The Flow Cytometry Marker for Early Detection of Irradiation Injury in NOG Mice Bone Marrow: A Short Report

doi:10.21203/rs.3.rs-892573/v1

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Short report

The Flow Cytometry Marker for Early Detection of Irradiation Injury in NOG Mice Bone Marrow: A Short Report

https://doi.org/10.21203/rs.3.rs-892573/v1

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Background

Radiation is used for myeloablation in mice's bone marrow (BM) for hematopoietic stem cells (HSC) engraftment experiments. To assess post-radiation damage to the BM, an accessible and convenient marker is needed. This study comparatively evaluated the flow cytometer's effectiveness in detecting the ROS and Annexin V-PI level.

Methods

We divided 30 NOG mice between irradiated (n = 20) and control groups (n = 10) for each time point. After sacrificing BM samples were collected, the percentage of annexin V, PI, and ROS were investigated at two different time points (Day 2 and 14 after exposure).

Results

At the first timepoint, the level of ROS was higher in the irradiated group than in the control group, and this difference was statistically significant (P < 0.05). Also, at the second timepoint, the mean differences of all markers in the irradiated group were significant compared to the control group (P < 0.05).

Conclusions

Thus, in this mice strain to assess the BM irradiation-induced injury, measurement of ROS level is helpful.

Oncology

Applied & Industrial Microbiology

General Cell Biology & Physiology

Flow Cytometry

Radiation

NOG Mice

Annexin V

Propidium Iodide

Bone Marrow

Apoptosis

Reactive Oxygen Species

The irradiation of rodents is a well-known useful tool in immunology and oncology studies (1). Radiation studies are used to understand biophysical and biological factors affecting animal models and improve experimental results (2). Biological targets, such as bone marrow (BM) and intestinal epithelial cells, are susceptible to irradiation (3). Rodents are irradiated to myeloablation BM either as immunosuppression or for donor graft regeneration (4). Sublethal systemic radiation is usually conducted before transplanting; this is useful for engrafting tumors or human hematopoietic stem cells (HSCs) to create humanized mice (5, 6). In immunodeficient rodents, such doses of radiation are often used to protect against innate immune rejection or to induce the development of chimeric animals expressing several types of HSC (7).

Irradiation leads to oxygen-derived free radicals and reactive nonradical molecules, so-called reactive oxidative species (ROS), and apoptosis resulting from DNA strand breaks (8). It is essential to wait for at least 4 to 12 weeks later to observe the levels of apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay or fibrosis by Hemotoxylin and Eosin (H&E) to assess the level of radiation-induced injury (6, 9). However, flow cytometry is a valuable tool for the assessment analysis of necrosis and apoptosis quickly. Apoptosis can be detected by measuring the release of phosphatidylserine from the plasma membrane that Annexin V attaches to it using fluorescently-tagged annexin V and the DNA-binding dye propidium iodide (PI), necrosis can be detected (10, 11). Also, flow cytometry can be used to detect intracellular ROS (12). In this study, we aimed to comparatively evaluate the flow cytometer's effectiveness in detecting the ROS and Annexin V-PI level in BM samples following irradiation exposure in NOG mice to find the best way to measure the amount of radiation-induced injury quickly.

A total of 30 female (12-week-old) NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) mice with uniform body weight were obtained from the Digestive Disease Research Institute of Tehran University of Medical Sciences animal facility. The mice were housed in a positive, individually ventilated caging (IVC) system under controlled temperature and humidity, 12h light/12h dark cycles, and fed a sterilized pellet diet and water ad libitum. Animals were divided into two groups; Group 1 was exposure total body irradiation (TBI) at a sub-lethal dose of (3.5 Gy, 2.5 Gy/min, Elekta Precise accelerators), Group 2 was used as a control group. Bone marrow samples were collected on days 2 and 14. All procedures were conducted under a protocol approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1399.227).

The mean fluorescence intensity (MFI) of dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was analyzed using flow cytometry to evaluate ROS production in BM samples. DCFH-DA is a stable, non-fluorescent, cell-permeable compound, which penetrates the cell and converts to DCFH by intracellular esterase and then trapped within the cell and stable for a few hours. On oxidation, ROS de-esterified product is transformed to the highly fluorescent 2′,7′ dichlorofluorescein (DCF), which emits green fluorescence when excited at 488 nm, proportional to the intracellular level of ROS. DCFH-DA (10 µM) was added to the BM-mononuclear cells and incubated at 37°C for 30 min in the dark. Subsequently, cells were washed, resuspended in PBS, and detected by flow cytometry. Data were analyzed using the FlowJo 7.6.1 software (Tree Star, Ashland, OR, USA).

High-quality staining is achieved by combining Annexin V with PI; a DNA intercalating counterstain often used to distinguish between apoptotic and necrotic cells in a population. To carry out the Annexin V-PI staining procedure (BD, Cat. No. 556547). After washing cells and adding Annexin V and PI, the cells were stored in the dark, and then flow cytometry was used to evaluate them within one hour. Untreated cells were gated at the FSC-SSC (forward scatter-side scatter) plot, and percentages felt into four zones were computed.

The implantation rates were compared to determine the effect of exposure to radiation on BM injury. The Mann-Whitney U test was used to compare differences between groups. Results are reported as the mean ± SD and P < 0.05 were considered statistically significant. Data analysis was performed with GraphPad Prism 9 for Windows (GraphPad Software, Inc; CA). The irradiation group consisted of 20 animals (n = 20), and the nonirradiated control group consisted of 10 animals (n = 10) then divided into two timepoints.

After exposure to 3.5 Gy of radiation, irradiated mice (n = 20) and the control group (n = 10) were sacrificed at two different time points (2 and 14 days after exposure). Apoptosis, necrosis, and ROS in BM samples were measured via the flow cytometry method. In time point 1, data showed mean differences in the intracellular amount of ROS was significant compared to the control group (P < 0.05) (Fig. 1). However, there was no significant difference between the irradiated group and control for early and late apoptosis or necrosis at this time point. Also, the results demonstrated that in time point 2 (day 14), apoptosis, necrosis, and ROS significantly increased in the irradiated group compared to control (P < 0.05) (Fig. 2).

Radiation is one of the BM myeloablation methods for HSC engraftment study in animal models (13). To assess the level of injury after radiation exposure, we determined ROS, apoptosis, and necrosis by flow cytometry method at two different time points. Our goal was to identify a suitable flow cytometry marker among them within a few days of irradiation. At the first time point, the mean difference ROS level was higher than other markers like Annexin V and PI, that this marker increased before apoptosis or necrosis induction. According to Lorimore et al.'s study, activated macrophages and inflammatory-type reactions occurred in the hemopoietic system after exposure to ionizing radiation. Based on their results, increased Nitric oxide synthase (NOS) enzyme activity and neutrophil infiltration in the tissue, activation of macrophages provide a mechanism for damage production through a "bystander" effect, which may contribute to radiation-induced genomic instability and leukemogenesis (14). In Burr et al. study, phosphorylated histone H2AX, Caspase 3, and TUNEL by immunocytochemistry 24h after radiation were assayed. So, their findings demonstrated that radiation exposure led to the production of a damaging BM microenvironment 24 h post-irradiation that macrophages had the potential to contribute secondary damage after the initial radiation-induced injury. This delayed bystander-type effect indicated the importance of tissue responses (15). Their study results were not in line with our study because macrophages have a significant role in this mechanism, but we used the immunodeficient NOG mice that have reduced macrophage function. Thus, this mechanism might cause the delay in increased apoptosis and necrosis, and in NOG mice, a better marker is needed for early determination.

In another study, irradiation at ranges of 2 to 20 Gy was used that did not cause direct cellular apoptosis or necrosis but induced mitochondrial damage in cells; thus, an easier and faster method is needed (16). In another study, the level of apoptosis in two mice strains (C57 and CBA) in different radiation doses was assessed. The results demonstrated that apoptosis levels differed between the strains with CBA that apoptosis levels were higher at 24 h than C57, but C57 showed a higher level of apoptosis at the delayed time point (17). However, in our study, NOG mice had a higher level of apoptosis on day 14 than two days after irradiation; this may be due to differences in the strains of the study mice from other studies. On the other hand, these mice have low irradiation tolerance, and basically, doses higher than 4 Gy cannot be used for this strain of mice (18).

Mukherjee et al. investigated the relationships between apoptotic responses of cells exposed and tissue cytotoxicity in irradiated (0.25-2 Gy) murine BM. Up to 24 h post-irradiation, BM cellularity showed reductions, but in vivo apoptosis measurements were not detected. There was ongoing cell death up to 24 h post-irradiation, while the level of apoptosis is not elevated and cytokines are produced in response to the initial tumor protein 53 (p53)-induced apoptosis. In the presence of low levels of measured apoptosis, intramedullary cell death and apoptotic processes associated with pro-inflammatory mechanisms contribute to additional ongoing cell death (19). In our study, due to the absence of immune cells (B-cells and T-cells, macrophages, and natural killer (NK) cells) and pro-inflammatory mechanisms, BM damage has been delayed in NOG mice (20, 21). So, determining intracellular ROS in BM samples could be a helpful marker to measure injury of irradiation for establishment HSC engraftment in NOG mice models.

Bone Marrow

Gray

NOG

NOD/Shi-scid/IL-2Rγnull

ROS

Reactive Oxidative Species

Ethics approval and consent to participate

This study was conducted under a protocol approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1399.227).

Consent for publication

All the authors have approved the manuscript and agree with submission to your journal.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that there is no conflict of interest.

Funding

This study was funded by a grant from the Tehran University of Medical Sciences (98-02-87-43338).

Authors' contributions

Conceptualization: [Maria Kavianpour] and [Javad Verdi]; Methodology: [Kobra Moradzadeh], [Sajjad Aghayan], [Amir Arsalan Khorsand] and [Maria Kavianpour]; Formal analysis and investigation: [Maria Kavianpour] and [Zahra Jabbarpour]; Writing-original draft preparation: [Maria Kavianpour]; Writing-review and editing: [Samad Muhammadnejad]; Funding acquisition: [Javad verdi]; Resources: [Mohammad Vasei], [Javad Verdi]; Supervision: [Javad verdi]. The authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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The Flow Cytometry Marker for Early Detection of Irradiation Injury in NOG Mice Bone Marrow: A Short Report

Status:

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References

Status:

Version 1