The authors have declared that no competing interests exist.
Edited the paper: TBE. Conceived and designed the experiments: JGK. Performed the experiments: JGK RF LHC NVG EDL TBE. Analyzed the data: JGK RF TBE. Wrote the paper: JGK RF.
Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg
Nuclear disasters such as detonation of weapons could cause serious injury to a human body by a combination of radiation exposure and other insults that include physical wounds and thermal burns, namely radiation combined injury (CI). CI generally leads to greater mortality than radiation injury (RI) alone, even though each injury separately may not be lethal
We previously reported an experimental model for CI, in which wound trauma increased RI-induced cell death, tissue damage, organ dysfunction, and finally mortality in mice
Ciprofloxacin (CIP) is an FDA-approved fluoroquinolone (FQ), which is widely used as an antimicrobial. CIP has been included in the Strategic National Stockpile, which is maintained by the U.S. Department of Health and Human Services, to control bacterial infection during a national emergency such as a nuclear detonation or other radiological incident. Besides the antimicrobial activity, several groups reported immunomodulatory effects that CIP exerts in rodent models and human clinical trials
Here we are the first to report that CIP modulates pathophysiological changes after CI. CIP significantly reduced levels of pro-inflammatory cytokines, while it potentiated IL-3 production, maintained GM-CSF production, and enhanced bone marrow repopulation. In the ilea of surviving mice, CIP limited apoptosis and autophagy, which may have prevented systemic infection from bacterial translocation. These CIP-mediated changes may have contributed to improve 10-day survivability in CI-mice compared to vehicle-treated control animals.
After CI, none of the CIP-treated mice died but two vehicle-treated mice died on days 2 and 6, respectively (
Mice received 9.75 Gy 60Co γ-photon radiation followed within 1 h by 15% TBSA skin wound (CI). Mice were given CIP (90 mg/kg
Because CI could alter concentrations of cytokines and chemokines in serum
On day 10 after CI, whole blood was collected from surviving mice and the sera were separated. Sera were tested for their cytokine and chemokine concentrations. n = 3−5 per group; For A-G, *
CI induced WBC and platelet depletion
On day 10 after CI, whole blood was collected from surviving mice and tested for numbers of WBCs and platelets. n = 4−5 per group; *
Bone marrow is very sensitive to irradiation because it contains proliferative hematopoietic stem cells, whose loss would result in severe hematologic decline including WBCs and platelets (
On day 10 after CI, femurs were collected from surviving mice and prepared for hematoxylin-eosin staining on paraffin sections. Representative histopathology photos are shown. n = 4−5 per group. CI: combined injury; CIP: ciprofloxacin; Veh: vehicle.
Previous studies showed that CI induced defects in intestinal wall, through which bacteria might have penetrated to cause systemic infection after CI
On day 10 after CI, ileal samples were collected from surviving mice and prepared for hematoxylin-eosin staining on paraffin sections. (A) Representative histopathology photos are shown, (B) An average mucosal injury grade was calculated as a damage index of 5 villa in 3 representative mice (
CIP evidently prevents and corrects the loss of ileal villi after CI. This action may be due to the adjustment in the process of cell death by apoptosis (type I death)
On day 10 after CI, ileal samples were collected from surviving mice and prepared for immunofluorescent staining on frozen sections. (A) TUNEL assay was performed to identify apoptotic population (green) and presented with (bottom) or without (top) nucleus (blue), and (B) LC3 was stained to identify autophagic population and presented with nucleus (blue). Representative pictures are shown. n = 4−5 per group. CI: combined injury; CIP: ciprofloxacin; Veh: vehicle.
To confirm ongoing apoptosis, we also measured the activity of caspase-3, a biomarker for apoptosis
On day 1 after CI, ileal samples were collected from surviving mice and total cell lysates were prepared for detection of caspase-3 activity. Data is presented by the levels equivalent to
We previously reported CI-induced systemic bacterial infection that might cause activation of iNOS and subsequently lead to increased mortality. iNOS is also known to be regulated by NF-κB, a transcription factor. We, therefore, investigated the effect of CIP on the CI-induced NF-κB activation and systemic bacterial infection. We first analyzed the level of the p65 subunit of NF-κB, also known as RelA, in the ileal samples. We found that CI significantly increased RelA in ileal villi of vehicle-treated CI-mice, which CIP treatment significantly reduced (
On day 10 after CI, ileal samples were collected from surviving mice and prepared for immunofluorescent staining on frozen sections. RelA expression was detected (green) and presented with nucleus (blue). Representative pictures are shown with their fluorescence intensities for green signals (bottom). n = 4−5 per group. CI: combined injury; CIP: ciprofloxacin; Veh: vehicle.
In an independent experiment, the ventricular heart blood, liver, and spleen were collected aseptically from moribund CI-mice to culture bacteria. Bacteria were found from all of such animals. In CI-vehicle mice, five Gram-positive and two Gram-negative bacterial species were detected. Gram–positive species were found in 6 out of 7 mice and Gram–negative species were found in 5 out of 7 mice; whereas in CI-CIP-treated mice, only three Gram-positive species were found in 3 out of 3 mice. The absence of Gram-negative bacteria suggested that CIP effectively eliminated target microorganisms. Of note, the bacterial species that caused sepsis after CI were always found in the ileal lumen of the same animals when tested (data available for the names of bacterial species).
Immunoglobulin A (IgA) is a unique antibody that is found on the intestinal mucosal surface as soluble dimers and protects it from bacterial and viral infection by this route
On day 10 after CI, ileal samples were collected from surviving mice and prepared for immunofluorescent staining on paraffin sections. IgA expression was detected (red) with counterstaining with nucleus (blue). Representative pictures are shown with their fluorescence intensities for red signals (bottom). n = 4−5 per group. CI: combined injury; CIP: ciprofloxacin; Veh: vehicle.
There is a pressing need to find countermeasures to treat RI and CI. At present, none of countermeasures yet evaluated to treat either condition has been approved by the U.S. FDA for the use in humans based upon evaluation in two suitable laboratory animal species. In the search for appropriate CI-treatment targets, it is necessary to understand the mechanisms underlying CI. Our research has been investigating many pathophysiological changes that occurred during the period of 30 days after CI, which identified increased DNA damage, stress-response gene expressions, serum cytokine and chemokine concentrations, ileal damage, bacterial translocation
CIP is a second-generation fluoroquinolone (FQ)
The wide use of CIP in clinical settings has helped the discovery of its immunomodulatory effect, apart from the antimicrobial activity. Since the stimulation of hematopoiesis highlights immunomodulation by CIP
On the other hand, it has also been reported that FQs including CIP inhibited mammalian topoisomerase II
In the present study, for the first time, we investigated whether CIP mitigated ionizing radiation combined injury (CI) through its effects on survival, cytokine levels, bone marrow cells, and ileum of surviving mice on day 10 after CI. The multiple effects of CIP were observed in several tissues. In the sera, CIP effectively reduced CI-induced pro-inflammatory cytokines IL-6, KC (
IL-3 is an essential mediator for myeloid lineage development and has been reported to be elevated by CIP treatment
CIP limited physical damages occurring in the ileum after CI (
CIP offers several advantages to be developed further as a drug to treat CI in a mass-casualty scenario: (1) it is included in the Strategic National Stockpile for bacterial infection control; (2) it can be taken orally so that patients can self-administer it; (3) it possesses not only antimicrobial but also favorable immunomodulatory activity; and (4) it is inexpensive.
In summary, CIP significantly increased survival, altered the serum cytokine and chemokine profile, accelerated bone-marrow recovery, inhibited cell death of ileum, and prevented systemic bacterial infection. These effects could be mediated by its capability of inhibiting NF-κB and caspase-3. Therefore, the results suggest that CIP may prove to be beneficial for treating critical sequelae of CI.
Research was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care-International (AAALACI). All procedures involving animals were reviewed and approved by the AFRRI Institutional Animal Care and Use Committee. Euthanasia was carried out in accordance with the recommendations and guidelines of the American Veterinary Medical Association.
Female B6D2F1/J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and were used when 33–36 weeks old with 25–35-g average weights. Male mice were not used in this study because of problems associated with aggression, which in these experiments could lead to further damage to wound sites and enhanced infection. All mice were randomly assigned to experimental groups. No more than 4 mice were housed per filter-topped polycarbonate cage (MicroIsolator) in conventional holding rooms. Rooms were provided with 10–15 changes per hour of 100% fresh air conditioned to 72±2°F with a relative humidity of 50±20%. Mice were maintained on a 12-h light/dark, full-spectrum light cycle with no twilight. Four or five days prior to the experiments, body weights were measured and hair of the dorsal surface was removed using electric clippers. On the day of experiments, mice were first irradiated and then wounded under anesthesia by methoxyflurane inhalation. All mice, including controls, received an intraperitoneal injection of 0.5 mL sterile isotonic 0.9% NaCl as fluid therapy immediately after combined injury or sham injury to avoid radiation-induced dehydration. After injuries mice were assigned to clean cages and provided with proper food and acidified water.
Mice were placed in well-ventilated acrylic restrainers and given 9.75 Gy of whole-body 60Co γ-photon radiation delivered at a dose rate of approximately 0.4 Gy/min. Dosimetry was performed using the alanine/electron paramagnetic resonance system. Calibration of the dose rate with alanine was traceable to the National Institute of Standards and Technology and the National Physics Laboratory of the United Kingdom. Sham-irradiated mice were placed in the same acrylic restrainers, taken to the radiation facility, and restrained for the time required for irradiation.
Within 1 h of irradiation, mice were anesthetized under methoxyflurane by inhalation; an experimental wound was administered 19±1.3 mm from the occipital bone and between the scapulae using a stainless steel punch on a Teflon®-covered board cleaned with 70% alcohol before each use. The panniculus carnosus muscle and overlying skin (23.5±1.1 mm long and 14.9±0.7 mm wide) were removed. Sham-wounded mice were treated identically to other groups except without wounding.
Veterinary-, oral-use ciprofloxacin tablets (500 mg/each) (Dr. Reddy’s laboratories, Hyderabad, India) were used to prepare fresh solution every week. Tablets were ground, dissolved in sterile water (vehicle) and after a brief centrifugation sterile-filtered using 0.22 µm CN (cellulose nitrate) filter system (Corning; Corning, NY). Each dose of 0.2 mL ciprofloxacin solution delivered 90 mg/kg based on average body weight. All mice received 0.2 mL of either ciprofloxacin or vehicle via oral route once per day for 11 days, starting within 2 h of CI and through day 10. The treatment regimen has been justified based on previous studies
Survival after CI and therapy with CIP was evaluated with 10 mice per group. The gross appearance, general health, and survival of each mouse were followed by visual inspection daily for 10 days in parallel with other assessments.
Ileal and femur specimens were collected for histopathology 10 days after CI (n = 4−5 per group). Specimens were immediately fixed in 10% phosphate-buffered formalin upon removal. The tissue was then embedded in paraffin, sectioned transversely and stained with hematoxylin and eosin (H&E). Tissue imaging and analysis were performed by the NanoZoomer 2.0 from HAMAMATSU PHOTONICS K.K. (Hamamatsu, Japan). The same acquisition setting, including scaling, applies to all images in the same figure. The mucosal damage of ileum for each slide was graded on a six-tiered index defined by Chiu et al.
The following antibodies were used for the analyses by immunofluorescent staining: mouse monoclonal to NF-κB p65 (F-6) and goat polyclonal to MAP LC3 (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA); rat monoclonal anti‐mouse IgA purified (eBioscience; San Diego, CA) provided by Ms. Kristen Gambles; Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 568 goat anti-rat IgG (Life Technologies Corporation; Grand Island, NY).
Ileal specimens were collected for immunofluorescent antibody staining 10 days after CI (n = 8−10 per group). Specimens were immediately fixed in phosphate-buffered 4% paraformaldehyde (FD NeuroTechnologies, Inc.; Baltimore, MD) at 4°C for overnight. They were then washed twice by ice-cold phosphate-buffered saline (PBS) before and after treatment with 20% sucrose in PBS at 4°C for 2 h. Resulting tissues were dried briefly on paper towels and embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek USA, Inc.; Torrance, CA) on dry-ice. Tissues were kept frozen at −80°C until sectioning on cryostat and used for immunofluorescent staining. In some studies, paraffin sections were also used for the staining as noted. Prepared slides were treated with Target Retrieval Solution and Protein Block Serum-Free (Dako North America, Inc.; Carpinteria, CA) according to the manufacturer’s protocol, and stained with respective primary and secondary antibodies with washing between and after with PBS with 0.1% Tween® 20. Resulting slides were briefly washed with PBS and desalted by soaking in distilled-deionized water and sealed by coverslips in mounting medium with DAPI (Life Technologies Corporation). Terminal deoxynucleotidyl transferase biotin–dUTP nick end labelling (TUNEL) staining and the following image processing were conducted with a provision of the manufacturer’s recommendations (EMD Millipore Corporation; Billerica, MA)
A Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss MicroImaging; Thornwood, NY) with EC Plan-Neofluar 10×/0.3, Plan-Apochromat 20×/0.8, and EC Plan-Neofluar 40×/0.75 objectives were used to scan the signals. Intensity of signals were also measured and shown as noted. The same acquisition setting, including scaling, applies to all images in the same figure.
Whole blood (0.7–1.0 mL) was collected by terminal cardiac puncture from mice anesthetized by methoxyflurane 10 days after CI. CapiJect tubes (Terumo; Somerset, NJ) were used to separate sera by centrifugation at 3,500 g for 90 seconds and stored at −70°C until assayed (n = 3−5 per group). Cytokine concentrations were analyzed using the Bio-Plex™ Cytokine Assay (Bio-Rad; Hercules, CA) following the manufacturer’s directions. Briefly, serum from each animal was diluted fourfold and examined in duplicate. Data were analyzed using the LuminexH 100™ System (Luminex Corp.; Austin, TX) and quantified using MiraiBio MasterPlexH CT and QT Software (Hitachi Software Engineering America Ltd.; San Francisco, CA), and concentrations were expressed in pg/mL unless otherwise noted. The cytokines analyzed were interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC (
Caspase-3 activity was determined using the Caspase-3 Colorimetric Activity Assay Kit, DEVD, (EMD Millipore Corporation). In brief, 10 µl of each sample lysate was added to wells of a 96-well plate already containing the substrate Ac-DEVD-
The detailed methods used for detecting bacteria in blood and tissues to assess bacterial translocation were previously described
Five mice per group were used for each survival experiment, and it was repeated to gain statistical significance by Mantel-Cox procedure (total n = 10/group). All other results are expressed as means ± SEM. One-way ANOVA, two-way ANOVA, studentized-range test, and χ2 test were used for comparison of groups; 5% was used as the level of significance.
We thank Ms. Joan T. Smith, HM1 Neil G. Agravante, USN, Mr. True M. Burns, Dr. Min Zhai, and Dr. David L. Bolduc for their technical assistance; CAPT David Lesser, USN and IACUC members for approval of the IACUC protocol; Veterinary Sciences Department staffs for high-quality animal care; Dr. Vitaly Nagy and radiation sources staffs for radiation dosimetry and source operation; and Ms. Lisa F.T. Meyers and Dr. Dennis P. McDaniel, Biomedical Instrumentation Center (BIC), Uniformed Services University of the Health Sciences, for histopathology and the confocal microimaging analysis. The views, opinions and findings contained in this report are those of the authors and do not reflect official policy or positions of the Armed Forced Radiobiology Research Institute, the U.S. Department of the Navy, the U.S. Department of Defense, the National Institutes of Health, or the United States Government.