Table 1.
Assessing morbidity.
Fig 1.
Diagram of experimental procedure.
Groups of 10 mice per treatment condition were irradiated or sham with 2 Gy of proton or gamma radiation and then hindlimb suspended or not. (A) Mice began treatment with enrofloxacin (10 mg/kg) subcutaneously 12 hrs before bacterial challenge and were dosed every 12 hrs. (B) Mice began treatment with G-CSF (600 μg/kg) subcutaneously after irradiation and suspension and were dosed every 3 days. Five days post-irradiation, mice were challenged with Pseudomonas aeruginosa (109 CFUs, IP). Mice were followed for 5 days and blood was obtained for quantitation of bacterial CFUs and granulocytes.
Fig 2.
Determination of bacterial challenge dose.
(A) The association between the OD600 of the bacterial culture and the number of CFUs/μl was determined for Pseudomonas aeruginosa by plating dilutions of bacterial culture and quantitating colonies. The OD600 quantitation of CFU/μl was then used to determine the numbers of CFUs in the bacterial culture in real time. (B) The amount of bacteria that untreated C3H/HeN mice could control was determined by challenging with increasing amounts of CFUs intraperitoneally and following for morbidity as defined in Table 1.
Fig 3.
Treatment with enrofloxacin reduces morbidity and increases bacterial clearance in irradiated and hindlimb suspension mice systemically challenged with Pseudomonas aeruginosa.
Groups of 10 mice per treatment group were irradiated with 2 Gy of gamma rays and hindlimb suspended or not and injected subcutaneously with either PBS or enrofloxacin (10 mg/kg) every 12 hrs starting 12 hrs before bacterial challenge. All groups were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed. Morbidity scores (Table 1) were calculated daily (A) and animals were considered morbid if their score increased by 3 points or remained 2 points elevated for more than 24 hrs. C = control, R + S = irradiated + hindlimb suspended. (B) Bacteremia was quantitated 5 days after challenge by diluting and plating blood followed by colony counting. Values are means ± SEM of log10 of colony-forming units (CFU). One-way ANOVA with Bonferroni’s correction for multiple comparisons was used to test statistical significance of changes in CFUs in blood. Statistical significance of Kaplan-Meier curves was measured by Mantel-Cox log rank test.
Fig 4.
Treatment with G-CSF reduces morbidity and increases bacterial clearance of irradiated and hindlimb suspension mice systemically challenged with Pseudomonas aeruginosa.
Sets of 10 mice per treatment group were irradiated with 2 Gy of protons or gamma rays and hindlimb suspended or not and injected with PBS or G-CSF (600 μg/kg) by the subcutaneous route every 3 days until the end of the experiment. All groups were exposed to Pseudomonas aeruginosa by intraperitoneal injection 5 days after irradiation and followed. Morbidity scores (Table 1) were calculated (A—proton and B—gamma rays) daily and animals were considered morbid if their score increased by 3 points or remained 2 points elevated for 24 hrs. C = control, R + S = irradiated + hindlimb suspended. Bacteria in blood were quantitated 5 days after infection (C—proton and D—gamma rays). Values are means ± SEM of log10 colony-forming units (CFUs). One-way ANOVA with Bonferroni’s correction for multiple comparisons was used to test the statistical significance of changes in CFUs. Kaplan-Meier curve statistical significance was measured by Mantel-Cox log rank test.
Fig 5.
Effect of G-CSF on peripheral blood granulocyte counts during systemic bacterial challenge of irradiated and hindlimb suspended mice.
Sets of 10 mice per treatment group were irradiated with 2 Gy of protons or gamma rays and hindlimb suspended or not and injected with PBS or G-CSF (600 μg/kg) every 3 days. All groups were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed. After 5 days, the number of granulocytes, Ly-6G high, CD14-, and F4–80- in peripheral blood was calculated using AccuCount fluorescent particles. (A) proton treated and (B) gamma ray treated. C = control, R + S = irradiated + hindlimb suspended. Values are means ± SEM. One-way ANOVA with Bonferroni’s correction for multiple comparisons was used to test statistical significance.