Glutamate-releasing BEST1 channel is a new target for neuroprotection against ischemic stroke with wide time window

Many efforts have been made to understand excitotoxicity and develop neuroprotectants for the therapy of ischemic stroke. The narrow treatment time window is still to be solved. Given that the ischemic core expanded over days, treatment with an extended time window is anticipated. Bestrophin 1 (BEST1) belongs to a bestrophin family of calcium-activated chloride channels. We revealed an increase in neuronal BEST1 expression and function within the peri-infarct from 8 to 48 h after ischemic stroke in mice. Interfering the protein expression or inhibiting the channel function of BEST1 by genetic manipulation displayed neuroprotective effects and improved motor functional deficits. Using electrophysiological recordings, we demonstrated that extrasynaptic glutamate release through BEST1 channel resulted in delayed excitotoxicity. Finally, we confirmed the therapeutic efficacy of pharmacological inhibition of BEST1 during 6-72 h post-ischemia in rodents. This delayed treatment prevented the expansion of infarct volume and the exacerbation of neurological functions. Our study identifies the glutamate-releasing BEST1 channel as a potential therapeutic target against ischemic stroke with a wide time window.

Modelagem celular in vitro da Doença de Huntington com ácido 3-nitropropiônico e ácido quinolínico & Avaliação das funções neuroprotetoras da adenosina / In vitro cell modeling of Huntington's Disease with 3-nitropropionic acid and quinolinic acid and evaluation of the neuroprotective functions of adenosine

A Doença de Huntington (Huntington's disease - HD) trata-se de uma patologia neurodegenerativa hereditária caracteriza por meio da expressão das proteínas huntingtinas mutantes (mHtt), das mortes dos neurônios espinhais médios (medium spiny neurons MSNs) GABAérgicos D2-positivos do striatum e da hipercinesia. Uma hipótese se refere à função das mHtts de potencializarem os efeitos excitotóxicos das estimulações dos receptores de NMDA (NMDAR) por meio da inibição da succinato desidrogenase, resultando em desequilibrio das [Ca2+]i, estresse oxidativo e apoptose. A adenosina agonista dos receptores purinérgicos P1 tem sido descrita por conta das suas funções neuroprotetoras e neuromodulatórias. Assim, estabelecemos dois modelos in vitro da HD fundamentados nas neurodiferenciações das linhagens murinas de célula-tronco embrionárias E14-TG2a e progenitoras neurais do hipocampo HT-22; seguidas pelos tratamentos com ácido quinolínico (QA) agonista seletivo dos NMDARs , na ausência e na presença do ácido 3-nitropropiônico (3-NP) inibidor irreversível da succinato desidrogenase. Estes modelos foram utilizados nas avaliações das funções neuroprotetoras da adenosina. Os neurônios pós-mitóticos das culturas de E14-TG2a diferenciadas foram caracterizados conforme os MSNs GABAérgicos do striatum; enquanto os neurônios HT-22 diferenciados foram caracterizados de modo inespecífico. Metodologia: imunofluorescência (microscopia e citometria); PCR em tempo real; análise das variações dos potenciais das membranas plasmáticas e das variações transientes das [Ca2+]i por microfluorimetria; e quantificações das reduções do AlamarBlue® (% de sobrevida celular) e das atividades extracelulares de LDH (U/L) (necrose) por espectrometria. Avaliamos a capacidade do 3-NP de potencializar os efeitos excitotóxicos do QA comparando dois grupos de neurônios HT-22 diferenciados: QA 8mM (EC50) (controle); e 3-NP 5mM/QA 8mM. Avaliarmos o potencial neuroprotetor da adenosina comparando quatro grupos de neurônios HT-22 diferenciados: QA 8mM; adenosina 250µM/QA 8mM; 3-NP 5mM/QA 8mM; 3-NP 5mM/adenosina 250µM/QA 8mM. Os neurônios pós-mitóticos derivados das E14TG2a foram classificados como MSNsGABAérgicos do striatum integrantes de uma cultura neuronal heterogênea semelhante às conexões nigroestriatais, corticoestriatais, striatonigral e striatopallidal. Os neurônios HT-22 diferenciados perfaziam uma cultura neuronal heterogênea, não totalmente madura, composta por neurônios glutamatérgicos, dopaminérgicos, colinérgicos e GABAérgicos. Os neurônios HT-22 diferenciados 3-NP 5mM apresentaram menores % de sobrevida celular após os tratamentos com QA 8mM por 24h (p<0.05); e maiores amplitudes das variações das [Ca2+]i dependentes do QA 8mM (p<0.05) (cinética 6 minutos). Por outro lado, os neurônios HT-22 diferenciados pré- tratados com 3-NP 5mM apresentaram menores atividades extracelulares de LDH após o tratamento com QA 8mM por 24h menor proporção de necrose. Os pré-tratamentos com adenosina 250µM indicaram uma tendência dos efeitos neuroprotetores (p>0.05) maiores % de sobrevida celular; menores atividades extracelulares de LDH; e menores amplitudes das variações transientes das [Ca2+]i. Em conjunto, nossos resultados indicam que a inibição da succinato desidrogenase potencializa os efeitos excitotóxicos dos NMDARs por meio da alteração das [Ca2+]i e, provavelmente, dos mecanismos de morte celular; enquanto a adenosina apenas tendeu à neuroproteção

Huntington's disease (HD) is a hereditary neurodegenerative pathology characterized by mutant huntingtin proteins (mHtt) expression, striatum D2-positive GABAergic medium spiny neurons (MSNs) cell death and hyperkinetic motor symptoms development. One hypothesis refers to the principle that mHtt potentiates the excitotoxic effects of NMDA receptor (NMDAR) stimulation by the inhibition of mitochondrial succinate dehydrogenase, resulting in [Ca2+]i imbalance, oxidative stress and apoptosis. Adenosine P1 purinergic receptor agonist is related to neuroprotective and neuromodulatory functions. Thus, we established two in vitro HD models based on the neurodifferentiation of murine embryonic stem cell lines E14-TG2a and hippocampal neuroprogenitor cell line HT-22 followed by treatment with quinolinic acid (QA) selective agonist of NMDARs , in the absence and in the presence of 3-nitropropionic acid (3-NP) irreversible inhibitor of succinate dehydrogenase. These models were used to assess the neuroprotective functions of adenosine. Post-mitotic neurons from differentiated E14-TG2a cultures were characterized according to striatum's GABAergic MSNs; while the differentiated HT-22 neurons were characterized in a non-specific way. Methodology included immunofluorescence (microscopy and cytometry); real-time PCR; analysis of variations in the plasma membrane potentials and of transient variations in the [Ca2+]i by microfluorimetry; and quantification of AlamarBlue® reductions (% cell survival) and of extracellular LDH activity (U/L) (necrosis) by spectrometry. We evaluated the ability of 3-NP to potentiate the excitotoxic effects of QA by comparing two groups of differentiated HT-22 neurons: 8mM QA (control); and 5mM 3-NP/8mM QA. We evaluated the neuroprotective potential of adenosine comparing four groups of differentiated HT-22 neurons: QA 8mM; 250µM adenosine/8mM QA; 5mM 3-NP/8mM QA; 5mM 3-NP/250µM adenosine/8mM QA. Postmitotic neurons derived from E14TG2a were classified as striatums GABAergic MSNs that are part of a heterogeneous neuronal culture similar to nigrostriatal, corticostriatal, striatonigral, and striatopallidal connections. Differentiated HT-22 neurons consisted of a heterogeneous neuronal culture and not fully mature glutamatergic,dopaminergic, cholinergic and GABAergic neurons. Differentiated HT-22 neurons following 5mM 3-NP treatment showed lower % cell survival after treatments with 8mM QA for 24h (p<0.05); and higher amplitudes of the variations of [Ca2+]i induced by 8mM QA (p<0.05) (kinetics 6 minutes). On the other hand, differentiated HT-22 neurons 5mM 3-NP showed lower extracellular LDH activities after treatment with 8mM QA for 24h indicating a lower proportion of necrotic cells. Pretreatments with 250µM adenosine indicated a trend towards neuroprotective effects, such as higher percentages of cell survival; lower extracellular LDH activities; and lower amplitudes of transient variations of [Ca2+]i. Taken together, our results indicate that succinate dehydrogenase inhibition potentiated the excitotoxic effects of NMDARs by altering [Ca2+]i and, probably, cell death mechanisms, while adenosine only to neuroprotection

Inhibition of intracellular proton-sensitive Ca2+-permeable TRPV3 channels protects against ischemic brain injury

Ischemic brain stroke is pathologically characterized by tissue acidosis, sustained calcium entry and progressive cell death. Previous studies focusing on antagonizing N-methyl-d-aspartate (NMDA) receptors have failed to translate any clinical benefits, suggesting a non-NMDA mechanism involved in the sustained injury after stroke. Here, we report that inhibition of intracellular proton-sensitive Ca2+-permeable transient receptor potential vanilloid 3 (TRPV3) channel protects against cerebral ischemia/reperfusion (I/R) injury. TRPV3 expression is upregulated in mice subjected to cerebral I/R injury. Silencing of TRPV3 reduces intrinsic neuronal excitability, excitatory synaptic transmissions, and also attenuates cerebral I/R injury in mouse model of transient middle cerebral artery occlusion (tMCAO). Conversely, overexpressing or re-expressing TRPV3 increases neuronal excitability, excitatory synaptic transmissions and aggravates cerebral I/R injury. Furthermore, specific inhibition of TRPV3 by natural forsythoside B decreases neural excitability and attenuates cerebral I/R injury. Taken together, our findings for the first time reveal a causative role of neuronal TRPV3 channel in progressive cell death after stroke, and blocking overactive TRPV3 channel may provide therapeutic potential for ischemic brain injury.

Expression of lncRNA Tsix in a mouse model of N-methyl-D-aspartic acid-induced retinal excitotoxicity and its significance / 中华实验眼科杂志

Objective:To investigate the damage effect of different concentrations of N-methyl-D-aspartic acid (NMDA) to retinal ganglion cells (RGCs) in mice and explore the expression of long noncoding RNA (lncRNA) Tsix in the retina of mice with excitotoxicity as well as the protective effect of lncRNA Tsix on retina and RGCs.Methods:A total of 105 C57B6/J mice at 7-8 weeks of age were selected and randomly divided into the normal control group, 2 mmol/L NMDA group, 10 mmol/L NMDA group, 20 mmol/L NMDA group and 40 mmol/L NMDA group using a random number table method, with 21 mice in each group.In the normal control group, the mice were intravitreally injected with 1 μl of sodium chloride solution in the right eye, and mice were given intravitreal injection of 1 μl of different doses of NMDA according to grouping.At one week after the injection, the thickness of each retinal layer, the number of ganglion cell layer (GCL) cells and the number of RGCs were analysed and compared among different groups through optical coherence tomography (OCT), hematoxylin-eosin staining, retinal whole mount staining and immunofluorescence staining.RNAscope in situ hybridization was used to verify the expression of lncRNA Tsix in the GCL of different groups.The quantitative real-time PCR was used to detect the transcript levels of Tsix in different groups.This study was approved by an Ethics Committee of Tianjin Medical University (No.SYXK2018-0004), and the use of experimental animals was in accordance with the regulations of Tianjin Medical University and ARVO statement. Results:The OCT results showed that the total retinal thickness of mice in the 2, 10, 20 and 40 mmol/L NMDA groups were (255.00±6.63), (252.40±6.41), (248.67±6.20) and (229.11±10.37)μm, respectively, which were thinner than (269.60±20.01)μm in the normal control group, and the differences were statistically significant (all at P<0.05). Hematoxylin-eosin staining showed that the cells in the GCL of the normal control group were uniform and compact, and arranged in a single layer with large and round nuclei.In the NMDA groups, the cells were uneven in volume with vacuoles and nuclear pyknosis.The cell density in the GCL was decreased significantly with the increasing NMDA doses in NMDA groups in comparison with the normal control group, and the differences were statistically significant (all at P<0.05). In the 20 mmol/L NMDA group, the cell density in the GCL was reduced to half of the normal control group.The results of retinal whole mount staining showed that the density of β3-tubulin-positive RGCs was decreased significantly as the dose of NMDA increased in NMDA groups, and the differences were statistically significant compared with the normal control group (all at P<0.05). The number of RGCs in the 10 mmol/L NMDA group was reduced to half of that in the normal control group.RNAscope results showed that lncRNA Tsix was mainly expressed in the cytoplasm of the GCL cells.The proportion of lncRNA Tsix-positive cells was significantly reduced with the increase of the NMDA dose ( F=13.670, P<0.01). The quantitative real-time PCR results verified that the trend of Tsix expression was consistent with the RNAscope result. Conclusions:NMDA exerts a dose-dependent damage to the layer thickness of mouse retina and RGCs.The expression of lncRNA Tsix in mouse retina is mainly enriched in the cytoplasm of the cells in the GCL, and the transcript level of Tsix is reduced with the increase of NMDA concentration and have a protective effect on RGCs.

Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury / 中国针灸

OBJECTIVE@#To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI.@*METHODS@#A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T@*RESULTS@#Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (@*CONCLUSION@#The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

Tongqiao Huoxuetang Improves Neurological Deficits in CIRI Rats by Regulating Glu-Gln Circulation to Reduce Glutamate Excitotoxicity of Astrocytes / 中国实验方剂学杂志

Objective:To observe and compare the protective effects of Tongqiao Huoxue decoction （TQHX） prepared by three methods against cerebral ischemia-reperfusion injury （CIRI）， and to explore its mechanism through the glutamate （Glu） metabolic pathway in astrocytes. Method:The male SD rats of SPF grade were subjected to CIRI model induction by the modified middle cerebral artery occlusion method. The model rats were randomly divided into a model group， a sham operation group， and water-decocted， wine-decocted， and alcohol-extracted TQHX （6.3 g·kg^-1·d^-1） groups. The rats were treated correspondingly for 7 days. Those in the sham operation group and the model group were treated with an equal volume of normal saline by gavage. After the final treatment， the neurological function of rats was assessed by the modified neurological severity score （mNSS）. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of ischemic brain tissues in rats. High-performance liquid chromatography （HPLC） was used to detect glutamate （Glu） in ischemic brain tissues. The expression of glutamate transporter-1 （GLT-1） and glial fibrillary acidic protein （GFAP） and co-expression of glutamine synthetase （GS） and GFAP in ischemic brain tissues were detected by immunofluorescence assay. Western blot was used to detect the protein expression of GFAP， GLT-1， and GS. Result:Compared with the sham operation group， the model group showed increased mNSS （<italic>P</italic><0.01）， large necrosis of cerebral cortex in ischemic brain tissues with disordered cell arrangement， obscure boundary， intracellular edema， and inflammatory infiltration， elevated Glu in ischemic brain tissues （<italic>P</italic><0.01）， declining GLT-1-GFAP co-expression and GS-GFAP co-expression （<italic>P</italic><0.01）， up-regulated expression of GFAP protein， and reduced protein expression of GLT-1 and GS（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. Compared with the model group， the TQHX groups showed decreased mNSS （<italic>P<</italic>0.01）， relieved injury in the cerebral cortex and hippocampal nerve cells in ischemic brain tissues， reduced Glu expression（<italic>P<</italic>0.05，<italic>P<</italic>0.01）， elevated co-expression of GLT-1 and GFAP （<italic>P<</italic>0.05，<italic>P<</italic>0.01）， and up-regulated protein expression of GFAP and GLT-1（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. The co-expression of GS and GFAP （<italic>P<</italic>0.05，<italic>P<</italic>0.01）and the expression of GS （<italic>P<</italic>0.01）were increased in the wine-decocted and alcohol-extracted TQHX groups. Compared with the water-decocted TQHX group， the alcohol-extracted group showed increased GLT-1-GFAP and GS-GFAP co-expression（<italic>P<</italic>0.05）； the wine-decocted and alcohol-extracted TQHX groups exhibited elevated GS protein expression （<italic>P<</italic>0.05）； the alcohol-extracted TQHX group displayed declining Glu content （<italic>P</italic><0.01） and increased protein expression of GFAP and GLT-1 （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. Compared with the wine-decocted TQHX group， the alcohol-extracted TQHX group showed increased protein expression of GFAP and GLT-1（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. Conclusion:TQHX prepared by three methods can improve neurological deficits in CIRI rats. The effect is presumedly achieved by promoting the further activation of astrocytes， increasing the expression of GLT-1 and GS， promoting the clearance of Glu accumulated in the synaptic cleft by astrocytes through the Glu-glutamine （Gln） circulation， and reducing the excitotoxicity of Glu. The alcohol-extracted TQHX group was superior to the water-decocted and wine-decocted TQHX groups in reducing the content of Glu in ischemic brain tissues， promoting the activation of astrocytes， and enhancing the protein expression of GLT-1 and GS.

Advances in genetics research in the pathogenesis of amyotrophic lateral sclerosis / 中南大学学报(医学版)

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease affecting the upper and lower motor neurons. It is characterized by progressive muscle weakness, atrophy and ultimate death due to dysphagia and dyspnea. There are many causes of ALS, among which the genetic factors show great relevance. Imbalance of protein homeostasis in neurons, prion-like proliferation and propagation of abnormal proteins, mitochondrial dysfunction, glutamate mediated excitotoxicity, and intraneuronal substance transport disorders are recognized as the pathogenesis.The study on gene mutation related to pathogenesis will bridge the molecular and cellular research of ALS, which can deepen the understanding of the occurrence and development of ALS and the role of gene mutation in ALS, and provide new ideas and enlightenment for the treatment of ALS.

Cell Death and Its Different Modes: History of Understanding and Current Trends

Discussions about what is life continue to struggle; there are pros and cons for whether a virus is alive. However, an opposite thing –cell death –appears to be tantamount important and equally not-easygoing to define. Nevertheless, our current knowledgeabout eukaryotic cell death has made a long way and resulted in a fruitful outcome: starting from three types of cell death (type I, II and III which are mainly applicable to eukaryotic cells of organisms from the biological kingdom animalia) in 1970s, Nomenclature Committee on Cell Death has named already twelve cell death forms in 2018, including the above mentioned apoptosis, autophagy and necrosis among them. How the scientific attitude towards cellular demise evolved and various aspects of different cell death modes are reviewed in this article.

Neuroprotective properties of RT10, a fraction isolated from Parawixia bistriata spider venom, against excitotoxicity injury in neuron-glia cultures

L-Glutamate (L-Glu), the major excitatory neurotransmitter in the mammalian Central Nervous System (CNS), is essential to cognitive functions. However, when L-Glu is accumulated in large concentrations at the synaptic cleft, it can induce excitotoxicity that results in secondary damage implicated in many neurological disorders. Current therapies for the treatment of neurological disorders are ineffective and have side effects associated with their use; therefore, there is a need to develop novel treatments. In this regard, previous studies have shown that neuroactive compounds obtained from the venom of the spider Parawixia bistriata have neuroprotective effects in vitro and in vivo. In this sense, this work aimed to evaluate potential neuroprotective effects of fraction RT10, obtained from this spider venom, on primary cultures of neuron and glial cells subjected to glutamate excitotoxicity insults. Methods: Primary cultures of neurons and glia were obtained from the cerebral tissue of 1-day-old postnatal Wistar rats. After 7 days in vitro (DIV), the cultures were incubated with fraction RT10 (0.002; 0.02; 0.2 and 2 µg/µL) or riluzole (100 µM) for 3-hours before application of 5 mM L-Glu. After 12 hours, the resazurin sodium salt (RSS) test was applied to measure metabolic activity and proliferation of living cells, whereas immunocytochemistry for MAP2 was performed to measure neuronal survival. In addition, the cells were immunolabeled with NeuN and GFAP in baseline conditions. Results: In the RSS tests, we observed that pre-incubation with RT10 before the excitotoxic insults from L-Glu resulted in neuroprotection, shown by a 10% reduction in the cell death level. RT10 was more effective than riluzole, which resulted in a cell-death reduction of 5%. Moreover, qualitative analysis of neuronal morphology (by MAP2 staining, expressed as fluorescence intensity (FI), an indirect measure of neuronal survival) indicate that RT10 reduced the toxic effects of L-Glu, as shown by a 38 % increase in MAP2 fluorescence when compared to L-Glu insult. On the other hand, the riluzole treatment resulted in 17% increase of MAP2 fluorescence; therefore, the neuroprotection from RT10 was more efficacious. Conclusion: RT10 fraction exhibits neuroprotective effects against L-Glu excitotoxicity in neuron-glia cultured in vitro.(AU)

Cordycepin protects against β-amyloid and ibotenic acid-induced hippocampal CA1 pyramidal neuronal hyperactivity

Cordycepin exerts neuroprotective effects against excitotoxic neuronal death. However, its direct electrophysiological evidence in Alzheimer's disease (AD) remains unclear. This study aimed to explore the electrophysiological mechanisms underlying the protective effect of cordycepin against the excitotoxic neuronal insult in AD using whole-cell patch clamp techniques. β-Amyloid (Aβ) and ibotenic acid (IBO)-induced injury model in cultured hippocampal neurons was used for the purpose. The results revealed that cordycepin significantly delayed Aβ + IBO-induced excessive neuronal membrane depolarization. It increased the onset time/latency, extended the duration, and reduced the slope in both slow and rapid depolarization. Additionally, cordycepin reversed the neuronal hyperactivity in Aβ + IBO-induced evoked action potential (AP) firing, including increase in repetitive firing frequency, shortening of evoked AP latency, decrease in the amplitude of fast afterhyperpolarization, and increase in membrane depolarization. Further, the suppressive effect of cordycepin against Aβ + IBO-induced excessive neuronal membrane depolarization and neuronal hyperactivity was blocked by DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A₁ receptor-specific blocker). Collectively, these results revealed the suppressive effect of cordycepin against the Aβ + IBO-induced excitotoxic neuronal insult by attenuating excessive neuronal activity and membrane depolarization, and the mechanism through the activation of A₁R is strongly recommended, thus highlighting the therapeutic potential of cordycepin in AD.

Effects of chemokine CCL2 on learning memory in rats and its mechanisms / 中国药理学通报

Aim To investigate the role of chemokine CC motif ligand 2(CCL2) in leaning memory of rats and its mechanisms. Methods Forty male SD rats were randomly divided into five groups, namely, control, sham, CCL2(0.5, 5, 50 ng) group. Except control group, stereotaxic technique was used in this study to perform bilateral hippocampal injection. Mor-ris water maze ( MWM ) was employed to assess the learning and memory ability of rats from day 3 to day 8. Hippocampus was removed on the 10th day. qPCR was used to detect the relative mRNA expression of caspase-8, caspase-3 and phosphate-activated glutami-nase(PAG). ELISA was used to calculate the content of tumor necrosis factor a ( TNF- A ) , acetylcholine (AChE) and the activity of glutamine synthetase (GS). Results Compared to sham group, the latency and swimming distance in each CCL2-treated group were significantly extended, and the crossing times and the percentage of distance in target quadrant of each CCL2-treated group were shorter; the relative mRNA expression of caspase-8, caspase-3 and PAG in each CCL2-treated group were higher than those of sham group; each CCL2-treated group had elevated expression of TNF-a and AChE while the activity of GS in each group decreased. Conclusions CCL2 can impair learning and memory in rats, which may involve in-flammation, excitotoxicity, decreased ACh expression and mediated cell apoptosis.

Synergic effect of Chuanxiong Rhizoma and borneol on protecting hippocampus and hypothalamus of rats from global cerebral ischemia reperfusion injury / 中国药理学通报

To research the synergic effect of Chuanxiong Rhizoma(CR) and borneol(BO) on pro-tecting hippocampus and hypothalamus of the rats from global cerebral ischemia reperfusion (GCIR) injury. Methods GCIR rats were divided into five groups, i. e. sham, model, CR(1.0 g kg"1), B0(0. 16 g kg"1) and CR + BO groups. The contents of Glu, Gly and GABA in brain microdialysis solution were detected by GC-MS. [Ca2+]i of the two regions were measured using a confocal microscope. The apoptotic rate and ultrastructure were obtained by TUNEL stain and transmission electron microscopy, respectively. Results The monotherapy of CR or BO attenuated Ca2 + over-load and improved ultrastructure in both areas. Their synergy showed a better effect. CR alone reduced the apoptotic rate, increased the levels of GABA in both brain areas, while BO only decreased that of Glu. The combination even enhanced the content of Gly in hypothalamus. Conclusions Obvious synergic effects between CR and BO exist in attenuating neuroceliular ex-citotoxicity and calcium overload, while the anti-apop-totic effect might be excluded.

Research progress on the mechanism of ischemic stroke / 中国药科大学学报

@#Stroke is one of the diseases that seriously endanger human health without effective treatment at present, due to the lack of in-depth understanding of the cellular and molecular mechanisms underlying neuronal cell death after ischemia. In the present article, in order to lay the foundations for the development of therapeutic regimens for ischemic stroke, we review the underlying pathophysiology of ischemic stroke including apoptosis, excitotoxicity, oxidative and nitrative stress, inflammation, innate and adaptive immune cells, the gutmicrobiota and intracranial atherosclerosis, and reveal the intertwinedpathways that are promising therapeutic targets.

Amburana cearensis seed extracts protect PC-12 cells against toxicity induced by glutamate

ABSTRACT Amburana cearensis (Allemão) A.C. Sm., Fabaceae, has been widely studied for its medicinal activities. Many neurodegenerative disorders are caused by oxidative stress, mitochondrial dysfunction, excitotoxicity induced by glutamate and ultimately cell death. This study describes the chemical profile of the ethanolic, hexane, dichloromethane, and ethyl acetate extracts obtained from seeds of A. cearensis. The objective of this study was to investigate the chemical profile of extracts obtained from seeds of A. cearensis, as well as their cytotoxicity and neuroprotective effects in cultures of neural PC12 cells. Metabolite profile was performed by GC–MS. PC12 cells were treated with increasing concentrations of the extracts (0.01–2000 µg/ml) and the cell viability was analyzed after 24 and 72 h using an MTT test. For the excitotoxicity assay, PC12 cells were pre-treated with glutamate (1 mM) for 6 h and treated with increasing concentrations (0.1–1000 µg/ml) of the extracts. The chromatographic analysis of the extracts detected various compounds with antioxidant properties, with the majority of peaks corresponding to the isoflavone coumarin. Only the hexane extract showed toxicity after 72 h exposure at the highest concentration (1000 µg/ml). By contrast, all extracts increased the cellular viability of PC12 cells against the toxicity caused by glutamate. Therefore, the extracts from the seeds of A. cearensis showed no toxicity and have neuroprotective potential against neuronal damage induced by glutamate, which may be related to their antioxidant properties.

Advance in Pathogenesis of Amyotrophic Lateral Sclerosis (review) / 中国康复理论与实践

@#Amyotrophic lateral sclerosis (ALS), including the familial and the sporadic, accounts for the most proportion of motor neuron disease. The pathogenesis of ALS covers gene mutation, oxidative stress, excitotoxicity, mitochondrial dysfunction, immune and inflammatory, and so on. With interplay and interrelation, these mechanisms, finally, caused multisystem lesion especially motor neural system.

Brief low Mg2+o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg2+]o to 0.1 mM can evoke repetitive [Ca2+]i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg2+]o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca2+ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg2+]o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca2+ channel antagonist nimodipine, which blocked 0.1 mM [Mg2+]o-induced [Ca2+]i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca2+]i spikes. The intracellular Ca2+ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca2+]i spikes. While Go6976, a specific inhibitor of PKCalpha had no effect on the tolerance, both the PKCepsilon translocation inhibitor and the PKCzeta pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca2+]i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg2+]o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca2+]i spike-induced activation of PKCepsilon and PKCxi, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.

Low Non-NMDA Receptor Current Density as Possible Protection Mechanism from Neurotoxicity of Circulating Glutamate on Subfornical Organ Neurons in Rats

The subfornical organ (SFO) is one of circumventricular organs characterized by the lack of a normal blood brain barrier. The SFO neurons are exposed to circulating glutamate (60~100 microM), which may cause excitotoxicity in the central nervous system. However, it remains unclear how SFO neurons are protected from excitotoxicity caused by circulating glutamate. In this study, we compared the glutamate-induced whole cell currents in SFO neurons to those in hippocampal CA1 neurons using the patch clamp technique in brain slice. Glutamate (100 microM) induced an inward current in both SFO and hippocampal CA1 neurons. The density of glutamate-induced current in SFO neurons was significantly smaller than that in hippocampal CA1 neurons (0.55 vs. 2.07 pA/pF, p0.05). These results demonstrate that glutamate-mediated action through non-NMDA glutamate receptors in SFO neurons is smaller than that of hippocampal CA1 neurons, suggesting a possible protection mechanism from excitotoxicity by circulating glutamate in SFO neurons.

Growth Differentiation Factor 15 Expression in Astrocytes After Excitotoxic Lesion in the Mouse Hippocampus

Growth differentiation factor 15 (GDF15) is, a member of the transforming growth factor beta (TGF-beta) superfamily of proteins. Although GDF15 is well established as a potent neurotrophic factor for neurons, little is known about its role in glial cells under neuropathological conditions. We monitored GDF15 expression in astrocyte activation after a kainic acid (KA)-induced neurodegeneration in the ICR mice hippocampus. In control, GDF15 immunoreactivity (IR) was evident in the neuronal layer of the hippocampus; however, GDF15 expression had increased in activated astrocytes throughout the hippocampal region at day 3 after the treatment with KA. LPS treatment in astrocytes dramatically increased GDF15 expression in primary astrocytes. In addition, LPS treatment resulted in the decrease of the IkappaB-alpha degradation and increase of the phosphorylation level of RelA/p65. These results indicate that GDF15 has a potential link to NF-kappaB activation, making GDF15 a valuable target for modulating inflammatory conditions.

Neuroprotective and Antioxidant Effects of Novel Benzofuran-2-Carboxamide Derivatives

In the present study, we synthesized a series of novel 7-methoxy-N-(substituted phenyl)benzofuran-2-carboxamide derivatives in moderate to good yields and evaluated their neuroprotective and antioxidant activities using primary cultured rat cortical neuronal cells and in vitro cell-free bioassays. Based on our primary screening data with eighteen synthesized derivatives, nine compounds (1a, 1c, 1f, 1i, 1j, 1l, 1p, 1q and 1r) exhibiting considerable protection against the NMDA-induced excitotoxic neuronal cell damage at the concentration of 100 muM were selected for further evaluation. Among the selected derivatives, compound 1f (with -CH3 substitution at R2 position) exhibited the most potent and efficacious neuroprotective action against the NMDA-induced excitotoxicity. Its neuroprotective effect was almost comparable to that of memantine, a well-known NMDA antagonist, at 30 muM concentration. In addition to 1f, compound 1j (with -OH substitution at R3 position) also showed marked anti-excitotoxic effects at both 100 and 300 muM concentrations. These findings suggest that -CH3 substitution at R2 position and, to a lesser degree, -OH substitution at R3 position may be important for exhibiting neuroprotective action against excitotoxic damage. Compound 1j was also found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit in vitro lipid peroxidation in rat brain homogenate in moderate and appreciable degrees. Taken together, our structure-activity relationship studies suggest that the compound with -CH3 substitution at R2 and -OH substitution at R3 positions of the benzofuran moiety might serve as the lead exhibiting potent anti-excitotoxic, ROS scavenging, and antioxidant activities. Further synthesis and evaluation will be necessary to confirm this possibility.

Consumo de aminoácido de cadeia ramificada na esclerose lateral amiotrófica: suplemento proteico ou substância neurotóxica? / Branched-chain amino acid consumption in amyotrophic lateral sclerosis: protein supplement or neurotoxic substance?

Estudos demonstraram efeito positivo principalmente no ganho ponderalem pacientes com esclerose lateral amiotrófica (ELA), suplementadoscom aminoácidos de cadeia ramificada (AACR). Achados recentes têm mostrado que o consumo excessivo e crônico de AACR pode contribuir para a progressão da doença, provavelmente devido a estes serem precursores do glutamato. O objetivo deste estudo foi avaliar a evidência acerca da utilização dos AACR por pacientes com ELA, a fim de elucidar questões pertinentes a sua ingestão. Foi feita busca em base de dados de artigos científicos relacionados ao consumo de AACR na ELA, no período de 1988 a 2013. Foram encontrados seis artigos relacionados ao consumo de AACR por pacientes com ELA. Desses, um referiu melhora da força, enquanto os outros relataram ganho ponderal ou não mostraram resultados significativosem relação aos desfechos. Além disso, foi possível observar uma estreita relação entre o consumo excessivo e crônico dos AACR com o agravamento da doença. À luz dos conhecimentos ora disponíveis, a suplementação com AACR não é recomendada devido aos possíveis efeitos nocivos. O consumo adequado de alimentos proteicos, fontes desses aminoácidos, deve ser utilizado pelos pacientes, respeitando as recomendações estabelecidas. No entanto, estudos adicionais devem ser desenvolvidos em virtude do escasso número de publicações disponíveis.

Studies have shown positive effect mainly in weight gain inpatients with amyotrophic lateral sclerosis (ALS) supplemented with branched chain amino acids (BCAA). However, recent studies have shown that excessive and chronic intake has contributed to the worsening of the disease progression, probably because the amino acids are glutamate precursors. The objective of this study was to assess the evidence about the use of BCAA by patients with ALS, with the aim to clarify pertinent issues for its intake. A search was conducted in data bases for scientific papers related to the intake of BCAA in ALS, between 1988 and 2013. For these review six articles related to the use of BCAA in ALS were found. Of these, one described strength improvement, while the remaining reported weight gain or no significant effects in relation to the outcome. Additionally, it waspossible to observe a close relationship between the excessive and chronic BCAA intake with the worsening of the disease. Considering the presente day available knowledge BCAA supplementation should not be indicated due to the possible harmful effect. The intake of appropriated protein foods should be consumed by these patients, respecting the suggested recommendation. However, more studies are necessary due to the scarce papers in this area.