Sttudy on ArterialA
Tissue CharacteCerizationwith Ultraasound
Hideyuki Haseegawa1,2 andd Hiroshi Kanai2,1
1 GraduateG
Schoool of Biomediccal Engineerinng, Tohoku Unniversity
2 GraduateG
Schoool of Engineerring, Tohoku UniversityU
E-m
mail: hasegawaa@ecei.tohokuu.ac.jp, hkanaii@ecei.tohokuu.ac.jp

Abbstract
The endotheelial dysfunctioon is considereed to be an
inittial step in atherosclerosis. Additionallly, it wasrepported that thee smooth musccle, which con
nstructs themeedia of the arteery, changes itss characteristiccs owing toathherosclerosis. Therefore,T
it iis essential too develop ameethod of assessing the regioonal endotheliial function
andd mechanical properties oof the arteriall wall. To
evaaluate the enndothelial fuunction, a coonventionaltechhnique of meeasuring the transient channge in the
diaameter of thee brachial arrtery caused by flowmeediated dilation (FMD) after the releaserofavaascularization is used. Howeever, this methhod cannot
evaaluate the meechanical propperties of thee wall. We
preeviously develloped a methood for the sim
multaneous
meeasurements off waveforms oof radial strainn and blood
preessure in thee radial arterry. In this study, theviscoelasticity off the arterial w
wall was estim
mated fromthee measured strress–strain relaationship using
g the leastsquuares method and the traansient chang
ges in themeechanical propeerties of the arrterial wall weere revealed.
Froom in vivo experimentalerresults, the stress-strainsrelaationship shhowed a hysteresis looplandviscoelasticity was
w estimated bby the proposeed method.
Thee slope of the loop decreaseed owing to FM
MD, whichresu
ulted in the decreasedin esttimated elasticc modulus.
Thee increase in the area of the loop occcurred after
reccirculation, whhich corresponnds to the incrrease in theratiio of the loss modulus (depends on visco
osity) to theelastic modulus when the Voiigt model is assumed.
aInthiss study, the variance in estiimates was ev
valuated byin vivov measurem
ment for 10 minn. The temporral decreasein static elasticitty after recircuulation due to FMD wasmuuch larger thann the evaluatedd variance. Th
hese resultsshoow a potentiaal of the prooposed metho
od for thethoorough analyysis of the transient changecinviscoelasticity duue to FMD.
In addition too the mechaniccal property an
nd functionof thet arterial waall, the conditiion of blood, suchs
as redblo
ood cell (RBCC) aggregation, a determinan
nt of blood
viscosity, plays an importannt role in bloodb
flow
rheeology. RBC aggregationais induced by th
he adhesionof RBCs when the electrosttatic repulsion betweenRB
BCs weakens owing to inncreases in protein
p
and
satu
urated fatty accid levels in bllood, and exceessive RBC
agg
gregation mayy lead to varioous circulatorry diseases.
Thiis study was conducted to establish a noninvasivenquantitaative method for the asssessment of RBC
aggregaation. The speectrum of nonaggregating RBCs
presentss Rayleigh beehavior, indicaating that the powerof a scaattered wave iss proportional to the fourth powerof freq
quency. By dividing thee measured power
spectrumm of echoes from scattereers by that fromfasiliconee plate reflecttor, the frequuency responses oftransmittting and recceiving transdducers are rem
moved
from thhe former spectrum. This normalized power
spectrumm changes linnearly with resspect to logarrithmic
frequency. In non-R
Rayleigh scatteering, on the other
hand, thhe spectral slope decreasees because a larger
scatterer behaves as a reflector aand echoes fromfareflector do not show frequency deppendence. Theerefore,
it is poossible to asssess RBC agggregation usin
ng thespectrall slope value. In this study, spherical scaatterers
with diaameters of 5, 11,
1 15, and 300 μm were measured
in basiic experimennts. The specctral slope ofo thenormaliized power spectrum of echhoes from the lumenof the vein in the dorsumdmanuus of a 24-yeear-old
healthy male was close to that fromm microspherees witha diameeter of 15 μm, and the typicaal RBC diametter was
smaller than this value.
v
The ffrequency-depeendent
attenuattion of ultrassound duringg propagation in abiomediical tissue was considered tto be one reasson for
this. Fu
urthermore, duuring avascularization, the slope
graduallly decreased owingoto the agggregation of RBCs.
R
These results show
w the possibiility of usin
ng theproposeed method forr the noninvaasive assessm
ment of
RBC agggregation.

1. Intro
oduction
1.1. Meeasurementss of flow med
diated dilatioon
The mainmcause off circulatory diiseases is conssidered
to be atherosclerossis. Thereforee, the quanttitative
assessm
ment of atherossclerosis is essential for makking anearly diagnosis of these diseases.
The endothelialedyysfunction is cconsidered to be an
initial stepsin atherossclerosis [1]. A
Additionally, it wasreportedd that the smoooth muscle, w
which construccts themedia ofo the artery, chhanges its chaaracteristics ow
wing toatheroscclerosis [2]. Consequently, iit is important for an
early prreventive treattment to noninnvasively asseess theendotheelial function and mechaniccal properties of themedia mainlymcompossed of smooth muscle.
Endoothelial cells reeact to the sheear stress caussed byblood flowfand prodduce nitric oxxide (NO), whhich isknown asa a vasodepreessor material. The smooth musclemis relaxed as a result of the response to the produced
NO. This function is important for maintaining thehomeostasis of the vascular system. Smooth muscle
cells in the media are classified into two types withdifferent functionalities [3]. The composite type isproliferative, and the contractional type contracts andrelaxes as responses to chemical and mechanicalstimuli.
When the blood vessel is initially formed, smoothmuscle cells change their type from composite tocontractional and control blood flow and bloodpressure. On the other hand, after the vascular systemis established, smooth muscle cells change theircharacteristics from contractional to composite owing
to atherosclerosis. The composite type is related to thegrowth factor and accelerates the migration of smoothmuscle cells to the intimal layer. Therefore, as
described above, the evaluation of the endothelial
function and characteristics of smooth muscle cells is
important for the early diagnosis of atherosclerosis.
For the evaluation of the endothelial function, there
is a conventional technique of measuring the transientchange in the inner diameter of the brachial artery
caused by flow-mediated dilation (FMD) after the
release of avascularization [4]. For a more sensitiveand regional evaluation, we developed a method of
directly measuring the change in the elasticity of theintima–media region due to FMD [5].
We propose a method for the evaluation of FMD,
which was previously applied to the measurement of
the radial artery. There is an inversely proportional
relationship between the percent change in innerdiameter due to FMD and that in the inner diameter of
the artery at rest because the flow velocity, which
affects the shear stress, is inversely proportional to thesquare of the inner diameter when the pressure andflow volume are constant [6]. Additionally, the bloodpressure (stress) waveform can be continuously
measured in the radial artery, together with the minutechange in thickness (radial strain), which is measured
using the ultrasonic phased tracking method [7]. Fromsuch measurements, we could determine the stressstrain relationship during each heartbeat [8]. Therefore,
the radial artery would be a more suitable site for themeasurement of FMD.
In this study, from the stress-strain relationshipduring each heartbeat, the viscoelasticity of the intima–
media region was estimated using the least-squares
method, and the transient change in viscoelasticity due
to FMD was estimated. In addition, the viscoelasticity
at rest was measured for 10 min to evaluate thevariance in measurements [9].

1.2. Evaluation of red blood cell aggregation
Medical ultrasound is clinically used to make adiagnosis for various organs, and because it isnoninvasive and relatively stress free for patients, it canbe repeatedly employed to confirm time-dependentchanges. Ultrasound B-mode imaging is widely used
for the morphological diagnosis of the arterial wall. In
addition, methods for evaluating the viscoelasticity of
the arterial wall have recently been developed [8,10]

because the mechanical properties of the arterial wall
are related to the atherosclerotic change.
The condition of blood is an important factor related
to various circulatory diseases. However, conventional
ultrasonic images cannot be used to evaluate thecondition of the blood in the blood vessel because red
blood cells (RBCs), which are the main components ofblood, are much smaller than the wavelength ofultrasound and the variation in acoustic impedance
between blood plasma and RBCs is very small.
However, the condition of blood is related to various
circulatory diseases, and the evaluation and diagnosis
of such a condition are important for the detection of adisease at an early stage.
As one of the determinants of blood viscosity, RBC
aggregation plays an important role in blood flowrheology. The adventitia of healthy RBCs is charged
with negative electricity, which impedes RBC
adherence by electrostatic repulsion [11]. However,owing to increases in protein and saturated fatty acidlevels in blood, such repulsion between RBCs isgradually weakened and RBC aggregation is inducedby the overlapping of RBCs. The main function ofblood is to transport nutrients, oxygen, and essential
elements to tissues and to remove metabolic products,
such as carbon dioxide and lactic acid, produced bythose tissues [12]. However, RBC aggregation
significantly degrades this function because of thedecrease in the superficial area used to transportmaterials. Excessive RBC aggregation may promotevarious circulatory diseases, such as atherosclerosis,
hypercholesterolemia, diabetes, thrombosis, and so on[13-16]. Therefore, the assessment of RBC aggregation
is essential [17]. The micro channel array flow analyzer(MCFAN) method is a recently developed technique
for the assessment of RBC aggregation by determining
whether red blood cells pass through gaps in silicon
substrates simulating blood capillaries [18]. However,this method is invasive and is not quantitative. The
present study was conducted to establish a noninvasive
quantitative method for the assessment of RBC
aggregation [19].

2. Materials and Methods
2.1. Measurement of viscoelasticity of radial
arterial wall
2.1.1. Measurement of minute change inthickness of arterial wall for measurement ofstress-strain relationship
The arterial wall is composed of three layers, namely,
intima, media and adventitia. The smooth muscle,
which constructs the media, is the main source of theviscoelasticity of the vessel wall. Therefore, thedilation and contraction of the artery depend on thecharacteristics of the media. The detailed analysis of
the change in the viscoelasticity of the arterial wall due
to FMD requires the in vivo measurement of the stressstrain relationship, which has not been measured
noninvasively thus far.

The
T blood pressure waveforrm can only bee measured
nonninvasively in vivo at the raddial artery. Hoowever, it is
imp
possible to measure the strrain of the raddial arterial
wall by ultrasounnd at a typicall frequency (110 MHz) in
con
nventional equuipment. Thereefore, we con
nstructed an
acqquisition systeem for high-frrequency ultraasound [8].
Thiis system imprroved the spattial resolution in the axial
direection. Thereffore, this systeem allows moore detailed
meeasurements annd analyses. Figure 1 sho
ows the Bmo
ode images of the radial arteery along the lo
ongitudinalaxiis and in the planepperpenddicular to the axis of thearteery.

g. 1. B-mode images of thee radial arteryy along theFiglon
ngitudinal axiss (left) and in the plane perrpendicular
to thet arterial lonngitudinal direcction (right).
To
T determinee the stress-strain relatio
onship, theminnute change ini the thickneess (strain) Δh(t) of therigh
ht radial arterrial wall duriing a cardiac cycle wasmeeasured usingg the phasedd tracking method
m
[7].
Tog
gether with thhe measuremeent of RF sign
nals for theestiimation of Δhh(t), the waveeform of bloo
od pressure(strress) p(t) in thhe left radial artery was coontinuously
meeasured with a sphygmometeer.
To
T obtain the change in thiickness, the veelocities ofarteery-wall bouundaries (lummen-intima an
nd mediaadv
ventitia) weree estimated. The velocityy v(t) was
estiimated from thet phase shift
ft Δθ(t) of echhoes in two
con
nsecutive frammes. The phasee shift Δθ(t) waas obtainedusin
ng the compplex cross-corrrelation applied to thedemmodulated signnals of RF echhoes. From thee estimatedphaase shift Δθ(t), the averagge velocity ̅of thearteerial wall at the pulse reepetition interv
val T wasobttained as
∆

,

(1)

wh
here ω0 and c0 area the center angular frequeency of theultrrasound wave and the speedd of sound, reespectively.
Thee change in thhickness, Δh(tt), between tw
wo different
deppths, A and B (correspondiing to the bou
undaries ofthee arterial wall),, in the arteriall wall along an
n ultrasonicbeaam was obtaained from tthe differencee between
dispplacements, xA(t) and xB(t), at these two positions as
∆
.

(2)

The changecin thicckness, Δh(t), corresponds to theincremeental strain in the arterial raadial direction at thetime t due
d to the prressure increm
ment Δp(t) fro
om thediastolicc pressure.

2.1.2. Estimation of viscoelassticity of arrterial
uares methoodwall ussing least-squ
The smoothsmuscle constructs thhe media and is theof the arterial wall.
main soource of the viscoelasticityv
We asssumed a viscoelastic modeel as a mechhanicalmodel ofo the arterial wall
w to estimatte its viscoelassticity.
Theree are many tyypes of modells for describinng theviscoelaasticity of thhe arterial waall. We seleccted aviscoelaastic model withw
two com
mponents to simplysunderstaand the viscooelastic behavvior of the arterialawall; onne component corresponds to elasticity anda theother too viscosity. In this study, thhe Voigt modeel wasused beecause the deformationdoof the arteriall wall
during one
o cardiac cyycle is reproduucible among cardiacccycles, and there is no permanennt deformation
n. TheMaxwelll model, whhich is anotheer model witth twocomponnents, was nott used because it should bee usedfor visscoelastic maaterials show
wing a perm
manentdeformaation.
By assumingathe Voigt modeel as a visco
oelastic
model of the intimaa–media regioon, the stress––strainrelationship is given by
b
,
̂

(3)

is the streess modeled bby the Voigt model
where ̂and γ ,
, Es, anda η are thee strain, strain
n rate,static ellasticity, and viscosity, resspectively. In the invivo measurement,mtthemeasuredd strain γ(t) is theincremeental strain duue to the pulse pressure, whereasw
the meaasured stress includesithe bbias stress (diastolicblood pressure).
p
Theerefore, τ0 is added to the righthand sid
de of eq. (3) asa the bias streess correspondding todiastolicc pressure.
The parameterspin eq.
e (3), Es, η, and τ0 are estiimatedusing thhe least-squarees method by mminimizing thee meansquaredd error α betw
ween the measuured τ(t) and modelτ streesses and definned by
α

E

̂

,

(4)

where Et[···] indicattes the averagiing operation during
a cardiaac cycle. Thee parameters, η , and τ thatminimizze α are determined by setting the partial
derivatives of α with respect to Es, ηη, and τ0 to zeero as
0,

0,

0.

(5)

To solvve the simulttaneous equattions, the optimum
parametters that minim
mize α are deteermined.

2.1
1.3. Procedurre for in vivoo measuremeent
The
T right radiaal arteries of tw
wo healthy maale subjects(suubject A: 35 yearsyold and subject B: 22 years old)
were measured. In these meassurements, ultrasonic RF
echhoes (transmit:: 22 MHz) werre acquired at a sampling
freq
quency of 66.5MHz for 2 s and the framme rate wasaboout 160 Hz [8]]. The acquisittion for the ev
valuation ofFM
MD was repeatted every 20 s for 2 min at rest before
avaascularization and every 12 s for 3 min after
reccirculation. Toogether with tthe measurem
ment of RF
signnals, the waveeform of bloodd pressure p(t)) in the leftraddial artery wasw
continuouusly measureed with asphhygmometer (CColin Jentow--7700). Additiionally, forthee evaluation off reproducibilitty, RF data and the blood
preessure waveforrm at rest werre measured ev
very 1 minfor 10 min. In this study, a sphygmomeeter, whichauttomatically opptimizes the poosition of the sensor forblo
ood pressure measurementmbby detecting th
he regional
pullsation of thhe radial arteery, was useed for theconntinuous meaasurement off the bloodd pressure
waveform p(t) for about 110 min. How
wever, thesphhygmometer always requiress the arterial pulsationptoopttimize the position of the sensor.. In thismeeasurement, therefore, tthe sensor of thesphhygmometer was
w placed inn the left arm
m, in whichavaascularization was not induced.
The
T experimenntal apparatuss employed inn this study
sho
owed undesirabble time delayys. We corrected the timedellay between strress and strainn to determine the stress–
straain relationshhip that deepended on only theviscoelasticity of the arterial wall [8]. Th
he transientchaange in stresss-strain relatioonship during
g a cardiac
cyccle due to FM
MD was obtaiined from thee measured
chaange in the thhickness Δh(t) of the arteriaal wall andfrom the blood prressure Δp(t).

where k is the wave number of ulltrasonic wavees (k =2π f /c0, c0 is the avverage sound velocity), ρ0 is theaveragee density of thet medium, G(f) is the electricecharacteeristic of thee transducer, Rs is the staandard
deviatioon of the ampplitude reflecction coefficieents ofscatterers, S0 is the arrea of the transducer aperturre, and
w0 is thetdistance betweenb
the transducer annd thescatterers. Furthermorre, g(f) is exprressed as
.

,

4

(7)
m
where d is the thicknness of the laayer of the medium
includinng scatterers, σb and σT are the beam radiius (σb= 80.5w0/ka) and a/20.55 (a: aperture rradius), respecctively,and σ is the corrrelation lenggth of contiinuous
inhomogeneous meddia [21], beiing related tot thediameteer of the scatterer [20]. In this casee, thecorrelation length is much smaller than the diaameterand beam width off the transducer. Thereforre, thefollowinng approximattion holds:
1
≅

.

(8)

Using eq. (8), eq. (6) is modified ass
|

|

.

2

,
(9)

2.2
2. Evaluationn of red bloood cell aggreg
gationAn
A RBC is a very small ulltrasonic scatterer whose
diaameter is 8 μm
m at most, andd thus, the am
mplitudes ofscaattered RF echhoes are very ssmall. To assess the levelof RBCR
aggregattion, the poweer spectrum off the echoesfrom RBCs is calculated uusing the faast Fourier
tran
nsform (FFT), and the scaattering properrties of RFechhoes are evaluuated in the frrequency dom
main. In thepreesent study, itt was assumedd that the diaameter of ascaatterer increases depending on the degreee of RBC
agg
gregation.
The
T
scattering strength can be measured
quaantitatively in terms of a bacckscattering cooefficient ifthee tissues can be modeled as a random
m medium.
Figgure 2 showss the geomeetrical conditiion in themeeasurement of echoes from a random med
dium with acirccular transduccer [20]. The average poweer spectrumSi(ff) of incoherennt componentss of the echo scatteredsbythee random mediium is given byy
S

|4

|

,

(6)

on in
Fig. 2. Illustration of geometrrical conditio
measureement of echooes from scatteerers with a circulartransduccer.
where Z0 is the average specific acoustic impedaance ofthe meddium. When scatterer diameeters are sufficciently
smaller than the ultrasonic w
wavelength [kσ«1,≅1,∝ σ ], echooes from scaatterers
show RayleighRbehhavior, i.e., tthe power Si(f) of
scattered waves is prroportional to the fourth pow
wer offrequency, f 4 [20,22,23]. Echoes ffrom scattererrs with

larg
ger diameters include the components off reflection,whhich have no frequencyfdeppendence. Theerefore, thespeectral slope deecreases whenn the sizes off scatterers
inccrease and the components oof reflection arre includedin thet echoes [23].
The
T measuredd power spectrrum Ps(f) of th
he received
ultrrasonic echo signalses(t) conntains both thee scatteringprooperty Si(f) fromfr
a microoscopic spherre and thefreq
quency responnses G(f) of trransmitting andd receiving
tran
nsducers. Theerefore, by nnormalizing the powerspeectrum Ps(f) off echoes fromm scatterers byy the powerspeectrum Pr(f) fromfa silicone plate, the frequency
resp
ponses G(f) of the trannsmitting and receiving
tran
nsducers are reemoved [25,266].

Fig
g. 3. System for measurem
ment of echoes from thesiliicone plate.

an in vivovmeasurem
ment. Figure 4 shows RF echoesereflected
d by the surrface of the silicone platte andscattered from the innside of the ssilicone plate. Threelines in Fig. 4 show RF
R signals fromm different pooints ofthe silicone plate. The
T
scatterer distribution in thesiliconee plate is assuumed to be ranndom, and on
nly theecho reeflected from the surface ccan be obtainned byaveraginng the echoess measured at different points on
the surfface. Thereforre, echoes fromm the siliconee plate
were accquired at 10000 different pooints by moving thetransduccer. In Fig. 4, there are echoes with muchsmaller amplitudes ofo around 10..4 μs. Such echoesewere su
upposed to be caused by the interferen
nce ofultrasonnic waves duuring focusing by the cooncavetransduccer used. SuchS
undesiraable echoes were
suppressed by applyiing Hanning w
windows centeered at
10.8 μss when the Fourier transfoorm was perfo
ormed.
Figure 5 shows thee power specctrum Pr(f) fromfrasiliconee plate that was
w obtained by averaging
g 1000power spectrasof RF echoesefrom a number of diffferent
points on the surfaace of the ssilicone platee. The
frequency range used for normalization was limitedlbecausee the ultrasoniic pulse used in this study had afinite frrequency banddwidth. In thee present studdy, weestimateed the slope ofo the normalizzed power speectrum
Ps(f)/Pr(s)(using thhe weighted least-mean-sq
quaredmethod by considerinng the signal-tto-noise ratio (SNR)of the echoeat each frequencyf[277]. Let us defiine themean squared diffeference betweeen the measured
spectrumm and a linear model asαFig
g. 4. Three RF
R echoes froom the siliconne plate atdifffferent points ono the surface.

∑∑,where y(fi) is the normalized logarithmic power
spectrumm log10Ps(f)/PPr(f) at a frequuency of fi, xi is thelogarithhmic frequencyy log10fi, and wi is the weiighting
functionn. In this studdy, the weighting function wi wasdefined by
rmax

Fig
g. 5. Means annd standard devviations of pow
wer spectraof the ultrasounnd echoes refllected from th
he silicone
plaate.
FigureF
3 show
ws a system forr measuring an
n echo er(t)from a silicone plate.
p
The disttance betweenn the probe
d the silicone plate was eqqual to that between theandproobe and the miiddle part of thhe intravasculaar lumen in

(10)

(11)

where Prmax is the maximum vaalue of the powerm Pr(fi) from a silicone platee.
spectrum
Figurre 6 shows thetweightingg function wi. It is
assumedd that the echooes due to refflection do nott show
frequency dependencce and that only the scattering
propertyy of a target remainsrin thee normalized power
spectrumm Ps(f)/Pr(f).
In RayleighRscatttering, the normalized power
spectrumm Ps(f)/Pr(f) chhanges linearlyy with respectt to thelogarithhmic frequencyy. In non-Rayyleigh scatterinng, onthe otheer hand, the spectral slopee decreases withw
anincreasee in scatterer diameter, suchh an increase being
equal too that in the coorrelation lengtth σ of the Gaaussiancorrelation function. In this study, the diameter of thescatterer was estimaated to assesss RBC aggreegationfrom thee spectral sloppe by assumingg that the correelation
length σ of the GaussianG
correelation functiion of

conntinuous inhom
mogeneous m
media correspo
onds to the
diaameter of the sccatterer.

Fig
g. 6. Weightinng function oobtained fromm averaged
pow
wer spectrum
m of the ultraasound echoes reflectedfrom the siliconee plate.

3. ResultsR
3.1
1. In vivo measurementsms of viscoela
asticity ofartterial wallRF
R data for 2 s obtained by eeach acquisitio
on included
at leastlan entiree cardiac cyclee. Therefore, the changes
in thickness Δhh(t) and bloood pressure p(t) were
obttained for att least one cardiac cyclee in eachmeeasurement forr estimating the viscoelastic parametersof thet radial arterrial wall.
To
T reveal the change in the stress-strain relationshiprof thet arterial waall due to FMD
D, the blood prressure p(t)andd the change in the thicknness of the inttima-media
reggion Δh(t) duriing a cardiac cycle during FMD weremeeasured. Figurre 7 shows tthe transient change instreess-strain rellationship beetween the changecinthicckness and thhe blood presssure of subjeect A. The
streess-strain relaationship show
wed hysteresiis property
andd gradually changedcits shhape. The slo
ope of thehyssteresis loop decreaseddowinng to FMD, whhich shows
thaat the elastic modulusmdecreeased. In addiition to thechaange in slope, the area of thhe loop clearlyy increased
afteer recirculatioon. The area oof the loop deepended onthee ratio of the looss modulus ωωη (ω: angularr frequencyof strain, η: visccosity) to the static elastic modulusmEs
whhen the Voigt modelm
was assuumed.

Fig. 7. Transient chhange in relattionship (hysteresis)
betweenn blood pressuure p(t) and cchange in thicckness
Δh(t) measuredmin thee radial artery iin vivo.
Figurres 8(a) and 8((b) show the ttime sequence of theblood flow
flvelocitiess and stress-strrain relationshhips ofsubject A, respecttively. Bloodd velocity clearlycincreaseed immediateely after reccirculation annd thestress-sttrain relationnship started to change. Sixty
secondss after recircullation, blood velocity return
ned toits origiinal value andd the stress-straain relationshiip alsocame arround graduallly.
Figurre 9(a) shows thet transient changes in the means
and stanndard deviatioons (SDs) of thhe static elasticcity Es
and visccosity η averaaged by 5 ultraasonic beams. These
parametters were esstimated fromm the stresss-strain
relationships obtaineed from subbject A (thee firstmeasureement). The trransient changge in static elaasticity
Es was similar to thaat in a differeent elastic paraameter
reportedd in the literrature [5]. Thhe minimum static
elasticitty Es was meaasured at 35 s after the releease of
the cuff
ff. The maxim
mum percentagge change in static
elasticitty Es was abouut 57% (770 kkPa). Moreover, theviscosity η, which waas evaluated nnoninvasively by the
proposeed method, inncreased afterr recirculation
n. Themeasureed viscosity was in the ssame range asa theviscosity at the carotiid artery reporrted in the liteerature(in vitroo measurement) [28]. The m
maximum visco
osity ηat 35 s after recirculaation was aboout 21 kPa·s, which
was aboout 94% (10 kPPa·s) larger thhan the mean at
a rest.

Fig. 8. Time sequennce of transientt change in thee blood velocitty (a) and stresss–strain hysteeresis (b).

temporaal decrease in elasticity Es and the increease inviscosity η were obseerved also in annother subject.

0. Transient change in mmeans and SD
Ds ofFig. 10
estimateed static elastiicity Es and viiscosity η measured
in anothher subject (subbject B).

Fig
g. 9. Transient change in meaans and SDs ofo estimatedstattic elasticity Es and viscositty η measured
d in subject
A: (a) first, (b) seecond, and (c) third measureements.
FiguresF
9(b) anda 9(c) show the transient changes inthee means and SDs of the static elasticiity Es andviscosity η meassured in the samme subject onn other days
(thee second and thirdtmeasuremments in subjeect A). The
maaximum perceentage changee in static ellasticity Es
obsserved at 11 s after recirculaation was abou
ut 36% (360kPaa) in the second measuremeent, and that at
a 7 s after
reccirculation waas about 50% (490 kPa) inn the third
meeasurement. Thhe maximum inncrease in visccosity at 35s afterarecirculattion was about 73% (11 kPPa·s) in theseccond measurem
ment and that aat 73 s after reecirculation
was about 240%
% (23 kPa·s) inn the third meeasurementa rest.
from the means at
FigureF
10 shoows the transieent change in the means
andd SDs of thee static elasticcity Es and viscosityvη
meeasured in anotther subject (suubject B). Thee maximum
perrcentage changge in static ellasticity Es at 19 s after
reccirculation wass about 55% (5570 kPa). Thee maximum
inccrease in viscoosity η at 44 s after recircu
ulation wasaboout 571% (233 kPa·s) fromm the mean at
a rest. The

Fig. 11. Means and SDs of statiic elasticity Es andviscosity η for 10 minn in vivo measuurements.

Table I. Means and SDs of param
meters for 10 min
m invivo meeasurements.
For thet evaluationn of reproduciibility, RF data andthe bloood pressure waveformw
at rrest were measured
every 1 min for 10 min.
m Figure 111 shows the means
and SD
Ds of the measured staticc elasticity Es andviscosity η. The SDs were obtainedd from 30 ultrrasonicbeams. Horizontal daashed lines shoow the means of theparametters.
Figurre 11 showss the means and SDs ofo theparametters. Table I showss
the meeans and SDs of theparrameters averaaged for 10 minn. For the statiic elasticity
Es, the maximum
m difference frrom the meann was about
200
0 kPa (17% of mean). Thhe maximum change instattic elasticity due
d to FMD ((770, 360, 490, and 570
kPaa) was much largerlthan thiss value. For th
he viscosity
η, the variation in mean was 5 kPa·s. Thee maximum
MD (10, 11, 23,
2 and 23chaange in viscossity due to FM
kPaa·s) was also much larger thhan this valuee. However,thee patterns of thhe changes in tthe static elastiicity Es andviscosity η aftter recirculatiion, and the time forobsserving the maximalmchangges in these parameters
were different ammong these meeasurements.

larger scatterer is smallersthan that of a smallersscatterer. This resullt shows thatt the slope ofo thenormaliized logarithm
mic power specctrum log10Ps(ff)/Pr(f)is relateed to the diameeter of the scattterer.

3.2
2 Evaluationn of red bloodd cell aggreg
gation3.2
2.1 Basic exp
periments using microspheres
TheT ultrasound diagnostic equipment (TTomey UD10000) employed is equipped with a mechaanical scanproobe at a center frequency f0 oof 40MHz (waavelength isaboout 40 μm). RF
R echoes were acquired at a sampling
freq
quency of 1 GHz at a 116-bit resolutiion by theosccilloscope (Teektronix DPO
O7054), and th
heir powerspeectra {P(f)} weere obtained bby Fourier tran
nsform witha HanningHwindoow of 1.024 μ
μs length. To reduce theeffefect of randomm noise, 10000 power spectra wereaveeraged. The leeft-hand colum
mn of Table III shows thedifffferent diameeters of mmeasured miccrospheres.
Microspheres (1)) and (3) were made of copo
olymer with
Cl2, and microsspheres (2) aand (4) weree made ofcoppolymer withoout Cl2. Micrrospheres withh different
diaameters, whicch were mixxed with water
w
at a
con
ncentration of 3.00 g/l, simuulated nonaggrregated andagg
gregated RBCss. This concenntration is loweer than thatof RBCsR
in actuaal blood, preveenting the agg
gregation of
miccrospheres. RF
F echoes fromm the spheres around thefoccal point werre acquired ffrom spatiallyy different
reggions by mechanical linnear scan as
a in themeeasurement off a silicone plate to acq
quire many
stattistically indeependent dataa points. Effeects of thedifffference
bettween
acouustic
impedaancesof
miccrospheres annd RBCs weree not consideered in thepreesent study, although suuch effects should beinvvestigated in fuuture studies.

Fig. 12.. (a) Averagedd power specttra of the ultrrasonicechoes scattered fromm microspherres of variouss sizesand (b)) normalized power spectraa and the weeighted
least-meean-squared regressionrlines of microspheres(1)–(4) in Table II.

Fig. 13
3. Mean and the standardd deviation ofo theestimateed spectral sllopes as a fuunction of sccatterer
diameteer.

3.2.2 Inn vivo experiiments
Tab
ble II. Microosphere diammeters and ex
xperimental
resu
ults.
FigureF12(a) showssthe aveeraged power spectra ofechhoes from thee microspheress, and Fig. 122(b) showsthee normalized power spectra. The numbers correspondto the microspheere numbers iin Table II. The
T spectralslop
pe and interceept values are shown in the right-handcollumn of Tablee II. Figure 113 shows the mean andstanndard deviattion of speectral slopes for ten
meeasurements ofo each microosphere. In Fiig. 12, theslop
pe of the norm
malized powerr spectrum Ps(ff)/Pr(f) of a

The mainmcomponnents of normaal blood are RBCs,
R
which dod not aggregaate in large veiins because off bloodflow [229,30]. To measurem
ultrasonic echoes fromaggregaated RBCs in addition to those fromnonaggrregated RBCss, blood flow
w was stopped bypressuriizing a cuff suurrounding thhe upper arm at 250
mmHg. Ultrasonic ecchoes were accquired at restt for 2
min, duuring avascullarization for 5 min, andd afterrecirculation for 3 min. Figure 14 shows B-modeBimages of a vein at thhe dorsum mannus of a 24-yeear-old
healthy male. B-modee images were measured at restr (1),
at the beginningbof avascularizatioaon (2), at the end of

avaascularization (3), at the beeginning of reecirculation(4), and 120 s afterarecirculattion (5). It is possible toredduce the attennuation of higgh frequency ultrasoundbeccause the veinn at the dorsumm manus is a superficial
blo
ood vessel. Thhe vein measuured in this sttudy had alarg
ge diameter annd a high steaddy blood flow rate, which
affefect RBC agggregation, and thus, the meeasurementcou
uld be assumeed to be perforrmed with resspect to thenonnaggregated RBCs
R
at rest. IIn addition, wee measured
thee change in sppectral slope ccaused by thee change inblo
ood flow rate dued to avasculaarization usingg a cuff. RF
echhoes from thee focal point positioned in the vessellum
men were acquuired from maany different positions
pbymeechanical lineaar scan to reeduce the efffect of theinccrease in scatterer diameeter variationn due toagg
gregation.

dB at 35 MHz. Accordingly,A
the spectral slope
decreasees by 1.37 in thet frequency bband of 25-35 MHz.
Therefoore, the measuured spectral sslope of RBC
Cs wasshown tot be decreasedd.

pspectra and the least--meanFig. 15. Normalized powersquaredd regression linnes.

6. Transient changecin thee estimated sp
pectralFig. 16
slope duuring avasculaarization for 5 m
min.
Fig
g. 14. B-mode images and w
windows of the vein at thedorrsum manus: (1) at rest, ((2) at the beeginning ofavaascularization, (3) at the endd of avascularrization, (4)at thet beginningg of recirculattion, and (5) 120 s after
reccirculation.
FigureF
15 shoows the normaalized logarith
hmic powerspeectrum log10Ps(f)/Pr(f) obtaiined from thee lumen ofthee vein at thee dorsum maanus of the 24-year-old
2
heaalthy male annd the weighhted least-meean-squaredreggression line. FigureF
16 show
ws the transien
nt change inestiimated spectraal slope due to avascularizzation. The
slop
pe of the noormalized pow
wer spectrum of echoesfrom RBCs at rest was close too that of microosphere (3)(diaameter: 15 μmm) in Table II.. The RBC diaameter is 8mm
m at most annd this diametter is between
n those of
miccrospheres (1)) (diameter: 5 μm) and (2) (diameter:
11 μm). The spectral slope mmeasured for RBCs was
slig
ghtly greater thantthat of a sphere whosse diameter
corrresponds to thhat of an RBC
C. One of the reasons forthiss was considdered to be thhe frequency dependent
atteenuation of ultrasound during propaagation inbio
ological tissuess. If the attenuuation coefficient and thedep
pth to vein aree assumed to bbe 1 dB·MHz-1·cm-1 and
1 mm, respectiively, the diifferences bettween two
log
garithmic norm
malized poweer spectra log10Ps(f)/Pr(f)witth and withoutt attenuation arre 5 dB at 25 MHz
M and 7Furthhermore, the spectral slope gradually deccreased
during avascularizatioon and returnned to the levvels ofrest in th
he in vivo meaasurement. Altthough there area stillmany factorsfto bee investigated, such as accoustic
impedanncesofmethods
microspheress,fornormaliization, and caalibration of thhe system [30], theseresults showedsthat thhe slope of thhe normalized power
spectrumm changes witth scatterer diaameter.

5. Conclusion
In this study, methodsm
for measuremen
nt ofviscoelaasticity of arterial wall andd evaluation of red
blood cell aggregatioon were develooped. Such methods
would beb useful for eaarly diagnosis of atherosclerrosis.

Acknow
wledgments
ould like to thhank Tomey Corporation for its
We wo
cooperaation on the usee of the diagnoostic equipmentt.

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