The rapid development of computer vision and deep learning has enabled these technologies to be applied to the automated classification and counting of microscope images, thereby relieving of some burden from pathologists in terms of performing tedious microscopic examination for analysis of a large number of slides for pathological lesions. Recently, the use of these digital methods has expanded into the field of medical image analysis. In this study, the Inception-v3 deep learning model was used for classification of chondrocytes from knee joints of rats. Knee joints were extracted, fixed in neutral buffered formalin, decalcified, processed and embedded in paraffin, and hematoxylin and eosin (H&E) stained. The H&E stained slides were converted into whole slide imaging (WSI), and the images were cropped to 79 × 79 pixels. The images were divided into training (60.42%) and test (39.58%) sets (46,349 and 30,360 images, respectively). Then, images containing chondrocytes were classified by Inception-v3 and accuracy was calculated. We visualized the images containing chondrocytes in WSIs by adding colored dots to patches. When images of chondrocytes in knee joints were evaluated, the accuracy was within the range of 91.20 ± 8.43%. Therefore, it is considered that the Inception-v3 deep learning model was able to distinguish chondrocytes from non-chondrocytes in knee joints of rats with a relatively high accuracy. The above results taken together confirmed that this deep learning model could classify the chondrocytes and this promising approach will provide pathologists a fast and accurate analysis of diverse tissue structures.

Traditionally, pathologists examine tissue slides under a microscope to find pathological lesions, and have the burden of finding the lesions among so many histopathology slides. Furthermore, inconsistency of diagnoses results differ corresponding to training among researchers. Therefore, accumulated research experience has led to the use of novel tools for increasing accuracy and consistency of diagnoses. With rapid transition from analog to digital methods and new developments in digital pathology, it is possible to use whole slide imaging (WSI) by scanning glass slides. Artificial intelligence (AI), including machine learning and deep learning using WSI, is starting to be applied to automatically classify and count microscope images, and this method has been expanded to include the field of medical image analysis. This review aims to define current trends toward AI application in the biomedical area, especially in the field of toxicopathology, outline current future business trends, and discuss multiple issues of diagnosis, quantification, three-dimensional reconstruction, molecular pathological research, and the future direction of AI in toxicopathology. Big data systems including a large amount of welldefined toxicopathological information will be highly useful for accuracy and corrections of diagnoses. In addition, the need for critical peer review is profound in the continuing educational process. Taken together, it is highly promising that AI model based on big data in the toxicopathological field could classify, detect, and segment pathological lesions in numerous organs of experimental animals and could help explain various biological mechanisms. This promising approach will provide an accurate and fast analysis of tissue structure and biological pathways using AI algorithms and big data.

The purpose of this study was to examine the characteristics of acetaminophen (APAP)-induced liver damage, using fluorescence bioimaging, serum biochemistry, and histopathology. At six weeks of age, eighteen mice were divided into three groups as group 1 (G1) as control, group 2 (G2) as fluorescence probe control and group 3 (G3) as APAP-treated. G3 mice were orally treated with APAP (800 mg/kg b.w.), while G1 and G2 mice were treated with 0.9% saline. Twenty-two hours after APAP treatment, G2 and G3 mice were intravenously treated with Annexin-Vivo 750 as probe, while G1 mice were treated with saline. Fluorescence bioimaging was performed at two hours after probe treatment. The mice were sacrificed and serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase were analyzed. Liver damage was examined by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. In vivo bioimaging, fluorescence intensity of the region of interest (ROI) was significantly increased in the livers of G2 and G3 mice compared with those in G1 mice (p<0.05 and p<0.01). In addition, ex vivo bioimaging confirmed that the fluorescence intensity of the ROI was significantly increased in the livers of G2 and G3 mice compared with those in G1 mice (p<0.05 and p<0.01). All examined serum parameters of G3 were significantly increased compared with G1 and G2 (p<0.05 and p<0.01). H&E examination showed acute hepatic cell necrosis in the livers of G3 mice, while there was no cell death in the livers of G1 and G2 mice. TUNEL staining also showed many cell death features in G3 mice, whereas no pathological findings were shown in G1 or G2 mice. In summary, fluorescence bioimaging showed the possibility of cell death detection in the livers of mice treated with APAP, and this was corroborated by serum chemistry and histopathological examination.

This study aimed to examine the effect of a mild elevation in serum cholesterol level in a porcine coronary overstretch restenosis model using a balloon angioplasty catheter or drug-eluting coronary stent. Pigs were divided into two groups and were fed a commercial normal diet (CND, n = 4) or a high-fat diet (HFD, n = 4) for 5 weeks. Coronary overstretch injury by balloon angioplasty or stent implantation was induced in the left anterior descending and left circumflex artery after 1 week of feeding. Histopathological analysis was performed at 4 weeks after coronary injury. During the experiment, the total cholesterol level in the HFD group increased by approximately 44.9% (from 65.9 ± 3.21 mg/dL at baseline to 95.5 ± 9.94 mg/dL at 5 weeks). The lumen area in the CND group was reduced in comparison with that in the HFD group after balloon angioplasty. After stent implantation, the injury score showed no significant difference. There were significant differences in the neointimal area (2.7 ± 0.33 mm2 in the CND group vs. 3.3 ± 0.34 mm2 in the HFD group, p<0.05), lumen area (2.6 ± 0.54 mm2 in the CND group vs. 2.0 ± 0.33 mm2 in the HFD group, p<0.05), and percent area stenosis (52.0 ± 7.96% in the CND group vs. 62.4 ± 5.15% in the HFD group, p<0.05). Body weight change was not different between the two groups. Increased serum cholesterol level activated vascular smooth muscle cell proliferation in the porcine coronary overstretch model.

The purpose of this study was to investigate diethylnitrosamine (DEN)-induced liver damage in zebrafish. Zebrafish larvae were divided into five groups after seventy-two hours fertilization: group 1 (G1) as control, group 2 (G2) as probe control, groups 3, 4, and 5 (G3, G4, and G5) as DEN treated at doses of 25, 50, and 100 μg/mL, respectively. At twenty-two hours after DEN treatment, groups 2, 3, 4, and 5 were treated with ApoFlamma H 675 at a dose of 100 μM/zebrafish. They were examined by fluorescence stereomicroscope at twenty-four hours after DEN treatment. After fixation, the zebrafish were processed, embedded, sectioned and stained with hematoxylin and eosin (HE) and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. Fluorescence intensity of the livers of G3, G4, and G5 was significantly increased compared with those of G1 (p<0.01). Furthermore, fluorescence intensity of the livers of G3 and G5 was significantly increased compared with those of G2 (p<0.05 and p<0.01). HE staining showed cell deaths in the livers of DEN-treated zebrafish and TUNEL staining confirmed cell death in the same location. Taken together, in vivo fluorescence bioimaging detected cell death in the liver of DEN-treated zebrafish. This outcome was confirmed with histopathological examination. The results of this study provide confidence for using zebrafish as a liver carcinogenesis model.

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of α-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

This study was carried out to investigate the protective effect of prednisolone in rabbit primary cultured articular chondrocytes treated with sodium nitroprusside (SNP), a nitric oxide donor. After a cell phenotype was determined, the MTT assay and Western blot analysis of type II collagen, cylooxygenase-2 (COX-2) and phosphorylated extracellular regulated kinase (pERK) were performed in the control, SNP (298 μg/ml) alone or SNP plus prednisolone (0.05-50 μg/ml)-treated rabbit articular chondrocytes. Immunofluorescence staining of type II collagen was also performed. Cell morphology indicated that SNP treatment induced cytotoxicity, and that the SNP-induced cytotoxicity was inhibited by prednisolone treatment. MTT assay showed that the SNP treatment resulted in a significant decrease in the level of cell viability compared with that of control (p<0.01), and that the prednisolone treatment resulted in a decrease in the SNP-induced cytotoxicity. SNP treatment resulted in a decrease in the level of type II collagen, compared with the control chondrocytes. The prednisolone treatment recovered the down-regulated expression of type II collagen induced by SNP, showing a significant level in 5 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p<0.05). A significant increase in COX-2 was significantly induced by the SNP treatment compared to control chondrocytes (p<0.01). The COX-2 expression was decreased by the prednisolone treatment, showing a significant level in 50 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p<0.05). These phenomena was confirmed by immunofluorescence staining. Furthermore, the SNP treatment significantly induced a decrease of pERK expression compared to the control chondrocytes (p<0.01). The prednisolone treatment recovered its expression, showing a significant level in 0.5 μg/ml of the prednisolone treatment group compared to the SNP treatment group (p<0.05). Taken the above results together, prednisolone is considered to inhibit SNP-induced cell death and dedifferentiation, and modulated expression of COX-2 and pERK in rabbit articular chondrocytes.

The purpose of this study was to investigate the lesions of a mouse collagen antibody-induced arthritis (CAIA) model using fluorescence bioimaging and micro-computed tomography (micro-CT) and to compare it with histopathological examination. Twelve mice were randomly divided into three groups: group 1 (G1) as control, group 2 (G2) as fluorescence probe control and group 3 (G3) as collagen antibodyinduced arthritis. The mice of G3 intravenously received anti-type II collagen 5-clone antibody cocktail (2 mg/mouse) on day 0 and intraperitoneally received lipopolysaccharide (50 μg/mouse) on day 3. On the while, the mice of G1 and G2 received 0.9% saline in equal volumes at equivalent times. Fluorescence bioimaging and micro-CT analysis were carried out to assess arthritis. Treatment with the collagen antibody cocktail increased the paw thickness of mice compared to those in both the control and probe-treated groups. Fluorescence bioimaging using a near infrared imaging agent showed high intensity in the joints of collagen antibody- treated mice, whereas those of control mice showed no signal. Micro-CT analysis of the knee joints of collagen antibody-treated mice showed rough and irregular articular appearance, whereas those of control mice showed normal appearance. Histopathological examination of the knee joints of collagen antibody-treated mice revealed destruction of cartilage and bony structure, synovial hyperplasia and infiltration of inflammatory cells. No cartilage destruction or inflammation was observed in control or probe control mice. Taken together, it is concluded that analyses of fluorescent bioimaging made it possible to evaluate CAIA lesions, comparable with those by micro-CT and histopathological examination in mice.

To clarify the role of stem cells in hepatocarcinogenesis, octamer-binding transcription factor 4 (Oct4) expression was investigated in mouse liver and embryonic cell lineages. In vivo, at 14 days of age, ten ICR mice were divided into two groups and treated with saline or diethylnitrosamine (DEN), and were sacrificed at 6 h after treatment. Livers were fixed in 10% neutral phosphate buffered formalin, embedded in paraffin, sectioned to a thickness of 5 μm, and immunohistochemical analysis of Oct4 was performed. In vitro, mouse embryonic stem cells, hepatic progenitor cells and hepatocytes, representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and RNA was isolated; Oct4 and Gadd45a mRNA were investigated. In vivo, Oct4 expression was not detected in saline-treated livers. However, its expression was observed in hepatocytes of mice treated with DEN, showing cytoplasmic staining. In vitro, Oct4 expression differed significantly for G4 on day 0 (P<0.05) and for G2 on day 22 (P<0.01) and G3 and G4 on day 40 (P<0.05 and P<0.01, respectively) compared with G1 at each time point. Gadd45a expression differed significantly in G4 (P<0.01) on day 0 and G4 on day 40 (P<0.01), compared with that of G1 at each time point. Taken together, Oct4 expression was increased by treatment with DEN in hepatocytes, however, not in embroyonic stem cells and hepatic progenitor cells. This finding suggests that Oct4 expression may be modulated in hepatocarcinogenesis induced by DEN.

Expression of epithelial cell adhesion molecule (EpCAM) in the early phase of hepatocarcinogenesis induced by diethylnitrosamine (DEN) was investigated. At 14 days of age, 60 ICR mice were divided into two groups and treated with saline (group 1) or DEN (group 2, 10 mg/kg of body weight, i.p. injection), and were sacrificed at 6 h and 1, 2, 3, 7, and 28 days after treatment with saline or DEN. During necropsy, half of the liver from saline- or DEN-treated mice was processed for histopathological examination and immunohistochemical staining of EpCAM and apoptosis. The remaining liver tissue was snap-frozen in liquid nitrogen for RNA extraction and analysis of EpCAM mRNA expression. Immunohistochemical examination showed that EpCAM expression was detected only in a small number of hepatocytes from saline-treated mice and its expression was detected in bile duct cells and round cells around portal areas, as well as hepatocytes in the livers of DEN-treated mice. In addition, multiple apoptotic cells were found in the livers of mice treated with DEN. EpCAM mRNA expression was significantly higher in DEN-treated mice at 1, 7, and 28 days compared to saline-treated mice at 6 h (P<0.01). Taken together, EpCAM expression and apoptosis were increased in liver by DEN treatment.

Liver cancer represents a major health problem with steadily increasing incidence rates. Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death. This study was conducted in order to investigate the gross findings following treatment with diethylnitrosamine (DEN) in mice. Sixteen male and female mice (B6C3F₁), initially 20 days of age, received intraparietal injection (20 mg/kg three times for a period of two weeks, IP) or were given drinking water (DW) containing 50 ppm DEN; all mice were sacrificed at the 80^th week of experiments. Hepatocellular adenoma (HCA) and HCC were induced in B6C3F₁), mice by administration of DEN. The numbers of HCA and HCC were 7.4±1.72 (IP) and 7.2±0.86 (DW) in male mice. However, no significant difference was observed between the DW and IP groups. The numbers of HCA and HCC were 0.67±0.33 (IP) and 2.0±0.63 (DW) in female mice. This study showed a tendency for high incidences of liver tumor with long-term exposure of newborn animals by drinking water.

“Younbaek”, a new noodle making wheat cultivar, was developed from the cross between “Keumkang” with white grain color and “Tapdongmil” by the Honam Agricultural Research Institute(HARI), National Institute of Crop Science (NICS), RDA, Korea in 2005. Amon