"青春期"大鼠前额皮质内BDNF与trkB的表达

目的:探讨"青春期"大鼠前额皮质内BDNF与trkB的表达。方法:出生后35天大鼠作为"青春期"大鼠,出生后15天、75天大鼠分别作为幼年期与成年期对照,每组大鼠各6只,用ABC免疫组织化学方法与图像分析相结合技术检测前额皮质内BDNF与trkB免疫反应的强度和免疫阳性产物平均光密度值的变化。结果:各时间点大鼠前额皮质内均可见BDNF与trkB免疫阳性产物。35天组BDNF与trkB免疫反应最强,阳性产物平均光密度值最高,与其它两组相比差异有显著性(p<0.05)。结论:"青春期"大鼠前额皮质内BDNF与trkB高表达,提示在此时期内前额皮质对BDNF的需求最多。     

Chronic Administration of Mood Stabilizers Upregulates BDNF and Bcl-2 Expression Levels in Rat Frontal Cortex

Brain-derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2) proteins are neuroprotective factors involved in neuronal signaling, survival and plasticity. Both can be regulated by cyclic AMP response element binding (CREB) protein. Decreased levels of BDNF and Bcl-2 are implicated in the pathogenesis of bipolar disorder. The present study investigated whether chronically administered mood stabilizers would increase BDNF and/or Bcl-2 levels in rat brain. Real time RT-PCR, sandwich ELISA and Western blotting were used to measure BDNF and Bcl-2 mRNA and protein levels in the frontal cortex of rats chronically administered carbamazepine (CBZ) or lamotrigine (LTG) to produce plasma concentrations therapeutically relevant to bipolar disorder. Chronic CBZ and LTG significantly increased BDNF and Bcl-2 mRNA and protein levels in the frontal cortex. A common mechanism of action of mood stabilizers in the treatment of bipolar disorder may involve neuroprotection mediated by upregulation of brain BDNF and Bcl-2 expression.     

Expression of TrkB and BDNF in human cochlea-an immunohistochemical study

Surgical human cochlear specimens were obtained during the removal of large posterior cranial fossa meningioma by a transcochlear approach in which the cochlea was removed for maximal exposure of the tumor and protection of important structures, such as the brainstem, cranial nerves, and pivotal blood vessels. The cochlear tissue was fixed and cryo-sectioned for tyrosine kinase receptor B (TrkB) and brain-derived neurotrophic factor (BDNF) immunohistochemistry. TrkB receptor protein was expressed in both neuronal somata and the processes of human spiral ganglion neurons (SGNs). In the human organ of Corti, TrkB immunoreactivity was mainly present in nerve fibers underneath outer hair cells. BDNF expression was found neither in the organ of Corti nor in the spiral ganglion of human cochlea. For antibody specificity and for control and comparative purposes, TrkB immunocytochemistry was performed in primary cultures of cochlear neuron/glia from adult guinea pig. Confocal laser scanning microscopy showed that TrkB was homogeneously distributed in the cytoplasm of both neuronal somata and axons. Knowledge of the expression of TrkB receptor in human cochlea should help to determine the target structures for neuron preservation in hearing-impaired patients. Our results indicate that the regeneration of SGNs under pathological conditions can be enhanced with BDNF/TrkB-based pharmaceutical or genetic strategies.     

NGF,BDNF and Arc mRNA Expression in the Hippocampus of Rats After Administration of Morphine

Morphine can influence immediate early genes (IEG) of activity-regulated cytoskeletal-associated protein (Arc) and brain-derived neurotrophic factor (BDNF) which are activated in response to physiological stimuli during learning, as well as the nerve growth factor (NGF) gene which increases the expression of several IEGs for memory formation. The purpose of the current study was first to evaluate the effect of acute (1-day) and subchronic (15-days) morphine administration on memory retrieval of rats and second to determine the hippocampal expression of NGF, BDNF and Arc genes as potential contributors in the observed effects in each setting. The effects of morphine (intraperitoneal, 10, 15 and 20 mg/kg) on memory function and gene expression were assessed using inhibitory avoidance test and real-time polymerase chain reaction, respectively. We found that a single dose of morphine at the highest dose of 20 mg/kg decreases the post-training step-through-latency, while repeated administration of the same dose for 15 successive days increases this indicator of memory retrieval. We did not detect a significant change in the hippocampal expression of Arc, BDNF or NGF genes after a single episode of morphine treatment. However, subchronic morphine administration (15 and 20 mg/kg) increased the expression of Arc and BDNF genes in a dose dependent manner. A higher mRNA expression for the NGF was observed at the higher dose of 20 mg/kg. We hypothesize that the subchronic effects were morphine-induced behavioral sensitization which may have been enhanced through increased hippocampal Arc expression.

     

Expression of BDNF and TrkB Phosphorylation in the Rat Frontal Cortex During Morphine Withdrawal are NO Dependent

Nitric oxide (NO) mediates pharmacological effects of opiates including dependence and abstinence. Modulation of NO synthesis during the induction phase of morphine dependence affects manifestations of morphine withdrawal syndrome, though little is known about mechanisms underlying this phenomenon. Neurotrophic and growth factors are involved in neuronal adaptation during opiate dependence. NO-dependent modulation of morphine dependence may be mediated by changes in expression and activity of neurotrophic and/or growth factors in the brain. Here, we studied the effects of NO synthesis inhibition during the induction phase of morphine dependence on the expression of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and insulin-like growth factor 1 (IGF1) as well as their receptors in rat brain regions after spontaneous morphine withdrawal in dependent animals. Morphine dependence in rats was induced within 6 days by 12 injections of morphine in increasing doses (10–100 mg/kg), and NO synthase inhibitor L-N^G-nitroarginine methyl ester (L-NAME) (10 mg/kg) was given 1 h before each morphine injection. The expression of the BDNF, GDNF, NGF, IGF1, and their receptors in the frontal cortex, striatum, hippocampus, and midbrain was assessed 40 h after morphine withdrawal. L-NAME treatment during morphine intoxication resulted in an aggravation of the spontaneous morphine withdrawal severity. Morphine withdrawal was accompanied by upregulation of BDNF, IGF1, and their receptors TrkB and IGF1R, respectively, on the mRNA level in the frontal cortex, and only BDNF in hippocampus and midbrain. L-NAME administration during morphine intoxication decreased abstinence-induced upregulation of these mRNAs in the frontal cortex, hippocampus and midbrain. L-NAME prevented from abstinence-induced elevation of mature but not pro-form of BDNF polypeptide in the frontal cortex. While morphine abstinence did not affect TrkB protein levels as well as its phosphorylation status, inhibition of NO synthesis decreased levels of phosphorylated TrkB after withdrawal. Thus, NO signaling during induction of dependence may be involved in the mechanisms of BDNF expression and processing at abstinence, thereby affecting signaling through TrkB in the frontal cortex.     

CFA注射致炎性疼痛过程中海马内BDNF的表达及作用研究

目的:探索完全弗氏佐剂(Complete Freund’s Adjuvant,CFA)致炎性疼痛后大鼠海马内脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的变化及其作用。方法:雄性SD大鼠随机分为溶媒对照组和实验组,实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100μL,建立炎性疼痛模型,注射后大鼠分别存活1天、4天、7天、14天(n=20只/组)。在建模前和建模后不同时间点采用Von Frey纤维检测大鼠50%机械缩足阈值(paw withdrawal threshold,PWT)的变化。采用强迫游泳实验和糖水偏好实验检测大鼠抑郁样行为,采用RT-PCR检测不同时间点大鼠海马中BDNF m RNA的水平,采用ELISA和免疫组织化学法检测BDNF的表达变化。结果:足底注射CFA后1天50%PWT下降,持续至14天仍低于基础值。在强迫游泳实验中,注射CFA7天后的大鼠不动时间百分比增加,一直持续到CFA注射后14天。糖水偏好实验中注射CFA后7天组、14天组大鼠表现出对糖水偏好的降低,提示注射CFA后7天大鼠出现抑郁样行为并持续到第14天。在足底注射CFA第7天后BDNF m RNA和BDNF的水平达到高峰,第14天表达下调并基本恢复正常水平。结论:结果提示,足底注射CFA能诱导大鼠产生炎性痛,CFA注射7天后出现抑郁样行为,此时海马内BDNF m RNA和BDNF蛋白水平均上调,可能与足底注射CFA后出现的抑郁样行为的发生密切相关。     

Microarray Analysis of Gene Expression in Rat Hippocampus After Chronic Ethanol Treatment

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD⁺-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.     

Effect of Oral Resveratrol on the BDNF Gene Expression in the Hippocampus of the Rat Brain

Resveratrol is a plant polyphenolic compound. Evidence indicates that resveratrol has beneficial effects against aging and neurodegenerative diseases. The goal of our study was in vivo examination of the effects of resveratrol on the abundance of mRNA encoding Brain Derived Neurotrophic Factor (BDNF) in the hippocampus of rat brain. Rats were administrated orally by different doses (2.5–20 mg/kg bwt) of resveratrol for 3, 10 and 30 days. Saline was used as control and 10% ethanol in saline was used as vehicle for resveratrol. Measurement of BDNF mRNA by Real-time RT–PCR showed that levels of the mRNA for BDNF were significantly and dose dependently elevated in the hippocampal tissues of rats. The findings suggest that the neuroprotective effects of resveratrol may be at least partly due to its inducing effects on the expression levels of the BDNF mRNA.     

Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.     

Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li⁺ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [³²P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more ³²P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.     

Intracerebroventricular Administration of Ouabain to Rats Changes the Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus

Ouabain exerts neurotoxic action and activates the population of NMDA receptors. Herein the effect of ouabain on the expression of NMDA subunits was evaluated. Adult Wistar rats were administered intracerebroventricularly with 0.1, 10 and 100 nmol ouabain or saline solution (control). Two days later, membranes of cerebral cortex and hippocampus were isolated. Western blots with antibodies for the NMDA receptor subunits: NR1; NR2A; NR2B; NR2C and NR2D were carried out. In cerebral cortex, NR2D subunit increased 30% with 10 nmol ouabain dose. With 100 nmol ouabain, NR1 and NR2D subunits enhanced 40 and 20%, respectively. In hippocampus, with the dose of 0.1 nmol ouabain, NR1 subunit enhanced roughly 50% whereas NR2B subunit decreased 30%. After administration of 10 nmol ouabain dose, NR2A, NR2B and NR2C subunits decreased 40, 50 and 30%, respectively. With the dose of 100 nmol of ouabain, NR1, NR2A and NR2B subunits diminished 10–20%. It is concluded that ouabain administration led to a differential regulation in the expression of NMDA subunits. These results may be correlated with the modulatory action of ouabain on NMDA receptor.     

慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin的表达变化

目的:观察慢性吗啡处理及戒断后大鼠杏仁核中Parvalbumin(PV)的表达变化,为其功能的研究提供形态学依据。方法:将30只健康雄性SD大鼠随机分为吗啡依赖组和生理盐水对照组。吗啡依赖组大鼠腹膜腔注射吗啡,2次/d,起始剂量为5 mg/kg,逐日递增5mg,至第10d为50mg/kg;对照组注射同体积的生理盐水。于末次注射后动物分别存活3h、3 d和14d。用免疫组化方法和相对平均灰度值检测杏仁核内PV的表达。结果:在生理盐水处理组各存活时间点,杏仁核内PV的表达相同。和生理盐水对照组相比,3h时杏仁核内PV的表达明显增加(P<0.05)。第3d时,杏仁核内PV的表达减少,明显低于第3 h组(P<0.05)。至第14d时,PV的表达又开始增加,明显高于第3 d组(P<0.05)。结论:本结果提示慢性吗啡处理及戒断后杏仁核PV的表达具有时相特异性;这种变化在戒断早期可能主要与躯体依赖相关,而戒断晚期主要与精神依赖相关。     

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.     

Seizure Activity Produces Differential Changes in Adenosine A1 Receptors within Rat Hippocampus

Specific ligand binding to rat hippocampal adenosine A₁ receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-³H adenosine] ([³H]CCPA), a potent and selective A₁ receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [³H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.     

Effect of Chronic Lead Exposure on Pro-Apoptotic Bax and Anti-Apoptotic Bcl-2 Protein Expression in Rat Hippocampus In Vivo

Despite reduction in environmental lead, chronic lead exposure still possess a public health hazard, particularly in children, with devastating effects on developing CNS. To investigate the mechanism of this neurotoxicity, young and adult rats were used to study whether exposure to 500 ppm concentrations of lead could induce apoptosis in hippocampus. 2–4 and 12–14-week-old rats received lead acetate in concentration of 500 ppm for 40 days. Control animals received deionized distilled water. In lead-treated groups, the blood lead levels were increased by 3–4 folds. Light and electron microscopical study of hippocampus revealed increased apoptotic cells. Western blot analysis of Bax and Bcl-2 (pro- and anti-apoptotic gene products, respectively) indicated higher expression of Bax protein and no significant change in bcl-2 expression and accordingly increased the Bax/Bcl-2 ratio compared to control group, confirming the histological study. In conclusion, these data suggest that neurotoxicity of chronic lead exposure in hippocampus in vivo may partly be due to facilitation of apoptosis.     

Repeated Estradiol Treatment Attenuates Chronic Cerebral Hypoperfusion-Induced Neurodegeneration in Rat Hippocampus

Although a substantial number of pre-clinical and experimental studies have investigated effects of 17β-estradiol, its precise molecular mechanism of action in the early state of chronic cerebral hypoperfusion remains controversial. The present study attempted to verify whether post-ischemic estradiol treatment (33.3 μg/kg for seven consecutive days) affects previously reported number of hippocampal apoptotic cells and amount of DNA fragmentation characteristic for apoptosis as well as the expression of key elements within synaptosomal Akt and Erk signal transduction pathways (NF-κB, Bax, Bcl-2, cytochrome C, caspase 3, and PARP). Additionally, alterations of aforementioned molecules linked to protection in various neurodegenerative disorders were monitored in the cytosolic, mitochondrial, and nuclear fractions associating investigated kinases and NF-κB with gene expression of their downstream effectors—Bcl-2, Bax, and caspase 3. The results revealed that an initial increase in the number of apoptotic cells and amount of DNA fragmentation induced by chronic cerebral hypoperfusion was significantly reduced by 17β-estradiol. In synaptic regions, an altered profile with respect to the protein expression of Bcl-2 and phosphorylated Akt was detected, although the level of other examined proteins was not modified. In other investigated sub-cellular fractions, 17β-estradiol elicited phosphorylation and translocation of Akt and Erk along with modulation of the expression of their subsequent effectors. Our findings support the concept that repeated post-ischemic 17β-estradiol treatment attenuates neurodegeneration induced by chronic cerebral hypoperfusion in hippocampus through the activation of investigated kinases and regulation of their downstream molecules in sub-cellular manner indicating a time window and regime of its administration as a valid therapeutic intervention.