Dr. Anilkumar Reddy of the Baylor College of Medicine presents data from his research outlining the importance of blood flow velocity measurement and shows examples of translational data. He provides an overview of Doppler flow velocity measurement technology and compares data obtained from complimentary devices such as 3D echo ultrasound and transit-time flow systems. Several models are presented showing how many selected measurements scale up in translational research from mice to mammals. During this presentation the audience learned how Flow Velocity measurements can reliably assess the following parameters in rodents: Systolic and diastolic cardiac function Myocardial perfusion & coronary reserve Pressure overload Aortic stiffness Peripheral perfusion

1. Utilizing Noninvasive Blood Flow Velocity
Measurements for Cardiovascular Phenotyping
in Small Animals
A webinar for cardiovascular researchers interested in using
noninvasive blood flow velocity measurements to quantify
changes in hemodynamics and characterize cardiac disease
without the need for complex surgery or imaging.

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3. Utilizing Noninvasive Blood Flow Velocity
Measurements for Cardiovascular Phenotyping
in Small Animals
Anilkumar K. Reddy, PhD
Assistant Professor
Medicine - Cardiovascular Sciences
Baylor College of Medicine
Consultant – Indus Instruments
areddy@bcm.edu

4. Most measurements and
parameters are functions of
time, so we need waveforms
• Pulsed Doppler Technology
• Why is it needed?
• How does it work?
• How does it compare to other similar systems?
• Applications
• Cardiac function
• Aortic/arterial stiffness
• Pressure overload - cardiac hypertrophy
• Coronary flow reserve
• Peripheral vascular function
Presentation Outline

5. Most measurements and
parameters are functions of
time, so we need waveforms
• Rodents are animals of choice in basic research
• Undergo genetic (and/or other) manipulations
• Resulting conditions affect cardiovascular system
• To study these conditions phenotyping is needed
Small Animals: Noninvasive phenotyping
“Have a nice day
at the lab, dear?”
But, the challenge is
to be Noninvasive

6. Pulsed Doppler Ultrasound: How does it work?
Relationship
between blood
velocity and Doppler
shift is given as:
V = (c Δf)/(2fo cos θ)
where…
V = flow velocity (cm/sec)
c = velocity of sound (cm/sec)
Δf = Doppler shift (Hz)
fo = transmission frequency
(Hz)
θ = angle between velocity
vector & beam vector

7. Using Pulsed Doppler Ultrasound
7
• Noninvasive - longitudinal studies
• Knowledge of anatomy
• Shapes of waveforms are distinct
• Possible to achieve small angles
• Can be measured at various locations
• Short signal acquisition times
• Signals from 2 sites can be combined
• Different from laser Doppler measurement

8. Pulsed Doppler
Noninvasive
Flow velocity (FV)
Small system foot print
Small probes (2.4mm)
Peripheral vessels - easy
Signal acquisition - fast
Cost - relatively low
Echocardiography
Noninvasive
Dimension & FV
Large system foot print
Larger probes
Peripheral vessels - challenging
Signal acquisition - slower
Cost - very high
How do technologies compare?

9. Pulsed Doppler
Noninvasive
Flow velocity (FV)
Small system foot print
Small probes (2.4mm)
Peripheral vessels - easy
Signal acquisition - fast
Cost - comparable
Multiple sites each time
Transit-Time Flow
Invasive - extravascular
Volume flow
Small system foot print
Small probes, but extravascular
Peripheral vessels - not easy
Signal acquisition - fast post-surg.
Cost - comparable
Limited to 1-2 sites
Why not measure volume flow?

10. • Cardiac systolic and diastolic
function
• Pressure overload - cardiac
hypertrophy
• Coronary flow reserve
• Aortic/arterial stiffness
• Peripheral perfusion
• Tail cuff flow/pressure
Challenge is to be noninvasive with
high spatial and temporal resolution
Mouse Heart Mouse Aorta
Applications of Pulsed Doppler Ultrasound

11. Why Blood Velocity ?

12. Scaling in mammals from elephants to mice
General allometric equation: Y = a.BW b
Parameter Relationship to BW (kg)* Value (BW=0.025kg)
Heart weight (mg) a BW1 4.3 BW 112 mg
LV volume (μl) a BW1 2.25 BW 56 ml
Stroke volume (μl) a BW1 0.95 BW 24 ml
Heart rate (bpm) a BW-1/4 230 BW-1/4 578 bpm
Cardiac output (ml/min) a BW3/4 224 BW3/4 14 ml/min
Aortic diameter (mm) a BW3/8 3.6 BW3/8 0.9 mm
Arterial pressure (mmHg) a BW0 100 100 mmHg
Aortic velocity (cm/s) a BW0 100 100 cm/s
PW velocity (cm/s) a BW0 500 500 cm/s
*T.H. Dawson, “Engineeringdesign of the cardiovascular system of mammals” , Prentice Hall, 1991.

13. Doppler Flow Velocity and
Rodent Monitoring Systems
[click to learn more]

14. Set-up for Noninvasive Doppler
Measurements in Mice
[click to learn more]

15. • Maintain anesthesia
• Monitor ECG and respiration
• Monitor body temperature
• Maintain board or body temperature
• Perform noninvasive measurements
• Perform surgery
• Perform invasive measurements
ECG
Respiration
With this configuration we can:
RA LA
LLRL
ECG/Resp
Electrodes
Mouse ECG &
Warming Pad
Warming
Zone
ECG/Resp Amplifier Temp Control

16. 10 MHz
pulsed
Doppler
ECG
CardiacDopplerMeasurementsinMice
+90
+60
+30
cm/s
-0
-30
-60
Aortic
Mitral
A----
mo
|
mc
|
|
ao
------P
Accel
------E
|
ao
|
ac
+12-
+8-
+4-
kHz
0-
-4-
-8- R
|
ECG
Cardiac Signals and Timing
Probe
The magnitude and shapes of the inflow and outflow velocities in mice are identical to humans.
380 ms

17. Mouse Cardiac Doppler Signals
Using 10 MHz
Doppler Probe
ECG
Aortic Velocity

18. Cardiac Systolic Function
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

19. Mouse Cardiac Doppler Signals
Using 10 MHz
Doppler Probe
ECG
Mitral Velocity

20. Cardiac Diastolic Function
÷ =

21. Cardiac Function in Myocardial Ischemic & Reperfusion
Sham operation (○), 2-h occlusion followed by reperfusion (●), & permanent occlusion (▵).
Data are % of preoperative values and are expressed as means±SE.
Peak Early Filling Velocity
*P < 0.05, permanent occlusion vs. sham;
*P < 0.05, reperfusion vs. sham.
Michael et al., Am J Physiol 277:H660-8, 1999
Peak Aortic Flow Velocity

22. Doppler Probe
mm
Sites on aorta & arteries
from where Doppler
signals can be measured
noninvasively in a mouse
Probe positions as shown with tip
placed on the skin
Anatomy is similar in shape &
structure to that of humans

23. right carotid
right renal
| 250 ms |
Velocities are similar in magnitude and shape to those from humans
Doppler Signals From Aorta and Arteries In a Mouse
left renal
aortic
arch
left carotid
descending
aorta
abdominal
aorta
ascending
aorta
100 -
50 -
0 -
coronary
Hartley et al., ILAR J 43:147-8, 2002

24. Pulse-Wave Velocity Measurements in Mice
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 =

25. Pulse-Wave Velocity Measurements in Mice
ECG
Aortic Arch Velocity

26. Abdominal Aortic Velocity
ECG
Pulse-Wave Velocity Measurements in Mice

27. Pulse-Wave Velocity Measurements in Mice
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

28. PWV in Knockout Mice and Responses To Phenylephrine
Hartley et al., ILAR J 43:147-8, 2002
Reddy et al., J Geron Biol Sci 62A:1319-25, 2007
Reddy et al., Am J Physiol 285:H1464-70, 2003
Hartley et al., Am J Physiol HCP 279:H2326-34, 2000
Grimes et al., Am J Physiol HCP 307:H284-91, 2014
Publication Links

29. Hartley et al., ILAR J 43:147-8, 2002
Reddy et al., J Geron Biol Sci 62A:1319-25, 2007
Reddy et al., Am J Physiol 285:H1464-70, 2003
Hartley et al., Am J Physiol HCP 279:H2326-34, 2000
Grimes et al., Am J Physiol HCP 307:H284-91, 2014
Publication Links
PWV in Knockout Mice and Responses To Phenylephrine
These values are similar to those measured in humans

30. Pulse wave velocity in mouse aorta:
at baseline and with interventions
Effect of anesthetic agents
on PWV & HR
Effect of phenylephrine
on PWV & HR
Effect of zatebradine
on PWV & HR
Hartley et al., Am J Physiol 273:H494-500, 1997

31. Conclusions - 1
• Blood velocity signals from the heart and most arteries of mice
can be obtained noninvasively
• Cardiac systolic and diastolic function can be monitored longitudinally
• Pulse wave velocity can be determined from velocity signals obtained
from two arterial sites
• Blood velocity and pulse wave velocity in mice are similar to those
measured in humans both in magnitude and shape
• The arterial time constants are scaled to heart period such that the wave
reflections return to the heart at similar times during the cardiac cycle

32. Aortic
band
-500
cm/s
-0
-20
-0
-160
cm/s
-0
ECG
Aortic Arch Jet Velocity - 10 MHz Doppler
Left Carotid Artery Velocity - 20 MHz Doppler
Right Carotid Artery Velocity - 20 MHz Doppler
msec
ΔP~75 mmHg
mm scalePeripheralVascularDopplerSignals
FromaBanded(TAC)Mouse
Hartley et al., Ultrasound Med Biol 34:892-901, 2008

33. Right
Carotid
Velocity
Left
Carotid
Velocity
Stenosis
Jet
Velocity
EffectsofTransverseAorticBanding
onBloodFlowPatternsinMice
-100
-
-50
cm/s
-0
-100
-
-50
cm/s
-0
-300
-
-150
cm/s
-0
| 0.5 sec |
P=4V2 =49mmHgP=4V2 =15mmHg
No band Loose Band Tight Band
PI=5.6; RI=1.1; M=9.1
PI=6.7; RI=1.1; M=8.4
PI=11.8; RI=1.3; M=10.3
PI=0.8; RI=0.5; M=7.2
PI=8.3; RI=1.1; M=9.6
PI=3.6; RI=0.8; M=7.8
P=4V2 =4mmHg

34. Effects of transverse aortic banding on
right and left carotid velocities in mice
Hartley et al., Ultrasound
Med Biol 34:892-901, 2008

35. Problems:
1. Coronary arteries are small, ≈200µm
2. They are close to many other vessels
3. They move along with the heart
4. Seems impossible to measure ....
Coronary Blood Flow in Mice?

36. Method to sense coronary blood flow
noninvasively in mice
20 MHz Doppler Probe(((
-50cm/s

37. Noninvasive coronary Doppler signals from a mouse
anesthetized at low and high levels of isoflurane gas
-90-
-
-
-60-
-
-
-30-
-
cm/s
- 0 -
| 400 ms |
24-
16-
8-
kHz
0-
ECG HR = 450
Vlow
low =1.0% high =2.5%
CFR = H/B = Vhigh/Vlow = 4.2
HR = 465
Hartley et al., Ultrasound Med Biol 33:512-521, 2007
Vhigh

38. CoronaryReserve(H/B)inyoung,
adult,oldandApoE-/-mice
0
20
40
60
80
100
120
140
6 wk 3 mo 2 yr ApoE
Base
Hyper
H/Bx
140-
120-
100-
80-
60-
40-
20-
cm/s
0-
H/B
-4
-3
-2
-1
-0
6 wk 3 mo 2 yr 2 yr ApoE-/-
B
H
H/B
B - Baseline Peak Diastolic Velocity (1.0 % Iso)
H - Hyperemic Peak Diastolic Velocity (2.5 % Iso)
Mean±SEM
Hartley et al., Ultrasound Med Biol 33:512-521, 2007

39. CoronaryBloodVelocityinaBandedMouse
Hartley et al., Ultrasound Med Biol 34:892-901, 2008

40. 0
20
40
60
80
Pre 1 d 7 d 14d 21d
Pre 1 day 7 day 14 day 21 day
80-
60-
40-
20-
mmHg
0-
-4
-3
-2
-1
H/B
-0
2 3.2 51 2.2 62 1.7 67 1.4 74 1.1
P
CFR(H/B)
PressureDropandH/BAfterAorticBanding
Hartley et al., Ultrasound Med Biol 34:892-901, 2008

41. Tail-cuff Doppler Pressure Sensing In Mice
Reddy et al., Ultrasound Med Biol 29:379-85, 2003
Hartley et al., ILAR J 43:147-8, 2002

42. • Aortic banding (or TAC) causes significant alterations in both
aortic and carotid hemodynamics in mice
• Severity of banding can be determined through the
measurement of left and right carotid artery velocities
• Pulsatility and resistivity indices can be determined to
understand the response of the vascular system
• Stenotic jet velocity can be measured and the peak jet velocity
can be used to estimate pressure gradient across the band
Conclusions - 2

43. Conclusions - 2 …
• Coronary velocity can be noninvasively measured in mice
• Coronary reserve can be estimated as H/B by varying the
concentration of isoflurane between 1.0% (B) and 2.5% (H)
• Without CAD, hyperemic velocity is consistent, but baseline
velocity is a function of age, anesthesia, and cardiac work
• CFR (H/B) is progressively reduced after banding as cardiac
work increases and the heart hypertrophies and remodels
before becoming decompensated and eventually failing
• Most of the signals and parameters measured in mice are
altered by age and disease in similar ways as in humans

44. Summary: Pulse Doppler Capabilities & Applications
 cardiac function (systolic - aortic FV; diastolic - mitral FV)
 myocardial perfusion (coronary flow reserve - coronary FV)
 pressure overload by stenosis (left & right carotid FVs and stenotic jet FV)
 aortic stiffness (aortic arch & abdominal FVs for pulse wave velocity)
 peripheral perfusion (renal, carotid, iliac, femoral, saphenous vein FVs)
 Noninvasive – allows for serial studies
 Shapes of waveforms are distinct
 Possible to achieve small angles
 Can be measured at various locations
 Signals from two sites can be combined
 Short signal acquisition times

45. Acknowledgements
Craig Hartley
Lloyd Michael
George Taffet
Mark Entman
Yong Xu
Thuy Pham
Jennifer Pocius
Jim Brooks
Ross Hartley
Technicians: Faculty Collaborators:
Sridhar Madala - Indus Instruments
Yi-Heng Li - NCK University, Taiwan
Jim Wang - Berlex Biosciences (now at Crown Biosciences)
Rochelle Buffenstein - UT San Antonio (now at Calico Labs)

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