Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles

Abstract

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.

Keywords: biomarkers; extracellular vesicles (EVs); real-time PCR; traumatic brain injury (TBI).

Figure 1

Transmission electron microscope and western blot images and NanoSight images. (a) Representative transmission electron microscopy of EVs isolated from saliva. EVs were viewed by JEOL Jem 1010 electron microscope (original magnification ×100,000; inset original magnification ×150,000; black lines = 100 nm). (b) Representative western blot analysis of CD63 from saliva EVs. (MW = standard molecular weight markers). EVs: extracellular vesicles

Figure 2

Transmission electron microscope and western blot images and NanoSight images. (a) Salivary EVs size distribution in nanometer (nm) of each control (n = 7), concussion clinic patients (CCPT) (n = 8), and ED head trauma patients (EDPT) (n = 13). (b) EVs concentration for each patient by NanoSight analysis showing the number of EVs per milliliter of saliva derived from controls (n = 7), CCPT (n = 8), and EDPT (n = 13). (c) Mean salivary EVs size with standard deviation by NanoSight analysis showing the mode size of EVs in 1 ml of saliva derived from controls (n = 7), CCPT (n = 8), and EDPT (n = 13). (d) Mean EVs concentration with standard deviation by NanoSight analysis showing the mode size of EVs in 1 ml of saliva derived from controls (n = 7), CCPT (n = 8), and EDPT (n = 13). ED: emergency department; EVs: extracellular vesicles [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Gene expression information of ED patients. (a) Number of genes upregulated and downregulated. Upregulated gene expression in three tiers: fold increase 50‐fold higher than controls in red, 20‐fold higher in yellow, and fold change of two in green. Downregulated gene expression <0.5 in blue. (b) Genes with a two‐fold increase in gene expression or higher. Error bars represent standard deviation. ED: emergency department [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Gene expression information of concussion clinic patients. (a) Number of genes upregulated and downregulated. Upregulated genes are shown in three tiers: fold increase 50‐fold higher than controls in red, 20‐fold higher in yellow, and a fold change of two in green. Downregulated gene expression <0.5 shown in blue. (b) All the genes that had a two‐fold increase in expression or higher. Error bars represent standard deviation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Upregulated Genes in experimental groups. Wilcoxon analysis was done comparing delta CT of EDPT and CCPT. Twenty‐three Alzheimer's disease genes significantly (p < 0.05) changed in EDPT (n = 15) compared with CCPT (n = 16). (a) Fold change of significant genes of each patient is compared. (b) Three genes found in both patient groups. No statistical difference of three genes between EDPT and CCPT (p > 0.05) [Color figure can be viewed at wileyonlinelibrary.com]