Development of hematopoietic syndrome mice model for localized radiation exposure

Abstract

Current models to study the hematopoietic syndrome largely rely on the uniform whole-body exposures. However, in the radio-nuclear accidents or terrorist events, exposure can be non-uniform. The data available on the non-uniform exposures is limited. Thus, we have developed a mice model for studying the hematopoietic syndrome in the non-uniform or partial body exposure scenarios using the localized cobalt⁶⁰ gamma radiation exposure. Femur region of Strain 'A' male mice was exposed to doses ranging from 7 to 20 Gy. The 30 day survival assay showed 19 Gy as LD₁₀₀ and 17 Gy as LD_50. We measured an array of cytokines and important stem cell markers such as IFN-γ, IL-3, IL-6, GM-CSF, TNF-α, G-CSF, IL-1α, IL-1β, CD 34 and Sca 1. We found significant changes in IL-6, GM-CSF, TNF-α, G-CSF, and IL-1β levels compared to untreated groups and amplified levels of CD 34 and Sca 1 positive population in the irradiated mice compared to the untreated controls. Overall, we have developed a mouse model of the hematopoietic acute radiation syndrome that might be useful for understanding of the non-uniform body exposure scenarios. This may also be helpful in the screening of drugs intended for individuals suffering from radiation induced hematopoietic syndrome.

Figure 1

Representative image of animal exposure with γ-rays in a field size of 2 cm × 35 cm.

Figure 2

Effect of various doses of radiation (localized exposure to both femurs) on mice survival. All the animals survived in 7, 9, 10, 12, 15 Gy localized irradiation group with LD₅₀ and LD₁₀₀ as 17 and 19 Gy respectively. For survival assay, mice were randomly divided into seven groups of six mice in each group and observed for a period of 30 days for radiation induced morbidity and mortality. Survival experiment was independently repeated thrice. Data obtained was statistically analyzed and represented by a Kaplan–Meier survival curve.

Figure 3

Cell counts in mice peripheral blood after 15 Gy localized radiation exposure at different time points. (A) Indicate WBC counts in the blood collected at various time points from mice. (B,C) represent lymphocytes and granulocytes percentage respectively. (D) Bars reflect RBC counts, (E) shows haemoglobin quantity in dL of blood, and (F) indicates the platelets counts at different time interval. Cells were counted using an automated haematology analyzer. The bars represent the mean ± SD of 6 animals. A value of p < 0.05 is considered statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p > 0.05).

Figure 4

Represents the bone marrow counts and differential bone marrow counts in terms of nucleated and non-nucleated cells at different time points (A) Bone marrow counts of untreated and treated mice at various time intervals. (B 1–7) Representative images of bone marrow smear which shows nucleated and non-nucleated cells of untreated and treated samples at various time points. (C) The bars represent the nucleated and non-nucleated cells population post-irradiation. The bars represent the mean ± SD of 6 animals. A value of p < 0.05 is considered statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p > 0.05).

Figure 5

Effect of 15 Gy localized dose on the splenocyte and thymocyte counts in mice post-irradiation. (A) Splenocytes counts of untreated and treated mice at various time interval. (B) Thymocytes counts of untreated and treated mice at various time intervals. The bars represent the mean ± SD of 6 animals. A value of p < 0.05 is considered statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p > 0.05).

Figure 6

Hematopoietic stem cell marker expression in mice bone marrow cells after 15 Gy localized radiation exposure. (A,B) represents the flow cytometry data in the form of histograms of CD 34 and Sca 1 respectively. (C) Bars indicate CD 34 expression in mouse bone marrow at various time intervals. (D) Bars indicate Sca 1 expression in mouse bone marrow at various time intervals. The results were analyzed at BD-FACS LSR II using BD FACSDiva V7 program. The bars represent the mean ± SD of 6 animals. A value of p < 0.05 is considered statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p > 0.05).

Figure 7

Changes in cytokines levels after localized 15 Gy exposure. (A–H) Bars indicate expression of IL-3, IL-6, G-CSF, GM-CSF, TNFα, IFNγ, IL-1α, and IL-1β. The results were analyzed at BD LSR II using BD FCAP Array V3 software. The bars represent the mean ± SD of 6 animals. A value of p < 0.05 is considered statistically significant *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant (p > 0.05).