Identification of serum microRNA signatures for diagnosis of mild traumatic brain injury in a closed head injury model

Abstract

Wars in Iraq and Afghanistan have highlighted the problems of diagnosis and treatment of mild traumatic brain injury (mTBI). MTBI is a heterogeneous injury that may lead to the development of neurological and behavioral disorders. In the absence of specific diagnostic markers, mTBI is often unnoticed or misdiagnosed. In this study, mice were induced with increasing levels of mTBI and microRNA (miRNA) changes in the serum were determined. MTBI was induced by varying weight and fall height of the impactor rod resulting in four different severity grades of the mTBI. Injuries were characterized as mild by assessing with the neurobehavioral severity scale-revised (NSS-R) at day 1 post injury. Open field locomotion and acoustic startle response showed behavioral and sensory motor deficits in 3 of the 4 injury groups at day 1 post injury. All of the animals recovered after day 1 with no significant neurobehavioral alteration by day 30 post injury. Serum microRNA (miRNA) profiles clearly differentiated injured from uninjured animals. Overall, the number of miRNAs that were significantly modulated in injured animals over the sham controls increased with the severity of the injury. Thirteen miRNAs were found to identify mTBI regardless of its severity within the mild spectrum of injury. Bioinformatics analyses revealed that the more severe brain injuries were associated with a greater number of miRNAs involved in brain related functions. The evaluation of serum miRNA may help to identify the severity of brain injury and the risk of developing adverse effects after TBI.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. H&E stained sections of the brain.

Histological evaluation of the brain tissue at the site of injury (arrow) was done to check for the lesion in the brain tissue following mTBI. No significant difference was observed in brain tissue sections between the sham (A), IS1 (B), IS2 (C), IS3 (D) and IS4 (E). Scale is 1 mm.

Figure 2. NSS-R for animals at day 1 post injury.

Change scores between day 1 post injury and baseline were calculated. A gradual, but significant increase in the NSS-R scores were observed day 1 post injury with the increased severity of the injury within the mild spectrum. As expected, naïve and sham groups did not show any increase in their NSS-R scores. No change was observed in the IS1 group. NSS-R score of IS2, IS3, and IS4 groups increased and was the highest among the IS4 group. * P value <0.05.

Figure 3. Neurobehavioral Activity.

OFL test was conducted to evaluate animals' various activities in an open field as a measurement of behavioral deficits. Overall, day 1 showed significant reductions in the activities of injured animals as compared to the naïve and sham controls except for the IS1 group, which was not significantly different from that of naïve and sham groups. (A) Horizontal activity was evaluated as an indicator of the overall health. Activity was significantly reduced in the IS3 and IS4 groups. Activity in the IS2 group was significantly reduced as compared to the activity of IS1. Activity of IS1 and IS2 groups were not significantly different from the sham group. (B) Time spent in the center of the open field was evaluated as an indicator of anxiety-like behavior. Center time of the animals in IS3 and IS4 groups was reduced as compared to the 246 g injury groups (i.e., IS1 and IS2), sham and naïve animals. The reduction in activity reached significance in the IS3. (C) Vertical activity was measured as an indicator of depression-related behavior. Vertical activity of the injured animals was significantly less than that of the sham and naïve animals except the IS1 group. * P value <0.05. (D) Startle response was measured as an indicator of emotional distress and potential sensory gating impairments. Startle responses of all injured groups, except the IS1 group, were reduced when compared to naïve and sham groups at day 1 post injury. There were no differences between groups at days 14 and 30 post injury, except in the IS4 group where the startle response was significantly higher than all other groups at day 14 post injury. Values are expressed as Mean ± SEM. * P value <0.05.

Figure 4. MiRNA expression pattern.

(A) MiRNAs that show Ct<36 were considered as expressed. The number of miRNAs expressed ranged from 315.83−362.17 with a maximum and minimum numbers detected in IS4 and IS1 respectively. The SD ranged from 19.28−43.37, the maximum being in IS2 and the minimum in IS1. The values are presented as Mean ± SD of the number of miRNAs detected in each group. (B) The number of miRNAs that were significantly modulated (≥1.5 fold; P<0.05) following the injury over the time point matched sham controls increased with the height and weight of the free falling metal rod except in the IS3 group where the number of significantly modulated miRNAs was marginally greater than the IS4 group. (C) Overlapping miRNA data analysis for significantly modulated miRNAs in the injury groups was done using the online Venn diagram generation tool [Oliveros (2007). VENNY. An interactive tool for comparing lists with Venn Diagrams. http://bioinfogp.cnb.csic.es/tools/venny/index.html].

Figure 5. Involvement of significantly modulated miRNA in axon guidance pathway.

Quantitative bioinformatics analysis on the number of miRNA involved in the axon guidance pathway was done using IPA. The number of the significantly modulated miRNAs that were predicted to modulate axon guidance pathway increased with the severity of the injury.

Figure 6. IPA Analysis for effect of significantly modulated miRNAs common among all four injury groups on brain functions related pathways.

MiRNAs common to all four injury groups were taken and their targets and pathway analysis in relation to brain related functions were performed. My Pathway tool of IPA software was used to build the custom pathway using miRNAs that show experimentally validated targets by overlaying with 1) depression, 2) sensorimotor function and the 3) axon guidance pathway molecules. Of the 13 common modulated miRNAs, only 6 miRNAs were found to have experimentally validated targets that are predicted to modulate axon guidance, synaptic depression and sensorimotor impairments.

Figure 7. Expression of miRNAs in individual real time PCR assay.

The fold upregulation of three miRNAs, miR-376a, miR-214 and miR-199a-3p, in the injury groups over the sham mice was validated using the individual real time PCR assays. Similar to the miRNA arrays, expression of the three miRNAs was found to be up regulated in the injured groups over the sham animals. Data presented is the fold up regulation (± SEM; *P<0.05) calculated from the mean DDCt value obtained from three biological samples for each injury group. Statistical significance was calculated using the individual Ct values obtained from the three biological replicates for each injury group.