Hindlimb suspension and SPE-like radiation impairs clearance of bacterial infections

Abstract

A major risk of extended space travel is the combined effects of weightlessness and radiation exposure on the immune system. In this study, we used the hindlimb suspension model of microgravity that includes the other space stressors, situational and confinement stress and alterations in food intake, and solar particle event (SPE)-like radiation to measure the combined effects on the ability to control bacterial infections. A massive increase in morbidity and decrease in the ability to control bacterial growth was observed using 2 different types of bacteria delivered by systemic and pulmonary routes in 3 different strains of mice. These data suggest that an astronaut exposed to a strong SPE during extended space travel is at increased risk for the development of infections that could potentially be severe and interfere with mission success and astronaut health.

Figure 1. Diagram of experimental procedure.

Groups of mice received radiation or not and were then hindlimb suspended or not (black bar). Five days later, all groups were challenged with bacteria and followed for 5 days. Depending on the experiment, blood was obtained with mice remaining in suspension at various times pre and post bacterial challenge (inverted lollipop).

Figure 2. Determination of bacterial challenge dose.

The association between the OD₆₀₀ of the bacterial culture and the number of CFUs/µl was determined for Pseudomonas aeruginosa (A) and Klebsiella pneumoniae (B) bacteria by plating dilutions of culture and quantitating colonies. The OD₆₀₀ quantitation of CFU/µl was then used to determine the amount of bacteria that untreated mice could control with minimal morbidity in ICR mice with Pseudomonas aeruginosa delivered IP (C) and Klebsiella pneumoniae delivered by inhalation (D). The numbers of CFUs of Klebsiella pneumoniae were found to be the same for Balb/c and C3H/HeN mice as for ICR mice. The maximal number of CFUs of Pseudomonas aeruginosa that resulted in the ability of Balb/c (E) and C3H/HeN (F) mice to control the infection with minimal morbidity was determined.

Figure 3. Proton radiation and hindlimb suspension of ICR mice impairs their ability to clear a lung challenge with Klebsiella pneumoniae or a peritoneal challenge with Pseudomonas aeruginosa.

Groups of 10 female ICR mice per treatment group were not treated, irradiated with 2/or placed in hindlimb suspension. Five days later, mice were exposed to Klebsiella pneumoniae by inhalation and followed. Lung tissue (A) and whole blood (B) was plated to quantitate CFUs of bacteria 5 days later. Similarly treated mice were delivered Pseudomonas aeruginosa IP and followed. After 5 days, bacteria in blood was quantitated (C). C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Median values are indicated by a barbell. The Friedman ANOVA, degrees of freedom in parentheses, with Wilcoxon signed-rank post-hoc analysis with Bonferroni correction was used to test statistical significance.

Figure 4. C3H/HeN mice treated with hindlimb suspension and 2 Gy of proton irradiation fail to control a challenge with Pseudomonas aeruginosa bacteria.

Groups of 10 C3H/HeN mice per treatment group were not treated, proton irradiated (2 Gy), and/or placed in hindlimb suspension. Five days later, mice were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed for 5 days. Morbidity scores (Table 1) were calculated daily and animals were considered morbid if their score increased by 3 points or remained 2 points elevated for 24 hrs. C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Statistical significance was measured by log rank analysis of Kaplan-Meier curves and expressed in the table as column versus rows.

Figure 5. Gamma irradiated mice treated with hindlimb suspension fail to control a challenge with Pseudomonas aeruginosa.

Groups of 10 C3H/HeN male (A) and female (B), Balb/c female (C) and ICR female (D) mice were gamma irradiated (2 Gy) and/or placed in hindlimb suspension. Five days later, mice were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed for 5 days. Morbidity scores (Table 1) were calculated daily and animals were considered morbid if their score increased by 3 points or remained elevated 2 points for 24 hrs. C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Statistical significance was measured by log rank analysis of Kaplan-Meier curves and expressed in the table as column versus rows.

Figure 6. Proton irradiated mice treated with hindlimb suspension fail to control a challenge with Pseudomonas aeruginosa.

Groups of 10 C3H/HeN male (A) and female (B) and Balb/c male (C) and female (D) mice were proton irradiated (2 Gy) and/or placed in hindlimb suspension. Five days later, mice were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed for 5 days. Morbidity scores (Table 1) were calculated daily and animals were considered morbid if their score increased by 3 points or remained elevated 2 points for 24 hrs. C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Statistical significance was measured by log rank analysis of Kaplan-Meier curves and expressed in the table as column versus rows.

Figure 7. Dose response of gamma radiation with hindlimb suspension on the ability to control a challenge with Pseudomonas aeruginosa.

Groups of 10 C3H/HeN female mice were gamma irradiated with 2.0, 1.5, or 1.0 Gy and placed in hindlimb suspension. Five days later, mice were exposed to Pseudomonas aeruginosa by intraperitoneal injection and followed for 5 days. Morbidity scores (Table 1) were calculated daily and animals were considered morbid if their score increased by 3 points or remained elevated 2 points for 24 hrs. Statistical significance was measured by log rank analysis of Kaplan-Meier curves.

Figure 8. Hindlimb suspension impairs the increase in peripheral blood granulocytes during systemic bacteremia.

C3H/HeN mice were hindlimb suspended and irradiated with 2 Gy of gamma radiation. Five days later, mice were challenged with Pseudomonas aeruginosa by intraperitoneal injection. The number of granulocytes, Ly-6G^high, CD14⁻, and F4-80⁻ in peripheral blood was calculated using AccuCount fluorescent particles at the indicated time points before and after bacterial challenge. Data from 10 mice in each group were averaged and error bars are standard error of the mean. C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Statistical significance of the effect of hindlimb suspension and irradiation and the interaction between them was calculated by 2-way ANOVA for the indicated days post bacterial challenge.

Figure 9. Hindlimb suspension and/or 2 Gy of proton radiation increases serum total corticosterone levels.

Groups of 10 C3H/HeN female mice per treatment group were placed in hindlimb suspension and/or proton irradiated (2 Gy). Five days later, serum was obtained and analyzed for total corticosterone by ELISA. Data from 10 mice in each group were averaged and error bars are standard error of the mean. C =  control, S =  hindlimb suspended, R =  irradiated, and R+S =  irradiated and suspended. Statistical significance of the effect of hindlimb suspension and irradiation and the interaction between them was calculated by 2-way ANOVA.