Experimental Study Of Ultrasound Diagnosis On Hepatic Fibrosis Rabbits

Liver biopsy is the golden standard for hepatic fibrosis or cirrhosis, but its clinical application is limited because of its invassiveness. Ultrasound diagnosis is a non-invasive method. If ultrasound measurement could diagnose chronic liver disease before cirrhosis is pathologyically occurred (stage of hepatic fibrosis), it would be helpful to investigate prognosis to prevent cirrhotic process or reverse the process of hepatic fibrosis.Purpose: (1) To assess the value of quantitative gray ultrasound on the hepatic fibrosis by computer associatied imaging analysis. (2) Hemodynamic changes at different stages of hepatic fibrosis were evaluated with color Doppler flow imaging (CDFI). Then the hepatic circulation index (HCI) at different stages of experimental hepatic fibrosis was calculated to assess liver reserve function, comparing with the result of indocyanine green (ICG) excretion study. (3) To find out the relationship between portal vein pressure and peripheral blood level of vasoactive substances (endothelin-1 ET-1, nitric oxide NO) within different stages of experimental hepatic fibrosis. (4) To assess the value of ultrasound bone strength in diagnosing hepatic bone disease (HBD). This study aimed to evaluate diagnostic values of quantitative two-dimension ultrasound imaging and hepatichemodynamic changes by CDFI at different stages of experimental hepatic fibrosis models to help clinical treatment and prognosis on chronic hepatic disease.Material and method: (1) Experimental hepatic fibrosis models of 35 adult healthy New Zealand rabbits were induced by thioacetamide (TAA 1.2g/L). The control group of 5 healthy New Zealand rabbits was fed normally. The liver of rabbit were obtained at random during the course of 0th week, 8th week, 12th week, 16th week and 20th week, then fixed by 10% formaldehyde and stained by hematoxylin eosin (HE) and Masson three colors.(2)Peripheral blood plasma ET-1 level and blood serum bone Gla-containing protein (BGP) levels were measured by radioimmunoassay (RIA) in control group and study groups. Blood serum NO levels of five groups were measured by Nitrate reductase method, at same time, peripheral blood serum liver function markers (total bilirubin TB, alanine aminotransferase ALT, aspartate aminotransferase AST, serum albumin ALB and serum globulin GLO) were examined by automatic biochemical analyzing system.(3) Liver parenchyma echo, thickness of gallbladder wall and portal vein diameter were measured at different stages of hepatic fibrosis, at same time, hemodynamic parameters of portal vein , superior mesenteric artery/vein and spleen artery/vein were obtained by automatic CDFI diagnostic ultrasound equipment. HCI was calculated with the formula (HCI=VphaxVPPv/SPPI , Vpha: peak velocity of hepatic artery , VPPv:peak velocity of portal vein , SPPI: spleen pulsatility index) at different stages of hepatic fibrosis.(4) 15min indocyanine green (ICG 15) concentration and clearance ratio of indocyanine green (KICG) were measured at the 0th week, 8th week ,12th week,16 week and 20th week by indocyanine green excretion study. Liver reserve function was assessed by HCI comparing with indocyanine green excretion study.(5) Portal vein pressures were directly measured by portal puncturation pressure measureing equipment. Then the relationships between portal vein pressure and HCI were analyzed at different stages of experimental hepatic fibrosis.(6) Speed of sound (SOS) of femoral bone in five groups were measured by quantitative ultrasound strength (QUS) equipment, then analyze the relationships between SOS and HCI or BGP.(7) ET-1 and nitricoxide synthase (NOS) at hepatic cell or hepatic sinusoid were expressed by immunohistochemistry at different stages of hepatic fibrosis. At the same time, ultramicrostructures of liver sinusoidal endothelial cells and hepatic sinusoid were viewed with transmission electron microscope.Result: (1) There was significant difference in mean liver parenchyma grey scale intensity between stage S4 and So-3(P<0.01). There was no significant difference within stage So to S3. In the process of hepatic fibrosis, liver parenchyma grey scale variances were significantly different at stage S2 S3 S4 and S0-1. There were significant difference about liver parenchyma grey scale within S3-4 to S2, S3 to S4 (P<0.01). Gallbladder thickness at stage S3n S4 was thicker than that at stage So-There was an increasing tendency about portal vein pressure with development of hepatic fibrosis, and portal vein pressures at stage S2-4 were higher than that at stage Sq (P<0.05). There was a good positive relationship between gallbladder thickness and portal vein pressure (r = 0.799).(2)HCI decreased and ICG15 increased with the development of experimental hepatic fibrosis. HCI at stage So> S^ S2, S3^ S4 was 470±52> 439±47> 355±62> 278±35> 225±59, respectively. There was significant difference between stage S4and stage Si-2 (P<0.05). The correlation between HCI and ICG15 were excellent (r= - 0.866 ). Portal vein pressure increased with the development of hepatic fibrosis, and portal vein pressures at stage S2-4 were higher than that at stage So (P <0.05). Portal vein pressure was significantly correlated to HCI and IGC15 (r= -0.773, r=0.837, respectively).(3) Peripheral plasma ET-1 level at 12th week was obviously raised comparing to beginning stage. ET-1 level at stage S2-4 was higher than that at stage So. On the early stage (8th week), peripheral serum NO level was decreased, but it increased from 12th week to 20th week. NO level at stage S3^ S4 was higher than that at stage So- On the other hand, there was significant difference in ET-1/NO ratio between stage Si^and stage So. ET-l/NO ratio was related to gallbladder wall thickness and portal vein pressure (r=0.534, r=0.542, respectively).(4) Serum biochemical markers (TB, ALT, AST, ALB, GLO) showed that there was significant difference in ALT between study groups and control group, but there was not difference among study groups. There was significantly positive correlation between TB and portal vein pressure (r=0.51).(5) SOS of rabbit femoral bone at stage S3-4 was lower than that at stage So. With development of experimental hepatic fibrosis, BGP concentration was decreased. There were significant difference comparing S34 to Si.2, S4 to S3, respectively (P <0.05). SOS was related to BGP (r=0.617, P<0.01). For HBD prediction, BGP concentration decreased earlier than SOS changes. BGP was a sensitive prediction marker for hepatic bone disease. SOS and BGP were related to HCI (r=0.694, 0.789 respectively).(6) ET-1 was negatively expressed at hepatic cell or hepatic sinusoid cells in control group. In study groups, ET-1 was high positively expressed at hepatic...