In situ quantification of endothelial cell damage caused by iodinated contrast media using a rat vena cava model.

PURPOSE: Quantification of vascular endothelial cell damage induced by iodinated contrast media using an in situ perfused rat vena cava model. MATERIALS AND METHODS: The institutional review board approved this study protocol prior to the commencement of all studies. A laparotomy was performed in 90 rats divided into 18 groups of five, and an 18 G-catheter was inserted into the abdominal vena cava (mean length: ca 8mm). After sacrificing, a thoracotomy was done and the outflow perfusate was emitted via a polyethylene tube inserted into the thoracic vena cava through the right atrium for an open system. Iopamidol (300 or 370 mgI/mL, 50 or 100mL) was injected via the abdominal vena cava at a rate of 1, 4, or 8 mL/s. The abdominal vena cava was removed for histological analysis (n=5). Physiological saline was injected as a negative control. The detachment percentage of endothelial cells was calculated by measuring the circumference and detachment section of the endothelium. The difference of the detachment percentage and circumference between each group was compared with Tukey's range test. RESULTS: In contrast media groups, the severity of damage to the vascular endothelial cell was direct proportional to the increase of injection rate. The detachment percentage at 4 or 8 mL/s was significantly higher than that at 1 mL/s. As a result, the correlation between the injection rate and severity of cell damage was significant; however, the detachment percentage among contrast media groups was not significant at any injection rate. CONCLUSION: The in situ vena cava model was able to quantify contrast media injection related endothelial damage based on histopathological endpoints. Moreover, our results indicate that mechanical shear stress besides physico-chemical properties such as osmolality or viscosity cause endothelial damage.

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.

Unenhanced fat fraction ratios obtained by MR and enhanced T2* values with liver-specific MR contrast agents for diagnosis of non-alcoholic steatohepatitis in rats.

BACKGROUND: Non-invasive MR imaging is expected to be used for accurate diagnosis and quantification of non-alcoholic steatohepatitis (NASH), because NASH is a progressive fatty liver disease. New MR techniques, such as fat fraction ratio (FFR) and T2* value measurement, have attracted an increasing attention, because those techniques can measure quantitative parameters of fibrosis, fat and iron deposition in the liver. PURPOSE: To investigate the potential of FFR and T2* value in NASH with pre-enhancement, gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) or super-paramagnetic iron oxide (SPIO)-enhanced MRI. MATERIAL AND METHODS: Twenty-eight rats were divided equally into four groups (one control group and three NASH groups). All rats underwent unenhanced, Gd-EOB-DTPA, and SPIO-enhanced MRI. The T2* value of the liver was measured for each image sequence, and then changes in T2* values before and after each injection were analyzed using Dunnett's test. The reduction rate of T2* value before and 13 min after injection of Gd-EOB-DTPA or SPIO was analyzed using Mann-Whitney's U test. Moreover, FFR of the liver was measured before enhancement, and the relationship between fat fraction and the calculated fat area percentage on a pathological specimen was examined using Spearman's correlation test. RESULTS: On pre-enhancement, FFR and T2* value were 26.0% ± 12.0% and 21.5 ± 4.2 ms for all NASH groups, and 0.9% ± 0.5% and 30.8 ±-5.5 ms for control, respectively. Both FFR and T2* values were significantly different between the NASH and control groups. The reduction rate of T2* value was significantly lower in the NASH groups than in the control group on SPIO-enhanced MRI, though there was no significant difference on Gd-EOB-DTPA-enhanced MRI. FFR was correlated with the calculated fat area percentage for the pathological specimen. CONCLUSION: Pre-enhancement FFR, T2* value measurement and reduction rate of T2* value on SPIO-enhanced MRI may help estimate the progress of liver fat deposition and fibrosis in NASH.

Effect of change in transporter expression on gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging during hepatocarcinogenesis in rats.

BACKGROUND AND AIMS: To analyze the difference in signal intensity on gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance imaging (MRI) among various hepatocellular nodules during hepatocarcinogenesis as correlated with the expressions of the transporters of Gd-EOB-DTPA. METHODS: We received institutional animal review board approval prior to the commencement of all studies. Forty rats were divided into three groups. The rats in the tumor groups received N-nitrosomorpholine solution (n = 16), and rats in the cirrhosis group (thioacetamide [TAA] group) received thioacetamide solution (n = 12). As a control, the remaining 12 rats were fed normal water. Each group was divided into two sub-groups: Group 1 for Gd-EOB-DTPA-enhanced MRI (0.025 mmol Gd/kg, n =7) and Group 2 for reverse transcription-polymerase chain reaction to compare transporter (oatp1 and mrp2) expressions (n = 5 for control and TAA groups, n = 9 for tumor groups). RESULTS: Signal enhancement of tumors decreased according to the progress of hepatocarcinogenesis. Although the relative enhancement of each tumor group was significantly lower than that of the control group (P < 0.01), and there was no significant difference between TAA, hyperplastic nodules (HPN), and HCC(well) groups. The relative enhancement of the HCC(mod) group was significantly lower than the other groups (P < 0.01). The oatp1 expression of HPN tended to be higher than those of HCC(well) and HCC(mod). The mrp2 expression of TAA was significantly higher than those of HCC(well), HCC(mod), HPN and control (P < 0.01). The mrp2 expression of HPN tended to be higher than those of HCC(well ) and HCC(mod). CONCLUSION: It was suggested that the signal enhancement on Gd-EOB-DTPA-enhanced MRI would correlate with the transporter expression in various hepatocellular nodules during hepatocarcinogenesis.

Cirrhotic rat liver: reference to transporter activity and morphologic changes in bile canaliculi--gadoxetic acid-enhanced MR imaging.

PURPOSE: To analyze the difference in signal intensity on gadoxetic acid-enhanced magnetic resonance (MR) images between normal and cirrhotic livers in rats as correlated with the expressions of the transporters of gadoxetic acid and the morphopathologic change in bile canaliculi. MATERIALS AND METHODS: Institutional animal review board approval was received prior to the commencement of all studies. Fifteen rats received thioacetamide (TAA) in their drinking water for 12 weeks to induce liver cirrhosis. As a control, 15 rats were given normal water. After 12 weeks, seven rats in the TAA group and seven rats in the control group underwent gadoxetic acid-enhanced MR imaging (0.025 mmol gadolinium per kilogram of body weight). Five rats in each group were used for comparison of transporter (organic anion-transporting polypeptide 1 [oatp1] and multidrug resistance-associated protein 2 [mrp2]) activities. Three rats in each group were used for morphologic examination. The Wilcoxon rank sum test was used for statistical analysis. RESULTS: Signal enhancement of the liver in the TAA group significantly decreased in comparison with that in the control group, although the signal enhancement of the kidney in both groups was almost identical. The oatp1 and mrp2 activities were as follows: 2.17 +/- 0.71 (standard deviation) for oatp1 in the TAA group, 2.58 +/- 0.35 for oatp1 in the control group, 3.37 +/- 1.04 for mrp2 in the TAA group, and 0.93 +/- 0.34 for mrp2 in the control group (P = .175 for oatp1, P = .009 for mrp2). At morphologic examination, enlargement of bile canaliculi and hyperplasia or elongation of microvilli were observed in the TAA group. CONCLUSION: Findings showed that liver cirrhosis promotes the elimination of gadoxetic acid mediated by mrp2; therefore, mrp2 up-regulation may explain the significant signal intensity loss noted on gadoxetic acid-enhanced MR images in the rat. In addition, the up-regulation of mrp2 may be accompanied by morphologic changes in bile canaliculi and microvilli.

New proposal for the staging of nonalcoholic steatohepatitis: evaluation of liver fibrosis on Gd-EOB-DTPA-enhanced MRI.

PURPOSE: We investigated whether the gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MRI was useful for nonalcoholic steatohepatitis (NASH) staging based on the severity of liver fibrosis. MATERIALS AND METHODS: Twenty-one male Sprague-Dawley rats aged 7 weeks, weighing about 150g in NASH group were fed a choline-deficient diet for 4, 7 or 10 weeks, and seven rats in the control group were fed a standard diet (n=7). After the feeding period, the rats were subjected to contrast-enhanced MRI (2D-FLASH; TR/TE=101/2.9ms, flip angle 90 degrees ). Gd-DTPA (0.1mmol Gd/kg) and Gd-EOB-DTPA (0.025mmol Gd/kg) were injected at 24-h intervals, and the speed of contrast injection was 1mL/s. Signal intensities of the liver were measured and the relative enhancement (RE), the time of maximum RE (T(max)) and elimination half-life of RE (T(1/2)) in the liver were compared. The fibrosis rate (%) was calculated with the following formula: fibrosis/whole areax100. RESULTS: The fibrosis rates of each group were as follows: 0.52, 0.79, 2.84, and 0.50% (4, 7, 10 weeks and control groups). The fibrosis rate of the 10 weeks group was significantly higher than the control and 4 or 7 weeks groups. Although there was no difference between the T(max) and T(1/2) of each group after Gd-DTPA injection, the T(max) and T(1/2) of the 10 weeks group were significantly prolonged in comparison with the control and 4 or 7 weeks groups after Gd-EOB-DTPA injection (p<0.01). There was a significant correlation between the fibrosis rate and T(max) or T(1/2) after Gd-EOB-DTPA injection (r=0.90 or 0.97). CONCLUSION: It was possible to assess the progress of liver fibrosis in NASH by evaluating the signal intensity-time course on Gd-EOB-DTPA-enhanced MRI.

Potential of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) for differential diagnosis of nonalcoholic steatohepatitis and fatty liver in rats using magnetic resonance imaging.

RATIONALE AND OBJECTIVES: To investigate the potential of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) for the differential diagnosis of nonalcoholic steatohepatitis (NASH) and fatty liver (FL). METHODS: Twenty-one male rats were divided into 3 groups. Seven rats in the NASH group were fed a choline-deficient diet for 10 weeks, and the 7 rats in the FL group were fed a standard diet also containing 1% (wt/wt) orotic acid for 4 weeks. As a control, 7 rats were fed a standard diet. After the feeding period, all rats were subjected to contrast-enhanced dynamic and delayed MRI with a 2D-FLASH technique. Gd-DTPA (0.1 mmol Gd/kg) and Gd-EOB-DTPA (0.025 mmol Gd/kg) were injected into the tail vein at 24-hour intervals. Signal intensities of the liver were measured for each MR image and the relative enhancement (RE) was calculated. In addition, the time of maximum RE (Tmax) and the half-life of RE (T1/2) in liver were compared. After MRI, the liver was histologically analyzed to evaluate steatosis, hepatitis, and fibrosis. RESULTS: Diffuse macrovesicular steatosis and severe fibrosis were observed in the NASH group, whereas diffuse microvesicular steatosis and rare fibrosis were observed in the FL group. Immediately after the Gd-DTPA injection, the RE in the liver of each group temporarily increased, and thereafter, rapid RE reduction was observed. However, a continuous increase and subsequent slow reduction of RE were induced after the Gd-EOB-DTPA injection. Although there was no difference between the Tmax and T1/2 of each group after the Gd-DTPA injection, Tmax and T1/2 of the NASH group were significantly prolonged in comparison with FL and control groups after the Gd-EOB-DTPA injection (P < 0.01). CONCLUSIONS: It was possible to differentiate NASH and FL by evaluating the SI time course on Gd-EOB-DTPA enhanced MRI.

Early decline of hemoglobin correlates with progression of ribavirin-induced hemolytic anemia during interferon plus ribavirin combination therapy in patients with chronic hepatitis C.

BACKGROUND: The aim of this study was to examine the factors correlated with the progression of ribavirin-induced hemolytic anemia in patients with chronic hepatitis C treated by interferon and ribavirin combination therapy. METHODS: This study was conducted on 505 patients by the Osaka Liver Disease Study Group. A decline of hemoglobin (Hb) concentration by 2 g/dl at the end of 2 weeks from the start of the treatment ("2 by 2" standard) was adopted as a predictive factor for progression to severe anemia. The ribavirin apparent clearance (CL/F) was also examined. RESULTS: Of 482 patients whose Hb value was more than 12 g/dl before the treatment, 68 patients (14%) had to discontinue ribavirin owing to severe anemia. Patients in the "2 by 2"-positive group (Hb decline over 2 g/dl) and the group with lower CL/F were significantly more likely to discontinue ribavirin owing to severe anemia. Discontinuation was more common among patients aged 60 years or older than for those under 60 years old (21% vs. 9%, P < 0.001). Among patients aged 60 years or older, only the "2 by 2" standard was significantly associated with the discontinuance of ribavirin owing to severe anemia in a multivariate analysis (odds ratio, 4.18; P < 0.001). CONCLUSIONS: The "2 by 2" standard of Hb decline can be used to identify patients likely to develop severe anemia. The early reduction of ribavirin can help prevent progression to severe anemia, thus allowing ribavirin therapy to be completed even in older patients.

Should aged patients with chronic hepatitis C be treated with interferon and ribavirin combination therapy?

UNLABELLED: The aim of this study was to investigate the efficacy and safety of combination therapy of interferon and ribavirin for aged patients with chronic hepatitis C. METHODS: This study was conducted at Osaka University Hospital and institutions participating in the Osaka Liver Disease Study Group on 329 patients with chronic hepatitis C receiving interferon and ribavirin combination therapy (group A, under 60 year old, n=199; group B, 60-64 year old, n=64; group C, over 65 year old (mean age, 67.8+/-2.2 year old, n=66)). Of the 293 patients who were tested for HCV serotype and HCV viral loads, 215 had HCV-RNA with serotype 1 and high viral loads (1H) and the other 78 had HCV-RNA with serotype 2 or low viral loads (non-1H). RESULTS: In per-protocol analysis, the overall SVR rate of 1H patients was 28% (51/184). Among the 1H patients, the SVR rate was significantly lower in group C (16%) and group B (17%) than in group A (34%) (p<0.05). The overall SVR rate of non-1H patients was 85% (57/67). No significant difference was found in the SVR rate among group C (79%), group B (100%), and group A (84%). On the other hand, the discontinuance of both drugs due to side effects was 29% (19/66) in group C, 20% (13/64) in group B, and 11% (21/199) in group A, with the discontinuance rates being higher in the older group (p=0.002). CONCLUSIONS: In aged chronic hepatitis C patients, interferon and ribavirin combination therapy can be recommended for the non-1H patients who showed a high SVR rate of approximately 65%, but not for the 1H patients.

Superparamagnetic iron oxide (SPIO) MRI contrast agent for bone marrow imaging: differentiating bone metastasis and osteomyelitis.

PURPOSE: We explored appropriate scan timing for bone marrow imaging enhanced using superparamagnetic iron oxide (SPIO) and evaluated the usefulness of SPIO in differentiating metastasis and osteomyelitis in patients. METHODS: To determine the adequate scan timing after administration of SPIO, 5 healthy subjects were examined using a 1.5T magnetic resonance (MR) imaging scanner. Sagittal images of their lumbar spines were obtained using short-TI inversion recovery (STIR) sequence before and 3, 6, 9, 24, and 48 hours after intravenous injection of 8 micromol Fe/kg SPIO (ferucarbotran). MR signal intensities (SIs) were evaluated. Based on the results, 12 patients, five with bone metastasis and seven with vertebral osteomyelitis, were examined using the same procedure before and 3 hours after intravenous injection of ferucarbotran at the same dose. SIs of the bone metastases, osteomyelitis, and surrounding normal bone marrow were measured, and relative enhancement (RE) was calculated for each lesion. RESULTS: In the healthy volunteers, maximum reduction in signal was observed 3 to 24 hours (P<0.05) after administration of SPIO; thereafter and up to 48 hours, the SI gradually recovered. In the patients, the RE of the bone metastases was -12.2%, which was significantly higher than that in the osteomyelitis (-35.0%, P<0.001) and normal bone marrow (-46.6%, P<0.0005). CONCLUSION: Maximum suppression of signal intensity in bone marrow was seen 3 hours after injection of ferucarbotran, the point at which ferucarbotran allows differentiation of bone metastasis from ostoemyelitis.

Potential of superparamagnetic iron oxide in the differential diagnosis of metastasis and inflammation in bone marrow: experimental study.

RATIONALE AND OBJECTIVES: The utility of ferucarbotran for the diagnosis of bone metastases was investigated using tumor-implanted rabbits. The potential of ferucarbotran in the differential diagnosis of metastasis and inflammation was also investigated. METHODS: Twelve rabbits were divided into 2 groups (tumor and inflammation groups). Six rabbits of tumor group were inoculated with VX2 tumor cell suspension, and the 6 rabbits of the inflammation group were inoculated with 10% croton oil in the bone marrow of the right femur. All rabbits were imaged using a clinical MRI system. Signal intensity in the bone marrow of the right femur was measured in each rabbit before and after the intravenous injection of 8 micromol Fe/kg of ferucarbotran. As a control, the signal intensity in the bone marrow of the left femur (the normal, intact femur) was measured in each rabbit. The change in signal intensity of each group was compared statistically. After MRI imaging, the femora were removed, and sections were prepared for microscopic examination. RESULTS: Signal intensity in the right femur of the tumor group did not change after injection, although that of the inflammation group and the control group decreased. In the histologic findings, tumors were widely spread in the right femur of the tumor group. The infiltration of pseudoeosinocytes was induced in the right femur of the inflammation group. CONCLUSIONS: This animal study showed that ferucarbotran was useful to detect bone marrow tumors. In addition, ferucarbotran may have potential in the differential diagnosis of bone metastasis and some kinds of inflammation.

Interstitial magnetic resonance lymphography using gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid in rabbits with lymph node metastasis.

RATIONALE AND OBJECTIVES: To investigate the dose dependency of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) for interstitial magnetic resonance (MR) lymphography and the detection of lymph node metastasis in rabbits. METHODS: Eighteen VX2 tumor-bearing rabbits were subjected to MR lymphography using clinical MRI equipment. The enhancement of popliteal lymph nodes was studied in these rabbits before and at 2.5 to 10 minutes after the subcutaneous administration of 4, 8, or 17 micromol Gd/kg of Gd-EOB-DTPA in TSE and 3D-FLASH (n = 6). Signal-to-noise ratio and contrast-to-noise ratio were statistically compared between each group by the Tukey test. After MR imaging, the popliteal lymph nodes were removed, and sections were prepared for microscopic examination. RESULTS: In the histologic findings, all metastases (3-12 mm) in the popliteal lymph nodes were detected by 3D-FLASH images. Gd-EOB-DTPA-enhanced T1WI showed a hypointense region for metastasis and a hyperintense region for nontumor regions, although the lymph nodes containing metastasis were detected as a hyperintense region by conventional PDWI and T2WI. Signal enhancement of the nontumor regions and contrast between the nontumor regions and metastasis showed dose dependency and reached a plateau at 8 micromol Gd/kg on T1WI (signal-to-noise ratio: 13.9 +/- 1.6; contrast-to-noise ratio: -12.7 +/- 1.7). CONCLUSIONS: This study showed that interstitial MR lymphography with Gd-EOB-DTPA can detect metastasis and that the optimal dose in rabbits is 8 micromol Gd/kg as a subcutaneous application.

Long-term clinical and virological outcomes of chronic hepatitis C after successful interferon therapy.

Clinical relevance of occult hepatitis C virus (HCV) and/or hepatitis B virus (HBV) infection(s) remains uncertain years after interferon (IFN) therapy for chronic hepatitis C. By 1993, 38 sustained virological responders (SVRs) showing HCV RNA clearance at 6 months post-treatment and 37 biochemical responders (BRs) with end-of-treatment alanine aminotransferase (ALT) normalization and subsequent 6-month stabilization within 2 x the upper limit of normal (ULN) were enrolled. They were monitored for 4.4-12 years (median 6.8), then 15 SVRs and 15 BRs underwent paired liver biopsies. Biopsy samples were tested for positive and negative HCV RNA strands, and HBV DNA surface and X sequences. All SVRs showed sustained serum HCV RNA clearance during follow-up, but hepatocellular carcinoma (HCC) developed in 4 (11%) SVRs. On paired liver biopsies, histological improvement was significant, but mild inflammation persisted in 87% of SVRs. Nonetheless, no HCV RNA sequence was amplified from liver tissues, and HBV DNA sequences were found in only one SVR. As for BRs, biochemical flare-up of >2 x ULN occurred at a 5-year risk of 41% (95% CI 24.7-56.4). The event was unpredictable but controllable by retreatment in 70%. Liver tissues after follow-up contained positive and negative HCV RNA strands, but no HBV DNA sequence was amplified. These results suggest that SVRs, albeit free of occult HCV and/or HBV infection(s) over a decade, retain mild liver inflammation and the risk of HCC. Occult HBV was also shown uninvolved in flare-up during follow-up of BRs.

Potential for differential diagnosis with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging in experimental hepatic tumors.

RATIONALE AND OBJECTIVES: To investigate whether dynamic and delayed magnetic resonance imaging (MRI) with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA), a hepatobiliary MRI contrast agent, has potential for the differential diagnosis of experimental hepatic tumors. METHODS: Twelve male rats received N-nitrosomorpholine solution as drinking water to induce hepatic tumors. After injection of Gd-EOB-DTPA, rats were subjected to dynamic and delayed MRI. The relative enhancement (RE) was calculated, and the time of the maximum RE (Tmax) was evaluated. After MRI, liver was histologically analyzed. RESULTS: One hundred sixty-three hepatic tumors 3-12 mm in diameter were induced after 18 weeks of treatment with 0.01 wt/vol% of N-nitrosomorpholine, and 81 of them were evaluated. The RE in hyperplastic nodules (HPNs) was significantly higher than that in moderately or poorly differentiated hepatocellular carcinomas (HCCs) in the late phase, whereas there was no significant difference in RE between well-differentiated HCCs and HPNs. The average Tmax in HPNs was about 13 minutes, whereas that of each differentiated HCCs was about 1 minute. CONCLUSIONS: It was possible to differentiate benign HPNs and malignant HCCs (especially well-differentiated HCCs) by evaluating the change of RE or comparison of Tmax with Gd-EOB-DTPA-enhanced MRI.