twelve adult beagle dogs , 110 years old and weighing 9.515.8 kg , were used in this study . 

dogs were divided into a conscious group ( n=6 ) and a sedated group ( n=6 ) .
all dogs were 
 healthy based on physical examination and normal cbc and serum biochemistry including 
 alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase , 
 gamma - glutamyltranspeptidase , ammonia , and fasting and post - prandial total bile acid levels . 

prior to the ceus study , b - mode us was performed on all the dogs , and no focal or diffuse 
 hepatic abnormalities were noted .
all b - mode us and ceus examinations were performed by the 
 same sonographer ( km ) with 10 years of experience in performing liver ultrasound .
an ultrasound scanner ( aplio xg , toshiba medical systems , otawara , japan ) with a 511 mhz 
 broadband linear probe ( plt-704 at , toshiba medical systems ) suitable for pulse subtracting 
 imaging was used for ceus .
imaging was performed with a low mechanical index of 0.21 and a 
 frame rate of 23 frames per sec .
the contrast imaging gain was set at 80 db , and the focus 
 was set at a depth of 4 cm .
scanning in the 
 sedated group was performed under anesthesia with propofol at an induction dosage of 6 mg / kg 
 and a maintenance rate of 0.40.6 mg / kg / min .
the 
 dogs were positioned in left lateral recumbency , and the right hv was identified using an 
 intercostal approach ( fig .
1.color doppler and ceus images of the right hv obtained using a right intercostal 
 approach .
( a ) right hv displayed in blue with color doppler flowed into the caudal 
 vena cava ( cvc ) .
( b ) at 8 sec , the hv , outlined by a dashed line , was not enhanced , although 
 surrounding liver parenchyma was slightly enhanced .
region of interest ( roi ) was 
 manually placed in the hv ( circle ) to measure the tissue intensity .
( c ) hv reached its 
 pi , which was similar in intensity to the liver parenchyma ( shown here 23 sec after 
 bolus injection of contrast agent ) .
( d ) at the end of the examination ( 120 sec ) , the 
 contrast agent in the hv appeared washed - out and hypoechoic compared to the liver 
 parenchyma . ) .
the right hv was imaged to maintain clear visualization of the confluence with the 
 caudal vena cava ( cvc ) as much as possible .
perfusion of the hv was evaluated after 
 intravenous bolus injection of microbubble contrast agent ( sonazoid , daiichi 
 sankyo , tokyo , japan ) . according to our previous report , we administered 0.01 ml / kg sonazoid through a 21 
 g butterfly catheter attached to a 22 g intravenous catheter placed in the cephalic vein , 
 flushed by 2 ml of heparinized saline .
immediately after bolus injection , 
 continuous scanning of the right hv was performed for 2 min .
the images were recorded in 
 40-sec cine - loops to a hard disk for further off - line analysis .
ceus examinations were 
 performed three times in each dog by using the same scanning plan , with a period of more 
 than 48-hr between examinations .
color doppler and ceus images of the right hv obtained using a right intercostal 
 approach .
( a ) right hv displayed in blue with color doppler flowed into the caudal 
 vena cava ( cvc ) .
( b ) at 8 sec , the hv , outlined by a dashed line , was not enhanced , although 
 surrounding liver parenchyma was slightly enhanced .
region of interest ( roi ) was 
 manually placed in the hv ( circle ) to measure the tissue intensity .
( c ) hv reached its 
 pi , which was similar in intensity to the liver parenchyma ( shown here 23 sec after 
 bolus injection of contrast agent ) .
( d ) at the end of the examination ( 120 sec ) , the 
 contrast agent in the hv appeared washed - out and hypoechoic compared to the liver 
 parenchyma .
two operators ( km and ah ) performed the quantitative analysis of the ceus images by using 
 an off - line image analysis system ( imagej , us national institutes of health , bethesda , md , 
 u.s.a . ) . this system measures intensity using a gray - scale level ranging from 0 to 255 mean 
 pixel value ( mpv ) .
one image per sec for the first 60 sec followed by 1 image at an interval 
 of every 5 sec until 120 sec from the start of microbubble contrast agent infusion was 
 analyzed .
the region of interest ( roi ) was drawn in the right hv within a 1.5-cm distance 
 from the confluence with the cvc as large as possible without including adjacent structures 
 ( fig .
hepatic vein arrival time ( hvat ) was the 
 time when the tic first reached 20% of peak intensity ( pi ) .
time to peak ( ttp ) and 
 time to peak phase ( ttpp ) were defined as the time when tic reached to pi and to 90% 
 pi , respectively .
wash - out ratio ( wr ) indicated the attenuation rate from pi to the 
 end of the study . ) . the hepatic vein arrival time ( hvat )
was the time from contrast agent injection to 
 20% of peak intensity ( pi ) .
time to peak ( ttp ) was the time taken from 20% of pi to pi .
time 
 to peak phase ( ttpp ) was the time taken from 20% to 90% of pi , which reflects the initial 
 upslope of tic better than ttp .
wash - out ratio ( wr ) was defined as the attenuation rate from 
 pi to the intensity at the end of a ceus study .
hepatic vein arrival time ( hvat ) was the 
 time when the tic first reached 20% of peak intensity ( pi ) .
time to peak ( ttp ) and 
 time to peak phase ( ttpp ) were defined as the time when tic reached to pi and to 90% 
 pi , respectively .
wash - out ratio ( wr ) indicated the attenuation rate from pi to the 
 end of the study .
all data were expressed as the median and range . statistical comparisons and calculations 
 of coefficient of variation ( cv ) of each parameter
when distribution approached normality , measured parameters 
 from the conscious and sedated groups were compared using student s t - test . 

the examinations were successfully performed , and the tic of the hv could be measured 
 clearly in both groups .
no adverse events were noted during or after injection of the 
 microbubble contrast agent .
after contrast agent administration , the microbubbles first reached the ha and then the pv . 

the contrast 
 effect of the hv developed more gradually than that of the pv ; it took over 10 sec to reach 
 pi . during the pi phase ,
1c ) and was followed by a gradual wash - out of the 
 contrast agent with gradual loss of enhancement ( fig . 

the intensity of the contrast agent in the hv dropped to almost 20% of pi at 
 the end of the examination , while the contrast agent in the pv retained its intensity .
the tics derived from the median pixel intensity in the hv were similar in the two groups , 
 but pi was higher in the conscious group ( fig . 

the line 
 graphs in the two groups are nearly identical , but the peak intensity was higher in 
 the conscious group . and 3b ) .
the line 
 graphs in the two groups are nearly identical , but the peak intensity was higher in 
 the conscious group .
the measured parameters and cvs for each variable are summarized in table 1table 1.median values and cvs of the characteristic parameters of the time intensity 
 curve in six healthy dogsconscious groupsedated groupp - valuemedianrangecv ( % ) medianrangecv ( % ) hvat ( sec)13.5(922)25.312(817)11.80.242ttp ( sec)12.5(624)29.712.5(917)14.80.854ttpp ( sec)8(613)27.19(713)12.40.377wr ( % ) 78.0(60.791.7)7.684.1(63.094.4)12.30.689 .
cvs 
 were < 20% ( range , 11.8 to 14.8% ) for all parameters in the sedated group . on the other 
 hand ,
cvs for hvat , ttp and ttpp in the conscious group were > 20% ( range , 25.3 to 29.7% ) 
 and were higher than sedated group with the exception of wr ( 7.6% ) .
our goal was to develop a new , non - invasive method that could assess intrahepatic 
 hemodynamic changes related to underlying progressive hepatic disease . in this study , we 
 characterized contrast - enhanced ultrasonography findings of the hv in normal dogs and 
 evaluated the repeatability of this examination .
we chose the right hv for analysis , because 
 it can be imaged constantly while using an intercostal approach without compressing the 
 upper abdomen which can affect the hepatic hemodynamics .
there was a delay of several 
 seconds between these two vessels , and the tic of the hv rose gradually .
this delayed and 
 gradual enhancement of the hv was similar to that observed in the previous human study 
 [ 1 , 10 , 19 ] .
because the ha carries a small amount of 
 microbubbles , the hv is enhanced only after the microbubbles reach the sinusoid from the pv 
 ( which has more blood flow than the ha ) .
since the sonazoid used in this study 
 is phagocytosed by kupffer cells when it passes through the sinusoid , it takes longer for 
 the hv to reach the pi , due to the escape of microbubbles via phagocytosis . after the pi
, 
 there is a gradual wash - out of the contrast agent in the hv , while the contrast effect in 
 the pv is still present at the end of the examination . the number of microbubbles that 
 re - enter the sinusoid through the general circulation decreases as time goes on due to 
 phagocytosis by kupffer cells , which could contribute to the gradual loss of enhancement of 
 the hv .
we established reference values both in conscious and sedated dogs , and also evaluated the 
 repeatability of this examination .
although there was no statistically significant 
 difference in each parameter between the two groups , median value of hvat was slightly 
 longer in the conscious group .
reported that the time to peak 
 enhancement of the hepatic parenchyma , calculated from the time of injection , was 
 significantly shorter in dogs anesthetized with propofol , as compared to that in 
 non - anesthetized dogs .
propofol has been found 
 to increase hepatic arterial blood flow despite having no effect on portal venous flow 
 , and the authors speculated that shortened 
 time to peak enhancement was related to the effect of propofol on the vascular system . 

although this was observed in the hepatic parenchyma , it is possible that using propofol 
 also affected the hvat results in the current study .
however , statistical differences were 
 not detected between the two groups , possibly because of the relatively low repeatability in 
 the conscious group ( cv ; 25.3% ) .
obtaining stable images was slightly difficult in the 
 conscious group compared with the sedated group , and this could have led to the low 
 repeatability .
in addition , changes in cardiac output and blood pressure related to the 
 dog s excitation level may have affected the hepatic circulation . recent research demonstrated shortening of the hvat with the development of liver fibrosis 
 in a ccl4-induced canine liver fibrosis model . however , the hvat was much shorter in the current study compared with that of 
 the baseline value ( 18.22  0.82 sec ) reported in the ccl4-induced canine liver 
 fibrosis model .
we speculate that the difference 
 between our value and the previously reported value is due to differences in contrast agent , 
 the volume of saline flush and/or how rapidly it was administered , and the method used for 
 quantitative analysis .
therefore , the reference values should be applied cautiously , and the 
 methods used to obtain these values should be considered .
ttp and ttpp , which also showed low repeatability in conscious dogs , were not different 
 between the two groups .
even if propofol increases arterial blood flow , the main blood 
 supply associated with the initial upslope is presumably portal blood flow .
in addition , 
 because these parameters reflect only the intrahepatic circulation , they may be less 
 vulnerable than the hvat to extra hepatic factors .
although these parameters are not as 
 common as hvat in human medicine , sugimoto et al . reported that the hv 
 rising rates in cirrhotic patients were significantly higher than those in the control group 
 and in non - cirrhotic patients .
therefore , the 
 ttp and ttpp , as a reflection of the hv rising rates , could be useful to assess the 
 arterialization of the hv in dogs .
in contrast to the hvat , ttp and ttpp , wr showed favorable repeatability in both groups .
wr 
 can be measured with only two values ( the peak intensity and the intensity at the end of the 
 examination ) , and this simple calculation could contribute to good repeatability .
wr may 
 also be less affected by systemic hemodynamic changes , because it is not a time - related 
 parameter , which might have contributed to the lack of difference in this parameter between 
 the two groups . to the author s knowledge
, wr of the hv has never been measured in human studies . in our 
 preliminary research ,
although multiple factors could affect wr , reduced kupffer cell phagocytic function may be 
 one of the most conceivable reasons .
it was reported that nonalcoholic steatohepatitis in a 
 rat model showed reduced kupffer cell phagocytic function , with no changes in the numbers of 
 kupffer cells .
more recently , park et 
 al . reported that elimination of sonazoid from the hepatic parenchyma 
 became slower with progression of cirrhotic disease .
the application of ceus to assess hemodynamic changes , as well as liver 
 function , should be investigated in the future .
quantitative assessment of portal pressure by using ceus was performed in a 
 ccl4-induced canine liver fibrosis model . in that study ,
the roi was set on the hepatic parenchyma , and modified 
 parameters based on the area under the curve of the tic were generated . compared with this 
 previous report , the perfusion parameters utilized in the current study can be measured more 
 simply . in addition , the ability of ceus of the hv to detect intrahepatic shunt flow that 
 directly bypassed the sinusoid may be superior to that of ceus of the liver parenchyma . on 
 the other hand , because a large roi can not be drawn on the hv , the repeatability of ceus of 
 the hv might be inferior to that of parenchyma - targeted ceus analysis .
this could be a major 
 limitation of the current method , especially if adequate imaging of the hv can not be 
 maintained .
second , the dogs enrolled in this research were all beagles , and therefore , 
 we did not evaluate the influence of body size on hepatic hemodynamics .
the differences in 
 body size may affect the repeatability of each measured parameter , because roi depends on 
 the diameter of the hv , which is associated with the dog s size .
finally , we used sonazoid , 
 because it is the only second - generation contrast agent available in japan . however , other 
 vascular - specific contrast agent might be better for assessing time - related parameters , 
 because they would purely reflect hemodynamic changes related to liver disease . in conclusion , this research characterized image enhancement of the normal canine hv using 
 sonazoid . established quantitative parameters may serve as a reference in the 
 future assessment of liver function as related to hemodynamics .
a further study into the 
 application of this technique to evaluate intrahepatic hemodynamic changes associated with 
 canine chronic hepatic disease is warranted .
 contrast - enhanced ultrasonography ( ceus ) of the hepatic vein can assess intrahepatic 
 hemodynamic changes and has been studied as a noninvasive method to assess the severity of 
 portal hypertension and hepatic fibrosis in humans . 
 however , few reports have described 
 its usefulness in veterinary medicine . the purpose of this study was to characterize ceus 
 findings of the hepatic vein in normal dogs and assess the repeatability of this method 
 both in a conscious group ( n=6 ) and a sedated group ( n=6 ) . 
 sonazoid ( 0.01 
 ml / kg ) was used as a contrast agent , and scanning of the hepatic vein 
 was performed for 2 min . 
 time - intensity curves were generated from regions of interest in 
 the hepatic vein . 
 four perfusion parameters were measured for quantitative analysis : 
 hepatic vein arrival time ( hvat ) , time to peak ( ttp ) , time to peak phase ( ttpp ) and 
 wash - out ratio ( wr ) . 
 ceus examinations were performed three times in each dog . the median 
 ( range ) 
 values of hvat , ttp , ttpp and wr in the conscious group were 13.5 sec ( 922 sec ) , 
 12.5 sec ( 624 sec ) , 8 sec ( 613 sec ) and 78.0% ( 60.791.7% ) , respectively . 
 median ( range ) 
 values of hvat , ttp , ttpp and wr in the sedated group were 12 sec ( 817 sec ) , 12.5 sec 
 ( 917 sec ) , 9 sec ( 713 sec ) and 84.1% ( 63.094.4% ) , respectively . 
 the coefficients of 
 variation of these parameters in the conscious and sedated groups were 7.629.7% and 
 11.814.8% , respectively . 
MATERIALS AND METHODS
RESULTS
DISCUSSION