Establishment of repeated liver biopsy technique in experimental mice.

Biopsy is a commonly used method for determining pathological diagnoses by directly using human tissues and cells. Biopsies are widely used to determine disease progression and treatment efficacy. Although organs and tissues are usually obtained by sacrifice during animal experiments, it is theoretically possible to use the same biopsy techniques in humans. In the present study, we examined the feasibility of performing four repeated liver biopsies in a spontaneous metabolic syndrome mouse model. Even though a small number of mice died accidently, most mice were able to undergo four liver biopsies without significant adverse events. We also performed three liver biopsies in mouse liver tumor carcinogen models at 4, 8, and 12 weeks of age. In addition to the sample collected at 16 weeks of age during sacrifice, we successfully collected four liver samples from the same mice at different stages of disease progression. The application of this liver biopsy technique might make it possible for direct evaluation of pathological conditions in the same individual over time, thereby reducing the number of experimental animals.

Spontaneous Occurrence of Various Types of Hepatocellular Adenoma in the Livers of Metabolic Syndrome-Associated Steatohepatitis Model TSOD Mice.

Male Tsumura-Suzuki Obese Diabetes (TSOD) mice, a spontaneous metabolic syndrome model, develop non-alcoholic steatohepatitis and liver tumors by feeding on a standard mouse diet. Nearly 70% of liver tumors express glutamine synthetase (GS), a marker of hepatocellular carcinoma. In contrast, approximately 30% are GS-negative without prominent nuclear or structural atypia. In this study, we examined the characteristics of the GS-negative tumors of TSOD mice. Twenty male TSOD mice were sacrificed at 40 weeks and a total of 21 tumors were analyzed by HE staining and immunostaining of GS, liver fatty acid-binding protein (L-FABP), serum amyloid A (SAA), and beta-catenin. With immunostaining for GS, six (29%) tumors were negative. Based on the histological and immunohistological characteristics, six GS-negative tumors were classified into several subtypes of human hepatocellular adenoma (HCA). One large tumor showed generally similar findings to inflammatory HCA, but contained small atypical foci with GS staining and partial nuclear beta-catenin expression suggesting malignant transformation. GS-negative tumors of TSOD mice contained features similar to various subtypes of HCA. Different HCA subtypes occurring in the same liver have been reported in humans; however, the diversity of patient backgrounds limits the ability to conduct a detailed, multifaceted analysis. TSOD mice may share similar mechanisms of HCA development as in humans. It is timely to review the pathogenesis of HCA from both genetic and environmental perspectives, and it is expected that TSOD mice will make further contributions in this regard.

Neonatal streptozotocin treatment rapidly causes different subtype of hepatocellular carcinoma without persistent hyperglycemia in 4CS mice fed on a normal diet.

Although diabetes mellitus (DM) is a well-known risk factor for hepatocellular carcinoma (HCC), the underlying mechanisms have not yet to be defined. We previously reported that DIAR mice fed with standard murine diet developed type 1 diabetes and HCC at age of 16 weeks old with a neonatal streptozotocin treatment (n-STZ). Because DIAR mice did not manifest obesity nor develop steatohepatitis, hyperglycemia with streptozotocin trigger or streptozotocin alone might turn on the hepato-carcinogenesis. An insulin-recruitment to DIAR-nSTZ mice showed an increased frequency of HCC during the first 12 weeks of age, although the diabetic indications notably improved. To elucidate the role of hyperglycemia in hepato-carcinogenesis, we performed a head-to-head comparative study by using 4CS mice and DIAR mice with n-STZ treatment. Newborn 4CS mice and DIAR mice were divided into STZ treated group and control group. The blood glucose levels of DIAR-nSTZ mice increased at age of eight weeks, while that of 4CS-nSTZ mice were maintained in the normal range. At eight weeks old, three out of five DIAR-nSTZ mice (60%) and one out of ten 4CS-nSTZ mice (10%) developed multiple liver tumors. At age of 12 weeks old, all eight of DIAR-nSTZ mice (100%) and two of 10 4CS-nSTZ mice (20%) developed multiple liver tumors. At 16 weeks old, all animals of DIAR-nSTZ and 4CS-nSTZ mice occurred liver tumors. DIAR-nSTZ showed hyperglycemia and HCC, and 4CS-nSTZ developed HCC without hyperglycemia. These results were interpreted that the onset of HCC maybe not related to the presence or absence of hyperglycemia but nSTZ treatment. On the other hand, since the carcinogenesis of 4CS-nSTZ is delayed compared to DIAR-nSTZ, hyperglycemia may play a role in the progression of carcinogenesis. Histologically, the liver tumor appeared irregularly trabecular arrangements of hepatocytes with various degrees of nuclear atypia. By immunohistochemical analyses, all liver tumors showed positive staining of glutamine synthetase (GS), an established human HCC marker. The expression pattern of GS was divided into a strong diffuse pattern and weak patchy pattern, respectively. The liver tumor showing the weak GS-patchy pattern expressed biliary/stem markers, EpCAM, and SALL4, partially. Because 4CS-nSTZ mice did not show any metabolic complications such as gaining body weight or high blood glucose level, it is a unique animal model with a simple condition to investigate hepatic carcinogenesis by excluding other factors.