The effect of nicotinamide on gene expression in a traumatic brain injury model

Abstract

Microarray-based transcriptional profiling was used to determine the effect of nicotinamide on gene expression in an experimental traumatic brain injury (TBI) model. Ingenuity Pathway Analysis (IPA) was used to evaluate the effect on relevant functional categories and canonical pathways. At 24 h, 72 h, and 7 days, respectively, 70, 58, and 76%, of the differentially expressed genes were up-regulated in the vehicle treated compared to the sham animals. At 24 h post-TBI, there were 150 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (82%) down-regulated. IPA analysis identified a significant effect of nicotinamide on the functional categories of cellular movement, cell-to-cell-signaling, antigen presentation and cellular compromise, function, and maintenance and cell death. The canonical pathways identified were signaling pathways primarily involved with the inflammatory process. At 72 h post-cortical contusion injury, there were 119 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (90%) was up-regulated. IPA analysis identified a significant effect of nicotinamide on cell signaling pathways involving neurotransmitters, neuropeptides, growth factors, and ion channels with little to no effect on inflammatory pathways. At 7 days post-TBI, there were only five differentially expressed genes with nicotinamide treatment compared to vehicle. Overall, the effect of nicotinamide on counteracting the effect of TBI resulted in significantly decreased number of genes differentially expressed by TBI. In conclusion, the mechanism of the effect of nicotinamide on secondary injury pathways involves effects on inflammatory response, signaling pathways, and cell death.

Keywords: cortical contusion model; gene expression; nicotinamide; traumatic brain injury.

Figure 1

Histology plate. Shown are representative dorsal images of the injury in each group prior to the harvesting of the tissue for analysis. Image bar is 2.0 mm.

Figure 2

The Venn diagrams show the number of genes whose expression was up or down-regulated more than 1.5-fold (p < 0.05) in the NAM/Sham and Vehicle/Sham contrasts at the 24 h, 72 h, and 7-day time points. Venn diagrams were generated with the Bioconductor limma package

Figure 3

The top 10 functional categories and canonical pathways involving significantly regulated genes in the nicotinamide treated compared to vehicle treated CCI injured animals for the 24-h time point. The length of the bar is the negative log of the p-value and is significant if it extends to the right of the threshold line −log(p < 0.05). The ratio number, is defined as the number of significant molecules in the given pathways divided by the total number of molecules that are included in the pathway.

Figure 4

The top 10 functional categories and canonical pathways involving significantly regulated genes in the nicotinamide treated compared to vehicle treated CCI injured animals for the 72-h time point. The length of the bar is the negative log of the p-value and is significant if it extends to the right of the threshold line [−log(p < 0.05)]. The ratio number, is defined as the number of significant molecules in the given pathways divided by the total number of molecules that are included in the pathway.

Figure 5

Cell-to-cell signaling network generated by ingenuity pathway analysis of differentially expressed genes: Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Knowledge Base. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Top: the nicotinamide treated injured animals compared to vehicle treated injured animal. Bottom: the vehicle treated injured animals compared to sham controls highlighting only the genes affected by nicotinamide treatment shown in the top figure.

Figure 6

TaqMan based RT-PCR validation of the microarray data for the selected genes: Cxc12, chemokine (C-X-C motif) ligand 2; Hmox1, heme oxygenase 1; Hspa1b, heat shock protein 1b; Il1b, interleukin 1beta; Il1rn, interleukin 1 receptor antagonist; Il8rb, interleukin 8 receptor beta; Mmp8, matrix metallopeptidase 8; Mmp9, matrix metallopeptidase 9; Niacr1, niacin receptor 1; Nrsn1, neurensin 1; Ptgs2, prostaglandin-endoperoxide synthase 2; S100a9, S100 calcium binding protein A9; Tnfrsf1b, tumor necrosis factor receptor superfamily, member 1b. The RT-PCR data was normalized to the housekeeping gene β-actin. In order to compare the microarray data to the RT-PCR data, the microarray data for each gene was divided by the beta-actin data as it was measured by the microarray analysis. The gray bars show the microarray data and the black bars display the RT-PCR data for the contrast. (A,B) Show the data from the 24 and 72-h time point for the contrasts nicotinamide treated injured animals compared to the vehicle treated injured animals.