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 limbs1,2.
In skeletal muscle, ischemia reduces the aerobic capacity of cells, leading to acute inflammation and adverse biochemical alterations. These effects are worsened by reperfusion, which stimulates the recruitment of neutrophils and the production of free radicals, further damaging the skeletal muscle. This can occur from vascular occlusion, whether the result of injury or the intentional use of a tourniquet to prevent hemorrhage. Some of the key mediators in this process are myeloperoxidase (MPO), an enzyme expressed by neutrophils that is integral to the respiratory burst function and production of free radicals3, and chemokines such as CXCL1 and CXCL5 that serve to recruit neutrophils to sites of acute inflammation4.
The femoral artery was not dissected to mimic an open tourniquet in an emergency. This approach is also based on the reproducibility of creating ischemia and reperfusion as well as a consistent blood-free area. Previous research has demonstrated that exposure to non-thermal infrared (NIR) light with a wavelength of 670 nm can increase vascular collateralization in a mouse ischemic hindlimb with NIR exposure over days, mitigating the effects of IRI5. Additionally, prior research has demonstrated that NIR light can induce the polarization of macrophages into either proinflammatory (M1) or prohealing (M2) phenotypes6.
Any treatment that can minimize tissue damage and cellular death following hypoxia and reperfusion would be beneficial in increasing the success of limb salvage following vascular injuries. Therefore, the overall goal is to improve IRI by introducing 670 nm light treatment as a viable option to other treatment modalities. This paper is based on the hypothesis that exposure to NIR light during a period of ischemia decreases inflammation and tissue necrosis by decreasing the secretion of chemoattractant proteins and influx of inflammatory cells by inducing macrophages to take on an M2 phenotype.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the Department of Orthopedic Surgery for financing this study. We also thank Brian Lindemer and Grant Broeckel for their technical support.
Materials
| 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 |
References
- Drysch, M., et al. An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: Inducing acute ischemia reperfusion injury in C57BL/6 wild type mice. PLoS One. 14 (1), 0210961 (2019).
- Bonheur, J. A., Albadawi, H., Patton, G. M., Watkins, M. T. A noninvasive murine model of hind limb ischemia-reperfusion injury. Journal of Surgical Research. 116 (1), 55-63 (2004).
- Whiteman, M., Spencer, J. P. Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo. Biochemical and Biophysical Research Communications. 371 (1), 50-53 (2008).
- Becker, S., Quay, J., Koren, H. S., Haskill, J. S. Constitutive and stimulated MCP-1, GRO alpha, beta, and gamma expression in human airway epithelium and bronchoalveolar macrophages. American Journal of Physiology. 266 (3), 278-286 (1994).
- Zaidi, M., et al. Transient repetitive exposure to low level light therapy enhances collateral blood vessel growth in the ischemic hindlimb of the tight skin mouse. Photochemistry and Photobiology. 89 (3), 709-713 (2013).
- Souza, N. H. C., et al. Photobiomodulation and different macrophages phenotypes during muscle tissue repair. Journal of Cellular and Molecular Medicine. 22 (10), 4922-4934 (2018).
- Tang, G. L., Kim, K. J. Laser doppler perfusion imaging in the mouse hindlimb. Journal of Visualized Experiments: JoVE. (170), e62012 (2021).
- Ionescu, A., Zahavi, E. E., Gradus, T., Ben-Yaakov, K., Perlson, E. Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance. European Journal of Cell Biology. 95 (2), 69-88 (2016).
- Adamovich, Y., Ezagouri, S., Dandavate, V., Asher, G. Monitoring daytime differences in moderate intensity exercise capacity using treadmill test and muscle dissection. STAR Protocols. 2 (1), 100331 (2021).
- Wang, C., Yue, F., Kuang, S. Muscle histology characterization using H&E staining and muscle fiber type classification using immunofluorescence staining. Bio-Protocol. 7 (10), 2279 (2017).
- Keszler, A., Lindemer, B., Hogg, N., Weihrauch, D., Lohr, N. L. Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light. Archives of Biochemistry and Biophysics. 649, 47-52 (2018).
- Weihrauch, D., et al. Vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice. Journal of Visualized Experiments: JoVE. (121), e55036 (2017).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72, 248-254 (1976).
- Li, Z., Jiang, J., Gao, S. Potential of C-X-C motif chemokine ligand 1/8/10/12 as diagnostic and prognostic biomarkers in idiopathic pulmonary arterial hypertension. Clinical Respiratory Journal. , (2021).
- Weihrauch, D., et al. Intralipid increases nitric oxide release from human endothelial cells during oxidative stress. JPEN. Journal of Parenteral and Enteral Nutrition. 45 (2), 295-302 (2021).
- Liebert, A., Krause, A., Goonetilleke, N., Bicknell, B., Kiat, H. A role for photobiomodulation in the prevention of myocardial ischemic reperfusion injury: A systematic review and potential molecular mechanisms. Scientific Reports. 7, 42386 (2017).
- Asghari, A., Takhtfooladi, M. A., Hoseinzadeh, H. A. Effect of photobiomodulation on ischemia/reperfusion-induced renal damage in diabetic rats. Lasers in Medical Science. 31 (9), 1943-1948 (2016).
- Quirk, B. J., Sonowal, P., Jazayeri, M. A., Baker, J. E., Whelan, H. T. Cardioprotection from ischemia-reperfusion injury by near-infrared light in rats. Photomedicine and Laser Surgery. 32 (9), 505-511 (2014).
- Smith, E. R., Shapiro, G. L. Totally tourniquets. The facts & details about different types of tourniquets. JEMS: A Journal of Emergency Medical Services. 38 (11), 48-50 (2013).
- Yu, G., et al. Inhibition of myeloperoxidase oxidant production by N-acetyl lysyltyrosylcysteine amide reduces brain damage in a murine model of stroke. Journal of Neuroinflammation. 13 (1), 119 (2016).
- de Brito Sousa, K., et al. Differential expression of inflammatory and anti-inflammatory mediators by M1 and M2 macrophages after photobiomodulation with red or infrared lasers. Lasers in Medical Science. 35 (2), 337-343 (2020).
- Iida, N., Grotendorst, G. R. Cloning and sequencing of a new gro transcript from activated human monocytes: expression in leukocytes and wound tissue. Molecular and Cellular Biology. 10 (10), 5596-5599 (1990).
- Jani, S., Bergmann, S. R. Metabolic modulation of myocardial ischemia. Current Cardiology Reports. 8 (2), 123-130 (2006).
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