Two-photon imaging of oxidative stress in living erythrocytes as a measure for human aging

Abstract

According to the "oxidative stress theory" of aging, this process is accompanied by a progressive and irreversible accumulation of oxidative damage caused by reactive oxygen species (ROS). This, in turn, has a deleterious impact on molecular mechanisms in aging thereby altering the physiological function of the organism, increasing the risk of different aging-related diseases, as well as impacting the life span. The aim of the current study was to investigate oxidative stress in living red blood cells (RBCs) in human aging as an oxidative stress-related pathological condition. Two-photon laser scanning and light microscopy techniques were applied to analyze the oxidative stress in RBCs and the cell viability. Spectrophotometric analyzes were performed to determine the percentage of RBC hemolysis, activities of superoxide dismutase and catalase in RBCs, as well as the ferroxidase activities of ceruloplasmin in blood plasma samples. The studies included three human aging groups, young, middle-aged, and elderly. According to the results, the two-photon fluorescence of carboxy-DCFDA, indicating the intensity of oxidative stress, significantly increase in RBCs by the increase of age (P < 0.05), and these intensities are in statistically significant positive correlation with age (P < 0.001) and a strong negative correlation (P < 0.05) with the activity of catalase in RBCs and ferroxidase activity of ceruloplasmin in plasma. In conclusion, two-photon fluorescent imaging of oxidative stress in human living RBCs is a valuable and accurate method for the determination of aging processes in humans and can be suggested as a novel indicator for human aging processes in individual aging.

Fig. 1.

Carboxy-DCFDA fluorescence mean intensities in three age groups, young, middle aged and elderly. Fluorescent intensities are represented in arbitrary units (AU; mean ± SEM). *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig. 2.

Representative two-photon fluorescence intensity images of RBCs from young, middle aged and elderly people treated (B, D, F) and non-treated (intact) with H₂O₂ (A, C, E).

Fig. 3.

The cell count (A) in 1 µL of blood and percentage of hemolysis (B) of RBCs of all three age groups exposed (RBCs + H₂O₂) and non-exposed (RBCs) to additional oxidative stress (M ± SEM). *P < 0.05 (number of participants, n = 15 in each group).

Fig. 4.

The activities of superoxide dismutase (A) and catalase (B), as well as the ferroxidase activity of ceruloplasmin in the blood plasma of young, middle aged and elderly people (M ± SEM). *P < 0.05; **P < 0.01; ****P < 0.0001 (number of participants, n = 15 in each group).

Fig. 5.

Correlation analysis of two-photon fluorescent intensity in human RBCs vs. age, activity of catalase and ferroxidase activity of ceruloplasmin (number of participants, n = 15 in each group).