Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression in response to axotomy in organotypical slices co-cultures

ABSTRACT: Brain slice cultures offer advantages over other in vitro methods, as they mimic numerous in vivo aspects. For most purposes, slices of the developing brain, termed organotypic slice cultures , preserve a high degree of cellular differentiation and tissue organization. The entorhino-hippocampal connection (EHP) is the main entrance of information to the hippocampus proper and the dentate gyrus. Also it has some specific features that make them particularly interesting in studies of axonal regeneration: (i) the culture method obviates the need for extensive animal surgery and requires less time than other in vivo approaches; (ii) the EHP is reproduced easily in vitro in cultures with a degree of laminar specificity similar to that found in vivo; (iii) the EHP is myelinated both in vitro and in vivo; and (iv) most of the cellular and molecular barriers to axon regeneration are present after the axotomy of the EHP in vitro. Altogether this model is useful to evaluate axon regeneration and putative estrategies to promote axonal growth. Keywords: organotypic slice cultures; axonal lession; gene expression To evaluate the genes whose transcription was regulated after 1, 3 and 7 days after EHP (Entorhino-Hippocampal Pathway) axotomy, RNA samples were analyzed with Agilent whole genome rat long oligonucleotide (44 K base) probe based microarrays.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Lauro Sumoy

PROVIDER: E-GEOD-20528 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The neurite outgrowth inhibitory myelin protein Nogo-A has been well studied in the context of central nervous system (CNS) injury and disease. We studied the effects of the application of neutralizing anti-Nogo-A antibodies (11C7 and 7B12) in intact CNS tissue in vitro using rat organotypic hippocampal slice cultures. This study had the purpose of elucidating the role of Nogo-A in the adult intact CNS and determining the consequences of its neutralization through antibody application. In vitro cultures treated with anti-Nogo-A antibody showed an elicited growth response. The results also gave indications that hippocampal circuitry might be altered due to the regulation at the synaptic and neurotransmission level. Experiment Overall Design: Nogo-A function in the intact CNS tissue is not well known, but its neutralization in vivo produced a transitory growth response of Purkinje axons and of the corticospinal tract in intact adult rats (Buffo et al., 2000; Bareyre et al., 2002; Gianola et al., 2003). Nogo-A is relatively highly expressed in oligodendrocytes and some neurons of the hippocampus (Huber et al., 2002; Meier et al., 2003; Gil et al., 2006; Trifunovski et al., 2006). Organotypic hippocampal slice cultures are a good in vitro model to study hippocampal function and structure (Stoppini et al., 1991; 1993; Bahr, 1995; Gahwiler et al., 1997; Hakkoum et al., 2006). They mature in vitro and retain many in vivo features from a structural and functional perspective. We chose this model to study the effects of acute Nogo-A neutralization, using two function blocking monoclonal antibodies, 11C7 and 7B12 (Oertle et al., 2003; Wiessner et al., 2003; Liebscher et al., 2005), exclusively targeted against the Nogo-A specific region. Hippocampal slices from P7 Wistar rats were cultured for 21 DIV. Control untreated cultures where cultured for additional 5days for a total of 26DIV, while control IgG, and 11C7 and 7B12 were added to 21DIV cultures which were then further cultured for 5days, changing medium every 2 days. All the conditions were repeated in triplicates with separate cultures from different animals. For each condition and experimental replicate 24 cultures were pooled together before being processed for RNA extraction. Data analysis was performed by GeneSpring 7.2 (Silicon Genetics, Agilent, CA, US) comparing 11C7 and 7B12 treated samples versus Not treated and IgG treated, as controls. A present call filter (2 out of 3 present calls in at least one out of the 3 experimental replicates) was applied. Normalization was run per chip as well as per gene to the median of the control replicates. Data were statistical restricted through a 1-way Anova (pâ?¤0.05). A final threshold of â?¥1.2 fold of increase or decrease in the expression level of each single transcript was applied. Regulated transcripts have been assigned to functional categories according to GeneOntology as well as literature and database mining (Pubmed; Bioinformatics Harvester EMBL Heidelberg; Rat Genome Database).

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Gene expression in response to axotomy in organotypical slices co-cultures

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