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

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

Myocardial microsphere concentrations and venous drainage of microspheres.

<p><b>A</b> Comparision of myocardial microsphere concentrations after injection <i>ex vivo</i> (EVMI) and <i>in vivo</i> (IVMI). <b>B</b> Myocardial fluorescence analysis unveils venous drainage (arrows) from the injection zone (*) to the right atrium (**).</p

Overview of fluorescence intensitites of standard microsphere dilutions.

<p>Overview of fluorescence intensitites of standard microsphere dilutions.</p

Overview of microsphere quantification in the IVMI group.

<p>Of half a million microspheres which were up taken by the syringe, only few (<75.000) remain in the injection site after 10 minutes of beating heart action. Additionally, further homogenization will lead to lower particle amounts. This phenomenon should be considered when quantifying microspheres in homogenate dilutions. The overall summation of fluorescence signals, retained in filters and organ homogenates respectively, resulted in comparable microsphere counts to whole organ IVIS measurements before homogenization.</p

Distribution of microspheres after injection in vivo (IVMI).

<p>The IVMI-group showed distribution to both lungs (right lobe: 1 – left lobe: 3) 10 minutes after injection into the heart (2). Additionally, the homogenization (Organ – Filter – Homogenate) process involved filter retention of microspheres leading to lower microsphere counts in homogenate dilutions.</p

Ex vivo microsphere injection (EVMI).

<p><b>A</b> Macroscopic fluorescence imaging of a murine heart following injection of 5×10⁵ microspheres. The injection zone (blue circle) shows low (dark red) to high (yellow) microsphere concentrations. For proper fluorescence analysis, background fluorescence (dotted blue circle) must be assessed on every image. <b>B</b> Histological assessment of a murine heart after microsphere injection <b>C</b> Augmentation of the microsphere distribution passage inside the left ventricle (LV).</p

Fluorescence analysis of standard microsphere dilutions.

<p><b>A</b> Correlation between microsphere amounts and fluorescence signals, which allows the calculation of microsphere concentrations in the function of fluorescence signals. <b>B</b> Standard dilutions between 250.000 (a) and 15.625 (e) were arranged on 96-well plates.</p