Figure 1.
To evaluate the correlation between the bacterial PI and the number of bacteria in the infectious process in vivo, the positive area of gram-stained MRSA on the each tissue section from the different time-point animals (3, 7, and 28 days after inoculation) were calculated. Six axial sections which cover whole abscess in SGM were obtained from each animal. The total numbers of pixels in gram-positive area (blue stains) were measured in five fields from six sections (A). Image obtained from software (B) shows that gram-positive blue area has been replaced to red pixel dots (C).
Figure 2.
Correlation between the number of bacteria and bacterial PI in vitro and in vivo.
BLI sensitivity was assessed using a CCD-based macroscopic detector to quantify the bacterial photon intensity (PI = photons/sec/cm2/steradian) for various numbers of bacteria (1.25×105 to 2.0×106 CFU per well). (A) In vitro study: the bacterial PI from colonies of bioluminescent MRSA was significantly correlated with the number of bacterial CFUs (R2 = 0.9912). (B) In vivo study: various numbers of bacteria (1.25×105–2.0×106 CFU per inoculation) were inoculated into the SGM, and bioluminescence in the region of interest (ROI) was monitored by BLI. There was a significant correlation between the number of inoculated bacteria and the bacterial PI in vivo (R2 = 0.9882).
Figure 3.
Changes in bacterial PI in the mouse SGM model and correlation between the number of inoculated bacteria and bacterial PI in the infection process.
We inoculated MRSA Xen31 (3×107 CFU/µl) in 5µl of medium into the SGM, and measured the bacterial PI in the ROI immediately after inoculation, and on days 1, 3, 7, 14, 21, and 28 (N = 6). (A) The mean bacterial PI in the SGM peaked immediately after inoculation (2.486×104 PI) and remained high for at least 28 days (2.016×104 PI). Means and SEM are shown. (B) There was a significant correlation between the pixels number of gram-positive area and the bacterial PI during 28 days in vivo (R2 = 0.9788).
Figure 4.
Dual optical imaging in the mouse SGM model.
(A) Bioluminescence and (B) fluorescence images in the same animal, 10 days after bacterial inoculation, are shown. (A) MRSA inoculated into the left SGM was sufficient to produce an observable bioluminescence signal. (B) A Cy5.5-conjugated inflammation probe accumulated at the inoculation site only 5 minutes after it was injected into the tail vein, and was monitored for over 2 weeks in the same animal. (C) In the merged image, the accumulation of the inflammation probe (yellow) appeared as an area of fluorescence that completely covered the entire region of bacterial bioluminescence signal (multi-color).
Figure 5.
Mean serum levels of IL-6, IL-1β, and CRP in MRSA-inoculated animals at various time points are shown (N = 4 each). Compared to the pre-inoculation level, the IL-6 level was significantly higher 12 hours after inoculation (P<0.05). The IL-6 level gradually decreased, becoming normal by day 21. IL-1β was significantly elevated after 12 hours, and remained high for 7 days (P<0.05). CRP was significantly higher than the pre-inoculation level 0.5, 1, 3, and 21 days after inoculation (P<0.05). The means and SEM are shown.
Figure 6.
Changes in histology over time in the SGM from infected mice. Axial sections of the infected SGM were prepared at 7 (A and B), 28 (C and D), and 42 (E and F) days after bacterial inoculation and stained with hematoxylin and eosin. Lower images: magnified views of the boxed regions in the upper images. On day 7, marked neutrophil infiltration and muscle destruction were detected at the injection sites. On day 28, an abscess that contained necrotic materials (asterisk) and covered by fibrous tissue was observed in the SGM. On day 42, scaring tissue (asterisk) observed in the subcutaneous tissue. Bars = 200 µm (A, C, E), 10 µm (B, D, F).