Impacts of Subchronic and Mild Social Defeat Stress on Plasma Putrefactive Metabolites and Cardiovascular Structure in Male Mice.

Psychosocial stress precipitates mental illnesses, such as depression, and increases the risk of other health problems, including cardiovascular diseases. In this study, we observed the effects of psychosocial stress on the histopathological features of systemic organs and tissues in a mouse psychosocial stress model, namely the subchronic and mild social defeat stress (sCSDS) model. There were several pathological findings in the tissues of both sCSDS and control mice. Mild fibrosis of the heart was observed in sCSDS mice but not in control mice. Extramedullary hematopoiesis in the spleen and hemorrhage in the lungs were observed in both the control and sCSDS mice. Focal necrosis of the liver was seen only in control mice. Furthermore, putrefactive substances in the blood plasma were analyzed because these metabolites originating from intestinal fermentation might be linked to heart fibrosis. Among them, plasma p-cresyl glucuronide and p-cresyl sulfate concentrations significantly increased owing to subchronic social defeat stress, which might influence cardiac fibrosis in sCSDS mice. In conclusion, several pathological features such as increased cardiac fibrosis and elevated plasma putrefactive substances were found in sCSDS mice. Thus, sCSDS mice are a potential model for elucidating the pathophysiology of psychosocial stress and heart failure.

Anterior segment dysmorphogenesis of the eye and glaucoma in MG-W gerbils.

Unilaterally swollen eyes were histopathologically characterized in four MG-W gerbils. The primary lesions resided in the anterior segment of the eye where neural crest cells play a critical role in embryonic development. They included indistinct filtration angle, unformed canal of Schlemm, hypoplastic iris, and ciliary body. The findings noted in the retina, optic nerve, optic tract, and lateral geniculate nucleus were consistent with the lesions induced following the persistent elevation of intraocular pressure as a result of insufficient drainage of aqueous humor. Thus, the present cases observed in the eyes of MG-W gerbils exemplified the anterior segment dysmorphogenesis associated with inadequate neural crest migration or differentiation, leading to subsequent glaucoma.

Drastic hypothermia after intraperitoneal injection of okadaic acid, a diarrhetic shellfish poisoning toxin, in mice.

The mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins had been used as the official method in Japan and also used in the world. In this study, hypothermia, one of the symptoms observed in mice after inoculation with DSP toxins, were characterized. Lethal and sublethal doses of okadaic acid (OA), a representative component of DSP toxins, were inoculated intraperitoneally into mice. Body-temperature changes over time were measured by an electronic thermometer or monitored by an infrared camera. Drastic hypothermia (<30°C in some mice) was observed in a few hours after administration of a lethal dose of OA. Dose-dependency was clearly seen between doses of OA inoculated and body-temperature decrease. Drastic hypothermia was also detected by using an infrared camera. These results suggest that hypothermia could be used as an index for the humane endpoint in experimental animal toxicological studies.

[Comparison of the Susceptibility to Tetrodotoxin in ddY Strain Mice from Different Breeder Companies].

The mouse bioassay for tetrodotoxin has been used in Japan and ddY strain mice are designated to be used in the assay. ddY strain is a closed-colony outbred mouse strain, originally from the National Institute of Health, however, the ddY mouse stocks of 3 breeders were divided 30 or more years ago. In this study, we investigated the differences in susceptibility to tetrodotoxin in ddY strain mice from 3 different breeders in Japan. No statistically significant differences were found among the ddY strain mice of 3 breeders, however, the coefficient of variation were different among the breeders. The results using the mice from one breeder were much stable compared with those using the mice from other 2 breeders.

Comparative toxicity of dinophysistoxin-1 and okadaic acid in mice.

The mouse bioassay for diarrhetic shellfish poisoning toxins has been used worldwide. In this study, dinophysistoxin-1 (DTX-1) and okadaic acid (OA) were compared for toxicity. The lethality rate increased and the median survival time decreased in a dose-dependent manner in both DTX-1 and OA. The median lethal dose value was 150.4 µg/kg (95% confidence interval=130.1-171.2 µg/kg) for DTX-1 and 185.6 µg/kg (95% confidence interval=161.2-209.6 µg/kg) for OA. The toxicity equivalent factor 1:1 has been used for OA and DTX-1 in the EU and Japan. Thus, it may be considered that toxicity potential of DTX-1 has remained underestimated as compared to that of OA and DTX-1 might be more toxic than OA.

Novel haemodialysis (HD) treatment employing molecular hydrogen (H2)-enriched dialysis solution improves prognosis of chronic dialysis patients: A prospective observational study.

Recent studies have revealed unique biological characteristics of molecular hydrogen (H2) as an anti-inflammatory agent. We developed a novel haemodialysis (E-HD) system delivering an H2 (30-80 ppb)-enriched dialysis solution by water electrolysis, and conducted a non-randomized, non-blinded, prospective observational study exploring its clinical impact. Prevalent chronic HD patients were allocated to either the E-HD (n = 161) group or the conventional HD (C-HD: n = 148) group, and received the respective HD treatments during the study. The primary endpoint was a composite of all-cause mortality and development of non-lethal cardio-cerebrovascular events (cardiac disease, apoplexy, and leg amputation due to peripheral artery disease). During the 3.28-year mean observation period, there were no differences in dialysis parameters between the two groups; however, post-dialysis hypertension was ameliorated with significant reductions in antihypertensive agents in the E-HD patients. There were 91 events (50 in the C-HD group and 41 in the E-HD group). Multivariate analysis of the Cox proportional hazards model revealed E-HD as an independent significant factor for the primary endpoint (hazard ratio 0.59; [95% confidence interval: 0.38-0.92]) after adjusting for confounding factors (age, cardiovascular disease history, serum albumin, and C-reactive protein). HD applying an H2-dissolved HD solution could improve the prognosis of chronic HD patients.

Possible clinical effects of molecular hydrogen (H2) delivery during hemodialysis in chronic dialysis patients: Interim analysis in a 12 month observation.

BACKGROUND AND AIM: It is supposed that enhanced oxidative stress and inflammation are involved with the poor clinical outcomes in patients on chronic dialysis treatment. Recent studies have shown that molecular hydrogen (H2) is biologically active as an anti-inflammatory agent. Thus, we developed a novel hemodialysis (E-HD) system which delivers H2 (30 to 80 ppb)-enriched dialysis solution, to conduct a prospective observational study (UMIN000004857) in order to compare the long-term outcomes between E-HD and conventional-HD (C-HD) in Japan. The present interim analysis aimed to look at potential clinical effects of E-HD during the first 12 months observation. SUBJECTS AND METHOD: 262 patients (140, E-HD; 122, C-HD) were subjected for analysis for comprehensive clinical profiles. They were all participating in the above mentioned study, and they had been under the respective HD treatment for 12 consecutive months without hospitalization. Collected data, such as, physical and laboratory examinations, medications, and self-assessment questionnaires on subjective symptoms (i.e., fatigue and pruritus) were compared between the two groups. RESULTS: In a 12-month period, no clinical relevant differences were found in dialysis-related parameters between the two groups. However, there were differences in the defined daily dose of anti-hypertensive agents, and subjective symptoms, such as severe fatigue, and pruritus, which were all less in the E-HD group. Multivariate analysis revealed E-HD was an independent significant factor for the reduced use of anti-hypertensive agents as well as the absence of severe fatigue and pruritus at 12 months after adjusting for confounding factors. CONCLUSION: The data indicates E-HD could have substantial clinical benefits beyond conventional HD therapy, and support the rationale to conduct clinical trials of H2 application to HD treatment.

High-density Association Mapping and Interaction Analysis of PLA2R1 and HLA Regions with Idiopathic Membranous Nephropathy in Japanese.

Although recent studies showed anti-PLA2R antibody plays a crucial role in idiopathic membranous nephropathy (IMN), detailed HLA mapping and interaction between the HLA genes and PLA2R1 have not been investigated in IMN. We genotyped across the PLA2R1 gene and the HLA region, using 183 IMN patients and 811 healthy controls. Five SNPs around the PLA2R1 gene were significantly associated with IMN. In addition to the two SNPs previously reported to be strongly associated with IMN, rs3749119 and rs35771982 (OR 3.02 and 2.93, P = 3.24E-14 and 4.64E-14, respectively), two novel intronic SNPs (rs2715928 and rs16844715) were also identified as IMN-associated SNPs (OR = 2.30 and 2.51, P = 3.15E-10 and 5.66E-13, respectively). In the HLA gene analysis, DRB1*1501 and DQB1*0602 were strongly associated with IMN (P = 1.14E-11 and 1.25E-11, respectively). The interaction was strongest between HLA-DRB1*15:01 - HLA-DQB1*06:02 and the intronic SNP rs2715928 (OR = 17.53, P = 4.26E-26). Furthermore, positive interaction was also observed between HLA-DRB1*15:01 - HLA-DQB1*06:02 and the missense SNP rs35771982 (OR = 15.91, P = 2.76E-29), which is in strong linkage disequilibrium with 5'UTR SNP rs3749119, and intronic SNP rs16844715 (OR = 15.91, P = 2.30E-26) for IMN. Neither HLA-DRB1*15:01 nor HLA-DQB1*06:02 was associated with steroid responsiveness, overall survival and renal survival during the observation period of mean 11 years though limited number of analysis.

Differences in susceptibility of mouse strains to tetrodotoxin.

The mouse bioassay for tetrodotoxin has been used for many years in Japan. To the best of our knowledge, however, there have only been a few reports that have specifically investigated differences in susceptibility to tetrodotoxin among mouse strains. In this study, we investigated the response of various mouse strains to tetrodotoxin. Tetrodotoxin solution was injected intraperitoneally into male mice of 5 inbred strains (A/J, BALB/c, C3H/He, C57BL/6, and DBA/2) and male and female mice of 2 non-inbred strains (ddY and ICR). Significant differences in susceptibility to tetrodotoxin were found among the mouse strains tested. In comparison to the ddY male mice, which are designated to be used in the Japanese reference method, the 5 inbred strains of mice tested were significantly more resistant to tetrodotoxin. However, no significant differences in tetrodotoxin susceptibility were observed between ddY male and female mice or between ddY male mice and ICR male and female mice. These results indicate that the users of the mouse bioassay should pay attention to differences in mouse strain in susceptibility to tetrodotoxin.

Comparison of toxicity between saxitoxin and decarbamoyl saxitoxin in the mouse bioassay for paralytic shellfish poisoning toxins.

The mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins has been used in the AOAC Official Method and the official Japanese method. In the AOAC Official Method, the saxitoxin (STX) standard provided by the U.S. Food and Drug Administration (FDA) is used, but no standard is used in the official Japanese method. The objective of this study was to compare the toxicity of decarbamoyl STX (dcSTX), one of the derivatives of STX and a candidate standard for the MBA for PSP toxins in Japan, to that of FDA STX in the MBA platform. In this study, the toxicity of dcSTX was 918.0 ± 44.9 mouse units/µmol, and the relative toxicity ratio of dcSTX to FDA STX based on moles was 0.478.

Bacterial translocation in alymphoplasia (aly/aly) mice.

Bacterial translocation (BTL) is defined as the passage of viable bacteria from the gastrointestinal tract to the organs. This study was to elucidate the roles of Peyer's patches (PPs) and/or mesenteric lymph nodes (MLNs) in BTL. Alymphoplastic mutant mice and phenotypically normal heterozygous micewere dominantly colonized with streptomycin-resistant Escherichia coli and BTL was examined. In PP- and MLN-competent mice, BTL to MLNs was detected in 100% of mice, but BTL to organs was rare (25%). On the other hand, in PP- and MLN-deficientmice, BTL to organs was detected in 91% of mice. The results clearly indicate that PPs are not the only site for bacterial entry.

Skin autofluorescence predicts cardiovascular mortality in patients on chronic hemodialysis.

Tissue accumulation of advanced glycation end products (AGE) is thought to contribute to the progression of cardiovascular disease (CVD). Skin autofluorescence, a non-invasive measure of AGE accumulation using autofluorescence of the skin under ultraviolet light, has been reported to be an independent predictor of mortality associated with CVD in Caucasian patients on chronic hemodialysis. The aim of this study was to assess the predictive value of skin autofluorescence on all-cause and cardiovascular mortality in non-Caucasian (Japanese) patients on chronic hemodialysis. Baseline skin autofluorescence was measured with an autofluorescence reader in 128 non-Caucasian (Japanese) patients on chronic hemodialysis. All-cause and cardiovascular mortality was monitored prospectively during a period of 6 years. During the follow-up period, 42 of the 128 patients died; 19 of those patients died of CVD. Skin autofluorescence did not have a significant effect on all-cause mortality. However, age, carotid artery intima-media thickness (IMT), serum albumin, high-sensitivity C-reactive protein (hsCRP), skin autofluorescence and pre-existing CVD were significantly correlated with cardiovascular mortality. Multivariate Cox regression analysis showed skin autofluorescence (adjusted hazard ratio [HR] 3.97; 95% confidence interval [CI]1.67-9.43), serum albumin (adjusted HR 0.05; 95% CI 0.01-0.32), and hsCRP (adjusted HR 1.55; 95% CI 1.18-2.05) to be independent predictors of cardiovascular mortality. The present study suggests that skin autofluorescence is an independent predictor of cardiovascular mortality in non-Caucasian (Japanese) patients on chronic hemodialysis.

Influence of body weight of mice on the susceptibility to okadaic acid, a diarrhetic shellfish poisoning toxin.

The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. However, different body weight ranges of mice are designated to be used in the Japanese official method and European Union procedure. In this study we investigated whether and to what extent the body weights of the mice affect the susceptibility to DSP toxins. A lethal dose of okadaic acid, one of the representative DSP toxins, was injected intraperitoneally into mice of five different body weight range groups, from 14 to 24 g. The mice were observed until 24 h after injection. The lethality was 100% in the 14-15 and 16-17 g groups, 80% in the 19-20 g group, 50% in the 21-22 g group, and 40% in the 23-24 g group, with significant differences. Survival analysis indicated a relationship between body weights of mice and susceptibility to okadaic acid. These results would be quite useful not only for the MBA, but also to improve understanding of the biological responses to DSP toxins.

Skin autofluorescence is associated with the progression of chronic kidney disease: a prospective observational study.

BACKGROUND: Advanced glycation end product (AGE) accumulation is thought to be a measure of cumulative metabolic stress that has been reported to independently predict cardiovascular disease in diabetes and renal failure. The aim of this study was to evaluate the association between AGE accumulation, measured as skin autofluorescence, and the progression of renal disease in pre-dialysis patients with chronic kidney disease (CKD). METHODS: Skin autofluorescence was measured noninvasively with an autofluorescence reader at baseline in 449 pre-dialysis patients with CKD. The primary end point was defined as a doubling of serum creatinine and/or need for dialysis. RESULTS: Thirty-three patients were lost to follow-up. Forty six patients reached the primary end point during the follow-up period (Median 39 months). Kaplan-Meier analysis showed a significantly higher risk of development of the primary end points in patients with skin autofluorescence levels above the optimal cut-off level of 2.31 arbitrary units, derived by receiver operator curve analysis. Cox regression analysis revealed that skin autofluorescence was an independent predictor of the primary end point, even after adjustment for age, gender, smoking history, diabetes, estimated glomerular filtration rate and proteinuria (adjusted hazard ratio 2.58, P = 0.004). CONCLUSIONS: Tissue accumulation of AGEs, measured as skin autofluorescence, is a strong and independent predictor of progression of CKD. Skin autofluorescence may be useful for risk stratification in this group of patients; further studies should clarify whether AGE accumulation could be one of the therapeutic targets to improve the prognosis of CKD.

Growth of Listeria monocytogenes in refrigerated ready-to-eat foods in Japan.

The ability of L. monocytogenes to grow in a series of Japanese ready-to-eat (RTE) foods, including boiled baby sardine and Japanese pickle, was tested at two different refrigeration temperatures. In RTE foods in which L. monocytogenes can grow, growth was significantly higher at 10°C than that at 4°C during their shelf lives and growth patterns varied extensively among the different types of foods. However, growth did not occur at 4°C within the shelf life of certain RTE foods, such as broiled squid. The patterns of growth were varied extensively with different sample types. These results suggest that some types of traditional Japanese RTE foods stored at 10°C may be potential sources of listeriosis. To reduce the risk of food-borne listeriosis, studies to determine the contamination levels in RTE foods and the effects of storage temperature on their shelf lives are needed.

Sequence of busulfan-induced neural progenitor cell damage in the fetal rat brain.

The sequence of neural progenitor cell (NPC) damage induced in fetal rat brain by transplacental exposure to busulfan, an antineoplastic bifunctional-alkylating agent, on gestational day 13 was examined by immunohistochemical and real-time RT-PCR analyses. Following busulfan treatment, pyknotic NPCs first appeared in the medial layer and then extended to the dorsal layer of the ventricular zone (VZ) of the telencephalon. Pyknotic NPCs that were immunohistochemically positive for cleaved caspase-3, i.e. apoptotic NPCs, began to increase at 24 h after treatment, peaked at 48 h, and returned to the control levels at 96 h. On the other hand, the index (%) of phospho-histone H3-positive NPCs, i.e. mitotic NPCs, and that of BrdU-positive NPCs, i.e. S-phase cells, decreased in accordance with the increase in the index of apoptotic NPCs. Prior to the peak time of apoptotic NPCs, the indices of p53- and p21-positive NPCs peaked at 36 h. In addition, the expression levels of p21 and Puma (p53-target genes) mRNAs were elevated in real-time RT-PCR analysis. These findings indicated that busulfan not only induced apoptosis through the p53-mediated intrinsic pathway but also inhibited cell proliferation in NPCs, resulting in a reduction of the width of the telencephalon. On the other hand, in spite of up-regulation of p21 expression, the expression of cyclin D1, part of the cell cycle machinery of the G1/S transition, and the expression levels of Cdc20 and cyclin B1 which are involved in G2/M transition, showed no changes, giving no possible information of busulfan-induced cell cycle arrest in NPCs.

Differences in susceptibility to okadaic acid, a diarrhetic shellfish poisoning toxin, between male and female mice.

The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. In the Japanese and EU methods, male mice are designated to be used for MBA. Female mice were described to be less susceptible than male mice. To the best of our knowledge, however, there have been no reports on the details of sex differences in susceptibility to DSP toxins. In this study, we investigated whether, and to what extent, female mice are less sensitive to DSP toxins. A lethal dose of okadaic acid (OA), one of the representative DSP toxins, was injected intraperitoneally into mice. The mice were observed until 24 hours after injection. Both male and female mice of ICR and ddY strains, which are designated in the Japanese official method, were compared. All the mice were four weeks old and weighed 18-20 g. The experiments were repeated twice. The lethality was 70%-100%. Survival analysis showed no sex differences in susceptibility to OA, but ICR female mice showed significant resistance compared with other groups in one out of two trials. These results indicate that sex differences were not clear but, nonetheless, male mice showed more stable results.

Susceptibility of different mice strains to okadaic acid, a diarrhetic shellfish poisoning toxin.

The mouse bioassay is widely used to detect diarrhetic shellfish poisoning (DSP) toxins. To the best of our knowledge, however, there have been no reports specifically on strain differences in susceptibility to DSP toxins. In this study, we investigated the susceptibility of different mice strains to okadaic acid (OA), one of the representative DSP toxins. A lethal dose of OA was injected intraperitoneally (i.p.) into mice. The mice were observed until 24 h after injection. Five inbred strains (A/J, BALB/c, C3H/He, C57BL/6, and DBA/2) and two non-inbred strains (ddY, and ICR) of mice were compared. All the mice were male, weighed 16-20 g, and were 4-5 weeks old. The lethality was 90-100% in the A/J, BALB/c, ddY, and ICR strains, 70-80% in the C3H/He and C57BL/6 strains, and 40% in DBA/2 strain. Survival analysis showed that the BALB/c, C57BL/6, ddY, and ICR strains died earlier and the A/J, C3H/He and DBA/2 strains survived longer. These results indicate that significant differences may exist in the susceptibility of mice strains to OA.

Age-dependent changes in intraepithelial lymphocytes (IELs) of the small intestine, cecum, and colon from young adult to aged mice.

We previously reported the regional differences in the IELs present in the proximal (P), middle (M), and distal (D) parts of the small intestine, cecum (Ce), and colon (Co) of mice. In this study, we investigated the age-dependent changes in the regional differences of IELs from young adult to aged mice. In this experiment, 3-, 6-, 12-, 18-, and 24-month-old mice were examined. IELs were separately isolated from 5 parts of the intestines and analyzed by flow cytometry. Regional differences in the number and phenotype of IELs showed the same trends in all age groups. The number of IELs was highest in 6-month-old mice and then gradually decreased with age. As to IEL subsets, age-related changes were not seen except for a few subsets among the age groups. We conclude that age-related decreases in IELs in mouse small intestine may be one of the aging phenomena of the intestinal immune system. Such age-related decreases in IELs may be concerned with the increased liability to intestinal infections in the elderly.

Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan.

The antimicrobial susceptibility of 201 Listeria monocytogenes isolates from foods, environments, animals and human patients in Japan was determined. All isolates were susceptible to ampicillin, the first choice of drug for listeriosis treatment, chloramphenicol, dihydrostreptomycin, erythromycin, enrofloxacin, gentamicin, kanamycin, lincomycin, nosiheptide, salinomycin, vancomycin, and virginiamycin. A human strain was resistant to oxytetracycline. The Minimum Inhibitory Concentration (MIC) for 50% of the strains and the MIC for 90% of the strains were comparable in all the isolates. This is the first investigation to compare antibiotic resistances between isolates from foods and isolates from human patients in Japan. The result showed that most of the isolates were susceptible to antibiotics used in this study.