Selective detection of early inflammation in a porcine model of ischemia/reperfusion injury targeting P-selectin with molecular MRI

Introduction: Ischemia and Inflammation are driving forces of wound healing after myocardial infarction. Early reperfusion preserves cardiac function. Although cardiac MRI has become the gold standard for non-invasive characterization of myocardial tissue, current MRI techniques such as T1-, T2-, T2* mapping and LGE only indirectly represent cardiac inflammation. Molecular MRI can overcome this limitation. In this study, an iron-labeled P-selectin contrast agent is tested in a pig model for its potential to assess the early cardiac inflammatory response after ischemia/reperfusion and compared with standard MRI techniques.

Methods: We used a closed-chest model of ischemia-reperfusion injury by temporary balloon-occlusion of the circumflex artery in juvenile farm pigs. 3T MRI was repetitively performed 2 – 4 hours after ischemia and lesions were characterized using injury (T1 mapping; late gadolinium enhancement) and edema (T2 imaging and T2 mapping) sensitive sequences. For molecular imaging, monoclonal P-selectin antibody was functionalized with microparticles of iron oxide (MPIO) and injected into the coronary artery under MRI guidance. Specific binding of the contrast agent to the epitope of interest was confirmed by FACS and in vitro flow chamber using activated porcine coronary endothelial cells or platelets. Additionally, we developed a procedure to extract tissue samples from ex vivo porcine hearts using a 3D printing method.

Results: In vivo cardiac imaging showed no impaired wall motion and no late enhancement after 40 minutes of ischemia. In addition, no significant difference was seen in T2 between ischemic left ventricular segments (38.8 ± 2.7) ms and remote segments (38.4 ± 2.0) ms (p = 0.25), whereas T1 was significantly increased in segments with ischemia (1301 ± 53) ms over remote segments (1225 ± 28) ms (p < 0.05). Animals that received P-selectin targeting contrast agent showed increased R2* values in the ischemic segments compared to the control group. The measured R2* values were normalized to the values in remote segments. Ex vivo imaging supported the finding of higher amount of MPIOs in the ischemic segments in the P-selectin group. Here, the number of high R2* values in the ischemic segments was measured. Additionally, we detected increased inflammatory activity by means of higher numbers of innate immune cells circulating the blood within the first hours after MI. Using a 3D printing method allowed for extracting tissue samples from ex vivo porcine hearts with a sub-milli­meter pre­cision: hearts were first imaged with ultra-high resolution MRI and biopsy target locations were defined in the 3D data sets, then exactly matching holders were 3D-printed individually for each heart, and in the hol­ders specific trajectories were integrated for a sub­se­quent biopsy procedure. In total, 61 tissue samples were extracted with an average offset of the tissue sample location from the target location of 0.59 ± 0.36 mm. All offsets were less than 2.5 mm and the targeted myocardial tissue was extracted in all samples.

Conclusion: Using a P-selectin-MPIO contrast agent allowed for sensitive detection of early myo­cardial inflammation after I/R beyond the capabilities of traditional edema sensitive imaging in pigs by balloon-occlusion for 40 minutes. The 3D-printed Needle Guide method enables extraction of myocardial tissue samples that are selected by ex vivo MRI with submillimeter precision.