Combined Therapy of Pegylated G-CSF and Alxn4100TPO Improves Survival and Mitigates Acute Radiation Syndrome after Whole-Body Ionizing Irradiation Alone and Followed by Wound Trauma

Abstract

Exposure to ionizing radiation alone or combined with traumatic tissue injury is a crucial life-threatening factor in nuclear and radiological incidents. Radiation injuries occur at the molecular, cellular, tissue and systemic levels; their mechanisms, however, remain largely unclear. Exposure to radiation combined with skin wounding, bacterial infection or burns results in greater mortality than radiation exposure alone in dogs, pigs, rats, guinea pigs and mice. In the current study we observed that B6D2F1/J female mice exposed to ⁶⁰Co gamma-photon radiation followed by 15% total-body-surface-area skin wounds experienced an increment of 25% higher mortality over a 30-day observation period compared to those subjected to radiation alone. Radiation exposure delayed wound healing by approximately 14 days. On day 30 post-injury, bone marrow and ileum in animals from both groups (radiation alone or combined injury) still displayed low cellularity and structural damage. White blood cell counts, e.g., neutrophils, lymphocytes, monocytes, eosinophils, basophils and platelets, still remained very low in surviving irradiated alone animals, whereas only the lymphocyte count was low in surviving combined injury animals. Likewise, in surviving animals from radiation alone and combined injury groups, the RBCs, hemoglobin, hematocrit and platelets remained low. We observed, that animals treated with both pegylated G-CSF (a cytokine for neutrophil maturation and mobilization) and Alxn4100TPO (a thrombopoietin receptor agonist) at 4 h postirradiation, a 95% survival (vehicle: 60%) over the 30-day period, along with mitigated body-weight loss and significantly reduced acute radiation syndrome. In animals that received combined treatment of radiation and injury that received pegylated G-CSF and Alxn4100TPO, survival was increased from 35% to 55%, but did not accelerate wound healing. Hematopoiesis and ileum showed significant improvement in animals from both groups (irradiation alone and combined injury) when treated with pegylated G-CSF and Alxn4100TPO. Treatment with pegylated G-CSF alone increased survival after irradiation alone and combined injury by 33% and 15%, respectively, and further delayed wound healing, but increased WBC, RBC and platelet counts after irradiation alone, and only RBCs and platelets after combined injury. Treatment with Alxn4100TPO alone increased survival after both irradiation alone and combined injury by 4 and 23%, respectively, and delayed wound healing after combined injury, but increased RBCs, hemoglobin concentrations, hematocrit values and platelets after irradiation alone and only platelets after combined injury. Taken together, the results suggest that combined treatment with pegylated G-CSF and Alxn4100TPO is effective for mitigating effects of both radiation alone and in combination with injury.

FIG. 1

Peg-G-CSF and Alxn4100TPO improved survival after 9.5 Gy whole-body irradiation alone or combined with skin wound. Mice were treated with vehicle (veh) or peg-G-CSF and Alxn4100TPO (P + A; panel A), peg-G-CSF alone (panel B) or Alxn4100TPO alone (panel C) after irradiation alone or combined injury. Their survival was monitored for 30 days. N = 16–22 per group. *P < 0.05 vs. sham-treated and wound-alone vehicle treatment groups; ^P < 0.05 vs. irradiation-alone vehicle-treated group; ^#P < 0.05 vs. respective vehicle group.

FIG. 2

Peg-G-CSF and Alxn4100TPO significantly mitigated body-weight loss after 9.5 Gy whole-body irradiation alone or combined with skin wound. Mice were treated with vehicle or peg-G-CSF and Alxn4100TPO (P + A; panel A), peg-G-CSF alone (panel B) or Alxn4100TPO alone (panel C) after sham, wounding, irradiation or combined injury. Body weights were measured on days 1, 3, 7, 14, 21 and 28. N=18–22 per group. Data are presented as mean ± SEM. *P < 0.05 vs. sham and wound, vehicle-treated groups; ^P < 0.05 vs. irradiation, vehicle-treated group; ^#P < 0.05 vs. respective vehicle group.

FIG. 3

Peg-G-CSF and Alxn4100TPO significantly improved water consumption after 9.5 Gy whole-body irradiation alone or combined with skin wound. Mice were treated with vehicle (veh) or peg-G-CSF and Alxn4100TPO (P + A; panel A), peg-G-CSF alone (panel B) or Alxn4100TPO alone (panel C) after irradiation or combined injury. Water intakes were measured daily for the first 7 days after sham, wounding irradiation or combined injury. N=16–22 per group. Data are presented as mean ± SEM. *P < 0.05 vs. sham vehicle-treated group; ^P < 0.05 vs. sham and irradiated vehicle-treated groups; ^#P < 0.05 vs. respective vehicle group.

FIG. 4

Peg-G-CSF and Alxn4100TPO did not improve the wound healing rate after 9.5 Gy whole-body irradiation combined with skin wound. Mice were treated with vehicle (panel A), peg-G-CSF and Alxn4100TPO (P + A; panel B), peg-G-CSF alone (panel C) or Alxn4100TPO alone (panel D) after irradiation or combined injury. Their wound areas were measured on days 1, 3, 7, 14, 21 and 28 after wounding or combined injury. N=16–22 per group. Data are presented as mean ± SEM. *P < 0.05 vs. the wound group; ^P < 0.05 vs. respective drug-treated wound groups.

FIG. 5

Peg-G-CSF and Alxn4100TPO significantly improved bone marrow cellularity after 9.5 Gy whole-body irradiation alone or after irradiation combined with skin wound. Data are presented as mean ± SEM. Panels A–D: Histology with H&E staining of bone marrow samples 30 days after irradiation or combined injury. N=4 per group. Panel E–F: Fat cells and megakaryocytes per 20×field were counted. For panel E: *P < 0.05 vs. sham and wound groups; ^P < 0.05 vs. irradiation vehicle-treated group. For panel F: *P < 0.05 vs. sham and wound groups; ^P < 0.05 vs. irradiation or combined injury groups treated with vehicle.

FIG. 6

Peg-G-CSF and Alxn4100TPO significantly mitigated WBC depletion after whole-body irradiation alone or combined with skin wound. Mice were treated with either vehicle, peg-G-CSF and Alxn4100TPO, peg-G-CSF alone or Alxn4100TPO alone after 9.5 Gy irradiation alone or combined with skin wound (combined injury). Panels A–F: WBC profiling of blood samples collected 30 days after irradiation or combined injury. N= 6 per group. Data are presented as mean ± SEM. *P < 0.05 vs. sham group; ^P < 0.05 vs. irradiation vehicle-treated group. ^#P < 0.05 vs. combined injury vehicle-treated group. P+A: peg-G-CSF+Alxn4100TPO; P: pegylated G-CSF; A: Alxn4100TPO

FIG. 7

Peg-G-CSF and Alxn4100TPO fully recovered RBC counts, hemoglobin levels and hematocrit readings after 9.5 Gy whole-body irradiation alone or combined with skin wound. Mice were treated with vehicle or peg-G-CSF and Alxn4100TPO, peg-G-CSF alone or Alxn4100TPO alone after irradiation or combined injury. Panels A–C: RBC profiling of blood samples collected 30 days after irradiation or combined injury. N = 6 per group. Data are presented as mean ± SEM. *P < 0.05 vs. sham group; ^P < 0.05 vs. irradiation vehicle-treated group. ^#P < 0.05 vs. combined injury vehicle-treated group.

FIG. 8

Peg-G-CSF and Alxn4100TPO significantly mitigate platelet counts after 9.5 Gy whole-body irradiation alone or combined with skin wound. Mice were treated with vehicle or peg-G-CSF and Alxn4100TPO, peg-G-CSF alone or Alxn4100TPO alone after irradiation or combined injury. Panel A: Platelet counts on day 30 in surviving sham, wounded, irradiation and combined injury groups. N = 6 per group. Data are presented as mean ± SEM. Panel B: Correlation between platelet counts in peripheral blood and megakaryocyte counts in bone marrow. N=31. *P < 0.05 vs. sham group; ^P < 0.05 vs. irradiation vehicle group; ^#P < 0.05 vs. combined injury vehicle group; ^&P < 0.05 vs. wounded only group.

FIG. 9

Peg-G-CSF and Alxn4100TPO significantly improved ileal morphology after 9.5 Gy whole-body irradiation alone or combined with skin wound. Panels A–D: Histology slides with H&E staining of ileums collected 30 days after irradiation and combined injury. N = 4 per group. Panels E–I: Villus heights, villus width, crypt depth, crypt counts and mucosal injury scores were measured. Data are presented as mean ± SEM. *P < 0.05 vs. sham group; ^P < 0.05 vs. irradiation (irrad.) vehicle group; ^#P < 0.05 vs. respective vehicle group.