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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Utilizing Noninvasive Blood Flow Velocity Measurements for Cardiovascular Phenotyping in Small Animals

Anilkumar K. Reddy, PhDAssistant Professor

Medicine - Cardiovascular SciencesBaylor College of MedicineConsultant – Indus Instrumentsareddy@bcm.edu

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

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

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 velocityvector & beam vector

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

Pulsed Doppler

NoninvasiveFlow velocity (FV)Small system foot printSmall probes (2.4mm)Peripheral vessels - easy Signal acquisition - fast Cost - relatively low

Echocardiography

NoninvasiveDimension & FVLarge system foot printLarger probes Peripheral vessels - challengingSignal acquisition - slower Cost - very high

How do technologies compare?

Pulsed Doppler

NoninvasiveFlow velocity (FV)Small system foot printSmall probes (2.4mm)Peripheral vessels - easy Signal acquisition - fast Cost - comparableMultiple sites each time

Transit-Time Flow

Invasive - extravascularVolume flowSmall system foot printSmall probes, but extravascularPeripheral vessels - not easySignal acquisition - fast post-surg. Cost - comparableLimited to 1-2 sites

Why not measure volume flow?

• 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

Why Blood Velocity ?

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 mgLV volume (μl) a BW1 2.25 BW 56 mlStroke volume (μl) a BW1 0.95 BW 24 mlHeart rate (bpm) a BW-1/4 230 BW-1/4 578 bpmCardiac output (ml/min) a BW3/4 224 BW3/4 14 ml/minAortic diameter (mm) a BW3/8 3.6 BW3/8 0.9 mmArterial pressure (mmHg) a BW0 100 100 mmHgAortic velocity (cm/s) a BW0 100 100 cm/sPW velocity (cm/s) a BW0 500 500 cm/s

*T.H. Dawson, “Engineering design of the cardiovascular system of mammals” , Prentice Hall, 1991.

Doppler Flow Velocity and

Rodent Monitoring Systems

[click to learn more]

Set-up for Noninvasive Doppler

Measurements in Mice

[click to learn more]

• 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/RespElectrodes

Mouse ECG &Warming Pad

WarmingZone

ECG/Resp Amplifier Temp Control

10 MHzpulsed

Doppler

ECG

Car

dia

c D

op

ple

r M

easu

rem

ents

in M

ice

+90

+60

+30cm/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

Mouse Cardiac Doppler Signals

Using 10 MHzDoppler Probe

ECG

Aortic Velocity

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

Mouse Cardiac Doppler Signals

Using 10 MHzDoppler Probe

ECG

Mitral Velocity

Cardiac Diastolic Function

÷ =

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

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

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 carotiddescending

aorta

abdominal aorta

ascending aorta

100 -

50 -

0 -

coronary

Hartley et al., ILAR J 43:147-8, 2002

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 =

Pulse-Wave Velocity Measurements in Mice

ECG

Aortic Arch Velocity

Abdominal Aortic Velocity

ECG

Pulse-Wave Velocity Measurements in Mice

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

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

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

Pulse wave velocity in mouse aorta: at baseline and with interventions

Effect of anesthetic agents on PWV & HR

Effect of phenylephrineon PWV & HR

Effect of zatebradineon PWV & HR

Hartley et al., Am J Physiol 273:H494-500, 1997

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

Aorticband

-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 scalePe

rip

her

al V

ascu

lar

Do

pp

ler

Sign

als

Fro

m a

Ban

ded

(TA

C)

Mo

use

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

RightCarotidVelocity

LeftCarotidVelocity

StenosisJet

Velocity

Effe

cts

of

Tran

sver

se A

ort

ic B

and

ing

on

Blo

od

Flo

w P

atte

rns

in M

ice

-100--50cm/s-0

-100--50cm/s-0

-300--150cm/s-0

| 0.5 sec |

P=4V2 =49mmHgP=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

Effects of transverse aortic banding on right and left carotid velocities in mice

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

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?

Method to sense coronary blood flow noninvasively in mice

20 MHz Doppler Probe(((

-50cm/s

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

Co

ron

ary

Res

erve

(H

/B)

in y

ou

ng,

ad

ult

, old

an

d A

po

E-/-m

ice

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

-06 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

Co

ron

ary

Blo

od

Vel

oci

ty in

a B

and

ed M

ou

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

0

20

40

60

80

Pre 1 d 7 d 14d 21dPre 1 day 7 day 14 day 21 day

80-

60-

40-

20-

mmHg

0-

-4

-3

-2

-1

H/B

-02 3.2 51 2.2 62 1.7 67 1.4 74 1.1P

CFR(H/B)

Pre

ssu

re D

rop

an

d H

/B A

fter

Ao

rtic

Ban

din

g

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

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

• 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

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

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

Acknowledgements

Craig HartleyLloyd Michael George TaffetMark Entman

Yong Xu

Thuy PhamJennifer Pocius

Jim BrooksRoss 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)

Thank You

Anilkumar K. Reddy, PhDAssistant Professor

Medicine - Cardiovascular SciencesBaylor College of MedicineConsultant – Indus Instrumentsareddy@bcm.edu

For additional information on the products and applications presented during this webinar please visit, www.indusinstruments.com