Novel preclinical murine model of trauma-induced elbow stiffness

Abstract

Background: Peri-articular injury may result in functional deficits and pain. In particular, post-traumatic elbow stiffness is a debilitating condition, precluding patients from performing activities of daily living. As such, clinicians and basic scientists alike, aim to develop novel therapeutic interventions to prevent and treat elbow stiffness; thereby reducing patient morbidity. Yet, there is a paucity of pre-clinical models of peri-articular stiffness, especially of the upper extremity, necessary to develop and test the efficacy of therapeutics. We set out to develop a pre-clinical murine model of elbow stiffness, resulting from soft tissue injury, with features characteristic of pathology observed in these patients.

Methods: A soft tissue peri-elbow injury was inflicted in mice using cardiotoxin. Pathologic tissue repair was induced by creating an investigator-imposed deficiency of plasminogen, a protease essential for musculoskeletal tissue repair. Functional testing was conducted through analysis of grip strength and gait. Radiography, microcomputed tomography, and histological analyses were employed to quantify development of heterotopic ossification.

Results: Animals with peri-elbow soft tissues injury in conjunction with an investigator-imposed plasminogen deficiency, developed a significant loss of elbow function measured by grip strength (2.387 ± 0.136 N vs 1.921 ± 0.157 N, ****, p < 0.0001) and gait analysis (35.05 ± 2.775 mm vs 29.87 ± 2.075 mm, ***, p < 0.0002). Additionally, plasminogen deficient animals developed capsule thickening, delayed skeletal muscle repair, fibrosis, chronic inflammation, and heterotopic ossification; all features characteristic of pathology observed in patients with trauma-induced elbow stiffness.

Conclusion: A soft tissue injury to the peri-elbow soft tissue with a concomitant deficiency in plasminogen, instigates elbow stiffness and pathologic features similar to those observed in humans. This pre-clinical model is valuable for translational studies designed to investigate the contributions of pathologic features to elbow stiffness or as a high-throughput model for testing therapeutic strategies designed to prevent and treat trauma-induced elbow stiffness.

Keywords: Elbow stiffness; Elbow trauma; Murine model; Plasmin; Plasminogen; Preclinical model.

Fig. 1

Preclinical Model of Upper Extremity Injury. a To reliably induce soft tissue injury around the elbow, three cardiotoxin injections were applied to the triceps, brachialis/brachioradialis, and the pronosupinators/hand extrinsics. b Injection of either blue dye or c) barium sulfate solution with subsequent radiographic analysis demonstrated well dispersed injection areas, fully surrounding the elbow joint and adjacent soft tissue. d Investigator induce plasminogen deficiency is attained by the time of upper extremity injury on both a protein (inset box) and RNA level following weekly administration of plasminogen ASO (330 mg/kg/wk) beginning two weeks before injury. Graphical representation of mean +/−SD

Fig. 2

Upper extremity functional assessment following per-elbow soft tissue injury. To assess changes in upper extremity function in our model following injury with or without an investigator induce plasminogen (PLG) deficiency, a Treadscan analysis was utilized to measure active motion in longitudinal direction. Points (n) represent individual limbs, left and right per mouse. N = 7; uninjured controls, 7; Injury + Control ASO, 7; Injury + PLG ASO. n = 14 points per group. Mean ± SD: Uninjured control- 35.05 ± 2.776; Muscle Injury + Control ASO- 32.20 ± 3.294; Muscle Injury + PLG ASO- 29.87 ± 2.075. b Grip strength analysis plotted as an average per mouse. N = 7; uninjured controls, 7; Injury + Control ASO, 7; Injury + PLG ASO. Total of 7 data points per group. Mean ± SD: Uninjured control- 2.387 ± 0.136; Muscle Injury + Control ASO- 2.238 ± 0.076; Muscle Injury + PLG ASO- 1.921 ± 0.157. Data represented in all plots as mean +/− SD. Statistical difference was assessed by an ordinary two-way ANOVA with a Tukey’s multiple comparison test. P values reported are adjusted for multiplicity

Fig. 3

Histological analysis of frontal and transverse sections of injured peri-elbow soft tissues 28 DPI. H/E staining of frontal sections from a) Control ASO or b) plasminogen ASO treated animals. Gross morphology of whole limb at 1×, scale bar represents 1 mm. Zoomed in sections of the injured triceps muscle or capsule immediately adjacent to the capitulum and radial head (20× and 10× respectively, scale bar represents 100 μm). Black asterisk indicates thickening of the capsule observed in plasminogen ASO treated animals. White arrows indicate focal chondrocytic lesions indicative of heterotopic ossification formation. c Transverse sections from control ASO or plasminogen ASO treated animals, stained with H/E, MSB, immunofluorescent staining for fibrin(ogen), or immunohistochemical staining for F4/80+ cells (macrophage/monocytes). All transverse images 20× magnification, scale bar represents 100 μm

Fig. 4

Characterization of skeletal muscle calcification following peri-elbow injury. a Longitudinal radiographic analysis of control ASO or plasminogen (PLG) ASO treated mice at 7, 14, 21, and 28 DPI. b Quantification of the soft tissue calcification within the triceps and muscle of the forearm as scored by the modified STiCSS for upper extremity. Error bar represent median and interquartile range. Statistical analyses between groups were analyzed by a non-parametric Mann-Whitney test. ****, p < 0.0001. c μCT analysis and 3D reconstruction of uninjured and injured upper extremities treated with control ASO or plasminogen ASO. d Von kossa staining of transvers sections of injured triceps 28 days post injury. e Gross morphology of PLG ASO treated animals at 1× (scale bar represents 1 mm) stained with MSB. b Zoomed in section of the injury triceps (20×, scale bar represents 100 μm). Black arrow heads indicate hypertrophic chondrocytes. Yellow arrow heads indicate erythrocytes