Micro-CT technique is well suited for documentation of remodeling processes in murine carotid arteries

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Abstract

Background: The pathomechanisms of atherosclerosis and vascular remodelling are under intense research. Only a few in vivo tools to study these processes longitudinally in animal experiments are available. Here, we evaluated the potential of micro-CT technology. Methods: Lumen areas of the common carotid arteries (CCA) in the ApoE -/- partial carotid artery ligation mouse model were compared between in vivo and ex vivo micro-CT technique and serial histology in a total of 28 animals. AuroVist-15 nm nanoparticles were used as in vivo blood pool contrast agent in a Skyscan 1176 micro-CT at resolution of 18 μmeter voxel size and a mean x-ray dose of 0.5 Gy. For ex vivo imaging, animals were perfused with MicroFil and imaged at 9 μmeter voxel size. Lumen area was evaluated at postoperative days 7, 14, and 28 first by micro-CT followed by histology. Results: In vivo micro-CT and histology revealed lumen loss starting at day 14. The lumen profile highly correlated (r = 0.79, P<0.0001) between this two methods but absolute lumen values obtained by histology were lower than those obtained by micro-CT. Comparison of in vivo and ex vivo micro-CT imaging revealed excellent correlation (r = 0.83, P<0.01). Post mortem micro-CT yielded a higher resolution than in vivo micro-CT but there was no statistical difference of lumen measurements in the partial carotid artery ligation model. Conclusion: These data demonstrate that in vivo micro-CT is a feasible and accurate technique with low animal stress to image remodeling processes in the murine carotid artery. Copyright:

Figures

  • Fig 1. Experimental design. Partial ligation of the LCCA in ApoE-/- mice at day 0. Timeline of high fat diet (HFD) treatment and overview showing analysis methods. Comparison of in vivo micro-CT and histology was performed by in vivo micro-CT scan and subsequent histological examination (d7, n = 4, d14, n = 10, d28, n = 9). Comparison of in vivo micro-CT and ex vivo micro-CT was performed by in vivo micro-CT scan and subsequent perfusion with the post mortem contrast agent MicroFil (n = 5); H/E, hematoxylin/eosin.
  • Fig 2. Lumen profile evaluation by in vivo micro-CT and histology (A) Volume rendering of a micro-CT angiography of a ApoE-/- mouse infused with AuroVist nanoparticles at postoperative day 14 after partial LCCA (left common carotid artery) ligation. A virtual elastic sphere (yellow) is fitted through the segmented LCCA lumen (left panel) from the aortic arch to the bifurcation. The LCCA was segmented into nine equidistant parts (right panel). (B) Side dependent vascular lumen profile of the LCCA from the aortic arch (1) till the bifurcation (9) Shown are the averages of all mice (n = 10) in orange and mouse M1, corresponding to (A), in blue. (C) H/E stainings of LCCA at postoperative day 14 after partial ligation of the LCCA. Lumen, tunica intima (red dashed line), tunica media, tunica externa, inner layer of tunica media (yellow dashed line) and plaque are clearly seen. (D) H/E stainings of LCCA (M1) at postoperative day 14 near the aortic arch (1), the bifurcation (9) and each 500 μm in between. (E) Histological evaluation of LCCA lumen (upper panel) and plaque area (lower panel) from the aortic arch (1) to the bifurcation (9) (n = 10). Squares show the mean ± SEM of all mice. Dashed blue line shows one individual mouse (M1). Scale bars in (C) and (D) represent 100 μm.
  • Fig 3. Comparison of in vivomicro-CT and histology vascular lumen profile. LCCA and RCCA lumen profiles evaluated by in vivo micro-CT and histology at postoperative days 7 (n = 4), 14 (n = 10) and 28 (n = 9) after LCCA ligation. (A) In vivo micro-CT evaluation of LCCA (left panel) and RCCA (middle panel) lumen area starting from the aortic arch (1) to the bifurcation (9). Average lumen areas evaluated by micro-CT (right panel). (B) Histological evaluation of LCCA (left panel) and RCCA (middle panel) lumen area profiles. Averages of carotid lumen areas evaluated by histology (right panel). (C) Comparison between mean lumen areas of LCCA and RCCA of each mouse evaluated by in vivo micro-CT and histology. Linear regression and correlation coefficient were calculated (C, left panel). Correlation of lumen areas of LCCA and RCCA of each mouse for each time point (C, middle panel). Bland-Altman plot depicting the degree of agreement between micro-CT and histology (C, right panel). Bars indicate the mean ± SEM. **, p<0.01; ***, p<0.001; LCCA left common carotid artery; RCCA right common carotid artery.
  • Fig 4. Comparison of in vivo and ex vivomicro-CT. Lumen area profile after partial ligation of the LCCA. (A, B) Lumen profile of the LCCA and RCCA was evaluated by in vivo (AuroVist Nanoparticles) and ex vivo (MicroFil) micro-CT at postoperative day 14 (n = 5). Lumen area was evaluated from the aortic arch (1) to the bifurcation (9). Error bars indicate the mean ± SEM. (C) Correlation between mean lumen area of LCCA and RCCA of each mouse examined by in vivo and ex vivo micro-CT. Linear regression parameters and correlation coefficients were calculated. (D) Volume rendering of the LCCA and RCCA examined by in vivo and post mortemmicro-CT with corresponding visualization of the virtual elastic sphere path (grey). BA, brachiocephalic artery; LCCA, left common carotid artery; RCCA right common carotid artery; RSA, right subclavian artery branch; STA, superior thyroid artery.

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Schürmann, C., Gremse, F., Jo, H., Kiessling, F., & Brandes, R. P. (2015). Micro-CT technique is well suited for documentation of remodeling processes in murine carotid arteries. PLoS ONE, 10(6). https://doi.org/10.1371/journal.pone.0130374

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