Protective Effects of Anmeidan on Cell Structure Against Neuronal Damage in Hippocampal CA1 Region of Sleep-deprived Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Anmeidan （AMD） on neuronal structure and neuronal marker protein expression in the hippocampal CA1 region of sleep-deprived （SD） rats. MethodRats were randomly divided into control group， model group， an AMD group （9.09 g·kg-1·d-1）， and melatonin group （0.27 g·kg-1·d-1）. Rats in the control group and the model group received equal volumes of physiologicol saline. The SD model was induced by the self-made sleep deprivation box for four weeks. Ethovision XT system detected and analyzed the spontaneous behaviors of rats. The histomorphology of neurons in the hippocampal CA1 region was observed by hematoxylin-eosin （HE） staining and Nissl staining， and the changes in Nissl bodies were observed by Nissl staining. The ultrastructure of hippocampal cells was observed by transmission electron microscopy （TEM）. Immunohistochemistry was used to detect the expression of glial fibrillary acidic protein （GFAP）， microtubule-associated protein 2 （MAP2）， nestin， and neuronal nuclei （NeuN） in the CA1 region. ResultCompared with the control group， the model group showed longer distance， increased average activity speed， cumulative duration， average body fill， and higher activity frequency （P<0.01）. Besides， the neurons in the CA1 region were reduced in number with disorganized arrangement， wrinkled nuclei， deeply stained cytoplasm， reduced Nissl bodies， swollen and deformed mitochondria， shortened cristae， and swollen Golgi vesicles. Furthermore， the mean integral absorbance （IA） value of GFAP increased and those of MAP2， nestin， and NeuN decreased （P<0.01）. Compared with the model group， the AMD group showed shortened distance traveled， lower average activity speed， shorter cumulative duration， decreased average body fill， and reduced activity frequency （P<0.05， P<0.01）. Moreover， the neurons in the CA1 region were relieved from damage with increased cell number， clear nuclei and cytoplasm， increased Nissl bodies， and relieved mitochondrial damage. The IA value of GFAP decreased and those of MAP2， nestin， and NeuN increased （P<0.05， P<0.01）. ConclusionAMD can improve structural damage of neurons in the hippocampal CA1 region of sleep-deprived rats， which may be achieved by decreasing GFAP expression and increasing MAP2， nestin， and NeuN expression.

Effect and mechanism of total flavonoids of Lichi Semen on CCl_4-induced liver fibrosis in rats, and prediction of Q-marker / 中国中药杂志

This paper was to investigate the effect of total flavonoids of Lichi Semen(TFL) on carbon tetrachloride(CCl_4)-induced liver fibrosis in rats, analyze and predict its mechanism of action and potential quality markers(Q-marker). Firstly, male SD rats were taken and injected subcutaneously with a 40% CCl_4-vegetable oil solution twice a week for 8 consecutive weeks to establish a rat model of liver fibrosis. The rats with liver fibrosis were randomly divided into model group, silybin group(43.19 mg·kg~(-1)), Fuzheng Huayu Capsules group(462.75 mg·kg~(-1)), and TFL groups(100 mg·kg~(-1) and 25 mg·kg~(-1)), with normal rats as a blank group, 10 rats in each group. Except for the blank group, the rats in the other groups were subcutaneously injected with 40% CCl_4-vegetable oil solution of a maintenance dose, once a week. The rats in various treatment groups received corresponding doses of drugs, while the rats in the blank group and model group received the same volume of normal saline once a day for 4 weeks. At the end of the experiment, blood was collected from the abdominal aorta and the liver tissues were collected. The levels of total bilirubin(TBiL), direct bilirubin(DBiL), indirect bilirubin(IBiL), alanine aminotransferase(ALT), and aspartate aminotransferase(AST) in serum were detected by using an automatic biochemical detector. Masson staining was used to observe the histopathological changes of rat liver. Then, the chemical compositions of TFL were collected, and the action targets of these chemical compositions were predicted through SWISS database and reverse molecular docking server(DRAR-CPI). After screening of disease targets of liver fibrosis by Gene Cards database, the protein-protein interaction was analyzed with use of STRING database, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) enrich analysis were also carried out. Moreover, an iTRAQ proteomics technology was used to determine protein expression in liver tissues of rats in TFL, model and blank groups to verify the targets. Furthermore, Cytoscape software was used to establish and visualize the network of chemical components, targets and pathways, and predict the potential Q-marker of TFL. The results showed that the levels of TBiL, DBiL, IBiL, ALT, and AST in the model group were significantly higher than those in the blank normal group(P<0.05), and the above levels in the treatment groups were lower than those in the model group, but with no significant differences. Masson staining showed that the liver damage and the degree of fibrosis were severe in the model group, and were relieved to different degrees in the treatment groups. Then, 74 chemical components were screened, which could act on 865 targets such as EGFR and SRC, participating in the regulation of cancer pathways, PI3 K-Akt signaling pathway, HIF-1 signaling pathway and other signaling pathways closely related to liver fibrosis. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin showed the highest correlation with liver fibrosis-related targets and pathways. Proteomics results showed that a total of 18 proteins among the 45 proteins predicted by internet pharmacology were identified, among which 6 proteins were significantly expressed, including 5 up-regulated proteins and 1 down-regulated protein. The protein expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1 was significantly returned to a normal state in the TFL treatment groups. In conclusion, TFL may demonstrate the anti-hepatic fibrosis and potential hepatoprotective effects by regulating the expression of ALB, PLG, HSP90 AA1, EGFR and MAP2 K1, which may be associated with the regulation of multiple signaling pathways related to liver fibrosis such as PI3 K-Akt pathway. Pinocembrin, quercetin, epicatechin, procyanidin A2, naringenin, nobiletin, phlorizin and rutin could be regarded as potential Q-markers of TFL for quality control.

Effect of magnolol on hippocampal neuroplasticity in unpredictable chronic mild stress treated rats / 中草药

Objective To investigate the effect and mechanism of magnolol on hippocampal neuroplasticity in depression model rats. Methods In this study, depression model rats were prepared with unpredictable chronic mild stress (UCMS), and was given different doses of magnolol (20, 40 mg/kg) for 28 d. The rats in the positive control group were given fluoxetine (20 mg/kg) for 28 d. The ameliorative effects of magnolol on symptom of depression were investigated through behavior tests including open-field test, sucrose preference test, and forced-swimming test. The mRNA levels of Map-2, Gap43, and SYP in the hippocampus, cortex and striatum of rats in each group were detected by qRT-PCR, and the localization and expression of MAP-2, GAP43, and SYP in the hippocampus were observed by immunohistochemical staining analysis. The quantitative analysis of MAP-2, p-MAP-2, and p-ERK in hippocampus of rats in each group were further analyzed by Western blotting. Results UCMS was able to decrease the sucrose preference index, reduce locomotor activity and increase the immobility time in the forced swimming test. Compared with model group, magnolol significantly increased the spontaneous activity of rats, increased the consumption of sugar and water, and decreased the immobility time of chronic stress rats in forced swimming (P < 0.05, 0.01). Magnolol reversed MAP-2 mRNA and protein level in the hippocampus, increased phosphorylated MAP-2 expression in the hippocampus (P < 0.05), and significantly restored the p-ERK expression (P < 0.05). Conclusion Magnolol can affect the phosphorylation of MAP-2 through ERK pathway and increase the expression of MAP-2, thus affecting the neuronal plasticity and exerting its antidepressant effect.

The effects of paired associative stimulation on sensorimotor function and the expression of MAP-2 and GAP-43 after focal cerebral ischemia and reperfasion / 中华物理医学与康复杂志

Objective To observe the effect of paired associative stimulation ( PAS) on the recovery of sensorimotor function and to explore the mechanism in terms of neural plasticity. Methods Ninety male adult Sprague-Dawley rats were randomly divided into a sham operation group (Sham group), a model group (Model group) and a paired associative stimulation group ( PAS group) , each of 30. Each group was then subdivided into 7-, 14-and 28-day subgroups with 10 rats in each. A model of focal cerebral ischemia and reperfusion was estab-lished using the Longa suture method in the Model and PAS groups. The rats in the Sham group underwent the same surgical procedure except for the occlusion of the middle cerebral artery. The rats received 30 minutes of paired pe-ripheral nerve stimulation and transcranial magnetic stimulation comprising 90 pairs at 0.05 Hz beginning 24 h after the occlusion. The impulse wave width of the peripheral nerve stimulation was 200 μs and the intensity was 6 mA. The intensity of the transcranial magnetic stimulation was 120％ of the resting motor threshold. The other two groups weren't given any intervention. Neurological function was tested using Garcia scores on the 1st, 7th, 14th and 28th day after surgery. The rats were then sacrificed and the expression of MAP-2 and GAP-43 in the ischemic penumbra were detected using western blotting and immunohistochemistry. Results No neurological dysfunction was ob-served in the Sham group at any time. Compared with the Sham group at the same time points, the average Garcia scores of the Model and PAS groups were significantly lower (P≤0.05). However, the average Garcia scores on the 7th, 14th and 28th day were significantly higher in the PAS group compared with the Model group at the same time points ( P≤0.05) . The average Garcia scores of the Model and PAS groups on the 28th day after surgery were significantly higher than those on the 1st day (P≤0.05), but only the PAS group's average Garcia score on the 28th day was significantly higher than that on the 7th day. Compared with the Sham group at the same time points, the expression of MAP-2 and GAP-43 protein in the Model and PAS groups was significantly higher, but with that of the Model group significantly lower than that of the PAS group ( all P≤0.05) . The protein expression of MAP-2 and GAP-43 protein in the PAS group on the 14th day was significantly higher than on the 7th and 28th day ( P≤0.05 for both) . Conclusions PAS can promote the recovery of sensorimotor function after cerebral thrombosis, at least in rats. That may be due to its promoting the expression of the neuroplasticity-associated proteins MAP-2 and GAP-43 in the ischemic penumbra.

Haplotype Association of the MAP2K5 Gene with Antipsychotics-Induced Symptoms of Restless Legs Syndrome among Patients with Schizophrenia

OBJECTIVE: Restless legs syndrome (RLS) is considered a genetic disease and, following a genome-wide association study conducted in 2007, the mitogen-activated protein kinase 5 (MAP2K5) gene has been regarded as the promising candidate gene for RLS. The present study investigated whether polymorphisms of MAP2K5 are associated with antipsychotics-induced RLS in schizophrenia. METHODS: We assessed antipsychotics-induced RLS symptoms in 190 Korean schizophrenic patients using the diagnostic criteria of the International Restless Legs Syndrome Study Group. Five single-nucleotide polymorphisms (SNPs) of MAP2K5 were genotyped. We investigated genetic and haplotypic associations of these five SNPs with the risk of antipsychotics-induced RLS symptoms. RESULTS: We divided the 190 subjects into 2 groups: 1) those with RLS symptoms (n=96) and 2) those without RLS symptoms (n=94). There were no significant intergroup differences in the distributions of the genotypes and alleles of the rs1026732, rs11635424, rs12593813, rs4489954, and rs3784709 SNPs. However, the haplotype analysis showed that the G-G-G-G-T (rs1026732-rs11635424-rs12593813-rs4489954-rs3784709) haplotype was associated with RLS symptoms (permutation p=0.033). CONCLUSION: These data suggest that a haplotype of MAP2K5 polymorphisms confers increased susceptibility to antipsychotics-induced RLS symptoms in schizophrenic patients.

Immunohistochemical overexpression of MAP-2 in the cerebral cortex of rabies-infected mice / Aumento en la expresión inmunohistoquímica de MAP-2 en la corteza cerebral de ratones infectados con rabia

The microtubule-associated protein MAP-2 is an integral part of the cytoskeleton and plays an important role in neural morphogenesis. This protein is an essential component of the dendritic cytoskeleton, especially in the adult brain, and its expression can be altered under experimental or pathological conditions. The purpose of this study was to evaluate the effect of infection with the rabies virus on MAP-2 immunoreactivity in the cerebral cortex of mice. The mice were inoculated with the rabies virus and the animals were sacrificed when the disease reached its advanced stage, together with uninfected animals of the same age. The brains were extracted after being previously perfusion-fixed with paraformaldehyde; coronal sections were obtained with a vibratome. The coronal sections were processed by immunohistochemistry to reveal the presence of the MAP-2 protein in neurons of the motor area of the cerebral cortex. Rabies-infected mice showed an increase in the immunoreactivity of the somata and apical dendrites in pyramidal neurons of the motor cortex. This is an unexpected result, as dendritic pathology has been previously demonstrated in rabies, and some studies on neurological disorders associate dendritic alterations with loss of expression of the MAP-2 protein. Therefore, whatever the alteration in the expression of this protein, decrease or increase, it could be causing a biochemical imbalance in the integrity and stability of the neuronal cytoskeleton.

La proteína asociada a microtúbulos MAP-2 es una parte integral del citoesqueleto y juega un papel importante en la morfogénesis neuronal. Esta proteína es un componente esencial del citoesqueleto de las dendritas, especialmente en el cerebro adulto, y su expresión puede ser alterada en condiciones experimentales o patológicas. El propósito de este estudio fue evaluar el efecto de la infección con el virus de la rabia sobre la inmunorreactividad de MAP-2 en la corteza cerebral de ratones. Ratones inoculados con el virus de la rabia fueron sacrificados cuando la enfermedad alcanzó su fase avanzada, junto con animales no infectados de la misma edad. Los cerebros se extrajeron después de que los animales fueron tratados con paraformaldehído mediante perfusión intracardiaca. En un vibrátomo se obtuvieron cortes coronales y estos se procesaron mediante inmunohistoquímica para revelar la presencia de la proteína MAP-2 en las neuronas de la zona motora de la corteza cerebral. Los ratones infectados con rabia mostraron un aumento en la inmunorreactividad de los somas y dendritas apicales en las neuronas piramidales de la corteza motora. Este es un resultado inesperado, ya que previamente se ha demostrado patología dendrítica en rabia, y algunos estudios sobre los trastornos neurológicos asocian las alteraciones dendríticas con pérdida de expresión de la proteína MAP-2. Por lo tanto, cualquiera que sea la alteración en la expresión de esta proteína, disminución o aumento, podría ser la causa de un desequilibrio bioquímico en la integridad y estabilidad del citoesqueleto neuronal.

Effects of Gastrodiae rhizoma on proliferation and differentiation of human embryonic neural stem cells

OBJECTIVE@#To investigate the effects of Gastrodiae rhizoma, a dried root of Gastrodia elata Blume, on proliferation and differentiation of human NSCs derived from embryonic stem cells.@*METHODS@#A 70% ethanol extract of Gastrodiae rhizoma (EEGR) was estimated with 4-hydroxybenzyl alcohol as a representative constituent by HPLC.@*RESULTS@#MTT assay showed that the treatment with EEGR increased the viability of NSCs in growth media. Compared to control, EEGR increased the number of dendrites and denritic spines extended from a differentiated NSC. Whereas EEGR decreased the mRNA expression of Nestin, it increased that of Tuj1 and MAP2 in NSCs grown in differentiation media. Immunocytochemical analysis using confocal microscopy also revealed the increased expression of MAP2 in dendrites of EEGR-treated NSCs. Furthermore, EEGR decreased mRNA expression of Sox2 in NSCs grown even in growth media.@*CONCLUSIONS@#In conclusion, our study demonstrates for the first time that EEGR induced proliferation and neuronal differentiation of NSCs, suggesting its potential benefits on NSC-based therapies and neuroregeneration in various neurodegenerative diseases and brain injuries.

Effects of Gastrodiae rhizoma on proliferation and differentiation of human embryonic neural stem cells

Objective: To investigate the effects of Gastrodiae rhizoma, a dried root of Gastrodia elata Blume, on proliferation and differentiation of human NSCs derived from embryonic stem cells. Methods: A 70% ethanol extract of Gastrodiae rhizoma (EEGR) was estimated with 4-hydroxybenzyl alcohol as a representative constituent by HPLC. Results: MTT assay showed that the treatment with EEGR increased the viability of NSCs in growth media. Compared to control, EEGR increased the number of dendrites and denritic spines extended from a differentiated NSC. Whereas EEGR decreased the mRNA expression of Nestin, it increased that of Tuj1 and MAP2 in NSCs grown in differentiation media. Immunocytochemical analysis using confocal microscopy also revealed the increased expression of MAP2 in dendrites of EEGR-treated NSCs. Furthermore, EEGR decreased mRNA expression of Sox2 in NSCs grown even in growth media. Conclusions: In conclusion, our study demonstrates for the first time that EEGR induced proliferation and neuronal differentiation of NSCs, suggesting its potential benefits on NSC-based therapies and neuroregeneration in various neurodegenerative diseases and brain injuries.

Study of Inhibition Fibrosis of MicroRNA-25 on Diabetic Nephropathy by Regulating Expression of MAP2 K4 / 医药导报

Objective To determine the expression level of microRNA-25 in animal models of diabetic nephropathy and human renal tubular epithelial cells (HK-2) cultured in different conditions, and to explore its regulating effect on the fibrosis in diabetic nephropathy. Methods The expression of microRNA-25 was detected by real-time PCR. The downstream target protein of microRNA-25 was verified by bioinformatic prediction, transient transfection of cells and Western blotting. Results MicroRNA-25 was down-regulated in animal models of diabetic nephropathy and HK-2 cells which were cultured in high-glucose medium (P<0. 01). MAP2K4 might be the downstream target protein of microRNA-25. Overexpression of microRNA-25 reduced the protein expression of MAP2K4 and α-SMA (P<0. 01). Conclusion MicroRNA-25 inhibits the fibrosis in diabetic nephropathy by regulating the expression of MAP2K4.

The Combined Effects of Ginkgo Biloba Extracts and Aspirin on Viability of SK-N-MC, Neuroblastoma Cell Line in Hypoxia and Reperfusion Condition

OBJECTIVE: The purpose of this study is to investigate the combined effects of ginkgo biloba extract, ginkgolide A and B and aspirin on SK-N-MC, human neuroblastoma cell viability and mRNA expression of growth associated protein43 (GAP43), Microtubule-associated protein 2 (MAP2), B-cell lymphoma2 (Bcl2) and protein53 (p53) gene in hypoxia and reperfusion condition. METHODS: SK-N-MC cells were cultured with Dulbecco's Modified Eagle's Medium (DMEM) media in 37degrees C, 5% CO2 incubator. The cells were cultured for 8 hours in non-glucose media and hypoxic condition and for 12 hours in normal media and O2 concentration. Cell survival rate was measured with Cell Counting Kit-8 (CCK-8) reagent assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to estimate mRNA levels of GAP43, MAP2, Bcl2, and p53 genes. RESULTS: The ginkgolide A and B increased viable cell number decreased in hypoxic and reperfused condition. The co-treatment of ginkgolide B with aspirin also increased the number of viable cells, however, there was no additive effect. Although there was no increase of mRNA expression of GAP43, MAP2, and Bcl2 in SK-N-MC cells with individual treatment of ginkgolide A, B or aspirin in hypoxic and reperfused condition, the co-treatment of ginkgolide A or B with aspirin significantly increased GAP43 and Bcl2 mRNA levels. In MAP2, only the co-treatment of ginkgolide A and aspirin showed increasing effect. The mRNA expression of p53 had no change in all treating conditions. CONCLUSION: This study suggests that the combined treatments of Ginkgo biloba extracts and aspirin increase the regeneration of neuroblastoma cells injured by hypoxia and reperfusion.

Caracterización de un modelo organotípico de cultivos de neuronas corticales de humano derivadas de trauma craneoencefalico / Organotypic human neuronal culture derived from traumatic brain injury

Introducción: El trauma craneoencefalico (TEC) es un problema de salud global que puede generar en los pacientes que lo padecen, muerte, discapacidad y/o alteraciones psiquißtricas con gran impacto sobre su desempe±o posterior y sobre su ßmbito familiar. En los últimos años se ha avanzado en el conocimiento de los mecanismos fisiopatológicos que subyacen al TCE. Sin embargo, esto no estß completamente entendido, como tampoco hay claridad sobre los mecanismos de neuroprotección. Por esta razón cada vez mas se buscan modelos que permitan aproximarse al estudio de este síndrome y de esta manera aproximarse a la neuroprotección. Objetivo: Caracterizar un modelo de cultivo organotípico de neuronas corticales humanas obtenidas de personas que sufrieron TCE y a las cuales se les practicó remoción de la contusión. Metodología: Se utilizó tejido cortical humano procedente de 4 individuos que sufrieron TCE y a los cuales se les removió la contusión. Se obtuvieron tajadas de corteza cerebral de 1,500-2,000 mm, las cuales se mantuvieron en un flujo continuo de LCRa a 2 ml/min y una mezcla gaseosa de O2 al 95 por ciento y CO2 al 5 por ciento con burbujeo permanente durante 2, 8 y 14 horas. Se tomó como tiempo cero el momento de obtención de la muestra. Después de cada tiempo se tomaron las tajadas, se cortaron en un vibratomo de medio líquido a 50 mm y se procesaron inmunohistoquímicamente con los marcadores neuronales de degeneración NeuN y MAP2. Resultados: Los resultados indican que las muestras de corteza cerebral se pudieron mantener con cierto grado de integridad celular y laminar hasta las 2 horas de cultivo. Se observó que a partir de este tiempo se inicia un proceso de alteración de la citoarquitectura neuronal y laminar, determinada por la pérdida y alteración de la inmunorreactividad a los marcadores NeuN y MAP2. Ademas se encontró que hay vulnerabilidad celular que compromete en mayor medida a las neuronas localizadas en las laminas corticales III y V.

Introduction: Traumatic brain injury is a global medical problem whose survivors may show disability and neurological or psychiatric sequelae. In the last few years the knowledge of physiopathological mechanisms of TBI has increase but still it is not entirely known. For this reason the research has turn over in one´s mind in new strategies to study this pathology looking for neuroprotection. Objective: The aim of this work is to develop an organotypic culture of cortical human neurons derived from a contusion tissue obtain from patients that suffered TBI. Methodology: We used contused brain tissue from 4 TBI patients. Sections between 1,500-2,000 mm were kept in a continuous flow of aCSF 2 ml/min in a mixture of 95 percent O2 and 5 percent CO2 for 2, 8 and 14 hours. The initial time (0 hours) was the tissue extraction time. From blocks, sections of 50 mm were obtained and processed for immunocytochemistry to NeuN and MAP2. Results: The results show that organotypic cultures keep neuron integrity and laminar organization in the cerebral cortex slices from 0 to 2 hours. From this time ahead neuronal morphology and laminar organization is altered especially in neurons located on layers III and V.

Alteración de la organización laminar y de la dendroarquitectura de la corteza cerebral del humano post-trauma craneoencefalico / Changes in laminar and dendritic organization of the cerebral cortex after traumatic brain injury

Introducción: El trauma craneoencefalico (TCE)es un fenómeno heterogéneo desde el punto de vista molecular, celular y en la respuesta clínica. Se considera que esta diversidad se debe a la intensidad de la injuria primaria, eventos secundarios asociados (hipoxia, isquemia, edema, inflamación), al estado metabólico del paciente, su base genética, edad, género, etc. Para determinar la integridad anatomo-funcional de las células nerviosas es importante verificar el estado de la cito, dendroarquitectura y preservación laminar como un requisito para garantizar conectividad. Objetivo: Valorar la respuesta de las neuronas al trauma con dos marcadores neuronales selectivos sensibles a la lesión NeuN y MAP2. Materiales y métodos: Se utilizaron muestras (4 de lóbulo temporal y 2 de lóbulo frontal) de 6 pacientes que habían sufrido TCE. Las muestras se fijaron en PLP, cortadas en vibrßtomo a 50 µm, incubadas con los anticuerpos NeuN y MAP2 y procesadas con el sistema avidina-biotina. Como control se utilizó tejido humano post-mortem. Resultados: La inmunorreactividad (IR) para NeuN fue anormal en todas las muestras, con sectores que mostraron IR ligeramente alterada, otros con perdida parcial de las capas supragranulares, sobre todo la lßmina III y otros con pérdida drastica de todas las laminas. La IR para MAP2 se alteró en todas las muestras con diferentes grados de compromiso. Los procesos dendríticos fueron difíciles de seguir, especialmente los procedentes de la lßmina V, los cuales se observaron tortuosos, fragmentados y con orientación aberrante. Conclusiones: Con el propósito de conocer el estado de las neuronas después de un evento lesivo se recomienda el uso de los marcadores NeuN y MAP2 complementarios a los métodos clasicos. El presente trabajo muestra la diversidad de respuestas histopatológicas en sectores adyacentes de una misma muestra con ambos marcadores, como un indicador de los diferentes estados de neurodegeneración.

Introduction: Traumatic brain injury (TBI) is a heterogeneous phenomenon from a molecular, cellular and pathological perspective. Clinical outcome is also extremely variable. It is considered that such a diversity response to TBI is related to the primary injury intensity, associated secondary events (hypoxia, ischemia, oedema and inflammation), metabolic patient state, genetic background, age, gender, etc. After injury the histopathological outcome is variable in time and space. In order to determine the anatomofunctional integrity of nerve cells in the cerebral cortex, it is important to verify the state of the cito and dendroarchitecture and the laminar preservation as a requisite to guarantee connectivity. Objective: The aim of the present work was to evaluate the response of human cortical neurons using two selective neuronal markers, NeuN and MAP2, which recognize citoarchitecture and dendritic arrangement, respectively. Materials and methods: In the present study we utilized six tissue samples (4 temporal and 2 frontal cortices) from TBI patients. Tissues from four post-mortem human brains were used as controls. Tissue samples were fixed in PLP, cut at 50 um in a vibratome, incubated with NeuN and MAP2 and processes with the avidin/biotin complex. Results: NeuN-IR was abnormal in all samples analyzed with some sectors showing slight NeuN-IR, others with NeuN-IR partial loss in supragranular layers, especially layer IIII, and other with a drastic reduction in staining in all cortical layers. MAP2-IR was altered across sections with sectors showing different degrees of changes in the normal pattern of MAP2-IR. Dendritic processes were difficult to follow because of its discontinuity. Layer V apical dendritic processes appear tortuous and its IR was fragmented in some cases they take aberrant orientations.

Effects of exercise on expression of MAP-2 and MAP-2 mRNA after intracerebral hemorrhage / 中华物理医学与康复杂志

Objective To investigate the effects of exercise on expression of microtube associated protein-2 (MAP-2) and MAP-2 mRNA in neuronal cells after intracerebral hemorrhage (ICH) in rats. Methods Ninety-six male Sprague-Dawley rats (weighing 270 to 300 g) were divided into 3 groups, a trial group (ICH-induction and ex-ercise, n=32), a control group (ICH-induction only, n=32) and a sham-operated group (no ICH and no exercise, n=32). The brains of the rats were sampled at 7, 14, 21, and 28 days after the operation for establishing ICH mod-el. Another 64 rats were divided into 8 groups (6 h, 12 h, 24 h, 48 h, 72 h, 7 d after ICH, no ICH group and nor-mal group). MAP-2 and MAP-2 mRNA activity were measured by immunohistochemical methods and RT-PCR. The rats in the trial group began cage-running exercise 72 h after the operation. The others were reared in the standard ca-ges. Results ①MAP-2-positive cells appeared around the hematoma in the cortex. The number of MAP-2-positive cells was very small in the sham-operation group. There was an up-regulation of MAP-2 from 24 h to 7 d after ICH, and significant difference was found between the non-ICH group and the normal group. The expression of MAP-2 showed an up-regulation trend in the trial and control groups. There was a significant difference compared with the sham operated group, and the trial group had significantly higher expression levels than the control group. ②RT-PCR showed up-ragulation of MAP-2 mRNA 12 to 24 h after ICH. There was a significant difference between the no ICH and normal groups. The trial and control groups showed up-regulation of MAP-2 mRNA at 14 to 28 d after ICH, with was significantly different from that of the sham group, and the trial group had significantly higher levels than the con-trol group. Conclusion MAP-2 might participated in neural cells plasticity after ICH, and exercise training (cage-running) can up-regulate MAP-2 and MAP-2-mRNA expression.

Influence of hypothermia on the expression of calpain Ⅱ and microtubule associated protein 2 mRNA in the lateral fluid percussion injury rats / 中国综合临床

Objective To study the early expression of ca]pain Ⅱ and microtubule associated protein 2 (MAP2) mRNA in the hippocampus of the lateral fluid percussion injury rats. Methods 18 Male Sprague-Dawley rats were randomly divided into 3 groups. The changes of Calpaln Ⅱ and MAP2 mRNA in hippocampus 3 h after injury were detected by real-time PCR. Results Compared to the control group (n = 6), the expression for Ca]pain Ⅱ mRNA increased obviously(P <0.01)in the lateral fluid percussion injury group(n=6) ,the expression for MAP2 mRNA degraded obviously(P <0.01). Compared with the lateral fluid percussion injury group(n =6) ,the expression for calpuin Ⅱ mRNA in the mild hypothermia group degraded obviously (n = 6), the expression for MAP2 mRNA increased obviously(P <0.01). Conclusion Mild hypothermia may act as neuroprotection by inhibiting the expression of Ca]pain Ⅱ and easing the degradation of cytoskeleton.

Induction of retinoic acid on PC12 cells differentiated into neuron-like cells / 吉林大学学报(医学版)

Objective To study the condition of pheochromocytoma cells(PC12 cells) differentiated into neuron-like cells induced by retinoic acid(RA) and to observe the lengh of neurite and max diameter and the expression of MAP2 in PC12 cells during their differentiation into neuron-like cells.Methods There were seven groups :0.1,0.3,0.5,1.0,2.0 and 5.0 mg?L-1RA groups and control group which contained 10% fetal calf serum.PC12 cells were exposed to RA with different concentrations for 72 h and the lengh of neurite and max diameter of PC12 cells were observed at 24,48,and 72 h respectively.The expression of MAP2 was detected by immunocytochemistry after PC12 cells were exposed to RA for 72 h.Results The number of MAP2 positive cells was significantly increased in 0.3,0.5,1.0,2.0 mg?L-1RA groups compared with control group and the most significant concentration was 1.0 mg?L-1(P

Neural Antigen Expressions in Cultured Human Umbilical Cord Blood Stem Cells in vitro

OBJECTIVES: Cord blood stem cells have been widely used as donor cells for bone marrow transplantation recently. These cells can give rise to a variety of hematopoietic lineages to repopulate the blood. Recent observations reveal that some bone marrow cells and bone marrow stromal cells(MSCs) can grow to become either neurons or glial cells. It is, however, unclear whether or not there exists stems cells which can differentiate into neurons in the blood during the early stages of postnatal life. METHODS: Human cord blood stem cells were prepared from human placenta after full term delivery. To induce neuronal differentiation of stem cells, beta-mercaptoethanol was treated. To confirm the neuro-glial characteristics of differentiated stem cells, immunocytochemical stain for NeuN, neurofilament, glial fibrillary acidic protein(GFAP), microtubule associated protein2(MAP2) was performed. RT-PCR was performed for detecting nestin mRNA and MAP2 mRNA. RESULTS: We showed in this experiment that neuro-glial markers(NeuN, neurofilament, MAP2, GFAP) were expressed and axon-like cytoplasmic processes are elaborated in the cultured human cord blood stem cells prepared from new born placenta after full term delivery. Nestin mRNA was also detected in fresh cord blood monocytes. Conclusions: These results suggest that human cord blood derived stem cells may be potential sources of neurons in early postnatal life.

Cytoskeletal Changes in Cortical Dysplasia

Cortical dysplasia is a cause of intractable epilepsy and a candidate for surgical resection to control epileptic attacks. The neuronal cytomegaly and balloon cell change are the diagnostic hallmarks of cortical dysplasia. Little research has been performed about the normal-sized dysplastic neuron which has complex arborizing dendrites and lacks in its polarity. The aim of this study was to define the histopathologic characteristics of the neurons in cortical dysplasia. Twelve cases of cortical dysplasia who underwent partial lobectomy for intractable seizures were selected and immunohistochemical staining for NF-M/H, MAP2, tau, and ubiquitin was performed. The perikarya and dendrite of dysplastic neurons were more intensely labeled with antibodies for the high and medium molecular weight neurofilament proteins (NF-M/H) than normal neurons. Immunoreactivity with the MAP2 antibody expressed mainly within the somatodendritic regions was present in the dysplastic or normal neurons without any significant difference in intensity. The complex arborizing dendrites of dysplastic neurons were easily identified due to pronounced immunoreactivity within the somatodendritic regions. Immunoreactivity with the primary antibody against tau and ubiquitin was present in the normal-looking neurons as well as the dysplastic neurons. This study suggests that the dysplastic neurons in cortical dysplasia are accompanied by changes of cytoskeletal neurofilaments, and the immunohistochemical stains for NF-M/H, MAP2, tau, and ubiquigin are useful to detect them.

NR1/NR2B receptor Activation induces Cytoskeletal Proteolysis in Partial Ischemic Rat Hippocampal Formation / 대한해부학회지

In the rat brain, partial ischemia causes a delayed neuronal degeneration that occurs hours to days after reoxygenation. It is generally thought that the ischemic damage is initiated by neurotoxicity mediated through glutamate receptors, particulaly NMDA subtypes. Calcium entry through the NMDA receptor is responsible for the synaptic plasiticity and neuronal pathology. Degradation of MAP-2 and NF200, a major components of neuronal cytoskeleton, by Ca2+-dependent protease after NMDA receptor activation has been postulated in delayed neuronal damage. Calcium-activated protease calpain, excessive degradation of MAP-2, together with the calpain-sensitive microtubule and neurofilaments, would be expected to disrupt intracellular transport- and membrane-related functions that is vital to neurons. Changed of NR subunit 2A, 2B, MAP2 and NF200 in rat hippncampal postsynaptic density[PSD] after partial ischemic injury were investigated though immunoblot analyses. To understand the effect of Ca2+, influx through NMDA receptors on neuronal damage which is manifested by cytoskeletal disruption, morphological change was examined through immunohistochemistry and routine staining method. We found that immunoreactivity to NR2B receptor subuit in the hippocampal formation PSD was upregulated while MAP2 and NF200 was down-regulted at 18 hours after initial partial ischemic insult. On the other hand, morphological changes of neuronal cell in partial ischemic conditions were manifested as eosinophilic inclusion bodies in the cytoplasm which is progression of neuronal damage after 6 days. Calcium influx through NR1/NR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which cause neuronal cell death.

Enhancement of N-methyl-D-aspartateNMDA Receptor 2B-mediated Neurotoxi-city after Hypoxia in the Rat Hippocampal Formation / 대한해부학회지

Ischemic brain hippocampal formation has been developed to understand the relationship between delayed neuronal damage and the expression of NMDA receptor subunits[NR2A, NR2B], MAP2, and NF200 in ttle conditions of hypoxia. Changes of NR subunits[NR2A, 2B], MAP2 6nd NF200 in rat brain postsynaptic density[PSD] after hypoxic injury were investigated through immunoblot analyses. To understand the effect of Ca2+ influx through NMDA receptors on neuronal damage which is manifested by morphological change, cytoskeletal disruption was examined through H & E, toluidine blue and immunohistochemical studies. The expression of NR2B was increased than normal at 30 hours after hypoxia. At this time, the expression of MAP2 and NF200 was markedly decreased and their morphology was more eosinophilic than normal and then became darker with expanded perineuronal space. Irreversible neuronal cell damage in hypoxic hippocampal formation is most prominent in CA3 region of hippocampus and the process is triggered by Ca2+ influx through NR1/MR2B receptor channel at 30 hour after initial hypoxic insult. Ca2+ influx through NR1/MR2B receptor channel may activate intracellular proteases which would degrade cytoskeleton. Proteolysis of cytoskeleton leads to its reorganization and eventually damages normal function of cell membrane which causes neuronal cell death. And, morphological changes of neuronal cells in hypoxic conditions were manifested as red neurons in the stage of reactive change, and as dark neuron in the stage of late hypoxic cell damage.

Changing features of microtubule associated protein 2 expression in hippocampal dentate gyrus of epileptic rats and the intervention results after using Diazepam / 临床神经病学杂志

Objective To investigate the changing features of microtubule associated protein 2 (MAP2) expression, which was a neural dendrite marker, of epileptic rat and the intervention results after using Diazepam. The relations between MAP2 and epileptogenesis were also explored.Methods Model of epileptic rat was established by Pentylenetetrazol (PTZ) and divided into PTZ-NA group,PTZ-Diazepam group, Diazepam-PTZ group and normal control group. Immunohistochemistry method was applied on hippocampus of epileptic rats to determine the change of MAP2 immunoreactivity (MAP2-IR) with or without Diazepam intervention at various time points. MAP2-IR was showed by mean optical density (COD).Results In the PTZ-NA group and PTZ-Diazepam group, MAP2-IR in molecular cell layer of hippocampal dentate gyrus increased after 3 days (all P