Untitled - MendelNet 2013 - Mendelova zemědělská a lesnická ...

MENDELNET 2013Heart preparationFirst, deep inhalation anesthesia by isoflurane was introduced. The chest of rat was then openedwith scissors and the heart with sufficiently long piece of aorta quickly cut out. It was placed into abeaker with cold Tyrode solution of following composition: 8 g/l NaCl, 0.2 g/l CaCl 2 , 1 g/lNaHCO 3 , 0.05 g/l Na 2 HPO 4 , 0.1 g/l MgCl 2 , 1 g/l glucose. The heart was then connected to theminiaturized Langendorff setup inside the In vivo imaging instrument and perfused under constantperfusion flow with oxygenated Tyrode solution. The coronary flow was kept by the peristalticpump at the rate of 9 ml/min.Fluorescence ex vivo imaging of rat heartThe fluorescence of doxorubicin was registered by Carestream In vivo Xtreme Imaging System(Rochester, USA) under following conditions: exposition time: 2 s, binning: 2x2 pixels, f-Stop: 1.1,excitation/emission 480 nm/600 nm. Camera is a cooled back-thinned, back illuminated cameradesigned for maximum sensitivity. The camera utilizes a two-stage thermo-electric cooler that coolsdown the CCD below -55 °C absolute. The camera collects the image data on a 2048 x 2048 pixelCCD. Single frame image data is digitized at 16-bits, and presented in software as a 32-bit floatingpoint image. The images were processed by Carestream molecular imaging software (Carestream)and the Carestream Multispectral software was used to eliminate the autofluorescence of the tissue.RESULT AND DISCUSSIONCommercially available in vivo instrument is a highly sensitive machine combining the X-ray andfluorescence imaging modality to give precise spatial visualization of targeted area. The chamberwith the imaging area of 20×20 cm provides enough space for miniaturized Langendorff perfusionsystem. The tubing supplying the isolated heart with the oxygenated Tyrode solution was slid intothe imaging chamber through the aperture commonly used to supply the anesthetic gas duringanimal imaging. Tyrode solution was oxygenated and heated to 37 °C outside the chamber prior toapplication. In order to prevent its cooling the length of the tubing was minimized. Constantperfusion flow ensured by peristaltic pump was set to 9 ml/min, which is sufficient value for the ratheart. The heated environment of the imaging chamber provided friendly environment for isolatedheart. The perfusate was drained by running down the side of the inclined Petri dish placed underthe isolated heart.Finally, the heart isolated from the doxorubicin administered rat was investigated. In Fig. 1 is thephotography of heart in the chamber, and the X-ray, fluorescence images and combined X-ray andfluorescence images are. The autofluorescence of the tissue was deducted. According to detectedfluorescence intensity of the doxorubicin in the heart the amount of doxorubicin in the heart wasdetermined to 1 µg. The in vivo imaging system is useful for the detection of fluorescencecompounds in the Langendorff-perfused heart.693 | P age