*3.2. Impact of Anaesthesia on the FDG-Uptake Pattern in the Infarcted Heart*

First, we examined if the myocardial glucose uptake suppression protocol established by Thackeray et al. [5] for C57BL/6 J mice can be applied in the mouse strain 129Sv used in our study on healthy mice (Figure 4).

**Figure 4.** Representative images of healthy mice anaesthetized with isofluorane (upper row) and ketamine/xylazine (lower row). Uptake of 18F-FDG is effectively suppressed by the use of ketamine/xylazine as can be seen in axial, coronal, and sagittal planes, as well as the polar maps.

Secondly, 18F-FDG PET was performed five days after permanent LAD ligation. Under anaesthesia with isofluorane, the highest tracer accumulation was detected in the viable myocardium, whereas ketamine/xylazine (KX) led to accentuated tracer accumulation within the border zone (Figure 5).

**Figure 5.** Sample axial, coronal, and sagittal myocardial 18F-FDG images at five days post-surgical myocardial infarction (MI) induction. Isofluorane leads to high tracer accumulation in the healthy myocardium, whereas the infarcted area can be clearly identified as an area of low glucose metabolism (upper row). When using ketamine/xylazine, the most intense tracer accumulation is detected in the area of the border zone, whereas 18F-FDG uptake in healthy myocardium remains suppressed.

The 18F-FDG uptake was significantly reduced by the use of ketamine/xylazine as compared with isofluorane for anaesthesia in the whole heart (5.2 ± 0.7% ID/g vs. 46.1 ± 11.2% ID/g; *p* = 0.02) and both remote (4.1 ± 0.6% ID/g vs. 79.3 ± 22.7% ID/g; *p* < 0.0001) and infarcted myocardium (4.35 ± 0.4% ID/g vs. 11.6 ± 6.0% ID/g, *p* = 0.002) (Figure 6).

**Figure 6.** Regional quantitative analysis of 18F-FDG uptake five days after myocardial infarction in the whole heart, the infarct and the remote myocardium. Mice were anaesthetized with isofluorane or ketamine/xylazine, respectively. Values are presented as mean ± SD. *p*-value was calculated using the student *t*-test.

With KX, there was no difference in tracer uptake between remote and infarcted myocardium (4.1 ± 0.6% ID/g vs. 4.4 ± 0.4% ID/g; *p* = 0.5). In contrast isoflurane lead to significantly higher 18F-FDG uptake in the remote as compared with the infarcted myocardium (79.3 ± 22.7% ID/g vs. 11.6 ± 6.0% ID/g; *p* = 0.001).

#### *3.3. FDG-Uptake Pattern is Fundamentally Changed by Cell Transplantation*

We then compared 18F-FDG uptake patterns in untreated to animals treated with CiC therapy (MIC) using the ketamine/xylazine protocol (Figures 7 and 8). Transplantation of embryoid bodies containing 10<sup>6</sup> syngeneic cardiac induced cells following acute myocardial infarction led to an increase in 18F-FDG uptake in the remote myocardium as compared with the MI group (10.7 ± 4.3% ID/g vs. 4.1 ± 0.6% ID/g; p = 0.003) (Figure 9). Interestingly, tracer accumulation in the center of the infarcted area was not altered by cell therapy (4.3 ± 1.4% ID/g in MIC vs. 4.4 ± 0.4% ID/g in MI; *p* = 0.9). Furthermore 18F-FDG uptake in the whole heart was significantly increased in the MIC group (8.0 ± 2.9% ID/g vs. 5.2 ± 1.1% ID/g; *p* < 0.05).

**Figure 7.** Sample axial, coronal, and sagittal myocardial 18F-FDG images at five days post-surgical MI induction and MI induction plus cell transplantation (MIC). Mice were anaesthetized with ketamine/xylazine. The MI group showed intense tracer accumulation in the border zone, whereas in cell-treated animals the highest tracer accumulation was found in the remote area.

**Figure 8.** Representative 17-segment tomographic polar maps visualizing distinctive differences of FDG-distribution in the left ventricle between the groups. The apex is in the middle and the anterior wall at top, the inferior wall at bottom, the septum in left and the lateral wall in the right. High tracer uptake is visualized by the yellow colors, lower upake by red and black. (**A**) MI group anaesthetized with isofluorane, the infarct region shows low FDG uptake as compared with remote myocardium; (**B**) MI group anaesthetized with ketamine/xylazine, the most intense FDG uptake came in the border zone; and (**C**) MI + cardiac induced cells (CiC) group anaesthetized with ketamine/xylazine shows a change of the FDG uptake to a pattern, which is similar to (A) with low FDG accumulation within the infarct region and highest uptake in the remote area.

**Figure 9.** Regional quantitative analysis of 18F-FDG uptake five days after myocardial infarction in the whole heart, infarct, and remote myocardium. Both MI and MIC group were anaesthetized using ketamine/xylazine. Values are presented as mean ± SD. *p*-value was calculated using the student *t*-test.
