Murine model of Peri-articular injury to the elbow
All animal procedures were approved by the Vanderbilt University Institutional Animal Care and Use Committee (M1600225) and carried out in strict accordance with the recommendation in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All skeletal muscle injuries were performed under anesthesia, and all efforts were made to minimize suffering.
Male C57BL/6 J mice were purchased from Jackson Laboratory and housed at Vanderbilt University in a 12-h light/dark cycle with food and water provided ad libitum. At 6 weeks of age, a cardiotoxin-induce injury was administered to the soft tissues surrounding the elbow (Garry et al. 2016; Mignemi et al. 2017; Moore et al. 2016). Following anesthetization with Isoflurane, mice were placed in the Trendelenburg position, supinating the forearm, while flexing the forearm over the surface of the nose cone (Fig. 1a). From this position, 20 μL of 10 μM cardiotoxin was injected with a 28.5 G 0.5 mL insulin syringe into the posterior compartment of the arm to infiltrate the elbow extensors (triceps), the lateral compartment of the arm/forearm to infiltrate the elbow flexors (brachialis) and mobile wad (brachioradialis), and the medial aspect of the proximal forearm (pronosupinators and hand extrinsics), resulting in a total of three injection regions (20 μL each) surrounding the elbow joint (Fig. 1b and c). Both the left and right upper limbs were injured.
To induce an investigator-imposed plasminogen deficiency, a plasminogen specific antisense oligonucleotide (ASO) or a non-targeting control ASO was injected subcutaneously (330 mg/kg/week) beginning two weeks before injury and continuing through the duration of the study (Fig. 1d) (Mignemi et al. 2017). All antisense oligonucleotides used in this study were developed in collaboration with Ionis Pharmaceuticals (Carlsbad, CA).
Quantification of elbow function following Peri-articular injury
To assess changes in elbow function following peri-articular injury, grip strength and gait analysis were performed 28 days following injury. Grip strength was assessed with an animal grip strength system force meter (San Diego Instruments, San Diego, CA). Briefly, a mouse is placed on the wire grid and allowed to grab on with its forepaws. Once secure, the mouse’s tail is gently pulled backwards and the maximum force of the grip is recorded in Newtons. This test was performed three times per mouse with 5–10 min of rest between measurements. The data from each trial is then averaged before the final analysis between experimental groups. Gait analysis was assessed with a Treadscan System (Clever Sys Inc. Reston, VA) to evaluate changes in gait disturbances. Briefly, mice were placed in a clear acrylic box above a treadmill monitored by a high-speed camera. The treadmill was then equilibrated to a speed of 13.7 cm/s and a 20 s video of the mouse’s walking pattern was obtained. Tredscan Software was then utilized to assess active range of motion in the longitudinal direction per limb. Results are presented as a mean step distance (mm) per animal.
Histological analysis
Following sacrifice at 28 days post peri-articular injury, the upper extremity was disarticulated at the glenohumeral joint, fixed in 10% neutral buffered formalin, decalcified in 0.5 M EDTA for one week, processed, and embedded in paraffin prior to frontal or transverse sectioning. Six micron frontal section of the upper extremity were produced and stained with hematoxylin and eosin (H/E) and Martius Scarlet Blue (MSB). Additionally, at the time of sacrifice, the triceps were dissected, fixed in 10% Neutral buffered formalin, processed, and embedded in paraffin prior to transverse sectioning. Six micron sections were then stained with H/E, MSB, or Von Kossa to visualize deposits of calcification. Additionally, immunofluorescence and immunohistochemistry were performed to detect the presence of fibrin or F4/80+ cells within damaged tissues, respectively. Whole slide imaging of frontal sections was performed in the Digital Histology Shared Resource at Vanderbilt University.
Hematoxylin and Eosin (H/E) staining was performed according to standard protocols to assess tissue morphology as previously described(Mignemi et al. 2017; Moore et al. 2016). Martius Scarlet Blue (MSB) staining was performed according to standard protocols to assess for the presence of fibrin and collagen deposits within damaged tissues. Von Kossa staining to assess calcific deposits was performed according to standard protocols as previously described. (Mignemi et al. 2017; Moore et al. 2016) Immunohistochemical staining of F/480+ cells were performed according to standard protocols by the Vanderbilt University Medical Center Tissue Pathology Shared Resource. Immunofluorescent staining for the presence of fibrin was performed as previously described (Mignemi et al. 2016).
Quantification of heterotopic ossification
Longitudinal radiographic analysis was performed weekly following injury to visualize and quantify heterotopic ossification within the injured tissues of the upper extremity. Briefly, following adequate anesthesia, digital radiographs (Faxitron LX60, Tucson, AZ) were collected weekly at an exposure of 35 kV for 4 s. Digital radiographs were standardized and the extent of muscle calcification was quantified using a modified version of the Soft Tissue Calcification Scoring System (STiCSS) for the upper extremity (Additional file 1: Figure S1) (Moore et al. 2016). All radiographic images were scored in a blinded manner by three independent observers found to be in substantial to almost perfect agreement (Kappa = 0.706–0.901, Additional file 2: Table S1) per the Landis and Koch criteria (Cohen 1968). As such, results reported represent the total score for a single limb (either left or right) as scored by a single observer.
In addition to radiographic analysis, microcomputed tomography (μCT) was utilized to visualize the formation of soft tissue calcification around the elbow joint. μCT images were acquired (μCT 40, Scanco Medical AG, Bassersdorf Switzerland) of injured forelimbs at 55kVp, a45uA, 200 ms integration, 500 projections per 180-degree rotation with a 20 μm isotropic voxel size. After scanning, the volume of interest containing the entire forelimb was selected the calcified tissues were segmented form soft tissues using a threshold of 220 per thousand (or 450.7mgHA/cm3), a Gaussian noise filter of 0.2, and support of 1.
Statistical analysis
Statistical analyses of functional changes between groups were evaluated using an ordinary two-way ANOVA with a multiple comparison test. P values reported are adjusted for multiplicity. STiCSS upper extremity scores were statistically evaluated between groups using a non-parametric Mann-Whitney test. For all analysis, alpha = 0.05.