Effects of Sublethal Irradiation on Murine Bone Marrow

Abstract 2243

Lethal irradiation leads to hematopoietic system effects including decreased red blood cells, white cells and platelets. Death is attributable in part to infections associated with lymphopenia and neutropenia. To determine the magnitude of the hematopoietic effects of sublethal irradiation, we irradiated 25 C57BL/6NTac female mice to a whole body dose of 9.0 Gy irradiation resulting in 90% survival at 30 days. At 0, 5 or 30 days after irradiation, five mice were sacrificed, bone marrow removed and assayed for clonogenic survival, blood analyzed for CBC, and femur assayed for histologic cellularity. Single cell suspensions, were plated in Metho-Cult GF3434 at cell concentrations ranging from 20,000 to 100,000 cells per well, incubated at 37°C for 10 days and multilineage CFU-GEMMs counted. Femur bone marrow cellularity, was scored on fixed and decalcified (Formical-4), sectioned sagittally and H&E stained samples. The sections were observed under a microscope at 20 × and the percent bone marrow cellularity determined. Five days after irradiation, there was a significant decrease in peripheral blood white blood cells × 1000 compared to day 0 (Day 0 = 2.0 + 2.2 to Day 5 = 0 + 0, p = 0.001), lymphocytes (Day 0 = 1.3 + 1.7 to Day 5 = 0 + 0, p = 0.006), neutrophils (Day 0 = 371.9 + 384.3 to Day 5 = 0 + 0, p = 0.002), and granulocytes (Day 0 = 0.5 + 0.5 to Day 5 = 0 + 0, p = 0.008). By 30 days after irradiation, there was still a significant decrease in the number × 1000 of monocytes (0.2 + 0.2, p = 0.011), but there was a significant increase in neutrophils (1221.0 + 866.9, p = 0.013) and primitive granulocytes (1.5 + 1.1, p = 0.013). Red cell numbers had returned to control levels. Marrow CFU-GEMMs were significantly decreased at 5 days compared to nonirradiated controls (p < 0.0001). Colonies were adjusted to the average number of colonies per 100,000 cells plated. At day 5, the CFU-GM numbers decreased from 373.1 + 42.7 to 12.4 + 12.5 colonies. The number of BFU-E colonies decreased from 66.6 + 14.2 to 0.8 + 1.2, while CFU-GEMM colonies went from 15.6 + 5.7 to 0 + 0. The total number of colonies decreased from 455.4 + 46.8 to 13.2 + 13.7. At 30 days all CFU categories had increased but were still significantly decreased compared to nonirradiated mice: CFU-GM at 202.3 + 48.0 (p < 0.0001), BFU-E at 37.7 + 7.0 (p = 0.013), CFU-GEMM at 3.0 + 2.2 (p < 0.0001) and total colonies at 243.0 + 46.8 (p < 0.0001). Bone marrow cellularity decreased from 93.5 + 2.5% to 4.8 + 2.1%, (p < 0.0001) by day 5 after 9.0 Gy, but by 30 days after irradiation, returned to control levels (94.8 + 0.4%). Therefore, while the LD10/30 irradiation dose results in a rapid depletion of all categories of clonogenic marrow cells, the rapid proliferative response of rare surviving hematopoietic stem cells leads to recovery by 30 days. Mitigator drugs for use in higher dose irradiated subjects must be safe and non-toxic in lower dose recovering subjects.

Supported by NIAID grant U19AI068021.