Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50MHz ultrasound

Abstract

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT1, 5- LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT1. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT1. Taken together, the current study reports the feasibility of a noninvasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing.

Role of Excessive Autophagy Induced by Mechanical Overload in Vein Graft Neointima Formation: Prediction and Prevention

“…Our recent publication 21 suggests that the increases in echogenicity in injured bone are positively correlated to tissue inflammatory responses, particularly COX-2- or 5-LO-mediated signal pathways. In the present study, immunohistological staining revealed significant increases in protein expression of COX-2, but not BLT1 (receptor of leukotriene B4 for 5-LO signaling), in the venous endothelium at 3 weeks after surgery ( Supplementary Fig.…”

“…S2A ). The HFU system comprised of a 30-MHz transducer was used to generate and receive ultrasound signals as described in a previous study 21 . The series of cross-sectional images were acquired to monitor the dynamic changes in vascular structures from proximal to distal sites of vessels with 50-μm intervals at post-surgery weeks 1, 2, and 3 (W1, W2, W3).…”

“…High-frequency ultrasound (HFU) in the range of 20–50 MHz provides enhanced image resolutions at the sub-tissue level (approximately 10 −5 m), which allows for visualizing detailed anatomical structures, and can be used for diagnostic, therapeutic, and surgical applications, especially in relation to cardiovascular interventions 20 . In addition, tissue inflammatory responses after fracture injury in mice are highly correlated with the alteration of echogenicity in HFU images 21 . Due to the increase in demand for following the prognosis of stenosis, the American Society of Echocardiography (ASE) and Society of Vascular Medicine and Biology have published the guidelines for using hemodynamic parameters acquired from color doppler to predict vein graft failure 22 .…”

Cross-Sectional Nakagami Images in Passive Stretches Reveal Damage of Injured Muscles

“…Echogenicity increases with increasing density and with decreasing homogeneity [ 16 ]. Our recent findings suggest that echogenicity can also be used as an indicator for tissue inflammation during fracture healing [ 17 ]. Numerous studies have examined backscattered signals analyzed by Nakagami distribution analysis in ultrasonic pulse-echo images; these studies have found that the signals reflect the structure of the tissue and can predict tissue composition and complexity [ 18 , 19 ].…”

Ultrasound characterization of the mastoid for detecting middle ear effusion: A preliminary clinical validation

“…Recently, the Nakagami parameter has been widely used in soft-tissue characterization of, for example, cataracts26, blood27, liver2829, breast30, and thrombus31. The Nakagami parameter is also used in modeling the echo amplitude distribution of bony tissues3233. Therefore, we assumed that the Nakagami parameter can quantify the acoustic structure of the mastoid and correlated it with clinical MEE.…”