Nuclei are stained in blue with Dapi
Nuclei are stained in blue with Dapi. resistant to the apoptotic effects of irradiation (the number of BM cells is usually significantly higher in TNF- KO mice BM 3 days after irradiation, p 0.05). Open in a separate window Physique 3 Sub-lethal irradiation reduces the number of total BM cells, an effect partially dependent on TNF-. A. WT and TNF- KO mice were sub-lethally irradiated and the total number of BM cells was counted as explained in Methods. As shown, TNF- KO mice were more resistant to the apoptotic effects of irradiation. On day 3 following irradiation, the number of total BM cells is usually significantly higher in TNF- KO mice than in WT mice. The results shown were obtained from two impartial experiments, using 12 animals per experimental group and 3 animals per time point. *: p 0.05. B. WT mice were injected with PBS (control) or with antibody anti-TNF prior to sub-lethal irradiation and total BM cells were counted. Like for TNF- KO mice, anti-TNF neutralized mice were more resistent to irradiation; here, the number of total BM cells is usually significantly higher (*: p 0.02) by 24 hours after irradiation than in controls. C. The percentage of apoptotic cells 24 hours after irradiation was obtained in control and neutralized mice by flow cytometry. Both precursor (Sca1+) and myeloid (CD11b+) cells were guarded from irradiation-induced apoptosis in the anti-TNF- treated mice, where the number of cells positive for annexin V is lower than in the controls. *: p 0.01 Rabbit polyclonal to NGFRp75 for CD11b+; for Sca1+ a p value could not be calculated due to the absence of Sca1+Annexin+ cells in neutralized mice. The results shown in B and C were obtained from one experiment, using 12 animals per experimental group and 3 animals per time-point. To overcome possible differences between the BM microenvironment of TNF- KO and WT mice, we also tested the effects of irradiation in the BM content of WT mice treated with anti-TNF- Ab. In Physique 3B, a marked decrease in BM cell numbers (higly significant: p 0.02) was observed in untreated (control) mice 24 hours after irradiation, comparing StemRegenin 1 (SR1) with Ab-treated animals. In parallel with these results, flow cytometry analysis for apoptotic cells showed that the number of Sca1/Annexin V- and CD11b/Annexin V-positive cells is usually significantly higher (p 0.01) in control mice in comparison with anti-TNF- treated mice, confirming the apoptotic effect of TNF- in both precursor and mature BM cells (Physique 3C). For the other time-points, also a minor protection from irradiation-induced apoptosis is usually observed in anti-TNF- treated mice (data not shown). Taken together, these experiments suggest irradiation induces TNF- production in the BM, and that the released TNF- is usually partially responsible for the increase in BM cell apoptosis following irradiation. 3-Cycle Irradiation Protocol Induces BM Dysfunction, Suggestive of MDS Next, we developed a 3-cycle irradiation protocol (to test the long term effects of irradiation), and characterized its effects in inducing BM dysfunction. First, we showed the irradiation protocol induces loss of microsatellite markers by BM cells, suggesting it had carcinogenic/transforming capacity (Supplementary Physique S1). Concerning the haematological phenotype induced by the irradiation protocol, as shown in Fig 4, three months after the last irradiation, 3x irradiated mice showed a significant decrease in circulating white blood cells (WBC), platelets and red blood cells (RBC). In addition, 3x irradiated mice showed a significant increase in MCH-pg Hemoglobin per RBC (Physique 4D), a phenotype usually seen in macrocytic anemia. Taken together, the concomitant clinical presentation of cytopenias, StemRegenin 1 (SR1) thrombocytopenia and anemia, and the incidence of cytogenetic abnormalities in 3x irradiated mice, strongly suggests this may be considered StemRegenin 1 (SR1) a model of BM dysfunction with clinical features of secondary MDS. Open in a separate window Physique 4 A 3-cycle irradiation protocol induces cytopenias and macrocytic anemia in WT mice. A. The WBC is usually significantly reduced in 3x irradiated mice. B. The number of platelets is usually reduced in 3x irradiated mice. C. The number of RBC is usually significantly reduced in 3x irradiated mice..