Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation

Summary

We describe the reduction of reperfusion injury by 670 nm irradiation in a mouse model of ischemia and reperfusion by tourniquet placement. This 670 nm irradiation reduced the inflammatory response, decreased the number of proinflammatory macrophages, and increased the protective macrophages.

Abstract

Tissue damage and necrosis from inflammatory processes are a consequence of ischemia reperfusion injury (IRI). In skeletal muscle, ischemia reduces the aerobic energy capacity of muscle cells, leading to adverse biochemical alterations and inflammation. The goal of this study is to show that exposure to near-infrared light (NIR) during a period of ischemia reduces IRI by decreasing necrosis and inflammation in addition to decreasing proinflammatory M1 and increasing protective M2 macrophages. C57/Bl6 mice underwent unilateral tourniquet-induced hindlimb ischemia for 3 h followed by reperfusion for either 15 or 30 min. Mice were randomly assigned to 3 groups. Group 1 underwent IRI with 30 min reperfusion. Group 2 underwent IRI with a 15 min reperfusion. Each group consisted of 50% no-NIR and 50% NIR-treated mice with exposure of 50 mW/cm² for 5 min/1 h after tourniquet closure. Group 3 were sham animals anesthetized for 3 h omitting IRI.

Laser doppler flow imaging was performed on all mice to confirm ischemia and reperfusion. Flow data were expressed as the ratio of ischemic limb and the contralateral control. The mice were euthanized after reperfusion, and the quadriceps and gastrocnemius were harvested. Immunoprecipitation and western blot of macrophage-markers CD68 (M1) and CD206 (M2) were performed and normalized to CD14 expression. The expression of the inflammatory markers CXCL1 and CXCL5 was significantly reduced by NIR in the IRI group. A significant decrease in CD68 and an increase in CD206 expression was observed in animals receiving IR and NIR. Tissue necrosis was decreased by NIR in the IRI group, as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The findings demonstrate that exposure to NIR reduced IRI and improved tissue survival. NIR reduced inflammation, decreased proinflammatory M1, and increased protective M2 macrophages. Exposure to NIR reduced inflammation and enhanced regeneration, leading to tissue protection following ischemia.

Introduction

Ischemia reperfusion injury (IRI) is a clinical challenge seen following vascular injuries and the prolonged use of surgical tourniquets. Previous studies have shown that 60-90 min is the upper threshold for warm ischemia time, beyond which irreversible tissue damage can occur. More than any other single factor, the limitations of warm ischemia time limit the success and salvage of reimplantation of dysvascular limbs^1,2.

In skeletal muscle, ischemia reduces the aerobic capacity of cells, leading to acute inflammation and adverse biochemical alterations. These effects are worsened by....

Protocol

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol: AUA#1517). All research involving mice was conducted in conformity with PHS policy. 

1. Tourniquet placement

NOTE: A tourniquet was placed to induce ischemia and achieve a blood-free surgical fie.......

Discussion

This paper describes one of the first studies to focus on the reduction of reperfusion injury by NIR light treatment by changing the inflammatory response in the hindlimb. Ischemia reperfusion injury and NIR light treatment are not entirely novel. Other studies focused on ischemia reperfusion by NIR light. NIR light treatment has been successfully used in the reduction of myocardial infract size and reduction of renal damage after ischemia reperfusion injury. Quirk et al. reported a reduction in myocardial infarct size a.......

Materials

Name	Company	Catalog Number	Comments
2,3,5-Triphenyltetrazolium	Sigma Aldrich	17779-10X10ML-F	1% solution
4–15% Criterio TGX Stain-Free Protein Gel	BioRad	#5678084	Tris-glycine extended gels
4x Laemmli Sample Buffer - 1610747	BioRad	16110747
670 nm light source	NIR Technologies		custom made
BCA Protein Assay Kit	Thermo Fisher	23227
BioRad ChemiDoc	Bio-Rad		Imaging system
Bio-Rad
β-mercaptoethanol	BioRad	1610710
CXCL1 ELISA	R&D Systems	DY453-05
CXCL5 ELISA	R&D Systems	DX000
Forane	Baxter	1001936040	isoflurane inhalant
goat anti-rabbit IgG-HRP	Santa Cruz Biotechnologies	sc-2004	1:10,000 dilution
Ice Accu ice pack
Laser doppler Imager	Moor	MOORLDI2-HIR
monoclonal CD14 antibody	Santa Cruz Biotechnologies	sc-515785	1:200 dilution
monoclonal CD206 antibody	Santa Cruz Biotechnologies	sc-58986	1:200 dilution
monoclonal CD68 antibody	Santa Cruz Biotechnologies	sc-20060	1:200 dilution
Pierce Protein free (TBS) blocking buffer			blocking buffer
polyclonal Chlorotyrosine Antibody	Hycult	HP5002	1:1,000 dilution
Protein A/G PLUS-Agarose	Santa Cruz Biotechnologies	sc-2003
Super Signal West Femto	ThermoFisher	34095	enhanced chemiluminescence reagent