Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Correlation of Sarcopenic Obesity on Various Cardiometabolic Risk Factors and Fracture Risk in Mid-Aged Korean Women.

OBJECTIVES: This study aimed to investigate the correlation of sarcopenic obesity with various cardiometabolic risk factors and fracture risk in middle-aged Korean women. METHODS: In this cross-sectional study, the medical records of 1,775 women who had visited Pusan National University Hospital for routine health screenings from 2010 to 2016 were reviewed. The patients were divided into four groups as follows: group 1, nonsarcopenic, nonobese (NS-NO); group 2, nonsarcopenic, obese (NS-O); group 3, sarcopenic, nonobese (S-NO); and group 4, sarcopenic, obese (S-O). Each patient was assessed based on self-reported questionnaires and individual interviews with a healthcare provider. The Fracture Risk Assessment Tool (FRAX) was used to assess bone fracture risk. RESULTS: Postmenopausal women accounted for 68.5% of the total patient population. The proportion of each group was as follows: NS-NO, 71.2%; NS-O, 17.9%; S-NO, 10.2%; and S-O, 0.7%. Statistical analysis of various parameters associated with metabolic and cardiovascular risks revealed that the S-O group had more patients with hypertension, diabetes, osteopenia, and metabolic syndrome. The FRAX scores were significantly higher in the S-O group than in other groups. CONCLUSIONS: Middle-aged women with obesity and reduced muscle mass, known as sarcopenic obesity, are at increased risk of hypertension, diabetes, and metabolic syndrome. Furthermore, sarcopenic obesity, individual cardiometabolic risks, and menopause can increase the bone fracture risk.

The Clinical Usefulness of Ultrasonographic Measurement Technique in Patients with Lower Extremity Lymphedema.

Background: A previous study reported a new ultrasonography (US) measurement technique to evaluate the cross-sectional area (ΔCSA) of lymphedema in the upper extremity. This ΔCSA correlated well with parameters, such as the circumference, volumetry, and bioimpedance analysis (BIA) in healthy people and upper extremity lymphedema patients. This study examined whether a US measurement technique is clinically useful in patients with lymphedema in the lower extremity. Methods and Results: Forty patients diagnosed with unilateral lower extremity lymphedema were enrolled in this study. The subjects' leg circumference, BIA, isokinetic strength, and ΔCSA were examined on the same day. The leg circumference was measured at 15 cm above the knee (AK) and below the knee (BK) crease using a tape measure. BIA was performed by a trained physical therapist, and the data of impedance (Z) at 1 and 5 kHz of each side of the lower limbs and extracellular water (ECW) were used. A fully experienced physician measured soft tissue thickness, the distance between the skin and the fascia of the muscle, three times each at the anterior, medial, posterior, and lateral aspects of the bilateral legs by US at 15 cm AK and BK. The amount of soft tissue in the ΔCSA was calculated using the designed formula from the mean values of the thicknesses. Each parameter was calculated as the ratio of the sound side to the lesion side. The Pearson and Spearman correlation coefficients were used to assess the significance of these parameters. The ratio of ΔCSA measured at 15 cm AK and BK showed strong positive correlations with the circumference difference at the same level (rho = 0.790, p = 0.000, and rho = 0.882, p = 0.000, respectively). In addition, it showed moderate or strong correlations with the ratio of Z at 5 and 1 kHz in the BIA of the lower limbs (AK15, r = -0.511, p = 0.001 and r = -0.497, p = 0.001; BK15, r = -0.780, p = 0.000 and r = -0.756, p = 0.000, respectively). Although ECW and body mass index showed weak positive correlations with the ratio of ΔCSA measured at 15 cm BK, there was no significant correlation between the ratio of ΔCSA and the isokinetic muscle strength. Conclusion: The ΔCSA results showed moderate-to-strong correlations with other conveniently used methods except for the isokinetic muscle strength. As the US ΔCSA technique could measure lymphedema status with a structural consideration, it could also be recommended as a conventional measurement method in patients with upper and lower extremity lymphedema.

Anti-stress effects of Fameyes in in vitro and in vivo models of stresses.

BACKGROUND: Fameyes (a mixture of Clematis mandshurica Rupr. extract (CMRE) and Erigeron annuus (L.) Pers. extract (EAPE)) containing scutellarin and chlorogenic acid as major components has been reported to relieve mental stress in human subjects, which is reflected in improved scores in psychometric tests measuring levels of depression, anxiety, well-being, and mental fitness. The aim of this study was to examine the anti-stress activity of Fameyes and to investigate the mechanisms of the anti-stress activity using in vitro and in vivo models of stresses. RESULTS: First, we tested the effect of Fameyes on corticosterone-induced cytotoxicity in SH-SY5Y cells (human neurofibroma cell lines). Corticosterone induced apoptosis and decreased cell viability and mitochondrial membrane potential, but treatment with Fameyes inhibited these cytotoxic effects in a dose-dependent manner. However, CMRE and EAPE (components of Fameyes) did not inhibit the cytotoxic effect of corticosterone individually. Next, we tested the effects of Fameyes on rats that were exposed to different kinds of stresses for four weeks. When the stressed rats were treated with Fameyes, their immobility time in forced swim and tail suspension tests decreased. A reduction was also observed in the serum levels of adrenocorticotropic hormone (ACTH) and corticosterone. Furthermore, upon oral administration of Fameyes, serum serotonin levels increased. These in vitro and in vivo results support the anti-stress effects of Fameyes. CONCLUSIONS: In vitro experiments showed anti-stress effects of Fameyes in cell viability, apoptosis, and mitochondrial membrane potential. In addition, in vivo experiments using rats showed anti-stress effects of Fameyes in blood and tissue levels of ACTH, corticosterone, and serotonin, as well as the immobility time in the forced swim and tail suspension tests. However, we did not specifically investigate which ingredient or ingredients showed anti-stress effects, although we reported that Fameyes contained chlorogenic acid and scutellarin major ingredients.

In Vitro and In Vivo Anti-Inflammatory Effects of TEES-10®, a Mixture of Ethanol Extracts of Ligularia stenocephala Matsum. & Koidz. and Secale cereale L. Sprout, on Gingivitis and Periodontitis.

Gingivitis and periodontitis are inflammatory disorders caused by dental plaque and calculus. These disorders often lead to tooth loss if not treated properly. Although antibiotics can be used, it is hard to treat them due to the difficulty in supplying effective doses of antibiotics to lesion areas and side effects associated with long-term use of antibiotics. In the present study, attempts were made to provide in vitro and in vivo evidence to support anti-inflammatory activities of TEES-10®, a mixture of ethanol extracts of Ligularia stenocephala (LSE) and Secale cereale L. sprout (SCSE) toward gingivitis and periodontitis by performing the following experiments. TEES-10® with a ratio of 6:4 (LSE:SCSE) showed the best effects in both stimulating the viability and inhibiting the cytotoxicity. In in vitro experiments, TEES-10® showed an ability to scavenge 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals and remove ROS generated in periodontal ligament cells treated with lipopolysaccharide. TEES-10® also enhanced the viability of stem cells from human exfoliated deciduous teeth and stimulated the osteogenic differentiation of deciduous teeth cells. In in vivo experiments using rats with induced periodontitis, TEES-10® significantly decreased inflammatory cell infiltration and the numbers of osteoclasts, increased alveolar process volume and the numbers of osteoblasts, decreased serum levels of IL-1ß and TNF-α (pro-inflammatory cytokines), and increased serum levels of IL-10 and IL-13 (anti-inflammatory cytokines). These results strongly support the theory that TEES-10® has the potential to be developed as a health functional food that can treat and prevent gingival and periodontal diseases and improve dental health.

Dichloromethane fractions of Calystegia soldanella induce S­phase arrest and apoptosis in HT­29 human colorectal cancer cells.

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT­29 was examined. First, five solvent fractions [n­hexane, dichloromethane (DCM), ethyl acetate, n­butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose­ and time­dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT­29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose­dependent manner, which was associated with the protein expression levels of apoptosis­related proteins, including increased Fas, Bad and Bax, and decreased pro­caspase­8, Bcl­2, Bcl­xL, pro­caspase­9, pro­caspase­7 and pro­caspase­3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose­dependent manner in HT­29 cells treated with DCM. In addition, cell cycle analysis confirmed S­phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle­related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT­29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S­phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT­29 colorectal cells. Further research on the composition of the DCM fraction is warranted.

Calystegia soldanella Extract Exerts Anti-Oxidative and Anti-Inflammatory Effects via the Regulation of the NF-κB/Nrf-2 Pathways in Mouse Macrophages.

Plant polyphenols are widely used to treat various inflammatory diseases, owing to their ability to suppress reactive oxygen species production and the expression of inflammatory cytokines. Herein, we investigated phenolic compounds from Calystegia soldanella using UPLC Q-TOF MS/MS and their antioxidative and anti-inflammatory activities were analyzed. The C. soldanella ethyl acetate fraction (CsEF) had the strongest antioxidative activity, given its high polyphenol compound content. It also exhibited anti-inflammatory effects, inhibiting the production of inflammatory cytokines such as NO, PGE2, IL-1ß, IL-6, and TNF-α in LPS-stimulated mouse macrophages. CsEF activated the nuclear transcription factor Nrf-2, thereby upregulating antioxidant enzymes such as HO-1 and NQO-1 and inhibiting NF-κB expression, which in turn, suppressed the expression of COX-2, iNOS, and inflammatory cytokines, ultimately exerting anti-inflammatory effects. Further, UPLC-Q-TOF-MS/MS was used to analyze the polyphenol compound contents in CsEF. The quercetin glycosides isoquercitrin and quercitrin were the primary flavonoid compounds, while the caffeic acid derivatives, chlorogenic acid and dicaffeoylquinic acid, were the primary phenolic acids. Thus, C. soldanella, which had only a limited use thus far as a medicinal plant, may serve as a natural medicinal resource for treating inflammatory diseases.

Aquatic Exercise and Land Exercise Treatments after Total Knee Replacement Arthroplasty in Elderly Women: A Comparative Study.

Background and Objectives: Early intensive exercise after total knee replacement arthroplasty (TKRA) has become increasingly popular due to its ability to enhance knee physical function and reduce pain. When implemented exclusively, aquatic exercise (AE) appears to be more advantageous than land exercise (LE), particularly in the early phase after TKRA. Our study aimed to compare the clinical efficacy of AE and LE with respect to their effects on pain and physical function after TKRA. Materials and Methods: Between February 2008 and January 2020, 100 female patients who underwent TKRA were enrolled in this retrospective study. We measured the range of motion (ROM) of the knee, the isokinetic strength of the knee joint (function), and pain both initially and one month after TKRA. Two weeks after TKRA, the participants were enrolled in either the AE or the LE program for a total of two weeks. Two 30 min sessions of intensive ROM and knee strengthening exercises and balance training were provided to the AE and LE groups for 10 days. The home exercise group (HE) only received information on ROM and strengthening exercises. There were 33, 21, and 46 patients allocated to the AE, LE, and HE groups, respectively. Results: The ROM of the side on which surgery was performed improved significantly in all groups, as did the pain scores. In the AE group, the knee flexor strength showed a tendency toward improvement. Contrastingly, there was no significant improvement in the knee extensor strength in the AE group. Conclusions: Overall, the AE and LE groups showed superior outcomes compared with HE. In addition, the AE group demonstrated some improvement in knee muscle strength even with a short hospital stay. Further study with long-term follow-up should be performed to better define the outcomes.

Effect of Incorporating a Dimethacrylate Monomer on the Shear Bond Strength of Two Adhesive Resin Cements to Zirconia Ceramic.

Self-assembled nano-layering resulting from interaction of the phosphate functional group of adhesive monomers with zirconia ceramic surface has been proposed. The purpose of this study was to investigate the bond strengths of two adhesive resin cements (Panavia F 2.0 and BisCem) containing phosphate monomers added with various concentrations (0.0, 1.0, 3.0, and 5.0 wt%) of triethylene glycol dimethacrylate (TEGDMA) to air-abraded zirconia ceramic. The polished/air-abraded zirconia plates (KaVo Everest® ZS-Ronde) were imaged using atomic force microscopy and the average surface roughness (Ra) values were calculated (n = 5). The surface energy parameters of the zirconia plates and the resin cements were calculated based on the extended Fowkes theory. All resin-bonded (diameter: 2.38 mm) zirconia specimens were stored in water at 37 °C for 24 h and then half of them additionally thermocycled 10,000 times before the shear bond strength (SBS) test (n = 10). Air-abrasion of zirconia surface significantly increased the γhS (hydrogen bonding component) value (p < 0.001), as well as greatly increasing the surface area (p < 0.001). For both resin cements, the γhS (dipole-dipole component) gradually increased with increasing incorporated TEGDMA concentrations, whereas the γhS gradually decreased. Overall, the addition of 3.0 wt% of TEGDMA consistently resulted in higher SBS values even after thermocycling. Under the tested condition, reducing the concentration of the adhesive monomers with phosphate functional group by adding the dimethacrylate monomer (up to 3.0 wt%) increased the bond strength between the resin cements and zirconia ceramic.

PYP1-4 peptide from Pyropia yezoensis protects against acetaminophen-induced hepatotoxicity in HepG2 cells.

Acetaminophen (APAP) is a widely used analgesic and antipyretic. It is safe at normal treatment doses; however, APAP overdose is a major cause of acute liver and kidney failure. A variety of methods to reduce the damage caused by APAP overdose have previously been evaluated. The protein-rich seaweed Pyropia yezoensis has antioxidant, antitumor and anti-inflammatory activities, and protects against cytotoxicity. However, little is known regarding the protective effects of P. yezoensis peptide against APAP-induced hepatotoxicity. The present study investigated the ability of P. yezoensis peptide (PYP1-4) to ameliorate the damage caused by APAP-induced hepatotoxicity using HepG2 as the model cell line in addition to the signaling pathways involved. Briefly, cell viability, nitric oxide, reactive oxygen species and apoptosis assays were performed in conjunction with western blot analysis and reverse transcription-quantitative PCR. First, the present study revealed the minimum toxic concentration of APAP (15 mM) and the resting concentration of PYP1-4 (0-500 ng/ml). Administration of PYP1-4 to APAP-induced cells decreased the nitric oxide and reactive oxygen species levels, and restored the levels of antioxidant-associated proteins (catalase, heme oxygenase 1, superoxide dismutase 2 and quinone oxidoreductase 1). PYP1-4 increased the translocation of nuclear factor, erythroid 2 like 2 to the nucleus and the activities of glycogen synthase kinase-3ß, Akt and AMP-activated protein kinase. In addition, APAP induced apoptosis; however, PYP1-4 inhibited apoptosis by modulating the levels of pro-apoptotic markers (Bad), anti-apoptotic markers (Bcl-2 and BH3 interacting domain death agonist), caspases and poly (ADP-ribose) polymerase 1. Subsequently, the insulin-like growth factor 1 receptor signaling pathway was investigated to determine whether PYP1-4 treatment restored the levels of cell growth-associated factors during APAP-induced hepatotoxicity. PYP1-4 treatment impacted the levels of components of the insulin receptor substrate 1/PI3K/Akt and Ras/Raf/ERK signaling pathways, and promoted cell survival. Therefore, the P. yezoensis peptide PYP1-4 may be useful for preventing APAP-induced hepatotoxicity.

Formation of Nano/Micro Hierarchical Structures on Titanium Alloy Surface by a Novel Etching Solution.

A new effective oxidative solution for titanium (Ti) surface etching was recently developed. The present in vitro study was aimed at determining the influence of shorter (than 240 min) treatment time on the surface characteristics of the Ti nano/micro hierarchical structures. Cylinder-shaped Ti grade 5 alloys were etched for 30, 60, 120, and 240 min at room temperature and cleaned successively with acetone, ethanol, and distilled water in an ultrasonic bath. The micro- and nanostructures, surface roughness, dynamic wettability, and the surface elemental composition of the etched surfaces were evaluated. Nano/micro hierarchical structures, composed of micro-pits and nano-channels, were formed on the Ti surface through simple immersion in the oxidative solution. The findings suggest that the 120-min immersion yielded significant enhancement in the roughness and wettability of the Ti surfaces.

Down-regulation of cyclin-dependent kinase 5 attenuates p53-dependent apoptosis of hippocampal CA1 pyramidal neurons following transient cerebral ischemia.

Abnormal activation of cyclin-dependent kinase 5 (Cdk5) is associated with pathophysiological conditions. Ischemic preconditioning (IPC) can provide neuroprotective effects against subsequent lethal ischemic insult. The objective of this study was to determine how Cdk5 and related molecules could affect neuroprotection in the hippocampus of gerbils after with IPC [a 2-min transient cerebral ischemia (TCI)] followed by 5-min subsequent TCI. Hippocampal CA1 pyramidal neurons were dead at 5 days post-TCI. However, treatment with roscovitine (a potent inhibitor of Cdk5) and IPC protected CA1 pyramidal neurons from TCI. Expression levels of Cdk5, p25, phospho (p)-Rb and p-p53 were increased in nuclei of CA1 pyramidal neurons at 1 and 2 days after TCI. However, these expressions were attenuated by roscovitine treatment and IPC. In particular, Cdk5, p-Rb and p-p53 immunoreactivities in their nuclei were decreased. Furthermore, TUNEL-positive CA1 pyramidal neurons were found at 5 days after TCI with increased expression levels of Bax, PUMA, and activated caspase-3. These TUNEL-positive cells and increased molecules were decreased by roscovitine treatment and IPC. Thus, roscovitine treatment and IPC could protect CA1 pyramidal neurons from TCI through down-regulating Cdk5, p25, and p-p53 in their nuclei. These findings indicate that down-regulating Cdk5 might be a key strategy to attenuate p53-dependent apoptosis of CA1 pyramidal neurons following TCI.

Pretreated Oenanthe Javanica extract increases anti-inflammatory cytokines, attenuates gliosis, and protects hippocampal neurons following transient global cerebral ischemia in gerbils.

Recently, we have reported that Oenanthe javanica extract (OJE) displays strong neuroprotective effect against ischemic damage after transient global cerebral ischemia. However, neuroprotective mechanisms of OJE have not been fully identified. Thus, this study investigated the neuroprotection of OJE in the hippocampal CA1 area and its anti-inflammatory activity in gerbils subjected to 5 minutes of transient global cerebral ischemia. We treated the animals by intragastrical injection of OJE (100 and 200 mg/kg) once daily for 1 week prior to transient global cerebral ischemia. Neuroprotection of OJE was observed by immunohistochemistry for neuronal nuclear antigen and histofluorescence staining for Fluoro-Jade B. Immunohistochemistry of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 was done for astrocytosis and microgliosis, respectively. To investigate the neuroprotective mechanisms of OJE, we performed immunohistochemistry of tumor necrosis factor-alpha and interleukin-2 for pro-inflammatory function and interleukin-4 and interleukin-13 for anti-inflammatory function. When we treated the animals by intragastrical administration of 200 mg/kg of OJE, hippocampal CA1 pyramidal neurons were protected from transient global cerebral ischemia and cerebral ischemia-induced gliosis was inhibited in the ischemic hippocampal CA1 area. We also found that interleukin-4 and -13 immunoreactivities were significantly increased in pyramidal neurons of the ischemic CA1 area after OJE pretreatment, and the increased immunoreactivities were sustained in the CA1 pyramidal neurons after transient global cerebral ischemia. However, OJE pretreatment did not increase interleukin-2 and tumor necrosis factor-alpha immunoreactivities in the CA1 pyramidal neurons. Our findings suggest that pretreatment with OJE can protect neurons and attenuate gliosis from transient global cerebral ischemia via increasing expressions of interleukin-4 and -13. The experimental plan of this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) in Kangwon National University (approval No. KW-160802-1) on August 10, 2016.

The protective effects of ethanolic extract of Clematis terniflora against corticosterone-induced neuronal damage via the AKT and ERK1/2 pathway.

Chronic stress induces neuronal cell death, which can cause nervous system disorders including Parkinson's disease and Alzheimer's disease. In this study, we evaluated the neuroprotective effects of Clematis terniflora extract (CTE) against corticosterone-induced apoptosis in rat pheochromocytoma (PC12) cells, and also investigated the underlying molecular mechanisms. At concentrations of 300 and 500 µg/ml, CTE significantly decreased apoptotic cell death and mitochondrial damage induced by 200 µM corticosterone. CTE decreased the expression levels of endoplasmic reticulum (ER) stress proteins GRP78, GADD153, and mitochondrial damage-related protein BAD, suggesting that it downregulates ER stress evoked by corticosterone. Furthermore, our results suggested that these protective effects were mediated by the upregulation of p-AKT and p-ERK1/2, which are involved in cell survival signaling. Collectively, our results indicate that CTE can lessen neural damage caused by chronic stress. [BMB Reports 2018; 51(8): 400-405].

Brain ischemic preconditioning protects against moderate, not severe, transient global cerebral ischemic injury.

Ischemic preconditioning (IPC) in the brain increases ischemic tolerance to subsequent ischemic insults. In this study, we examined whether IPC protects neurons and attenuates microgliosis or not in the hippocampus following severe transient global cerebral ischemia (TCI) in gerbils. Gerbils were assigned to 8 groups; 5- and 15-min sham operated groups, 5-min and 15-min TCI operated groups, IPC plus 5- and 15-min sham operated groups, and IPC plus 5- and 15-min TCI operated groups. IPC was induced by subjecting animals to 2-min transient ischemia 1 day before 5-min TCI for a typical transient ischemia and 15-min TCI for severe transient ischemia. Neuronal damage was examined by cresyl violet staining and Fluoro-Jade B histofluorescence staining. In addition, microglial activation was examined using immunohistochemistry for Iba-1 (a marker for microglia). Delayed neuronal death and microgliosis was found in the CA1 alone in the 5-min TCI operated group at 5 days post-ischemia, and, in the 15-min TCI operated group, neuronal death and microgliosis was shown in all CA areas (CA1-3) and the dentate gyrus. IPC displayed neuroprotection and attenuated microglial activation in the 5-min TCI operated group. However, in the 15-min TCI operated group, IPC did not show neuroprotection and not attenuate microglial activation. Our present findings indicate that IPC hardly protect against severe transient cerebral ischemic injury.

Vanillin improves scopolamine­induced memory impairment through restoration of ID1 expression in the mouse hippocampus.

4-Hydroxy-3-methoxybenzaldehyde (vanillin), contained in a number of species of plant, has been reported to display beneficial effects against brain injuries. In the present study, the impact of vanillin on scopolamine­induced alterations in cognition and the expression of DNA binding protein inhibitor ID­1 (ID1), one of the inhibitors of DNA binding/differentiation proteins that regulate gene transcription, in the mouse hippocampus. Mice were treated with 1 mg/kg scopolamine with or without 40 mg/kg vanillin once daily for 4 weeks. Scopolamine­induced cognitive impairment was observed from 1 week and was deemed to be severe 4 weeks following the administration of scopolamine. However, treatment with vanillin in scopolamine­treated mice markedly attenuated cognitive impairment 4 weeks following treatment with scopolamine. ID1­immunoreactive cells were revealed in the hippocampus of vehicle­treated mice, and were hardly detected 4 weeks following treatment with scopolamine. However, treatment with vanillin in scopolamine­treated mice markedly restored ID1­immunoreactive cells and expression 4 weeks subsequent to treatment. The results of the present study suggested that vanillin may be beneficial for cognitive impairment, by preventing the reduction of ID1 expression which may be associated with cognitive impairment.

Fucoidan downregulates insulin-like growth factor-I receptor levels in HT-29 human colon cancer cells.

Fucoidan, a sulfated polysaccharide present in brown seaweed, has demonstrated anticancer activity in lung, breast, liver and colon cells. The insulin-like growth factor (IGF) signaling pathway regulates growth in HT-29 cells through the insulin receptor substrate-1 (IRS-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Ras/Raf/extracellular signal-regulated kinase (ERK) pathways. The aim of the present study was to investigate whether fucoidan downregulates the IGF-IR signaling pathway in HT-29 human colon cancer cells. Fucoidan treatment (0-1,000 µg/ml) was administered for 24 h in HT-29 cells. First, we investigated IRS-1/PI3K/AKT pathway-related protein expression levels following treatment with fucoidan (0-500 µg/ml) using western blot analysis. Fucoidan significantly inhibited the expression of IGF-IR, PTEN, PI3K and AKT as well as their phosphorylated forms (p-IRS-1, p-PI3K and p-AKT). Next, we investigated the effects of fucoidan on Ras/Raf/ERK pathway­related protein expression levels in HT-29 cells. Fucoidan significantly inhibited the expression of IGF-IR, Shc, Ras, SOS, Raf and MEK. HT-29 cells were then incubated in the presence of fucoidan (0 or 250 µg/ml), and IGF-I (10 nM) was added for 0 to 60 min. Immunoprecipitation (IP) experiments showed that fucoidan inhibited IGF-I-induced phosphorylation of IGF-IR, PI3K, Shc (IP, IGF-IR), and phosphorylated IRS-1 and PI3K (IP, IRS-1) compared to the control group. Western blot analysis showed that fucoidan inhibited the expression of IGF-I-induced p-IGF-IR/IGF-IR and p-AKT/AKT, but not p-ERK/ERK. In conclusion, the inhibition of cell viability by fucoidan in HT-29 cells may be due to the downregulation of IGF-IR signaling through the main IRS-1/PI3K/AKT pathway. Fucoidan also partially impacted Ras/Raf signaling in the Ras/Raf/ERK pathway. Therefore, we suggest that fucoidan may be a suitable candidate chemopreventive agent in HT-29 colon cancer cells.

Long-term treadmill exercise improves memory impairment through restoration of decreased synaptic adhesion molecule 1/2/3 induced by transient cerebral ischemia in the aged gerbil hippocampus.

Exercise improves cognitive impairments induced by transient cerebral ischemia, and modulates synaptic adhesion molecules. In this study, we investigated effects of long-term treadmill exercise on cognitive impairments and its relation to changes of synaptic cell adhesion molecule (SynCAM) 1/2/3 in the hippocampus after 5 min of transient cerebral ischemia in aged gerbils. Animals were assigned to sedentary and exercised groups, given treadmill exercise for 4 consecutive weeks from 5 days after transient ischemia, and evaluated cognitive function through passive avoidance test and Morris water maze test. SynCAM 2 protein levels were determined in the hippocampus by western blot. In addition, neuronal and synaptic changes were examined by NeuN immunohistochemistry, and SynCAM 1/2/3 and MAP2 double immunofluorescence, respectively. We found that transient cerebral ischemia led to neuronal death in the CA1 area and dentate gyrus, and impaired -memory function; however, 4 weeks of treadmill exercise improved ischemia-induced memory impairment. In addition, SynCAM 1/2/3 and SynCAM 2 expression in the hippocampus was significantly decreased in the sedentary group after transient cerebral ischemia; however, SynCAM 1/2/3 expressionand and SynCAM 2 protein level was significantly increased in the ischemic group with exercise. These results suggest that long-term treadmill exercise improves memory impairment through the restoration of decreased SynCAM 1/2/3 expression in the hippocampus induced by transient cerebral ischemia in the aged gerbil.

Melatonin Improves Cognitive Deficits via Restoration of Cholinergic Dysfunction in a Mouse Model of Scopolamine-Induced Amnesia.

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT-, and M1R-immunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

Effects of chronic scopolamine treatment on cognitive impairment and neurofilament expression in the mouse hippocampus.

Neurofilaments (NFs) including neurofilament­200 kDa (NF­H), neurofilament­165 kDa (NF­M) and neurofilament­68 kDa (NF­L) are major protein constituents of the brain, and serve important roles in the regulation of axonal transport. NF alteration is a key feature in the pathogenesis of neurological disorders involving cognitive dysfunction. In the present study, cognitive impairments were investigated, via assessments using the Morris water maze and passive avoidance tests, in mice following chronic systemic treatment with 1 mg/kg scopolamine (SCO) for 4 weeks. SCO­induced cognitive impairments were significantly observed 1 week following the SCO treatment, and these cognitive deficits were maintained for 4 weeks. However, the NF immunoreactivities and levels were altered differently according to the hippocampal subregion following SCO treatment. NF­H immunoreactivity and levels were markedly altered in all hippocampal subregions, and were significantly increased 1 week following the SCO treatment; thereafter, the immunoreactivity and levels significantly decreased with time. NF­M immunoreactivity and levels gradually decreased in the hippocampus and were significantly decreased 4 weeks following SCO treatment. NF­L immunoreactivity and levels gradually decreased in the hippocampus, and were significantly decreased 2 and 4 weeks following SCO treatment. In conclusion, the results of the present study demonstrated that chronic systemic treatment with SCO induced cognitive impairment from 1 week following SCO treatment, and NF expression was diversely altered according to the hippocampal subregion from 1 week following SCO treatment. These results suggest that SCO­induced changes in NF expression may be associated with cognitive impairment.