Effects of Gestational and Lactational Lead Exposure and High Fat Diet Feeding on Cerebellar Development of Postnatal Rat Offspring.

Obesity and heavy metals, such as lead (Pb), are detrimental to the adult brain because they impair cognitive function and structural plasticity. However, the effects of co-administration of Pb and a high-fat diet (HFD) on the developing cerebellum is not clearly elucidated. We investigated the effects of Pb exposure (0.3% lead acetate) on developing cerebellum in the pups of an HFD-fed obese rat model. One week before mating, we fed a chow diet (CD) or HFD to the rats for one week and additionally administered Pb to HFD-fed female SD rats. Thereafter, treatment with Pb and a HFD was continued during the gestational and lactational periods. On postnatal day 21, the pups were euthanized to sample the brain tissue and blood for further analysis. Blood Pb levels were significantly higher in HFD-fed rats than in CD-fed rats. Histologically, the prominent degeneration of Purkinje cells was induced by the co-administration of Pb and HFD. The calbindin-28Kd-, GAD67-, NMDAR1-, and PSD95-immunopositive Purkinje cells and inhibitory synapse-forming pinceau structures were significantly decreased following Pb and HFD co-administration. MBP-immunoreactive myelinated axonal fibers were also impaired by HFD but were significantly damaged by the co-administration of HFD and Pb. Oxidative stress-related Nrf2-HO1 signaling was activated by HFD feeding, and Pb exposure further aggravated oxidative stress, as demonstrated by the consumption of endogenous anti-oxidant in HFD-Pb rats. The pro-inflammatory response was also increased by the co-administration of HFD and Pb in the cerebellum of the rat offspring. The present results suggest that HFD and Pb treatment during the gestational and lactational periods are harmful to cerebellar development.

Transthyretin is a key regulator of myoblast differentiation.

Transthyretin (TTR) is a known carrier protein for thyroxine (T4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd) did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.

Neurobehavioural deficits correlate with the cerebral infarction volume of stroke animals: a comparative study on ischaemia-reperfusion and photothrombosis models.

The study investigated the correlation between infarction areas and behavioural deficits in middle cerebral artery occlusion (MCAO) and photothrombosis stroke models. In the MCAO model, a 0.38 mm-diameter silicone-coated thread was introduced through the left external carotid artery and advanced 18 mm via the internal carotid artery to the origin of middle cerebral artery of male Sprague-Dawley rats weighing 300-350 g. The thread was removed for reperfusion after occlusion for 0.5, 1 or 2h. In the photothrombosis model, after a midline incision on the scalp, a focused light (10,000 lux, 6 mm-diameter) was delivered 1mm anterior to the bregma and 3mm left of the midline for 5, 10 or 20 min. During the first 2 min of irradiation, Rose Bengal dye (30 mg/kg) was injected intravenously. Twenty four hours post-surgery, the animals were subjected to neurological scoring and behavioural performances, and were sacrificed for macroscopic and microscopic examinations of brain injury. Total infarction volumes in the MCAO model rats increased in an occlusion time-dependent manner, while the infarction volumes in photothrombosis model rats plateaued relatively quickly with no time-dependent increase. The MCAO model displayed neurological scores and behavioural deficits that correlated well with infarction volumes, while relatively poor correlation between infarction volume and neurobehavioural abnormalities was evident in the photothrombosis model. The results indicate the suitability of the MCAO model for studies on preventive or therapeutic compounds related to functional recovery, although the photothrombosis model might be useful to generate focused lesions leading to the location-related behavioural changes.

Antiteratogenic effect of resveratrol in mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effect of resveratrol, an aryl hydrocarbon receptor antagonist, on the teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated. Pregnant C57BL/6J mice were orally administered resveratrol (50 mg/kg) for 6 consecutive days, from gestational day (GD) 8 to GD13, followed by an oral challenge with TCDD (14 mug/kg) on GD12. TCDD caused severe fetal malformations including cleft palate (40.7%), renal pelvic dilatation (100%, mean score 3.060), and ureteric dilatation (100%, mean score 3.210) and tortuosity (95.1%). Resveratrol significantly reduced both the incidence of TCDD-induced cleft palate to 18.4% and the degrees of renal pelvic and ureteric dilatations caused by TCDD. The results suggest that pretreatment with resveratrol might bring a beneficial outcome for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.

Immune responses and pathogenesis in immunocompromised chickens in response to infection with the H9N2 low pathogenic avian influenza virus.

The H9N2 low pathogenic avian influenza (LPAI) viruses have often caused moderate mortality with severe clinical signs in domestic poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans, but the basis for their pathogenesis remains unclear especially in chickens. To better understand the effect of immunosuppression on the risk of H9N2 viral infection, the pathogenesis and host immune responses of the H9N2 LPAI virus in a T-cell-suppressed chicken model were investigated. Cyclosporin A (CsA) treatment led to suppression of cell-mediated immunity such as CD8+ T-cells and reduced expression of IFN-gamma mRNA. T-cell suppression correlated with high viral load in the oropharynx and cloaca of H9N2 LPAI virus-infected specific pathogen free (SPF) chickens. Elevated level of viral RNA in the peripheral blood lymphocytes was found only in immunocompromised chickens. Viral protein and associated cellular apoptosis were observed only in the kidney of the immunocompromised chickens, particularly in those that had died. Our findings suggest that T-cell-mediated responses are important in influenza viral clearance and may help to explain in part the reasons for the increased mortality in chickens infected with H9N2 LPAI viruses in domestic poultry farms.

Effect of maternal restraint stress on fetal development of ICR mice.

The present study was conducted to elucidate the susceptibility of embryos and fetuses at different gestational stages to the maternal stress in mice. Groups of pregnant ICR mice were subjected to daily 12-h restraint stress, taped in the supine position on a plastic board, on gestational days (GD) 1-4, 5-8, 9-12 and 13-16, respectively. Caesarean sections were performed on gestational day 18, and the fetuses were weighed and examined for morphological defects. During the daily restraint for 4 days, the maternal body weights markedly decreased. Although the body weights recovered gradually after termination of the stress, the recovery was not full until the final stage of pregnancy. Interestingly, restraint stress caused growth retardation of the fetuses, leading to a significant decrease in their body weights, and increased early and late resorptions of embryos and fetuses according to the stress periods. Although the preceding (GD1-4) and concurrent (GD5-8) stresses did not affect embryonic implantation, restraint stress on GD9-12 caused cleft palate. Whereas vertebral abnormalities, mainly bipartite ossification, were observed only in animals stressed on GD5-8, abnormalities of sternebrae, exhibiting asymmetric or bipartite ossification, were enhanced by the stress at all of the gestational stages. On the other hand, the incidence of other malformations including renal malposition and costal abnormalities was not increased by stress at any of the 4 stages. Taken together, the results suggest that intensive restraint stress influences the maternal body weight resulting in growth retardation and increased mortality of embryos and fetuses, in addition to gestational stage-specific ventricular dilatation, cleft palate and sternal abnormalities.

trans-Resveratrol relaxes the corpus cavernosum ex vivo and enhances testosterone levels and sperm quality in vivo.

We examined the effects of trans-resveratrol on male reproductive functions; ex-vivo penile erection and in-vivo sperm counts and quality. For the ex-vivo study, the relaxation effects of resveratrol on isolated New Zealand white rabbit corpus cavernosum, precontracted by phenylephrine (5x10(-5) M) were measured. The in-vivo study measured reproductive organ weights, blood testosterone levels, testicular histopathology, sperm counts, as well as the epididymal sperm motility and deformity of male ICR mice given an oral dose of resveratrol (50 mg/ kg) for 28 days. Resveratrol elicited a concentration-dependent relaxing effect on corpus cavernosum, leading to a median effective concentration (EC50) of 0.29 mg/mL. Repeated treatment with resveratrol (50 mg/kg) did not cause an increase in body weight, reproductive organ weight or testicular microscopic findings; however, resveratrol did elicit an increase in blood testosterone concentration, testicular sperm counts and epididymal sperm motility by 51.6%, 15.8% and 23.3%, respectively, without influence on sperm deformity. In conclusion, we propose that resveratrol has a positive effect on male reproductive function by triggering a penile erection, as well as enhancing blood testosterone levels, testicular sperm counts, and epididymal sperm motility.

Debilitating stresses do not increase blood-brain barrier permeability: Lack of the involvement of corticosteroids.

The involvement of corticosteroids in stress-induced change in blood-brain barrier (BBB) permeability was investigated. Mice were adrenalectomized and administered with pyridostigmine bromide (PB) or Evan's blue, markers of BBB penetration, followed by 18-h cold-restraint stress (CRS). Rats were administered with mifepristone, a corticosteroid receptor blocker, and the markers, followed by 4-h water immersion-restraint stress (WIRS). Separately, soman was administered to induce seizures-mediated BBB opening. CRS did not induce PB and Evan's blue penetration, which were not affected by adrenalectomy. Also, the markers were not detected in the brain of rats subjected to WIRS, regardless of the treatment of mifepristone. In comparison, 1-h epileptic seizures increased the penetration of Evan's blue by 875%. The results suggest that in contrast to seizure-related BBB opening, profound stresses do not practically increase the BBB permeability, and that corticosteroids are not involved in the stress-induced BBB penetration of charged chemicals and albumin-dye complex.

The spatio-temporal expression pattern of cytoplasmic Cu/Zn superoxide dismutase (SOD1) mRNA during mouse embryogenesis.

The cytoplasmic Cu/Zn-superoxide dismutase (SOD1) represents along with catalase and glutathione peroxidase at the first defense line against reactive oxygen species in all aerobic organisms, but little is known about its distribution in developing embryos. In this study, the expression patterns of SOD1 mRNA in mouse embryos were investigated using real-time RT-PCR and in situ hybridization analyses. Expression of SOD1 mRNA was detected in all embryos with embryonic days (EDs) 7.5-18.5. The signal showed the weakest level at ED 12.5, but the highest level at ED 15.5. SOD1 mRNA was expressed in chorion, allantois, amnion, and neural folds at ED 7.5 and in neural folds, notochord, neuromeres, gut, and primitive streak at ED 8.5. In central nervous system, SOD1 mRNA was expressed greatly in embryos of EDs 9.5-11.5, but weakly in embryos of ED 12.5. At EDs 9.5-12.5, the expression of SOD1 mRNA was high in sensory organs such as tongue, olfactory organ (nasal prominence) and eye (optic vesicle), while it was decreased in ear (otic vesicle) after ED 10.5. In developing limbs, SOD1 mRNA was greatly expressed in forelimbs at EDs 9.5-11.5 and in hindlimbs at EDs 10.5-11.5. The signal increased in liver, heart and genital tubercle after ED 11.5. In the sections of embryos after ED 13.5, SOD1 mRNA was expressed in various tissues and especially high in mucosa and metabolically active sites such as lung, kidney, stomach, and intestines and epithelial cells of skin, whisker follicles, and ear and nasal cavities. These results suggest that SOD1 may be related to organogenesis of embryos as an antioxidant enzyme.

Antiteratogenic effects of alpha-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice in utero.

The effects of alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, on the reproductive toxicity and teratogenicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated. Pregnant C57BL/6J mice were orally administered alpha-naphthoflavone either once on gestational day 12 (GD12; 50 microg/kg) or for 6 days (GD8-GD13; 5 mg/kg/day) followed by an oral challenge with TCDD (14 microg/kg) on GD12. Cesarean section was performed on GD18 for the evaluation of maternal and fetal toxicities. TCDD caused severe fetal malformations including cleft palate (43.7%) and renal pelvic and ureteric dilatations (100%). The administration of alpha-naphthoflavone either in a single treatment or 6-days remarkably reduced the incidence of cleft palate to 27.6% and 26.5%, respectively. In addition, the degree of renal pelvic and ureteric dilatations caused by TCDD were significantly attenuated by repeated treatment of alpha-naphthoflavone. These results suggest that AhR antagonists such as alpha-naphthoflavone could be promising candidates for reducing the incidence and severity of fetal malformations caused by TCDD exposure in utero.