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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.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Aging is а complex process resulting in alterations of physiological functions and in elevated mortality rate. It is accompanied with different modifications in genetic, molecular, cellular, organ and system levels, leading to the susceptibility to many aging-related pathological diseases [1,2].
Reactive oxygen species (ROS) have been shown to be one of the primary determinants of aging. According to the “oxidative stress theory” of aging [2–4], during aging a progressive and irreversible accumulation of oxidative damage caused by ROS takes place, which has 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 [2]. Oxidative stress occurs due to a violation of prooxidant-antioxidant homeostasis which in turn leads to the generation of ROS that have deleterious effects on cell membranes, proteins, nucleic acids and other organic compounds [5,6].
Although the age is a continuous variable, which cannot be changed anyhow, however, its phenotypic occurrence is very heterogeneous and is changed from individual to individual, which is reflected in the individual’s susceptibility to different age-related diseases, death, life span and quality of life [7]. Particularly, aging is one of the leading factors for such diseases as anemia, Alzheimer’s disease, cardiovascular diseases, osteoporosis, dementia, diabetes, etc. [8]. Moreover, the phenomenon of physiological aging explains why different people with the same chronological age are more susceptible to different microbial infections [9]. Therefore, for the treatment of different diseased conditions, it is very important not only to rely on the chronological age in years, but also take into account the physiological aging, including the molecular and/or cellular phenotypes of age.
Thus, there is an urgent need to develop appropriate approaches for the detection of aging processes and risk of mortality, which would also help to prevent the development of age-related disorders and not only extend the life span, but also increase the quality of life [10]. Currently, several molecular and clinical methods exist for the determination of the appearance of phenotypic age, i.e. is the individual younger or older than physiological age on molecular level. Among these methods of the determination of aging are the assessment of telomere length [11,12], DNA methylation [13], as well as comparison of the of the individual’s observable characteristics (such as functioning) with those observed in general population. Although these methods are very precise in studies including large sample size, however, they not always associate with the phenotype in case of small sample size [7].
In our recent study we demonstrated a novel approach for investigation of oxidative stress in human living red blood cells (RBCs) using two photon laser scanning imaging technique and showed that two-photon laser scanning imaging is a valuable tool for studying oxidative stress in living RBCs under oxidative stress-related pathological conditions [14]. The combined benefits of two-photon microscopy, including deeper penetration depth in thick, highly scattering biological specimens, and decreased overall fluorophore photobleaching and photodamage compared with confocal microscopy, are particularly useful for lifetime studies that require the maintenance of viability to detect sequential events in the same specimen over extended periods of time [15]. Thus, two-photon microscopy allows to conduct long-term imaging studies in living cells and tissues to reconstruct dynamic processes [16].
RBCs represent the first line of cells in an organism subjected to both endogenous and exogenous sources of ROS [17,18]. Meanwhile, they are the biggest pool in the organism containing the powerful antioxidant capacity of the whole blood including non-enzymatic and enzymatic antioxidants. Due to this capacity, RBCs provide the maintenance of the oxidant/antioxidant balance of the organism [19]. Therefore, different oxidative stress-related pathological conditions directly alter the RBCs and the activities and/or levels of antioxidant enzymes existing in these cells [20–22], making RBCs attractive for studying oxidative stress processed in pathological conditions. Although several enzymes in RBCs, such as superoxide dismutase (SOD) and catalase, are proposed as reliable biomarkers for oxidative stress-related pathological conditions and aging [10,23], there are no data on the oxidative stress in human RBCs in healthy aging.
Hence, taking into account the “oxidative stress theory” of aging and the urgent need to develop sensitive methods and approaches for the assessment of the risk of aging mortality, the aim of current study was to investigate oxidative stress in living RBCs in human aging as an oxidative stress-related pathological condition, as well as to suggest two-photon imaging of living RBCs as a novel indicator for aging processes.
2. Materials and methods
2.1 Study population
Healthy volunteers without any serious diagnosis (neurological/psychiatric disorders or major medical illness) were enrolled in this study (Table 1). Only active and living independently healthy subjects were recruited. The participants were categorized into three age groups: young age group involving participants aged 20-40 years, middle-aged group involving participants aged 40-60 years, and elderly group involving participants at more than 60 years [24–26].
Table 1. Demographic Characteristics of the Healthy Volunteers Enrolled in the Study.
2.2 Blood collection and sample preparation
Venous blood samples (2 ml) of healthy volunteers were collected in EDTA-containing tubes to prevent coagulation [27]. RBCs were isolated from the fresh blood samples using isotonic phosphate-buffered saline (PBS; NaH2PO4 × 2H2O 123 mmol/l, Na2HPO4 27 mmol/l, NaCl 123 mmol/l; pH 7.4) solution according to the method described elsewhere [14,28–30] and were used for two-photon microscopy imaging immediately to avoid any alterations in erythrocytes’ morphology.
To obtain erythrocyte hemolysates, 100 µL of whole blood samples were centrifuged at 3000 g for 10 min at + 4 °C, and the erythrocyte-containing sediment was washed by 1 mL of 0.1% NaCl for three times, after which the sediment was resuspended in distilled Н2О with the ratio 0.6 mL/1 mL (Н2О/blood), providing 100% erythrocyte hemolysis. The hemolysate samples were stored at -30 °C until further use.
2.3 Generation of an in vitro model of oxidative stress
To have additional control groups imitating the aging process, the RBCs from all the three age groups were exposed to additional in vitro oxidative stress by their incubation in 0.3% peroxide solution at 37 °C for 40 minutes, after which the samples were washed twice in PBS at 2000g at 4 °C for 5 minutes [14,31,32].
2.4 Two-photon laser scanning microscopy imaging of oxidative stress in RBCs
For the detection of oxidative stress inside living cells, all the samples were treated with a membrane-permeable 5(6)-carboxy-2’,7'-dichlorofluorescein diacetate (carboxy-DCFDA, Sigma-Aldrich Chemie GmbH, Germany) fluorescent dye [14,33–36]. Microscopic slides of living RBCs for two-photon microscopy imaging were prepared according to the standard procedure described earlier [14]. All measurements were made at room temperature (20-22 °C).
Two-photon imaging was performed using a diode-pumped Yb:KGW ultrafast oscillator (“t-pulse”, Amplitude Systems, France) available at the AREAL facility attached to a two-photon laser scanning upright microscope (MOM- Movable Objective Microscope, Sutter Instruments, USA) with 20× water immersion objective and numerical aperture of 1.0 and 2.0 mm working distance was used to capture microscopy images of RBCs. Two-channel system with green filter was used providing 70 nm of full width at half maximum, 525 nm of maximum transmission and 92% of average transmission. А photomultiplier with 185-900 nm bandwidth (R6357; Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) was used to detect the carboxy-DCFDA fluorescence. A final power of 300 mW was maintained at the sample. Images were obtained by x,y galvanometric scanner in standard (512 × 512 pixels; 3.05 fps frame rate) modes on 12 bits photomultiplier with pixel clock of 1000 ns [14]. The mentioned parameters were maintained similar all over the experiments.
2.5 Cell viability test
The cell viability was tested by Trypan blue exclusion test [29], where the cells were stained with 0.4% Trypan blue solution for 5 minutes in room temperature and counted in a hemocytometer under a light microscope.
2.6 Calculation of in vitro hemolysis of erythrocytes
The calculation of the percentage of the hemolysis (%H) of the samples with intact RBCs and those exposed to oxidative stress, the samples were undergoing to 100% hemolysis by adding the same amount of water. The spectrophotometric measurement of the hemoglobin absorption in these three samples (the supernatant of the suspension of intact RBCs in PBS, the supernatant of the suspension of RBCs exposed to oxidative stress and 100% hemolysed samples) was done at 540 nm. The determination of the percentage of the hemolysis was done using the following equation: %H = A/B × 100, with “A” as absorbance of the supernatant and “B” as absorption of the same sample exposed to 100% hemolysis [14,37].
2.7 Detection of ceruloplasmin ferroxidase activity
The ferroxidase activity of ceruloplasmin was determined colorimetrically using аmmonium iron(II) sulfate (Mohr's salt; (NH4)2Fe(SO4)2 × 6H2O) as a substrate [38]. Due to its ferroxidase activity, the ceruloplasmin is able to oxidize the bivalent iron contained in Mohr's salt in the complex with o-phenanthroline to a trivalent form, iron(III) ammonium sulfate, with a characteristic orange colored chromagen. Briefly, 230 µL of 0.45 M acetate buffer, pH 5.8, and 50 µL of 367 µM was added to the 5 µL plasma samples, then 20 µL of 5 mg/mL o-phenanthroline was added after 10 min. incubation at 37 оС. The optical density was detected at 420 nm (A420) using a microtiter plate reader (Stat Fax 3200, Awareness Technology Inc.). The concentration of the substrate at the end of the reaction, which is inversely proportional to A420, was calculated based on the standards. For the control, the plasma and Mohr's salt were replaced by distilled water. For the two standards the plasma was replaced by distilled water or by 25 mM EDTA, respectively. The first standard is zero (A420 = 0 U/L), which represents the total ferrous concentration and the initial concentration of the substrate in the reaction mixture. In case of the second standard, EDTA binds all the ferrum imitating the total substrate oxidation (A420 = 2400 U/L). The ferroxidase activity of ceruloplasmin was expressed in activity units per 1 µL of the product, iron(III) ammonium sulfate, per 1 L of plasma per 1 minute (µM/min/L) and calculated using the following equation: FAC = (C1-C2)/t × V1/V2, where FAC is the ferroxidase activity of ceruloplasmin, C1 is the concentration of the substrate (Fe2+) at the beginning of the reaction (120 µM/L), C2 is the concentration of the substrate at the end of the reaction, t is the incubation time (10 minutes), V2 is the total volume of the reaction mixture (350 µL) and V1 is the plasma volume (5 µL).
2.8 Determination of SOD activity
The activity of SOD in the hemolysates was evaluated colorimetrically by measuring the inhibition level of adrenaline auto-oxidation by SOD in alkaline conditions and in presence of superoxide radicals [39]. Thus, 200 µL of 0.2 M bicarbonate buffer, pH 10.65, and 10 µL of 5.46 mM adrenaline hydrochloride was added to 10 µL of erythrocyte hemolysate with the subsequent incubation at 37 °С for 3 minutes. The optical density was detected at 347 nm, as A347 represents the maximum absorption of the intermediate product of adrenaline auto-oxidation. The inhibition level was calculated using the following equation: [1-(А347sample/А347control)] × 100%. For the control, the erythrocyte hemolysate was replaced by distilled water. For the calibrators, the erythrocyte hemolysate was replaced by commercial SOD (Sigma-Aldrich) in concentrations of 300, 240, 120, 60, 30 and 15 U/mL. The SOD activity was calculated according to the calibration curve representing the dependence of the level of inhibition of adrenaline oxidation from the SOD activity, which was expressed in units per mL of hemolysate (U/mL), where one unit represents the enzyme amount necessary to catalyze the formation of 1 µM of product or to cleave 1 µM of substrate for 1 minute in the conditions optimal for the enzyme activity.
2.9 Determination of catalase activity
The activity of catalase in the erythrocyte hemolysates was evaluated colorimetrically by the decrease of the H2O2 content in the reaction mixture and in the presence of the enzyme. Briefly, 50 µL of 0.08% H2O2 and 100 µL of 0.02 M ammonium orthomolybdate was added to the 50 µL erythrocyte hemolysate with the subsequent incubation at 37 °С for 10 minutes. The optical density was detected at 420 nm, which is the maximum absorption of the H2O2 - ammonium orthomolybdate complex. For the control, the erythrocyte hemolysate was replaced by distilled water. For the calibrators, the erythrocyte hemolysate was replaced by commercial catalase (Sigma-Aldrich) in concentrations of 50, 25, 10 and 5 U/mL. The catalase activity was calculated by the decrease in the H2O2 concentration according to the following equation: [1-(А420sample/А420control)] × 100%. The catalase activity was calculated according to the calibration curve representing the dependence of the absorption (A420) from the catalase activity, expressed in U/mL.
2.10 Image processing and statistical analysis
The images were processed using the Fiji/ImageJ software (ImageJ 1.50 i NIH, Bethesda, MD, USA) [40] as described previously [14]. The 0-255 normalized scale was used for the selected images, where 0 corresponds to the black and 255 corresponds to the white color. The image intensity was calculated as a sum of intensities of all the cells from the ROI. The following formula was used to calculate the corrected total cell fluorescence (CTCF). CTCF = Integrated density – (Area of selected cell х Mean fluorescence of background readings).
Statistical analysis was performed using “Graphpad Prism 8.0.1” (GraphPad Software Inc., USA) and one-way or two-way ANOVA followed by Tukey post-tests. Data is presented in bar graphs showing mean ± SEM (standard error of the mean). The degree of linear regression was assessed for correlation analysis between the studied parameters using Pearson correlation coefficient. Subsequently, Pearson’s r and P values were calculated to evaluate the effects of any difference and the correlation equations were developed. P-values less than 0.05 were considered statistically significant.
3. Results
3.1 Two-photon fluorescence imaging of human living erythrocytes
The results of two-photon fluorescence imaging of oxidative stress in human living erythrocytes in three age groups, young, middle aged and elderly, presented in Figs. 1 and 2 clearly shows that there is a clear evidence in increasing oxidative stress by aging. According to the results obtained, there is 3.8 (P < 0.0001) and 1.9 (P < 0.05) times statistically significant increase in Carboxy-DCFDA fluorescence mean intensities in elderly group compared to young people and middle aged group, respectively. Despite the obvious 2 times increase in the Carboxy-DCFDA fluorescence mean intensity between the middle aged and young groups, this difference appeared to be statistically not significant.
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).
The same tendency is registered in the modeling of aging, where H2O2 was used to mimic the aging process. Here, 2.4 (P < 0.01) and 2 (P < 0.05) times statistically significant increase was registered in mimicked pathological elderly (Elderly RBCs + H2O2) and mimicked elderly aged group (Middle Aged RBCs + H2O2) compared to mimicked middle aged (“Young RBCs + H2O2”) group, respectively. However, no any statistically significant differences were detected between the mimicked pathological elderly (Elderly RBCs + H2O2) and mimicked elderly aged groups (Middle Aged RBCs + H2O2). Also, despite the obvious differences between the groups exposed and non-exposed to additional oxidative stress in all age categories, these differences were not statistically significant.
3.2 Cell counting and cell viability
According to the results of Trypan blue staining, the cell viability of both the intact RBCs and those exposed to additional oxidative stress (H2O2), varies from 99.89% to 100% in all three age groups (Table 2). The reason of such a high percentage of living cells may be the fact that the RBCs are very delicate and sensitive, and in case of any damaging action they disrupt very fast, therefore we do not see any dead cells. We did not do MTT assay due to its long incubation time, 4 hours, because we do not keep the RBCs for such a long time.
Table 2. The Percentage of Living Cells in All Studied Groups.
The results of cell counting are presented in Fig. 3(A), which provides a clear evidence in decreasing the number of RBCs with the age. However, 1.4 times statistically significant decrease (P < 0.05) in the number of RBCs was revealed only between the young groups exposed (Young + H2O2 or mimicked middle aged) and non-exposed to additional oxidative stress (Young). Interestingly, despite the obvious tendency of decrease in the number of RBCs both exposed and non-exposed to additional oxidative stress (RBCs + H2O2) in other age categories, these differences were found to be statistically non-significant.
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).
3.3 In vitro hemolysis of erythrocytes
The results of the analysis of in vitro hemolysis of intact RBCs and those exposed to additional oxidative stress are presented in Fig. 3(B). Here also it is obvious that as higher is the age as higher is the percentage of hemolysis of RBCs. However, the only statistically significant difference was found between the elderly and young groups (2.3 times; P < 0.05).
3.4 Analysis of antioxidant system
The results of the activities of superoxide dismutase and catalase, as well as the ferroxidase activity of ceruloplasmin are presented in Fig. 4. Despite the decrease in the activity of superoxide dismutase with the increase of the age, this decrease was found to be non-significant. In case of the activity of catalase, a small however statistically significant 1.2 times (P < 0.01) decrease was observed in blood plasma of elderly people compared to young people. In contrast highly significant results have been obtained for the ferroxidase activity of ceruloplasmin. Thus, small however statistically significant (P < 0.05 and P < 0.0001, respectively) decreases of the ferroxidase activity of ceruloplasmin were observed in the blood plasma of middle aged and elderly people compared to young people, with the statistically significant (P < 0.0001) difference between the middle aged and elderly people.
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).
Moreover, a strong, statistically significant positive correlation was found between the two-photon fluorescent intensities of oxidative stress in RBCs and age (P < 0.001), as well as a strong, statistically significant negative correlation (P < 0.05) was found between the two-photon fluorescent intensities of oxidative stress in RBCs and both activity of catalase and ferroxidase activity of ceruloplasmin in plasma (Fig. 5).
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).
4. Discussion
It is proved that aging is a process involving different and complex molecular and cellular mechanisms bringing to degradation of age-regulating cellular processes [41]. However, despite the existing models of aging representing the application of different molecular methods and techniques for studying aging, the research in aging often faces hurdles in the correlation of molecular and cellular mechanisms and aging phenotype.
In this manuscript, we provided an evidence-based approach for aging research for the application of the two-photon fluorescence imaging of oxidative stress in human RBCs in the research of human aging, which very precisely illustrates the aging processes clearly reflecting the oxidative stress increase with the increase of the age. For this purpose, 6 groups were examined for two-photon fluorescence imaging of oxidative stress in human aging: young intact RBCs, young RBCs with H2O2, middle age intact RBCs, middle age RBCs with H2O2, as well as elderly intact RBCs and elderly RBCs with H2O2.
In young people the additional H2O2 causes quite high changes in the intensities, while in elderly people the changes are very low indicating an interesting tendency or phenomenon of reaching the saturation level when additional H2O2 do not cause more oxidative stress but already brings to death conditions.
To confirm the appropriateness and sensibility of this method for the determination of oxidative processes in human aging, as well as to have control experiments confirming the accuracy and the high sensitivity of the two-photon fluorescence imaging method for detection of aging processes, we studied also the cell viability, cell count and hemolysis of RBCs, as well as antioxidant system.
The reason of the high percentage of living cells obtained in our studies may be the fact that the RBCs are very delicate and sensitive, and in case of any damaging action they disrupt very fast, therefore we do not see any dead cells (We did not do MTT assay due to its long incubation time, 4 hours, because we do not keep the RBCs for such a long time.). Therefore, in case of RBCs, it is more relevant and informative to assess the percentage of hemolysis rather than the cell viability, although in our case it was very important to claim that all the cells visualized in the two-photon fluorescence images are viable cells. Our results are in compliance with the previously reported studies, which also demonstrate decrease of the number and the percentage of hemolysis of RBCs [10], as well as decline of antioxidant system with the increase of age [10]. However, these studies have been done in bigger cohorts, while our study indicates that in case of these parameters the statistical significance is not detected in smaller groups. Thus, these enzymes previously proposed as reliable biomarkers for aging [10,23] do not adequately reflect the aging processes in case of individual aging in contrast to the two-photon fluorescence intensities, which provide statistically significant changes even in smaller groups, thereby providing evidence that this approach can be used as an indicator for physiological aging. In addition, we demonstrate that the two-photon fluorescence intensities reflecting the oxidative stress in human RBCs not only positively correlate with the age, but also negatively correlate with the activity of catalase and ceruloplasmin, one more time confirming the appropriateness and sensitivity of this method.
In general, there are two types of aging measures, the population-level and the individual-level [42]. There are several measures of population aging, including chronological-age measures mainly based on current chronological-age structure and period life tables, economic, physical-health, functional, subjective measures and biomarkers [43]. The situation is difficult with the individual aging measures, where despite the chronological age, the individual’s physiological and biological decline should be taken into account. And in this case the algorithms and models developed for measuring population aging not always reflect and interpret the individual aging [44]. From this point of view, the finding of molecular and cellular biomarkers of aging are of most interest.
5. Conclusion
In conclusion, two-photon fluorescent imaging of oxidative stress in human erythrocytes is a valuable and accurate method for the determination of aging processes in human aging and can be applied not only for testing of different antiaging agents or strategies for slowing down the aging processes, but also for the regulation or forestalling the deleterious cellular mechanisms underlying aging processes and progressively leading to death, thereby both extending the life span of the organism, as well as increase the vitality and the quality of life over the entire life span of individuals.
Funding
State Committee of Science (14AR-1f09, 16GE-040, 17A-1F009, 19YR-1F045).
Acknowledgments
The authors would like to acknowledge Dr. Bagrat Grigoryan and Dr. Arsham Yeremyan for excellent technical assistance in the adjustment of the laser for two-photon microscopy, Dr. Nelli Babayan for discussion of the cell viability data, and Dr. Roza Izmailyan for literature assistance, as well as all the volunteer blood donors for their readiness to kindly contribute to this study.
Disclosures
The authors declare no conflicts of interest.
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