Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor α-/- Transgenic Mouse Model Using Targeted Microbubbles

Abstract

Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing-remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MB_Selectin) in an interleukin-2 receptor α deficient (IL-2Rα^-/-) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Rα^-/- (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MB_Selectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Rα^-/- mouse between 6-30 weeks of age. USMI signals were compared between IL-2Rα^-/- vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Rα^-/- mice between 6-30 weeks (onset at 6-14 weeks) compared to wild-type mice. Both male and female IL-2Rα^-/- mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Rα^-/- mice between 6-30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Rα^-/- mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.

Keywords: E-selectin; P-selectin; chronic disease; colitis; flare; inflammatory bowel disease (IBD); interleukin 2 receptor α deficient (IL-2Rα−/−) mice; remission; transgenic mouse model; ultrasound molecular imaging (USMI).

Figure 1

Overall experimental design.

Figure 2

Axial dual P- and E-selectin targeted ultrasound molecular images of proximal, middle, and distal colon in a representative IL-2Rα^−/− mouse from week 6 to 26 and in a representative wild-type mouse at 6 weeks of age. Compared to the wild-type mouse, the IL-2Rα^−/− mouse showed substantially higher imaging signals from week 6 to 26, and the imaging signals fluctuated with being higher at week 14 and 26 than other time points. The imaging signals did not show a substantial locational difference between proximal (top row), middle (middle row) and distal colon (bottom row). Scale bars, 1 mm.

Figure 3

IL-2Rα^−/− mice showed spontaneous colitis without sex or locational difference between 6–30 weeks of age. (A) The mean USMI signals of all the imaging acquisitions from proximal, middle and distal colon in both male and female IL-2Rα^−/− mice were significantly higher than wild-type mice, and there was no significant difference in USMI signals between male and female IL-2Rα^−/− mice. (B) When the imaging acquisitions were pooled according to proximal, middle and distal colon in both male and female IL-2Rα^−/− mice, the mean USMI signals of proximal, middle, and distal colon were significantly higher than wild-type mice, and there was no significant difference in USMI signals observed between proximal, middle and distal colon in both male and female IL-2Rα^−/− mice. (C) When USMI signals of proximal, middle, and distal colon were further divided according to male and female IL-2Rα^−/− mice, mean USMI signals of proximal, middle, or distal colon in either male or female IL-2Rα^−/− mice were significantly higher than wild-type mice, and did not show a significant difference in USMI signals between the 3 locations or between the sexes. Each box in the plot represents the 25th and 75th quartiles while the line inside the box identifies the median. The small square within the box indicates the mean. ♦ represents outliers; * indicates p-value < 0.05 between the two boxes under the bar (A) or between the box of wild-type mice and any box in IL-2Rα^−/− mice (B,C).

Figure 3

IL-2Rα^−/− mice showed spontaneous colitis without sex or locational difference between 6–30 weeks of age. (A) The mean USMI signals of all the imaging acquisitions from proximal, middle and distal colon in both male and female IL-2Rα^−/− mice were significantly higher than wild-type mice, and there was no significant difference in USMI signals between male and female IL-2Rα^−/− mice. (B) When the imaging acquisitions were pooled according to proximal, middle and distal colon in both male and female IL-2Rα^−/− mice, the mean USMI signals of proximal, middle, and distal colon were significantly higher than wild-type mice, and there was no significant difference in USMI signals observed between proximal, middle and distal colon in both male and female IL-2Rα^−/− mice. (C) When USMI signals of proximal, middle, and distal colon were further divided according to male and female IL-2Rα^−/− mice, mean USMI signals of proximal, middle, or distal colon in either male or female IL-2Rα^−/− mice were significantly higher than wild-type mice, and did not show a significant difference in USMI signals between the 3 locations or between the sexes. Each box in the plot represents the 25th and 75th quartiles while the line inside the box identifies the median. The small square within the box indicates the mean. ♦ represents outliers; * indicates p-value < 0.05 between the two boxes under the bar (A) or between the box of wild-type mice and any box in IL-2Rα^−/− mice (B,C).

Figure 4

USMI signals fluctuated between 6–30 weeks of age in IL-2Rα^−/− mice. First, the USMI signals from three USMI imaging acquisitions of the proximal, middle, and distal colon in an IL-2Rα^−/− mouse in the same imaging session were averaged out as a single value of imaging signal per mouse per time point. Afterwards, all the single values from all the IL-2Rα^−/− mice imaged at multiple time points between 6–30 weeks were pooled together. (A) The mean USMI signals in IL-2Rα^−/− mice between 6–30 weeks were higher than wild-type mice, and fluctuated between the 2 consecutive time points. Black * indicates p value < 0.05 between the box of wild-type mice and the box of IL-2Rα^−/− mice; olive ** indicate p value < 0.05 between the two boxes under the bar. (B) The evolution of inflammation degree at multiple time points from the six representative IL-2Rα^−/− mice. Most of the UMSI signals were higher than 1.0 a.u., while some of them fell below 1.0 a.u. There was at least one peak of USMI signals in each mouse, suggesting the recapitulation of remission-flare pattern in patients with IBD. The frequencies and duration of remission-flare vary in each mouse.

Figure 4

USMI signals fluctuated between 6–30 weeks of age in IL-2Rα^−/− mice. First, the USMI signals from three USMI imaging acquisitions of the proximal, middle, and distal colon in an IL-2Rα^−/− mouse in the same imaging session were averaged out as a single value of imaging signal per mouse per time point. Afterwards, all the single values from all the IL-2Rα^−/− mice imaged at multiple time points between 6–30 weeks were pooled together. (A) The mean USMI signals in IL-2Rα^−/− mice between 6–30 weeks were higher than wild-type mice, and fluctuated between the 2 consecutive time points. Black * indicates p value < 0.05 between the box of wild-type mice and the box of IL-2Rα^−/− mice; olive ** indicate p value < 0.05 between the two boxes under the bar. (B) The evolution of inflammation degree at multiple time points from the six representative IL-2Rα^−/− mice. Most of the UMSI signals were higher than 1.0 a.u., while some of them fell below 1.0 a.u. There was at least one peak of USMI signals in each mouse, suggesting the recapitulation of remission-flare pattern in patients with IBD. The frequencies and duration of remission-flare vary in each mouse.

Figure 5

Hematoxylin and eosin (H&E) stained sections of colon in representative IL-2Rα^−/− mice and wild-type mice. Wild-type (A,B) mice show no evidence of inflammation in the colon. Inflammation in the colon increases over time in IL-2Rα^−/− mice. At 14 weeks (when all the IL-2Rα^−/− mice had developed colitis), the colon of IL-2Rα^−/− animals shows minimal inflammatory cell infiltration expanding the lamina propria (C). Inflammatory cell infiltrate generally increases over time in IL-2Rα^−/− animals from 18 weeks (D), 23 weeks (E), 26 weeks (F), to 30 weeks (G,H). Edema, fibrosis, and ulceration were not observed, and neutrophilic inflammation was only observed at 30 weeks (G,H). Magnification: 40×. Scale bar: 20 μm.

Figure 6

P- and E-selectin expression of colon from representative IL-2Rα^−/− and wild-type mice. Representative wild-type mice (left column) showed background expression of P-selectin (top row) and E-selectin (bottom row) in the colon. Representative IL-2Rα^−/− mice (right column) demonstrated strong expression (red arrows) of P-selectin (top row; highlighted in red) and E-selectin (bottom row; highlighted in red) on CD31-positive vascular endothelial cells (highlighted in green) in the inflamed colon. Confocal micrographs were overlaid on differential interference contrast (DIC) images. Scale bars, 100 μm.