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Objective To investigate the effects of melatonin on oxidative stress and neuronal apoptosis in hippocampus of epileptic rats and the mechanism. Methods Seventy-two adult male Sprague-Dawley rats were equally divided into control group, model group, low dose group and high dose group. The model group was injected coriamyrtin 50μg/kg in the lateral ventricle, while the low dose group and high dose group were injected melatonin 20 mg/kg and 60 mg/kg, respectively, 30 minutes before injection of coriamyrtin. The contents of malo-ndialdehyde (MDA) and superoxide dismutase (SOD) were detected with ultraviolet spectrophotometer, the apoptosis was detected with TU-NEL, and the ultrastructural changes of neurons and mitochondria in hippocampal CA3 region were observed with electron microscopy, af-ter 60 minutes of epilepsy. Results The neurons and mitochondria in hippocampus were damaged, the number of apoptotic cells significant-ly increased (P0.05), SOD increased (P<0.001), and MDA decreased (P<0.001), compared with the model group. Conclusion Exogenous melatonin may signifi-cantly reduce neuronal apoptosis in rat hippocampal after epilepsy, and high dose is more effective, which may relate with resistance of oxi-dative stress, alleviate neuronal mitochondrial damage.
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BACKGROUND: NOV protein encoded by nephroblastoma overexpression gene(NOV) is IGF(insulin-like growth factor) -binding protein. What is its impact on human neural stem cell(hNSC) proliferation and differentiation?OBJECTIVE: To investigate the impacts of NOV protein on hNSCs proliferation and differentiation.DESIGN: A single factor analysis of variance experimental study using cells as subjectsSETTING: Department of histology and embryology, and department of neurobiology in a military medical university.MATERIALS: Study was conducted in the Department of Histology and Embryology of the Third Military Medical University of Chinese PLA. Subjects were hNSCs cultured from 10 to 14 weeks human embryo cerebral cortex.INTERVENTIONS: COS-7 cells were transfected by NOV gene recombined plasmid. COS-7 cell and COS-7 cell modified by NOV gene conditioned culture media(COS-CM and NOV-CM) were collected and reacted with the cultured HNSCs.MAIN OUTCOME MEASURES: hNSCs proliferation was detected by 3H-TdR scintillation analysis, and hNSCs differentiation was detected by immunocytochemistry and flow cytometer(FCM).RESULTS: Both COS-CM and NOV-CM could significant promote the intake of 3H-TdR by HNSCs, of which the 1/minute of NOV-CM group was significantly higher than that of COS-CM group(P ＜ 0.05), which indicated that NOV-CM contained component that could facilitate hNSCs proliferation, and moreover, there was certain dose-effect relationship in NOV-CM' s facilitation of cellular proliferation. The results of immunocytochemistry and FCM revealed that there were more NF-200 positive cells in NOV-CM group, while many glial fibrillary acidic protein positive cells could be seen in COS-CM group.CONCLUSION: NOV protein might have facilitative effects on hNSCs proliferation and differentiation into neurons.
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Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma overexpression gene (NOV) and detect its expression in COS-7 cells. Methods A 1 165-bp cDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(+)/lacZ. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes HindⅢ and BamHⅠ. The recombinant plasmid was transfected into COS-7 cells with liposome. The expression of NOV gene was detected by Western blotting and immunocytochemistry. Results Eukaryotic expression vectors containing 1 165 -bp coding region of NOV gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of NOV protein in cytoplasm. Conclusion That eukaryotic expression vectors containing coding region of NOV gene was constructed can provide a strong molecular tool for the studies of effect of NOV gene.
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Objective To explore the fluorescent distribution, and the relation between fluorescent intensity and time after injecting rear-thigh muscles with DiI. Methods Sixty-five new born mice were equally and randomly divided into 13 groups. Fluorescent distribution and intensity were investigated at 2, 4, 6, 8, 12, 24 h, and 2, 4, 7, 14, 28 d and 2, 3 months after injecting the left posterior limb rear thigh muscles with 1.5 ?l 4 mg/ml DiI for one mouse. Results The labeling neurons were scattered from L2 level to S2 level and associated dorsal root ganglion (DRG), but the most were located at L4 to L6 section. The faint red fluorescence neurons were observed at dorsal root ganglion and cornu anterius medullae spinalis 6 h post-injection. The labeling neurons increased up to the 4th day. The fluorescent intensity enhanced gradually from 6 h to 24 h, then kept the intensity for 3 months. Conclusion It is a quick, precise, persistent method to trance and label the dorsal root ganglions sensory neuron and spinal motoneuron by injecting rear-thigh muscles with DiI, and the rate of labeling neurons can be improved by prolonging the tracing time properly. It is also provide basic data for clinical or experimental neuron label and location.
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Objective To observe the distribution of dopaminergic neurons in SNpc and to establish standard curve in normal mice so as to measure the changes of dopaminergic neurons in number in SNpc of the mice toxicated with MPTP. Methods Ten male C57BL6 mice aged 8-12 weeks, weight 20-22 g, were randomized to receive 20 mg/kg MPTP or physical saline every 3 h for 4 times, then killed 7 d later. The mouse mesocerebrum was taken out and fixed, frozen, sectioned. All sections containing SNpc were observed under the guide of mouse brain atlas. Every other sections were chosen to stain tyrosine hydroxylase (TH) to show dopaminergic neurons immunohistochemically. The TH positive cells in SNpc were counted in each section and the standard curve was established. Results The standard curve of SNpc compact part position and TH positive cells was established. By comparing the standard curves for the MPTP intoxicated mice and the saline mice, TH positive cells in SNpc from MPTP toxicated mice decreased significantly, which confirmed the validity and feasibility of the standard curve. Conclusion The establishment of standard curve greatly facilitates the comparison of specimen from different groups and makes the assessment of dopaminergic neuron loss more accurate and efficient. The standard curve can serve as an excellent reference curve for the assessment of dopaminergic neurons in SNpc in normal C57BL6 mice.
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Objective To observe the effects of different membrane proteins and dimethylsulfoxide on neurite outgrowth of cerebellum granule cells(CGC).Methods Membrane proteins were extracted from the liver,sciatic nerve and brain white matter of adult rats and coated on the cover slips.CGC were dissociated from newborn rats and inoculated on the coated cover slips,while dimethylsulfoxide(DMSO) was added into the CGC suspension.Results The neurite outgrowth was inhibited by membrane protein of brain white mater and the effect was concentration-dependent.Low concentration(