Ciprofloxacin enhances stress erythropoiesis in spleen and increases survival after whole-body irradiation combined with skin-wound trauma.

Severe hematopoietic loss is one of the major therapeutic targets after radiation-combined injury (CI), a kind of injury resulting from radiation exposure combined with other traumas. In this study, we tested the use of ciprofloxacin (CIP) as a treatment, because of recently reported immunomodulatory effects against CI that may improve hematopoiesis. The CIP regimen was a daily, oral dose for 3 weeks, with the first dose 2 h after CI. CIP treatment improved 30-day survival in mice at 80% compared to 35% for untreated controls. Study of early changes in hematological parameters identified CI-induced progressive anemia by 10 days that CIP significantly ameliorated. CI induced erythropoietin (EPO) mRNA in kidney and protein in kidney and serum; CIP stimulated EPO mRNA expression. In spleens of CI mice, CIP induced bone morphogenetic protein 4 (BMP4) in macrophages with EPO receptors. Splenocytes from CIP-treated CI mice formed CD71⁺ colony-forming unit-erythroid significantly better than those from controls. Thus, CIP-mediated BMP4-dependent stress erythropoiesis may play a role in improving survival after CI

CIP increases survival, accelerates wound closure, and ameliorates body weight loss after CI.

<p>Mice received 9.25/or 15% TBSA wounding to establish following four treatment groups: CI, RI, Wound, or Sham. Mice in each group received daily, oral doses of CIP or Veh for 3 weeks starting 2 hours after injury unless otherwise noted. *<i>p</i><0.05 for the values between groups. (A) Top: Daily survival rates for RI (left) and CI (right) mice treated with CIP or Veh. Bottom: Daily survival rates for RI (left) and CI (right) mice treated with CIP (+3 d) or Veh (+3 d) regimens where CIP treatment was same except initiated as late as 3 days after CI. No mice died in Sham and Wound groups (data not shown). <i>n</i> = 20/group. Mantel-Cox test was used to evaluate statistical significance. (B) Average wound closure rates for Wound (left) and CI (right) mice treated with CIP or Veh. Data are presented as percentage of day 1 average wound size. (C) Average body weight rates for RI (left) and CI (right) mice treated with CIP or Veh. Data are presented as percentage of day 0 average body weight. CIP: ciprofloxacin; Veh: vehicle; CI: combined injury; RI: radiation injury; Wound: wounding alone; Sham: free of any injury.</p

Enhanced ability of stress BFU-E formation from the splenocytes of CIP-treated CI mice.

<p>Mice received CI or Sham and were treated with daily, oral doses of CIP or Veh starting 2 hours after injury. Splenocytes were isolated and inoculated from mice 10 days after injury. (A and B) Representative images of BFU-E and stress BFU-E that formed in early stage (4–6 days after <i>ex vivo</i> inoculation; A) and late stage (10–12 days after <i>ex vivo</i> inoculation; B). (C and D) Images of dishes containing splenocytes in early (C) and late (D) stages after <i>ex vivo</i> inoculation. Boxed dish in (D) was magnified in (F) inset. “X” signifies dishes that demonstrated bacterial growth. (E) Numbers of BFU-E and stress BFU-E in the early stage (day 4). *<i>p</i><0.01, Sham vs. CI BFU-E. **<i>p</i><0.01, Sham vs. CI sBFU-E. There were no sBFU-E in Sham-CIP, and no BFU-E in CI-Veh and CI-CIP. (F) Numbers of BFU-E and hemoglobinized BFU-E in late stage (day 10). Sham-Veh and Sham-CIP were studied. *<i>p</i><0.1 compared to BFU-E in other group. CIP: ciprofloxacin; Veh: vehicle; CI: combined injury; Sham: free of any injury; sBFU-E: stress BFU-E; Hem: hemoglobinized.</p

CIP enhances stress erythropoiesis after CI.

<p>Mice received CI or Sham and were treated with daily, oral doses of CIP or Veh starting 2 hours after injury. Spleens isolated from mice 10 days after injury were used for splenocyte inoculation followed by immunoblot analysis (A) or immunofluorescent staining followed by microimaging analysis (B). (A) Presence of CD71, a CFU-E marker, in colonies harvested in early (top) and late (bottom) stages. Insufficient number of splenocyte colonies prevented analysis of Sham at day 6; analysis was successful at day 12. (B) Top left: Sham-Veh; top right: Sham-CIP; bottom left: CI-Veh; and bottom right: CI-CIP. In each panel, upper left quadrant: DAPI; upper right quadrant: CD71; lower left quadrant: F4/80; and lower right quadrant: overlay of other three quadrants. Annotation in top-left image applies to all other images unless otherwise noted. Staining for CI-CIP was magnified and presented at bottom of figure as indicated. CIP: ciprofloxacin; Veh: vehicle; CI: combined injury; Sham: free of any injury.</p

CIP improves anemia induced by CI.

<p>Mice received CI and were treated with daily, oral doses of CIP or Veh starting 2 hours after injury. Erythrocytes and hemoglobin levels were analyzed over course of 10 days. (A) Body weight rates for CI mice 5 and 10 days after CI. Individual and average body weights from each group are shown. Data are presented as percentage of day 0 average body weight. (B) Erythrocyte counts at 1, 2, 3, 7 and 10 days after CI. *<i>p</i><0.05 between CI+Veh and CI+CIP; **<i>p</i><0.1 between Sham+Veh and Sham+CIP. (C) Hemoglobin levels at 1, 2, 3, 7 and 10 days after CI. *<i>p</i><0.05 between CI+Veh and CI+CIP. (D) Erythrocyte counts at 1 day after wounding with increasing doses of radiation. *<i>p</i><0.2. CIP: ciprofloxacin; Veh: vehicle; CI: combined injury; Wound: wounding alone; Sham: free of any injury.</p