Silibinin Alleviates the Learning and Memory Defects in Overtrained Rats Accompanying Reduced Neuronal Apoptosis and Senescence

Neurochemical Research - Excessive physical exercise (overtraining; OT) increases oxidative stress and induces damage in multiple organs including the brain, especially the hippocampus that plays...     

Aging Differentially Affects the Loss of Neuronal Dendritic Spine,Neuroinflammation and Memory Impairment at Rats after Surgery

It is known that age is an important factor for postoperative cognitive dysfunction (POCD) and the patients with POCD suffer from the impairment of multiple brain regions and multiple brain functions. However currently animal studies of POCD mainly focus on hippocampus region, therefore in this study we performed partial hepatectomy in young adult and aged rats to test the questions (1) whether POCD in animals involves other brain areas besides hippocampus; (2) how age influences POCD of young adult and aged animals. We found that (1) in young adult rats, the memory was not significantly affected (P>0.05) 1d, 3d and 7d after partial hepatectomy, but was significantly impaired (p<0.001) in aged rats 1d and 3d post-surgery; (2) in young adult rats, the surgery did not significantly affect the densities of dendritic spines of neurons at CA1, dentate gyrus (DG) and cingulate cortex (P>0.05, respectively) 1d and 3d post-surgery, but the spine densities at CA1 and DG of aged rats were significant reduced 1d and 3d post-surgery (p<0.001, respectively), however this didn’t happen at cingulate cortex (P>0.05); (3) In young adult rats, surgery didn’t affect the activation of microglia and levels of TNF-α and IL-1β at hippocampus (P>0.05), but significantly activated microglia and increased levels of TNF-α and IL-1β at hippocampus of aged rats (P<0.05). Our data suggest that (1) partial hepatectomy-induced POCD mainly involves hippocampus impairments, and (2) differential loss of neuronal dendritic spines and neuroinflammation at hippocampus are most likely the mechanism for the formation of POCD in aged rats.     

高压氧预处理对SPS暴露大鼠学习记忆能力以及海马神经元凋亡的影响

目的:研究高压氧(HBO)预处理对SPS暴露大学学习记忆能力及其大脑海马神经元细胞凋亡的影响.方法:48只雄性Sprague-Dawley大鼠(体重220-260 g)随机分为4组(n=12):对照(sham)组,高压氧(HBO)组,SPS组以及高压氧+SPS组.高压氧组每天1小时高压氧预处理(2.5个大气压,100%O2)连续5天;SPS组采用单次延长应激模型;高压氧+SPS组每天l小时高压氧预处理连续5天于最后一次预处理后24小时,制作SPS模型.4组大鼠于SPS暴露后72小时进行TUNEL染色,第15天经行水迷宫测试.结果:水迷宫实验中大鼠逃避潜伏期及游泳路径四组之间有明显统计差异[F0.01(3,28)=4.88＞4.57,P＜0.01;F0.01(3,28)=5.31＞4.57,P＜0.01].SPS组明显长于Sham组(P＜0.01),而高压氧预处理能够逆转这种效应(P＜0.01).游泳速度四组之间无明显统计差异[F0.05(3,28)=2.23＜2.95,P＞0.05]. SPS暴露后海马神经元细胞数量和密度明显减少,给予高压氧预处理后,神经元形态明显好转,但仍不及对照组.结论:高压氧预处理可以减少海马神经元细胞凋亡从而改善SPS暴露后大鼠认知功能障碍.     

Epigallocatechin-3-Gallate Ameliorated Iron Accumulation and Apoptosis and Promoted Neuronal Regeneration and Memory/Cognitive Functions in the Hippocampus Induced by Exposure to a Chronic High-Altitude Hypoxia Environment

We aimed to explore the protective effects and potential treatment mechanism of Epigallocatechin-3-gallate (EGCG) in an animal model of chronic exposure in a natural high-altitude hypoxia (HAH) environment. Behavioral alterations were assessed with the Morris water maze test. Iron accumulation in the hippocampus was detected by using DAB enhanced Perls’ staining, MRI, qPCR and colorimetry, respectively. Oxidative stress (malondialdehyde, MDA), apoptosis (Caspase-3), and neural regeneration (brain-derived neurotrophic factor, BDNF) were detected by using ELISA and western blotting. Neural ultrastructural changes were evaluated by transmission electron microscopy (TEM). The results showed that learning and memory performance of rats decreased when exposure to HAH environment. It was followed by iron accumulation, dysfunctional iron metabolism, reduced BDNF and the upregulation of MDA and Caspase-3. TEM confirmed the ultrastructural changes in neurons and mitochondria. EGCG reduced HAH-induced cognitive impairment, iron deposition, oxidative stress, and apoptosis and promoted neuronal regeneration against chronic HAH-mediated neural injury.

     

Extracellular Somatostatin Measured by Microdialysis in the Hippocampus of Freely Moving Rats: Evidence for Neuronal Release

Abstract: Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect <1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 μl/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 ± 6.4% ( p < 0.05) when Ca²⁺ was omitted and 1 m M EGTA added to the Krebs-Ringer solution and by 65.5 ± 3.2% ( p < 0.05) in the presence of 1 μ M tetrodotoxin. Depolarizing concentrations of the Na⁺ channel opener veratridine (6.25, 25, 100 μ M ) induced 11 ± 2 ( p < 0.05), 17 ± 2 ( p < 0.05), and 21 ± 5 ( p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 μ M veratridine was blocked by coperfusion with 5 μ M tetrodotoxin ( p < 0.01) and reduced by 79% ( p < 0.01) in the virtual absence of Ca²⁺. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 m M KCl and proportionally lowered Na⁺ increased the dialysate SLI 4.4 ± 1 ( p < 0.05) and 17 ± 3 ( p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na⁺,K⁺-ATPase, increased the dialysate SLI 15-fold baseline, on average ( p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

Facilitated Neurogenesis in the Developing Hippocampus After Intake of Theanine,an Amino Acid in Tea Leaves,and Object Recognition Memory

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. In this study, cognitive function and the related mechanism were examined in theanine-administered young rats. Newborn rats were fed theanine through dams, which were fed water containing 0.3% theanine, and then fed water containing 0.3% theanine after weaning. Theanine level in the brain was under the detectable limit 6 weeks after the start of theanine administration. Theanine administration did not influence locomotor activity in the open-field test. However, rearing behavior was significantly increased in theanine-administered rats, suggesting that exploratory activity is increased by theanine intake. Furthermore, object recognition memory was enhanced in theanine-administered rats. The increase in exploratory activity in the open-field test seems to be associated with the enhanced object recognition memory after theanine administration. On the other hand, long-term potentiation (LTP) induction at the perforant path-granule cell synapse was not changed by theanine administration. To check hippocampal neurogenesis, BrdU was injected into rats 3 weeks after the start of theanine administration, and brain-derived neurotropic factor (BDNF) level was significantly increased at this time. Theanine intake significantly increased the number of BrdU-, Ki67-, and DCX-labeled cells in the granule cell layer 6 weeks after the start of theanine administration. This study indicates that 0.3% theanine administration facilitates neurogenesis in the developing hippocampus followed by enhanced recognition memory. Theanine intake may be of benefit to the postnatal development of hippocampal function.     

Inhibiting Matrix Metalloproteinase 3 Ameliorates Neuronal Loss in the Ganglion Cell Layer of Rats in Retinal Ischemia/Reperfusion

It has been demonstrated that matrix metalloproteinase 3 (MMP3) is integrally involved in the neuronal degeneration of the central nervous system by promoting glial activation, neuronal apoptosis and damage to the brain–blood barrier. However, whether MMP3 also contributes to the neuronal degeneration induced by retinal ischemia/reperfusion is still uncertain. In the present study, we detected the cellular localization of MMP3 in adult rat retinae and explored the relationship of its expression with neuronal loss in the ganglion cell layer (GCL) in retinal ischemia/reperfusion. We found that MMP3 was widely expressed in many cells throughout the layers of the rat retinae, including Vertebrate neuron-specific nuclear protein (NeuN)-, parvalbumin-, calbindin-, protein kinase C-α-, glial fibrillary acidic protein-, glutamine synthetase- and CD11b-positive cells. Furthermore, all rats were treated with high intraocular pressure (HIOP) for 1 h (h) and sacrificed at 6 h, 1 day (d), 3 d, and 7 d after HIOP. Compared to the normal control, the expression of both proenzyme MMP3 and active MMP3 were significantly up-regulated after HIOP treatment without alteration of the laminar distribution pattern. Moreover, inhibiting MMP3 ameliorated the loss of NeuN-positive cells in the GCL following HIOP. In summary, our data demonstrates that MMP3 is expressed in multiple types of neurons and glial cells in normal rat retinae. Simultaneously, the up-regulation of its expression and activity are closely involved in neuronal loss in the GCL in retinal ischemia/reperfusion.     

Intranasal Administration of Interferon Beta Attenuates Neuronal Apoptosis via the JAK1/STAT3/BCL-2 Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy

Neonatal hypoxic-ischemic encephalopathy (HIE) is an injury that often leads to detrimental neurological deficits. Currently, there are no established therapies for HIE and it is critical to develop treatments that provide protection after HIE. The objective of this study was to investigate the ability of interferon beta (IFNβ) to provide neuroprotection and reduce apoptosis after HIE. Postnatal Day 10 rat pups were subjected to unilateral carotid artery ligation followed by 2.5 hr of exposure to hypoxia (8% O₂). Intranasal administration of human recombinant IFNβ occurred 2 hr after HIE and infarct volume, body weight, neurobehavioral tests, histology, immunohistochemistry, brain water content, blood–brain barrier permeability, enzyme-linked immunosorbent assay, and Western blot were all used to evaluate various parameters. The results showed that both IFNβ and the Type 1 interferon receptor expression decreases after HIE. Intranasal administration of human recombinant IFNβ was able to be detected in the central nervous system and was able to reduce brain infarction volumes and improve neurological behavior tests 24 hr after HIE. Western blot analysis also revealed that human recombinant IFNβ treatment stimulated Stat3 and Bcl-2 expression leading to a decrease in cleaved caspase-3 expression after HIE. Positive Fluoro-Jade C staining also demonstrated that IFNβ treatment was able to decrease neuronal apoptosis. Furthermore, the beneficial effects of IFNβ treatment were reversed when a Stat3 inhibitor was applied. Also an intraperitoneal administration of human recombinant IFNβ into the systemic compartment was unable to confer the same protective effects as intranasal IFNβ treatment.     

镉诱导小鼠睾丸细胞凋亡与Caspase-3、Bcl-2和Bax mRNA的表达

一个月大雄性小鼠24只,随机分为6组,用30 μmol/kg CdCl2作用小鼠睾丸不同的时间(3 h、6 h、12 h、18 h、24 h)后,利用DNA电泳、免疫组化和半定量RT-PCR技术,分析生殖细胞凋亡过程中三种关键物质Caspase-3、Bcl-2、Bax蛋白和mRNA的表达量变化.结果显示:1) DNA各组 (除对照组外)均出现不同程度断裂.2)Caspase-3蛋白表达量一直上升,与对照组相比差异极显著;Bax蛋白在12 h前一直上升,与对照组相比差异显著,12 h后又开始下降,且与对照组相比无显著差异;Bcl-2蛋白在下降,与对照组相比差异显著.3)RT-PCR结果显示Caspase-3基因表达量减少;Bax基因表达量逐渐上升;Bcl-2基因表达量波动很大.综上所述,Caspase-3、Bcl-2和Bax三个基因可能参与了镉应激状态下小鼠睾丸组织细胞的凋亡过程.     

The Involvement of Upregulation and Translocation of Phospho-Rb in Early Neuronal Apoptosis Following Focal Cerebral Ischemia in Rats

The aim of this study was to investigate the temporal and spatial relationship between phospho-Rb (ser 795) and neuronal apoptotic death in rats subjected to transient focal cerebral ischemia. We found increased phosphorylation of Rb and translocation from neuronal nucleus to cytoplasm in the penumbra zone at 12 h, 1 day, 3 days and 7 days after middle cerebral artery occlusion (MCAO)/reperfusion, compared with sham-operated controls. At 12 h and 1 day, phospho-Rb appeared to be colocalizated with TUNEL staining in neurons, but staining was not colocalizated at 3 days and 7 days. These results demonstrated that cytoplasmic translocation of phospho-Rb from nucleus of neurons occurs in potential apoptotic neurons in the early stages of ischemia/reperfusion, suggesting that the Rb pathway may only be involved in early neuronal apoptosis and may be not an apoptotic signal in the late stages of transient cerebral ischemia. Ying Yu and Xiang Luo contributed equally to this work.     

2R,4R-APDC,a Metabotropic Glutamate Receptor Agonist,Reduced Neuronal Apoptosis by Upregulating MicroRNA-128 in a Rat Model After Seizures

This study aimed to study the protective effect of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a selective metabotropic glutamate receptor agonist, against hippocampal neuronal apoptosis induced by seizures in a rat model of pilocarpine-induced epilepsy. The Morris water maze test was used to assess the spatial memory abilities of epileptic rats with or without 2R,4R-APDC treatment. TUNEL assay was performed to examine neuronal apoptosis in hippocampus. Western blot was conducted to evaluate changes in the levels of caspase-3 and caspase-9 in hippocampus. Real-time PCR was used to determine the levels of microRNA-128 (miR-128) in hippocampus. The results of the Morris water maze test showed that the 2R,4R-APDC treatment reduced the escape latencies and swimming lengths of rats after seizures. The TUNEL assay showed that 2R,4R-APDC significantly counteracted seizure-induced cell apoptosis. The western blot confirmed this finding, demonstrating that the levels of cleaved caspase-3 and cleaved caspase-9 were potently decreased by 2R,4R-APDC in rat hippocampus after seizures. In addition, 2R,4R-APDC upregulated miR-128 expression levels in the hippocampus. A miR-128 mimic or inhibitor decreased or increased the percentage of TUNEL-positive cells in rats after seizures and 2R,4R-APDC treatment, respectively. The levels of both cleaved caspase-3 and cleaved caspase-9 were decreased in hippocampus exposed to the miR-128 mimic, whereas they were markedly increased in miR-128 inhibitor-treated hippocampus. In conclusion, 2R,4R-APDC protected hippocampal cells from cell apoptosis after seizures, possibly by upregulating miR-128.     

Effect of Acute Administration of 3-Butyl-1-Phenyl-2-(Phenyltelluro)Oct-En-1-One on Oxidative Stress in Cerebral Cortex,Hippocampus, and Cerebellum of Rats

Organotellurium compounds have been synthesized since 1840, but pharmacological and toxicological studies about them are still incipient. Therefore, the objective of this study was to verify the effect of acute administration of the organochalcogen 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one on some parameters of oxidative stress in the brain of 30-day-old rats. Animals were treated intraperitoneally with a single dose of the organotellurium (125, 250, or 500 μg/kg body weight) and sacrificed 60 min after the injection. The cerebral cortex, the hippocampus, and the cerebellum were dissected and homogenized in KCl. Afterward, thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD), nitric oxide (NO) formation, and hydroxyl radical production were measured in the brain. The organotellurium enhanced TBARS in the cerebral cortex and the hippocampus, and increased protein damage (carbonyl) in the cerebral cortex and the cerebellum. In contrast, the compound provoked a reduced loss of thiol groups measured by the sulfhydryl assay in all the tissues studied. Furthermore, the activity of the antioxidant enzyme CAT was reduced by the organochalcogen in the cerebral cortex and the cerebellum, and the activity of SOD was inhibited in all the brain tissues. Moreover, NO production was increased in the cerebral cortex and the cerebellum by this organochalcogen, and hydroxyl radical formation was also enhanced in the cerebral cortex. Our findings indicate that this organotellurium compound induces oxidative stress in the brain of rats, corroborating that this tissue is a potential target for organochalcogen action.     

Down-Regulation of Neuregulin1/ErbB4 Signaling in the Hippocampus Is Critical for Learning and Memory

Hippocampal function is important for learning and memory, and dysfunction of the hippocampus has been linked to the pathophysiology of neuropsychiatric diseases such as schizophrenia. Neuregulin1 (NRG1) and ErbB4, two susceptibility genes for schizophrenia, reportedly modulate long-term potentiation (LTP) at hippocampal Schaffer collateral (SC)-CA1 synapses. However, little is known regarding the contribution of hippocampal NRG1/ErbB4 signaling to learning and memory function. Here, quantitative real-time PCR and Western blotting were used to assess the mRNA and protein levels of NRG1 and ErbB4. Pharmacological and genetic approaches were used to manipulate NRG1/ErbB4 signaling, following which learning and memory behaviors were evaluated using the Morris water maze, Y-maze test, and the novel object recognition test. Spatial learning was found to reduce hippocampal NRG1 and ErbB4 expression. The blockade of NRG1/ErbB4 signaling in hippocampal CA1, either by neutralizing endogenous NRG1 or inhibiting/ablating ErbB4 receptor activity, enhanced hippocampus-dependent spatial learning, spatial working memory, and novel object recognition memory. Accordingly, administration of exogenous NRG1 impaired those functions. More importantly, the specific ablation of ErbB4 in parvalbumin interneurons also improved learning and memory performance. The manipulation of NRG1/ErbB4 signaling in the present study revealed that NRG1/ErbB4 activity in the hippocampus is critical for learning and memory. These findings might provide novel insights on the pathophysiological mechanisms of schizophrenia and a new target for the treatment of Alzheimer’s disease, which is characterized by a progressive decline in cognitive function.