Differential expression of microRNAs in the brains of mice subjected to increasing grade of mild traumatic brain injury

<p><i>Objective</i>: To investigate the effect of heterogeneity in mTBI on miRNA expression in mouse brain and to identify molecular pathways targeted by the modulated miRNAs.</p> <p><i>Methods</i>: A weight drop device was used to induce four increasing grades of mTBI. MiRNA expression was evaluated using TaqMan rodent miRNA arrays. Bioinformatics analysis was done using the DIANA miRPath tool and Ingenuity Pathway Analysis software. Histology of brain sections was evaluated using H&E staining.</p> <p><i>Results</i>: No histologic lesions were observed in the brains of injured mice; however, significant modulation in miRNA expression profile was observed. Global miRNA profiling indicated a trend of decrease in the number of modulated miRNAs from 24 hours to day 7 post-injury, except for the most severe grade of mTBI. Canonical pathways like calcium signalling, synaptic pathways and axon guidance pathway were the major targets of the modulated miRNAs. Network correlation analyses indicated an interaction between the modulated miRNAs and putative protein biomarkers of TBI.</p> <p><i>Conclusions</i>: The data demonstrated that varying intensities of mTBI induced a differential miRNA expression profile in the brain post-injury. Pathways such as calcium and synaptic signalling were major targets of modulated miRNAs and may play a role in the pathophysiology of mTBI.</p

Identification of Serum MicroRNA Signatures for Diagnosis of Mild Traumatic Brain Injury in a Closed Head Injury Model

<div><p>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.</p></div

miRNAs in kidney of mice 24 h after CI.

<p>miRNAs in kidney of mice 24 h after CI.</p

Involvement of significantly modulated miRNA in axon guidance pathway.

<p>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.</p

Expression of miRNAs in individual real time PCR assay.

<p>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; *<i>P</i><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.</p

CI enhances bone marrow cell loss and whole caspase-3 increase.

<p><b>(A)</b> Bone marrow cells from femur were counted. <b>(B)</b> Bone marrow cell lysates on day 1 were immunoblotted for detecting whole caspase-3 protein (N = 6 per group at each time point). <b>(C)</b> Bone marrow cell lysates on day 1 were assayed for measuring activated caspase-3 protein (N = 6 per group at each time point). All values are means ± SEM. *p<0.05 vs. sham and Hemo; **p<0.05 vs. respective RI time point, determined by 2-way ANOVA and Bonferroni’s inequality for panel A and Student’s <i>t</i>-test for panels B and C. Sham: free of any injury; Hemo: 20% bleeding; RI: radiation injury; CI: RI+Hemo.</p

Significantly modulated miRNA in IS3.

<p>One hundred and sixteen miRNA were significantly modulated. Of these, 70 were up regulated and 46 were down regulated. Values are given as Log10 of the fold change (2^{−(mean ΔΔCt)}).</p><p>Significantly modulated miRNA in IS3.</p

CI alters the miRNAs in serum.

<p>Ingenuity Pathway Analysis filtered experimentally validated and direct gene targets of cytokines/chemokine in serum after CI.</p

Alteration of commonly present miRNAs in ileum after Hemo, RI, and CI.

<p>Hemo: hemorrhage; RI: 8.75 Gy; CI: 8.75 Gy+hemorrha.</p

CI alters the miRNAs.

<p>Ingenuity Pathway Analysis filtered experimentally validated and direct gene targets of <b>(A)</b> NF-κB, <b>(B)</b> HIF-1, and <b>(C)</b> EPO signaling.</p