Introduction Dyslipidemia, abnormal amounts of lipid in the blood, has been associated with chronic Achilles tendinopathy. Tendinopathy is a major cause of morbidity in athletic populations and in the work force,1 traditionally thought to occur as a consequence of an imbalance between damage (resulting from mechanical loading) and repair.2 However, one third of the cases for midportion Achilles tendinopathy occur in sedentary individuals, and recent data suggests an association between dyslipidemia and the occurrence of tendon rupture or tendinopathy.3 The goal of this study is to examine the missing biological link between elevated lipids and the development of tendinopathy, and establish a suitable model to examine the potential reversibility of tendinopathy associated with dyslipidemia. The study will use the ApoE-knockout mouse model, in which deficiency of ApoE leads to development of hypercholesterolemia. We hypothesise that ApoE-KO mice fed on a high fat diet, in comparison to those fed a normal diet and the wild type (non-atherosclerotic) mice, will demonstrate reduced collagen density and organisation, increased intracellular and extracellular lipid deposition, increased tendon cross-sectional area, and reduced tendon biomechanical function.
Methods The ApoE-KO mice are fed a high fat or regular diet at 7 weeks of age, and sacrificed at 0, 15, and 30 weeks. For collagen density and organisation, tendon tissue will be viewed using second harmonic generation microscopy to determine collagen density and organisation. Tendons will be systematically scanned, and collagen density will be determined and compared among different study groups. Lipid deposition in Achilles tendon will be observed with oil red O and filipin staining. Tissue sections will be viewed and photographed with brightfield, polarisation, and fluorescence microscopy. Filipin dye will be excited with epiillumination and viewed through a 510-nm barrier filter. Ultrasound imaging will be used to analyse patellar tendon cross-sectional area (ie, tendon thickening, an indicator of tendon pathology). The effect of dyslipidemia on tendon biomechanics will be determined by testing the tendon to failure at 0.003 mm/s in a Bose ElectroForce BioDynamic test instrument. Local strain will be measured and tendon stiffness calculated.
Anticipated results and significance Tendinopathy tendon in an H&E stained tissue section is expected to show disorganised collagen architecture with high cellularity of rounded tenocytes (as compared to parallel, longitudinal architecture with scattered elongated tenocytes of normal tendons).4 Tissue sections of Achilles tendon is expected to have wavy, discrete, band-like deposits of green filipin staining. Oil red O staining is to be observed intracellularly and extracellularly.5 If significant, these results will provide the first insights into the biology underlying dyslipidemia-induced tendinopathy, and could have direct impact on clinical care and preventive strategies.