Comparison of Two Procedures of Loading with Voltage-Sensitive Dye Di-4-ANEPPS in Rabbit Isolated Heart
| Název česky | Srovnání dvou způsobů barvení napěťově citlivým barvivem di-4-ANEPPS u izolovaného srdce králíka |
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| Autoři | |
| Rok publikování | 2008 |
| Druh | Článek ve sborníku |
| Konference | Computers in Cardiology |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Fyziologie |
| Klíčová slova | isolated rabbit heart;voltage sensitive dye;di-4-ANEPPS |
| Popis | Voltage-sensitive dyes (VSDs) undergo changes in their electronic structure, and consequently their fluorescence spectra, in response to changes in the surrounding electric field. VSD di-4-ANEPPS is used in our laboratory to record monophasic action potentials (MAPs) by optical method in isolated rabbit hearts perfused according to Langendorff (80mmHg) with Krebs-Henseleit solution (K-H, 37C, 1.25mM Ca2+). Each experiment consists of four phases: isolation of the heart, control perfusion, loading with the dye, washout and MAPs recording under various experimental conditions. In our experimental set-up, the heart is exposed to VSD diluted in K-H solution to the concentration of 2microM. The tissue is perfused with this mixture for 20-25 minutes and then washed out for the same time. In other laboratories, different way of loading is used - direct application of small amount of concentrated dye into the bubble trapper by means of a microsyringe or a pump followed by short washout. We studied these two procedures of loading in isolated rabbit hearts - some hearts were loaded by "slow" procedure – application of diluted dye into coronary system at speed given by coronary flow, in other hearts "fast" loading (instant application of 20micro of stock solution in the aortic cannula, 5min washout) was applied. After the heart was loaded by either procedure and the excess of VSD washed out from its coronary system, continuous exposure to light source and recording of MAPs was started. In each minute of recording, MAPs were manually marked in their maximum and signal-to-noise ratio (SNR) was evaluated according to formula: SNR = 20 log (signal/noise). Slow loading with low concentration of the dye applied into coronary system together with perfusion solution brought tissue with higher signal-to-noise ratio and also the obtained signal was smoother. Persistence of dye in the myocardium in this case was quite high in comparison with myocardium loaded with small amount of dye applied as a bolus into the perfusion setup. Also fading of the signal during continuous illumination of the heart loaded in this way was quite fast. In conclusion, both methods of loading of rabbit myocardium with VSD di-4-ANEPPS result in loaded heart where recording of MAPs is possible. However, in case of long-lasting exposure to light it is necessary to pay attention to persistence of the dye in the heart muscle. Although the slow procedure might affect electrophysiological properties of the myocardium, in case of rabbit heart it is negligible since it is quite resistant to side-effects of VSDs. |
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