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@#Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r = 0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.
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Background Paraquat (PQ) is one of the most widely used herbicides in the world and a risk factor for Parkinson's disease (PD), but the mechanisms underlying PD are poorly understood. Single-cell RNA sequencing (scRNA-seq) technology can study cellular heterogeneity at genetic level, providing insights into the pathogenesis of PQ-induced PD. Objective To analyze the brain cell grouping of PQ-infected mice and the biological processes involved in the subpopulation of PD-like changes cells by scRNA-seq, and to provide clues for revealing potential mechanisms of PQ-induced PD-like changes in mouse brains. Methods Six male 6-week-old C57BL/6 mice were randomly divided into a control group and an experimental group, three mice in each group, and were intraperitoneally injected with 0 (saline) and 10.0 mg·kg−1 PD respectively, once every two days, for 10 consecutive injections for modeling. After infection, mouse brains were taken and scRNA-seq was performed. Cell segmentation was performed according to gene expression characteristics of different cell types, PD-related cell subsets were screened by bioinformatics tools, and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), protein interaction network analysis, and transcription factor prediction were performed on their characteristic genes. Finally, GO and KEGG analyses were performed on the differential genes of PD-associated cell subsets between the PQ-treated group and the control group, and the biological processes in which these genes may participate were analyzed. Results The sequencing data met quality control standards, a total of 55779 cells were obtained, and all cell dimensionality reduction analysis results showed that they could be further divided into 37 clusters, including 5 major cell types. Based on the KEGG analysis of the top 20 characteristic genes of each subpopulation, the specifically expressed Cluster 33 subpopulation (dopaminergic neurons) was screened and found to be significantly associated with PD. The results of GO analysis showed that the biological function of this subpopulation mainly enriched neurotransmitter transport and regulation. The results of GSEA analysis showed that the tyrosine metabolic pathway and the ligand-receptor interaction pathway of neural activity in brain tissues were significantly enriched. The analysis of transcriptional regulatory networks showed that 39 transcription factors were expressed differently. The metabolic pathway of the dopamine neuronal subset, endocytosis, Ras-associated protein 1 (Rap1) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway were all affected by PQ exposure, according to further analysis of its effects on this subpopulation. The GO analysis showed that differential genes were involved in biological processes such as ion transport and synaptic assembly regulation, and were involved in the cellular component formation of cytoplasm and synapses. Conclusion This study has initially mapped the transcriptome of single cells in the mouse brain after PQ exposure, and screened out the specific expression of Cluster 33 subgroup (dopaminergic neurons), which is significantly correlated with PD, and its biological function changes may be one of the mechanisms of PD-like changes in the mouse brain induced by PQ.
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Aim To study the effect of H2S synthase CBS-derived H2S from mouse nerve cells on the proliferation and migration of the brain vascular endothelial cells and its relationship with VEGFR2. Methods CCK-8 method was used to detect cell proliferation, cell scratch method and Transwell method were used to detect cell migration, methyl blue method was used to detect H2S content, and calcium fluorescence imaging method was used to detect intracellular free Ca2 + concentration. HT22 cells were co-cultured with bEnd. 3 cells by using Transwell system. Results The H2S donor NaHS( 1 xlO"8-1 X 10"3'5 mol • L'1) significantly increased the proliferation and migration of HU- VEC cells and bEnd. 3 cells, while the VEGFR2 blocker SU5416 (10 (xmol • L"1) markedly inhibited the NaHS- increased proliferation and migration; in the co-culture system,the substrate of H2S synthase CBS, L-Cys (100 |xmol • L"1) , significantly promoted the proliferation and migration of bEnd. 3 cells, and increased intracellular Ca2 + fluorescence intensity in bEnd. 3 cells and the H2S content in the co-culture. However, CBS inhibitor AO A A (1 mmol • L"1 ) and SU5416 significantly attenuated the effects of L-Cys on the proliferation and migration and intracellular Ca2 + fluorescence intensity. Conclusions The CBS-derived H2S from neurons can promote the proliferation and migration of mouse cerebral vascular endothelial cells, which may be related to activation of VEGFR2 and subsequently increase of intracellular free Ca2+ concentration.
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AIM:To investigate the protective effect of Huoxue-Dingxuan capsule-medicated serum on mouse brain microvascular endothelial cell line bEnd .3 with hypoxic injury .METHODS:The Wistar rats were randomly divided into control group and Huoxue-Dingxuan capsule group .The serum was collected at the 7th day after drug administration . The bEnd.3 cells were divided into normal group , hypoxia serum model group and Huoxue-Dingxuan capsule serum group . After administration of the medicated serum , bEnd.3 cell were treated with hypoxia for 6 h.The cell morphology was ob-served under microscope , the apoptosis rate and cell cycle were detected by flow cytometry , and the activity of superoxide dismutase ( SOD) and the content of malondialdehyde ( MDA) were detected by ELISA .RESULTS:Compared with other groups, Huoxue-Dingxuan capsule-medicated serum significantly resisted the injury caused by hypoxia , obviously improved the morphology of bEnd .3 cells, significantly reduced the number of apoptotic cells induced by hypoxia , and effectively in-hibited the occurrence of hypoxia-induced G1/S phase arrest in the bEnd.3 cells.At the same time, the medicated serum inhibited MDA production , and increased SOD activity .CONCLUSION: Huoxue-Dingxuan capsule attenuates hypoxia-induced bEnd .3 cell damage by enhancing the antioxidant capacity of cells and inhibiting cell apoptosis .
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Objective: To investigate the effect of Huoxue Dingxuan Capsule (HDC) containing serum on gene expression profile in mice brain microvascular endothelial cell bEnd.3 and its molecular mechanism. Methods: Thirty rats were randomly divided into control group and HDC group, each group had 15 rats, and the serum was collected in two groups of rats. BEnd.3 was divided into blank serum group, hypoxia model group, and 10% HDC containing serum group. The cells were intervened in different conditions, and after oxygen deprivation for 6 h, the cytoskeleton was observed under laser microscope. And to detect the effect of HDC containing serum on gene expression profile in mice brain microvascular endothelial cell bEnd.3 by Affymetrix U133 plus2.0 gene expression microarray, and for clustering analysis by molecular annotation system MAS3.0. Results: Differentially expressed genes were identified based on P 3. There were 405 differentially expressed genes between blank group and model group, in which 176 genes were up-regulated and 229 genes were down-regulated. There were 368 differentially expressed genes between HDC sero group and model group, in which 146 genes were up-regulated and 222 genes were down-regulated. These differentially expressed genes had the functions of positive regulation of endothelial cell migration, process of acid metabolism, production of vascular endothelial growth factors, positive regulate activity of NF-κB transcription factors, oxidative stress-induced senescence, mitosis prophase, platelet aggregation (blockage formation), ect. And many signaling pathways were regulated by these genes including vascular endothelial growth factor signaling pathway, interleukin signal pathway, p53 pathway, TNF-signal pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, apoptosis signal pathway etc. Conclusion: HDC has significant inhibitory effects on damage of bEnd.3 cells induced by hypoxia. Its mechanism is related to oxidative stress, inhibition of apoptosis, promotion of angiogenesis, inflammation, and immune response. Huoxue Dingxuan Capsule can regulate the function of vascular endothelial cell on gene level by several signaling pathways.
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PURPOSE: By using the micro-imaging unit modified from NMR spectrometer, the high resolution MRI protocols of finer than 100 micron in 5 minutes, is sought for mouse, which plays a central role in animal studies MATERIALS AND METHODS: C57BL/6 mouse, lighter than 50 gram, is used for the experiments. The superconducting magnet is vertical type with 89 mm inner diameter at 4.9 Tesla. The diameter of rf-coil is 30 mm. Mostly used techniques are the fast spin echo and the gradient echo pulse sequence. RESULTS: For 2D images, proton density and T2 weighted images are obtained and their optimum experimental variables were sought. Minute structure of mouse brain can be recognized and 3D brain image is also obtained additionally. 3D image will be useful particularly for the dynamic contrast study using various contrast agents. CONCLUSION: Like the case of human and other small animals, the high resolution of mouse brain is enough to recognize the minute structure of it. Recently, similar studies are reported domestically, but it seems only a beginning stage. Due to easiness of breeding /control, mouse MRI study will soon play a vital part in brain study.
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Animals , Humans , Mice , Brain , Breeding , Magnets , ProtonsABSTRACT
Objective To investigate the effect of injectable Xuebijing on the proliferation of mouse brain microvascular endothelial cell in vitro.Methods Cultured mouse brain microvascular endothelM cell line bEnd. 3 was treated by injectable Xuebijing of different concentrations,0 (control),5,25 and 50 mg/ml.The regulatory. effect of Xuebijing on the proliferation of cell line bEnd.3 was observed and studied by means of MTT method and cell cycle analyzed with flow cytometry.Results Compared to the control group,MTT and proliferation index (PI) of 5 and 25 mg/ml groups were significantly increased at 12 and 24 h,and PI,but not MTT,of these 2 groups was decreased remarkably at 48 h.Meanwhile,50 mg/ml group showed significantly decreased MTT at 24 and 48 h,and PI of this group was increased obviously at 12 and 24 h,but decreased significantly at 48 h. Conclusions Injectable Xuebijing at certain concentrations might promote the proliferation of cultured mouse brain microvascular endothelial,cells within specific time frame.
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The immune humoral response induced by the rabies vaccine produced in suckling mouse brain was studied in 23 dogs. The mouse neutralization test (MNT) was used to evaluate the level of rabies antibodies. Ten dogs received vaccine stored at 2 to 8 degrees C, showing the following results: 30 days after vaccination, six samples (60%) responded to the MNT; 180 days after vaccination, 4 samples (40%); and 360 days after vaccination, only one sample (10%). The remaining 13 dogs received previously frozen vaccine and 30 days after vaccination, only two samples (l5.4%) responded to the MNT. No titers were detected 180 and 360 days after vaccination. Statistical analysis of each variable used Tuckey analysis of variance, which showed statistically significant differences between the two groups.
A resposta imune humoral induzida pela vacina contra a raiva produzida em cérebros de camundongos recém-nascidos foi estudada em 23 cães e o teste de soroneutralização em camundongos foi usado para avaliação dos níveis de anticorpos rábicos. Um grupo com 10 animais recebeu vacina conservada de 2 a 8oC e apresentou os seguintes resultados: após 30 dias da vacinação 6 (60%) amostras responderam ao teste; após 180 dias 4 (40%) e após 360 dias apenas 1 (10%). O outro grupo com 13 cães recebeu vacina previamente congelada e somente 2 (15,4%) amostras no dia 30 apresentaram resposta satisfatória; os demais períodos (180 e 360) após a vacinação, não foi encontrado título. A análise estatística dos dados referentes a cada uma das variáveis consideradas no estudo foi efetuada segundo a técnica de análise de variância seguida por Tuckey e indicaram diferenças estatisticamente significativas entre os grupos.
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Animals , Dogs , Mice , Antibodies, Viral/biosynthesis , Freezing , Rabies Vaccines , Rabies/immunology , Rabies/prevention & control , BrainABSTRACT
PURPOSE: To investigate the regulation of apoptosis and cell cycle in mouse brain irradiation. MATERIALS AND METHODS: 8-week old male mice, C57B1/6J were given whole body gamma-radiation with a single dose of 25 Gy using Cobalt 60 irradiator. At different times 1, 2, 4, 8 and 24hr after irradiation, mice were killed and brain tissues were collected. Apoptotic cells were scored by TUNEL assay. Expression of p53, Bcl-2, and Bax and cell cycle regulating molecules; cyclins B1, D1, E and cdk2, cdk4, p34cdc2 were analysed by Western blotting. Cell cycle was analysed by Flow cytometry. RESULTS: The peak of radiation induced apoptosis is shown at 8 hour after radiation. With a single 25 Gy irradiation, the peak of apoptotic index in C57B1/6J is 24.0+/-0.25 (p<0.05) at 8 hour after radiation. Radiation upregulated the expression of p53/tubulin, Bax/tubulin, and Bcl-2/tubulin with 1.3, 1.1 and 1.45 fold increase, respectively were shown at the peak level at 8 hour after radiation. The levels of cell cycle regulating molecules after radiation are not changed significantly except cyclin D1 with 1.3 fold increase. Fractions of Go-G1, G2-M and S phase in the cell cycle does not specific changes by time. CONCLUSIONS: In mouse brain tissue, radiation induced apoptosis is particularly shown in a specific area, subependyma. These results and lack of radiation induced changes in cell cycle offer better understanding of radiation response of normal brain tissue.
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Animals , Humans , Male , Mice , Apoptosis , Blotting, Western , Brain , Cell Cycle , Cobalt , Cyclin D1 , Cyclins , Flow Cytometry , In Situ Nick-End Labeling , S PhaseABSTRACT
BACKGROUND: The Central nervous system(CNS) plays a essential role in mediating~stress responses. However, the enact mechanism of the CNS in mediating stress responses has not been clarified sufficiently as yet. Stress may cause brain dysfunction including cognitive dysfunction which was most commonly found in Alzheimer's dementia. Amyloid precursor protein(APP) is a large, ubiquitously distributed and evolutionarily conserved molecule whose function remains unknown. Although the precise function of APP following injury to the CNS such as stab and kainic acid lesion. However, there have not been reports on the effects of stress on the expression of amyloid precursor protein in the brain. This study was undertaken to elucidate the effects of stress on the expression of APP in the mouse brain. METHODS: The several brain region was isolated from the mouse that was in the immoblization stress for 30 min, 1 hour, and 2 hours. The mouse brain was divided into 5 regions, cerebral cortex, cerebellum hippocampus, midbrain and thalamus, corpus striatum and brain stem. The change of mRNA was examined in the several brain regions using Northern blot hybridization. RESULTS: The amounts of APP mRNA in the cerebral cortex, hippocampus and brain stem were found to be significantly increased after stress for 30 minutes and to 1.each a maximum after stress for 1 hour and to normal range at stress for 2 hours. On the contray, the contents of APP mRNA in midbrain and thalamus were decreased after stress for 30 minutes and sustained after stress for 2 hours. CONCLUSION: These findings suggest that APP may not be static but functional protein reactive to stress and stress may increase the levels of APP mRNA especially in Alzheimer disease associated sites such as cerebral cortex and hippocampus, which may contriute to the pathogenesis of Alzheimer disease.
Subject(s)
Animals , Mice , Alzheimer Disease , Amyloid , Blotting, Northern , Brain Stem , Brain , Cerebellum , Cerebral Cortex , Corpus Striatum , Dementia , Hippocampus , Immobilization , Kainic Acid , Mesencephalon , Negotiating , Reference Values , RNA, Messenger , ThalamusABSTRACT
No abstract available.
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Animals , Mice , Adenosine Triphosphatases , Adenosine , Brain , Microsomes , PhenobarbitalABSTRACT
The animal model for the study of B encephalitis was established by injecting Jin Wei Yan 1 strain of B encephalitis virus into the peritoneal cavity of mice. All the mice injected demonstrated the histological features of the disease. Special attention was paid to the ultrastructural changes of the granular cells, the Purkinje cells and the Golgi cells in the cerebellum, and the findings in these cells were compared with those of the nerve cells in the cerebral cortex, dience-phalon and tnesencephalonIt was found that endoplasrnic reticulum could appear in the nerves infected by B encephalitis virus. A radiating structure was-usually formed by the viral replication multivesicular bodies with microvesiculo-tubular body as its center. The morphological changes and the developmental sequence as well as the significance of this radiating structure were discussed.In the late stage of the infection, viral particles were found in the nuclei of a part of the necrotic cells. It was likely that the viral particles entered the nuclei by way of cytoplasm. Our observation confirmed the Chen Liming's hypothesis that viral particles can be formed in the perinuclear space. The way that the viral particles were evacuated from the infected nerve cells observed in this study was in agreement with that reported by Chen Liming, but most of the viral particles left the infected nerve cells through the axons.