Figure 1.
Two-dimensional in vivo bioluminescence and fluorescence imaging to monitor the bacterial burden and neutrophil infiltration during an orthopaedic implant infection.
S. aureus or no bacteria (uninfected) (n = 8 mice per group) were inoculated into the knee joints of LysEGFP mice in the presence of a titanium K-wire implant and mice were imaged using the IVIS Spectrum® imaging system (Caliper). (A) Mean bacterial burden as measured by in vivo bioluminescence (mean total flux [photons/s] ± sem) (logarithmic scale). (B) Mean neutrophil infiltration as measured by in vivo fluorescence (mean total radiant efficiency [photons/s]/[µW/cm2] ± sem). *p<0.05, †p<0.01, ‡p<0.001 S. aureus-infected mice versus uninfected mice (Student's t-test [two-tailed]). (C) (Left) Representative images of in vivo bioluminescence (upper panels) and in vivo fluorescence (bottom panels) overlaid on a grayscale photograph of the mice from the three S. aureus-infected mice that had different patterns of the bioluminescent signals. (Right) In vivo bioluminescent signals (photons/s/cm2/sr) (logarithmic scale) (blue) and in vivo fluorescent signals ([photons/s]/[µW/cm2]) (red) for each of these three mice.
Figure 2.
Two-dimensional in vivo bioluminescence, fluorescence and X-ray imaging to monitor the bacterial burden and neutrophil infiltration during an orthopaedic implant infection.
S. aureus or no bacteria (uninfected) (n = 8 mice per group) were inoculated into the knee joints of LysEGFP mice in the presence of a titanium K-wire implant and mice were imaged using the IVIS Lumina XR® imaging system (Caliper). (A) Mean bacterial burden as measured by in vivo bioluminescence (mean total flux [photons/s] ± sem) (logarithmic scale). (B) Mean neutrophil infiltration as measured by in vivo fluorescence (mean total radiant efficiency [photons/s]/[µW/cm2] ± sem). *p<0.05, †p<0.01, ‡p<0.001 S. aureus-infected mice versus uninfected mice (Student's t-test [two-tailed]). (C) Representative images of in vivo bioluminescence (upper panels) and in vivo fluorescence (bottom panels) on a color scale overlaid on an X-ray image of a S. aureus-infected mouse and an uninfected mouse. (D) Representative osteolytic lesions (denoted by an arrow) and increased bone size observed on close-ups of the X-ray images at the distal end of the femur at the site of the implant infection in S. aureus-infected mice on day 48. These osteolytic lesions were not observed in any of the uninfected mice.
Figure 3.
Three-dimensional μCT images to visualize the changes in the bone during an orthopaedic implant infection.
S. aureus or no bacteria (uninfected) (n = 8 mice per group) were inoculated into the knee joints of LysEGFP mice in the presence of a titanium K-wire implant and mice were imaged using the Quantum FX® in vivo μCT system (Caliper). (A) Representative 3D μCT renderings of the femurs implanted with the titanium K-wire implant from S. aureus-infected (upper panels) and uninfected mice (lower panels). (B) Representative maximal intensity projection images of the femurs with the titanium K-wire implants taken from a lateral view from S. aureus-infected (upper panels) and uninfected mice (lower panels). (C) Percent outer bone volume change of the distal femurs normalized to the initial time point (mean ± sem). †p<0.01, ‡p<0.001 S. aureus-infected mice versus uninfected mice (Student's t-test [two-tailed]).
Figure 4.
Three-dimensional longitudinal and cross-sectional μCT images to visualize bone changes during an orthopaedic implant infection.
S. aureus or no bacteria (uninfected) (n = 8 mice per group) were inoculated into the knee joints of LysEGFP mice in the presence of a titanium K-wire implant and mice were imaged using the Quantum FX® in vivo μCT system (Caliper). Representative 3D longitudinal (left) and cross-sectional (right) μCT images (from the area within the dashed red lines on the longitudinal images) on days 14 and 48 of femurs implanted with a titanium K-wire implant from S. aureus-infected mice (A) and uninfected mice (B).
Figure 5.
Bacterial Counts and Histologic analysis.
S. aureus or no bacteria (uninfected) (n = 8 mice per group) were inoculated into the knee joints of LysEGFP mice in the presence of a titanium K-wire implant and mice. At the end of the experiment (on postoperative day 48), the infected and uninfected groups were divided, with half the implants and joint tissue harvested to count the numbers of bacteria and the remainder for histologic analysis. (A) Mean CFUs ± sem (logarithmic scale) isolated from the implants and joint tissue. (B) Representative photomicrographs of histologic sections (1 of 4 mice per group, with similar results). Left large panels: low magnification of hematoxylin and eosin (H&E) stained joint specimens with a line drawing of the location of the implant with the intramedullary canal seen within the femur. Upper right small panels: higher magnification of H&E- and Gram-stained joint specimens of the boxed area in the large left panel at the location of the cut end of the implant within the joint. Lower right small panels: higher magnification of H&E- and Gram-stained joint specimens of the boxed area in the upper right panels. n.d. = not detected. Scale bar = 100 µm.