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Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos.

Chan PK, Lin CC, Cheng SH - BMC Biotechnol. (2009)

Bottom Line: A significant decrease in heart rate was found by our method in treated embryos (p < 0.01).Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, HKSAR, PR China.

ABSTRACT

Background: Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.

Results: The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p < 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.

Conclusion: The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.

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A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of a 52-hpf control embryo. fbasic: first frequency component
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Figure 1: A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of a 52-hpf control embryo. fbasic: first frequency component

Mentions: Flow of blood cells is clearly seen inside blood vessels in zebrafish embryos once the circulation begins at 24 hpf. The flow is pulsatile, with a rhythm of fast and slow movement. With our image analysis software, this oscillatory movement of blood cells can be visualized by waveform of dynamic pixels. A typical example of waveform of dynamic pixels for a 52 hpf zebrafish embryo is shown in Figure 1A. The waveform exhibited a rapid upstroke of the amount of dynamic pixels followed by a drop, showing the rhythmic change of blood cell velocity. This rhythmic fluctuation of dynamic pixels was consistent with the direct observation of pulsatile flow of blood cells observed under stereo-microscope. Frequency characterization of this rhythmic flow of blood cells was done by transforming the waveform in time domain to frequency domain. Figure 1B showed the corresponding power spectrum for the waveform of dynamic pixels. Two peaks of frequency components (at 2.31 Hz and 4.69 Hz) were identified. The first peak (2.31 Hz) was the basic frequency (fbasic) component of the waveform of dynamic pixels. The second frequency component was the multiple of the first one and should be considered as the harmonic counterpart of the basic frequency component [25].


Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos.

Chan PK, Lin CC, Cheng SH - BMC Biotechnol. (2009)

A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of a 52-hpf control embryo. fbasic: first frequency component
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664803&req=5

Figure 1: A typical example of the signal of dynamic pixels (A) and its corresponding power spectrum (B) obtained from the caudal circulation of a 52-hpf control embryo. fbasic: first frequency component
Mentions: Flow of blood cells is clearly seen inside blood vessels in zebrafish embryos once the circulation begins at 24 hpf. The flow is pulsatile, with a rhythm of fast and slow movement. With our image analysis software, this oscillatory movement of blood cells can be visualized by waveform of dynamic pixels. A typical example of waveform of dynamic pixels for a 52 hpf zebrafish embryo is shown in Figure 1A. The waveform exhibited a rapid upstroke of the amount of dynamic pixels followed by a drop, showing the rhythmic change of blood cell velocity. This rhythmic fluctuation of dynamic pixels was consistent with the direct observation of pulsatile flow of blood cells observed under stereo-microscope. Frequency characterization of this rhythmic flow of blood cells was done by transforming the waveform in time domain to frequency domain. Figure 1B showed the corresponding power spectrum for the waveform of dynamic pixels. Two peaks of frequency components (at 2.31 Hz and 4.69 Hz) were identified. The first peak (2.31 Hz) was the basic frequency (fbasic) component of the waveform of dynamic pixels. The second frequency component was the multiple of the first one and should be considered as the harmonic counterpart of the basic frequency component [25].

Bottom Line: A significant decrease in heart rate was found by our method in treated embryos (p < 0.01).Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, HKSAR, PR China.

ABSTRACT

Background: Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis.

Results: The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p < 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot.

Conclusion: The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.

Show MeSH
Related in: MedlinePlus