Ciprofloxacin and pegylated G-CSF combined therapy mitigates brain hemorrhage and mortality induced by ionizing irradiation

Abstract

Introduction: Brain hemorrhage was found between 13 and 16 days after acute whole-body 9.5 Gy ⁶⁰Co-γ irradiation (IR). This study tested countermeasures mitigating brain hemorrhage and increasing survival from IR. Previously, we found that pegylated G-CSF therapy (PEG) (i.e., Neulasta^®, an FDA-approved drug) improved survival post-IR by 20-40%. This study investigated whether Ciprofloxacin (CIP) could enhance PEG-induced survival and whether IR-induced brain hemorrhage could be mitigated by PEG alone or combined with CIP.

Methods: B6D2F1 female mice were exposed to ⁶⁰Co-γ-radiation. CIP was fed to mice for 21 days. PEG was injected on days 1, 8, and 15. 30-day survival and weight loss were studied in mice treated with vehicles, CIP, PEG, or PEG + CIP. For the early time point study, blood and sternums on days 2, 4, 9, and 15 and brains on day 15 post-IR were collected. Platelet numbers, brain hemorrhage, and histopathology were analyzed. The cerebellum/pons/medulla oblongata were detected with glial fibrillary acidic protein (GFAP), p53, p16, interleukin-18 (IL-18), ICAM1, Claudin 2, ZO-1, and complement protein 3 (C3).

Results: CIP + PEG enhanced survival after IR by 85% vs. the 30% improvement by PEG alone. IR depleted platelets, which was mitigated by PEG or CIP + PEG. Brain hemorrhage, both surface and intracranial, was observed, whereas the sham mice displayed no hemorrhage. CIP or CIP + PEG significantly mitigated brain hemorrhage. IR reduced GFAP levels that were recovered by CIP or CIP + PEG, but not by PEG alone. IR increased IL-18 levels on day 4 only, which was inhibited by CIP alone, PEG alone, or PEG + CIP. IR increased C3 on day 4 and day 15 and that coincided with the occurrence of brain hemorrhage on day 15. IR increased phosphorylated p53 and p53 levels, which was mitigated by CIP, PEG or PEG + CIP. P16, Claudin 2, and ZO-1 were not altered; ICAM1 was increased.

Discussion: CIP + PEG enhanced survival post-IR more than PEG alone. The Concurrence of brain hemorrhage, C3 increases and p53 activation post-IR suggests their involvement in the IR-induced brain impairment. CIP + PEG effectively mitigated the brain lesions, suggesting effectiveness of CIP + PEG therapy for treating the IR-induced brain hemorrhage by recovering GFAP and platelets and reducing C3 and p53.

Keywords: Ciprofloxacin; G-CSF; ICAM1; glial fibrillary acidic protein; hemorrhage; mouse; platelet; radiation.

Figure 1

CIP enhances PEG efficacy on 30-day survival and mitigates body weight loss after IR. Animals were treated with CIP alone, PEG alone, or a combination of CIP + PEG. Survival was monitored daily for 30 days and body weight was measured on days 1, 3, 7, 14, 21, and 28. All sham-treated mice survived over 30 days. (A) Daily percent of the 30-day survival from each group is presented. (B) The end 30-day percent survival from each group is presented. (C) Body weights are shown. N = 20 per group. Data are presented as mean ± SEM. ^*p < 0.05 vs. S + V1 + V2; ^∧p < 0.05 vs. Sham + V1 + V2, RI + V1 + V2 and RI + PEG + CIP; ^#p < 0.05 vs. Sham + V1 + V2, IR + V1 + CIP, and IR + PEG + CIP; ^&p < 0.05 vs. Sham + V1 + V2, IR + PEG + V2, and IR + PEG + CIP. S, Sham; IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 2

CIP therapy with pegylated G-CSF mitigates hemorrhagic lesions on brain surfaces on day 15 after IR. Animals were treated with CIP alone, PEG alone, or a combination of CIP + PEG. Brains were collected on day 15 post-irradiation. N = 5–6 per group. Data are presented as mean ± SEM. (A) Representative images of brains from each group. (B) Brain surface hemorrhage counts. *p < 0.05 vs. respective sham group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 3

CIP therapy with PEG mitigates intracranial hemorrhagic lesions after IR. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Brains were collected on day 15 post-irradiation. N = 5–6 per group. Data are presented as mean ± SEM. (A) Representative sham brain histopathology image was presented. (B) The hemorrhages are indicated by the black arrows in each magnified area as labeled B1-B6. (C) Total intracranial hemorrhage count from each group was determined. *p < 0.05 vs. respective sham group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 4

Representative images of cerebellum/pons/medulla oblongata from each group are presented. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Brains were collected on day 15 post-irradiation. N = 5–6 per group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 5

CIP therapy with PEG recovers the IR-induced GFAP decrease but not ICAM1 increases or tight junction in cerebellum/pons/medulla oblongata. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Brains were collected on day 15 post-irradiation. Lysate samples with cerebellum/pons/medulla oblongata were prepared to detect ICAM1 (A), Claudian 2 (B), ZO-1 (C), and GFAP (D). N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. sham + V1 + V2 group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for ciprofloxacin; C or CIP, ciprofloxacin; P or PEG, pegylated G-CSF.

Figure 6

(A–E) CIP therapy with PEG recovers megakaryocyte counts after IR. Animals were treated with CIP alone, PEG alone, or a combination of CIP + PEG. Sternums were collected on days 2, 4, 9 and 15 post-IR for counting megakaryocytes in each group. N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. respective sham group. ^∧p < 0.05 vs. IR + V1 + V2; IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 7

(A–D) CIP therapy with PEG partially recovers platelet counts caused by IR. Animals were treated with CIP alone, PEG alone, or a combination of CIP + PEG. Blood was collected on days 2, 4, 9, and 15 post-IR for measuring platelet counts in each group. N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. Sham + V1 + V2 group; ^∧p < 0.05 vs. IR + V1 + V2 group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 8

(A–D) CIP therapy with PEG inhibits IR-induced increases in serum IL-18. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Blood was collected on days 2, 4, 9, and 15 post-IR for measuring IL-18 levels in each group. N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. Sham + V1 + V2 group; ^∧p < 0.05 vs. IR + V1 + V2 group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 9

(A–D) CIP therapy with PEG mitigates IR-induced increases in complement protein 3 (C3) in circulation. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Blood was collected at different time points post-irradiation for measuring C3 levels in each group. N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. Sham + V1 + V2 group. IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.

Figure 10

CIP therapy with PEG recovers IR-induced increases in p53 and phosphorylated p53 in cerebellum/pons/medulla oblongata. Animals were treated with CIP alone, PEG alone, or combination of CIP + PEG. Brains were collected on day 15 post-IR. Lysate samples with cerebellum/pons/medulla oblongata were prepared to detect p53 and p-p53. (A) Representative gel images were presented, (B) p53, (C) phosphorylated p53. N = 5–6 per group. Data are presented as mean ± SEM. *p < 0.05 vs. respective sham group. ^∧p < 0.05 vs. IR + V1 + V2. S: Sham; IR, ionizing radiation at 9.5 Gy; V1, vehicle for pegylated-G-CSF; V2, vehicle for Ciprofloxacin; CIP, Ciprofloxacin; PEG, pegylated G-CSF.