Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor la, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage There is little evidence of direct regulatory effects of hypoxia-inducible factor le on oligodendrocyte lineage gene-l. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxygen-glucose deprivation. Then, slices were transfected with hypoxia-inducible factor la or oligodendrocyte lineage gene-1. The expression levels of hypoxia-inducible factor la and oligodendrocyte lineage gene-1 were significantly up-regulated in rat brains prior to transfection, as detected by immunohistochemical staining. Eight hours after transfection of slices with hypoxia-inducible factor la, oligodendrocyte lineage gene-1 expression was upregulated, and reached a peak 24 hours after transfection. Oligodendrocyte lineage gene-1 transfection induced no significant differences in hypoxia-inducible factor la levels in rat brain tissues with oxygen-glucose deprivation. These experimental findings indicate that hypoxia-inducible factor la can regulate oligodendrocyte lineage gene-1 expression in hypoxic brain tissue, thus repairing the neural impairment.

Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage

Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

Seeing the wood for the trees:towards improved quantification of glial cells in central nervous system tissue

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.

Dissecting brain tumor growth and metastasis in vitro and ex vivo

Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system(CNS)tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors.A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes,to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness.These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments.However,the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel,physiologically more relevant assay systems including organotypic brain slice culture.In this review,using publicly available peer-reviewed primary research and review articles,we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors.Furthermore,we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors.We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.