Two-photon microscopy imaging of oxidative stress in human living erythrocytes.

Gohar Tsakanova,Elina Arakelova,Violetta Ayvazyan,Rouben Aroutiounian,Yeva Dalyan,Arsen Arakelyan,Stepan Tatikyan,Anna Ayvazyan,Vasili Tsakanov,Samvel Haroutiunian

doi:10.1364/boe.8.005834

Abstract

Red blood cells (RBCs) are known to be the most suitable cells to study oxidative stress, which is implicated in the etiopathology of many human diseases. The goal of the current study was to develop a new effective approach for assessing oxidative stress in human living RBCs using two-photon microscopy. To mimic oxidative stress in human living RBCs, an in vitro model was generated followed by two-photon microscopy imaging. The results revealed that oxidative stress is clearly visible on the two-photon microscopy images of RBCs under oxidative stress compared to no fluorescence in controls (P<0.0001). This novel approach for oxidative stress investigation in human living RBCs could efficiently be applied in clinical research and antioxidant compounds testing.

Highlights

Oxidative stress plays a crucial role in the development and progression of pathological conditions in age-related human diseases, such as diabetes, cancer, cardiovascular disorders, rheumatoid arthritis, neurological diseases, ulcers, pneumonia, cataract, glaucoma and human aging [1,2,3]
For optimization of the oxidative stress conditions 0.05%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5% final peroxide concentrations were tested by the comparison of the data obtained upon the two-photon microscopy imaging and in vitro hemolysis assays, and the optimal condition was used for the main experiments
To find the optimal H2O2 concentration that would cause the maximum oxidative stress to the cells and at the same time minimally affect the cell integrity, we compared the percentage of hemolysis of red blood cells (RBCs) and the intensity of oxidative stress under different oxidative stress conditions

Summary

Introduction

Oxidative stress plays a crucial role in the development and progression of pathological conditions in age-related human diseases, such as diabetes, cancer, cardiovascular disorders, rheumatoid arthritis, neurological diseases, ulcers, pneumonia, cataract, glaucoma and human aging [1,2,3]. The above-mentioned circumstances show the benefits of studying oxidative stress in RBCs in pathological conditions In this regard, there is a huge amount of methodologies and approaches for studying ROS in RBCs, that, are based on the measurement of either the end product of oxidation proteins or lipids, or the antioxidant activity of the cells, involving superoxide dismutase, catalase, glutathione, ceruloplasmin, xanthine oxidase, etc., and require the use of hemolysate of RBCs but not the cells themselves [10,11,12,13]. Chemical reactions that involve the generation of free radical compounds, form the basis of the most widely used methods These methods work via a single electron transfer reaction, such as the phosphomolybdenum method [17], a hydrogen atom transfer reaction between an oxidant and a free radical, or by oxidative coupling between these two species, such as the diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical assay [18]

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