Deletion of Protein Kinase D3 Promotes Liver Fibrosis in Mice.

BACKGROUND AND AIMS: Liver fibrosis (LF) is a central pathological process that occurs in most types of chronic liver diseases. Advanced LF causes cirrhosis, hepatocellular carcinoma, and liver failure. However, the exact molecular mechanisms underlying the initiation and progression of LF remain largely unknown. APPROACH AND RESULTS: This study was designed to investigate the role of protein kinase D3 (PKD3; gene name Prkd3) in the regulation of liver homeostasis. We generated global Prkd3 knockout (Prkd3-/- ) mice and myeloid-cell-specific Prkd3 knockout (Prkd3∆LysM ) mice, and we found that both Prkd3-/- mice and Prkd3∆LysM mice displayed spontaneous LF. PKD3 deficiency also aggravated CCl4 -induced LF. PKD3 is highly expressed in hepatic macrophages (HMs), and PKD3 deficiency skewed macrophage polarization toward a profibrotic phenotype. Activated profibrotic macrophages produced transforming growth factor beta that, in turn, activates hepatic stellate cells to become matrix-producing myofibroblasts. Moreover, PKD3 deficiency decreased the phosphatase activity of SH2-containing protein tyrosine phosphatase-1 (a bona-fide PKD3 substrate), resulting in sustained signal transducer and activator of transcription 6 activation in macrophages. In addition, we observed that PKD3 expression in HMs was down-regulated in cirrhotic human liver tissues. CONCLUSIONS: PKD3 deletion in mice drives LF through the profibrotic macrophage activation.

Dose-dependency of clonidine's effects in ascitic cirrhotic rats: comparison with α1-adrenergic agonist midodrine.

BACKGROUND & AIMS: Sympathetic nervous system (SNS) activation decreases response to diuretics, but both α1-adrenoceptor agonists and sympatholytic α2-adrenoceptor agonists are recommended in the management of ascitic cirrhosis. We intend to compare the effects of increasing doses of clonidine (α2-agonist) vs. midodrine (α1-agonist) in advanced cirrhosis. METHODS: Renal function, mean arterial pressure (MAP), and hormonal status were measured in rats with ascitic cirrhosis due to 13-week CCl(4) administration (groups G1-G5), in control rats (Gc), and in rats with ascitic cirrhosis untreated (G6) or treated with daily diuretics (0.5 mg/kg furosemide plus 2 mg/kg K(+) -canrenoate during the 11(th) -13(th) weeks of CCl(4)) (G7). G1-G5 cirrhotic rats received daily, during the 11(th)-13(th) CCl(4) weeks: clonidine 0.3 µg only (G1), diuretics + clonidine 0.2 (G2), 0.5 (G3) or 1 µg (G4), and diuretics + midodrine 1 mg/kg b.w. (G5). RESULTS: Cirrhotic rats in G1 or G2 had higher glomerular filtration rate, renal plasma flow and natriuresis than cirrhotic rats treated with diuretics (G7) (all P < 0.05). The addition of clonidine 0.2 µg to diuretics (G2 vs. G7) reduced serum norepinephrine (169 ± 71 ng/L vs. 523 ± 88 ng/L) and plasma renin activity (12 ± 3 ng/ml/h vs. 25 ± 5 ng/ml/h) (all P < 0.05). Midodrine did not improve the renal performance in ascitic rats treated with diuretics. In comparison to absolute cirrhotic controls (G6), MAP was lower in G4 and higher in G5 (all P < 0.05). CONCLUSION: Low-dose α2-agonists improve natriuresis and reduce SNS function and hyper-aldosteronism without affecting arterial pressure in experimental ascitic cirrhosis treated with diuretics.

Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats.

AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats. METHODS: A total of 75 male Wistar rats were randomly divided into a normal control group (A), a CCl4-induced hepatic fibrosis model group (B), a natural recovery group (C), a low-dose DHC-treated group (D), and a high-dose DHC-treated group (E), with 15 rats in each group. Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride (CCl4) and a high-lipid/low-protein diet for 8 wk, except for the rats in group A. Then, the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks, respectively. By the end of the experiment, the level of transforming growth factor ß1 (TGF-ß1) in the liver homogenate was determined by an enzyme-linked immunosorbent assay. The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reverse-transcription polymerase chain reaction, an immunohistochemical assay, and Western blot analysis. RESULTS: Compared with group A, the level of TGF-ß1 and the mRNA and protein expression of Gremlin were significantly higher in group B (TGF-ß1: 736.30 ± 24.40 µg/g vs 284.20 ± 18.32 µg/g, P < 0.01; mRNA of Gremlin: 80.40 ± 5.46 vs 49.83 ± 4.20, P < 0.01; positive protein expression rate of Gremlin: 38.46% ± 1.70% vs 3.83% ± 0.88%, P < 0.01; relative protein expression of Gremlin: 2.81 ± 0.24 vs 0.24 ± 0.06, P < 0.01), and the mRNA and protein expression of BMP-7 was significantly lower in group B (mRNA: 54.00 ± 4.34 vs 93.99 ± 7.03, P < 0.01; positive protein expression rate: 28.97% ± 3.14% vs 58.29% ± 6.02, P < 0.01; relative protein expression: 0.48 ± 0.31 vs 1.05 ± 0.12, P < 0.01). Compared with groups B and C, the degree of hepatic fibrosis was significantly improved, and the level of TGF-ß1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHC-treated groups (TGF-ß1: 523.14 ± 21.29 µg/g, 441.86 ± 23.18 µg/g vs 736.30 ± 24.40 µg/g, 651.13 ± 15.75 µg/g, P < 0.01; mRNA of Gremlin: 64.86 ± 2.83, 55.82 ± 5.39 vs 80.40 ± 5.46, 70.37 ± 4.01, P < 0.01; positive protein expression rate of Gremlin: 20.78% ± 1.60%, 17.43% ± 2.02% vs 38.46% ± 1.70%, 29.50% ± 2.64%, P < 0.01; relative protein expression of Gremlin: 1.95 ± 0.26, 1.65 ± 0.20 vs 2.81 ± 0.24, 2.22 ± 0.63, P < 0.01), and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups (mRNA: 73.52 ± 4.56, 81.78 ± 5.38 vs 54.00 ± 4.34, 62.28 ± 4.51, P < 0.01; positive protein expression rate: 41.44% ± 4.77%, 47.49% ± 4.59% vs 28.97% ± 3.14%, 35.85% ± 3.50%, P < 0.01; relative protein expression: 0.71 ± 0.06, 0.81 ± 0.07 vs 0.48 ± 0.31, 0.60 ± 0.37, P < 0.01). CONCLUSION: The therapeutic mechanism of DHC for hepatic fibrosis in rats may be associated with inhibition of the expression of Gremlin and up-regulation of the expression of BMP-7.

qFibrosis: a fully-quantitative innovative method incorporating histological features to facilitate accurate fibrosis scoring in animal model and chronic hepatitis B patients.

BACKGROUND & AIMS: There is increasing need for accurate assessment of liver fibrosis/cirrhosis. We aimed to develop qFibrosis, a fully-automated assessment method combining quantification of histopathological architectural features, to address unmet needs in core biopsy evaluation of fibrosis in chronic hepatitis B (CHB) patients. METHODS: qFibrosis was established as a combined index based on 87 parameters of architectural features. Images acquired from 25 Thioacetamide-treated rat samples and 162 CHB core biopsies were used to train and test qFibrosis and to demonstrate its reproducibility. qFibrosis scoring was analyzed employing Metavir and Ishak fibrosis staging as standard references, and collagen proportionate area (CPA) measurement for comparison. RESULTS: qFibrosis faithfully and reliably recapitulates Metavir fibrosis scores, as it can identify differences between all stages in both animal samples (p<0.001) and human biopsies (p<0.05). It is robust to sampling size, allowing for discrimination of different stages in samples of different sizes (area under the curve (AUC): 0.93-0.99 for animal samples: 1-16 mm(2); AUC: 0.84-0.97 for biopsies: 10-44 mm in length). qFibrosis can significantly predict staging underestimation in suboptimal biopsies (<15 mm) and under- and over-scoring by different pathologists (p<0.001). qFibrosis can also differentiate between Ishak stages 5 and 6 (AUC: 0.73, p=0.008), suggesting the possibility of monitoring intra-stage cirrhosis changes. Best of all, qFibrosis demonstrates superior performance to CPA on all counts. CONCLUSIONS: qFibrosis can improve fibrosis scoring accuracy and throughput, thus allowing for reproducible and reliable analysis of efficacies of anti-fibrotic therapies in clinical research and practice.

[Values of magnetic resonance spectrum imaging in the diagnosis of hepatic fibrosis of rats].

OBJECTIVE: To explore the values of magnetic resonance spectrum (MRS) in early diagnosis, quantization analysis and staging of hepatic fibrosis. METHODS: A rat model of hepatic fibrosis was established by the method of carbon tetra carbon (CCl4). A total of 47 SD rats were divided into model (n = 40) and control (n = 7) groups. 1H-MRS was performed. The model rats of hepatic fibrosis were grouped according to their pathological stages. The ratio of peak height and peak area of metabolites and lipid (Cho/Lip, Glx/Lip, Lac/Lip and Cr/Lip) were calculated and compared respectively. RESULTS: The ratios of peak height of metabolites and lipid were as follows: ratio of Cho and Lip: significant differences existed between control and grades 3 and 4 model groups (P < 0.05); ratio of Glx and Lip: significant differences existed between control and grades 2, 3 and 4 model groups (P < 0.05); ratio of Cr and Lip: significant differences existed between control and grade 3 model groups (P < 0.05). The peak area ratio of main metabolites and lipid of liver were as follows: ratio of Cho and Lip: significant differences existed between control and grade 4 model groups (P < 0.05); ratio of Glx and Lip: significant differences existed between control and other groups (P < 0.05); ratio of Cr and Lip: significant differences existed between control and grade 4 model groups (P < 0.05); ratio of Lac and Lip: no significant differences existed between these groups (P > 0.05). CONCLUSION: The ratios of peak height and peak area of Cho/Lip, Glx/Lip and Cr/Lip are important for the staging of hepatic fibrosis.

ED50 and recovery times after propofol in rats with graded cirrhosis.

BACKGROUND: Patients with end-stage liver disease have increased sensitivity to general anesthetics. In this study, we sought to quantify sensitivity to propofol as a function of the degree of liver disease, in a rat model of cirrhosis. METHODS: Liver disease was induced by carbon tetrachloride (CCl(4)) injections for 6, 9, or 12 weeks in 3 study groups. Control rats received saline injections on the same schedule as CCl(4)-injected rats. A second control (comparison) group was treated with phenobarbital for a week followed by 9 weeks of phenobarbital and 10% ethanol in drinking water. Liver function was assessed by liver function tests and pathologic scoring of liver histology. RESULTS: Progressively worse cirrhosis was associated with longer CCl(4) treatment by histologic criteria, by hypersplenism, liver to body weight ratios, and liver function tests. The major findings were that mild liver disease (either steatosis or fibrosis) was not associated with increased propofol sensitivity, but recovery times after propofol bolus and propofol infusion were significantly increased in rats with more severe liver fibrosis. CONCLUSION: Propofol sensitivity is not significantly affected in the setting of mild liver disease, similar to clinical observations, but end-stage liver disease (fibrosis) is associated with significantly prolonged time to recovery after propofol infusion. The progressive liver disease model used in these studies is useful for rigorously studying anesthetic sensitivity as a function of degree of hepatocellular-fibrotic liver disease.

A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Development of a large animal model of cirrhosis and portal hypertension using hepatic transarterial embolization: a study in swine.

PURPOSE: To develop a clinically relevant porcine model of liver cirrhosis with portal hypertension by means of hepatic transarterial embolization. MATERIALS AND METHODS: Institutional animal care and use committee approval was obtained for all experiments. Pigs received transcatheter arterial infusion of a 3:1 mixture of iodized oil and ethanol into the hepatic artery in volumes of 16 mL in group 1 (n = 4), 28 mL in group 2 (n = 4), and 40 mL in group 3 (n = 4) with intent of bilobar distribution. Hepatic venous pressure gradient (HVPG) measurement, liver function tests, and volumetry were performed at baseline, at 2 weeks, and before necropsy. RESULTS: Cirrhosis was successfully induced in three animals that received 16 mL of the embolic mixture and in all four animals that received 28 mL. The animals in the 40-mL group did not recover from the procedure and were euthanized within 48 h. Increases in HVPG after 6-8 weeks versus baseline reached statistical significance (P < .05). Correlation between degree of fibrosis and volume of embolic agent did not reach statistical significance, but there was a trend toward increased fibrosis in the 28-mL group compared with the 16-mL group. CONCLUSIONS: Transcatheter hepatic arterial embolization can be used to create a reliable and reproducible porcine model of liver cirrhosis and portal hypertension.

Signal profile on Gd-EOB-DTPA-enhanced MR imaging in non-alcoholic steatohepatitis and liver cirrhosis induced in rats: correlation with transporter expression.

OBJECTIVES: To compare the transporter expression and signal profile on Gd-EOB-DTPA-enhanced MRI between non-alcoholic steatohepatitis (NASH) and cirrhotic liver induced in rats, and investigate the correlation of the transporter expression and fibrosis rate in both diseases. METHODS: Forty-eight rats were divided into four groups of 12: TAA (cirrhosis), NASH 7- and 10-week, and control groups. Each group was divided into two subgroups: Group 1 for MRI and Group 2 for transporter examinations. RESULTS: The relative enhancement of the TAA group was significantly lower than those of other groups (p < 0.01). The T(max) and T(1/2) of the NASH 10-week group was significantly prolonged in comparison with the TAA group (p < 0.01). There was no significant difference in the oatp1 expression, whereas the mrp2 expression of the TAA group was significantly higher than those of other groups (p < 0.01). There was no significant correlation between the fibrosis rate and oatp1 expression, whereas a paradoxical correlation was found between the fibrosis rate and mrp2 expression (NASH: negative correlation, r = 0.91, p < 0.01; TAA: positive correlation, r = 0.85, p < 0.01). CONCLUSIONS: Our findings showed that the mrp2 expression in cirrhosis increases in comparison with NASH, and there was a paradoxical correlation between the fibrosis rate and mrp2 expression.

Therapeutic effects of vitamin A on experimental cholestatic rats with hepatic fibrosis.

PURPOSE: The aim of this study is to investigate the role of hepatic stellate cells (HSCs) and the effect of vitamin A administration on liver damage induced by bile duct ligation (BDL) and administration of CCl(4). METHODS: Two types of animal model were used; one was BDL as a model of biliary atresia, the other was CCl(4)-induced hepatic fibrosis. Pathological changes of the liver with or without administration of vitamin A were compared by light and electron microscopy with focusing on HSCs in each experimental group. Immunohistochemical examination was performed with anti-keratinocyte growth factor (KGF), anti-alpha-smooth muscle actin (α-SMA), and anti-glial fibrillary acidic protein (GFAP) antibodies, as markers of fibrosis. RESULTS: On light microscopic findings, periportal inflammation with bile ductular proliferation was obvious in BDL group and pericentral necrosis with fatty degeneration was observed in CCl(4) group, both of which were ameliorated by subcutaneous injection of vitamin A. Electron microscopy showed lipid droplets were almost depleted in the HSCs treated with BDL or CCl(4), which improved with vitamin A administration. Immunohistochemistry demonstrated that enhanced expression of all three fibrotic markers in the BDL group was diminished by vitamin A administration. CONCLUSIONS: Although most of our data are qualitative observation, vitamin A may ameliorate hepatic fibrosis in the BDL model by restoring vitamin A in the HSCs.

Diffusion tensor imaging of liver fibrosis in an experimental model.

PURPOSE: To characterize changes in diffusion properties of liver using diffusion tensor imaging (DTI) in an experimental model of liver fibrosis. MATERIALS AND METHODS: Liver fibrosis was induced in Sprague-Dawley rats (n = 12) by repetitive dosing of carbon tetrachloride (CCl(4)). The animals were examined with a respiratory-gated single-shot spin-echo echo-planar DTI protocol at 7 T before, 2 weeks after, and 4 weeks after CCl(4) insult. Apparent diffusion coefficient (ADC), directional diffusivities (ADC(//) and ADC(⊥)), and fractional anisotropy (FA) were measured. Liver histology was performed with hematoxylin-eosin staining and Masson's trichrome staining. RESULTS: Significant decrease (P < 0.01) in ADC was found at 2 weeks (0.86 ± 0.09 × 10(-3) mm(2)/s) and 4 weeks (0.74 ± 0.09 × 10(-3) mm(2)/s) following CCl(4) insult, as compared with that before insult (0.97 ± 0.08 × 10(-3) mm(2)/s). Meanwhile, FA at 2 weeks (0.18 ± 0.03) after CCl(4) insult was significantly lower (P < 0.01) than that before insult (0.26 ± 0.05), and subsequently normalized at 4 weeks (0.26 ± 0.07) after the insult. Histology showed collagen deposition, presence of intracellular fat vacuoles, and cell necrosis/apoptosis in livers with CCl(4) insult. CONCLUSION: DTI detected the progressive changes in water diffusivities and diffusion anisotropy of liver tissue in this liver fibrosis model. ADC and FA are potentially valuable in detecting liver fibrosis at early stages and monitoring its progression. Future human studies are warranted to further verify the applicability of DTI in characterizing liver fibrosis and to determine its role in clinical settings.

Evaluation of the 13C-octanoate breath test as a surrogate marker of liver damage in animal models.

BACKGROUND: Octanoate (also known as sodium octanoate), a medium-chain fatty acid metabolized in the liver, is a potential substrate for non-invasive breath testing of hepatic mitochondrial beta-oxidation. METHODS: We evaluated the 13C-octanoate breath test (OBT) for assessing injury in acute hepatitis and two rat models of liver cirrhosis, first testing octanoate absorption (per os or intraperitoneally (i.p.)) in normal rats. We then induced acute hepatitis with thioacetamide (300 mg/kg/i.p., 24-h intervals). Liver injury end points were serum aminotransferase levels and 13C-OBT (24 and 48 h following initial injection). Thioacetamide (200 mg/kg/i.p., twice per week, 12 weeks) was used to induce liver cirrhosis. OBT and liver histological assessment were performed every 4 weeks. Bile duct ligation (BDL) was used to induce cholestatic liver injury. We completed breath tests with 13C-OBT and 13C-methacetin (MBID), liver biochemistry, and liver histology in BDL and sham-operated rats (baseline, 6, 14, 20 days post-BDL). RESULTS: Octanoate absorbs well by either route. Peak amplitudes and cumulative percentage dose recovered at 30 and 60 min (CPDR30/60), but not peak time, correlated with acute hepatitis. Fibrosis stage 3 at week 8 significantly correlated with each OBT parameter. Cholestatic liver injury (serum bilirubin, ALP, gamma-GT, liver histology) was associated with significant suppression of the maximal peak values and CPDR30/60, respectively (P<0.05),using MBID but not 13C-octanoate. CONCLUSIONS: OBT is sensitive for potentially evaluating liver function in rat models of acute hepatitis and thioacetamide-induced liver cirrhosis but not in cholestatic liver injury. The MBID test may be better for evaluation of cholestatic liver disease in this model.

[Establishment of liver fibrosis in rabbit model and quantitative study on hepatic perfusion with dynamic whole-liver 3D MR imaging].

OBJECTIVE: To investigate the ideal approach in creating rabbit model of hepatic fibrosis and to evaluate the feasibility and value of dynamic whole-liver 3D magnetic resonance (MR) perfusion-weighted imaging (PWI) in the quantitative study on the staging of hepatic fibrosis. METHODS: Rabbit model of hepatic fibrosis was created by intraperitoneal injection of 5% and 100% carbon tetrachloride (0.1 ml/kg, once a week) respectively. MR perfusion weighted imaging was performed at the 6th, 8th, 10th and 12th week since injection. The time of peak (TOP), the time to peak (TTP), the maximum slope of increase(MSI) and the maximal relative signal increase (MRSI) of portal vein and hepatic parenchyma were analyzed quantitatively, and were compared with pathological results. Comparison of different concentrations of CCl4 was analyzed using chi-square test. Inter-group comparison of perfusion parameters was analyzed using one-way ANOVA P less than 0.05 was regarded as statistically significant. RESULTS: 40% of the rabbits treated with 5% carbon tetrachloride developed hepatic fibrosis, while 75% of the rabbits treated with 100% carbon tetrachloride developed hepatic fibrosis; the mortality rate is significantly different between these two groups (X2=5.013, P less than 0.05). PWI examination was successfully achieved in 31 rabbits, liver perfusion baseline was stable, and good TIC curve was obtained. With the progress of hepatic fibrosis, TOP and TTP of portal vein and hepatic parenchyma were increased, and MSI and MRSI were decreased. There were significant differences among stage of S0-S2, S3 and S4. CONCLUSIONS: The method (100% carbon tetrachloride intraperitoneal injection, 0.1 ml/kg, once a week) has high success rate of creating rabbit model of hepatic fibrosis. The stage of hepatic fibrosis could be evaluated quantitatively with dynamic whole-liver 3D MR perfusion-weighted imaging.

[Serum hyaluronic acid level: correlation with quantitative measurement of hepatic fibrosis in a cirrhotic rat model].

BACKGROUNDS/AIMS: The serum level of hyaluronic acid (HA) has been suggested as a useful serologic marker for hepatic fibrosis. However, the relationship between serum HA levels and quantitative markers of fibrosis from liver tissue has not been reported. The aim of this study was to determine the correlation between serum HA level and quantitative measurement of hepatic fibrosis in a cirrhotic rat model. METHODS: Cirrhosis was produced by common bile duct ligation (BDL) in adult Sprague-Dawley rats. The animals were classified into four groups: (1) G1, sham operated (n=5); (2) G2, BDL for 2 weeks (n=6); (3) G3, BDL for 3 weeks (n=6); and (4) G4, BDL for 4 weeks (n=6). Hepatic fibrosis was analyzed histomorphologically using the Batts and Ludwig scoring system. Serum HA level and hepatic hydroxyproline content were quantified. The gene expressions in the liver of procollagen, collagen, and transforming growth factor-beta (TGF-beta) were measured by reverse transcriptase-polymerase chain reaction. RESULTS: In groups G1, G2, G3, and G4, the Batts and Ludwig scores (mean+/-SD) were 0, 1.3+/-0.5, 2.6+/-0.5, and 3.4+/-0.5, respectively (P<0.05), serum HA levels were 12.5+/-3.2, 30.0+/-4.3, 228.6+/-157.7, and 391.3+/-207.7 ng/mL (P<0.05), and the concentration of hydroxyproline was 12.4+/-2.8, 17.6+/-3.8, 17.9+/-2.4, and 33.4+/-3.4 microg/g liver tissue, and it was significantly higher in group G4 than in the other groups (P<0.05). The gene expressions of collagen, procollagen, and TGF-beta1 in the liver were also significantly higher in group G4 compared with the other groups (P<0.05). Direct linear correlations were observed between serum HA level and hepatic hydroxyproline content, hepatic gene expressions of collagen, procollagen, TGF-beta1, and histomorphological grade of hepatic fibrosis (P<0.001). CONCLUSIONS: These results indicate that serum HA is a useful and noninvasive serologic marker for the evaluation of advanced hepatic fibrosis.

[A pilot study of hepatic fibrosis with magnetic resonance diffusion-weighted imaging in a rabbit model].

OBJECTIVE: To evaluate the role of MRI diffusion imaging in the diagnosis of hepatic fibrosis. METHODS: Fifty-eight rabbits were divided randomly into a blank control group (n = 10) and a model group (n = 48). Carbon tetrachloride was injected intraperitoneally into the animals of the model group to induce liver fibrosis. SE-EPI sequence was used in diffusion weighted imaging for all the rabbits. Then apparent diffusion coefficient (ADC) and exponential apparent diffusion coefficient (EADC) of their livers were obtained with Functool 2.0 software. The degrees of liver fibrosis of the rabbits were graded with histological examinations. Statistical analysis was performed by SPSS 11.0 statistical software. One-way analysis of variance was used to compare every rank data respectively. P less than 0.05 was considered statistically significant. RESULTS: When the b value was 300 s/mm2, ADC of diffusion-weighted imaging (DWI) was (2.460+/-0.424) x 10(-3) in the control group (S0). ADCs were (2.170+/-0.311) x 10(-3), (1.950+/-0.248) x 10(-3), (1.635+/-0.296) x 10(-3), (1.566+/-0.353) x 10(-3) in the model group (S1, S2, S3 and S4). EADC of DWI was 0.102+/-0.044 in the control group and were 0.167+/-0.047, 0.183+/-0.042, 0.216+/-0.054, 0.219+/-0.048 in the model group (S1, S2, S3 and S4). ADC and EADC of the control group and model group had significant differences (F = 12.13, P = 0.0006; F = 10.06, P = 0.004 respectively). When the b value was 500 s/mm2, ADC of DWI was (2.044+/-0.215) x 10(-3) in the control group, ADC were (1.907+/-0.223) x 10(-3), (1.785+/-0.232) x 10(-3), (1.542+/-0.268) x 10(-3), (1.312+/-0.212) x 10(-3) in the model group (S1, S2, S3 and S4). EADC of DWI was 0.1106+/-0.069 in the control group and EADCs of DWI were 0.1764+/-0.073, 0.1889+/-0.056, 0.2421+/-0.079, 0.2657+/-0.037 in the model group (S1, S2, S3 and S4). ADCs and EADCs of the control group and model group had significant differences (F = 14.57, P = 0.0002; F = 10.42, P = 0.003 respectively). There was a significant difference of ADCs between S1 and S4 of the model group when b value were 300 s/mm2 and 500 s/mm2 (P = 0.03, P = 0.044 respectively). No differences were found between other subgroups in the model group. CONCLUSION: Our preliminary study shows that measuring ADCs and EADCs has a good potential in diagnosing and quantifying hepatic fibrosis, especially when using b values of 300 sec/mm2 and 500 sec/mm2.

Serum hyaluronic acid level: Correlation with quantitative measurement of hepatic fibrosis in a cirrhotic rat model / 대한간학회지

BACKGROUNDS/AIMS: The serum level of hyaluronic acid (HA) has been suggested as a useful serologic marker for hepatic fibrosis. However, the relationship between serum HA levels and quantitative markers of fibrosis from liver tissue has not been reported. The aim of this study was to determine the correlation between serum HA level and quantitative measurement of hepatic fibrosis in a cirrhotic rat model. METHODS: Cirrhosis was produced by common bile duct ligation (BDL) in adult Sprague-Dawley rats. The animals were classified into four groups: (1) G1, sham operated (n=5); (2) G2, BDL for 2 weeks (n=6); (3) G3, BDL for 3 weeks (n=6); and (4) G4, BDL for 4 weeks (n=6). Hepatic fibrosis was analyzed histomorphologically using the Batts and Ludwig scoring system. Serum HA level and hepatic hydroxyproline content were quantified. The gene expressions in the liver of procollagen, collagen, and transforming growth factor-beta (TGF-beta) were measured by reverse transcriptase-polymerase chain reaction. RESULTS: In groups G1, G2, G3, and G4, the Batts and Ludwig scores (mean+/-SD) were 0, 1.3+/-0.5, 2.6+/-0.5, and 3.4+/-0.5, respectively (P<0.05), serum HA levels were 12.5+/-3.2, 30.0+/-4.3, 228.6+/-157.7, and 391.3+/-207.7 ng/mL (P<0.05), and the concentration of hydroxyproline was 12.4+/-2.8, 17.6+/-3.8, 17.9+/-2.4, and 33.4+/-3.4 microgram/g liver tissue, and it was significantly higher in group G4 than in the other groups (P<0.05). The gene expressions of collagen, procollagen, and TGF-beta1 in the liver were also significantly higher in group G4 compared with the other groups (P<0.05). Direct linear correlations were observed between serum HA level and hepatic hydroxyproline content, hepatic gene expressions of collagen, procollagen, TGF-beta1, and histomorphological grade of hepatic fibrosis (P<0.001). CONCLUSIONS: These results indicate that serum HA is a useful and noninvasive serologic marker for the evaluation of advanced hepatic fibrosis.

Molecular determination of liver fibrosis by synchrotron infrared microspectroscopy.

Liver fibrosis is an adaptive response to various injuries and may eventually progress to cirrhosis. Although there are several non-invasive methods available to monitor the progression of liver fibrogenesis, they cannot reliably detect fibrosis in its early stages, when the process can be stopped or reversed by removing or eliminating the underlying etiological agent that cause the hepatic injury. In this study, early fibrosis alterations were characterized biochemically, morphologically, and spectroscopically in a rat bile duct ligation (BDL) model. Progressive elevations in serum alanine transaminase (ALT), aspartate transaminase (AST), and bilirubin levels in the BDL rats were found indicating the dynamic deterioration of hepatocellular function. Immunofluorescence microscopy using monoclonal anti-collagen III antibody further revealed abnormal intertwined networks of collagen fibres surrounding the portal areas and extending into the lobules towards the central veins in all BDL samples starting from week one. Synchrotron infrared microspectroscopy of liver sections was exploited to generate false color spectral maps based upon a unique and strong collagen absorption at 1340 cm(- 1), revealing a collagen distribution that correlated very well with corresponding images provided by immunofluorescence imaging. We therefore suggest that infrared microspectroscopy may provide an additional and sensitive means for the early detection of liver fibrosis.

[Study on pathogenesis of CCl4 induced cirrhosis formation in rats based on the recipe used].

OBJECTIVE: To investigate the recipe-based pathogenesis and effects of Xiayuxue Decoction (XD), Yinchenhao Decoction (YcD), Yiguanjian Decoction (YgD) and Huangqi Decoction (HD) on carbon tetrachloride (CCl4) induced liver cirrhosis formation in rats on the basis of the recognition of basic pathogenesis of liver cirrhosis in TCM and train of thoughts of detecting the TCM syndrome by recipe. METHODS: Model rats of liver cirrhosis were established by subcutaneous injecting of 100% CCl4 3ml/kg followed by 50% CCl4 olive solution 2ml/kg, twice a week for 12 weeks. They were randomly divided into the model group, the XD treated group, the YcD treated group, the YgD treated group and the HD treated group. Rats in the three treated group received the treatment starting from the 9th week of modeling with the corresponding decoctions. All animals were sacrificed by the end of the 12th week, and their hepatic function, liver pathological changes and hydroxyproline (Hyp) content of hepatic tissue were detected. RESULTS: (1) Typical chronic liver injury and fibrosis became evident in the model rat at the 8th week and cirrhosis came into being at the 12th week. (2) Compared with the rats in the model group, hepatic pathological changes were alleviated significantly, content of Hyp in hepatic tissue was decreased markedly and hepatic function improved remarkably in the XD group and YgD group. The improvement in the XD group was superior to that in the YgD group, while the serum albumin level elevated more significant in the YgD group. CONCLUSION: The main pathological changes during CCl4 induced liver cirrhosis formation in rats is the rapid hyperplasia of hepatic fibrous connective tissue and obstruction of collaterals by blood stasis, thus induced reconstruction of the tissue structure, which could be treated with XD effectively, while the severe injury of liver parenchyma in this phase is another pathological change of Gan-yin deficiency syndrome, which could be effectively treated with YgD by its Yin-nourishing action.