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Macroscopic fluorescence imaging: a novel technique to monitor retention and distribution of injected microspheres in an experimental model of ischemic heart failure.

Martens A, Rojas SV, Baraki H, Rathert C, Schecker N, Hernandez SR, Schwanke K, Zweigerdt R, Martin U, Saito S, Haverich A, Kutschka I - PLoS ONE (2014)

Bottom Line: Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly.Measurements were repeated after a reduction to homogenate dilutions.Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany; Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Hannover, Germany.

ABSTRACT

Background: The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections.

Methods and results: A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5 × 10(5)) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78 × 10(5) ± 0.31 × 10(5) in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74 × 10(5) ± 0.18 × 10(5); p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups.

Conclusions: We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs.

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Fluorescence analysis of standard microsphere dilutions.A Correlation between microsphere amounts and fluorescence signals, which allows the calculation of microsphere concentrations in the function of fluorescence signals. B Standard dilutions between 250.000 (a) and 15.625 (e) were arranged on 96-well plates.
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pone-0101775-g001: Fluorescence analysis of standard microsphere dilutions.A Correlation between microsphere amounts and fluorescence signals, which allows the calculation of microsphere concentrations in the function of fluorescence signals. B Standard dilutions between 250.000 (a) and 15.625 (e) were arranged on 96-well plates.

Mentions: Automated particle count of standardized dilutions with a nominal amount of 1×106 revealed a mean of 0,99×106±7.0×104 microspheres. IVIS quantification was performed using reference dilutions: The correlation coefficient (r) between absolute microsphere counts (CASY) and fluorescence intensity was r = 0,997 (p<0.001) for fluorescence analysis. The mean fluorescence values of microsphere standard dilutions are shown in Table 1 and Fig. 1.


Macroscopic fluorescence imaging: a novel technique to monitor retention and distribution of injected microspheres in an experimental model of ischemic heart failure.

Martens A, Rojas SV, Baraki H, Rathert C, Schecker N, Hernandez SR, Schwanke K, Zweigerdt R, Martin U, Saito S, Haverich A, Kutschka I - PLoS ONE (2014)

Fluorescence analysis of standard microsphere dilutions.A Correlation between microsphere amounts and fluorescence signals, which allows the calculation of microsphere concentrations in the function of fluorescence signals. B Standard dilutions between 250.000 (a) and 15.625 (e) were arranged on 96-well plates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121070&req=5

pone-0101775-g001: Fluorescence analysis of standard microsphere dilutions.A Correlation between microsphere amounts and fluorescence signals, which allows the calculation of microsphere concentrations in the function of fluorescence signals. B Standard dilutions between 250.000 (a) and 15.625 (e) were arranged on 96-well plates.
Mentions: Automated particle count of standardized dilutions with a nominal amount of 1×106 revealed a mean of 0,99×106±7.0×104 microspheres. IVIS quantification was performed using reference dilutions: The correlation coefficient (r) between absolute microsphere counts (CASY) and fluorescence intensity was r = 0,997 (p<0.001) for fluorescence analysis. The mean fluorescence values of microsphere standard dilutions are shown in Table 1 and Fig. 1.

Bottom Line: Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly.Measurements were repeated after a reduction to homogenate dilutions.Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany; Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Hannover, Germany.

ABSTRACT

Background: The limited effectiveness of cardiac cell therapy has generated concern regarding its clinical relevance. Experimental studies show that cell retention and engraftment are low after injection into ischemic myocardium, which may restrict therapy effectiveness significantly. Surgical aspects and mechanical loss are suspected to be the main culprits behind this phenomenon. As current techniques of monitoring intramyocardial injections are complex and time-consuming, the aim of the study was to develop a fast and simple model to study cardiac retention and distribution following intramyocardial injections. For this purpose, our main hypothesis was that macroscopic fluorescence imaging could adequately serve as a detection method for intramyocardial injections.

Methods and results: A total of 20 mice underwent ligation of the left anterior descending artery (LAD) for myocardial infarction. Fluorescent microspheres with cellular dimensions were used as cell surrogates. Particles (5 × 10(5)) were injected into the infarcted area of explanted resting hearts (Ex vivo myocardial injetions EVMI, n = 10) and in vivo into beating hearts (In vivo myocardial injections IVMI, n = 10). Microsphere quantification was performed by fluorescence imaging of explanted organs. Measurements were repeated after a reduction to homogenate dilutions. Cardiac microsphere retention was 2.78 × 10(5) ± 0.31 × 10(5) in the EVMI group. In the IVMI group, cardiac retention of microspheres was significantly lower (0.74 × 10(5) ± 0.18 × 10(5); p<0.05). Direct fluorescence imaging revealed venous drainage through the coronary sinus, resulting in a microsphere accumulation in the left (0.90 × 10(5) ± 0.20 × 10(5)) and the right (1.07 × 10(5) ± 0.17 × 10(5)) lung. Processing to homogenates involved further particle loss (p<0.05) in both groups.

Conclusions: We developed a fast and simple direct fluorescence imaging method for biodistribution analysis which enabled the quantification of fluorescent microspheres after intramyocardial delivery using macroscopic fluorescence imaging. This new technique showed massive early particle loss and venous drainage into the right atrium leading to substantial accumulation of graft particles in both lungs.

Show MeSH
Related in: MedlinePlus