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Objective To study the pathological alterations,such as oxidative stress,cell proliferation and insulin resistance,especially autophagy,in Alzheimer' s disease (AD) complicated with type 2 diabetes (AD + T2DM).Methods The mouse models of T2DM,AD and AD + T2DM were used in the study,and totally 80 mice were divided into four groups:control group,T2DM group,AD group and AD + T2DM group.Morris water maze was applied to test the ability of learning and memory among the above mentioned groups.In the meantime,insulin resistance index,the expression of insulin receptor substrate 2,oxidative stress,cell proliferation and autophagy were observed with chemical analysis,immunofluorescent labeling,transmission electron microscopy and Western blotting.Results On day 4,the difference of time to find Morris water maze in control group,T2DM group,AD group and AD + T2DM group ((26.08 ±4.93) s,(38.46 ± 4.07) s,(47.32 ± 5.86) s,(53.01 ± 6.12) s,F =2.454,P =0.025) was statistically significant,and the time in AD + T2DM group was longer than that in AD group (t =-3.624,P =0.033).Compared with control group,insulin resistance occurred in T2DM group,AD group and AD + T2DM group (4.35 ± 0.48,16.12 ± 3.57,7.03 ± 3.11,18.78 ± 5.06,F =5.602,P =0.009),and the reduction of insulin receptor substrate 2 expression,the oxidative stress reaction,neural proliferative suppression and autophagy (F =418.344,222.514,436.250,113.934,23.772,35.469,all P ＜ 0.05) were induced in T2DM group,AD group and AD + T2DM group,which were more serious in AD + T2DM group than in AD group (t =-2.796,21.723,-8.041,9.037,-4.403,-32.011,-26.593,all P ＜0.05).Conclusion AD + T2DM mice suffered more serious cognitive impairment than AD and T2DM mice.The oxidative stress levels of AD + T2DM mice were upregulated,and thus led to the inhibition of cell proliferation,eventually leading to promotion of autophagy.
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Objective Our purpose is to investigate the expression of natriuretic peptide receptor A (NPR-A) in the retina and to understand the NPR-A’ s functions during the mouse development .Methods Mice eyes were harvested from E16 ( embryonic day 16 ) to P90 ( postnatal day 90 ) . Total of 127 eyes were used in the study . Immunohistochemistries of NPR-A were carried out .Results During development , NPR-A was widely expressed in the retinal neurons .In the outer nuclear layer , NPR-A began to appear in the inner and outer projections of cone and rod cells at P7, but decreased at P14.From P30 afterward, it continued to express weakly .In the inner nuclear layer , NPR-A expressed in the dendrites of bipolar cells weakly from P 7 to adulthood , whereas no expression in horizontal cells .In the ganglion cell layer, NPR-A started highly to express in the ganglion cell bodies at E 16, and in the meantime, in the nerve fiber layer , ganglion cell axons , NPR-A was expressed highly from embryonic to adult .In the inner and outer plexiform layers, NPR-A was highly expressed at P14, but decreased gradually after P30.In addition, NPR-A also widely expressed in the inner protrusions of Müller cells.Conclusion NPR-A participates in the development of the retina , and may be the key molecule in the developing retinal neurons .Moreover, it plays an important regulatory role in the functional activity of Müller cells .
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Objective To investigate the neural proliferation , differentiation and apoptosis of the developing spinal cord of the mouse and to discuss the mechanism of spinal cord ’ s development .Methods 5-Bromodeoxyuridine ( BrdU) assay was used to mark the proliferative neural stem cells , and the immunofluorescent stainings ( DCX, NeuN and Caspase8) were carried out to visualize the newborn neurons , mature cells and apoptotic cells in the spinal cord with 173 mice arrange from E18 to P90.Results BrdU positive neural stem cells appeared evenly in the spinal cord at early days . With age increasing , the neural stem cells differentiated into neuroglial cells and neurons .The newborn neurons in the subventricular zone migrated toward the intermediate zone ( putative gray matter ) and differentiated into mature neurons gradually .With neurons ’ concentrating towards the center , the gray matter formed an “H” shape .In the meantime , with neural differentiation , some apoptotic neurons appeared among the newborn neurons and mature neurons . Double immunostaining showed that most apoptotic neurons were newborn neurons , suggesting the neuroapoptosis more likely occurred in newborn neurons .The statistical data showed that the number of DCX , NeuN and Caspase-8 positive cells reduced with age increasing , suggesting neural differentiation and neuroapoptosis decreased during spinal cord ’ s development .Conclusion Neural proliferation , neural differentiation and neuroapoptosis occur in developing spinal cord . They work together to regulate the formation and development of the spinal cord .
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In order to understand the alcohol's toxicity to the quantitative alternations of synapses in mouse visual cortex, the expression of synaptophysin after prenatal alcohol exposure was investigated. In present study, the experimental mice at P0, P7, P14 and P30 were grouped, as control, 2 g x kg(-1) alcohol treatment and 4 g x kg(-1) alcohol treatment. The pre-synaptic elements which were used to represent synapses were marked with synaptophysin (a synaptic vesicle associated protein) by immunocytochemistry technique. The synaptophysin positive boutons in layer VI of visual cortex were imaged under laser confocal microscope. With stereological methods, the number cal density of synapse in visual cortex was calculated in different groups at various ages. Moreover, Western blotting was carried out to detect the expression of synaptophysin in visual cortex. The results showed that prenatal alcohol exposure could cause synaptic loss with long-term effect and in a dose dependent manner. For instance, there were significant difference among the different treatment groups of P0, P14 and P30 as well (P < 0.05). Western blotting supported the results of immunofluorescent labeling. In conclusion, prenatal alcohol exposure can induce the synaptic loss dose dependently and with long-term effect. Our findings implicate that the synaptic loss with long-term effect in CNS probably contributes to the lifelong mental retardation and memorial lowliness associated with childhood FAS.
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The prenatal ethanol exposure induced the alterations of dendritic spine and synapse in visual cortex and their long-term effect would be investigated in mice from P0 to P30. Pregnant mice were intubated ethanol daily from E5 through the pup's birth to establish mode of prenatal alcohol abuse. The dendritic spines of pyramidal cells in visual cortex of pups were labeled with DiI diolistic assay, and the synaptic ultrastructure was observed under transmission electron microscope. Prenatal alcohol exposure was associated with a significant decrease in the number of dendritic spines of pyramidal neurons in the visual cortex and an increase in their mean length; ultrastructural changes were also observed, with decreased numbers of synaptic vesicles, narrowing of the synaptic cleft and thickening of the postsynaptic density compared to controls. Prenatal alcohol exposure is associated with long-term changes in dendritic spines and synaptic ultrastructure. The changes were dose-dependent with long term effect even at postnatal 30.
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ObjectiveIn order to compare the alteration of reelin-immunoreactive Cajal-Retzius cells (CR cells) in molecular layer of dentate gyrus of APPswe transgenic mice with wild type, the histochemical and developmental characteristics of CR cells were studied, therefore, the roles of CR cells in Alzheimer's disease would be revealed further.Methods The Thioflavine S staining, reelin immunofluorescence with or without reelin/glutamate and reelin/GABA immuno-double staining were carried out in the study. In the meantime, Western blotting was used to study the expression of reelin in hippocampi of the both wild type and transgenic mice. Results Reelin positive CR cells could be double-labeled with either glutamate or GABA immunostaining. Caspase-3 immunofluorescence demonstrated that some CR cells went through apoptosis during their development. Compared with wild type, CR cells in APPswe transgenic mice had significantly decreased in the molecular layer of the dentate gyrus. The result was supported with Western blotting analysis of reelin expression in hippocampus. Conclusion Reelin could be co-expressed with either glutamate or GABA, suggesting CR cells would be glutamatergic exciting neurons and GABAergic interneurons. The loss of CR cells during development probably was caused by the neuroapoptosis. Significant decrease of CR cells in hippocampus of APPswe transgenic mice indicated reelin may play an important role in AD pathological alterations.
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BACKGROUND: The augmenter of liver regeneration (ALR) is a kind of important secondary liver regeneration factors, and it can particularity promote the liver regeneration. OBJECTIVE: To clone the RNA extracted from neonatal rat liver to ALR gene, and to analyze the gene using the bioinformatics. DESIGN, TIME AND SETTING: The study, gene molecule in vitro experiment, was performed at the Laboratory of Neurobiology, College of Life Science of Henan University between March 2008 and May 2008. MATERIALS: The liver tissues were harvested from 3-day-old Wistar rat of SPF grade. METHODS: Total RNA was exacted from rat liver tissues using guanidinium isothiocyanate-phenol-chloroform extraction method. According to the guideline of molecular biology experiment, the target fragment was amplified using reverse transcription-polymerase chain reaction. Alter purified, the target gene was recovered with gel recovery kit and subcloned to pGEM-T easy, and then was transformed to E.coli. The extracted piasmid was sequenced by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd. The sequence was analyzed with molecular biological software. MAIN OUTCOME MEASURES: Polymerase chain reaction amplification band; analysis of gene sequence; sedne, threonine and tyrosine phosphorylation predictions; phytogenetic analysis of ALR. RESULTS: The potymerase chain reaction product was in concordance with target fragment. The analysis of the gene sequence proved it is the ALR gene. The overall length of the ALR gene was 378 bp, it was highly conservative in many species, for instance the Homo sapiens, the Mus musculus, the Bos Taurus and the Danio redo. CONCLUSION: In this experiment, we have cloned the ALR gene successfully.
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Objective In order to investigate the influence of reelin deficiency on the hippocampal development, the histochemical characteristics of hippocampal pyramidal cells and granule cells of reeler mice were analyzed, therefore, reelin's function would be better understood with more morphological evidences. Methods With immunofluorescent double labeling, the pyramidal cells, granule cells and mossy cells in hippocampi between wild type and reeler mice were labeled. Results The development and lamination of hippocampal cortical plate were in disorder. Pyramidal cells and granule cells dispersed, and moreover, granule cells proliferated rapidly and migrated into hilus, so that the bound between granule layer and hilus disappeared. Conclusion As a stop signal and regulatory signal, reelin plays important roles in neuronal migration of developing cortical plate, especially in the regulation of granule cell proliferation.
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Objective In order to make a epileptic model in organotypic slice culture and to further investigate the epileptic pathology. Methods Immunohistochemistry, Fluoro-Jade B staining and BrdU labeling were carried out in the hippocampal slices with domoic acid treatment after 7 days of culture.Results Domoic acid could induce a series of pathological changes, such as, dispersion of granule cells(0.105±0.063) mm vs (0.057±0.012) mm, t = 4.8, P<0.01), neurogenesis in granular layer and cell loss of pyramidal cell and Mossy cell as well. Conclusions In hippocampal organotypic slices, domoic acid induced such pathological changes as human temporal lobe epilepsy and epileptic animal model. It is an ideal epileptic model for pathological, pharmacological and electronic physiological studies with great applicable prospect.
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Objective The entorhino-hippocampal pathway is the major excitatory input from neurons of the entorhinal cortex on both ipsilateral and contralateral hippocampus/dentate gyrus. This fiber tract consists of the alvear path, the perforant path and a crossed commissural projection. In this study, the histogenesis and development of the various subsets of the entorhino-hippocampal projection have been investigated. Methods DiI, DiO and fast blue tracing as well as anti-calretinin immunocytochemistry were carried out with prenatal and postnatal rats at different ages. Results The alvear path and the commissural pathway started to develop as early as embryonic day (E) 16, while the first perforant afferents reached the stratum lacunosum-moleculare of the hippocampus at E17 and the outer molecular layer of dentate gyrus at postnatal day (P) 2, respectively. Retrograde tracing with DiI identified entorhinal neurons in layer II to IV as the origin of entorhino-hippocampal pathway. Furthermore, anti-calretinin immunocytochemistry revealed transitory Cajal-Retzius (CR) cells in the stratum lacunosum-moleculare of the hippocampus from as early as E16. DiI labeling of entorhinal cortex fibers and combined calretinin-immunocytochemistry showed a close association between CR cells and entorhinal afferents. Conclusion The subsets of entorhino-hippocampal pathway appear in the developmental hippocampus during E16-P2. The temporal and spatial relationship between CR cell and perforant afferent suggests the role of this cell type as a guiding cue for entorhinal afferents at early cortical development.
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The methods and experiences of anatomy,histology and embryology teaching in English for foreign students are discussed in this article to exchange experiences with each other and progress together.
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Aim To study ethanol-induced changes in the development and neuronal number of visual cortex in C57BL /6 mice. Methods Female mice were fed with ethanol during pregnancy . The neuron density (ND) and cortical thickness (CT) in visual cortex of off spring mice were measured at either P0, P7 and P14 with hematoxylin and eosin (H.E) and Nissl staining. Results Embryonic death and malformationswere found in the ethanol-treated groups. Malformations, such as microcephaly,anencephaly and myeloschisis with spinabifida, etc were found in late-term embryos. The malformation rate was 12%. Compared with control group, the development of visual cortex in ethanol-treated groups was delayed, and its lamination was in disorder. The neuron polarity was disturbed. Neuron loss was found after ethanol exposure. At various ages, the neuron density in ethanol-treated groups was lower than that in control group(P
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The problems about scientific research design,data disposal and paper writing of medical scientific research are described in the article.
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Objective The diolistic assay has been modified to make it simpler and more efficient in labeling neurons and glia. Methods CNS neurons and glial cells were labeled with DiI diolistic assay in fixed tissue and living brain slices of C57/B6J mice. Results The method allowed the visualization of the fine structure of neurons and glia including synaptic structures such as dendritic spines. Conclusion With the method, the labeling efficacy of cell's fine structure is improved, making it preferable for the analysis of dendritic spine. In addition, the ability to label the living neuron and glia will extend its application vastly.
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Objective To study the effects of matrine on inducing autophagy and autophagic death in BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI1640 alone or exposed to different concentration of matrine. Cell growth inhibition was assessed by MTT assay. Apoptosis was evaluated by flow cytometry. Autophagosome marker LC3 was examined by immunofluorescence microscopy. The ultrastructure change of cells was analyzed by transmission electron microscope. Results The cell proliferation was inhibited by matrine in a dose dependent manner. Its IC50 value was 1.1 mg/mL at 48 h. Treatment with 0.6-1.6 mg/mL matrine for 12 h induced apoptosis of BEL-7402 cells and the apoptosis rate reached (44.88?0.78)% at concentration of 1.2 mg/mL. The autophagy positive cells were greatly increased after 1.2 mg/mL matrine treated in BEL-7402 cells and the number of cells reached (63.16?0.29)%. Morphological features of typical autophagic death were apparent in the cytoplasm at the ultrastructural level. Conclusion Matrine could induce autophagy and autophagic death of BEL-7402 cells in vitro.
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Objective To study ethanol-induced the neuroapoptosis of visual cortex in offspring mice. Methods Pregnant female mice were fed by intubating alcohol daily,beginning on E5(embryonic,E) and continuing through the pup's birth.The neuroapoptosis in P0,P7 and P14 visual cortex was visualized by Caspase-3 activity immunohistochemistry and Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) staining. Results Usually,the pup's birth days would delay one or two days after ethanol exposure.Moreover,ethanol induced reabsorption of fetus and malformations,such as microcephaly,anencephaly and myeloschisis with spinabifida and so on,were found in the study.Apoptosis index in ethanol treatment groups was obviously higher than that in control at either P0,P7 or P14(P
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The neuroendoerine cells (NE cell) of the lung have been studied in 61 human fetuses by silver stain, immunocytochemical method and electron microscopic technique. The NE cells are mainly located in epithelium of conducting portion with various appearance. Some NE cells are neuron like with several processes. The 5-HT positive NE cells are often found in the epithelium of the primitive alveoli with pyramidal, squamous and tadpole-like appearance. Based upon their ultrastructure, the NE cells are rich in organelles and dense core vesicles (DCV). The NEB is often covered by a layer of cuboidal cells, but no nerve fibers are found among the cells in the NEB.The argyrophil cells usually appear in the 12th week, and reach the maximum number by 20th week. Before the 19th week, the argyrophil cells in the upper segment of the conducting portion are more numerous than those in the lower segment, while after 19th week the condition is just reverse. A regression curve made from the number of argyrophil cells and the thickness of intrapulmonary arteries in different fetal ages shows positive correlation.