Effects of combined hypoxia and irradiation on mouse bone marrow hematopoietic cells

Objective‍ ‍To determine the effects of hypoxia pre-treatment combined with radiation damage on the hematopoietic cells in the bone marrow of mice. Methods‍ ‍A total of 165 male C57BL/6 mice (10~12 weeks old, weighing 20~25 g) were randomly divided into 7 groups: normal control (Control, n=33), 6 Gy irradiation (6-Gy, n=43), 7 d hypoxia-6 Gy irradiation (Hy-7 d+6 Gy, n=43), 7 Gy irradiation (7 Gy, n=12), 7 d hypoxia-7 Gy irradiation (Hy-7 d+7 Gy, n=12), 7 Gy continuous hypoxia treatment (Hy-7 d+7 Gy+Hy, n=12), and 6 Gy continuous hypoxia treatment (Hy-7 d+6 Gy+Hy, n=10). The mice of the hypoxia treatment groups were given 7-day hypoxic pretreatment (12% oxygen) in a normobaric hypoxic chamber, while those of the other groups were housed in normoxic condition. After pretreatment, the mice of the irradiation groups were exposed to a single 6 or 7 Gy of whole-body 60Co γ-irradiation in normoxia. The mice of the hypoxia and irradiation groups were kept in hypoxic condition in 24 h post-irradiation followed by being resumed to normoxia, while those of the continuous hypoxia treatment groups were remained in hypoxia. After bone marrow cell suspensions were prepared from the Control, 6 Gy, and Hy-7 d+6 Gy groups, bone marrow nucleated cells (BMNCs) were counted via automated cell counter. HE staining was employed to observe pathologic changes in medullary cavity, and flow cytometry was used to assess Lin-Sca1⁺c-Kit⁺ (LSK) hematopoietic stem/progenitor cells, myeloid progenitors (MPs), and mature T/B/myeloid cells. The mice of the 7 Gy, Hy-7 d+7 Gy, and Hy-7 d+7 Gy+Hy groups were monitored for 30-day survival after hypoxic pretreatment. The dynamic changes in the counts of red blood cells (RBC), white blood cells (WBC) and platelets (PLT), and hemoglobin (HGB) level were observed in the 6 Gy, Hy-7 d+6 Gy, and Hy-7 d+6 Gy+Hy groups with aid of a fully automatic blood analyzer. Single-cell RNA sequencing was performed on bone marrow cell suspension derived from the mice euthanized in 17 d after irradiation from the Control, 6 Gy, and Hy-7 d+6 Gy groups. Results‍ ‍①Compared to the Control group, the 6 Gy group showed significantly reduced BMNCs (P<0.01), dilated bone marrow sinusoids, and erythrocyte extravasation. The Hy-7 d+6 Gy group exhibited higher cellular density and attenuated BMNC loss than the 6 Gy group (P<0.01). ②Flow cytometry revealed less LSK, MP, and mature T/B/myeloid cells in the 6 Gy group than the Control group (P<0.05), and the reduced counts of LSK and MP were mitigated in the Hy-7 d+6 Gy group (P<0.01). ③The Hy-7 d+7 Gy group demonstrated improved 30-day survival than the 7 Gy group (P<0.01), while continuous hypoxia (Hy-7 d+7 Gy+Hy) failed to enhance the survival. No statistical difference was seen in the survival rate between the 2 groups (P=0.12), though the Hy-7 d+7 Gy group showing higher survival rate. ④Routine blood test revealed that the Hy-7 d+6 Gy group showed faster WBC recovery (vs the 6 Gy and Hy-7 d+6 Gy+Hy groups, P<0.05), higher pre-irradiation RBC/HGB levels, and accelerated PLT restoration (P<0.05). ⑤Single-cell RNA sequencing indicated that hypoxia pretreatment suppressed the numbers of long-term hematopoietic stem cells/short-term hematopoietic stem cells (LT-HSC/ST-HSC) depletion in the Hy-7 d+6 Gy group when compared with the 6 Gy group, which was consistent with the results of flow cytometry. Pseudotime trajectory aligned the Hy-7 d+6 Gy group, as the Control group, showed enriched undifferentiated LSKs. Differential gene analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that oxidative phosphorylation pathway was strongly activated in the 6 Gy group, while the Hy-7 d+6 Gy group had enriched in chromatin remodeling and mRNA surveillance pathways. Conclusion‍ ‍Hypoxic preconditioning alleviates radiation-induced bone marrow injury, and post-irradiation normoxia restoration promotes hematopoietic recovery in acute radiation-exposed mice.