Effects of transcutaneous vascular photobiomodulation on the peripheral and bone marrow leukocytes determination during the inflammatory process and muscle repair in a cryoinjury animal model.

• Grant number: 23/04948-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: January 01, 2024
• End date: December 31, 2026
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Fabio Daumas Nunes
• Grantee: Tainá Caroline dos Santos Malavazzi
• Host Institution: Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Photobiomodulation (PBM) using a low-level laser (LLL) in an intravascular form (ILIB) or transcutaneously and non-invasively on blood vessels (vascular photobiomodulation, VPBM) are shown in studies in the literature with positive effects on the inflammatory process and chronic conditions, with the premise of systemic effect. Thus, inciting the need of understanding the non-invasive systemic VPBM on the muscle repair process and, mainly, understanding the mechanisms of action involved, the aim of this project is to evaluate the effect of VPBM using LLL on the inflammatory process and skeletal muscle repair of rats applied before and after acute muscle injury induced by cryoinjury in the tibialis anterior (TA) muscle. Eighty-five Wistar rats will be used, and evaluated in the following experimental groups: (1) Control; (2) Injured; (3) Non-injured + VPBM; (4) Previous VPBM + Injury; (5) Injury + post-VPBM. VPBM will be performed punctually, in transcutaneous contact over the vein/artery located at the base of the tail of the animals, using the Aluminum Gallium Arsenide laser (780 nm; 40 mW; 0.04 cm2; 80 J/cm2). The animals will be euthanized at 1, 2, 5 and 7 days after induction of the lesions. TA muscle will be collected for mRNA analysis covering 92 genes related to the inflammatory process and 83 genes referring to vascular endothelial growth factors (VEGF) by qPCR "Array" and validation by gene expression and protein analysis of IL-6, TNF-a, IL-1b and MCP-1 by qPCR and ELISA, respectively. The TA muscle will also be subjected to muscle trophism analysis of mRNA by qPCR of the muscle regulatory factors Myostatin, Calcineurin, the myosin heavy chain isoforms MyHC-I, MyHC-IIa, MyHC-IIx, MyHC-IIb, the myogenic regulatory factors MyoD, Myogenin and the atrophy marker MuRF1. To determine the effects on the signaling pathway involved in muscle regeneration, the mRNA of the somatotrophic endocrine hormone IGF-1 and its respective cellular receptor IGF1R will also be assessed, as well as the important signaling pathway in the muscle regeneration process PI3K/Akt/mTOR. Tissue oxidative stress will be determined by quantifying protein oxidation and the activity of the enzymes CAT, SOD and GPx. Differential immunolabeling of neutrophils, M1 and M2 macrophages will be performed on the muscle tissue samples. Blood samples will be collected for hematological analysis of leukocytes, erythrocytes, hemoglobins and platelets. Bone marrow samples will be used for the evaluation of leukocyte recruitment and quantification by flow cytometry. The data will be submitted for statistical analysis.