Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz 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 non-invasive 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.

Keywords: 3,3′-Diaminobenzidine; 3D; 5-LO; 5-lipoxygenase; 50MHz ultrasound; AA; Asp; BLT(1); COX; COX-2; DAB; Echogenicity; EtOH; FBS; Fracture; H&E; Hematoxylin and eosin; IACUC; IB; IHC; IND; Inflammation; Institutional Animal Care and Use Committee; Leukotriene B4 receptor 1; MRI; Magnetic resonance imaging; Max. Adj. R(2); NCKU; NSAIDs; National Cheng Kung University; PGE2; R(2) value; R-square value; RF; ROI; SD; STIR; Short TI Inversion Recovery; TBST; TUNEL; Tris-buffered saline with tween 20; arachidonic acid; aspirin; cyclooxygenase; cyclooxygenase-2; ddH(2)O; deoxynucleotidyl transferase dUTP nick end-labeling; double distilled H(2)O; ethanol; fetal bovine serum; immunohistochemistry; indomethacin; integrated backscatter; maximum value of adjusted R-square; non-steroidal anti-inflammatory drugs; prostaglandin E2; radio-frequency; region of interest; standard deviation; three-dimensional.