Quantitative and antioxidative behavior of Trolox in rats' blood and brain by HPLC-UV and SMFIA-CL methods.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10(3) antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0-360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood-brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified.

Impacts of dosing and drug withdrawal period on tacrolimus-based triple therapy in a non-human primate renal transplantation model.

Non-human primate (NHP) renal transplantation models are widely used vivo models for researching new immunosuppressive therapies including allograft tolerance strategies. To enroll animals into a tolerance study, an immunosuppressive regimen that efficiently establishes stable renal function in NHPs is needed. Here, we assessed the effect of triple therapy comprising 2.0 mg/kg tacrolimus, mycophenolate mofetil and a steroid and its success rate for achieving stable renal function. In addition, to predict the pathophysiological consequences of withdrawing immunosuppressants, an indispensable process after induction of tolerance, we also assessed changes in the stable renal state maintained by triple therapy after drug withdrawal. Six cynomolgus monkeys were used. The median survival time was >176 days over the dosing period and 45 days after drug withdrawal. The triple therapy successfully induced stable graft function without calcineurin inhibitor nephrotoxicity in three of six recipients, although adopting trough-dependent tacrolimus dose adjustment rather than a preset dose regimen could improve on the present strategy. Further, drug withdrawal led to deterioration of renal function, de novo donor specific antibody production and increased the memory/naïve T cell ratio within two weeks post drug withdrawal. We expect that these findings contribute to establish one of the choices for animal model for evaluating future tolerance therapy for renal transplantation.

Sedative and physiological effects of alfaxalone intramuscular administration in cynomolgus monkeys (Macaca fascicularis).

To evaluate the sedative and physiological effects of alfaxalone intramuscular (IM) administration, 12 healthy cynomolgus monkeys were administered single IM doses of alfaxalone at 0.625 mg/kg (ALFX0.625), 1.25 mg/kg (ALFX1.25), 2.5 mg/kg (ALFX2.5), 5 mg/kg (ALFX5), 7.5 mg/kg (ALFX7.5), or 10 mg/kg (ALFX10); saline was used as the control (CONT). The sedative effects were subjectively evaluated using a composite measure scoring system in six animals. Changes in respiratory rate, pulse rate, non-invasive blood pressure, percutaneous oxygen-hemoglobin saturation (SpO2), and rectal temperature were observed after IM treatments in the other six animals. All animals were allowed to lay down following the ALFX5, ALFX7.5, and ALFX10 treatments, whereas lateral recumbency was achieved in only two animals after ALFX2.5 treatment and none after the CONT, ALFX 0.625, and ALFX1.25 treatments. The median time (interquartile range) to lateral recumbency was 6.5 min (5.3-7.8), 4.0 min (4.0-4.0), and 3.0 min (3.0-3.8), and the duration of immobilization was 27.5 min (19.0-33.8), 56.0 min (42.3-60.8), and 74.5 min (62.8-78.0) after the ALFX5, ALFX7.5, and ALFX10 treatments, respectively. Endotracheal intubation was achieved in all six animals after the ALFX7.5 and ALFX10 treatments. Dose-dependent decreases in respiratory rate, non-invasive blood pressure, SpO2, and rectal temperature were observed, and the quality of recovery was smooth in all animals after the ALFX5, ALFX7.5, and ALFX10 treatments. Thus, alfaxalone IM induced a dose-dependent sedative effect in cynomolgus monkeys, but at higher doses, hypotension, hypoxemia, and hypothermia could be induced.

Effects of housing conditions on behaviors and biochemical parameters in juvenile cynomolgus monkeys (Macaca fascicularis).

To investigate the effects of environmental enrichment on laboratory monkeys, we studied behavioral and physiological differences following changes in housing conditions. Ten male and female juvenile cynomolgus monkeys were first housed in pairs for 8 weeks after quarantine/acclimatization (singly housed) and subsequently housed alone for the next 8 weeks. Monkeys were subjected to evaluations of body weight gain, stereotypic or affiliative behaviors, cortisol, 4-ethylphenyl sulfate (4EPS) and catecholamine concentrations in biological samples, and blood chemistry tests under both housing conditions. Under paired housing, the stereotypic behavioral score decreased in both sexes, and the affiliative behavioral score increased in males and showed an increasing trend in females. Under single housing, the stereotypic score increased in both sexes, and the affiliative score decreased in males. Paired housing decreased serum calcium and urine cortisol concentrations in both sexes and decreased plasma cortisol in males and plasma 4EPS concentrations in females. The stereotypic score was positively correlated with serum calcium, plasma and urine cortisol, and plasma 4EPS concentration and negatively correlated with the affiliative score. The feces painting score, affiliative score, and plasma cortisol and serum calcium concentrations showed sex differences, suggesting differences in responsiveness to environmental changes between males and females. In conclusion, paired housing improved behavioral abnormalities in juvenile cynomolgus monkeys, suggesting that it may be an effective environmental enrichment paradigm. Calcium, cortisol, and 4EPS concentrations in biological samples may be useful indices for evaluating the effects of environmental enrichment.

Effect of road transportation on the serum biochemical parameters of cynomolgus monkeys and beagle dogs.

To determine the effect of long-distance (approximately 600 km) road transportation on the blood biochemistry of laboratory animals, we investigated the changes in serum biochemical parameters in healthy cynomolgus monkeys and beagle dogs transported by truck from Osaka to Tsukuba, Japan. The concentrations of serum cortisol, total bilirubin and aspartate aminotransferase in monkeys increased during transportation. Serum cortisol and total bilirubin levels in dogs also increased during transportation, but serum triglyceride decreased. Serum parameter values in truck-transported monkeys and dogs returned to baseline levels within two weeks following arrival. Taken together, these results suggest that a two-week acclimation period is the minimum duration required for adaptation following road transportation.

Effect of new training technique on affinity of cynomolgus monkeys for animal care personnel.

To confirm our hypothesis that the sex and age of cynomolgus monkeys influences the effect of training, we employed a new training technique designed to increase the animal's affinity for animal care personnel. During 151 days of training, monkeys aged 2 to 10 years accepted each 3 raisins/3 times/day, and communicated with animal care personnel (5 times/day). Behavior was scored using integers between -1 and 5. Before training, 35 of the 61 monkeys refused raisins offered directly by animal care personnel (Score -1, 0 and 1). After training, 28 of these 35 monkeys (80%) accepted raisins offered directly by animal care personnel (>Score 2). The mean score of monkeys increased from 1.2 ± 0.1 to 4.3 ± 0.2. The minimum training period required for monkeys to reach Score 2 was longer for females than for males. After 151 days, 6 of the 31 females and 1 of the 30 males still refused raisins offered directly by animal care personnel. Beneficial effects of training were obtained in both young and adult monkeys. These results indicate that our new training technique markedly improves the affinity of monkeys for animal care personnel, and that these effects tend to vary by sex but not age. In addition, abnormal behavior and symptoms of monkeys were improved by this training.

An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1-7 Cells) for Evaluation of the Neuroendocrine Effects of Essential Oils.

Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer's disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1-7 cells). In this study, we evaluated the effects of essential oils on neuronal death induced by hydrogen peroxide (H2O2), aluminum, zinc, or the antagonist of estrogen receptor (tamoxifen). Among tests of various essential oils, we found that H2O2-induced neuronal death was attenuated by the essential oils of damask rose, eucalyptus, fennel, geranium, ginger, kabosu, mandarin, myrrh, and neroli. Damask rose oil had protective effects against aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zinc-induced neurotoxicity. In contrast, geranium oil and ginger oil enhanced the neurotoxicity of tamoxifen. Our in vitro assay system could be useful for the neuropharmacological and endocrine pharmacological studies of essential oils.

Protective activity of carnosine and anserine against zinc-induced neurotoxicity: a possible treatment for vascular dementia.

Carnosine (ß-alanyl-L-histidine) is a small dipeptide with numerous beneficial effects, including the maintenance of the acid-base balance, antioxidant properties, chelating agent, anti-crosslinking, and anti-glycation activities. High levels of carnosine and its analogue anserine (1-methyl carnosine) are found in skeletal muscle and the brain. Zinc (Zn)-induced neurotoxicity plays a crucial role in the pathogenesis of vascular dementia (VD), and carnosine inhibits Zn-induced neuronal death. Here, the protective activity of carnosine against Zn-induced neurotoxicity and its molecular mechanisms such as cellular Zn influx and Zn-induced gene expression were investigated using immortalised hypothalamic neurons (GT1-7 cells). Carnosine and anserine protected against Zn-induced neurotoxicity not by preventing increases in intracellular Zn(2+) but by participating in the regulation of the endoplasmic reticulum (ER) stress pathway and the activity-regulated cytoskeletal protein (Arc). Accordingly, carnosine and anserine protected against neurotoxicity induced by ER-stress inducers thapsigargin and tunicamycin. Hence, carnosine and anserine are expected to have future therapeutic potential for VD and other neurodegenerative diseases.

Involvement of trace elements in the pathogenesis of prion diseases.

Prion diseases are progressive neurodegenerative diseases that are associated with conformational changes that convert normal cellular prion protein (PrP(C)) into an abnormal pathogenic prion protein (PrP(Sc)). It is widely recognized that prion diseases are forms of transmissible amyloidosis and are considered to be protein-misfolding diseases (conformational diseases), a category that also includes Alzheimer's disease. Trace elements play crucial roles in the conformational change affecting PrP(C), and increasing evidence suggests that PrP(C) is a metal-binding protein that is involved in the homeostasis of Cu, Zn, and Fe. In this article, we review the current understanding of links between trace elements and the conformational change to PrP(Sc), based on our studies using synthetic prion peptides, as well as other new findings. We also focus on PrP(Sc)-induced disruption of Ca homeostasis as a molecular mechanism for neurodegeneration in prion diseases. Possible roles of carnosine (ß-alanyl histidine) as a candidate neuroprotective substance use in prion diseases are also discussed.

Disruption of zinc homeostasis and the pathogenesis of senile dementia.

Zinc (Zn) is an essential trace element that is abundantly present in the brain. Although Zn plays crucial roles in learning and memory, numerous studies have indicated that the disruption of Zn homeostasis, namely both depletion and excess Zn, causes severe damage to neurons and is linked with various neurodegenerative diseases including Alzheimer's disease and vascular dementia. Here, we review the current understanding of the role of Zn in the pathogenesis of these neurodegenerative diseases. Based on our findings and other numerous studies, Zn acts as a contributor to Alzheimer's disease in the oligomerization, and as a protector in the neurotoxicity of Alzheimer's ß-amyloid protein. Furthermore, Zn plays a central role in ischemia-induced neuronal death and the pathogenesis of vascular dementia. Involvement of Ca(2+) dyshomeostasis and endoplasmic reticulum (ER) stress in the mechanism of Zn-induced neurotoxicity are suggested. We also discuss the possible role of carnosine (ß-alanyl histidine), a dipeptide that is present in the brain, as a protective substance for neuronal injury.

Anesthetic effect of a combination of medetomidine-midazolam-butorphanol in cynomolgus monkeys (Macaca fascicularis).

The anesthetic effect of a combination of medetomidine, midazolam and butorphanol (Me-Mi-Bu) was evaluated in healthy cynomolgus monkeys. The Me-Mi-Bu combination was intramuscularly administered as follows: Dose 1, Me 0.015 mg/kg-Mi 0.1 mg/kg-Bu 0.15 mg/kg; Dose 2, Me 0.02 mg/kg-Mi 0.15 mg/kg-Bu 0.2 mg/kg; and Dose 3, Me 0.04 mg/kg-Mi 0.3 mg/kg-Bu 0.4 mg/kg. The combination rapidly induced immobilization, and lateral recumbency was reached within 15 min. The duration of anesthesia for each dose administered was follows: Dose 1, 47 ± 27 min; Dose 2, 113 ± 31 min; and Dose 3, 190 ± 24 min. The anesthetic effect of the combination was abolished by the α2-adrenoceptor antagonist atipamezole. No marked changes in the levels of hematologic or serum biochemical parameters were noted in cynomolgus monkeys administered the combination plus atipamezole. Taken together, these results suggest that the Me-Mi-Bu combination exhibits reversible anesthetic effect and may be useful for studies involving cynomolgus monkeys.

D-histidine and L-histidine attenuate zinc-induced neuronal death in GT1-7 cells.

Although zinc (Zn) is an essential trace element, excess Zn causes neuronal death following transient global ischemia and plays a central role in the pathogenesis of vascular-type dementia. In this study, we developed a rapid and convenient screening system for substances that prevent Zn-induced neurotoxicity by using GT1-7 cells (immortalized hypothalamic neurons), with the aim of identifying a treatment for vascular-type dementia. Among tested, we found a protective substance in the extract of round herring (Etrumeus teres), and determined its structure as l-histidine. Analysis of the structure-activity relationship by using histidine analogues revealed that both l-histidine and d-histidine exhibit the same neuroprotective activity. Furthermore, we investigated the molecular mechanisms underlying the protective effect of histidine on Zn-induced neurotoxicity using Zn imaging and gene expression analysis, and found that histidine protects against Zn-induced neurotoxicity not by inhibiting Zn chelation, thereby preventing increases in intracellular Zn(2+). Moreover, it is also suggested that endoplasmic reticulum (ER) stress and activity-regulated cytoskeleton associated protein (Arc) are implicated in Zn-induced degeneration of neurons.

Effects of transport stress on serum alkaline phosphatase activity in beagle dogs.

Here, to determine the effects of transport stress on blood parameters in dogs, we investigated the changes in hematologic and serum chemical parameters in healthy beagle dogs transported from Beijing, China, to Osaka, Japan, to obtain the background data. Only the activity of serum alkaline phosphatase increased clearly upon arrival, a change attributed to transport stress, but the activity gradually reduced afterward. No marked changes in levels of other blood parameters were noted. Our findings here suggest that alkaline phosphatase is a useful tool for studying transport stress.

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126.

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of ß-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited ß-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (ß-alanyl histidine) significantly inhibited both the ß-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.

Stimulation of butyrate production by gluconic acid in batch culture of pig cecal digesta and identification of butyrate-producing bacteria.

Gluconic acid reaches the large intestine to stimulate lactic acid bacteria. However, the fermentation pattern of gluconic acid has yet to be elucidated. Accordingly, we examined the fermentation properties induced by gluconic acid in the pig cecal digesta in vitro. We also tested sorbitol and glucose, substrates for which the fermentation rate and patterns are known. The gluconic acid-utilizing bacteria were further isolated from pig cecal digesta and identified to examine the effect of gluconic acid on hind gut fermentation. Gluconic acid was fermented more slowly than were the other two substrates. Gluconic acid stimulated butyrate production; the butyrate molar percentage reached 26%, which is considered a high butyrate production. The majority of gluconic acid fermenters were identified as lactic acid bacteria, such as Lactobacillus reuteri and L. mucosae, and acid-utilizing bacteria, such as Megasphaera elsdenii and Mitsuokella multiacida. The gluconic acid fermented by lactic acid bacteria, and the lactate and acetate that were produced were used to form butyrate by acid-utilizing bacteria, such as M. elsdenii. Gluconic acid may be useful as a prebiotic to stimulate butyrate production in the large intestine.

Dietary sodium gluconate protects rats from large bowel cancer by stimulating butyrate production.

Butyrate has an antitumorigenic effect on colorectal cancer cell lines. Dietary sodium gluconate (GNA) promotes butyrate production in the large intestine. Accordingly, we examined the effect of dietary GNA on tumorigenesis in the large intestine in rats. Male Fisher-344 rats (n = 32) were divided into 4 groups: 2 diets (with or without 50 g GNA/kg basal diet) x 2 treatments (with or without carcinogen administration). Colonic tumors were induced by 3 intraperitoneal injections of azoxymethane (15 mg/kg body wt, 1 time/wk) and dietary deoxycholic acid (2 g/kg basal diet). The experiment was conducted for 33 wk except for a few rats. Ingestion of GNA increased cecal butyrate concentration at the end of experiment (P < 0.01). No tumor development occurred in the untreated groups. Ingestion of GNA decreased the incidence of tumors in rats administered the carcinogen (37.5 vs. 100%, P < 0.05). Ingestion of GNA also decreased the mean number of tumors per rat (0.5 +/- 0.8 vs. 2.8 +/- 1.5, P < 0.01). beta-Catenin accumulation and TdT-mediated dUTP nick end labeling (TUNEL) positive cells in tumors were histochemically examined. The results of this study suggested that the antitumorigenic effect of GNA may involve the stimulation of apoptosis through enhanced butyrate production in the large intestine.

Megasphaera elsdenii JCM1772T normalizes hyperlactate production in the large intestine of fructooligosaccharide-fed rats by stimulating butyrate production.

Hyperlactate production is related to disorders of the large intestine such as inflammatory bowel diseases. Lactate, an intermediate in hindgut fermentation, is metabolized to SCFA. Megasphaera elsdenii can convert lactate to butyrate, a physiologically important organic acid for the hindgut mucosa. This experiment was conducted to determine whether M. elsdenii normalizes hyperlactate production and stimulates butyrate production in the rat large intestine. Specific pathogen-free Sprague-Dawley male rats (n = 12) were fed a fructooligosaccharide (FOS)-supplemented (100 g/kg), semipurified diet to induce lactate production. Lactate excretion in all rats was >30 mmol/kg fresh feces on d 2 of FOS-feeding. The rats were divided into two groups on the morning of d 4. One group (n = 5) was dosed orally with M. elsdenii JCM1772T (1.3 x 10(13) cells) for 3 d. The other group was treated with a vehicle solution. Fecal lactate was significantly lower in rats administered M. elsdenii than in controls. An increase in fecal butyrate compensated for the decrease in lactate. The number of cecal epithelial cells was greater in rats administered M. elsdenii than in controls. M. elsdenii has the potential to normalize hyperlactate accumulation in the large intestine, and lactate-utilizing butyrate producers may be useful probiotics when hyperlactate fermentation in the large intestine is a problem.