ReviewQuantitative proteomics analysis to identify diffuse axonal injury biomarkers in rats using iTRAQ coupled LC–MS/MS
Graphical abstract
Introduction
Diffuse axonal injury (DAI), which is predominantly caused by vehicular accidents or other traumas, accounts for up to 70% of all traumatic brain injury (TBI) cases [1] and is associated with high mortality [2]. DAI was initially described as diffuse degeneration of cerebral white matter by Strich, but now the definition of widespread damage of axons in the white matter of the brain is generally accepted [3]. Rotational accelerations are seen as the main contributor to DAI in a traumatic brain injury [2]. Microscopically DAI is characterized by various degrees of derangement of axons and the form of axonal retraction balls. Axonal retraction balls, which are formed in 12–24 h after the injury, are the most striking feature of morphologic changes to axons [4]. It has long been assumed that the direct shear or tensile forces generated by the injury physically disrupt the axons leading to the disconnection of the axons and finally, the form of axonal retraction balls, but recent study showed that the subsequent biochemical cascades post-injury play an important role in the processes of axonal damage [5]. While many studies have examined the pathophysiological mechanisms of DAI, it is still difficult for clinicians to make an early diagnosis, determine appropriate therapeutic interventions, or provide accurate prognostic evaluations. These difficulties are in part attributed to the microscopic and disseminated nature of axons, making detection via imaging very difficult, and the fact that patients show no specific neurological symptoms and can rapidly fall unconscious [6]. Therefore, a need to elucidate the mechanisms leading to axonal injury after a head trauma and identifying reliable biomarkers is necessary to enable early DAI diagnosis.
Currently, trauma severity and potential DAI are assessed using magnetic resonance imaging (MRI) and computed tomography (CT). While traditional MRIs are significantly more sensitive than CT scans for detecting TBI associated damage, particularly nonhemorrhagic damage [7], [8], histological studies have demonstrated that axonal injuries can still be present with normal MRI results [9], [10]. However, histopathological studies cannot identify DAI in patients that die prior to the formation of axonal retraction balls [11]. Therefore, gaining an understanding of the molecular mechanisms driving DAI and identifying differentially expressed proteins associated with axonal injury may facilitate improved DAI diagnoses and clinical management. Cerebral tissue was chosen in our proteomic analysis work for the possibility that it may elucidate more of the mechanisms of axonal injury in DAI than cerebrospinal fluid or blood.
In contrast to genomics and transcriptomics, proteomic analysis can provide insight into the signal transduction events that directly impact the biochemical processes of life. Modern proteomic platforms offer the advantages of high-throughput, simultaneous detection under a single condition and a reduced time and cost. Furthermore, these platforms have already been successfully applied to identify biomarkers associated with many diseases such as colorectal cancer [12], epileptogenesis [13], and oligodendroglioma [14]. Of these approaches, this study utilized tandem mass spectrometry (MS/MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling to enable the identification and quantification of differentially expressed proteins in a DAI rat model. Bioinformatic analysis was employed to identify differently expressed proteins associated with the mechanisms of axonal injury after head trauma. Overall, these findings will further an understanding of the pathophysiological mechanisms underlying the substantial mortality and specific symptoms in patients with DAI. Furthermore, the identified differentially expressed proteins may serve as reliable DAI biomarkers and aid in early diagnosis and treatment.
Section snippets
Ethics statement
The procedures for this study were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Care and Use Committee of Chong Qing Medical University.
Animals
A total of 90 adult male Sprague–Dawley rats (350–375 g, housed under 12-h light and dark conditions with food and water available ad libitum) were used in this study. All animals were provided by the C.Q. Medical University Laboratory Animal Center.
Experimental model
DAI was induced in animals using an
Identification of differentially expressed proteins via iTRAQ coupled LC–MS/MS
DAI animal models were first histopathological validated (Fig. 1) and three cerebral tissue samples from each subgroup were randomly selected for iTRAQ coupled MS/MS as a means to identify candidate proteins associated with the pathophysiological mechanisms of axonal injury. A total of 1858 proteins were identified and quantified overall, with GO analysis groupings including biological processes (BP), cellular component association (CC), and molecular function (MF; Fig. 2A–C). Proteins with
Discussion
TBI biomarkers from proteomic studies can provide clinicians with a powerful tool for early diagnosis, early assessment of severity, and accurate prognostic evaluation of patients with brain injury [20], [21], [22], [23], [24], especially for mild TBI (mTBI) which often shows no visible focal lesions detected by routine imaging techniques [25]. To date, some proteins have been identified as candidate TBI biomarkers, such as ubiquitin carboxy-terminal hydrolase L1 protein (UCHL1) [26],
Conflict of interest
The authors have declared that no competing interests exist.
Acknowledgments
This work was funded by the National Natural Science Foundation of China (81273344), the Open Project of Shanghai Key Laboratory of Forensic Medicine (KF1105) and Application Development Plan Project of Chongqing (cstc2014yykfA110003).
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