Orally Administered Plasmalogens Alleviate Negative Mood States and Enhance Mental Concentration: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Plasmalogens have been shown to improve neurodegenerative pathology and cognitive function. We hypothesized that plasmalogens work in small amounts as a kind of hormone interacting with a G protein-coupled receptor, and then explored the effects of scallop-derived purified plasmalogens on psychobehavioral conditions in a randomized placebo-controlled trial of college athletes in Japan. Methods and materials: Eligible participants were male students aged 18-22 years who belonged to university athletic clubs. They were randomly allocated to either plasmalogen (2 mg per day) or placebo treatment of 4 weeks' duration. The primary outcome was the T-score of the Profile of Mood States (POMS) 2-Adult Short, and the secondary outcomes included the seven individual scales of the POMS 2, other psychobehavioral measures, physical performance, and laboratory measurements. The trial was registered at the Japan Registry of Clinical Trials (jRCTs071190028). Results: Forty participants (20 in the plasmalogen group and 20 in the placebo group) completed the 4-week treatment. The Total Mood Disturbance (TMD) score of the plasmalogen group showed a greater decrease at 4 weeks than that of the placebo group while the between-group difference was marginally significant (p = 0.07). The anger-hostility and fatigue-inertia scores of the POMS 2 decreased significantly in the plasmalogen group, but not in the placebo group, at 4 weeks. Between-group differences in those scores were highly significant (p = 0.003 for anger-hostility and p = 0.005 for fatigue-inertia). The plasmalogen group showed a slight decrease in the Athens Insomnia Scale at 2 weeks, and the between-group difference was near-significant (p = 0.07). The elapsed time in minute patterns on the Uchida-Kraepelin test, which is a marker of mental concentration, revealed significantly greater performance in the plasmalogen group than in the placebo group. There were no between-group differences in physical and laboratory measurements. Conclusion: It is suggested that orally administered plasmalogens alleviate negative mood states and sleep problems, and also enhance mental concentration.

Early changes to oxidative stress levels following exposure to formaldehyde in ICR mice.

Formaldehyde (FA) is a commonly used chemical in everyday life and can react with many molecules in the human body. Although toxicity has been reported, exposure to FA has also been shown to have beneficial effects or no effect at all. In the present study, we examined the effect of FA inhalation on oxidative stress and inflammation in mice. Male adult ICR mice were exposed FA in gaseous form (0.1 ppm), and blood, urine, brain, lung and liver were obtained for 24 hr. Levels of 8-hydroxy-2'-deoxyguanosine (8OHdG) and NO(3)(-) were then determined by HPLC. A second group of mice were injected with 5 mg/kg lipopolysaccharide (LPS) after 24 hr of FA (3 ppm) inhalation and blood and organs were assayed for NO(3)(-) level and SOD activity. After exposure to a low dose of FA (0.1 ppm), the 8OHdG/dG ratio significantly increased in plasma. However, the ratio in urine and organs significantly decreased during 24 hr of FA exposure. The NO(3)(-) levels mirrored the 8OHdG/dG ratio. After 24 hr exposure to a high dose of FA (3 ppm), NO(3)(-) levels in plasma and liver were significantly lower than in control mice exposed to air only. The SOD activity of blood and urine were conversely increased in FA exposed animals. In the present study, we suggest that inhalation of FA at low doses influences the oxidative stress response in a tissue-specific manner. The FA may partially alleviate in some tissues like preconditioning in oxidative stress.

Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

It has been shown that molecular hydrogen (H(2)) acts as a therapeutic antioxidant and suppresses brain injury by buffering the effects of oxidative stress. Chronic oxidative stress causes neurodegenerative diseases such as Parkinson's disease (PD). Here, we show that drinking H(2)-containing water significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration-dependency of H(2) showed that H(2) as low as 0.08 ppm had almost the same effect as saturated H(2) water (1.5 ppm). MPTP-induced accumulation of cellular 8-oxoguanine (8-oxoG), a marker of DNA damage, and 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation were significantly decreased in the nigro-striatal dopaminergic pathway in mice drinking H(2)-containing water, whereas production of superoxide (O(2)*(-)) detected by intravascular injection of dihydroethidium (DHE) was not reduced significantly. Our results indicated that low concentration of H(2) in drinking water can reduce oxidative stress in the brain. Thus, drinking H(2)-containing water may be useful in daily life to prevent or minimize the risk of life style-related oxidative stress and neurodegeneration.

Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP(+/-) and PACAP(-/-) mice than in PACAP(+/+) animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP(+/-) and PACAP(-/-) mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP(+/-) mice was less than that in PACAP(+/+) animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP(+/-) and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP(+/-) mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.

Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells.

Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.

Selective activation of primary afferent fibers evaluated by sine-wave electrical stimulation.

Transcutaneous sine-wave stimuli at frequencies of 2000, 250 and 5 Hz (Neurometer) are thought to selectively activate Abeta, Adelta and C afferent fibers, respectively. However, there are few reports to test the selectivity of these stimuli at the cellular level. In the present study, we analyzed action potentials (APs) generated by sine-wave stimuli applied to the dorsal root in acutely isolated rat dorsal root ganglion (DRG) preparations using intracellular recordings. We also measured excitatory synaptic responses evoked by transcutaneous stimuli in substantia gelatinosa (SG) neurons of the spinal dorsal horn, which receive inputs predominantly from C and Adelta fibers, using in vivo patch-clamp recordings. In behavioral studies, escape or vocalization behavior of rats was observed with both 250 and 5 Hz stimuli at intensity of approximately 0.8 mA (T5/ T250), whereas with 2000 Hz stimulation, much higher intensity (2.14 mA, T2000) was required. In DRG neurons, APs were generated at T5/T250 by 2000 Hz stimulation in Abeta, by 250 Hz stimulation both in Abeta and Adelta, and by 5 Hz stimulation in all three classes of DRG neurons. However, the AP frequencies elicited in Abeta and Adelta by 5 Hz stimulation were much less than those reported previously in physiological condition. With in vivo experiments large amplitude of EPSCs in SG neurons were elicited by 250 and 5 Hz stimuli at T5/ T250. These results suggest that 2000 Hz stimulation excites selectively Abeta fibers and 5 Hz stimulation activates noxious transmission mediated mainly through C fibers. Although 250 Hz stimulation activates both Adelta and Abeta fibers, tactile sensation would not be perceived when painful sensation is produced at the same time. Therefore, 250 Hz was effective stimulus frequency for activation of Adelta fibers initiating noxious sensation. Thus, the transcutaneous sine-wave stimulation can be applied to evaluate functional changes of sensory transmission by comparing thresholds with the three stimulus frequencies.

Differentiation of skeletal muscle from pituitary folliculo-stellate cells and endocrine progenitor cells.

We previously reported the ectopic differentiation of skeletal muscle cells in a pituitary gland transplanted beneath a kidney capsule. Morphological observation suggested that the skeletal muscle cells may have differentiated from folliculo-stellate (FS) cells in the anterior pituitary gland. However, at that time, we did not confirm this directly with an in vitro system. To obtain direct evidence, we used the Tpit/F1 cell line. The Tpit/F1 cell line was recently established from the pituitary gland of a temperature-sensitive T antigen transgenic mouse and has the characters of pituitary FS cells. Using Tpit/F1 cells, we have found that FS cells of the pituitary are able to differentiate into muscle cells in vitro. Additionally, we showed that the cells have some characteristics of pituitary FS cells and also express pituitary endocrine cell-specific transcription factor (pit-1) and prolactin genes, and can differentiate into striated muscle cells. The anterior pituitary gland is known to be of ectodermal origin, so the differentiation of its cells into striated muscle is completely unexpected. This is the first report of direct evidence of ectopic differentiation of skeletal muscle cells from pituitary cells.

Endotoxin inhibitor blocks heat exposure-induced expression of brain cytokine mRNA in aged rats.

To investigate the age-related changes in the expression of interleukin-1beta (IL-1beta) and its related substances in the brain during heat stress, we measured amounts of mRNAs for IL-1beta, cyclooxygenase-2 (COX-2), and an inhibitor of nuclear factor (NF)-kappaB-beta (IkappaB-beta) that is known to reflect an activation of NF-kappaB, in the cortex, cerebellum, and hippocampus using a quantitative real-time capillary PCR method. The basal levels of IL-1beta mRNA in aged rats (108-110 weeks old) was significantly higher than those in young animals (10-11 weeks old) in these brain regions. Heat exposure (33 degrees C) for 1 h enhanced the expression of IL-1beta and COX-2 mRNAs in aged rats but not in young ones. The amount of lipopolysaccharide (LPS) assessed by its bioactivity in the cortex increased by heat exposure only in aged rats. To further examine an involvement of LPS in the increase in mRNAs, an endotoxin inhibitor (EI), a synthetic peptide that detoxifies LPS by binding to the toxic component of LPS, lipid A, was intraperitoneally injected before heat exposure in aged rats. An intraperitoneal injection of EI significantly attenuated the heat exposure-induced increases in mRNAs for IL-1beta, COX-2, IkappaB-beta, and the LPS activity. Administration of EI also debilitated the heat exposure-induced hyperthermia and responses of plasma ACTH and catecholamines. These findings, taken together, suggest that the bacterial translocation is involved in the mechanisms of the responses to heat exposure in aged rats including the increased expression of mRNAs for IL-1beta and its related substances in the brain.

Nucleoprotamine diet derived from salmon soft roe protects mouse hippocampal neurons from delayed cell death after transient forebrain ischemia.

The nutritional benefits of nucleoprotamine (NP), the main component of fish soft roe, have been rarely addressed. In the present study, the preventive effect of oral supplements of nucleoprotamine and its derivatives, DNA and protamine (PT), extracted from salmon soft roe, on survival rate and hippocampal cell death induced by transient brain ischemia, was evaluated in mice. Artificially formulated nucleoprotamine-free (NF) diet with/without nucleoprotamine, DNA or protamine was fed orally. One week after commencement of respective diets, animals were subjected to transient brain ischemia, which was performed by common carotid artery (CCA) occlusion for 25 (severe) or 15 min (mild). After severe ischemia, the survival rate of the NF group was lower than that in the group fed standard diet or NP. Morphological changes in the hippocampal CA1 region were estimated 48 h after mild ischemia. The NP and PT groups significantly decreased the neuronal damage compared with the NF group. The number of cell death in the DNA group, however, was affected similar to that of the NF group. Our data suggests that the nucleoprotamine content in salmon soft roe could be a useful nutritional resource for the prevention of cell damage caused by ischemia such as those occurring with cerebral and/or heart infarction.

In vivo response of neutrophils and epithelial cells to lipopolysaccharide injected into the monkey ileum.

Since few previous studies have investigated the in vivo response of intestinal mucosa to the luminally administered lipopolysaccharide (LPS), we examined the cellular localization of exogenously applied LPS in the intestinal mucosa and the expression of Toll-like receptor (TLR) and IL-1 receptor-associated kinase (IRAK) in the epithelial cells of monkey ileum. FITC-labeled LPS was injected into the lumen of monkey ileum. Thirty minutes after the LPS injection, the ileal tissue was fixed and localization of FITC fluorescence in the ileal mucosa was examined. We applied Factor C immunohistochemistry to demonstrate the bioactivity of LPS taken up by the mucosal tissue. The expression of TLR4 and IRAK-1 in the epithelial cells was also examined by immunohistochemistry. FITC fluorescence was detected in the cells migrated into the epithelium and those in the lamina propria. The FITC-labeling cells were completely overlapped with the Factor C immunoreactive cells. These FITC-labeling/Factor C-positive cells were identified as neutrophils by the immunoelectron microscopic analysis. TLR4 and IRAK-1 were expressed at the apical membrane of the epithelial cells in the ileum of both control and FITC-LPS injected animals. These results suggest that intraluminal injection of LPS stimulates the transmigration of neutrophils into the epithelium and these neutrophils may uptake luminally applied LPS and possibly inactivate the enterotoxin. Expression of TLR4 and IRAK-1 in the epithelial cells suggests that epithelial cells may react to LPS and produce chemoattractant mediator to induce the neutrophil chemotaxis.

Enteric lipopolysaccharide raises plasma IL-6 levels in the hepatoportal vein during non-inflammatory stress in the rat.

It has long been known that plasma interleukin (IL)-6 levels elevate during non-inflammatory, physico/psychological stresses such as immobilization (IMB) and electric foot shock (FS). We previously demonstrated that an IMB-induced rise in plasma IL-6 in the rat was caused, at least partly, by an increased production of IL-6 in hepatic reticulo-endothelial cells which were induced by enteric flora-derived lipopolysccharide (LPS). This study investigated whether such enteric flora-derived LPS may produce IL-6 also in the mesentery and the mesenteric lymphoid nodes (MLN) before it reaches the liver. We found a rise in the IL-6 levels in the hepatoportal vein (PV) within 30 min during FS, while the IL-6 levels in the jugular vein showed a smaller and delayed rise with slower recovery. Plasma IL-6 levels near the exit of the hepatic vein in the inferior vena cava was highest at both control and stressed conditions, compared with those in the PV and any other extra-hepatic circulation. The stress-induced IL-6 elevation in the PV was abolished by an in vivo neutralization of LPS with continuous infusion of polymyxin B. Furthermore, the amount of LPS as assessed by its bioactivity increased rapidly in the mesentery, the MLN and the liver within 15 min after the initiation of FS. Finally, fluorescent dye-labeled LPS infused into the lumen of the ileum was found in the extra-intestinal tissues and systemic vein, and FS increased the optical density in them. The findings suggest that lymphoid tissues in the gut associated lymphoid organs are continuously exposed to LPS at the basal condition, and that FS facilitates the LPS/bacterial translocation across the intestinal wall and thereby increases the production of IL-6 before LPS reaches the liver.

Reduced postischemic apoptosis in the hippocampus of mice deficient in interleukin-1.

The cytokine interleukin-1 (IL-1) has been implicated in ischemic brain damage, because the IL-1 receptor antagonist markedly inhibits experimentally induced neuronal loss. However, to date, no studies have demonstrated the involvement of endogenous IL-1alpha and IL- 1beta in neurodegeneration. We report here, for the first time, that mice lacking IL-1alpha/beta (double knockout) exhibit markedly reduced neuronal loss and apoptotic cell death when exposed to transient cardiac arrest. Furthermore, we show that, despite the reduced neuronal loss, phosphorylation of JNK/SAPK (c-Jun NH2- terminal protein kinase/stress activated protein kinase) and p38 enzymes remain elevated in IL-1 knockout mice. In contrast, the inducible nitric oxide (iNOS) immunoreactivity after global ischemia was reduced in IL-1 knockout mice as compared with wild-type mice. The levels of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in the hippocampus of wild-type mice were increased with time after ischemia-reperfusion, whereas the increase was significantly inhibited in IL-1 knockout mice. These observations strongly suggest that endogenous IL-1 contributes to ischemic brain damage, and this influence may act through the release of nitric oxide by iNOS.

Involvement of tumor necrosis factor alpha, rather than interleukin-1alpha/beta or nitric oxides in the heme oxygenase-1 gene expression by lipopolysaccharide in the mouse liver.

Heme oxygenase-1 (HO-1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO-1 by LPS is reported to be mediated through interleukin-1beta (IL-1beta), rather than other inflammatory cytokines in the mouse liver. However, we found that IL-1alpha/beta knockout (KO) mice responded well to LPS insult, as did wild-type mice with respect to HO-1 mRNA induction (about 30-fold increase). In contrast, tumor necrosis factor alpha KO (TNFalphaKO) mice responded very weakly to LPS in the HO-1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO-1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL-1KO or TNFalphaKO. In addition, the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular signal-regulated kinase, was very low in TNFalphaKO mice due to LPS administration. All of these findings indicate that TNFalpha is a major candidate to trigger HO-1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways.