Noninvasive in vivo tracking of mesenchymal stem cells and evaluation of cell therapeutic effects in a murine model using a clinical 3.0 T MRI

Cardiac cell therapy with mesenchymal stem cells (MSCs) represents a promising treatment approach for endstage heart failure. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work is to determine the feasibility of magnetic resonance imaging (MRI) and in vivo monitoring after transplantation into infarcted mouse hearts using a clinical 3.0 T MRI device. The labeling procedure of bone marrow-derived MSCs with micron-sized paramagnetic iron oxide particles (MPIOs) did not affect the viability of the cells and their cell type-defining properties when compared to unlabeled cells. Using a clinical 3.0 T MRI scanner equipped with a dedicated small animal solenoid coil, 105 labeled MSCs could be detected and localized in the mouse hearts for up to 4 weeks after intramyocardial transplantation. Weekly ECG-gated scans using T1-weighted sequences were performed, and left ventricular function was assessed. Histological analysis of hearts confirmed the survival of labeled MSCs in the target area up to 4 weeks after transplantation. In conclusion, in vivo tracking of labeled MSCs using a clinical 3.0 T MRI scanner is feasible. In combination with assessment of heart function, this technology allows the monitoring of the therapeutic efficacy of regenerative therapies in a small animal model. </jats:p

Importance of endothelial nitric oxide synthase for the hypothermic protection of lungs against ischemia-reperfusion injury

ObjectivesThe hypothesis that the protective effects of mild hypothermia against the pulmonary ischemia-reperfusion injury are mediated by endothelial nitric oxide synthase was tested.MethodsEndothelial nitric oxide synthase knock-out and wild-type mice were sham operated or underwent a 1-hour occlusion of the left pulmonary hilum, followed by 5 hours of reperfusion. Temperature in the left pleural cavity during ischemia was maintained at either 36°C (normothermia) or 32°C (hypothermia). Inflammatory response (myeloperoxidase activity), endothelial barrier function (extravasation of Evans blue–labeled albumin), and endothelial nitric oxide synthase expression and phosphorylation were determined at the end of reperfusion.ResultsAfter normothermic ischemia both strains had a similar mortality (wild-type, 22.9%; knock-out, 15.4%), which was completely abolished by hypothermia. Endothelial barrier function was disturbed after normothermic ischemia in both wild-type and knock-out mice. Mild hypothermia significantly reduced pulmonary Evans blue extravasation in wild-type mice, but not in knock-out mice. Myeloperoxidase activity increased after normothermic ischemia to the same degree in both strains. This response was significantly attenuated by hypothermia in wild-type mice, but not in knock-out mice. In wild-type mice, endothelial nitric oxide synthase expression and phosphorylation were higher after hypothermic ischemia than after normothermic ischemia. No effect of ischemia on expression of inducible nitric oxide synthase was found in wild-type or knock-out mice.ConclusionHypothermic protection against pulmonary ischemia-reperfusion injury is dependent on endothelial nitric oxide synthase and is associated with increased expression and phosphorylation of endothelial nitric oxide synthase

Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices

Background: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. Methods: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. Results: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. Conclusions: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases

Major complications during negative pressure wound therapy in poststernotomy mediastinitis after cardiac surgery

Objective: Negative pressure wound therapy is the first-line treatment modality for poststernotomy mediastinitis in many heart centers. The aim of this study was to analyze major complications and possible preventive methods during negative pressure wound therapy in patients with deep sternal wound infections. Methods: We retrospectively analyzed 69 consecutive patients treated with negative pressure wound therapy for poststernotomy mediastinitis between June 2006 and September 2009. Results: Five (7.2%) patients sustained major complications during negative pressure wound therapy. Bleeding from coronary artery venous bypass grafts was observed in 4 patients and fulminant bleeding from an infected homograft of the ascending aorta was observed in 1 patient during routine dressing changes of the negative pressure wound therapy system. Conclusions: Bleeding is the major complication during negative pressure wound therapy for poststernotomy mediastinitis. Covering the heart with several layers of paraffin gauze is a necessary protective maneuver but cannot completely prevent major complications during negative pressure wound therapy. All operative procedures, including dressing changes, should be performed in the operating room under optimal hygienic and monitoring conditions to increase the salvage rate and to guarantee optimal surgical and anesthesiologic conditions in case of negative pressure wound therapy-related complications. (J Thorac Cardiovasc Surg 2010;140:1133-6