Evidence for radiation-induced disseminated intravascular coagulation as a major cause of radiation-induced death in ferrets

Abstract

Purpose: The studies reported here were performed as part of a program in space radiation biology in which proton radiation like that present in solar particle events, as well as conventional gamma radiation, were being evaluated in terms of the ability to affect hemostasis.

Methods and materials: Ferrets were exposed to 0 to 2 Gy of whole-body proton or gamma radiation and monitored for 30 days. Blood was analyzed for blood cell counts, platelet clumping, thromboelastometry, and fibrin clot formation.

Results: The lethal dose of radiation to 50% of the population (LD50) of the ferrets was established at ∼ 1.5 Gy, with 100% mortality at 2 Gy. Hypocoagulability was present as early as day 7 postirradiation, with animals unable to generate a stable clot and exhibiting signs of platelet aggregation, thrombocytopenia, and fibrin clots in blood vessels of organs. Platelet counts were at normal levels during the early time points postirradiation when coagulopathies were present and becoming progressively more severe; platelet counts were greatly reduced at the time of the white blood cell nadir of 13 days.

Conclusions: Data presented here provide evidence that death at the LD50 in ferrets is most likely due to disseminated intravascular coagulation (DIC). These data question the current hypothesis that death at relatively low doses of radiation is due solely to the cell-killing effects of hematopoietic cells. The recognition that radiation-induced DIC is the most likely mechanism of death in ferrets raises the question of whether DIC is a contributing mechanism to radiation-induced death at relatively low doses in large mammals.

Figure 1

Delivery of the proton dose to ferrets. (A) Ferrets were given radiation to ensure a whole body dose by having 100% of the dose given within the cage, (B) Ferrets were constrained in order to deliver the full isodose region.

Figure 2. Kaplan-Meier mortality curves for ferrets

The ferrets were exposed to radiation doses from 0 – 2 Gy and survival was determined over the course of 30 days. Statistical significance was determined using a Mantel-Cox test; the 2 Gy curve was determined to be statistically significant with ***p < 0.001. (A) Ferrets exposed to 2 Gy of proton radiation (n=12) have a 0% survival rate by day 13. Exposures of 0, 0.5, and 1 Gy resulted in 100% survival over the course of 30 days. (B) Ferrets were exposed to gamma radiation at doses from 0 – 2 Gy.

Figure 3

Whole blood clotting is severely impaired by proton radiation. Whole blood was collected from animals exposed to 0.5–2 Gy at pre-irradiation to 30 days post-irradiation. Whole blood was analyzed by rotational thromboelastometry (ROTEM). Statistical significance was obtained using a Student’s paired t-test; significance is reported for *p < 0.05, **p < 0.005, and ***p < 0.001. (A) Coagulation time (CT) is significantly increased with ferret exposure to 2 Gy of protons. (B) Clot Formation Time (CFT) is significantly affected by all doses of proton radiation with increases observed at 0.5–30 days. (C) The proton dose significantly increased the time to generate a stable clot, as measured by the clot formation time (CFT). The results from the ferrets were plotted in an inverse survival curve in which the percentage of animals reaching the CFT at a specific time was measured (See Supplementary Section for details of this technique). By day 7, 100% of the animals reached the CFT at ~ 10 min. At day 13, 67% reached the CFT by 60 min, and 33% of animals never reached the CFT (i.e., never formed a clot). The trend of the curves indicating that there is an increased percentage of the animals having increased CFT values is statistically significant (at p<0.05), as determined by using a Mantel-Cox test. (D) A significant decrease in the Maximum Clot Firmness (MCF) was observed, with the nadir at day 13 for ≥ 1 Gy. The 2 Gy cohort decreased rapidly to a terminal MCF of 19 ± 2 mm.

Figure 4

White blood cell counts are severely affected by proton radiation exposure. Blood was collected prior to radiation (pre-irradiation), until 30 days post-irradiation. 2 Gy data were unavailable at days 21 and 30 due to 0% animal survival. Data were normalized to pre-irradiation counts and are reported as a fraction of control. The dotted line indicates the pre-irradiation count of 1.0. ANOVA was used to determine the dose response for each time-point (with three doses; if only two dose groups were available, a student’s t-test was used). Significance is reported for *p < 0.05, **p < 0.005, and ***p < 0.001. A statistically significant dose dependent decrease in WBC (A), lymphocyte (B), neutrophil (C), monocyte (D), and eosinophil (E) counts was observed at 0.5, 7, and 13 days post-irradiation.