Intracardiac Injection of Dental Pulp Stem Cells After Neonatal Hypoxia-Ischemia Prevents Cognitive Deficits in Rats.

Neonatal hypoxia-ischemia (HI) is associated to cognitive and motor impairments and until the moment there is no proven treatment. The underlying neuroprotective mechanisms of stem cells are partially understood and include decrease in excitotoxicity, apoptosis and inflammation suppression. This study was conducted in order to test the effects of intracardiac transplantation of human dental pulp stem cells (hDPSCs) for treating HI damage. Seven-day-old Wistar rats were divided into four groups: sham-saline, sham-hDPSCs, HI-saline, and HI-hDPSCs. Motor and cognitive tasks were performed from postnatal day 30. HI-induced cognitive deficits in the novel-object recognition test and in spatial reference memory impairment which were prevented by hDPSCs. No motor impairments were observed in HI animals. Immunofluorescence analysis showed human-positive nuclei in hDPSC-treated animals closely associated with anti-GFAP staining in the lesion scar tissue, suggesting that these cells were able to migrate to the injury site and could be providing support to CNS cells. Our study evidence novel evidence that hDPSC can contribute to the recovery following hypoxia-ischemia and highlight the need of further investigation in order to better understand the exact mechanisms underlying its neuroprotective effects.

Sex-dependent consequences of neonatal brain hypoxia-ischemia in the rat.

Neonatal hypoxia-ischemia (HI) is an important cause of neurological deficits in humans, and the Levine-Rice model of experimental HI in the rat mimics the human brain lesion and the following sensory motor deficits and cognitive disabilities. With the growing evidence that sex influences all levels of brain functions, this Mini-Review highlights studies in which sex was a controlled variable and that provided evidence of sexual dimorphism in behavioral outcome, extension of brain damage, mechanisms of lesion, and treatment efficacy in the rat neonatal HI model. It was shown that 1) females have greater memory deficits; 2) cell death is dependent mainly on caspase activation in females; 3) males are more susceptible to oxidative stress; and 4) treatments acting on distinct cell death pathways afford sex-dependent neuroprotection. These tentative conclusions, along with growing evidence from other fields of neurobiology, support the need for scientists to design their experiments considering sex as an important variable; otherwise, important knowledge will continue to be missed. It is conceivable that sex can influence the development of efficacious therapeutic tools to treat neonates suffering from brain HI. © 2016 Wiley Periodicals, Inc.

Chronic Stress Causes Sex-Specific and Structure-Specific Alterations in Mitochondrial Respiratory Chain Activity in Rat Brain.

Chronic restraint stress (CRS) induces a variety of changes in brain function, some of which are mediated by glucocorticoids. The response to stress occurs in a sex-specific way, and may include mitochondrial and synaptic alterations. The synapse is highly dependent on mitochondrial energy supply, and when mitochondria become dysfunctional, they orchestrate cell death. This study aimed to investigate the CRS effects on mitochondrial respiratory chain activity, as well as mitochondrial potential and mass in cell body and synapses using hippocampus, cortex and striatum of male and female rats. Rats were divided into non-stressed (control) and stressed group (CRS during 40 days). Results showed that CRS increased complex I-III activity in hippocampus. We also observed an interaction between CRS and sex in the striatal complex II activity, since CRS induced a reduction in complex II activity in males, while in females this activity was increased. Also an interaction was observed between stress and sex in cortical complex IV activity, since CRS induced increased activity in females, while it was reduced in males. Glucocorticoid receptor (GR) content in cortex and hippocampus was sexually dimorphic, with female rats presenting higher levels compared to males. No changes were observed in GR content, mitochondrial potential or mass of animals submitted to CRS. It was concluded that CRS induced changes in respiratory chain complex activities, and some of these changes are sex-dependent: these activities are increased in the striatal mitochondria by CRS protocol mainly in females, while in males it is decreased.

Mitochondrial and Oxidative Stress Aspects in Hippocampus of Rats Submitted to Dietary n-3 Polyunsaturated Fatty Acid Deficiency After Exposure to Early Stress.

Chronic dietary long-chain polyunsaturated fatty acids (PUFAs) deficiency may lead to changes in cortex and hippocampus neuronal membrane phospholipids, and may be linked to impaired central nervous system function. Particularly docosahexaenoic acid deficiency appears to be involved in neuropsychiatric disorders. On the other hand, adverse events early in life may also profoundly affect brain development, leading to long-lasting effects on neurophysiology, neurobiology and behavior. This research assessed if neonatal stress and a dietary n-3 PUFAs deficiency could interact to produce hippocampal alterations related to mitochondrial functions in adult rats. There were no effects of diet, neonatal intervention or interactions on superoxide dismutase or catalase enzymatic activities, mitochondrial membrane potential and respiratory chain complexes. Rats fed n-3 PUFAs deficient diet displayed higher levels of glutathione peroxidase and catalase activity, higher free radicals production and higher thiol content compared to rats fed n-3 PUFAs adequate diet. There were interactions among diets and neonatal stress, since glutathione peroxidase, free radicals production and thiol content were increased in groups that were subjected to neonatal interventions fed n-3 PUFAs deficient diet. Additionally, reduced mitochondrial potential was observed in handled animals. Total thiol revealed a neonatal stress effect, since animals subjected to neonatal interventions displayed lower thiol content. In conclusion, we observed that a chronic treatment with deficient n-3 PUFAs diet, from the puberty period on, increased free radicals production and imbalanced antioxidant enzymes activities, and these increases were higher in animals subjected to neonatal interventions.

In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways.

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

Neonatal handling impairs spatial memory and leads to altered nitric oxide production and DNA breaks in a sex specific manner.

Early life events lead to behavioral and neurochemical changes in adulthood. The aim of this study is to verify the effects of neonatal handling on spatial memory, nitric oxide (NO) production, antioxidant enzymatic activities and DNA breaks in the hippocampus of male and female adult rats. Litters of rats were non-handled or handled (10 min/day, days 1-10 after birth). In adulthood they were subjected to a Morris water maze or used for biochemical evaluations. Female handled rats showed impairment in spatial learning. They also showed decreased NO production, while no effects were observed in these parameters in male rats. No effects were observed on the number of hippocampal NADPH diaphorase positive cells. In the Comet Assay, male handled rats showed increased DNA breaks index when compared to non-handled ones. We conclude that neonatal handling impairs learning performance in a sex-specific manner, what may be related to NO decreased levels.

Diphenyl ditelluride effect on embryo/fetal development in mice: interspecies differences.

It is well established that diphenyl ditelluride, (PhTe)(2), is a potent teratogen in rats, however, little is known about its effects on embryo/fetal development in mice. The present study was undertaken to investigate whether any differences exist on embryo/fetal development of mice exposed to (PhTe)(2) during distinctive periods of gestation compared to rats. Dams were treated subcutaneously (s.c.) with 0.12 or 60.0 mg/kg (PhTe)(2) on gestational day (GD) 4, 8 or 14. Cesarean section was performed on GD18 and external and skeletal alterations were examined. The lower dose did not affect any parameter evaluated in mouse fetuses. The maternal body weight for 60 mg/kg (PhTe)(2) groups, at all periods studied, was not affected. Maternal liver and spleen weights were increased at GD8. At GD14, maternal relative weight of kidney was also increased. A significant reduction in the number of implantation sites at GD4 was found. At GD4 and GD14, there was a reduction in the fetal weight and biometry. A few signs of reduced ossification in sternebrae and limbs were observed at GD14 in (PhTe)(2) group. In conclusion, (PhTe)(2) was not toxic to dams and affected some fetal endpoints only at the dose about 500-fold higher than the dose that was teratogenic in rats, suggesting a different developmental toxicity induced by (PhTe)(2) among species. Thus, the mice were less susceptible to toxic effects induced by (PhTe)(2) than were rats.

Adult male rats sub-chronically exposed to diphenyl diselenide: Effects on their progeny.

The present study was conducted to evaluate the toxicity of diphenyl diselenide [(PhSe)2] exposure on the progeny of Wistar male rats. Male rats were exposed to (PhSe)2 subcutaneously for 4 weeks at the dose of 5.0 mg/kg and 8 weeks at the dose of 2.5 mg/kg, prior to mating with unexposed females. No lethality was noted in any group. At term of exposure period, 4-week exposed male rats presented significant decrease in the body weight. Sex organ weights were similar in (PhSe)2-exposed and control male groups. The number of implantation sites in females mated with males exposed to (PhSe)2 for 8 weeks was significantly higher than those of the respective control group. Male exposure to (PhSe)2, administered for 4 and 8 weeks, did not change fetal body weight. Gross examination of fetuses from 4- to 8-week exposed groups did not reveal the appearance of external anomalies. Examination of live fetuses for ossification centers did not show significantly difference between groups. No increase in the incidence of skeletal anomalies was observed in fetuses obtained from females impregnated with (PhSe)2-exposed males. The current study indicated that (PhSe)2 given sub-chronically (4 or 8 weeks) to male rats had no adverse effects on their progeny.

Sub-chronic exposure of adult male rats to diphenyl ditelluride did not affect the development of their progeny.

The present study was undertaken to evaluate the toxicity of diphenyl ditelluride [(PhTe)(2)] exposure on the progeny of Wistar male rats. Male rats were exposed to (PhTe)(2) subcutaneously for 4 weeks (wk) at the dose of 0.006 mg/kg and 8-wk at the dose of 0.003 mg/kg, prior to mating with unexposed females. The body and sex organ weights of male rats were not affected in both 4- and 8-wk (PhTe)(2)-exposed groups. The gravid uterus weight and the body weight gain (overall or corrected) during the pregnancy were not statistically different to those obtained from females mated with control males. The number of implantation sites, resorptions and live and dead fetuses were not affected by male exposure to (PhTe)(2). Fetal body weight and crown-rump length were not affected, as well. Examination of the fetuses from both exposed groups for external and skeletal changes did not reveal any male-mediated effect of (PhTe)(2). The current study indicated that (PhTe)(2) given sub-chronically (4- or 8-wk) to male rats had no adverse effects on their progeny.

Assessment of reproductive toxicity in male rats following acute and sub-chronic exposures to diphenyl diselenide and diphenyl ditelluride.

The present study was conducted to evaluate the toxicity of the exposure to diphenyl diselenide [(PhSe)2] and diphenyl ditelluride [(PhTe)2] on reproductive system in Wistar rats. Adult male rats were exposed intraperitonealy (acute) or subcutaneously (sub-chronic, during 4 or 8 weeks) to (PhSe)2 or (PhTe)2 prior to mating. A number of biochemical parameters in rat testes were examined, such as delta-aminolevulinate dehydratase (delta-ALA-D) activity, lipid peroxidation, glycogen content and components of the antioxidant defenses (superoxide dismutase (SOD) activity and ascorbic acid concentration). Furthermore, a possible effect on fertility and reproductive performance in male rats were studied. Sperm counts of caudal epididymis were also evaluated. No lethality was noted in any group. Reduction on body weight in rats which received (PhTe)2 was only evidenced in acute exposure, while (PhSe)2-exposed rats presented significant loss of body weight in acute and 4 week-exposure. Mating and fertility indexes were not affected after acute and sub-chronic exposure. Regarding other parameters studied, except for a decrease in testes glycogen content in acutely (PhSe)2-treated group, no alterations were found in treated groups. Sperm counts of rats treated acutely and sub-chronically were unaffected by drugs exposure. Histological evaluation revealed no modification on testicular tissue in rats exposed to (PhSe)2 and (PhTe)2. The results suggest the absence of the male reproductive toxicity induced by (PhSe)2 and (PhTe)2 administered intraperitonealy (acute) or subcutaneously (sub-chronical) to adult rats Wistar.

A new device for step-down inhibitory avoidance task--effects of low and high frequency in a novel device for passive inhibitory avoidance task that avoids bioimpedance variations.

BACKGROUND: Step-down inhibitory avoidance task has been widely used to evaluate aversive memory, but crucial parameters inherent to traditional devices that may influence the behavior analysis (as stimulus frequency, animal's bioimpedance) are frequently neglected. NEW METHOD: We developed a new device for step-down inhibitory avoidance task by modifying the shape and distribution of the stainless steel bars in the box floor where the stimuli are applied. The bars are 2 mm wide, with rectangular shape, arranged in pairs at intervals of 1cm from the next pairs. Each pair makes an electrical dipole where the polarity inverts after each pulse. This device also presents a component that acquires and records the exact current received by the animal foot and precisely controls the frequency of stimulus applied during the entire experiment. RESULT: Different from conventional devices, this new apparatus increases the contact surface with bars and animal's paws, allowing the electric current pass through the animal's paws only, drastically reducing the influence of animal's bioimpedance. The analysis of recorded data showed that the current received by the animal was practically the same as applied, independent of the animal's body composition. Importantly, the aversive memory was observed at specific stimuli intensity and frequency (0.35 or 0.5 mA at 62 and 125 Hz but not at 0.20 mA or 20 Hz). Moreover, with this device it was possible to observe the well-known step-down inhibitory avoidance task memory impairment induced by guanosine. CONCLUSION: This new device offers a substantial improvement for behavioral analysis in step-down inhibitory avoidance task and allows us to precisely compare data from different animals with distinct body composition.

Chronic brain hypoperfusion causes early glial activation and neuronal death, and subsequent long-term memory impairment.

Reduction of cerebral blood flow is an important risk factor for dementia states and other brain dysfunctions. In present study, the effects of permanent occlusion of common carotid arteries (2VO), a well established experimental model of brain ischemia, on memory function were investigated, as assessed by reference and working spatial memory protocols and the object recognition task; cell damage to the hippocampus, as measured through changes in immunoreactivity for GFAP and the neuronal marker NeuN was also studied. The working hypothesis is that metabolic impairment following hypoperfusion will affect neuron and glial function and result in functional damage. Adult male Wistar rats were submitted to the modified 2VO method, with the right common carotid artery being occluded first and the left one week later, and tested seven days, three and six months after the ischemic event. A significant cognitive deficit was found in both reference and working spatial memory, as well as in the object recognition task, three and six months after surgery. Neuronal death and reactive astrogliosis were already present at 7 days and continued for up to 3 months after the occlusion; interestingly, there was no significant reduction in hippocampal volume. Present data suggests that cognitive impairment caused by brain hypoperfusion is long - lasting and persists beyond the time point of recovery from glial activation and neuronal loss.