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Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Hein SJ, Lehmann LH, Kossack M, Juergensen L, Fuchs D, Katus HA, Hassel D - PLoS ONE (2015)

Bottom Line: We show that functional recovery of cryoinjured hearts occurs in three distinct phases.Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi).The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine III, Cardiology, Heidelberg University Hospital, 69120 Heidelberg, Germany and DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany.

ABSTRACT
Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

No MeSH data available.


Related in: MedlinePlus

Three plane echocardiography to assess heart function in adult zebrafish.(A) Overview of experimental setting and illustration of transducer positioning to image short axis view (SAX) (1), abdominal-cranial axis (ACX) for pulsed-wave Doppler (PWD) recordings (2), and long axis view (LAX) (3). (B) Lateral view of dorsally positioned adult zebrafish with three defined transducer positions illustrated. The red line indicates direction of ultrasound beam for PWD acquisition for cardiac inflow velocity imaging and the blue line indicates ultrasound beam direction for acquisition of cardiac outflow velocities. (C) Representative images of SAX view (1) and LAX view (2) acquisition (upper row). In the lower row the ventricle is outlined in red in (1) and (2). (D) PWD images derived from the ACX view (3) with the upper image showing representative signals attained from the AV-valve region with clearly visible positive A- and E- waves (in pink) and the bottom image displaying one representative V VTI PWD signal obtained from the bulbus arteriosus region. PWD, pulsed-wave Doppler.
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pone.0122665.g001: Three plane echocardiography to assess heart function in adult zebrafish.(A) Overview of experimental setting and illustration of transducer positioning to image short axis view (SAX) (1), abdominal-cranial axis (ACX) for pulsed-wave Doppler (PWD) recordings (2), and long axis view (LAX) (3). (B) Lateral view of dorsally positioned adult zebrafish with three defined transducer positions illustrated. The red line indicates direction of ultrasound beam for PWD acquisition for cardiac inflow velocity imaging and the blue line indicates ultrasound beam direction for acquisition of cardiac outflow velocities. (C) Representative images of SAX view (1) and LAX view (2) acquisition (upper row). In the lower row the ventricle is outlined in red in (1) and (2). (D) PWD images derived from the ACX view (3) with the upper image showing representative signals attained from the AV-valve region with clearly visible positive A- and E- waves (in pink) and the bottom image displaying one representative V VTI PWD signal obtained from the bulbus arteriosus region. PWD, pulsed-wave Doppler.

Mentions: To acquire short axis view (SAX) recordings the transducer was positioned in a 60° angle onto the zebrafish corpus starting from the cranial side and moving caudal until ventricular basis was reached and ventricular boundaries were detected as a round shape with circular contraction in its maximum extension (Fig 1A1 and 1B1). B-Mode imaging quality was optimized by adaption of focus depth, 2D gain, image width and depth according to the manufacturer’s recommendations. From each plane 2–3 representative image sequences were recorded. The examination table was then rotated 180° and the ultrasound beam was directed in a 45° angle towards the abdominal wall to obtain the abdominocranial view (ACX) using the B-Mode imaging (Fig 1A3 and 1B3). The correct image plane for the ACX view is characterized by a clear view onto the u-shaped ventricle and valvular plane located in the cranial direction. In a second step C-Mode imaging was conducted to locate the areas with maximal flow velocities by stepwise augmentation of the Doppler gain. Maximum cardiac inflow velocity was usually located at the tip leaflets of the atrioventricular valve. The pulsed-wave Doppler (PWD) sample volume was placed at the region of maximum inflow velocity identified using C-Mode imaging and the PWD signal was detected for at least three seconds. To attain the maximum PWD outflow signals the PWD sample was placed at the conus arteriosus adjacent to the bulboventricular valve after C-Mode imaging guided identification of maximal outflow velocities. Therefore, transducer position needed to be shifted 35–50 μm (depending on the animals size) to a more cranial plane displaying the upper thoracic aperture, as illustrated in Fig 1B3 by the blue line. Similar to recording of inflow PWD signaling, outflow PWD signals were recorded for at least three seconds. Finally the experimental platform was turned 90° to attain long axis (LAX) view. Now the transducer was placed strictly vertically at the median line of the zebrafish corpus along his longitudinal axis (Fig 1A2 and 1B2). After image optimization, equally as described for SAX, at least two B-Mode image sequences capturing 4–5 cardiac cycles were recorded. Repeated measurements deploying our method on four animals on three consecutive days assessing systolic and diastolic area revealed highly consistent and highly reproducible measurements with an interclass correlation coefficient (ICC) of 0.98 and 0.99, respectively.


Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Hein SJ, Lehmann LH, Kossack M, Juergensen L, Fuchs D, Katus HA, Hassel D - PLoS ONE (2015)

Three plane echocardiography to assess heart function in adult zebrafish.(A) Overview of experimental setting and illustration of transducer positioning to image short axis view (SAX) (1), abdominal-cranial axis (ACX) for pulsed-wave Doppler (PWD) recordings (2), and long axis view (LAX) (3). (B) Lateral view of dorsally positioned adult zebrafish with three defined transducer positions illustrated. The red line indicates direction of ultrasound beam for PWD acquisition for cardiac inflow velocity imaging and the blue line indicates ultrasound beam direction for acquisition of cardiac outflow velocities. (C) Representative images of SAX view (1) and LAX view (2) acquisition (upper row). In the lower row the ventricle is outlined in red in (1) and (2). (D) PWD images derived from the ACX view (3) with the upper image showing representative signals attained from the AV-valve region with clearly visible positive A- and E- waves (in pink) and the bottom image displaying one representative V VTI PWD signal obtained from the bulbus arteriosus region. PWD, pulsed-wave Doppler.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390243&req=5

pone.0122665.g001: Three plane echocardiography to assess heart function in adult zebrafish.(A) Overview of experimental setting and illustration of transducer positioning to image short axis view (SAX) (1), abdominal-cranial axis (ACX) for pulsed-wave Doppler (PWD) recordings (2), and long axis view (LAX) (3). (B) Lateral view of dorsally positioned adult zebrafish with three defined transducer positions illustrated. The red line indicates direction of ultrasound beam for PWD acquisition for cardiac inflow velocity imaging and the blue line indicates ultrasound beam direction for acquisition of cardiac outflow velocities. (C) Representative images of SAX view (1) and LAX view (2) acquisition (upper row). In the lower row the ventricle is outlined in red in (1) and (2). (D) PWD images derived from the ACX view (3) with the upper image showing representative signals attained from the AV-valve region with clearly visible positive A- and E- waves (in pink) and the bottom image displaying one representative V VTI PWD signal obtained from the bulbus arteriosus region. PWD, pulsed-wave Doppler.
Mentions: To acquire short axis view (SAX) recordings the transducer was positioned in a 60° angle onto the zebrafish corpus starting from the cranial side and moving caudal until ventricular basis was reached and ventricular boundaries were detected as a round shape with circular contraction in its maximum extension (Fig 1A1 and 1B1). B-Mode imaging quality was optimized by adaption of focus depth, 2D gain, image width and depth according to the manufacturer’s recommendations. From each plane 2–3 representative image sequences were recorded. The examination table was then rotated 180° and the ultrasound beam was directed in a 45° angle towards the abdominal wall to obtain the abdominocranial view (ACX) using the B-Mode imaging (Fig 1A3 and 1B3). The correct image plane for the ACX view is characterized by a clear view onto the u-shaped ventricle and valvular plane located in the cranial direction. In a second step C-Mode imaging was conducted to locate the areas with maximal flow velocities by stepwise augmentation of the Doppler gain. Maximum cardiac inflow velocity was usually located at the tip leaflets of the atrioventricular valve. The pulsed-wave Doppler (PWD) sample volume was placed at the region of maximum inflow velocity identified using C-Mode imaging and the PWD signal was detected for at least three seconds. To attain the maximum PWD outflow signals the PWD sample was placed at the conus arteriosus adjacent to the bulboventricular valve after C-Mode imaging guided identification of maximal outflow velocities. Therefore, transducer position needed to be shifted 35–50 μm (depending on the animals size) to a more cranial plane displaying the upper thoracic aperture, as illustrated in Fig 1B3 by the blue line. Similar to recording of inflow PWD signaling, outflow PWD signals were recorded for at least three seconds. Finally the experimental platform was turned 90° to attain long axis (LAX) view. Now the transducer was placed strictly vertically at the median line of the zebrafish corpus along his longitudinal axis (Fig 1A2 and 1B2). After image optimization, equally as described for SAX, at least two B-Mode image sequences capturing 4–5 cardiac cycles were recorded. Repeated measurements deploying our method on four animals on three consecutive days assessing systolic and diastolic area revealed highly consistent and highly reproducible measurements with an interclass correlation coefficient (ICC) of 0.98 and 0.99, respectively.

Bottom Line: We show that functional recovery of cryoinjured hearts occurs in three distinct phases.Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi).The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine III, Cardiology, Heidelberg University Hospital, 69120 Heidelberg, Germany and DZHK (German Center for Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany.

ABSTRACT
Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.

No MeSH data available.


Related in: MedlinePlus