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Oxytocin (OXT) is a neuropeptide that regulates memory, emotion, stress response, and behavior in the brain. In our previous study with cattle, we demonstrated the anti-stress effect of intracerebroventricularly administered OXT on the central nervous system. However, it is important to investigate the effects of this peptide after intranasal administration, as it offers convenience and non-invasiveness for practical use. Therefore, this study investigated the effects of intranasal OXT on the behavior and autonomic nervous system of Holstein steers. The experiment followed a within-subjects design, including a total of six steers. Each steer received intranasal administration of either 1 mL of saline (SAL), 100 µg OXT (OXT100), or 200 µg OXT (OXT200). However, due to some issues, the sample size for the OXT200 group was reduced to five. After these treatments, we conducted electrocardiography recordings to analyze heart rate variability (HRV) and also made behavioral observations for 90 min. OXT200 tended to increase the time spent ruminating while lying down (Steel's multiple comparison test; P=0.053). In contrast, OXT treatment did not affect HRV indices. In conclusion, the current OXT dosage did not significantly affects behavior or the autonomic nervous system. However, the observed tendency to increase rumination may suggest a central effect of OXT.

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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KOICHI TSUNEYAMA reports financial support was provided by JSPS-KAKENHI. KOICHI TSUNEYAMA reports a relationship with JSPS-KAKENHI that includes: funding grants., (© 2023 The Authors.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Dungubat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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.

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

This study was conducted to investigate the effect of tryptophan (TRP) supply on the thermoregulatory responses via brain serotonin (5-HT) in cattle. In period 1, 12 Holstein steers were kept under a constant room temperature (22°C) and were administered the intravenous (i.v.) infusion of saline or TRP (38.5 mg/kg/2 h). Changes in rectal temperature (RT), 5-HT concentration in the cerebrospinal fluid (CSF), and other factors involved in thermoregulation were measured. In period 2, the steers received the same treatments as in period 1; however, the room temperature was elevated from 22°C to 33°C during i.v. infusion and maintained at 33°C for 3 h. 5-HT concentration in CSF increased following TRP infusion in both periods, and RT significantly decreased following TRP infusion only in period 2. The effect of TRP on respiration rate and plasma prolactin and total triiodothyronine concentrations was not significant. These results suggest that increase in TRP supply can attenuate increase in RT in response to acute heat stress through the increase in brain 5-HT, followed by presumable increase in evaporative heat loss from the skin surface in cattle. It is possible that the increase in peripheral blood TRP metabolites could also participate in the hypothermic effect of TRP., (© 2018 Japanese Society of Animal Science.)

The effects of l-DOPA, a precursor of dopamine (DA), and sulpiride, a D 2 -type DA receptor blocker, on growth hormone (GH) and prolactin (PRL) secretion were investigated in steers. Eight Holstein steers (212.8 ± 7.8 kg body weight) were used. Lighting conditions were 12:12 L:D (lights on: 06.00-18.00 hours). Blood samplings were performed during the daytime (11.00-15.00 hours) and nighttime (23.00-03.00 hours). Intravenous injections of drugs or saline were performed at 12.00 hour for the daytime and 00.00 hour for the nighttime, respectively. Plasma GH and PRL concentrations were determined by radioimmunoassay. l-DOPA did not alter the GH secretion when it was injected at 12.00 hour (spontaneous GH level at its peak). On the other hand, l-DOPA increased GH secretion at 00.00 hour (GH level at its trough). Injection of sulpiride suppressed GH secretion at 12.00 hour but did not affect GH levels at 00.00 hour. l-DOPA inhibited and sulpiride stimulated PRL release during both periods. These results suggest that dopaminergic neurons have stimulatory action on GH secretion and inhibitory action on PRL secretion in cattle. In addition, injection time should be considered to evaluate the exact effects on GH secretion due to its ultradian rhythm of GH secretion in cattle., (© 2017 Japanese Society of Animal Science.)

We previously reported that Tsumura-Suzuki obese diabetic (TSOD) mice, a polygenic model of spontaneous type 2 diabetes, is a valuable model of hepatic carcinogenesis via non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). One of the characteristics of tumors in these mice is the diffuse expression of glutamine synthetase (GS), which is a diagnostic marker for hepatocellular carcinoma (HCC). In this study, we performed detailed histopathological examinations and found that GS expression was diffusely positive in >70% of the hepatic tumors from 15-month-old male TSOD mice. Translocation of β-catenin into nuclei with enhanced membranous expression also occurred in GS-positive tumors. Small lesions (<1 mm) in GS-positive cases exhibited dysplastic nodules, with severe nuclear atypia, whereas large lesions (>3 mm) bore the characteristics of human HCC, exhibiting nuclear and structural atypia with invasive growth. By contrast, the majority of GS-negative tumors were hepatocellular adenomas with advanced fatty change and low nuclear grade. In GS-negative tumors, loss of liver fatty acid-binding protein expression was observed. These results suggest that the histological characteristics of GS-positive hepatic tumors in TSOD mice resemble human HCC; thus, this model may be a useful tool in translational research targeting the NAFLD/NASH-HCC sequence.

This study was conducted to investigate the possibility of suppression of stress-induced cortisol (CORT) secretion by tryptophan (TRP) administration and to better understand its regulatory mechanisms by using a noradrenaline (NA) injection into the third ventricle (3V) as a stress model in cattle. A total of 25 Holstein steers with a cannula in the 3V were used. First, the increase in CORT secretion was observed following a NA injection into the 3V in a dose-dependent manner, verifying the appropriateness of this treatment as a stress model of CORT secretion (Experiment 1). The effect of prior-administration of TRP into peripheral blood with a dose that has been demonstrated to increase brain 5-hydroxytryptamine levels on the elevation of plasma CORT induced by NA or corticotropin-releasing hormone (CRH) was then examined (Experiment 2). The prior administration of TRP suppressed NA-induced, but not CRH-induced, CORT elevation. These results suggest that an increase in TRP absorption into peripheral blood could suppress the stress-induced CORT secretion in cattle via the attenuation of the stimulatory effect of NA on the hypothalamic CRH release., (© 2015 Japanese Society of Animal Science.)