Role of 5-lipoxygenase enzyme (5-LO) on the intramembranous and endochondral bone phenotype: comparative macroscopic and microscopic anatomy between 129/Sv-WT and 129/Sv 5-LOKO mice

Abstract

Bone formation and bone resorption can be influenced by different inflammatory factors, such as the products from Arachidonic Acid (AA) metabolism. In this context, the AA products metabolized by 5-lipoxigenase (5-LO), such as the leukotrienes LTB4 and CysLT, have a resorptive effect, since they stimulate the increase of osteoclastogenesis. This study aim to analyze the role of 5-lipoxygenase enzyme on the bone phenotype from different skeleton sites, considering bones from intramembranous and endochondral embryologic origins. The macro or microscopic analysis will be performed comparatively considering 129/Sv-WT and mice with genetic disruption of 5-LO enzyme (5LO knockout, or 129/Sv 5-LOKO). Then, 20 male mice (10 WT mice and 10 -5LOKO mice), 12 weeks old, will be euthanatized for sample collection of different bone specimens. Bone specimens (mandible, maxillae, skull bones, femur and L5 vertebrae) will be fixed in 10% formalin solution, then washed in water and storage in ethanol 70° for microCT scanning. Subsequently, bone specimens will be submitted to histological processing for staining methods: hematoxylin and eosin (HE), goldner thrichrome (GT), picrosirius red and Schmorl staining. After microCT scanning and reconstruction, the macro and microscopic characterization will be performed following tridimensional reconstructive methods, and also quantitative analysis of microtomographic parameters by considering cortical bone volume (BV), the propotion of bone volume by tissue (BV/TV, %), the trabecular microarchitecture (number of trabeculae [Tb.N], separation [Tb.Sp], and thicknes [Tb.Th]). For comparative evaluation of microscopic anatomy, will be used morphologic parameters of cellular and extracellular components, by observation of histological slices stained in HE, GT, picrosirius red (analysis of collagenous matrix by birefringence). The quantities of osteocytes and canaliculae will be analysed by means Schmorl staining. At the conclusion of quantitative analysis, the results will be submitted to statistical analysis of normal distribution, and then, suitable tests will be selected for significance analysis between groups, considering a p value d 0,05.