New Surgical Model for Bone–Muscle Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout Mouse

Abstract

Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the vastus lateralis (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO Biguetti et al. MSK Healing in 5LOKO Mice mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone-muscle crosstalk studies.

Alveolar socket healing in 5-lipoxygenase knockout aged female mice treated or not with high dose of zoledronic acid

“…Mutant mice for 5LO might present lower amount of osteoclasts and similar bone microarchitecture compared to WT ZL group, although, these differences resulted from distinct biological mechanisms 43 . Aged WT female mice appeared to decrease bone mass after 17 months compared to young controls, while aged 5LOKO mice presented a more robust phenotype with no major changes compared to 5LOKO young controls 29 . Since 5LO inhibition results in an osteopetrotic-like skeletal condition with few osteoclasts, consequently, the low bone turnover may contribute to the maintenance of their bone mass during their lives 36 This characteristic was the key point for the selection of this animal model for the present investigation.…”

“…Scans were reconstructed using NRecon software (Skyscan, Kontich, Belgium), realigned in Dataviewer (Skyscan, Kontich, Belgium) and three-dimensional images were obtained by CTVox (Skyscan, Kontich, Belgium). Quantitative variables were assessed using CTAn software (Skyscan, Kontich, Belgium) to examine of socket bone healing and also for skeletal phenotyping of femur and L5 vertebrae 25 , 29 , 54 . Evaluated variables comprised fraction of Bone Volume /Tissue Volume (BV/TV, %), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).…”

“…Considering inflammatory candidates to play a role in ONJ development, lipid signaling mediators derived from arachidonic acid might be considered as key players, since these pathways have been shown in bone healing and metabolism 29 . Both cyclooxygenase-2 (COX-2) and 5-lypoxigenase (5-LO) have been identified in specific sites during bone repair 30 , and are highly expressed in macrophages 31 .…”

“…COX-2 products (specifically PGE2) appears to be produced by osteocytes and protect these cells from apoptosis 32 . Furthermore, PGE2 are considered beneficial for bone healing 33 , 34 , while 5-LO and its products (i.e.LTB4 and the CysLTs), seems to exert a detrimental effect on this process, inducing osteoclastogenesis 29 , 35 , 36 . Mutant mice for 5-LO enzyme (5LOKO) show accelerated fractured healing 37 , and aged female 5LOKO mice also present improved bone regeneration in femur defects 29 .…”

“…Furthermore, PGE2 are considered beneficial for bone healing 33 , 34 , while 5-LO and its products (i.e.LTB4 and the CysLTs), seems to exert a detrimental effect on this process, inducing osteoclastogenesis 29 , 35 , 36 . Mutant mice for 5-LO enzyme (5LOKO) show accelerated fractured healing 37 , and aged female 5LOKO mice also present improved bone regeneration in femur defects 29 . These previous findings indicate that inhibition of 5LO seems to favor bone healing in endochondral bones of young and aged mice.…”