The role of 5-Lypoxigenase (5-LO) on the musculoskeletal system: a comparative analysis of regenerative and senescence processes in 129/Sv-WT vs. 129/Sv 5-LOKO

Abstract

Interactions between bone and muscular tissue as parts of the musculoskeletal system are beyond their structural and mechanical functions, including stimulation of inflammatory contexts. It has been demonstrated that both muscle and bone may act mutually regulating immune system, as the case in the development of chronic diseases related to ageing (osteoporosis and sarcopenia), and also along the tissue regeneration that takes place after bone and/or muscle injuries. In both conditions, destruction or regeneration, inflammatory factors (e.g. leukotriens from arachidonic acid metabolism by 5-lypoxigenase [5-LO]) are present in bone and muscle. However, the function of 5-LO in the musculoskeletal system remains poorly understood. The aim of this study is to analyze the role of 5-LO in bone and muscle, considering the process of regeneration of both tissues in 129/Sv-WT and 129/Sv 5-LOKO mice, male and female, at 12 weeks (adult mice) and 64 weeks old (elderly mice). To achieve this goal, bone defects and muscle injuries will be performed in the right side of craniofacial region (masseter muscle and bone from mandible), and also long bones and associated muscles (femur and vastus lateralis). Bone and muscle of left side will be used as controls. At 7 and 21 days post surgeries, the animals will be euthanized and the specimens will be removed for differential microscopic analyses, including microCT and birefringence of collagen fibers (for bone tissue), histochemical ATPase method for muscle, histopathological and morphometric analyses for bone and muscle (slides stained in H&E and TG), immunolabelling for different targets involved in regeneration of bone (Runx-2, TRAP, OCN, RANKL, OPG, and Col-1) and muscle (MyoD and myogenin). Additionally, immunostaining of inflammatory markers for arachidonic acid metabolism (COX-2, 5-LO) and macrophages (F4/80, CD80 e CD206) will be performed in muscle and bone. Collected specimens will also be used for molecular analysis by zymography for MMP2 and MMP9, and ELISA for TNF± and IL-6. Bone and muscle of other body regions (lumbar vertebrae, maxilla and cranium, and muscles gastrocnemius and soleus) will be used (such as control side of femur and mandibule) for phenotype characterization of these tissues in both sex (male and female), ages (12 weeks and 64 weeks) and experimental conditions (5-LOKO e WT). For these characterization, microCT will be used for bones, histological and immunohistochemical analyses for well-studied targets as well as atrophy markers for muscles like Atrogenin-1 and MuRF-1, zymography and ELISA, and histochemical ATPase analysis for muscle. Density of muscle mass will be obtained by evaluation the relation between muscle weight (gastrocnemius, soleus and masseter) and total body weight. In conclusion, the collective results from this project could provide useful insight for the mechanism of action of 5-LO in tissue regeneration and homeostasis of musculoskeletal system. (AU)