Quantitative diagnosis of early acute compartment syndrome using two-dimensional shear wave elastography in a rabbit model
Article information
Abstract
Purpose
This study explored the association of the elasticity modulus and shear wave velocity (SWV) of the tibialis anterior muscle, as measured by two-dimensional shear wave elastography (2D-SWE), with the intracompartmental pressure (ICP) determined using the Whitesides method in a New Zealand rabbit model of acute compartment syndrome (ACS). Additionally, it evaluated the viability of 2D-SWE as a noninvasive, quantitative tool for the early detection of ACS.
Methods
An ACS model was established through direct external compression by applying pressure bandaging to the lower legs of 15 New Zealand rabbits using neonatal blood pressure cuffs. Another five animals represented a non-modeled control group. To measure the elasticity modulus and SWV of the tibialis anterior muscles, 2D-SWE was employed. Blood oxygen saturation, serum creatine kinase (CK), and myoglobin levels were monitored. Subsequently, the anterior tibial compartment was dissected, and the tibialis anterior was removed for hematoxylin and eosin staining to assess muscle injury.
Results
The elasticity modulus and SWV of the tibialis anterior muscle increased with compression duration, as did serum CK and myoglobin levels. ICP was strongly positively correlated with these parameters, particularly mean velocity (r=0.942, P<0.001) and CK (r=0.942, P<0.001). Blood oxygen saturation was negatively correlated with ICP (r=-0.887, P<0.001). Histological analysis indicated progressive muscle cell swelling over time, with damage transitioning from reversible to irreversible and culminating in necrosis.
Conclusion
In a rabbit ACS model, ICP was strongly positively correlated with muscle elasticity modulus/SWV. Consequently, 2D-SWE may represent a novel tool for assessing early-phase ACS.
Introduction
Acute compartment syndrome (ACS) is a serious and urgent complication that can arise from traumatic injury or surgery [1,2]. Without prompt diagnosis and treatment, ACS can result in permanent dysfunction or amputation of the affected limb and may even be life-threatening [3,4]. Early and accurate diagnosis is not only the foundation for timely and effective treatment but also crucial for improving patient prognosis.
The diagnosis of ACS remains a topic of debate in the medical community due to the absence of universally recognized early diagnostic guidelines and standards at both national and international levels [5]. The evaluation of ACS is primarily based on clinical assessment and the measurement of intracompartmental pressure (ICP) [6]. Clinical judgment, which is subjective and can be delayed, depends largely on patient-reported symptoms and the experience of the clinician. ICP measurement is the standard method for diagnosing ACS [7]. However, its application in clinical practice is limited because the procedure is invasive, operationally complex, and has low repeatability. Consequently, clinicians are in urgent need of a non-invasive, objective, accurate, and reproducible diagnostic technique that can facilitate early monitoring and diagnosis of ACS. Such a method could minimize the risks associated with unnecessary or delayed interventions and improve patient outcomes.
As a non-invasive ultrasound imaging technique for the real-time quantitative assessment of tissue stiffness, ultrasound two-dimensional shear-wave elastography (2D-SWE) has been explored in the diagnosis of ACS [8]. This modality estimates the elastic modulus of tissues based on the shear wave speed induced by the acoustic radiation force of a focused ultrasound beam [9]. It can provide quantitative in vivo measurements of soft tissue elasticity or stiffness by measuring shear wave velocity (SWV), which is directly related to the elastic modulus [10]. The growing body of literature on the assessment and diagnosis of ACS using 2D-SWE suggests that this technology has considerable potential for clinical application [11,12]. Previous research conducted by this research group revealed a significant increase in the elasticity values of muscles within the affected compartment during ACS [13]. However, the correlation between the measured ICP and 2D-SWE data during the development of ACS has not been clearly established.
The aim of this study was to establish a New Zealand rabbit animal model of ACS using a neonatal blood pressure cuff for compression bandaging. The authors sought to investigate the correlation between ICP and 2D-SWE findings. The ultimate goal was to lay the groundwork for using 2D-SWE as a noninvasive, objective, and quantitative technique for the early monitoring and diagnosis of ACS.
Materials and Methods
Compliance with Ethical Standards
All procedures described in this study adhered to the ethical standards for laboratory animal care and were approved by the Animal Ethics Committee of the Air Force Medical University.
Animal Preparation and Anesthesia
A total of 20 New Zealand rabbits, aged 20 weeks and comprising both male and female animals, were selected for the study. These rabbits had a weight of 2.3 to 2.8 kg, with an average weight of 2.4±0.4 kg, and were sourced from the Experimental Animal Center of the Air Force Medical University. Approximately 8 to 12 hours prior to the experiment, the rabbits were fasted, and water was withheld for 4 hours. Before the experiment, the rabbits were anesthetized by administering a 3% pentobarbital sodium saline solution intravenously at a dose of 1 mL/kg. The solution was injected slowly into the ear vein, and the animal was then positioned on the platform. The hair on the lower limbs and dorsal toes of each rabbit was removed using hair shears and depilatory paste. Throughout the experiment, vital signs such as respiration and heart rate were monitored using an electrocardiograph.
ACS Model Preparation Method
The animal model of ACS was established using the direct external compression method [14,15]. Twenty rabbits were randomly assigned to four groups based on the duration of compression bandaging, with each group consisting of five rabbits. Group 1 served as the control group and did not receive compression bandaging. Group 2 underwent 2 hours of compression, group 3 underwent 4 hours, and group 4 underwent 6 hours. In brief, a neonatal blood pressure cuff, 7 cm in width, was wrapped around the lower leg of each rabbit. A bandage was applied over the cuff for reinforcement. The cuff was inflated to a pressure of 40 kPa, resulting in the disappearance or notable weakening of the arterial pulsation in the lower limb when compared to the opposite side (Fig. 1).
Establishment of rabbit models of acute compartment syndrome.
A. Intracompartmental pressure in the anterior tibial compartment was measured using the Whitesides method. B. Blood oxygen saturation was monitored. C. At the conclusion of the experiment, the tibialis anterior muscle was harvested for pathological observation.
2D-SWE Assessment
In this study, 2D-SWE was performed using an L15-4 high-frequency linear array ultrasound transducer (4-15 MHz) with an ultrasound system (Aixplorer, Supersonic Imagine, Aix-en-Provence, France) to measure the elastic modulus (E) and SWV (V) of the rabbit anterior tibialis muscle in each group before and after the application of pressure dressing. Each rabbit was positioned on its side on the platform, and due to the anesthesia, its lower extremities were naturally relaxed and extended. To minimize tissue compression, the probe was placed as lightly as possible, with a generous amount of coupling agent applied between the probe and the skin. Measurements were taken at the intermediate segment of the tibialis anterior muscle. The elastography function was activated after ensuring a clear grayscale image of the long axis section, allowing the elastography window to stabilize for 5 seconds to produce stable color-coded maps before the image was acquired. Data were analyzed using a single circular region of interest with a diameter of 4 to 5 mm, positioned at the center of the rectangular area. Each muscle was measured five times, and the mean of the three measurements with the lowest coefficients of variation within the circular region was used for the analysis.
ICP Measurement
In this study, the Whitesides method was used to evaluate the ICP of the anterior tibial compartment in the lower extremities (Fig. 1A) [16]. The setup involved a three-way valve: one end was connected to a mercury sphygmomanometer after removal of the cuff, another end to a 10-mL syringe, and the third to a disposable infusion set. Before measurements were taken, the syringe was filled with normal saline and connected to the needle end of the infusion set to ensure that all air was expelled from the system. The mercury sphygmomanometer was positioned at the same height as the limb being measured. The needle was inserted perpendicularly into the anterior tibial compartment of the rabbit, approximately 2 to 3 cm below the knee, to a depth of about 1 to 1.5 cm. Following the injection of 2 mL of normal saline into the compartment, pressure readings were taken once the mercury column had stabilized, which typically occurred after about 5 minutes. The puncture point, depth, and rate of injection had been established in preliminary experiments. To minimize the influence of puncture-related changes on the SWE examination, the SWE assessment of the target muscle was performed after the predetermined modeling duration for each group had elapsed. This was followed by the ICP measurement.
Monitoring of Blood Oxygen Saturation
After the toes of the New Zealand rabbits were depilated, an electrocardiogram monitor was connected to a disposable oxygen saturation probe. The red, luminescent segment of the probe was positioned on the nail cap of the rabbit’s toes, while the other end was secured around the base of the toes to prevent leakage of light (Fig. 1B). The electrocardiogram monitor provided continuous and stable readings of blood oxygen levels, and the oxygen saturation of the lower limbs was recorded before and after the modeling process.
Detection of Blood Biochemical Indices
In this study, serum creatine kinase (CK) and myoglobin (Mb) levels were measured due to their established significant correlation with ACS [17]. A 2-mL blood sample was drawn from the internal jugular vein and placed into a yellow-top blood collection tube both before and after the modeling process. The samples were allowed to stand for 2 hours at room temperature before being centrifuged at 3,000 rpm for 15 minutes. The resulting supernatant was collected and sent for analysis to the clinical laboratory at Xijing Hospital.
Histopathological Section Preparation and Hematoxylin and Eosin Staining
After all measurements were taken, the rabbits were euthanized via air embolization, in which 20 mL of air was rapidly injected into the heart. The tibialis anterior muscle tissues were carefully dissected, and the gross morphological features of the muscles were recorded (Fig. 1C). The tissues were then fixed in a 40% formalin solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Pathological examination of these sections was performed using a light microscope to evaluate muscle injury.
Statistical Analysis
SPSS version 23 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA) were utilized for data analysis. Continuous variables are presented as mean±standard deviation. The normality of the data distribution was verified using the Shapiro-Wilk test. To determine the relationship between measured ICP and various indicators, Pearson correlation was employed. An independent samples t-test was applied to compare various indicators before and after modeling in each group. P-values of less than 0.05 were considered to indicate statistical significance.
Results
Pre- and post-modeling measurements of SWE, SWV, blood oxygen saturation (SpO2), Mb, CK, and ICP in the New Zealand rabbits of each experimental group are presented in Tables 1 and 2 (Fig. 2). The findings revealed a gradual increase in ICP within the anterior compartment as the duration of compression extended. Analysis of the 2D ultrasound images showed that increasing pressure was associated with a thickening of the anterior tibial muscle, a loss of muscle texture clarity, and the appearance of a high echogenicity resembling ground glass. This phenomenon is believed to stem from an increase in muscle exudation and edema. Both SWE measurements of the tibialis anterior muscle—V and E—demonstrated progressive increases. Elastography images for each group are displayed in Fig. 3. The CK and Mb values for each experimental group demonstrated upward trends, with significant positive correlations noted between these monitoring indicators and ICP (Table 3). Notably, the correlation coefficient (r) values between ICP and Mb, CK, and Vmean were as high as 0.93 to 0.94. The Emean and Vmean values of each experimental group differed significantly before and after modeling, whereas the control group exhibited no significant difference (Fig. 2). Furthermore, the SpO2 of the limb on the compressed side decreased significantly over time, dropping from a baseline level of 98% to a hypoxic state of 80%. SpO2 was found to be negatively correlated with ICP (r=-0.887).
Baseline data for the monitoring indicators (n=5 per group)
Changes in monitoring indicators for each group after modeling (n=5 per group)
Correlation analysis.
Graphs show the correlations between intracompartmental pressure (ICP) and the maximum, minimum, and average values of shear wave velocity (Vmax, Vmin, and Vmean) (A-C), the correlations between ICP and the maximum, minimum, and average values of the Young modulus (Emax, Emin, and Emean) (E-G), and a comparison of Emean and Vmean in each group before and after modeling (D, H). No statistically significant difference was observed in the control group, while significant differences were noted in the experimental groups (***P<0.001).
Images of two-dimensional shear wave elastography and H&E staining for each group.
A-D. Compared with the control group (A), the elastic values of the experimental groups exhibited gradual increases, observed after various durations of compression (B, 2 hours; C, 4 hours; D, 6 hours). E-H. H&E staining analysis of pathological sections for each group is shown (E, control; F, 2 hours; G, 4 hours; H, 6 hours; ×400, scale bars=50 μm).
Correlation analysis between each index and ICP
Finally, cross-sections of the tibialis anterior muscle belly were obtained from each group for H&E staining. This was done to assess early pathological damage to the skeletal muscle in ACS. The staining revealed that the tibialis anterior muscle of the control (non-compression) animals contained well-preserved, orderly fibers with minimal intercellular space. As the duration of modeling lengthened, a gradual increase was noted in muscle fiber edema within the tibialis anterior of the experimental rabbits. The spaces between the muscle fibers became more pronounced, and interstitial edema was evident. Notably, in the 6-hour group, the muscle cell swelling intensified, leading to cell necrosis. This indicated a transition from early reversible damage to irreversible injury, as depicted in Fig. 3.
Discussion
Prior studies have indicated that the conversion of the Young modulus is based on the assumption that the target is a uniform (isotropic) elastic body, whereas muscle tissue exhibits anisotropy [18,19]. When the transducer is oriented perpendicular to the long axis of muscle fibers, recommendations favor reporting the shear wave speed rather than converting it to shear modulus (in kilopascals) and thus to tissue stiffness [10]. However, other research indicates that the Young modulus or SWV can be used to quantitatively evaluate muscle stiffness, with both methods demonstrating good feasibility and credibility [20,21]. In the current investigation, an animal model of ACS was established through the application of pressure bandaging with a neonatal blood pressure cuff. Then, the relationship between the measured ICP and the SWE findings (V and E) in the anterior compartment of the tibia was evaluated. To support the consistency and reliability of subsequent research, these authors documented the maximum, minimum, and average values of SWV and the Young modulus within the region of interest and performed appropriate statistical analyses. The resulting observations revealed linear increases in V and E with rising ICP, along with significant positive correlations between these variables, particularly between Vmean and ICP.
To prevent the serious complications associated with ACS, consensus indicates that fasciotomy should be performed before irreversible tissue necrosis occurs [3,22,23]. Unfortunately, fasciotomy can have serious complications, including the need for additional procedures for delayed wound closure, surgical reconstruction with skin grafting or vascularized flaps, aesthetic issues, pain, nerve injury, permanent muscle weakness, and chronic venous insufficiency [24]. Therefore, early, timely, and accurate diagnosis is crucial for prompt treatment and optimal patient outcomes. With the advancement of diagnostic technologies, illnesses may be identified earlier, enabling the performance of less invasive or non-surgical interventions.
At present, although ICP measurement is the standard method for diagnosing ACS [7], it is not always accurate or practical in clinical settings due to its complexity, invasiveness, poor repeatability, and increased risk of infection and patient discomfort [25]. Most clinicians rely on empirical diagnoses based on patients’ clinical signs and symptoms; however, for inexperienced physicians, particularly those in primary care, this approach can lead to missed or delayed diagnoses [26,27]. Consequently, many researchers have sought a more convenient and non-invasive diagnostic method for compartment syndrome. Near-infrared spectroscopy, which monitors changes in tissue oxygenation to reflect alterations in tissue perfusion pressure, has been shown to aid in the diagnosis of ACS [28-31]. However, its limitations include the inability to monitor the early stages of ACS in high-risk patients, and its diagnostic role for ACS remains undefined [32]. Steinberg employed a non-invasive device to assess the hardness of limb compartments with elevated ICP in volunteers, finding a strong correlation between compartment hardness and ICP [33]. Notably, however, these quantitative hardness measurement techniques typically require specialized equipment and complex mathematical formulas, limiting their clinical utility [11].
The 2D-SWE technique employs shear waves to quantitatively assess tissue stiffness and has been developed as a safe, real-time, and noninvasive method. Several studies have explored the utility of 2D-SWE for ACS. Toyoshima et al. [8] established a compartment syndrome-like model in a turkey hind limb using a vascular infusion technique to evaluate the correlation between the measured ICPs and SWV in superficial and deep compartments in vitro. They identified a strong correlation between the measured pressure and the mean shear wave speed in each compartment [8]. Notably, a cadaver ACS model may not fully replicate the complex pathophysiological conditions that influence SWE outcomes. In previous research, these authors applied 2D-SWE to measure the elasticity values of the same muscle on the affected and unaffected sides in patients suspected of having ACS, then conducted a comparative analysis [13]. This study revealed varying degrees of increased elasticity in the affected muscle compared to the unaffected side. These increases corresponded with clinical symptoms and the tension observed in the related anatomical areas. Consequently, 2D-SWE may hold potential as a reliable method for the early quantitative assessment and diagnosis of ACS.
In preliminary experiments, prolonged application of the cuff for over 6 hours resulted in significant necrosis of the muscle. Ultrasonographic images revealed pronounced muscle swelling, extensive exudation, and disrupted muscle texture. Consequently, a reliable SWE image could not be acquired in these cases. However, E and V values increased significantly as the compression time was prolonged, culminating in muscle necrosis. Therefore, these authors hypothesize that while SWE may not be suitable for assessing advanced compartment syndrome after severe irreversible damage has occurred, it could represent a sensitive and effective tool for evaluating the initial stages of ACS. Thus, the use of 2D-SWE to assess the early progression of ACS appears to have notable clinical implications.
In this study, SpO2, Mb, and CK levels were strongly correlated with ICP in an animal model of ACS. Clinical diagnosis of ACS currently depends on patient symptoms, with laboratory tests serving as supplementary diagnostic tools. Previous studies have indicated that elevated levels of CK and Mb are early indicators of muscle tissue ischemia [34,35]. Following skeletal muscle injury, Mb levels increase notably within 30 minutes and return to baseline within 24 hours, while CK levels peak at around 2 hours and remain elevated for at least 48 hours [36,37]. Consequently, monitoring the trends of these two biomarkers in high-risk patients can be useful for diagnosing ACS. However, clinical use requires caution, as these markers lack specificity and may not differentiate muscle injury from ACS, trauma, or even myocardial damage [17]. Additionally, monitoring these blood indicators necessitates repeated blood sampling from patients, and waiting for results can be time-consuming. Tuckey [38] utilized peripheral pulse oxygen saturation as an auxiliary diagnostic tool for ACS. However, SpO2 does not accurately or reliably indicate increased intrafascial pressure or tissue hypoperfusion.
ACS is characterized by elevated ICP, resulting in reduced perfusion pressure and tissue hypoxemia. Tolerance to ischemia varies among tissues, with muscle necrosis typically occurring after about 4 hours of complete ischemia [1]. It is unclear whether the diagnostic criterion for ACS in humans—that is, the differential pressure remaining below 30 mmHg for more than 2 hours—is applicable to rabbits [39]. In this study, the presence of muscle necrosis observed in H&E-stained sections was adopted as the diagnostic criterion. The findings illustrated the pathological changes in muscles induced by elevated ICP during the development of ACS. The experimental findings indicate that as ICP increases, myocyte edema progressively worsens and culminates in muscle cell necrosis. Furthermore, observations from the 2D-SWE images were congruent with the pathological findings. This concurrence validates the reliability and potential of 2D-SWE in the early assessment of ACS.
The present study has several limitations. First, the sample size was small, necessitating expansion in future research to better assess the reliability of the findings. Second, the compression injury was simulated using a specific modeling method; thus, the applicability of the research findings to ACS caused by other etiologies requires further investigation. Finally, after the removal of the pressure dressing, the study did not involve dynamic monitoring of the ICP changes and 2D-SWE imaging in each group to observe the progression and alterations of ACS. Addressing this gap is one objective of this group’s subsequent research. This study utilized an animal model, and one must acknowledge the differences in anatomical and pathophysiological characteristics between human patients with ACS and rabbits. Consequently, additional research is needed to determine the utility of 2D-SWE in the clinical monitoring, quantitative diagnosis, and guidance in formulating clinical treatment plans for ACS.
In a rabbit model of ACS, a strong positive correlation was observed between ICP and the elasticity modulus/SWV of muscle as measured by 2D-SWE. This finding indicates that 2D-SWE may serve as a valuable quantitative tool in supporting the clinical diagnosis and treatment of ACS. However, the precise diagnostic cut-off value for ACS using 2D-SWE requires further investigation. Advancing research to define this diagnostic threshold is crucial for improving the precision and reliability of 2D-SWE, facilitating its application in clinical settings.
Notes
Author Contributions
Conceptualization: Yu M, Guan H. Data acquisition: Zhang J, Duan K, Wei J, Zhang W, Zhou H, Sang L, Sun Y, Gong X. Data analysis or interpretation: Zhang J, Duan K, Wei J. Drafting of the manuscript: Zhang J, Duan K, Wei J, Zhang W, Zhou H, Sang L, Sun Y, Gong X. Critical revision of the manuscript: Yu M, Guan H. Approval of the f inal version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.
Acknowledgements
This research was supported by the Military Medicine Clinical Application Research Project of the First Affiliated Hospital of Air Force Medical University in China (JSYXM10). The authors express their gratitude to the Air Force Medical University Laboratory Animal Center for their support. The authors also wish to thank Dr. Mengyao Huang from the Department of Clinical Laboratory and Dr. Mengwei Xu from the Department of Pathology at the First Affiliated Hospital of Air Force Medical University for their technical assistance.
References
Article information Continued
Notes
Key point
The study demonstrates a strong positive correlation between the muscle elasticity modulus/shear wave velocity and intracompartmental pressure, along with significant associations with diagnostic markers such as myoglobin, creatine kinase, and SpO2. These f indings highlight the potential of two-dimensional shear wave elastography as a noninvasive and quantitative diagnostic method for the early detection of acute compartment syndrome.