Bilateral gene interaction hierarchy analysis of the cell death gene response emphasizes the significance of cell cycle genes following unilateral traumatic brain injury

Abstract

Background: Delayed or secondary cell death that is caused by a cascade of cellular and molecular processes initiated by traumatic brain injury (TBI) may be reduced or prevented if an effective neuroprotective strategy is employed. Microarray and subsequent bioinformatic analyses were used to determine which genes, pathways and networks were significantly altered 24 h after unilateral TBI in the rat. Ipsilateral hemi-brain, the corresponding contralateral hemi-brain, and naïve (control) brain tissue were used for microarray analysis.

Results: Ingenuity Pathway Analysis showed cell death and survival (CD) to be a top molecular and cellular function associated with TBI on both sides of the brain. One major finding was that the overall gene expression pattern suggested an increase in CD genes in ipsilateral brain tissue and suppression of CD genes contralateral to the injury which may indicate an endogenous protective mechanism. We created networks of genes of interest (GOI) and ranked the genes by the number of direct connections each had in the GOI networks, creating gene interaction hierarchies (GIHs). Cell cycle was determined from the resultant GIHs to be a significant molecular and cellular function in post-TBI CD gene response.

Conclusions: Cell cycle and apoptosis signalling genes that were highly ranked in the GIHs and exhibited either the inverse ipsilateral/contralateral expression pattern or contralateral suppression were identified and included STAT3, CCND1, CCND2, and BAX. Additional exploration into the remote suppression of CD genes may provide insight into neuroprotective mechanisms that could be used to develop therapies to prevent cell death following TBI.

Fig. 1

Overall functional analysis. Analysis of the top 15 molecular and cellular functions determined by IPA for the TBI-I (ipsilateral vs. naïve) dataset (a) and the TBI-C (contralateral vs. naïve) dataset (b) showed that cell death and survival was a top ranked function on both sides of the brain

Fig. 2

Fluoro-Jade® B staining of the cortex and hippocampus. Fluoro-Jade® B (FJB) staining showed a dense distribution of damaged neurons throughout all layers of the cortex near the sight of impact (a, b). Damaged neurons were also detected in the hippocampus ipsilateral to the injury (d). These neurons were sparsely distributed in the hippocampal CA regions. No FJB staining was detected in either brain region contralateral to the injury (C: cortex; E: hippocampus). FJB: green; Scale bars: 200 μm ( a , c - e ), 100 μm ( b )

Fig. 3

TUNEL staining of the cortex and hippocampus. TUNEL staining showed distribution of injured cells in the cortex similar to FJB as they were distributed throughout all layers of the cortex (a, b). However, no TUNEL staining was detected in the ipsilateral hippocampus (d). No TUNEL was observed on the contralateral side of the brain (C: cortex; E: hippocampus). TUNEL: red; Scale bars: 200 μm ( a , c - e ), 100 μm ( b )

Fig. 4

Breakdown of CD genes based on increased and decreased expression. a 361 CD genes changed uniquely on the ipsilateral side of the brain and 88 % (317 genes) of those increased in expression. b 136 CD genes changed uniquely on the contralateral side of the brain and 75 % (102 genes) of those decreased in expression. c There were 405 genes that changed more than 2-fold on both sides of the brain. Eighty-two percent of them (332 genes) changed similarly while the remaining 18 % (73 genes) changed differently (TBI-I/TBI-C ratio >2; see text)

Fig. 5

Canonical pathway analysis. The apoptosis signaling pathway with all gene families, groups and complexes expanded to show the member genes and showing the relative expression values of potential GOI for TBI-I (a) and TBI-C (b) included in this pathway. red: relative increase in expression; green: relative decrease in expression; white: no change in expression; gold connections and outlines: expansion of gene families, groups and complexes in the original pathway

Fig. 6

Examples of TBI-I networks. TBI-I CD networks 2 (a) and 4 (b) (see Table 2) with all gene families, groups and complexes expanded to show the member genes and showing the relative expression values of potential GOI for TBI-I. red: relative increase in expression; green: relative decrease in expression; white: no change in expression; gold connections and outlines: expansion of gene families, groups and complexes in the original network

Fig. 7

Examples of TBI-C networks. TBI-C CD networks 2 (a) and 4 (b) (see Table 3) with all gene families, groups and complexes expanded to show the member genes and showing the relative expression values of potential GOI for TBI-C. red: relative increase in expression; green: relative decrease in expression; white: no change in expression; gold connections and outlines: expansion of gene families, groups and complexes in the original network

Fig. 8

An example of calculating the number of direct connections for the TBI-I GOI network. In IPA, the gene in question was selected (MDM2 in this example). Then, its direct connections were selected by right clicking on MDM2 and using the “select nearest neighbors” option (highlighted in purple). A list of the selected genes was exported and MDM2 was removed from the list (upper right corner). The remaining genes were counted (26 in this example) and MDM2 was ranked in the TBI-I gene interaction hierarchy (primary tier) by this number

Fig. 9

An example of calculating the number of direct connections for the TBI-C GOI network. In IPA, the gene in question was selected (SOX2 in this example). Then, its direct connections were selected by right clicking on SOX2 and using the “select nearest neighbors” option (highlighted in blue). A list of the selected genes was exported and SOX2 was removed from the list (upper right corner). The remaining genes were counted (13 in this example) and SOX2 was ranked in the TBI-C gene interaction hierarchy (primary tier) by this number

Fig. 10

Functional analysis of GOI and top 2 GIH tiers. The top 10 molecular and cellular functions determined by IPA to be associated with the unranked GOI for TBI-I (a) and TBI-C (c) and the primary and secondary tiers of the TBI-I (b) and TBI-C (d) GIHs. Side by side comparison allowed for visualization of how functions changed in significance order once the genes were put into a ranked order. Notably, cell cycle moved up to be ranked second on both sides of the brain. The cell death and survival category was removed from this analysis because all genes were initially selected from that functional category

Fig. 11

Real-time PCR results for selected genes. SPP1, HSPB1, STAT3, and CCND1 were chosen for real-time PCR studies. Using the ΔΔCt method, the normalized target gene expression level was given by 2^-ΔΔCt. For all genes, ipsilateral (IPSI) expression was significantly different from naïve (a-d). Ipsilateral expression was also significantly different from contralateral (CONTRA) expression for SPP1 (a) and HSPB1 (b). The comparison of ipsilateral to contralateral expression for STAT3 (c; p = 0.088) and CCND1 (d; p = 0.063) fell short of statistical significance. Contralateral expression was not significantly different from naïve for any genes. The results are shown as mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.005