Portal hypertension is a severe complication of chronic liver disease. Clinical consequences include development of MAPSS, ascites, hepatic encephalopathy, or a combination of these, which cause substantial morbidity and fatalities.1 Therefore, assessment of portal pressure before the onset of such late-stage clinical conditions provides valuable information to improve the prognosis. However, direct or indirect measurement of portal pressure is seldom performed in veterinary medicine because of its invasiveness. Therefore, an alternative noninvasive method is needed.
Conventional B-mode ultrasonography and Doppler ultrasonography are useful imaging modalities for screening to detect portal hypertension. Several findings, including ascites or MAPSS, enlargement of the portal vein, decreased velocity of portal blood flow, and a dilated left gonadal vein, can indicate the presence of portal hypertension.2–4 However, because most of these variables have been evaluated in retrospective analyses, their correlation with portal pressure was not determined.
Contrast-enhanced ultrasonography is a candidate for the assessment of portal hypertension. There are several intrahepatic and extrahepatic hemodynamic changes in patients with cirrhosis, such as arterialization of the liver, intrahepatic shunts, pulmonary arteriovenous shunts, and a hyperdynamic circulatory state.5–8 These hemodynamic changes contribute to the early HVAT measured by use of CEUS, and it was reported9,10 that HVAT is negatively correlated with portal pressure. Thus, CEUS has been evaluated as an alternative noninvasive method for assessing the severity of portal hypertension in humans.
In veterinary medicine, CEUS is used mainly to characterize the vascularity of focal liver lesions,11,12 but CEUS also has been used on other organs such as the spleen,13 pancreas,14 and kidneys.15 However, with regard to the assessment of portal pressure with CEUS, the authors are aware of only 1 report16 in which CEUS was used to evaluate the intensity of the hepatic parenchyma in dogs with experimentally induced liver fibrosis. Therefore, fundamental data are lacking before CEUS can be used clinically.
The objectives of the study reported here were to determine the feasibility for the use of CEUS to detect hemodynamic changes associated with portal hypertension in dogs with experimentally induced portal hypertension and to evaluate the correlation between CEUS variables derived for the hepatic vein and portal pressure.
Materials and Methods
Animals
Six adult Beagles (3 males and 3 females) that were part of a research colony owned by our laboratory were used in this prospective study. Age of dogs ranged from 1 to 3 years, and body weight ranged from 9.2 to 12.0 kg. All dogs were considered healthy on the basis of results of a baseline physical examination, CBC, and serum biochemical analysis that included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyltransferase activities and total protein, albumin, total bilirubin, ammonia, and fasting and postprandial TBA concentrations. In addition, B-mode ultrasonography was performed on all dogs, and no focal or diffuse hepatic abnormalities were detected. All procedures were approved by the Hokkaido University Animal Care and Use Committee.
Establishment of portal hypertension
Food was withheld from each dog for 12 hours. Dogs then were premedicated by IV administration of midazolam (0.1 mg/kg) and butorphanol tartrate (0.05 mg/kg). Anesthesia was induced with propofol (6 mg/kg, IV) and maintained with 1.5% isoflurane in oxygen. A midline abdominal incision was made, and the right pancreatic lobe was identified. An implantable port devicea was surgically inserted in the portal vein, and the catheter tip of the device was inserted into the main trunk of the portal vein via the pancreaticoduodenal vein. The port was affixed to the subcutaneous tissues of the right abdominal wall. After surgery, buprenorphine (0.01 mg/kg, IM, q 12 h) was used as needed for analgesia, and cephalexin (20 mg/kg, PO, q 12 h for 3 days) was administered to prevent secondary bacterial infection. A liver specimen was obtained during the catheterization procedure and used for histologic examination.
One week after the catheter was surgically placed, intraportal injections (as described elsewhere17) were initiated. Dogs were anesthetized with propofol (induction dose, 6 mg/kg; maintenance rate, 0.4 to 0.6 mg/kg/min18) for each intraportal injection. Microspheresb (10 mg/kg) were administered at 5-day intervals to the first 2 dogs. However, portal hypertension was not adequately induced after 6 injections. Thereafter, the dose was increased to 15 mg/kg, which led to a gradual increase in portal pressure. The remaining 4 dogs were administered all injections of microspheres at a dose of 15 mg/kg. Injections of microspheres were continued until portal hypertension was successfully established. Establishment of portal hypertension was confirmed by formation of MAPSS as observed by use of B-mode ultrasonography and CT.
The PVP was determined by measuring intraport pressure with a disposable blood pressure–monitoring kitc in accordance with the manufacturer's directions. Briefly, anesthetized dogs were placed in dorsal recumbency, and a 20-gauge coreless needled connected to the transducer was inserted into the port. A PVP measurement was obtained immediately before each microsphere injection. The PVP obtained before the first microsphere injection was defined as the baseline value.
Computed tomography was performed before the first microsphere injection (defined as the baseline value) and was repeated at 30-day intervals or after MAPSS formation was suspected on the basis of ultrasonographic evaluation. Anesthetized dogs were positioned in dorsal recumbency as described previously. For CT angiography, iodinated contrast medium at a dose of 600 mg of I/kg was injected IV over a 30-second period, and the entire abdominal cavity was scanned with a 16-slice helical CT scanner.e
After establishment of MAPSS was confirmed via CT, dogs were euthanized (anesthetized with thiopental and then administered an IV injection of potassium chloride). Necropsy was performed to enable investigators to examine gross changes of intra-abdominal organs and to collect liver and lung specimens. Specimens were fixed in neutral-buffered 10% formalin, embedded in paraffin, stained with H&E stain, and examined by use of light microscopy.
CEUS
Contrast-enhanced ultrasonography (baseline value) was performed before the first microsphere injection and after MAPSS was confirmed via CT. An ultrasound scannerf with a 5- to 11-MHz broadband linear probeg suitable for pulse subtraction imaging was used for CEUS. Imaging was performed with a mechanical index of 0.21 and frame rate of 23 frames/s. Contrast imaging gain was set at 80 dB, and focus was set at a depth of 4 cm. Scanning was performed on dogs anesthetized by administration of propofol in the same manner as for microsphere injection. Anesthetized dogs were positioned in left lateral recumbency, and the right hepatic vein was identified by use of an intercostal approach. A bolus of microbubble contrast agenth (0.01 mL/kg) was administered IV through a 21-gauge butterfly catheter attached to a 22-gauge catheter that had been inserted in a cephalic vein, which was followed by flushing with 2 mL of heparinized saline (0.9% NaCl) solution. Perfusion of the hepatic vein was evaluated by scanning the hepatic vein for 2 minutes; images were recorded in 40-second cine loops to a hard disk for further off-line analysis.
Quantitative analysis of CEUS images was performed with an image analysis system.i The system measured intensity by use of a gray scale ranging from 0 to 255 mean pixel value. Images (1 image/s for the first 60 seconds followed by 1 image at 5-second intervals until 120 seconds after starting the infusion of microbubble contrast agent) were analyzed. A region of interest was drawn in the hepatic vein; the region was as large as possible without including adjacent structures. A time-intensity curve depicting tissue intensity over time was generated for each injection. Four perfusion variables were measured for each time-intensity curve. The HVAT was the interval from contrast agent injection to 20% of peak intensity. The TTP was the interval from 20% of peak intensity to peak intensity. The TTPP was the interval from 20% to 90% of peak intensity. Washout ratio was defined as the attenuation rate from peak intensity to the intensity at 2 minutes after contrast agent injection.
Statistical analysis
Statistical analysis was performed with commercially available software.j Normal distribution of data was confirmed by use of the Shapiro-Wilk test. Body weight, blood biochemical variables, PVP, diameter of the left gonadal vein measured by CT, and 4 CEUS variables were compared before and after induction of portal hypertension by use of a paired t test for parametric data and Wilcoxon rank sum test for nonparametric data. Significance was set at values of P < 0.05. For blood biochemical variables and CEUS variables, simple regression analyses were performed to investigate correlations with PVP.
Results
Establishment of portal hypertension
Dogs remained healthy throughout the experimental period. Portal hypertension was induced successfully in all dogs. Median total dose of microspheres was 170 mg/kg (range, 105 to 285 mg/kg). Body weight at the time of portal hypertension was not significantly different from baseline values. Two dogs had bacterial infection in the subcutaneous tissues around the port device, which resolved after administration of a course of cephalexin.
Results for blood biochemical analyses were summarized (Table 1). Alanine aminotransferase, alkaline phosphatase, and γ-glutamyltransferase activities and total bilirubin concentration increased significantly, compared with baseline values. Significant increases in both fasting and postprandial TBA concentrations were also detected, whereas there were no significant changes in ammonia concentrations.
Median (range) values for blood biochemical variables before and after induction of portal hypertension in 6 dogs.
| Variable | Before induction | After induction | P value* |
|---|---|---|---|
| Total protein (g/dL) | 5.7 (5.2 to 6.2) | 6.0 (5.7 to 7.2) | 0.094 |
| Albumin (g/dL) | 3.1 (2.6 to 3.2) | 2.8 (2.5 to 3.1) | 0.210 |
| ALT (U/L) | 34 (19 to 68) | 223 (54 to 488) | 0.014 |
| AST (U/L) | 36 (24 to 44) | 40 (23 to 86) | 0.147 |
| ALP (U/L) | 149 (78 to 381) | 602 (435 to 838) | 0.004 |
| GGT (U/L) | 5.5 (1.3 to 8.0) | 8.0 (4.0 to 10.0) | 0.045 |
| Total bilirubin (mg/dL) | 0.1 (0.1 to 0.2) | 0.3 (0.1 to 0.3) | 0.020 |
| Ammonia (μmol/dL) | 39 (38 to 45) | 60 (18 to 93) | 0.199 |
| Fasting TBA (μmol/dL) | 2.3 (1.0 to 11.8) | 17.4 (12.5 to 98.8) | 0.008 |
| Postprandial TBA (μmol/dL) | 8.4 (2.9 to 16.0) | 107.7 (73.5 to 186.2) | < 0.001 |
Values were considered significant at P < 0.05.
ALP = Alkaline phosphatase. ALT = Alanine aminotransferase. AST = Aspartate aminotransferase. GGT = γ-Glutamyltransferase.
The PVP after induction of portal hypertension could not be measured accurately in 2 dogs because of thrombi formation immediately cranial to the catheter tips. These dogs were excluded from the analysis of PVP after portal hypertension. Median baseline PVP was 6.5 mm Hg (range, 2 to 8 mm Hg [n = 6]), which increased significantly to 12.5 mm Hg (range, 9 to 15 mm Hg [4]) after MAPSS formation.
Small amounts of ascitic fluid were detected ultrasonographically in all dogs. Imaging with CT revealed multiple small tortuous vessels located caudal to the left kidney. Anastomoses between the left colic vein and left gonadal vein were visible in 4 dogs. In addition to this collateral circulation, a left splenogonadal shunt was present in 2 dogs. Median diameter of the left gonadal vein after induction of portal hypertension was 3.6 mm (range, 3.0 to 4.2 mm), which was significantly (P = 0.01) larger than the baseline value (1.9 mm; range, 1.5 to 2.0 mm).
Necropsy revealed multiple small tortuous collateral vessels in the left perirenal area in all dogs (Figure 1). These vessels were consistent with CT findings. Portal veins were markedly enlarged and tortuous. The liver had no apparent abnormalities with a smooth serosal surface. Small amounts of ascitic fluid were classified as transudates in all dogs. Histologic examination of the liver specimens revealed only slight atrophy and vacuolar degeneration of hepatocytes. The interlobular veins were dilated with microspheres. Proliferations of abundant epithelioid cells, macrophages, and multinucleated giant cells surrounding the microspheres were evident. Aggregation of macrophages was detected in the interlobular connective tissue, especially around the interlobular veins. Moreover, microspheres were also present in the pulmonary arteries; these microspheres were surrounded by a foreign body granuloma.
Photograph of the left perirenal area of a dog with experimentally induced portal hypertension. Notice the multiple small tortuous collateral vessels (arrows).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.465
CEUS findings
Contrast-enhanced ultrasonography performed before microsphere infusion revealed that the hepatic artery was enhanced first, followed by the portal vein and then the hepatic vein. The contrast effect of the hepatic vein was gradual until it reached peak intensity, whereas the hepatic artery and portal vein had a rapid increase in intensity. During the peak intensity phase, the hepatic vein could not be seen clearly because it was isoechoic with the liver parenchyma. Thereafter, there was gradual washout of the contrast agent with gradual loss of enhancement. At the end of the examination, the hepatic vein was clearly visible because its echo intensity decreased in contrast to that of the liver parenchyma, which gained contrast effect.
The enhancement pattern of the hepatic vein changed after the induction of portal hypertension. Subjectively, the hepatic vein had a rapid increase in echogenicity, which was represented as a rapid increase in the time-intensity curve (Figure 2). Results for each variable were summarized (Table 2). Among the 4 CEUS variables, TTP and TTPP were significantly shorter after induction of portal hypertension, which corresponded with the visual observations.
Time-intensity curve of the pixel intensity in the hepatic vein for CEUS before (A) and after (B) induction of portal hypertension in 6 dogs. Values reported are median and range. Notice that the initial increase in the time-intensity curve is steeper after induction of portal hypertension than before induction. MPV = Mean pixel value.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.465
Graph of the relationship between TTPP and PVP obtained for dogs before (circles; n = 6) and after (crosses; 4) induction of portal hypertension. The PVP could not be measured accurately in 2 dogs after induction of portal hypertension. Notice that there was a significant negative correlation between TTPP and PVP.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.465
Mean ± SD values for CEUS perfusion variables before and after induction of portal hypertension in 6 dogs.
| Variable | Before induction | After induction | P value* |
|---|---|---|---|
| HVAT (s) | 11.2 ± 1.9 | 11.7 ± 2.8 | 0.386 |
| TTP (s) | 15.0 ± 1.8 | 9.3 ± 4.9 | 0.008 |
| TTPP (s) | 10.8 ± 1.8 | 5.5 ± 3.8 | 0.003 |
| Washout ratio (s) | 79.5 ± 6.8 | 71.7 ± 13.7 | 0.082 |
See Table 1 for key.
Correlation between PVP and CEUS variables
Results for 2 dogs were excluded from the analysis. Therefore, correlation between PVP and CEUS variables was tested by use of 6 baseline values and 4 postinduction values. Simple regression analysis revealed a significant negative correlation between TTPP and PVP (R2 = 0.548; P = 0.014; Figure 3). This correlation was weaker than that for both fasting and postprandial TBA concentrations and PVP (Table 3).
Results of simple regression analyses to determine the correlation between PVP and CEUS variables or blood biochemical variables determined for 6 dogs before and after induction of portal hypertension.
| Variable | R2 | AICc | P value* |
|---|---|---|---|
| Postprandial TBA | 0.675 | 53.4 | 0.004 |
| Fasting TBA | 0.584 | 55.9 | 0.010 |
| TTPP | 0.548 | 56.7 | 0.014 |
| ALP | 0.468 | 58.3 | 0.029 |
| TTP | 0.375 | 60.0 | 0.060 |
| ALT | 0.269 | 61.5 | 0.125 |
| Total bilirubin | 0.216 | 62.2 | 0.176 |
| GGT | 0.070 | 63.9 | 0.461 |
Six values before and 4 values after induction of portal hypertension were used for analyses because PVP could not be measured accurately in 2 dogs after induction of portal hypertension.
AICc = Akaike information criterion correction.
See Table 1 for remainder of key.
Discussion
In the study reported here, changes in hepatic hemodynamics of dogs with experimentally induced portal hypertension were evaluated by use of CEUS. The enhancement pattern of the hepatic vein changed dynamically and was represented by shorter TTP and TTPP values.
Shorter time-dependent variables indicated that the initial increase in the time-intensity curve became steeper, compared with the increase for the baseline time-intensity curve, which probably was related to the development of intrahepatic shunts between branches of the portal vein or hepatic artery (or both) and hepatic vein. Intrahepatic shunts have been detected in rats with portal hypertension induced by intraportal injection of microspheres,19,20 which is a method similar to the one used in the present study. The development of intrahepatic shunts can contribute to the shorter TTP and TTPP because the microbubbles passing through them can reach the hepatic vein directly without passing into the sinusoids. A similar rapid increase in the time-intensity curve was also reported for cirrhotic patients in clinical studies.9,21,22
The TTPP had a significant negative correlation with PVP. However, this correlation was weaker than that for both fasting and postprandial TBA concentrations and PVP. Although TBA concentrations are helpful for determining the presence of clinically relevant hepatobiliary disease in dogs, many other pathological states can also increase TBA values.23,24 Because MAPSS formation induced by portal hypertension is one of the factors that can contribute to an increase in TBA concentration,24 monitoring of TBA concentrations would not be suitable for assessing the severity of portal hypertension in clinical cases. On the other hand, hemodynamic analysis by use of CEUS has been evaluated mainly for use in assessing the severity of portal hypertension or degree of hepatic fibrosis.9,10,21,25–27 However, many patients with metastases to the liver also have a left shift of the time-intensity curve similar to that seen for patients with cirrhosis.28 Consequently, TTPP should be evaluated with caution and with consideration of underlying disorders. Further studies are warranted to evaluate the clinical use of TTPP for dogs with portal hypertension.
In addition, HVAT, another time-dependent variable, did not change significantly from before to after the establishment of portal hypertension. This was an unexpected finding because it has been reported in several studies9,10,21 that HVAT is shorter in cirrhotic patients than in clinically normal volunteers and is negatively correlated with portal pressure. Furthermore, it has recently been reported29 that there is a similar shorter HVAT with the development of liver fibrosis in dogs with experimentally induced liver fibrosis. The difference between the experimentally induced portal hypertension in the present study and patients with hepatic cirrhosis or experimentally induced liver fibrosis might be related to differences in the pathological condition. Hepatic cirrhosis and experimentally induced liver fibrosis mainly cause sinusoidal portal hypertension, which involves intrahepatic and extrahepatic hemodynamic changes that contribute to a shorter HVAT. In contrast, the study reported here represented presinusoidal portal hypertension that involved only slight periportal inflammation with no fibrosis. The characterization of ascites also substantiated that we induced presinusoidal portal hypertension. In idiopathic portal hypertension, which is the most common cause of presinusoidal portal hypertension in humans, increased and dilated hepatic arterial branches that compensate for the reduced portal blood supply (such as that observed in patients with a cirrhotic liver) are not seen.30 In the same manner as for idiopathic portal hypertension, it is possible that the method described for the study reported here might not induce the same hemodynamic changes as those seen in patients with cirrhosis.
To our knowledge, the study reported here is only the second involving portal hypertension experimentally induced in dogs by intraportal administration of microspheres. Similar to results for that other study,18 we were able to induce abundant MAPSS that resulted from increased portal venous resistance in all dogs. Formation of MAPSS was obvious during diagnostic imaging and necropsy. Microspheres in the pulmonary beds, which presumably reached the lungs through MAPSS, were also confirmed histologically. In view of these results, we consider that this is a useful method to induce presinusoidal portal hypertension in dogs without clinically serious adverse events.
However, several factors in the present study differed from those in previous studies. First, we were not able to induce an adequate increase in portal pressure by administration of microspheres at 10 mg/kg. Second, the increase in portal pressure was relatively milder in the present study (median PVP after induction of portal hypertension was 12.5 mm Hg). Investigators of another study18 reported that administration of microspheres at 10 mg/kg at 5-day intervals (total of 6 injections) could induce portal hypertension, and the PVP increased to 24.3 mm Hg after 1 month. Although the reason for these differences is not clear, individual variability might be the most likely explanation because the total dose of microspheres injected into each dog was also widely variable in the present study.
In the study reported here, there was a significant increase in the fasting TBA concentration, but no significant changes in ammonia concentrations were detected. We speculated that ammonia concentrations did not increase because other tissues, including the kidneys, muscles, brain, and intestines, could detoxify ammonia that escaped hepatic metabolism via MAPSS.31 In addition, because normal hepatocytes have a remarkable functional reserve for detoxifying ammonia,32 even ammonia that passed through the intrahepatic shunts might have been cleared effectively.
The present study had some limitations. First, the number of dogs used was small. In addition, it would be more appropriate to compare CEUS variables between preinfusion and postinfusion values as well as values for a control group infused with saline solution (rather than microspheres). However, the method used required a surgical procedure to catheterize the portal vein; therefore, such a control group was not included on the basis of guidelines for use of laboratory animals.33 Second, we could not measure PVP after portal hypertension in 2 dogs, which lowered the statistical power of the study. Third, the effect of body size and positioning, which can influence CEUS variables, could not be evaluated because we used only Beagles positioned in left lateral recumbency. Fourth, the contrast agenth was selected because it was the only second-generation contrast agent available in Japan. However, other vascular-specific contrast agents might have been better for assessing time-related variables because they only reflect hemodynamic changes related to portal hypertension. Finally, this method required clear visual examination of the hepatic vein, and this could be a major limitation for dogs with microhepatia or dogs that are excessively obese. In fact, it has been reported10 that HVAT could not be measured in cirrhotic patients because of the poor echo window (failure rate, 11.5%). In case of failure to obtain adequate images of the hepatic vein, analysis of the liver parenchyma may have an advantage over the method used in the present study. Because the microbubbles passing through the sinusoids or bypassing the sinusoids would not make a major difference to the contrast effect of the liver parenchyma, CEUS of the hepatic vein could be superior for detection of intrahepatic shunt flow. Moreover, as previously reported,26,34 the combination of CEUS variables or the combination of CEUS and other assessments can improve the diagnostic ability of researchers and clinicians. Thus, further studies are warranted to determine the optimal CEUS variable or combination of variables, including assessments other than CEUS, for the evaluation of portal hypertension in dogs.
The study reported here revealed that CEUS can be used for detecting hemodynamic changes induced by presinusoidal portal hypertension in dogs. The TTPP, a quantitative variable measured from the time-intensity curve, was negatively correlated with PVP and can provide useful complementary information with regard to the presence of portal hypertension. These fundamental data derived by use of an experimentally induced condition may be valuable for conducting clinical trials.
Acknowledgments
Presented in abstract form at the 2014 American College of Veterinary Internal Medicine Forum, Nashville, Tenn, June 2014.
ABBREVIATIONS
| CEUS | Contrast-enhanced ultrasonography |
| HVAT | Hepatic vein arrival time |
| MAPSS | Multiple acquired portosystemic shunts |
| PVP | Portal vein pressure |
| TBA | Total bile acid |
| TTP | Time to peak |
| TTPP | Time to peak phase |
Footnotes
MRI port, C R Bard Inc, Murray Hill, NJ.
Sephadex G-50 medium, GE Healthcare UK Ltd, Chalfont St Giles, Buckinghamshire, England.
Disposable blood pressure–monitoring kit, Nihon Kohden Co, Tokyo, Japan.
Coreless needle, Nipro Co, Osaka, Japan.
Aquilion 16, Toshiba Medical Systems, Tochigi, Japan.
Aplio XG, Toshiba Medical Systems, Tochigi, Japan.
PLT-704 AT, Toshiba Medical Systems, Tochigi, Japan.
Sonazoid, Daiichi-Sankyo, Tokyo, Japan.
ImageJ, version 2.0, National Institutes of Health, Bethesda, Md. Available at: rsbweb.nih.gov/ij/index.html. Accessed Apr 15, 2016.
JMP Pro, version 11, SAS Institute Inc, Cary, NC.
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