Animal experiments were performed in accordance with the European Directive (2010/63/UE) and the Italian law (D.Lvo 26/2014), and it followed principles of laboratory animal care. The Local Ethical Approval Panel approved the study.
1. Imaging Procedure
2. Post-Processing
The proposed approach has been applied to mice abdominal aorta in a previous study11. The following figures show the results of the application of the described approach on real mice images. These data are from a single animal (wild type mice, 13 weeks old, strain: C57BL6, weight: 33 g) In particular, Figure 1 represents the result of the analysis of the US images. Edge detection and contour tracking techniques applied to B-mode images acquired with the high frame rate ECG-gated modality provide the diameter waveform; on the other hand, the identification of the PW-Doppler signal envelope leads to the single-beat mean velocity curve assessment. The evaluation of the single-beat mean velocity waveforms includes the average of data from different cardiac cycles. For the data shown, the standard deviation of the velocity curves (calculated as the average of the standard deviation obtained at each time point) is 0.0137 m/sec.
Single-beat diameter and mean velocity waveforms are interpolated in both the frequency and time domain and then time aligned (Figure 2A). The lnD-V loop is obtained by plotting the natural logarithm diameter values vs. the mean velocity measurements, as shown in Figure 2B. PWV is assessed by calculating the slope of the linear part of the loop, which is known to correspond to the early systolic phase. This portion is automatically identified as that corresponding to the upslope of the mean velocity curve. These figures indicate that the image processing operations required for the implementation of the proposed technique leads to a final lnD-V loop which is similar to that obtained in humans using a similar approach7. This suggests that this technique could represent a valid alternative for non-invasive PWV assessment in mice.
Figure 1: Processing of B-mode and PW-Doppler Images. B-Mode images (a) are processed using edge detection and contour tracking techniques. PW-Doppler images (b) are processed for the identification of the envelope signal from which the single-beat mean velocity waveform is obtained. Please click here to view a larger version of this figure.
Figure 2: Implementation of lnD-V Loop for PWV Calculation. Diameter and mean velocity waveforms obtained from B-mode and PW-Doppler image processing. (a). The lnD-V loop is obtained by plotting the natural logarithm of the diameter values against the mean velocity values (b). Please click here to view a larger version of this figure.
VEVO2100 | FUJIFILM VisualSonics Inc, Toronto, Canada | micro-ultrasound equipment | |
MS250 Ultrasound Probe | FUJIFILM VisualSonics Inc, Toronto, Canada | micro-ultrasound probe | |
EKV Software | FUJIFILM VisualSonics Inc, Toronto, Canada | Software | |
Matlab R2015a | MathWorks Inc, Natick, MA, USA | Software | |
Conductive Paste | Chosen by the operator | Laboratory material | |
Petroleum Jelly | Chosen by the operator | Laboratory material | |
Depilatory Cream | Chosen by the operator | Laboratory material | |
Acoustic Coupling Gel | Chosen by the operator | Laboratory material | |
Developed Matlab Software | The authors are willing to collaborate with those researchers who are interested in the software and to make the software available under their supervision |
Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.
Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.
Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.