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.