Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response

Abstract

Background: Traumatic brain injury (TBI) results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to secondary neural injury in the surrounding tissue. We used microarray analysis to determine which genes, pathways and networks were significantly altered using a rat model of TBI. Adult rats received a unilateral controlled cortical impact (CCI) and were sacrificed 24 h post-injury. The ipsilateral hemi-brain tissue at the site of the injury, the corresponding contralateral hemi-brain tissue, and naïve (control) brain tissue were used for microarray analysis. Ingenuity Pathway Analysis (IPA) software was used to identify molecular pathways and networks that were associated with the altered gene expression in brain tissues following TBI.

Results: Inspection of the top fifteen biological functions in IPA associated with TBI in the ipsilateral tissues revealed that all had an inflammatory component. IPA analysis also indicated that inflammatory genes were altered on the contralateral side, but many of the genes were inversely expressed compared to the ipsilateral side. The contralateral gene expression pattern suggests a remote anti-inflammatory molecular response. We created a network of the inversely expressed common (i.e., same gene changed on both sides of the brain) inflammatory response (IR) genes and those IR genes included in pathways and networks identified by IPA that changed on only one side. We ranked the genes by the number of direct connections each had in the network, creating a gene interaction hierarchy (GIH). Two well characterized signaling pathways, toll-like receptor/NF-kappaB signaling and JAK/STAT signaling, were prominent in our GIH.

Conclusions: Bioinformatic analysis of microarray data following TBI identified key molecular pathways and networks associated with neural injury following TBI. The GIH created here provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our findings can also provide a context for evaluating the potential of therapeutic agents currently in development.

Figure 1

Principal component and functional analyses. (A) PCA was applied to all microarray datasets and the resulting scores for the first 3 principal components were plotted. The first 3 principal components explain 91.267% of the variance in the data. This analysis revealed clustering of datasets by injury status: Ipsilateral (Ipsi), Contralateral (Contra), and Naïve. (B) Analysis of the top 15 biological functions determined by IPA for the TBI-I (Ipsilateral vs Naïve) dataset demonstrates that the inflammatory response is one of the primary functions of the genes expressed after TBI. Additionally, all of the functions IPA found to be more significant than inflammatory response also have an inflammatory component (see text).

Figure 2

Cortical histology following TBI. Damage to the ipsilateral cortex is demonstrated by FJB staining (A). FJB staining is absent in the contralateral cortex (B), consistent with the lack of trauma. The damaged cortex (C) contains many macrophages and activated microglia as demonstrated by ED-1 immunostaining (D). There were no activated macrophages in the contralateral cortex (E) and the brain anatomy appears intact (F). FJB: green; DAPI: blue; ED-1: red; Scale bars: 250 μm (A,B), 125 μm (C-F).

Figure 3

Hippocampal region histology following TBI. Damage to the ipsilateral hippocampal region was demonstrated by FJB staining (A). FJB staining was absent in the contralateral hippocampal region (B). While the integrity of the hippocampus appears intact ipsilaterally (C), many activated microglia and macrophages were present as demonstrated by ED-1 immunostaining (D). There were no activated macrophages in the contralateral hippocampal region (E) and the anatomical structure appears intact (F). FJB: green; DAPI: blue; ED-1: red. Scale bars: 250 μm (A,B), 125 μm (C-F).

Figure 4

Microglial activation in the injured brain. CD11b immunostaining demonstrated the ramified resting morphology of microglia on the uninjured, contralateral side of the brain (A &B). This same morphology was seen on the ipsilateral side of the brain in areas less affected by the trauma (C). In damaged brain regions, the microglia were activated and underwent a morphological change, becoming amoeboid in shape (D). Both morphologies can be seen in the hippocampal region where there are amoeboid microglia in the areas of damage and ramified microglia in the surround (E). The same section was counterstained with DAPI to demonstrate the overall cellular anatomy of the region (F). CD11b: green; DAPI: blue. Scale bars: 200 μm (A,B), 100 μm (C-F).

Figure 5

Breakdown of IR genes based on up- and downregulation in expression. (A) There were 146 genes that change more than 2-fold on both sides of the brain. Seventy-five percent of them (109 genes) changed similarly while the remaining 25% (37 genes) changed differently (ratio >1.75; see text). (B) 188 IR genes changed uniquely on the ipsilateral side of the brain and 95% (179 genes) of those increased in expression. (C) 38 IR genes changed uniquely on the contralateral side of the brain and 74% (28 genes) of those decreased in expression.

Figure 6

Canonical pathway analysis. The IL-6 signaling pathway showing the relative expression values for all TBI-I IR genes (A) and all TBI-C IR genes (B) involved in this pathway. red: relative increase in expression; green: relative decrease in expression; white: no change in expression.

Figure 7

Analysis of the highest scored IPA generated gene network. This was the highest scored gene network associated with the TBI-I/TBI-C union dataset. The relative expression values of the unique TBI-I IR genes (A) and unique TBI-C IR genes (B) included in this network are shown. red: relative increase in expression; green: relative decrease in expression; white: no change in expression; orange connections and outlines: direct connections with gene groups and complexes in the original network.

Figure 8

Analysis of a highly scored IPA generated gene network. This gene network was scored in the top three networks associated with the TBI-I/TBI-C union dataset. The relative expression values of all TBI-I IR genes (A) and all TBI-C IR genes (B) included in this network are shown. red: relative increase in expression; green: relative decrease in expression; white: no change in expression; orange connections and outlines: direct connections with gene groups and complexes in the original network.

Figure 9

Cytokine and growth factor network analysis. The gene network was created in IPA by seeding with the IR cytokines and growth factors expressed uniquely in the TBI-I analysis and “growing” those genes into a network by showing their direct connections with genes in the union of all TBI-I and TBI-C IR genes dataset. The relative expression values of all TBI-I IR genes (A) and all TBI-C IR genes (B) included in this network are shown. red: relative increase in expression; green: relative decrease in expression; white: no change in expression.