Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy

Abstract

Purpose To examine whether cardiac chemical exchange saturation transfer (CEST) imaging can be serially and noninvasively used to probe cell survival or rejection after intramyocardial implantation in mice. Materials and Methods Experiments were compliant with the National Institutes of Health Guidelines on the Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. One million C2C12 cells labeled with either europium (Eu) 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) or saline via the hypotonic swelling technique were implanted into the anterior-lateral left ventricular wall in C57BL/6J (allogeneic model, n = 17) and C3H (syngeneic model, n = 13) mice. Imaging (frequency offsets of ±15 parts per million) was performed 1, 10, and 20 days after implantation, with the asymmetrical magnetization transfer ratio (MTR_asym) calculated from image pairs. Histologic examination was performed at the conclusion of imaging. Changes in MTR_asym over time and between mice were assessed by using two-way repeated-measures analysis of variance. Results MTR_asym was significantly higher in C3H and C57BL/6J mice in grafts of Eu-HP-DO3A-labeled cells (40.2% ± 5.0 vs 37.8% ± 7.0, respectively) compared with surrounding tissue (-0.67% ± 1.7 vs -1.8% ± 5.3, respectively; P < .001) and saline-labeled grafts (-0.4% ± 6.0 vs -1.2% ± 3.6, respectively; P < .001) at day 1. In C3H mice, MTR_asym remained increased (31.3% ± 9.2 on day 10, 28.7% ± 5.2 on day 20; P < .001 vs septum) in areas of in Eu-HP-DO3A-labeled cell grafts. In C57BL/6J mice, corresponding MTR_asym values (11.3% ± 8.1 on day 10, 5.1% ± 9.4 on day 20; P < .001 vs day 1) were similar to surrounding myocardium by day 20 (P = .409). Histologic findings confirmed cell rejection in C57BL/6J mice. Estimation of graft area was similar with cardiac CEST imaging and histologic examination (R² = 0.89). Conclusion Cardiac CEST imaging can be used to image cell survival and rejection in preclinical models of cell therapy. ^© RSNA, 2016 Online supplemental material is available for this article.

Figure 1a:

(a, b) Longitudinal cardiac CEST images of the survival of Eu-HP-DO3A–labeled cells in C3H mice. Twenty-four hours after implantation (a), significantly increased MTR_asym values were observed adjacent to the inferior papillary muscle (arrow), corresponding to the location of Eu-HP-DO3A–labeled cells. After 20 days (b), increased MTR_asym was still observed in the same myocardial region surrounding the inferior papillary muscle (arrow). The proliferation of labeled cells and the likely dilution of Eu-HP-DO3A with cell division reduced the MTR_asym values of the graft relative to day 1. (c) Photomicrograph (hematoxylin-eosin stain; original magnification, ×4) of the corresponding histologic slice demonstrates a graft of proliferating cells (blue) adjacent to the inferior papillary muscle in a similar location to increased MTR_asym values seen on b. (d) Higher magnification photomicrograph (hematoxylin-eosin stain; original magnification, ×20) of the area on c enclosed within the black box demonstrates the presence of proliferating cells (arrows) near the endocardial surface at the boundary of the papillary muscle.

Figure 1b:

(a, b) Longitudinal cardiac CEST images of the survival of Eu-HP-DO3A–labeled cells in C3H mice. Twenty-four hours after implantation (a), significantly increased MTR_asym values were observed adjacent to the inferior papillary muscle (arrow), corresponding to the location of Eu-HP-DO3A–labeled cells. After 20 days (b), increased MTR_asym was still observed in the same myocardial region surrounding the inferior papillary muscle (arrow). The proliferation of labeled cells and the likely dilution of Eu-HP-DO3A with cell division reduced the MTR_asym values of the graft relative to day 1. (c) Photomicrograph (hematoxylin-eosin stain; original magnification, ×4) of the corresponding histologic slice demonstrates a graft of proliferating cells (blue) adjacent to the inferior papillary muscle in a similar location to increased MTR_asym values seen on b. (d) Higher magnification photomicrograph (hematoxylin-eosin stain; original magnification, ×20) of the area on c enclosed within the black box demonstrates the presence of proliferating cells (arrows) near the endocardial surface at the boundary of the papillary muscle.

Figure 1c:

(a, b) Longitudinal cardiac CEST images of the survival of Eu-HP-DO3A–labeled cells in C3H mice. Twenty-four hours after implantation (a), significantly increased MTR_asym values were observed adjacent to the inferior papillary muscle (arrow), corresponding to the location of Eu-HP-DO3A–labeled cells. After 20 days (b), increased MTR_asym was still observed in the same myocardial region surrounding the inferior papillary muscle (arrow). The proliferation of labeled cells and the likely dilution of Eu-HP-DO3A with cell division reduced the MTR_asym values of the graft relative to day 1. (c) Photomicrograph (hematoxylin-eosin stain; original magnification, ×4) of the corresponding histologic slice demonstrates a graft of proliferating cells (blue) adjacent to the inferior papillary muscle in a similar location to increased MTR_asym values seen on b. (d) Higher magnification photomicrograph (hematoxylin-eosin stain; original magnification, ×20) of the area on c enclosed within the black box demonstrates the presence of proliferating cells (arrows) near the endocardial surface at the boundary of the papillary muscle.

Figure 1d:

(a, b) Longitudinal cardiac CEST images of the survival of Eu-HP-DO3A–labeled cells in C3H mice. Twenty-four hours after implantation (a), significantly increased MTR_asym values were observed adjacent to the inferior papillary muscle (arrow), corresponding to the location of Eu-HP-DO3A–labeled cells. After 20 days (b), increased MTR_asym was still observed in the same myocardial region surrounding the inferior papillary muscle (arrow). The proliferation of labeled cells and the likely dilution of Eu-HP-DO3A with cell division reduced the MTR_asym values of the graft relative to day 1. (c) Photomicrograph (hematoxylin-eosin stain; original magnification, ×4) of the corresponding histologic slice demonstrates a graft of proliferating cells (blue) adjacent to the inferior papillary muscle in a similar location to increased MTR_asym values seen on b. (d) Higher magnification photomicrograph (hematoxylin-eosin stain; original magnification, ×20) of the area on c enclosed within the black box demonstrates the presence of proliferating cells (arrows) near the endocardial surface at the boundary of the papillary muscle.

Figure 2a:

Cardiac CEST images show how the rejection of implanted Eu-HP-DO3A–labeled cells in C57BL/6J mice leads to elimination of initial CEST contrast. (a) Implantation of Eu-HP-DO3A–labeled cells in a representative C57BL/6J mouse resulted in increased MTR_asym values in the lateral ventricular wall in close proximity to the inferior papillary muscle 24 hours after implantation (arrow), similar to that observed in C3H mice. (b) However, at 20 days after implantation, MTR_asym values in the same myocardial region (arrow) decreased and were indistinguishable from surrounding myocardium.

Figure 2b:

Cardiac CEST images show how the rejection of implanted Eu-HP-DO3A–labeled cells in C57BL/6J mice leads to elimination of initial CEST contrast. (a) Implantation of Eu-HP-DO3A–labeled cells in a representative C57BL/6J mouse resulted in increased MTR_asym values in the lateral ventricular wall in close proximity to the inferior papillary muscle 24 hours after implantation (arrow), similar to that observed in C3H mice. (b) However, at 20 days after implantation, MTR_asym values in the same myocardial region (arrow) decreased and were indistinguishable from surrounding myocardium.

Figure 3a:

Bar graphs illustrate the measurement of MTR_asym, which allows differentiation of (a) cell survival from (b) cell rejection. In C3H mice (a), MTR_asym values in the region that contains Eu-HP-DO3A–labeled cells (cell graft) were significantly higher compared with septal regions (septum) in the same hearts and compared with corresponding regions after implantation of saline-labeled control cells (saline graft) in genetically identical mice at all measured time points. By 20 days after implantation, the mean MTR_asym values in cell grafts were significantly lower when compared with values 1 day after implantation. In C57BL/6J mice (b), MTR_asym values were increased 1 day after implantation in areas of Eu-HP-DO3A–labeled cells when compared with septal regions and saline-labeled controls and remained significantly increased 10 days after implantation but were similar 20 days after implantation. By 10 days, MTR_asym values in the region of implanted Eu-HP-DO3A–labeled cells were significantly reduced compared with both the initial values 1 day after implantation and the corresponding values in C3H mice at identical time points. Bars represent P values less than .05 at identical time points. * = P < .05 versus day 1 in the same region, † = P < .05 versus cell graft in C3H mice at the same time point.

Figure 3b:

Bar graphs illustrate the measurement of MTR_asym, which allows differentiation of (a) cell survival from (b) cell rejection. In C3H mice (a), MTR_asym values in the region that contains Eu-HP-DO3A–labeled cells (cell graft) were significantly higher compared with septal regions (septum) in the same hearts and compared with corresponding regions after implantation of saline-labeled control cells (saline graft) in genetically identical mice at all measured time points. By 20 days after implantation, the mean MTR_asym values in cell grafts were significantly lower when compared with values 1 day after implantation. In C57BL/6J mice (b), MTR_asym values were increased 1 day after implantation in areas of Eu-HP-DO3A–labeled cells when compared with septal regions and saline-labeled controls and remained significantly increased 10 days after implantation but were similar 20 days after implantation. By 10 days, MTR_asym values in the region of implanted Eu-HP-DO3A–labeled cells were significantly reduced compared with both the initial values 1 day after implantation and the corresponding values in C3H mice at identical time points. Bars represent P values less than .05 at identical time points. * = P < .05 versus day 1 in the same region, † = P < .05 versus cell graft in C3H mice at the same time point.

Figure 4a:

Plots show the comparison of graft size according to cardiac CEST imaging and histologic findings. (a) The quantification of graft size measured as a percentage of left ventricular (LV) myocardial area according to cardiac CEST imaging across all mice in which Eu-HP-DO3A cells were implanted demonstrated association with corresponding histologic measurements. (b) Bland-Altman analysis demonstrated close agreement in graft size, with slight underestimation of graft size according to MR imaging in grafts larger than 12% of left ventricular area.

Figure 4b:

Plots show the comparison of graft size according to cardiac CEST imaging and histologic findings. (a) The quantification of graft size measured as a percentage of left ventricular (LV) myocardial area according to cardiac CEST imaging across all mice in which Eu-HP-DO3A cells were implanted demonstrated association with corresponding histologic measurements. (b) Bland-Altman analysis demonstrated close agreement in graft size, with slight underestimation of graft size according to MR imaging in grafts larger than 12% of left ventricular area.

Figure 5a:

Photomicrographs show macrophage staining in C3H cardiac tissue. (a) Nuclear staining (4ʹ6-diamidino-2-phenylindole·2HCl, or DAPI; original magnification, ×40 composite) of a cross-section of cardiac tissue from a mouse that underwent implantation of Eu-HP-DO3A–labeled C2C12 cells. (b) Photomicrograph with higher magnification of a region of interest within the cell graft (original magnification, ×400 composite) is shown after immunostaining for murine macrophages (F4/80, red) and cell nuclei (DAPI, blue). Murine macrophages (arrow) were not present in high numbers in C3H cardiac tissue 20 days after cell implantation. Corresponding images from a C57BL/6J mouse are found in Figure E4 (online).

Figure 5b:

Photomicrographs show macrophage staining in C3H cardiac tissue. (a) Nuclear staining (4ʹ6-diamidino-2-phenylindole·2HCl, or DAPI; original magnification, ×40 composite) of a cross-section of cardiac tissue from a mouse that underwent implantation of Eu-HP-DO3A–labeled C2C12 cells. (b) Photomicrograph with higher magnification of a region of interest within the cell graft (original magnification, ×400 composite) is shown after immunostaining for murine macrophages (F4/80, red) and cell nuclei (DAPI, blue). Murine macrophages (arrow) were not present in high numbers in C3H cardiac tissue 20 days after cell implantation. Corresponding images from a C57BL/6J mouse are found in Figure E4 (online).