Newborn Rat Brain Research Articles

Age-Dependent Vulnerability to Oxidative Stress of Postnatal Rat Pyramidal Motor Cortex Neurons.

Oxidative stress is one of the main proposed mechanisms involved in neuronal degeneration. To evaluate the consequences of oxidative stress on motor cortex pyramidal neurons during postnatal development, rats were classified into three groups: Newborn (P2–P7); infantile (P11–P15); and young adult (P20–P40). Oxidative stress was induced by 10 µM of cumene hydroperoxide (CH) application. In newborn rats, using the whole cell patch-clamp technique in brain slices, no significant modifications in membrane excitability were found. In infantile rats, the input resistance increased and rheobase decreased due to the blockage of GABAergic tonic conductance. Lipid peroxidation induced by CH resulted in a noticeable increase in protein-bound 4-hidroxynonenal in homogenates in only infantile and young adult rat slices. Interestingly, homogenates of newborn rat brain slices showed the highest capacity to respond to oxidative stress by dramatically increasing their glutathione and free thiol content. This increase correlated with a time-dependent increase in the glutathione reductase activity, suggesting a greater buffering capacity of newborn rats to resist oxidative stress. Furthermore, pre-treatment of the slices with glutathione monoethyl ester acted as a neuroprotector in pyramidal neurons of infantile rats. We conclude that during maturation, the vulnerability to oxidative stress in rat motor neurons increases with age.

Lipid Nanoparticles Vectorized with NFL-TBS.40-63 Peptide Target Oligodendrocytes and Promote Neurotrophin-3 Effects After Demyelination In Vitro.

Promoting remyelination in multiple sclerosis is important to prevent axon degeneration, given the lack of curative treatment. Although some growth factors improve this repair, unspecific delivery to cells and potential side effects limit their therapeutic use. Thus, NFL-TBS.40-63 peptide (NFL)-known to enter specifically myelinating oligodendrocytes (OL)-was used to vectorize 100nm diameter lipid nanoparticles (LNC), and the ability of NFL-LNC to specifically target OL from newborn rat brain was assessed in vitro. Specific uptake of DiD-labeled NFL-LNC by OL characterized by CNP and myelin basic protein was observed by confocal microscopy, as well as DiD colocalization with NFL and with Rab5-a marker of early endosomes. Unvectorized LNC did not significantly penetrate OL and there was no uptake of NFL-LNC by astrocytes. Canonical maturation of OL which extended compacted myelin-like membranes was observed by transmission electron microscopy in cells grown up to 9days with NFL-LNC. Endocytosis of NFL-LNC appeared to depend on several pathways, as demonstrated by inhibitors. In addition, vectorized NFL-LNC adsorbed on neurotrophin-3 (NT-3) potentiated the proremyelinating effects of NT-3 after demyelination by lysophosphatidyl choline, allowing noticeably decreasing NT-3 concentration. Our results if they were confirmed in vivo suggest that NFL-vectorized LNC appear safe and could be considered as putative carriers for specific drug delivery to OL in order to increase remyelination.

Neuroprotective Effects of Lacosamide and Memantine on Hyperoxic Brain Injury in Rats.

In neonates supraphysiological oxygen therapy has been demonstrated to cause neuronal death in hippocampus, prefrontal cortex, parietal cortex, and retrosplenial cortex. There is a need for the detection of novel neuroprotective drugs. Neuroprotective effects of lacosamide or memantine have been demonstrated in adult patients with ischemia, trauma and status epilepticus. The effects in immature brains may be different. This study aimed to evaluate neuroprotective effects of lacosamide and memantine treatment in a hyperoxia-induced brain injury model in immature rats. This study was performed in the Animal Experiments Laboratory of Dokuz Eylul University Faculty of Medicine. Neonatal Wistar strain rat pups were exposed to hyperoxia (80% oxygen + 20% nitrogen) for five days postnatally. They were divided into five groups; hyperoxia + lacosamide, hyperoxia + memantine, hyperoxia + lacosamide and memantine, hyperoxia + saline, control groups. After termination of the experiment, brain tissues were examined. Neuron counting in examined regions were found to be higher in hyperoxia + memantine and hyperoxia + lacosamide and memantine groups than hyperoxia + saline group. The presence of apoptotic cells evaluated with TUNEL and active Caspase-3 in hyperoxia + memantine and hyperoxia + lacosamide and memantine groups were found to be lower compared to hyperoxia + saline group. This study demonstrates that neuron death and apoptosis in newborn rat brains after hyperoxia is reduced upon memantine treatment. This is the first study to show the effects of memantine and lacosamide on hyperoxia-induced damage in neonatal rat brains.

Combination of lipopolysaccharide and hypoxia-ischemia attenuates cholinergic anti-inflammatory effect in newborn rat brains

Aim: Pharmacologic parasympathetic nerve stimulation markedly reduces brain damage in a newborn rat model of hypoxia-ischemia (HI). Our aim is to determine whether a brain-protective effect can be achieved by parasympathetic nerve stimulation even with the synergy effect of lipopolysaccharide (LPS) and HI. Study design: 7-day-old Wistar rats were used. Rats received intraperitoneal administration of LPS which was followed by left carotid artery ligation. After a two-hour recovery period rats were placed in a hypoxic environment (8% oxygen) for 1 hour. Carbachol, used as an acetylcholine receptor agonist, or saline was injected subcutaneously immediately prior to one-hour hypoxia to determine its neuroprotective effect. Seven days later, the degree of brain damage in the ligated side of the hemisphere was compared to that in the contralateral hemisphere, which acts as reference. The relative difference in hemisphere area ((ligated side/non-ligated side) × 100%) was employed as an indicator of brain damage. Microglial aggregation (cells/mm 2) on the cortex and cytokine production in the ligated side of the hemisphere were also evaluated. Results: Three pups died in the carbachol group. There was no significant difference in brain damage between the saline (n = 34) and carbachol (n = 30) groups (86.0 ± 12.1% vs. 90.8 ± 13.6%, respectively). There was no significant difference in microglial aggregation between the saline and carbachol groups (155.7 ± 201.1 cells/mm2 vs. 89.8 ± 149.1 cells/mm2, respectively). IL-1β production in the saline and carbachol groups was 1.8 ± 1.2 pg/dL and 1.8 ± 1.1 pg/dL, respectively, with no significant difference between the groups. Conclusion: Combination of LPS and HI attenuates cholinergic anti-inflammatory effect in newborn rat brains.

ЭФФЕКТ ПРЕНАТАЛЬНОГО ПАССИВНОГО КУРЕНИЯ НА ХОЛИНЕРГИЧЕСКИЕ МЕХАНИЗМЫ МОДУЛИРОВАНИЯ ДЫХАТЕЛЬНОГО РИТМА У НОВОРОЖДЕННЫХ КРЫС (IN VITRO)

Both active and passive smoking increases the risk of sudden death of the newborn. Researchers are actively studying the effect of chronic nicotine infusion, as one of the leading neurogenic factors of tobacco smoke on cholinergic mechanisms of respiratory control. In this paper, using a fumigation model of passive smoking, tested the assumption that second-hand smoke that is transferred in the prenatal period, changes the expression mediated by nicotinic receptors activating influence of the cholinergic system of the brain stem to the processes of the respiratory activity of the neural network generation. It is found that the fumigation of tobacco smoke pregnant rats decreases their progeny respiratory sensitivity to the action of a neural network and exogenous nicotine increases cholinergic part tonic effect mediated by nicotinic cholinergic receptors in the modulation of respiratory rhythm. The study uses data obtained from 40 brain stem-spinal cord preparations (BSP) of the newborn rats. The experimental group was 22, and the control group was 18 newborn rats. In the processing of neurograms, the duration of the cycle of respiratory activity, duration, and the amplitude of inspiratory discharges were measured. To describe the peaks of the respiratory discharge spectrum, the following parameters were used: the peak frequency and the peak power spectral density of the peak. Analysis of the statistical differences was made using Student’s t-test for mean values. Differences were considered significant at p<0.05. Our results confirm the presence for exogenous nicotine of powerful activating effect on the generation frequency, amplitude and duration of inspiratory discharges of the BSP of newborn rats in the control group. It is established that an increase in the amplitude of the inspiratory discharges is accompanied by an increase in the spectral power density in the mid-frequency range of their spectrograms. In the BSP of the brain of newborn rats with prenatal exposure to tobacco smoke, exogenous nicotine increased only the frequency of inspiratory discharge generation. The amplitude of the inspiratory discharges and the power of the mid-frequency oscillations under the influence of exogenous nicotine in the BSP of the experimental group was significantly reduced. Mecamylamine, a selective blocker of nAChR, added to the perfusate of the BSP of the control group, caused a significant increase in the amplitude and duration of the inspiratory discharges, without significantly changing the duration of the respiratory cycle. At the same time, in BSP of newborn rats subjected to prenatal exposure to tobacco smoke, nAChR blockade resulted in an increase in the duration of the respiratory cycle. Thus, our study showed that fumigation of pregnant rats with tobacco smoke reduces the sensitivity of the respiratory neural network to the action of exogenous nicotine in early postnatal period and increases the involvement of tonic cholinergic effect mediated by nicotinic cholinergic receptors in modulating the respiratory rhythm.

Effects of Ginsenoside Rg1 Regulating Wnt/β-Catenin Signaling on Neural Stem Cells to Delay Brain Senescence.

This is a study on the relationship between the protective effect of ginsenoside Rg1 on senescent neural stem cells and Wnt-β/catenin signaling pathway. Background. Recent studies have shown that overactivation of the Wnt/β-catenin signaling pathway is closely related to stem cell senescence. Whether Rg1 delays the senescence of NSCs is related to the regulation of this signaling pathway. Methods. The whole brain of Nestin-GFP transgenic newborn rat was extracted, and NSCs were extracted and cultured to P3 generation. The following indicators were detected: (1) NSC culture identification, (2) the effect of LiCl on the proliferation and survival rate of NSCs, (3) the effect of ginsenoside Rg1 on the proliferation and survival of NSCs, (4) the growth of NSCs in each group observed by an optical microscope, (5) the cell cycle of each group detected by flow cytometry, (6) the proliferative ability of each group detected by BrdU, (7) the fluorescence intensity of Nestin and Sox2 of NSCs in each group observed by a fluorescence microscope, (8) the positive rate of senescence staining analyzed by SA-β-Gal staining, (9) the localization of β-catenin in NSCs observed by laser confocal microscopy, and (10) the changes of the Wnt/β-catenin pathway-related proteins in each group detected by Western blotting. Results. LiCl activates the Wnt/β-catenin pathway and promotes mouse neural stem cell senescence. Ginsenoside Rg1 promotes proliferation of neural stem cells and inhibits Wnt/β-catenin pathway activation. Conclusions. LiCl can activate the Wnt/β-catenin signaling pathway of NSCs, and ginsenoside Rg1 can antagonize the senescence of NSCs caused by activation of the Wnt/β-catenin signaling pathway and delay brain aging.

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals.

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Remote Sensing of Ciliary Beating Using a SQUID Gradiometer

Beating of ependymal cilia in the mammalian brain ventricle causes cerebrospinal fluid circulation. Beating of ependymal cilia is studied in vitro in many laboratories but has not been monitored non-invasively in live animals. We have developed a new method for remote sensing of the magnetic flux density changes associated with the reciprocal movement of a magnetic particle bound to cilia in the lateral ventricle of a newborn rat brain with superconducting quantum interference device (SQUID). We detected an oscillatory signal (10–20 Hz and ca. 10 pT amplitude) from a small neodymium magnetic particle attached to cilia with the SQUID above the specimen; the distance between the cilia to the SQUID sensor was 12 mm. This successful detection provides a new sensing platform that enables the non-invasive measurement of beating of ependymal cilia from a live animal.

TARP mediation of accelerated and more regular locus coeruleus network bursting in neonatal rat brain slices

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca2+ (Cai) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 μM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2–5 mV depolarization and a modest Cai rise whereas Cai was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 μM), CNQX caused a 5–8 mV depolarization and also the Cai rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 μM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Cai rise was blocked by the L-type voltage-activated Ca2+ channel inhibitor nifedipine (100 μM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca2+ channel-mediated neuronal Cai rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K+ channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.

Effect of experimentally induced hypothyroidism during gestation period on activity dependent neurotrophic factor (ADNF) in newborn rat brain tissue.

Purpose The aim of the study was to evaluate the effects of prenatal hypothyroidism on neonatal rats by the way of activity-dependent neuroprotective factor (ADNF) expression. Methods Twenty-one Wistar albino neonatal rats were divided into two subgroups; a control group and neonatal rats with experimental maternal hypothyroidism. Hypothyroidism was induced by using propylthiouracil (PTU). Neonatal rats obtained PTU from breast milk continuously for 1 week after birth. The rats from the control group were fed only normal feed and water. After birth, body weight and blood thyroid hormone levels were tested. Glial fibrillary acidic protein (GFAP), Slug, Numb, Notch-1 and ADNF antibodies were used for immunohistochemical analysis. Real-time polymerase chain reaction (RT-PCR) and Western blotting analyses were used to evaluate ADNF gene expression levels from 1-week-old rat's brain. Results There was no difference between the two groups for birth weights. The thyroxine (T4) level from the experimental group was <0.4 ng/mL, and it was 0.8 ng/mL for the control group. It was shown that, the results from the experimental group samples had significantly lower ADNF mRNA levels than control group (p < 0.05). The increase from GFAP and Numb expression and decrease from Slug expression were shown in the experimental group. Local differences were identified for ADNF and a decrease was shown in both sides of brain. There was no difference for Notch-1 expression for both groups. Conclusion In this study, decreasing ADNF expression might contribute to developing neurological problems in congenital hypothyroidism.

Iron deficiency and iron treatment in the fetal developing brain \u2013 a pilot study introducing an experimental rat model

BackgroundIron deficiency is especially common in women during the reproductive age and it is estimated that 52% of pregnant women have iron deficiency anemia. Maternal iron deficiency with or without anemia in pregnancy may have consequences for the fetus, where it may have an impact on the cerebral development of the brain. Both animals and adult human studies support that iron deficiency affects psychomotor development, behavioral traits, and cognitive functions in the offspring. However, it has not yet been established whether the availability of sufficient iron is particularly important in certain phases during brain development, and whether possible damages are reversible if iron supplementation is provided during pregnancy. Here we report results from a pilot study in an experimental rat model suitable for introducing iron deficiency in the fetal rat brain.MethodsThe model was utilized for examination of the potential to reverse changes in fetal brain iron by maternal parenteral iron administration. Fertilized females subjected to iron deficiency without anemia were subcutaneously injected with iron isomaltoside at the day of mating (E0), 14 days into pregnancy (E14), or at the day of birth (Postnatal (P) 0). Blood, brain and liver in the offspring were examined on P0 or in adulthood on postnatal day P70.ResultsMaternal iron restriction during pregnancy led to significantly lower levels of iron in the brains of newborn rats compared to levels in pups of iron sufficient mothers. Females fed ID diet (5.2 mg/kg Fe) had offspring with significantly lower cerebral iron compared to a control group fed a standard diet (158 mg/kg Fe). Injection of IIM to pregnant ID females on E0 or E14 yielded normalization of Fe in the developing brain known to express elevated levels of capillary transferrin receptors, indicating that the administered iron passed the placenta and fetal blood brain barrier.ConclusionsIn future studies, this translational model may be applied to examine morphological and biochemical consequences of iron deficiency and iron deficiency treatment in the developing fetal brain.

Suction electrode recording in locus coeruleus of newborn rat brain slices reveals network bursting comprising summated non-synchronous spiking

The brainstem locus coeruleus (LC) controling behaviors like arousal, sleep, breathing, pain or opioid withdrawal is an established model for spontaneous action potential synchronization. Such synchronous ‘spiking’ might produce an extracellular field potential (FP) which is a crucial tool for neural network analyses. We found using ≥10 μm tip diameter suction electrodes in newborn rat brainstem slices that the LC generates at ∼1 Hz a robust rhythmic FP (rFP). During distinct rFP phases, LC neurons discharge with a jitter of ±33 ms single spikes that summate to a ∼200 ms-lasting population burst. The rFP is abolished by blocking voltage-gated Na+ channels with tetrodotoxin (TTX, 50 nM) or gap junctions with mefloquine (100 μM) and activating μ-opioid receptors with [D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin (DAMGO, 1 μM). Raising superfusate K+ from 3 to 7 mM either increases rFP rate or transforms its pattern to slower and longer multipeak bursts similar to those during early recovery from DAMGO. The results show that electrical coupling of neonatal LC neurons does not synchronize their spiking as previously proposed. They also indicate that both increased excitability (by elevated K+) and recovery from inhibition (by opioids) can enhance spike desynchronization to transform the population burst pattern. Both observations show that this gap junction-coupled neural network has a more complex connectivity than currently assumed. These new findings along with the inhibitory drug effects that are in line with previous reports based on single neuron recording point out that field potential analysis is pivotal to further the understanding of this brain circuit.

A transient insulin system dysfunction in newborn rat brain followed by neonatal intracerebroventricular administration of streptozotocin could be accompanied by a labile cognitive impairment

The early postnatal period is a critical period of hippocampus development, which is highly dependent on insulin receptor (IR) signaling and very important in cognitive function. The present study was conducted in order to present a model of neonatal transient brain insulin system dysfunction through finding an appropriate dose of injection of streptozotocin (STZ) during the neonatal period.Sixty male Wistar rat pups were divided into 4 groups of 15 and received intracerebroventricular saline or STZ (icv-STZ) (15, 20 and 25μg/kg) on postnatal day 7. Gene expression of IR and target genes for IR signaling (choline acetyltransferase (ChAT) and Tau) were measured at the ages of 2 and 7 weeks. Behavioral tests were performed at the ages of 3 and 6 weeks to assess short- and long-term cognitive function. 20μg/kg dose of icv-STZ was estimated as the optimal dose causing transient alteration in gene expression of IR, ChAT and Tau. Additionally, cognitive function of the animals restored to normal level at the age of 6 weeks. Therefore, 20μg/kg dose of icv-STZ is proposed as a new approach to generating transient brain insulin system dysfunction associated with transient cognitive impairments at a critical postnatal period of brain development.

Central Nervous System Infection with Borna Disease Virus Causes Kynurenine Pathway Dysregulation and Neurotoxic Quinolinic Acid Production.

ABSTRACTCentral nervous system infection of neonatal and adult rats with Borna disease virus (BDV) results in neuronal destruction and behavioral abnormalities with differential immune-mediated involvement. Neuroactive metabolites generated from the kynurenine pathway of tryptophan degradation have been implicated in several human neurodegenerative disorders. Here, we report that brain expression of key enzymes in the kynurenine pathway are significantly, but differentially, altered in neonatal and adult rats with BDV infection. Gene expression analysis of rat brains following neonatal infection showed increased expression of kynurenine amino transferase II (KATII) and kynurenine-3-monooxygenase (KMO) enzymes. Additionally, indoleamine 2,3-dioxygenase (IDO) expression was only modestly increased in a brain region- and time-dependent manner in neonatally infected rats; however, its expression was highly increased in adult infected rats. The most dramatic impact on gene expression was seen for KMO, whose activity promotes the production of neurotoxic quinolinic acid. KMO expression was persistently elevated in brain regions of both newborn and adult BDV-infected rats, with increases reaching up to 86-fold. KMO protein levels were increased in neonatally infected rats and colocalized with neurons, the primary target cells of BDV infection. Furthermore, quinolinic acid was elevated in neonatally infected rat brains. We further demonstrate increased expression of KATII and KMO, but not IDO, in vitro in BDV-infected C6 astroglioma cells. Our results suggest that BDV directly impacts the kynurenine pathway, an effect that may be exacerbated by inflammatory responses in immunocompetent hosts. Thus, experimental models of BDV infection may provide new tools for discriminating virus-mediated from immune-mediated impacts on the kynurenine pathway and their relative contribution to neurodegeneration.IMPORTANCE BDV causes persistent, noncytopathic infection in vitro yet still elicits widespread neurodegeneration of infected neurons in both immunoincompetent and immunocompetent hosts. Here, we show that BDV infection induces expression of key enzymes of the kynurenine pathway in brains of newborn and adult infected rats and cultured astroglioma cells, shunting tryptophan degradation toward the production of neurotoxic quinolinic acid. Thus, our findings newly implicate this metabolic pathway in BDV-induced neurodegeneration. Given the importance of the kynurenine pathway in a wide range of human infections and neurodegenerative and neuropsychiatric disorders, animal models of BDV infection may serve as important tools for contrasting direct viral and indirect antiviral immune-mediated impacts on kynurenine pathway dysregulation and the ensuing neurodevelopmental and neuropathological consequences.

The neuroprotective effect of melatonin on the hippocampus exposed to diclofenac sodium during the prenatal period

Melatonin (Mel) has strong antioxidant properties since it is a direct scavenger of oxygen-based free radicals and related species. The main aim of this study is to show whether the effects of Mel can prevent the potential adverse effects of diclofenac sodium (DS), used as a non-steroidal anti-inflammatory drug (NSAID) during the prenatal period, on the newborn experimental rat brain tissues using stereological methods Twenty-four male 12-week old Wistar albino rats were used. The study involved four groups (each containing six rats), those exposed, during the prenatal period, to saline 1ml/kg (Saline group), to diclofenac sodium 3.6mg/kg (DS group), or to diclofenac sodium+melatonin 50mg/kg (DS+Mel group), and a control group (Cont group). At the end of the experiment, the brains were removed from the cranium for histological and stereological analyses. Cell loss in the hippocampus exposed to DS was observed compared to the Cont group (p<0.01), and a similar side-effect was also seen in the Saline group (p<0.01). However, there was no significant difference in cell numbers between the Cont and DS+Mel groups (p>0.05). These results suggest that exposure to DS during pregnancy causes a decrease in the number of cells in the hippocampus and dentate gyrus in the postnatal period. Using Mel, a neuroprotective agent, reduced the toxic effects of DS.

Antenatal taurine supplementation in fetal rats with growth restriction improves neural stem cell proliferation by inhibiting the activities of Rho family factors

Objective: To investigate whether antenatal taurine supplementation improves neural stem cell proliferation in rats with fetal growth restriction (FGR) through regulating the activity of Rho family factors.Methods: FGR models were established via food restriction throughout pregnancy. Pregnant rats were randomly divided into the control group, the FGR group (given 40% of the normal daily feeding in the control group), and the Taurine group (FGR model treated with 300 mg/kg·d taurine from gestational day seven). Expression of fatty acid binding protein-7 (FABP-7), Rho-associated coiled coil-forming protein kinase (ROCK2), Ras homolog gene family member A (RhoA), and rac in the brains of newborn rats was detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blotting (WB).Results: Relative FABP7 mRNA levels, the optical density (OD) values of FABP7-positive cells and the expression levels of the tested proteins all demonstrated that the number of neural stem cells (NSCs) in brain tissue was lower in the FGR group than in the control group but was significantly increased after antenatal taurine supplementation (p < .05). Compared with the control group, the mRNA and protein levels of RhoA and ROCK2 were higher in the FGR group but lower in the Taurine group (p < .05). In contrast, the rac mRNA level was lower in the FGR group than in the control group but was higher in the Taurine group (p < .05).Conclusions: Taurine prenatal supplementation improved neural stem cell proliferation in rats with FGR by inhibiting the activity of Rho family factors.

Prenatal alcohol-induced neuroapoptosis in rat brain cerebral cortex: protective effect of folic acid and betaine.

Alcohol consumption in pregnancy may cause fetal alcohol syndrome (FAS) in the infant. This study aims to investigate prenatal alcohol exposure related neuroapoptosis on the cerebral cortex tissues of newborn rats and possible neuroprotective effects of betaine, folic acid, and combined therapy. Pregnant rats were divided into five experimental groups: control, ethanol, ethanol + betaine, ethanol + folic acid, and ethanol + betaine + folic acid combined therapy groups. We measured cytochrome c release, caspase-3, calpain and cathepsin B and L. enzyme activities. In order to observe apoptotic cells in the early stages, TUNEL method was chosen together with histologic methods such as assessing the diameters of the apoptotic cells, their distribution in unit volume and volume proportion of cortical intact neuron nuclei. Calpain, caspase-3 activities, and cytochrome c levels were significantly increased in alcohol group while cathepsin B and L. activities were also found to be elevated albeit not statistically significant. These increases were significantly reversed by folic acid and betaine + folic acid treatments. While ethanol increased the number of apoptotic cells, this increase was prevented in ethanol + betaine and ethanol + betaine + folic acid groups. Morphometric examination showed that the mean diameter of apoptotic cells was increased with ethanol administration while this increase was reduced by betaine and betaine + folic acid treatments. We observed that ethanol is capable of triggering apoptotic cell death in the newborn rat brains. Furthermore, folic acid, betaine, and combined therapy of these supplements may reduce neuroapoptosis related to prenatal alcohol consumption, and might be effective on preventing fetal alcohol syndrome in infants.

Synchronization of Mammalian Cell Cultures by Serum Deprivation.

Mammalian cells are amenable to the study of regulatory mechanisms dictating cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for the study of primary astrocytes and a glioma cell line, but is broadly applicable to other mammalian cells. Its application allows astrocytes to re-enter the cell cycle from a state of quiescence (G0) under carefully defined experimental conditions to move together into subsequent phases such as the G1 and S phases. A number of methods have been established to synchronize mammalian cell cultures, which include counterflow centrifugal elutriation, mitotic shake off, chemically induced cell cycle arrest, and newer live cell methods, such as cell permeable dyes. Yet, there are intrinsic limitations associated with these methods. In the present protocol, we describe a simple, reliable, and reversible procedure to synchronize astrocyte and glioma cultures from newborn rat brain by serum deprivation. The procedure is similar, and generally applicable, to other mammalian cells. This protocol consists essentially of two parts: (1) proliferation of astrocytes under optimal conditions in vitro until reaching desired confluence; and (2) synchronization and G0 phase arrest of cultures by serum down-shift. This procedure has been utilized to examine cell cycle control in astroglioma cells and astrocytes from injured adult brain. It has also been employed in precursor cloning studies in developmental biology, suggesting wide applicability.

Optimal timing of antenatal taurine supplementation for improvement of neuron and neural stem cell proliferation in fetal rats with intrauterine growth restriction

Objective To determine the optimal timing of antenatal taurine supplementation to improve neuron and neural stem cell proliferation in fetal rats with intrauterine growth restriction. Methods Twenty-five pregnant SD rats were randomly divided into five groups (five rats in each group): group A was the control group, group B to E were the fetal growth restriction (FGR) model groups with low-protein diet during the experiment, group C, D, and E were supplemented with taurine [300 mg/(kg·d)] at day 9, 11 and 15, respectively. The birth weight of newborn rats was measured after natural delivery. The rats with body weight two standard deviations lower than the average weight in group A were diagnosed as FGR. There were five litters of newborn rats in each group, and two were randomly selected from each litter, resulting in ten newborn rats in each group. Proliferating cell nuclear antigen (PCNA) and fatty acid binding protein 7 (FABP7) positive cell expression in newborn rat brain tissues were detected by immunohistochemistry. Single factor analysis of variance, LSD tests were used for statistical analysis. Results The average birth weight of newborn rats in group A, B, C, D and E were (6.61±0.45), (4.65±0.23), (5.37±0.17), (5.74±0.21), and (5.00±0.24) g, respectively. Average birth weight was lower in group B than in group A (t=2.447), higher in group D and E than in group B (t=2.306 and 2.306), higher in group D than in group C and E (t=2.306 and 2.306), and the differences were statistically significant (all P 0.05). The IOD in group D was higher than that in group E, and the difference was statistically significant (t=4.182, P<0.05). Conclusions Antenatal taurine supplementation can promote neuron and neural stem cell proliferation in rats with FGR. The effect is most obvious on the 11th day of pregnancy, and may lead to the promotion of brain development. Key words: Fetal growth retardation; Taurine; Neurons; Neural stem cells; Cell proliferation; Proliferating cell nuclear antigen; Nerve tissue proteins; Fatty acid-binding proteins; Disease models, animal