Utilizing Dynamic Phosphorous-31 Magnetic Resonance Spectroscopy for the Early Detection of Acute Compartment Syndrome: A Pilot Study on Rats

Introduction: Disasters, including terrorism and earthquakes, are significant threats to people and may lead to many people requiring rescue. The longer the rescue takes, the higher the chances of an individual contracting acute compartment syndrome (ACS). ACS is fatal if diagnosed too late, and early diagnosis and treatment are essential. Objective: To assess the ability of dynamic phosphorus magnetic resonance spectroscopy (31P-MRS) in the early detection of muscular damage in ACS. Materials and Methods: Six ACS model rats were used for serial 31P-MRS scanning (9.4 Tesla). Skeletal muscle metabolism, represented by the levels of phosphocreatine (PCr), inorganic phosphate (Pi), and adenosine triphosphate (ATP), was assessed. The PCr/(Pi + PCr) ratio, which decreases with ischemia, was compared with simultaneously sampled plasma creatine phosphokinase (CPK), a muscle damage marker. Results: The PCr/(Pi + PCr) ratio significantly decreased after inducing ischemia (from 0.86 ± 0.10 to 0.18 ± 0.06; p < 0.05), while CPK did not change significantly (from 89 ± 29.46 to 241.50 ± 113.28; p > 0.05). The intracellular and arterial pH index decreased over time, revealing significant differences at 120 min post-ischemia (from 7.09 ± 0.01 to 6.43 ± 0.13, and from 7.47 ± 0.03 to 7.39 ± 0.04, respectively). In the reperfusion state, the spectra and pH did not return to the original values. Conclusions: The dynamic 31P-MRS technique can rapidly detect changes in muscle bioenergetics. This technique is a promising non-invasive method for determining early muscular damage in ACS.


Introduction
Acute compartment syndrome (ACS) of the extremities, a syndrome that frequently occurs in disaster-prone countries such as Japan, can cause permanent damage to the muscles, nerves, and vasculatures [1][2][3]. Moreover, if left untreated for more than 8 h, limb salvage may be impossible, and it may even cause death [4][5][6]. Thus, early detection and monitoring of the disease progression are essential for evaluating skeletal muscle status, and differentiating reversible and irreversible tissue damage in skeletal muscles.
The initial suspicion of ACS is mostly inferred from observing its classical features, such as pain, pallor, pulselessness, paresthesia, poikilothermy, and paralysis [7]. It is verified either through invasive or non-invasive methods [8][9][10]. A typical invasive method is through measuring and calculating the difference between the mean arterial pressure and the intra-compartmental pressure (ICP) [11]. An absolute ICP of 30 mmHg indicates
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 [38]. Such conduct includes upholding ethical values of non-maleficence, among others.

Creation of the Fastened Zip-Tie Rat Model
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 magnetic resonance (MR) procedures as it does not impact the results obtained [43,44]. Respiratory functioning was monitored in real-time using an MR-compatible small animal monitoring and gating system (SA Instruments, Inc., Stony Brook, NY, USA). Once the anesthesia had taken effect, the rats were placed on a heating pad in a supine position. Then, two plastic zip ties (ELPA, Osaka, Japan; length: 150 mm, width: 3.6 mm) were loosely attached to the intended area to induce ischemia, and were sewn at three points (two points supine, one point on the lateral side) to the rat's inguinal region while avoiding vascular damage ( Figure 1). Then, pre-ischemic scanning was undertaken, followed by the placement of MR coils on the hindlimb's lateral side and tightening of the zip ties to induce ischemia.

Creation of the Fastened Zip-Tie Rat Model
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 magnetic resonance (MR) procedures as it does not impact the results obtained [43,44]. Respiratory functioning was monitored in real-time using an MR-compatible small animal monitoring and gating system (SA Instruments, Inc., Stony Brook, NY, USA). Once the anesthesia had taken effect, the rats were placed on a heating pad in a supine position. Then, two plastic zip ties (ELPA, Osaka, Japan; length: 150 mm, width: 3.6 mm) were loosely attached to the intended area to induce ischemia, and were sewn at three points (two points supine, one point on the lateral side) to the rat's inguinal region while avoiding vascular damage (Figure 1). Then, pre-ischemic scanning was undertaken, followed by the placement of MR coils on the hindlimb's lateral side and tightening of the zip ties to induce ischemia. After preparation of the fastened zip-tie rat model of ACS, the rats were placed in a supine position and immobilized on the MR cradle with the hindlimb in full extension. The MR cradle was connected to a small animal ventilator (SA Instruments, Inc., Stony Brook, NY, USA). The rat's respiration rate was continuously monitored (SA Instruments, Stony Brook, NY, USA) and controlled at 80-120/minute throughout the MR scanning. Body temperature was maintained at about 37 degrees Celsius by heating bath circulator equipment (CW-05G, Lab Companion, Daejeon, Korea) connected to the MR cradle. All equipment used is shown in Figure 2. After 120 min of ischemia, the zip ties were cut to resume perfusion. After preparation of the fastened zip-tie rat model of ACS, the rats were placed in a supine position and immobilized on the MR cradle with the hindlimb in full extension. The MR cradle was connected to a small animal ventilator (SA Instruments, Inc., Stony Brook, NY, USA). The rat's respiration rate was continuously monitored (SA Instruments, Stony Brook, NY, USA) and controlled at 80-120/minute throughout the MR scanning. Body temperature was maintained at about 37 degrees Celsius by heating bath circulator equipment (CW-05G, Lab Companion, Daejeon, Korea) connected to the MR cradle. All equipment used is shown in Figure 2. After 120 min of ischemia, the zip ties were cut to resume perfusion. , where the inhalation concentration could be adjusted accordingly. An ultrahigh field 9.4 Tesla magnetic resonance imaging (MRI) machine (C) and a surface coil capable of observing the biochemical kinetics of 1 H and 31 P (D) were fixed to the right lower leg region. The red circle (20 mm inner diameter) is the region of interest.

Experimental Procedure
We acquired 31 P-MRS spectra, hydrogen MRI images ( 1 H-MRI), and blood samples from rats induced with ACS through the use of zip ties. Pre-ischemic and ischemic states were monitored continuously from time zero to 120 min. After releasing the zip ties, the reperfusion state was followed for 90 min [26]. 31 P-MRS spectra and blood samples were acquired at rest, during muscle compression (120 min), and after inducing reperfusion (90 min) ( Figure 3). During the MR procedure, the concentration of pH and CPK were analyzed by blood samples obtained from the tail artery. The rats were able to walk normally at 2 days follow-up.

Assessing 1 H-MRI and 31 P-MRS
Measurements were performed on an MR scanner, Biospec, 9.4 Tesla (Bruker Optik GmbH, Ettlingen, Germany) and controlled using the Paravision 6.2 software package (Bruker Biospin). The rat's knee and leg were fully extended, with the knee centered over the surface coil, and Bruker dual resonance linearly polarized coils 1 H/ 31 P ( 1 H: 400.525 MHz and 31 P: 162.056 MHz) with 20 mm inner diameter, attached underneath the hind limb to perform shimming. The leg was secured to the 1 H/ 31 P dual coil covered with masking tape to avoid motion during the experiment.
Localizer 1 H-images were acquired to detect the leg's position within the sensitive area of the 1 H/ 31 P dual coil. Subsequently, wobble adjustment was performed that allowed for the radiofrequency (RF) coil's (measuring its absorption spectra) manual tuning/matching. We selected two coils (elements 1 H and 31 P, respectively) with an isotropic voxel of 48 × 48 × 48 mm 3 in the surface coil's sensitive field. The wobble curve was tuned and matched until the dip reached the center, and its minimum was close to zero. After that, the external magnetic field (B0) map, used to measure a field map of the object used in the 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

Experimental Procedure
We acquired 31 P-MRS spectra, hydrogen MRI images ( 1 H-MRI), and blood samples from rats induced with ACS through the use of zip ties. Pre-ischemic and ischemic states were monitored continuously from time zero to 120 min. After releasing the zip ties, the reperfusion state was followed for 90 min [26]. 31 P-MRS spectra and blood samples were acquired at rest, during muscle compression (120 min), and after inducing reperfusion (90 min) ( Figure 3). During the MR procedure, the concentration of pH and CPK were analyzed by blood samples obtained from the tail artery. The rats were able to walk normally at 2 days follow-up. result, a proton linewidth of 120-140 Hz was obtained. When the line width at half the 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 31 P nuclei. The entire procedure took about five minutes on average. 31 P spectra were acquired, followed by T2-weighted images, leading to a total acquisition time of 10 min during the pre-ischemic phase. The 31 P-MRS dynamic protocol consisted of 15 min for the pre-ischemic phase, 120 min for the ischemic phase, and 90 min for the recovery phase, respectively, to quantify phosphate metabolite changes. Each acquisition of 31 P-MRS (using non-localized spectra with a single pulse technique) was acquired with the following parameters: flip angle = 90°; repetition time (TR) = 2000 ms; average = 192. Total acquisition time was 6 min 24 s. The parameters used for axial slice T2-weighted images ( Figure 4) were as follows: "fast spin-echo sequence"; TR = 2000 ms; time to echo (TE) = 30.69 ms; refocusing angle = 143.7°; rare factor = 8; averages = 3, matrix = 256 × 256 pixels; field of view = 35 × 50 mm 2 ; slice thickness = 1.00 mm, slices = 18; scanning time = 3 min 12 s). The experimental procedure workflow is illustrated in Figure 3. White arrows indicate the timings of data collection for phosphorus magnetic resonance spectroscopy ( 31 P-MRS) and T2-weighted images. Red arrows designate the time of arterial blood sampling for creatine phosphokinase and pH concentration. 31 P-MRS spectra were acquired at rest, during muscle compression (for 120 min) and reperfusion (for 90 min). During the magnetic resonance (MR) procedure, the concentration of pH and creatine phosphokinase (CPK) were analyzed from blood samples obtained from the tail artery.
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 the 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 metabolite spectra that included Pi, PCr, and ATP. PCr and Pi peaks were fitted to Lorentzian line shapes, whereas µ-ATP, α-ATP, and β-ATP signals were Figure 3. The experimental protocol. White arrows indicate the timings of data collection for phosphorus magnetic resonance spectroscopy ( 31 P-MRS) and T2-weighted images. Red arrows designate the time of arterial blood sampling for creatine phosphokinase and pH concentration. 31 P-MRS spectra were acquired at rest, during muscle compression (for 120 min) and reperfusion (for 90 min). During the magnetic resonance (MR) procedure, the concentration of pH and creatine phosphokinase (CPK) were analyzed from blood samples obtained from the tail artery.

Assessing 1 H-MRI and 31 P-MRS
Measurements were performed on an MR scanner, Biospec, 9.4 Tesla (Bruker Optik GmbH, Ettlingen, Germany) and controlled using the Paravision 6.2 software package (Bruker Biospin). The rat's knee and leg were fully extended, with the knee centered over the surface coil, and Bruker dual resonance linearly polarized coils 1 H/ 31 P ( 1 H: 400.525 MHz and 31 P: 162.056 MHz) with 20 mm inner diameter, attached underneath the hind limb to perform shimming. The leg was secured to the 1 H/ 31 P dual coil covered with masking tape to avoid motion during the experiment.
Localizer 1 H-images were acquired to detect the leg's position within the sensitive area of the 1 H/ 31 P dual coil. Subsequently, wobble adjustment was performed that allowed for the radiofrequency (RF) coil's (measuring its absorption spectra) manual tuning/matching. We selected two coils (elements 1 H and 31 P, respectively) with an isotropic voxel of 48 × 48 × 48 mm 3 in the surface coil's sensitive field. The wobble curve was tuned and matched until the dip reached the center, and its minimum was close to zero. After that, the external magnetic field (B 0 ) map, used to measure a field map of the object used in the study to calculate shims, was set up. The shim was calculated based on a previously measured B 0 -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 the 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 31 P nuclei. The entire procedure took about five minutes on average. 31 P spectra were acquired, followed by T2-weighted images, leading to a total acquisition time of 10 min during the pre-ischemic phase. The 31 P-MRS dynamic protocol consisted of 15 min for the pre-ischemic phase, 120 min for the ischemic phase, and 90 min for the recovery phase, respectively, to quantify phosphate metabolite changes. Each acquisition of 31  T2-weighted images were analyzed using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institute of Health, Bethesda, MD, USA) [48]. The signal intensity was measured in two compartments corresponding to the tibialis anterior and the gastrocnemius muscles (locations indicated in Figure 4). 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 phase, from the pre-ischemic phase to the recovery phase, was quantified.

Examining Blood Samples
Blood samples were collected for ex vivo analysis. A 24-gauge catheter for invasive blood sampling collection was inserted into the ventral tail artery. After sampling arterial blood in the pre-ischemic state, heparinized physiological saline was flushed into the catheter. The catheter was kept in place until the end of the experiment. Each blood sample 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 the 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 metabolite spectra that included 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 [45]. PCr was used as an internal reference for calculating the absolute concentration of Pi and ATP [46,47]. The PCr/(Pi + PCr) ratio, a marker of the energy state level, was calculated from the Pi and PCr areas [27]. The intracellular pH was calculated from the chemical shift of Pi relative to PCr, utilizing the following equation: where δ is the chemical shift of the Pi peak in parts per million relative to PCr [21]. T2-weighted images were analyzed using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institute of Health, Bethesda, MD, USA) [48]. The signal intensity was measured in two compartments corresponding to the tibialis anterior and the gastrocnemius muscles (locations indicated in Figure 4). 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 phase, from the pre-ischemic phase to the recovery phase, was quantified.

Examining Blood Samples
Blood samples were collected for ex vivo analysis. A 24-gauge catheter for invasive blood sampling collection was inserted into the ventral tail artery. After sampling arterial blood in the pre-ischemic state, heparinized physiological saline was flushed into the catheter. The catheter was kept in place until the end of the experiment. Each blood sample (0.25 mL) was obtained and placed into the i-STAT CG4 + cartridge (Abbott product, Princeton, NJ, USA) and FUJI DRI-CHEM slides (Fujifilm Medical, Tokyo, Japan) of the blood test kit. Blood samples were obtained in the pre-ischemic and ischemic states (60 min and 120 min), and after pressure removal (5 min, 60 min, and 90 min). It was assessed at each time point for measurement of the inflammatory biomarker CPK.
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 min. The analysis included pH and CPK [13,49]. Because the blood analyzer cannot display values over 2000 U/L, we decided to declare results that exceeded the upper boundary as 2000 U/L.

Statistical Analysis
The results are presented as the mean ± SD. A repeated-measures nonparametric Friedman test was used to compare 31 P-MRS and CPK between time points. Potential relationships between the relative signal intensity of the T2-weighted images and CPK were assessed using nonparametric Spearman rank-order correlation. Nonparametric tests were done in adherence with conventional statistical guidelines, as the sample size was small [50]. Statistical significance was p < 0.05. Statistical analyses were performed using GraphPad Prism (version 8.0.2, GraphPad Software, Inc., SanDiego, CA, USA). Significant differences are indicated by p values in the figures.

Results
1 P-MRS: Upon inducing ACS, 31 P-MRS detected changes in the muscles at all time points, but assessing derived parameters such as spectra helped identify muscle changes, as shown in Figure 5. The metabolite concentration of PCr, Pi, and ATP was documented as baseline values. Typical T2-weighted fast spin-echo images are shown in Figure 6.

Results
1 P-MRS: Upon inducing ACS, 31 P-MRS detected changes in the muscles at all time points, but assessing derived parameters such as spectra helped identify muscle changes, as shown in Figure 5. The metabolite concentration of PCr, Pi, and ATP was documented as baseline values. Typical T2-weighted fast spin-echo images are shown in Figure 6. In the ischemic state, a phosphate monoester (PME) peak appeared at 30 min and gradually increased until 120 min of the ischemic state. At the beginning of the reperfusion state, the PME peak started to decrease, and completely disappeared at 185 min of the reperfusion state. The mean signal intensity in T2-weighted images. The mean signal intensities were calculated at two regions during the experiment (GA and TA muscles). The increasing relative signal intensity of T2-weighted images showed that the water content increased in the extracellular space. It demonstrated that the edema phenomenon gradually appeared in the reperfusion phase.
After muscle compression, the PCr/(Pi + PCr) ratio displayed a steep fall of 0.68 from the pre-ischemic value, and continued to decline for up to 120 min of ischemia (Table 1). The Friedman test revealed a significant difference in the PCr/(Pi + PCr) ratio between the pre-ischemic state, 60 min of ischemia, and 120 min of the ischemic state (p < 0.05, Figure   1  In the ischemic state, a phosphate monoester (PME) peak appeared at 30 min and gradually increased until 120 min of the ischemic state. At the beginning of the reperfusion state, the PME peak started to decrease, and completely disappeared at 185 min of the reperfusion state.
GraphPad Prism (version 8.0.2, GraphPad Software, Inc., SanDiego, CA, USA). Significant differences are indicated by p values in the figures.

Results
1 P-MRS: Upon inducing ACS, 31 P-MRS detected changes in the muscles at all time points, but assessing derived parameters such as spectra helped identify muscle changes, as shown in Figure 5. The metabolite concentration of PCr, Pi, and ATP was documented as baseline values. Typical T2-weighted fast spin-echo images are shown in Figure 6. In the ischemic state, a phosphate monoester (PME) peak appeared at 30 min and gradually increased until 120 min of the ischemic state. At the beginning of the reperfusion state, the PME peak started to decrease, and completely disappeared at 185 min of the reperfusion state. The mean signal intensity in T2-weighted images. The mean signal intensities were calculated at two regions during the experiment (GA and TA muscles). The increasing relative signal intensity of T2-weighted images showed that the water content increased in the extracellular space. It demonstrated that the edema phenomenon gradually appeared in the reperfusion phase.
After muscle compression, the PCr/(Pi + PCr) ratio displayed a steep fall of 0.68 from the pre-ischemic value, and continued to decline for up to 120 min of ischemia (Table 1). The Friedman test revealed a significant difference in the PCr/(Pi + PCr) ratio between the pre-ischemic state, 60 min of ischemia, and 120 min of the ischemic state (p < 0.05, Figure   1  After muscle compression, the PCr/(Pi + PCr) ratio displayed a steep fall of 0.68 from the pre-ischemic value, and continued to decline for up to 120 min of ischemia (Table 1). The Friedman test revealed a significant difference in the PCr/(Pi + PCr) ratio between the pre-ischemic state, 60 min of ischemia, and 120 min of the ischemic state (p < 0.05, Figure 6). Additionally, there was a significant change of intracellular pH after 120 min of ischemia (p < 0.05, Figure 7, Table 2). At 30 min of the ischemic state, a phosphate monoester (PME) peak was detected, which was not seen in the pre-ischemic phase, in the region of about 4.7-5.0 ppm to the left of the Pi peak ( Figure 5). The signal intensity of the tibialis anterior (TA) and gastrocnemius (GA) muscles gradually increased in the T2-weighted images.  The arterial blood pH level also revealed a minuscule change from pre-ischemia (7.47 ± 0.03) to 60 min of ischemia (7.42 ± 0.03). However, at 120 min of ischemia, a significant change was documented. At 90 min of reperfusion, it had not fully recovered to its preischemic value (Figure 8, Table 2).  Once the zip ties were released, the 31 P-MRS spectra displayed instantaneous gradual recovery to the pre-ischemic values. Nevertheless, the PCr/(Pi + PCr) ratio did not ultimately return to normal values, with a difference of 0.19 at 90 min of the reperfusion state (Table 1). The intracellular pH also increased during the reperfusion time, but did not recover to its baseline values ( Table 2). The PME peak gradually decreased and disappeared during the reperfusion state ( Figure 5).
After release, the muscles' signal intensities became inhomogeneous, wherein they were remarkably higher than those of other areas (Figure 6). At 90 min of the reperfusion state, the high signal intensity of both the TA and GA muscles was preserved. The signal intensity of the two regions was obtained as relative values to the mean baseline signal (Table 3).  Mean ± SD (n = 6). * Interquartile range p value in relation to the pre-ischemic state.
Blood samples: No significant changes were seen in the CPK concentration during the ischemic state when compared to the pre-ischemic level (p > 0.05, Figure 7); only slight changes were observed (86 ± 29.47). CPK gradually increased throughout the experiment period. At 90 min of reperfusion, the CPK increased more rapidly (1566.17 ± 493.30 U/L).
The arterial blood pH level also revealed a minuscule change from pre-ischemia (7.47 ± 0.03) to 60 min of ischemia (7.42 ± 0.03). However, at 120 min of ischemia, a significant change was documented. At 90 min of reperfusion, it had not fully recovered to its pre-ischemic value ( Figure 8, Table 2). There was a significant correlation between the relative signal intensity of the T2weighted image and CPK concentration from blood examination (R 2 = 0.1996, p < 0.05), revealing the presence of edema at the site of the ACS (Figure 9). There was a significant correlation between the relative signal intensity of the T2weighted image and CPK concentration from blood examination (R 2 = 0.1996, p < 0.05), revealing the presence of edema at the site of the ACS (Figure 9). There was a significant correlation of the relative signal intensity of T2-weighted images with the CPK concentration, based on blood examination. The gradual increase in the T2 signal intensity and CPK level demonstrated the edema phenomenon.

Discussion
The most important finding of this study is that 31 P-MRS can detect the change of muscle bioenergetics that occurs during the early ischemic state in rats. This could potentially be a reliable non-invasive method for the early detection of ACS in humans; more research is warranted. 31 P-MRS provided a reliable, sensitive measure of the muscle metabolites changes that occur during ACS. These data are consistent with previous data from the arterial occlusion model, wherein 31 P-MRS was found to provide early detection of muscle bioenergetic changes [40]. 31 P-MRS spectra displayed real-time responsiveness with an immediate detectable change at a constant level when plastic zip ties were fastened, suggesting a loss of perfusion to the tissues. These data revealed that the ratio of PCr/(Pi + PCr) was firmly lower than the baseline value during the ischemic state. In contrast to this, the CPK concentration only showed a slight change in the ischemic state. Thus, it could be suggested that 31 P-MRS can detect bioenergetic changes more rapidly and sensitively than CPK. However, CPK can still be used as a useful biomarker to indicate the extent of muscle damage in the sub-acute phase [51]. Moreover, CPK has long been used as a primary biomarker in recognizing trauma for patients with ACS [13]. It is essential to differentiate between invasive measures, such as blood CPK, and non-invasive measures, such as 31 P-MRS.
During the ischemic and recovery phase, we consistently found that the PME was peaking. This is an additional sign that can potentially help in detecting early muscle damage in ACS [52]. While it is possible to perform 31 P-MRS at a more clinically common field strength of 3 Tesla, differentiation of peaks in the spectra will be more difficult and less efficient [34]. Nowadays, a magnetic field MRI higher than 3 Tesla with 31 P-MRS is used to detect the early stage of disease [36,53]. Thus, 31 P-MRS has been applied more often in clinical practice to diagnose muscle diseases. The results of this study show there is merit There was a significant correlation of the relative signal intensity of T2-weighted images with the CPK concentration, based on blood examination. The gradual increase in the T2 signal intensity and CPK level demonstrated the edema phenomenon.

Discussion
The most important finding of this study is that 31 P-MRS can detect the change of muscle bioenergetics that occurs during the early ischemic state in rats. This could potentially be a reliable non-invasive method for the early detection of ACS in humans; more research is warranted. 31 P-MRS provided a reliable, sensitive measure of the muscle metabolites changes that occur during ACS. These data are consistent with previous data from the arterial occlusion model, wherein 31 P-MRS was found to provide early detection of muscle bioenergetic changes [40]. 31 P-MRS spectra displayed real-time responsiveness with an immediate detectable change at a constant level when plastic zip ties were fastened, suggesting a loss of perfusion to the tissues. These data revealed that the ratio of PCr/(Pi + PCr) was firmly lower than the baseline value during the ischemic state. In contrast to this, the CPK concentration only showed a slight change in the ischemic state. Thus, it could be suggested that 31 P-MRS can detect bioenergetic changes more rapidly and sensitively than CPK. However, CPK can still be used as a useful biomarker to indicate the extent of muscle damage in the sub-acute phase [51]. Moreover, CPK has long been used as a primary biomarker in recognizing trauma for patients with ACS [13]. It is essential to differentiate between invasive measures, such as blood CPK, and non-invasive measures, such as 31 P-MRS.
During the ischemic and recovery phase, we consistently found that the PME was peaking. This is an additional sign that can potentially help in detecting early muscle damage in ACS [52]. While it is possible to perform 31 P-MRS at a more clinically common field strength of 3 Tesla, differentiation of peaks in the spectra will be more difficult and less efficient [34]. Nowadays, a magnetic field MRI higher than 3 Tesla with 31 P-MRS is used to detect the early stage of disease [36,53]. Thus, 31 P-MRS has been applied more often in clinical practice to diagnose muscle diseases. The results of this study show there is merit in using 31 P-MRS for peripheral vascular disease detection as well, specifically ACS. The 31 P-MRS method exhibited potential for use in diverse settings.
Edema of the hindlimb's compartment, depending on the location in the muscles, was determined by 1 H-MR images. During the reperfusion phase, the elevated relative signal intensity of T2-weighted images showed that the water content increased in the extracellular space, similar to other studies [54]. Additionally, there is a correlation between the relative signal intensity of the T2-weighted images (TA and GA muscle regions) and CPK. After the release of muscle compression, it is understood that apparent edema around the skeletal muscle occurred due to the increased vascular permeability in the skeletal muscle induced by oxygen-derived free radicals. This result demonstrated that the muscle damage was consistent for edema symptoms, and thereby suggests that alterations in skeletal muscle high-energy phosphate metabolism occur early in the pathophysiology of ACS. Thus, 31 P-MRS can potentially be a useful non-invasive method for the early diagnosis of ACS, thereby increasing the probability of limb salvage in these patients.
In our study, there was an evident difference between the control pH of 31 P-MRS and the blood test during the ischemic state. The pH decreased more extensively in ischemic tissue than in the rest of the body. Anaerobic glycolysis lowered the pH in the ischemic area. However, as homeostasis tightly controls the systematic pH value, the value was almost unchanged (normal mammalian blood pH = 7.40 ± 0.02). Hence, this observation supports a previous study which asserted a difference between the arterial blood pH and the superficial pH value [55].
However, the present study has several limitations. First, as we only utilized six rats, the sample size was evidently small, which greatly affected the generalizability of the findings of this study due to a lack of variability. Nonetheless, this pilot study is useful to provide the groundwork for future studies [56]. Second, the surface coil's sensitivity area was limited. Thus, standardization of images was not possible, rendering the images incomparable. Regardless, it proved the presence of edema in the ACS model [40]. Third, similar to most cases of the reperfusion state, the blood analyzer equipment could not display the specific value of the CPK concentration as it exceeded the upper limit of measurement (2000 U/L). However, it was considered that detailed numerical measurement was unnecessary because it was sufficiently high when compared to the pre-ischemic level. Lastly, after the experiment, we did not conduct a follow-up on the rats' health status by MRS. We only observed the rats' behaviors. In the next phase of this study, we recommend conducting a follow-up on the health of the animals post-experiment.
There is merit in comparing the 31 P-MRS and blood samples of patients with ACS to further enhance the clinical utility of this technique. There is also tremendous research in the field of regenerative medicine, with possible application in emergency medicine. It has been reported that the clinical efficacy of mesenchymal stem cell therapy for ischemic diseases [57]. Therefore, mesenchymal stem cell therapy has the potential for ACS. By advancing this research, we hope to improve the survival of patients rescued from disasters, and to prevent the deterioration of quality of life due to unnecessary amputation of the lower limbs.

Conclusions
Our study showed the possibility of using 31 P-MRS as a non-invasive, reliable, and sensitive assessment of changes in muscle metabolites. Acquiring clinical data via 31 P-MRS with MRI ultra-field 9.4 Tesla may have the benefit of increased SNR and spectra quality. This research demonstrated that dynamic 31 P-MRS measurements may be faster and more accurate than blood sampling tests. Dynamic 31 P-MRS could be useful for real-time detection of early ischemic muscular damage in ACS.