During this Q&A webinar, Dr. Anilkumar Reddy of the Baylor College of Medicine and Ms. Tonya Coulthard, Product Manager at Indus Instruments, answer questions about how to apply pulsed Doppler flow velocity technology in the lab. This session provided an opportunity to review and delve deeper into important topics regarding technical and operational features of pulsed Doppler flow technology, including how and where to make measurements in order to obtain specific flow signals depending on your research objectives, tips for signal acquisition, waveform analysis and interpretation of data. For more information on this technology visit www.indusinstruments.com

1. Doppler Flow Velocity Measurements
for Cardiovascular Research
A special Question & Answers webinar for cardiovascular
researchers interested in learning how to apply and master the
use of noninvasive blood flow velocity measurements to study
cardiovascular function in rodents.

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3. Doppler Flow Velocity Measurements
for Cardiovascular Research
Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
areddy@bcm.edu
Tonya Coulthard, MSc.
Product Manager
Indus Instruments
tcoulthard@indusinstruments.com

4. We will address some of your specific questions, submitted during webinar registration:
• Doppler Flow Velocity System introduction and overview
• Pulsed Doppler vs. Echocardiography systems discussion
• Specific Application based discussion:
• Cardiac function
• Coronary flow reserve
• Vascular injury and Pulse Wave Velocity
• Peripheral vasculature
Due to time we may not be able to address all submitted questions, we will follow up with responses to those questions following the webinar
Presentation Outline

5. 1. Are the measurements invasive or non-invasive?
2. Is it possible to record from conscious animals if they are restrained? Is it possible to measure
blood flow without anesthesia?
3. How does control of body temperature (or lack thereof) effect cardiovascular parameters?
4. Are Doppler flow velocity measurements and resulting data translatable to humans?
5. How do Doppler flow velocity measurements translate to large animals?
6. Can this technology be used in large animals?
7. Have these measurements been validated?
8. In what other species of animals can the Doppler flow velocity technology be used?
9. How can the Doppler Flow Velocity System be incorporated with other technology to provide a
more comprehensive cardiovascular evaluation?
Doppler Flow Velocity System Introduction & Overview

6. The Need to Phenotype Various Animal Models
• Rodents are common experimental models in
basic research
• Genetic manipulations widely available
• Surgical interventions are well developed
• A wide array of experimental models are
available affecting the cardiovascular system
• Most measurements and parameters are
functions of time
• high temporal resolution waveforms are needed
The challenge is to remain
non-invasive…

7. Animal Species Investigated
• Non-invasive Doppler assessment – limited to 1cm depth of penetration
• Mice
• Rats
• Naked Mole Rats
• Similar sized rodents or primates
• Bats
• Fish
• Amphibians – frogs, salamanders, etc.
• Surgically implanted silicone cuff probes – leads are tunneled out to skin
for connection to Doppler system
• Above species
• Larger rodents, mammals and primates

8. Pulsed Doppler Ultrasound

9. Doppler Signals from Mouse Vasculature
| 250 ms |
ascending
aorta
100 -
50 -
0 -
right carotid
right renal
left renal
aortic
arch
left carotid
descending
aorta
abdominal
aorta
coronary
Hartley et al., ILAR J 43:147-8, 2002
cm/s
ECG

10. Doppler Flow Velocity System (DFVS)
• Basic System Includes:
• Rodent Surgical Monitoring
• 10MHz and 20MHz ultrasound
probes
• May be used on anaesthetized or
conscious (restrained) animals
• Two-channel transceiver and
Signal digitizer
• Ability to integrate third party
systems (i.e. blood pressure) via
BNC connection
• Options available:
• Cuff probes (various sizes) for
surgical implantation
• May be used on anaesthetized or
conscious (restrained) animals
Doppler Probe
mm

11. Rodent Surgical Monitor
• Features
• Heated platform
• Monitor ECG and respiration (mouse and rat)
• Monitor core body temperature
• Maintenance of physiological conditions
while under anesthesia
• heart rate and body temperature

12. DFVS Designed with Small Animals in Mind
• Data acquired non-invasively
• Spectrograms are displayed in real-time, and saved rapidly
• High temporal resolution is required
• High spatial resolution is required
• Blood velocities may be very high in surgical models
• DFVS can measure up to 9m/s
• Angle between blood flow and Doppler signal must be
minimized
• DVFS probes are 2-4mm in diameter
• Some measurements require simultaneous data acquisition
• DFVS can acquire data from two sites simultaneously
Mouse Aorta

13. Translation of Doppler Measurements to Larger Species:
Effect of Body Weight on Select Parameters
Parameter Relationship to Body Weight* Value (mouse; BW=0.025kg)
Heart Weight (mg) BW1 4.3 BW 112 mg
Left Ventricle Volume (l) BW1 2.25 BW 56 l
Stroke Volume (l) BW1 0.95 BW 24 l
Heart rate (bpm) BW-1/4 230 BW-1/4 578 bpm
Cardiac Output (ml/min) BW3/4 224 BW3/4 14 ml/min
Aortic Diameter (mm) BW3/8 3.6 BW3/8 0.9 mm
Arterial Pressure (mmHg) BW0 100 100 mmHg
Aortic Velocity (cm/s) BW0 100 100 cm/s
Pulse Wave Velocity (cm/s) BW0 100 500cm/s
* T.H. Dawson, “Engineering design of the cardiovascular system of mammals”, Prentice Hall, 1991

14. Validation of the Doppler Flow Velocity System
• Pulsed Doppler has been considered the gold standard for flow velocities in humans and
large animals for many years
• Numerous papers using DFVS with 10 and 20 MHz probes
• Similar peak velocities in mice
• Flow velocity waveforms have expected shapes
• Timing of events within the cardiac cycle are the same
• DFVS References:
• Hartley CJ et al. Am J Physiol. 268 (Heart Circ. Physiol. 37): H499-H505, 1995
• Hartley CJ et al. Am J Phsiol. 273 (Heart Circ. Physiol. 42): H494-H500, 1997
• Hartley CJ et al. ILAR Journal (43;3): 147 -158, 2002
• Li YH et al. Ultrasound in Med. & Biol. (29;9): 1281-1289, 2003
• Reddy AK et al. IEEE Transactions on Biomedical Engineering (52;10): 1764 - 1770, 2005
• Reddy AK et al. IEEE Transactions on Biomedical Engineering (52;10): 1771 - 1783, 2005

15. How does this system compare to an echo
system specifically designed for small animals,
and what are the advantages of using Doppler
Flow Velocity measurements?
Pulsed Doppler vs.
Echocardiography System Discussion

16. Pulsed Doppler vs. Echocardiography
Pulsed Doppler
• Non-invasive
• Flow velocity
• Small system footprint
• Small probes (2-4mm) – less
error and variability due to
angles
• Flow in Peripheral vessels -
easy
• Cost – relatively low
Echocardiography
• Non-invasive
• Dimensions/Images and Flow
velocity
• Large system footprint
• Larger probes – increased error
and variability due to angles
• Flow in Peripheral vessels –
challenging
• Cost - higher

17. Measurement Angle
• Pulsed Doppler may be considered complimentary to echocardiography
• Both systems have their strengths – use the right tool to collect the most accurate data
• Why are Doppler measurements more accurate on the Indus system than
traditional echo systems?
• Small probes allow for small angles are achieved between the Doppler probe and the
blood velocity
• DFVS ~ 0-15o; Echo ~60-90o
• Specifically for the ascending aorta, coronary artery and
peripheral vessels (such as the carotid and femoral/popliteal
arteries) small angles are easily achieved with the DFVS,
this is not so with echo
• Significant error, and difficulty in reproducibility over a longitudinal study are
introduced as the angle gets above 15o, due to cos from velocity equation
Right Carotid artery

18. Cardiac Function - Systolic
• Systolic cardiac function may be over or under estimated when using M-
mode (1 dimensional) or B-mode (2 dimensional) measures of fractional
shortening
• Regional variations occur in models such as myocardial infarction
• Variability introduced over longitudinal studies
• Doppler flow velocity, as measured at the aortic valve, takes into
account contractility of the entire left ventricle
• Locate the spectrogram at the aortic valve leaflets
• May use velocity or acceleration to assess cardiac function

19. Pulse Wave Velocity – Aortic Stiffness
• Most accurately measured with
2 probes simultaneously
• Simultaneous measurement is
possible with DFVS, not possible
with echocardiography systems
• The same pulse of blood is
observed at two sites along the
artery (i.e. aorta)
• Sequential measurements are
possible, however any variation
due to separate heart beats,
transient effects of drugs or other
metabolites, cannot be accounted
for

20. Ease of Use
• Some Doppler flow velocity spectrograms may be challenging for users
of an echocardiography machine
• Coronary artery (mouse ~200m)
• Mitral Valve (mouse, 525bpm)
20 MHz Doppler Probe(((
-50cm/s

21. System Cost
• The DFVS from Indus is ~ 10% of the cost of a small animal
echocardiography system
• Probes are single element, for Doppler only, rather than linear array probes
required for image acquisition
• Probes are highly durable and low cost to replace if required
• System electronics are equally simplified for Doppler only acquisition
• System footprint is significantly smaller, requiring less laboratory space

22. 1. What are the typical/standard Doppler flow measurement parameters that are used by
researchers?
2. Can you talk about the reproducibility of the assay? Is accuracy technician/user dependent?
3. What are the appropriate markers to ensure reproducibility between animals?
Cardiac Function
1. How does one measure cardiac function from Doppler Flow?
2. Can you measure aortic artery flow?
3. What are the advantages/disadvantages of using non-invasive vs. invasive techniques for measuring
blood pressure and other cardiovascular parameters?
Specific Application Based Discussion

23. Systolic Function – Aortic Outflow
Using 10 or 20 MHz
Doppler Probe
• Measurements:
• Peak Velocity
• Mean Velocity
• Peak
Acceleration
• Mean
Acceleration
• Pre-ejection time
• Ejection time
• Rise time
• Stroke Distance

24. Systolic Function – Various Models
Taffet et al., J Geron Biol Sci 52A:B285-90, 1997
Reddy et al., J Geron Biol Sci 62A:1319-25, 2007
Taffet et al., Am J Physiol 270:H2204-09, 1996
Grimes et al., Am J Physiol 307:H284-91, 2014
Reddy et al., IEEE TBME 52:1771-83, 2005
DeLaughter et al., FASEB J 13:1923-9, 1999
Publication Links

25. Diastolic Function – Mitral Inflow
• Measurements
• E-Peak Velocity
• E-Stroke Distance
• E-Time Duration
• E-Acceleration Time
• E-Deceleration Time
• E-Peak to ½ E-Peak Time
• E-Linear Deceleration Time
• E-Linear Deceleration Rate
• A-Peak Velocity
• A-Stroke Distance
• A-Time Duration
• E-A Peak Velocity Ratio
• Isovolumic Contraction Time
• Isovolumic Relaxation Time
Using 10 or 20 MHz
Doppler Probe

26. Diastolic Function – Various Models
÷ =

27. Aortic acceleration as a surrogate for LV contractility
• LV contractility is assessed with the derivative of invasively measured LV
pressure (+dP/dtmax) , but is a terminal procedure in small animals.
• Non-invasive methods to assess LV contractility have been reported in
patients,1 dogs,2 sheep,3 and in rats4
• Of all the above studies, the noninvasive measurement of aortic acceleration
in dogs was reported to have high correlation to Max dP/dt.
• Max dP/dt = ρc Max du/dt; ρ-density of blood, c-PWV, u-aortic flow velocity
• We are conducting a study to validate the use of acceleration of aortic flow
velocity (dV/dt) in lieu of +dP/dtmax to assess LV contractility in mice.
1. Hunt et al. Cath Cardiovasc Diag, 23:219-223, 1991. 2. Harada et al. Heart Vessels, 3:25-32, 1987.
3. Bauer et al. J Am Coll Cardiol, 40:1320-1327, 2002. 4. De Wildt and Sangster. J Pharmacol Meth, 10:55-64, 1983.

28. Aortic Outflow
Velocity (V)
Left Ventricular
Pressure (P)
dV/dt
dP/dt
Aortic outflow velocity (V) & its derivative (dV/dt) and
Left ventricular pressure (P) & its derivative (dP/dt)

29. Noninvasive surrogate measurements for peak +dP/dt
derived from Doppler aortic blood flow velocity waveform
Peak aortic acceleration Mean aortic acceleration

30. An Example of Aortic Acceleration Application
(Vincelette et al., Translational
Research, vol.148, 2006)

31. 1. What are the typical/standard Doppler flow measurement parameters that are used by
researchers?
2. Can you talk about the reproducibility of the assay? Is accuracy technician/user dependent?
3. What are the appropriate markers to ensure reproducibility between animals?
Coronary Flow Reserve
1. Can this technology be utilized to assess coronary flow reserve?
2. How does one measure coronary flow in a rodent ischemia reperfusion or myocardial infarction
model?
Specific Application Based Discussion

32. Coronary Artery Blood Flow
• Challenges in collecting data from
Coronary Artery
• Coronary arteries are small, ~200m
• Close to many other vessels
• Move along with the heart
20 MHz Doppler Probe(((
-50cm/s

33. Coronary Flow Reserve (CFR) using Isofluorane
Hartley et al., Ultrasound Med Biol 33:512-521, 2007, Baylor College of Medicine
24-
16-
8-
kHz
0-
-90-
-
-
-60-
-
-
-30-
-
cm/s
- 0 -
| 400 ms |
ECG HR = 450
Vlow
low =1.0% high =2.5%
HR = 465
Vhigh
𝑪𝑭𝑹 =
𝑽 𝒉𝒊𝒈𝒉
𝑽𝒍𝒐𝒘
= 𝟒. 𝟐

34. 1. What are the typical/standard Doppler flow measurement parameters that are used by
researchers?
2. Can you talk about the reproducibility of the assay? Is accuracy technician/user dependent?
3. What are the appropriate markers to ensure reproducibility between animals?
Vascular Injury and Pulse Wave Velocity
1. Has this technique been successfully applied to look at drug-induced vascular injury?
2. Is it feasible to measure the Pulse Wave Velocity in a Doppler mode using an equipment with only
one probe? And how trustful is it?
Specific Application Based Discussion

35. Vascular Injury
• Ferric chloride induced vascular injury
• Doppler flow velocity was used to assess time to occlusion in a thrombosis
mouse model
• Zhou et al., J Immunol, 197:288-295, 2016
• Drug induced changes
in aortic stiffness as assessed
by pulse wave velocity

36. Aortic stiffness is estimated using the velocity
of pulse wave. It is defined as:
The distance between 2 aortic sites (mm)
Transit time of the velocity pulse from
site 1 to site 2 (ms)
PWV =
Pulse Wave Velocity

37. Pulse Wave Velocity
• Measurements
• Peak Diastolic Velocity
• Peak Systolic Velocity
• Diastolic Area
• Systolic Area
• Velocity and Area Ratios
• Pulse Wave Velocity
PWV measured from signals acquired
non-simultaneously from aortic arch and
abdominal aortic sites
PWV measured from signals acquired simultaneously
from aortic arch and abdominal aortic sites

38. 1. What are the typical/standard Doppler flow measurement parameters that are used by
researchers?
2. Can you talk about the reproducibility of the assay? Is accuracy technician/user dependent?
3. What are the appropriate markers to ensure reproducibility between animals?
Peripheral Vasculature
1. Is the resolution and sensitivity of the Doppler Flow Velocity technology good enough to study
peripheral vasculopathy?
2. What is the resolution (minimum size) of blood vessel that can be selected to study blood flow?
Specific Application Based Discussion

39. Peripheral Vasculature
• Peripheral vasculature may be measured, provided the vessel can be
localized, sufficient flow velocity to obtain a Doppler signal
• 5-10 cm/s to establish the presence or absence of flow or at 15 cm/s to derive
pulsatility and resistive indices
• Examples of peripheral vasculature and smaller animals:
• Mouse coronary artery
• Mouse saphenous artery
• Zebrafish heart

40. Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
areddy@bcm.edu
Tonya Coulthard, MSc.
Product Manager
Indus Instruments
tcoulthard@indusinstruments.com
Thank You:
For additional information on the products and applications presented
during this webinar please visit www.indusinstruments.com