Establishing a murine model of the hematopoietic syndrome of the acute radiation syndrome

Abstract

The authors have developed a murine model of the Hematopoietic Syndrome of the Acute Radiation Syndrome (H-ARS) for efficacy testing of medical countermeasures (MCM) against radiation according to the FDA Animal Rule. Ten- to 12-wk-old male and female C57BL/6 mice were exposed to the LD50/30-LD70/30 dose of total body irradiation (TBI, (137)Cs, 0.62-0.67 Gy min(-1)) in the morning hours when mice were determined to be most radiosensitive, and they were assessed for 30-d survival and mean survival time (MST). Antibiotics were delivered in drinking water on days 4-30 post-TBI at a concentration based on the amount of water that lethally-irradiated mice were found to consume. The fluoroquinolones, ciprofloxacin and levofloxacin, as well as the tetracycline doxycycline, and aminoglycoside neomycin, all significantly increased MST of decedent mice, while ciprofloxacin (p = 0.061) and doxycycline + neomycin (p = 0.005) showed at least some efficacy to increase 30-d survival. Blood sampling (30 μL/mouse every fifth day) was found to negatively impact 30-d survival. Histopathology of tissues harvested from nonmoribund mice showed expected effects of lethal irradiation, while moribund mice were largely septicemic with a preponderance of enteric organisms. Kinetics of loss and recovery of peripheral blood cells in untreated mice and those treated with two MCM, granulocyte-colony stimulating factor and Amifostine further characterized and validated this model for use in screening studies and pivotal efficacy studies of candidate MCM for licensure to treat irradiated individuals suffering from H-ARS.

Figure 1. Lethally-irradiated C57BL/6 mice provided with wet feed consumed less water from sipper tube bottles than mice provided only sipper tube bottles

C57BL/6 mice were exposed to 7.96Gy and monitored for fluid consumption for 29 days. All mice were provided autoclaved acidified water for the duration of the study. Mice in the “sipper tube bottle + wet feed” group (broken line) were also provided wet feed set in Petri dishes on cage bottoms beginning on day 4 post-exposure. Data are from 1 to 2 separate experiments. *p<0.0001 comparing “Sipper tube bottle” to “Sipper tube bottle + wet feed”, n=29-35 mice/group. Data on days 2-4 and days 23-25 represent mean daily water intake over the weekend.

Figure 2. Chronosensitivity of C57Bl/6 mice

C57BL/6 mice were irradiated with the predicted LD50/30 at different times during the day and analyzed for survival time over 30 days. p≤0.019 comparing Midday group with either morning or afternoon. N=68 (morning group), n=10 (midday group), and n=41 (afternoon group).

Figure 3. Radiation dose-response relationship (DRR) of C57Bl/6 mice

C57BL/6 mice were exposed to different doses of radiation and provided support on days 4 through 30 after irradiation as follows: one group received ciprofloxacin-treated water and chow wetted with ciprofloxacin-water (blue line). A second group received neomycin-treated water plus doxycycline chow in cage hoppers and also as wet feed (black line). A third group received levofloxacin-treated water and chow wetted with levofloxacin-water, plus seven subcutaneous injections of PBS every other day beginning on day one post-exposure (green line). Controls received no antibiotic support but did receive regular wet feed on days 4-30 either without (red line) or with the addition of seven PBS injections (orange line). Panel A shows the DRR using probit models. Survival at day 30 was analyzed at each dose of radiation and is shown as percent mortality on the Y-axis. Panel B are Kaplan-Meier survival curves showing the proportion of mice surviving at each time point for each treatment condition using all of the radiation doses combined. P values for 30 day survival: p=0.005 comparing doxycycline and neomycin with No antibiotic support, p=0.061 comparing ciprofloxacin with No antibiotic support, p=0.461 comparing levofloxacin with No antibiotic support + 7 PBS injections. P values for overall survival time: p<0.0001 comparing doxycycline and neomycin with No antibiotic support, p<0.0001 comparing ciprofloxacin with No antibiotic support, p=0.278 comparing levofloxacin with No antibiotic support + 7 PBS injections.

Figure 3. Radiation dose-response relationship (DRR) of C57Bl/6 mice

C57BL/6 mice were exposed to different doses of radiation and provided support on days 4 through 30 after irradiation as follows: one group received ciprofloxacin-treated water and chow wetted with ciprofloxacin-water (blue line). A second group received neomycin-treated water plus doxycycline chow in cage hoppers and also as wet feed (black line). A third group received levofloxacin-treated water and chow wetted with levofloxacin-water, plus seven subcutaneous injections of PBS every other day beginning on day one post-exposure (green line). Controls received no antibiotic support but did receive regular wet feed on days 4-30 either without (red line) or with the addition of seven PBS injections (orange line). Panel A shows the DRR using probit models. Survival at day 30 was analyzed at each dose of radiation and is shown as percent mortality on the Y-axis. Panel B are Kaplan-Meier survival curves showing the proportion of mice surviving at each time point for each treatment condition using all of the radiation doses combined. P values for 30 day survival: p=0.005 comparing doxycycline and neomycin with No antibiotic support, p=0.061 comparing ciprofloxacin with No antibiotic support, p=0.461 comparing levofloxacin with No antibiotic support + 7 PBS injections. P values for overall survival time: p<0.0001 comparing doxycycline and neomycin with No antibiotic support, p<0.0001 comparing ciprofloxacin with No antibiotic support, p=0.278 comparing levofloxacin with No antibiotic support + 7 PBS injections.

Figure 4. Mortality and morbidity associated with periodic blood drawing

Mice were exposed to the LD90/30 dose of radiation and then bled by tail snips every 5 days (solid line), or not bled (dashed line). Mice were provided ciprofloxacin in their drinking water and wet feed ad libitum from day 4 through 30 post-exposure. Panel A shows Kaplan-Meier survival curves; panel B shows the mean euthanasia score per mouse in each group over time. n=60 mice in the bleed group and n=41 mice in the no bleed group. p=0.008 comparing 30 day survival of bled and not bled mice; p=0.031 comparing MST of bled and not bled mice, p=0.005 comparing overall survival time of bled and not bled mice.

Figure 4. Mortality and morbidity associated with periodic blood drawing

Mice were exposed to the LD90/30 dose of radiation and then bled by tail snips every 5 days (solid line), or not bled (dashed line). Mice were provided ciprofloxacin in their drinking water and wet feed ad libitum from day 4 through 30 post-exposure. Panel A shows Kaplan-Meier survival curves; panel B shows the mean euthanasia score per mouse in each group over time. n=60 mice in the bleed group and n=41 mice in the no bleed group. p=0.008 comparing 30 day survival of bled and not bled mice; p=0.031 comparing MST of bled and not bled mice, p=0.005 comparing overall survival time of bled and not bled mice.

Figure 5. Survival and CBC parameters in mice exposed to lethal radiation and treated with Amifostine or G-CSF

Groups of 30 mice were exposed to the LD70/30 dose of radiation and treated with either Amifostine (400mg/kg, green lines) 30 minutes prior to exposure, Neupogen (125ug/kg, red lines) beginning 24 hr post exposure and continuing daily to day 16, or remained untreated (blue lines). Black lines are from non-irradiated age-matched controls. Mice were bled by tail snips every 5th day for CBC analyses using a validated HEMAVET® 950FS Hematology Systems. Panel A shows the Kaplan-Meier survival curves. Panels B through F show the white blood cell count (WBC), absolute Neutrophil count (ANC), absolute Lymphocyte count (ALC), red blood cell count (RBC) and platelet count (plt), respectively. Green asterisks indicate statistically significant difference comparing Amifostine to untreated control, p<0.0001; red asterisks indicate statistically significant difference comparing Neupogen group to untreated control, p<0.0367. n=30 mice/group.