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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.
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Objective:To observe the effect of exercise on cognitive function and the expression of NeuN and SynapsinI in prefrontal cortex of rats after cerebral ischemia-reperfusion. Methods:Forty health male Sprague-Dawley rats were randomly divided into sham group (S group, n = 10), model group (M group, n = 10), sham & exercise group (SE group, n = 10) and model & exercise group (ME group, n = 10). The left middle cerebral artery was occluded for one hour and reperfused. SE and ME groups accepted treadmill training, for 14 days. The cognitive function of rats was evaluated with open field experiment and new object recognition experiment. The number and distribution of nerve cells in rat brain were observed with Nissl staining and immunofluorescence staining. The SynapsinI expression in serum was detected with ELISA. Results:Compared with S group, the activity, times of crossing, length and activity time in central area of open field experiment decreased in M group, as well as the cognitive index and length of new object recognition experiment (P < 0.05). Compared with M group, all the indexes increased in ME group (P < 0.05). The number of Nissl bodies decreased in M group (P < 0.05) and the arrangement was disordered, while the number of Nissl bodies increased in ME group (P < 0.05) compared with M group. The NeuN positive cells was more in S group than in M group (P < 0.05), and it was more in ME group than in M group (P < 0.05); as well as that of SynapsinI. The expression of SynapsinI decreased in M group (P < 0.05), and increased in ME group (P < 0.05). Most results of behavioral experiments positively correlated with the expression of Nissl, NeuN and SynapsinI (r > 0.221, P < 0.05). Conclusion:Cerebral ischemia-reperfusion can cause cognitive impairment in rats. Exercise can alleviate nerve injury and improve cognitive function, which may relate to promoting the expression of NeuN and SynapsinI in prefrontal cortex, to increase the number of neurons and synapses.
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@#Introduction: Obesity has been demonstrated to induce oxidative stress and inflammation processes that lead to senescence in brain cells. Obesity-induced cellular senescence in the brain is still widely investigated. This study aimed to investigate the expression of antioxidant and neuronal markers in the frontal lobes of obese rats. Methods: Eighteen adult rat Sprague Dawley divided into three groups: Control (SO), Obese-2 (DIO2), and Obese-4 (DIO4) were observed. Control rats were fed with a standard diet AIN 76A for two month. In contrast, DIO2 and DIO4 rats were fed with a high-fat diet daily for two and four months, respectively. After being sacrificed, the rats’ brains were dissected out then the frontal lobes were used for RNA extraction. Reverse transcriptase PCR of SOD2, GPx, BDNF, NeuN and beta-actin was performed to investigate the relative expression of the antioxidant and neuronal markers. Results: DIO2 and DIO4 groups had significantly increased body Weights, blood glucose level and triglyceride level after being fed with a high-fat diet for two and four months, respectively. The DIO4 group had the significantly lowest mRNA expressions of SOD2, GPx, BDNF and NeuN. Conclusion: Decreased antioxidant and neuronal markers in the rats frontal lobes were observed as the chronic effect of obesity.
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Objective To observe the expressions of NeuN and pCaMKⅡα in brain and test the spacial learning and memory in neonatal hypoxic?ischemia encephalopathy ( HIE ) model mice. Methods 7d ICR mice were randomly divided into sham group( n=19) and model group( n=23). HIE model was induced by right common carotid artery ligation followed by 8% oxygen hypoxia for 100 min. DAPI staining was used to examine brain pathological change,immunofluorescent staining was used to examine the expression of NeuN and pCaMKⅡα in the ipsilateral brain,and Morris water maze was used to test the spa?cial learning and memory. Results Mice in sham group showed that brain cells were arranged in a dense and orderly manner,the number of NeuN?positive cells and pCaMKⅡα?positive cells were (106.50±20.07), (87.17±16.55) respectively in the brain,and the escape latency was short. Compared with mice in sham group,mice in model group showed more cells loss,less NeuN?positive cells(19.17±3.60) and less pCaMKⅡα?positive cells(13.33±3.62) in the ipsilateral hemisphere,and longer escape latency(P<0.01). Conclu-sion The spacial learning and memory are impaired in hypoxia ischemia,which may be related to the de?creasing expression of pCaMKⅡα in neurons in ipsilateral brain.
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Rabies is a lethal disease caused by a neurotropic virus that produces inconspicuous morphological changes hardly observable with conventional histopathology. The fatal outcome caused by rabies could be attributed to specific biochemical changes that severely impact neuronal function. The neuronal nuclear protein (NeuN) has become a widely used neuronal marker for the research and the histopathological diagnosis of nervous system diseases. To evaluate the distribution of the protein NeuN in the motor cortex of normal and rabies-infected mice adult ICR mice were inoculated with rabies virus either intramuscularly or intracerebrally. Rabies-infected mice were sacrificed at the terminal stage of the disease. Control mice were also euthanized at the same age. The brains were removed and cut into coronal sections on a vibratome. Immunohistochemistry was used to study the expression of NeuN in the motor area of the cerebral cortex. Neuronal counts, cellular optical densitometry and neuronal diameter measurements were performed to analyze the immunoreactivity of the protein. All parameters revealed decreased immunoreactivity for NeuN in cortical neurons of mice intracerebrally infected with rabies. In contrast, the changes were not statistically significant in mice inoculated intramuscularly. Either the immunoreactivity of NeuN or its expression is affected by the presence of rabies virus in the cerebral cortex depending on the inoculation route. These results contribute to the knowledge of the dynamics of cellular infection on rabies pathogenesis.
La rabia es una enfermedad mortal causada por un virus neurotrópico que produce discretos cambios morfológicos difícilmente observables con la histopatología convencional. El desenlace fatal causado por la rabia puede atribuirse a cambios bioquímicos específicos que afectan gravemente la función neuronal. La proteína nuclear neuronal (NeuN) es un marcador ampliamente utilizado para la investigación y el diagnóstico histopatológico de enfermedades del sistema nervioso. Este trabajo se realizó con el propósito de evaluar la distribución de la proteína NeuN en la corteza motora de ratones normales y ratones infectados con virus de la rabia. Ratones ICR adultos fueron inoculados con virus de la rabia por vía intramuscular o por vía intracerebral. Los animales infectados con rabia fueron sacrificados en la etapa terminal de la enfermedad. Ratones de la misma edad no inoculados con el virus (controles) fueron sacrificados simultáneamente. Se extrajeron los cerebros y se obtuvieron cortes coronales en un vibrátomo. Mediante inmunohistoquímica se estudió la expresión de NeuN en el área motora de la corteza cerebral. Se realizaron conteos neuronales, densitometría óptica celular y mediciones del diámetro de los perfiles neuronales para analizar la inmunorreactividad de la proteína. En los ratones inoculados por vía intracerebral hubo disminución significativa de la inmunorreactividad de NeuN manifestada en los diferentes parámetros evaluados. En contraste, estos cambios no fueron estadísticamente significativos en los cerebros de ratones inoculados por la ruta intramuscular. La inmunorreactividad de NeuN o su expresión es afectada por la presencia del virus de la rabia en la corteza cerebral pero dependiendo de la vía de inoculación. Estos resultados contribuyen al conocimiento de las dinámicas de infección celular en la patogénesis de la rabia.
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Animals , Mice , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Rabies virus/pathogenicity , Rabies/metabolism , Cerebral Cortex/virology , Immunohistochemistry , Nerve Tissue Proteins/analysis , Nuclear Proteins/analysis , Rabies virus/metabolismABSTRACT
Objective To observe the influence of Xiaoshuan enteric-coated Capsule (XSECC) on neuronal damage in cortex of cerebral hypo-perfusion rats. Methods Rats were divided into a sham group (12), a model group (17), a XSECC large dose group (15), a medium dose group (15) and a low dose group (15) by a random number table. The cerebral hypo-perfusion model was produced by permanent bilateral common carotid artery ligation (2VO). The mixed suspension of XSECC was given orally to rats in the XSECC large dose group (420 mg/kg), the medium dose group (140 mg/kg) and the low dose group (47 mg/kg) once each day for 40 days from the beginning of two hour after ischemia. The expressions of NeuN and Caspase-3 were observed by immunofluorescence staining at 40 days after ischemia. The content of GSH-PX,SOD and MDA were measured by biochemical assay. Results Compared to the model group, the expressions of NeuN (8 716.86 ± 2 539.93, 9 549.31 ± 1 663.26 vs. 7 297.05 ± 1 932.49) were significantly increased in the XSECC large dose group and the medium dose group (P<0.05 or P<0.01);The content of GSH-PX (7.37 ± 1.08 U/mg, 7.77 ± 3.26 U/mg vs. 3.67 ± 2.52 U/mg) was increased in the XSECC large dose group and the medium dose group(P<0.05); The expressions of Caspase-3 (11.65 ± 2.68, 14.05 ± 4.55, 12.60 ± 4.56 vs. 16.80 ± 5.41) were obviously decreased in the XSECC large dose group, the medium dose group and the low dose group, compared with the model group (P<0.05 or P<0.01);The content of MDA (1.44 ± 0.40 nmol/mg, 1.96 ± 1.13 nmol/mg, 2.12 ± 1.19 nmol/mg vs. 3.19 ± 0.98 nmol/mg )was decreased in the XSECC large dose group, the medium dose group and the low dose group when compared with the model group (P<0.05 or P<0.01);The content of SOD (555.61 ± 92.45 U/mg, 607.90 ± 228.45 U/mg, 515.98 ± 184.01 U/mg vs. 348.12 ± 108.84 U/mg) was increased in the XSECC large dose group, the medium dose group and the low dose group when compared with the model group (P<0.05 or P<0.01). Conclusion XSECC could protect injured neurons, which is related to the improvement of free radical metabolism.
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Dexmedetomidine (Dex) has been demonstrated to provide neuroprotective effect against brain injury in the central nervous system. However, the underlying mechanism of this neuroprotection remains unclear. In this study, we explored whether Dex has the protective potential in rat models of traumatic brain injury(TBI). More importantly, our study further investigated the role of neuronic autophagy induced by PI3K/Akt/mTOR pathway in this neuroprotective action. Adult male Sprague-Dawley rats were subjected to a diffuse cortical impact injury caused by a modified weight-drop device and Dex (15ug/kg, i.v.) was administered immediately after TBI. Wet-dry weight method was used to evaluate brain edema. Motor function outcome was assessed by Neurologic Severity Score and the spatial learning ability was evaluated in a Morris water maze. The co-localization of microtubule-associated protein 1 light chain 3(LC3) and neuronal nuclei (NeuN), or LC3 and mammalian target of rapamycin (mTOR) were analyzed by immunofluorescence respectively. The expression of LC3, Phosphorylated protein kinase B (p-Akt) and p-mTOR were quantified using Western blot analysis. Our results showed treatment of rats exposed to TBI with Dex caused not only marked reduction in cerebral edema, motor and cognitive functions deficits, but also a decrease in LC3 levels and a increase in p-Akt and p-mTOR levels. Taken together, these findings indicated that treatment with Dex after TBI could inhibited neuronic autophagy in the hippocampus mediated by the activation of the PI3K/Akt/mTOR pathway, finally promoting neurological recovery.
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Objective To explore the effect of behavioral training on the differentiation of neural stem cells in the dental gyrus (DG) in rats with hippocampus infarction. Methods Seventy-eight Sprague-Dawley rats were randomized into infarction plus behavior training group, infarction group and control group. Photochemistry method was used to induce hippocampal infarction in rats of the infarction plus behavioral training group and infarc-tion group. At 1 day after surgery, Morris water maze training was used for infarction plus behavioral training group, free-movement without training was performed for infarction group. Double staining immunofluorescence was used to detect the co-expression of bromodeoxyuridine (BrdU) with neuronal nuclei ( NeuN ) or glia fibrillary acidic protein (GFAP) in the DG at different time points. Results Few BrdU/NeuN and BrdU/GFAP double staining cells were observed in the DG of control rats. In the infarction group and infarction plus behavioral training group the number of BrdU/NeuN and BrdU/GFAP double-stained cells increased in the DG on the opposite side compared with the control group on 14th, 21st, 28th and 35th days after surgery (P < 0.05 ). There observed significantly more BrdU/NeuN and BrdU/GFAP double-stained cells in the infarction plus behavioral training group than that in the infarction group on the 14th, 21st, 28th and 35th days after surgery ( P < 0.05 ). Conclusion Behavioral training can accelerate the differentiation of neural stem cells to neuron and astrocyte, by which to promote the re-covery of neural functions.
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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.
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Humans , Cerebral Cortex , Craniocerebral Trauma , NeuronsABSTRACT
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.
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Humans , Cerebral Cortex , Craniocerebral Trauma , Frontal Lobe , Laminar Flow , Temporal Lobe , Edema , Hypoxia , IschemiaABSTRACT
Medulloblastoma (MB) is the most common malignant brain tumor in childhood. The alterations found include: presence of oncoproteins p53 and HER2, elevated mitotic index, and presence of neuronal differentiation. The aim of this study was to determine the immunohistochemical expression of markers Ki-67, NeuN, synaptophysin, HER2 and p53 in 40 MB samples and their correlation with clinicopathologic parameters and survival. In 29 patients (72.5 percent), >20 percent of cells were positive for Ki-67. Males showed greater ki-67 expression (p=0.02) and smaller survival rates (p=0.002). NeuN and synaptophysin were negative in 16 (40 percent) and 8 (20 percent) cases, respectively. P53 was positive in 18 (45 percent) cases, with 11 (61 percent) weakly positive and 7 (39 percent) strongly positive. HER2 was positive in 23 (57.5 percent) of the samples and did not show statistical association with survival (p=0.07).
Meduloblastoma (MB) é o tumor maligno encefálico mais freqüente na infância. dentre as alterações encontradas estão: a presença das oncoproteínas p53 e HER2, elevado índice mitótico e presença de diferenciação neuronal. o objetivo deste estudo foi determinar a expressão imunoistoquímica (IMQ) dos marcadores Ki-67, NeuN, sinaptofisina, HER2 e p53 em 40 amostras de MB, correlacionando-as com parâmetros clinicopatológicos e com a sobrevida. Vinte e nove pacientes (72,5 por cento) apresentaram 20 por cento ou mais das células positivas para Ki-67. os pacientes do sexo masculino apresentaram maior expressão do Ki-67 (p=0,02) e também menor sobrevida (p=0,002). NeuN e sinaptofisina foram negativos em 16 (40 por cento) e 8 (20 por cento) casos, respectivamente. P53 foi positivo em 18 (45 por cento) casos, sendo 11 (61 por cento) fracamente positivos e 7 (39 por cento) fortemente positivos. HER2 foi positivo em 23 (57,5 por cento) das amostras e não demonstrou associação estatística com a sobrevida (p=0.07).
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Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male , Cerebellar Neoplasms/pathology , Medulloblastoma/pathology , Biomarkers, Tumor/metabolism , Antigens, Nuclear/metabolism , Brazil/epidemiology , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/mortality , Epidemiologic Methods , /metabolism , Medulloblastoma/metabolism , Medulloblastoma/mortality , Neoplasm, Residual , Nerve Tissue Proteins/metabolism , /metabolism , Synaptophysin/metabolism , /metabolismABSTRACT
BACKGROUND: The developing brain has a distinctive set of characteristics that make it have different susceptibility to excitotoxins. Using primary tissue cultures of rat hippocampus, we investigated the developmental susceptibility to N-methyl-D-aspartic acid (NMDA)-induced cell death at various days in vitro in relation to the appearance of Bcl-2 protein and NMDA receptor 2B subunit. METHODS: Six, 12, and 18 days-in-vitro (DIV) hippocampal tissue cultures derived from 7-day-old Sprague-Dawley rat pups were used. Each group was treated with 100 micrometer NMDA in 5% CO2 incubator at 36 degrees C for 30 min. A western blot was then performed for the NeuN, Bcl-2 and NMDA receptor 2B subunit and propidium Iodide (PI) staining. RESULTS: The NeuN and Bcl-2 were most highly expressed in 12 DIV tissues. The reductions of the NeuN and Bcl-2 protein expressions by NMDA were significant at the 12 and the 18 DIV tissues, but less at 6 DIV tissues (p<0.05). The PI staining showed that the area of fluorescence of the 7 DIV tissues after NMDA exposure was less than the DIV 13 and 19 tissues. Without NMDA treatment, the NMDA receptor 2B subunit protein expressions at the 6 DIV tissues were highest and decreased with maturation. CONCLUSIONS: These results suggest that the immature tissues were more resistant to NMDA toxicity than the mature tissues, and further studies are needed to establish its relationship with the Bcl-2 protein and NMDA receptor 2B subunit.
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Animals , Rats , Blotting, Western , Brain , Cell Death , Fluorescence , Hippocampus , Incubators , N-Methylaspartate , Neurotoxins , Propidium , Rats, Sprague-DawleyABSTRACT
PURPOSE: Transcranial electromagnetic stimulation(TMS) is a noninvasive method which stimulates the central nervous system through pulsed magnetic fields without direct effect on the neurons. Although the neurobiologic mechanisms of magnetic stimulation are unknown, the effects on the brain are variable according to the diverse stimulation protocols. This study aims to observe the effect of the magnetic stimulation with two different stimulation methods on the cultured hippocampal slices. METHODS: We obtained brains from 8-days-old Spague-Dawley rats and dissected the hippocampal tissue under the microscope. Then we chopped the tissue into 450 micrometer thickness slices and cultured the hippocampal tissue by Stoppini's method. We divided the inserts, which contained five healthy cultured hippocampal slices respectively, into magnetic stimulation groups and a control group. To compare the different effects according to the frequency of magnetic stimulation, stimulation was done every three days from five days in vitro at 0.67 Hz in the low stimulation group and at 50 Hz in the high stimulation group. After N-methyl-D-aspartate exposure to the hippocampal slices at 14 days in vitro, magnetic stimulation was done every three days in one and was not done in another group. To evaluate the neuronal activity after magnetic stimulation, the NeuN/beta-actin ratio was calculated after western blotting in each group. RESULTS: The expression of NeuN in the magnetic stimulation group was stronger than that of the control group, especially in the high frequency stimulation group. After N-methyl-D-aspartate exposure to hippocampal slices, the expression of NeuN in the magnetic stimulation group was similar to that of the control group, whereas the expression in the magnetic non-stimulation group was lower than that of the control group. CONCLUSION: We suggest that magnetic stimulation increases the neuronal activity in cultured hippocamal slices, in proportion to the stimulating frequency, and has a neuroprotective effect on neuronal damage.
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Animals , Rats , Blotting, Western , Brain , Central Nervous System , Magnetic Fields , Magnets , N-Methylaspartate , Neurogenesis , Neurons , Neuroprotective AgentsABSTRACT
BACKGROUND: Bcl-2 protein is essential for neurodevelopment and it is implicated in various neurodegenerative diseases. However, little is known about the normal developmental expression of Bcl-2 and NeuN protein in hippocampus slice cultures. We performed this study to assess their morphological changes and developmental expressions of Bcl-2 and NeuN protein. METHODS: We cultured the hippocampus of an eight days postnatal Sprague-Dawley (SD) rat in slices of 450 m. We observed the morphological development and differentiation of the hippocampus slices. Additionally, we measured the developmental expression of Bcl-2 and NeuN, -actin protein by western blotting. RESULTS: The hippocampal slice cultures revealed matured structures as early as 6 days in vitro (DIV) and a relatively high degree of tissue organization until 18 DIV. After 21 DIV, there were migration of the cells away from the margins of the slices. NeuN expressions at 7, 13, and 19 DIV were 1.42 +/- 0.38 (meanSD, NeuN/ beta-actin OD ratio, insert number=4, each insert had 5 culture slices), 2.95 +/- 0.53, 1.58 +/- 0.42 respectively. Bcl-2 expression at 6, 12, and 18 DIV were 0.56 +/- 0.18 (mean +/- SD, Bcl-2/beta-actin OD ratio, insert number=4, each insert had 5 culture slices), 1.22 +/- 0.28, 1.03 +/- 0.25. CONCLUSIONS: Organotypic slice cultures of a rat's hippocampus shows morphological maturation within 14 DIV and the highest NeuN and Bcl-2 level at about 13 DIV. It is expected that these findings would be useful as baseline data in the field of research on neuronal development and aging processes.