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.

Successful induction of pseudopregnancy using sonic vibration in mice.

Embryo transfer (ET) is an essential reproductive technology for the production of new animal strains and maintenance of genetic resources. We developed a method, named Easy-ET, to induce pseudopregnancy in female rats by artificial stimulation using sonic vibration instead of mating with vasectomized males. This study examined the application of this method for the induction of pseudopregnancy in mice. Offspring were obtained from two-cell embryos transferred into females with pseudopregnancy induced using sonic vibration in proestrus on the day before embryo transfer. Furthermore, high developmental rates of offspring were observed when pronuclear and two-cell embryos were transferred to females in estrus that were stimulated on the day of embryo transfer. Genome-edited mice were also obtained using frozen-warmed pronuclear embryos with clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) nucleases introduced using the technique for animal knockout system by electroporation (TAKE) method, which were transferred to females with pseudopregnancy induced on the day of embryo transfer. This study demonstrated that induction of pseudopregnancy by sonic vibration was also possible in mice.

Age-dependent sex difference of non-alcoholic fatty liver disease in TSOD and db/db mice.

According to previous clinical studies, the prevalence of non-alcoholic fatty liver disease (NAFLD) is higher in men than women only during the reproductive age. Animal models of NAFLD that reflect sex differences in humans have not been established. In this study, we examined sex differences in the hepatic lesions of Tsumura Suzuki obese diabetes (TSOD) and db/db mice, which are representative genetic models of NAFLD. Male and female TSOD and db/db mice were fed with a normal diet and tap water ad libitum. Six male and female mice of each strain were sacrificed at the ages of 3 and 9 months, respectively, and serum biochemical, pathological, and molecular analyses were performed. Serum aspartate aminotransferase (AST) levels were significantly higher in male than female mice of both strains at the age of 3 months; however, at 9 months, significant sex differences were not observed. Similarly, alanine aminotransferase (ALT) levels were significantly higher in male mice than in female TSOD mice at the age of 3 months; however, at 9 months, significant sex differences were not observed. Image analysis of histological slides revealed that the frequency of the steatotic area was significantly higher in male than female db/db mice at the age of 3 months; however, significant sex differences were not observed at 9 months. The frequency of Sirius red-positive fibrotic area was significantly higher in male than female mice in both strains at the age of 3 months; however, significant sex differences were not observed at 9 months. Serum AST and ALT levels and hepatic steatosis and fibrosis in TSOD and db/db mice showed age-dependent sex differences consistent with those observed in human NAFLD. These mice may be suitable for studying sex differences of the disease.

Iron-accumulating splenocytes may exacerbate non-alcoholic steatohepatitis through the production of proinflammatory cytokines and reactive oxygen species.

Non-alcoholic steatohepatitis (NASH) results from non-alcoholic fatty liver disease (NAFLD) via multiple-parallel events, including hepatic triglyceride accumulation, oxidative stress, and inflammation. The complex interaction between the liver and multiple other organs is involved in NASH development. Although spleen-derived humoral factors can directly contribute to NAFLD/NASH onset via the portal vein, the status of the spleen in the early stage of NASH remains unknown. Here, our aim was to investigate whether splenocytes may exacerbate NASH via the generations of reactive oxygen species (ROS) and proinflammatory cytokines. Iron accumulation was observed in the spleen but not the liver, and the proportion of phagocytic macrophages increased in the spleen of Tsumura Suzuki Obese Diabetes (TSOD) mice showing histological characteristics of NASH in the early stage. The splenocytes generated moderate amounts of ROS and released high amounts of tumor necrosis factor (TNF)-α in response to lipopolysaccharide, indicating excessive inflammatory cytokine released by activated macrophages in iron-accumulating spleens. Our results suggest that iron-accumulating splenocytes can easily induce inflammation and contribute to exacerbate NASH via the portal vein. Thus, the regulation of iron metabolism in the spleen should be considered in the development of novel therapeutic targets against NASH.

Successful pseudopregnancy of rats by short period artificial stimulation using sonic vibration.

Psuedopregnancy for embryo transfer (ET) is usually induced in rats by mating with vasectomized males. Previously, we successfully induced pseudopregnancy using sonic vibration instead (Easy-ET method). The transferred embryos developed normally. Conventionally, stimulation is performed 7 × 30 s with 5 min intervals at the day before ET. However, this protocol is time-consuming because it imitates natural mating behavior. Here, we investigated pseudopregnancy induction with shorter stimulation times. Stimulation was performed 2 × 30 s, with 30 s intervals at the proestrus stage at the day before ET. Of the transferred pronuclear or two-cell embryos, 43% or 62% developed normally, respectively. Furthermore, 67% or 68% of transferred pronuclear or two-cell embryos in rats at estrus stage stimulated on the day of ET developed normally, respectively. Pseudopregnancy was successfully induced with shorter stimulation. Furthermore, this protocol may be used to perform a single-day stimulation and ET operation at the estrus stage.