparametric Spearman rank-order correlation. Statistical significance was accepted at P <0.05. Statistical analyses performed using GraphPad Prism (version 8.0.2, GraphPad Software, Inc.)