Abstract

Introduction: High dose total body irradiation (TBI) causes severe damage to the hematopoietic system, resulting in ablation of both red and white blood cells and a high probability of infection, hemorrhage, and death. This is known as the hematopoietic syndrome of the acute radiation syndrome (HS-ARS). TBI is used therapeutically, and incidental exposure through nuclear accidents or radical terrorism is a current threat for which preparation is critical. Safe, effective, and pragmatic agents against HS-ARS are needed. We have identified prostaglandin E2 (PGE2) as a promising medical countermeasure to promote hematopoietic recovery and increase survival from lethal irradiation. Here, using the PGE2 analog 16,16-dimethyl PGE2 (dmPGE2), pre-irradiation dosing strategies were assessed in survival studies with an established mouse model of HS-ARS, and time course analyses were performed to elucidate the early cellular events associated with PGE2 radioprotection.

Methods: C57BL/6 mice (n = 20/group) were exposed to LD70/30 (872 cGy) or LD90/30 (904 cGy) and treated with one of the following four dmPGE2 dosing strategies: a single dose of 35 μg at time -30 min, -1 hr, or -3 hr, or a double-dose of 20 μg each at time -45 min and -15 min pre-irradiation. Thirty-day survival was evaluated. The double-dose regimen was further utilized to assess cellular effects over time following LD70/30. Peripheral blood (PB) and bone marrow (BM) cells were harvested on days 1, 2, 4, 7, and 10 post-irradiation (n = 3/group/day). BM was analyzed by flow cytometry with immunostaining for surface markers (Sca-1, c-Kit, CD150, CD48 and blood cell lineage markers), cell cycle (Hoechst/PyroninY), and apoptosis (AnnexinV/7-AAD). PB differentials were assessed by a veterinary hematology analyzer.

Results: All dmPGE2 regimens conferred 100% survival to mice receiving LD70/30, and all regimens provided a distinct survival advantage to mice receiving LD90/30 with the double-dose strategy conferring 100% survival at LD90/30. The double-dose was then chosen to assess cellular effects of dmPGE2 throughout the critical 10-day time period following LD70/30. Within the BM, marker-defined HSC numbers were significantly preserved by day 1 post-irradiation compared to vehicle controls, with a drop to vehicle levels observed on day 2 only. Cell cycling frequency among primitive hematopoietic cells was also conserved on day 1, decreased on day 2, then restored to non-irradiated control levels by days 7-10, while that of the unprotected mice remained significantly lower. Differentiated BM cells (lineage marker-positive) were substantially less apoptotic by day 2 and, in significant contrast to the untreated mice, returned to control levels of basal apoptosis by days 7-10. Overall BM cell populations were found to be largely replenished within one week as compared to the vehicle-treated mice, which remained significantly depressed. Further, 73-88% of detectable BM cells from the untreated mice were non-viable by day 4 post-irradiation based on sub-diploid DNA content and did not recover through day 10; dmPGE2-treated mice maintained significantly greater BM cell viability at each time point, returning to only 17-26% sub-diploid by day 7 (non-irradiated controls demonstrated ~5% sub-diploid DNA content). A corresponding effect on PB resulted in significant rescue from neutropenia, anemia, and thrombocytopenia by or before day 10.

Conclusions: A single treatment with PGE2 prior to lethal irradiation can facilitate virtually complete survival, though a double treatment regimen may be most effective; further work will test whether this may be primarily due to the combined higher dose or to the altered timing. This PGE2-mediated survival involves early protection of both primitive and differentiated hematopoietic BM cells, affecting cell number, cell cycling, and apoptosis. Early hematopoietic BM recovery subsequently rescues PB components, facilitating ultimate survival. With further study, PGE2 may serve to provide an effective radioprotectant with the potential to safeguard patients undergoing TBI therapy, as well as military personnel and first responders, from the risk of radiation-associated mortality.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.