Georgene W. Hergenroeder

Human Traumatic Brain Injury Alters Plasma microRNA Levels

Circulating microRNAs (miRNAs) present in the serum/plasma are characteristically altered in many pathological conditions, and have been employed as diagnostic markers for specific diseases. We examined if plasma miRNA levels are altered in patients with traumatic brain injury (TBI) relative to matched healthy volunteers, and explored their potential for use as diagnostic TBI biomarkers. The plasma miRNA profiles from severe TBI patients (Glasgow Coma Scale [GCS] score ≤8) and age-, gender-, and race-matched healthy volunteers were compared by microarray analysis. Of the 108 miRNAs identified in healthy volunteer plasma, 52 were altered after severe TBI, including 33 with decreased and 19 with increased relative abundance. An additional 8 miRNAs were detected only in the TBI plasma. We used quantitative RT-PCR to determine if plasma miRNAs could identify TBI patients within the first 24 h post-injury. Receiver operating characteristic curve analysis indicated that miR-16, miR-92a, and miR-765 were good markers of severe TBI (0.89, 0.82, and 0.86 AUC values, respectively). Multiple logistic regression analysis revealed that combining these miRNAs markedly increased diagnostic accuracy (100% specificity and 100% sensitivity), compared to either healthy volunteers or orthopedic injury patients. In mild TBI patients (GCS score > 12), miR-765 levels were unchanged, while the plasma levels of miR-92a and miR-16 were significantly increased within the first 24 h of injury compared to healthy volunteers, and had AUC values of 0.78 and 0.82, respectively. Our results demonstrate that circulating miRNA levels are altered after TBI, providing a rich new source of potential molecular biomarkers. Plasma-derived miRNA biomarkers, used in combination with established clinical practices such as imaging, neurocognitive, and motor examinations, have the potential to improve TBI patient classification and possibly management.

Biomarkers for the diagnosis, prognosis, and evaluation of treatment efficacy for traumatic brain injury

SummaryTraumatic brain injury (TBI) remains a serious health concern, and TBI is one of the leading causes of death and disability, especially among young adults. Although preventive education, increased usage of safety devices, and TBI management have dramatically increased the potential for surviving a brain injury, there is still a need to develop reliable methods to diagnose TBI, the secondary pathologies associated with TBI, and predicting the outcomes of TBI. Biomarkers (changes of amount or activity in a biomolecule that reflect injury or disease) have shown promise in the diagnosis of several conditions, including cancer, heart failure, infection, and genetic disorders. A variety of proteins, small molecules, and lipid products have been proposed as potential biomarkers of brain damage from TBI. Although some of these changes have been reported to correlate with mortality and outcome, further research is required to identify prognostic biomarkers. This need is punctuated in mild injuries that cannot be readily detected using current techniques, as well as in defining patient risk for developing TBI-associated secondary injuries.

Biomarkers for the Diagnosis and Prognosis of Mild Traumatic Brain Injury/Concussion

Mild traumatic brain injury (mTBI) results from a transfer of mechanical energy into the brain from traumatic events such as rapid acceleration/deceleration, a direct impact to the head, or an explosive blast. Transfer of energy into the brain can cause structural, physiological, and/or functional changes in the brain that may yield neurological, cognitive, and behavioral symptoms that can be long-lasting. Because mTBI can cause these symptoms in the absence of positive neuroimaging findings, its diagnosis can be subjective and often is based on self-reported neurological symptoms. Further, proper diagnosis can be influenced by the motivation to conceal or embellish signs and/or an inability of the patient to notice subtle dysfunctions or alterations of consciousness. Therefore, appropriate diagnosis of mTBI would benefit from objective indicators of injury. Concussion and mTBI are often used interchangeably, with concussion being primarily used in sport medicine, whereas mTBI is used in reference to traumatic injury. This review provides a critical assessment of the status of current biomarkers for the diagnosis of human mTBI. We review the status of biomarkers that have been tested in TBI patients with injuries classified as mild, and introduce a new concept for the discovery of biomarkers (termed symptophenotypes) to predict common and unique symptoms of concussion. Finally, we discuss the need for biomarker/biomarker signatures that can detect mTBI in the context of polytrauma, and to assess the consequences of repeated injury on the development of secondary injury syndrome, prolongation of post-concussion symptoms, and chronic traumatic encephalopathy.

Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure.

Brain injury biomarkers may have clinical utility in stratifying injury severity level, predicting adverse secondary events or outcomes, and monitoring the effectiveness of therapeutic interventions. As a biomarker source, serum offers several advantages over cerebrospinal fluid (CSF), including ease of accessibility and reduced risk to the patient. We screened pooled serum samples obtained from 11 severely injured traumatic brain injury (TBI) patients (Glasgow Coma Scale [GCS] <or= 8) and 11 age-, sex- and race-matched volunteers. Two time points-41.5 +/- 4.9 h and 66.3 +/- 6.6 h post-injury-were chosen for the initial screening analysis. Samples were immunodepleted for 12 highly abundant serum proteins, and then labeled with mass-balanced isobaric tags (iTRAQ), and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identification and quantification of 2455 iTRAQ-labeled peptides that mapped to 160 proteins revealed 31 candidate biomarkers whose serum abundance was altered after injury. Changes in three candidate biomarkers (serum amyloid A, [SAA], c-reactive protein [CRP], retinol binding protein 4 [RBP4]) were verified using independent TBI and healthy volunteer serum samples. Receiver operating characteristic (ROC) curve analysis of CRP and SAA indicated they were robust indicators of injury even at very acute time points. Analysis of serum RBP4 levels at 24-36 h post-injury indicates it may predict subsequent increases in intracranial pressure (ICP) with a sensitivity of 86% and specificity of 88% at 11.6 mug/mL [n = 7, ICP < 20 mm Hg; n = 8, ICP > 25 mm Hg). Our results support the use of serum as a source for discovery of TBI biomarkers, and indicate that serum biomarkers may have utility for predicting secondary pathologies (e.g., elevated ICP) associated with TBI.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury

BackgroundIncreased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patients risk for developing this pathology.MethodsIn the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCS≤8) with or without incidence of elevated intracranial pressure (ICP). De-identified samples and ELISAs were used to confirm the sensitivity and specificity of IL-6 as a prognostic marker of elevated ICP in both isolated TBI patients, and polytrauma patients with TBI.ResultsConsistent with previous reports, we observed sustained increases in IL-6 levels in TBI patients irrespective of their ICP status. However, the group of patients who subsequently experienced ICP ≥ 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained ≤20 mm Hg. When blinded samples (n = 22) were assessed, a serum IL-6 cut-off of <5 pg/ml correctly identified 100% of all the healthy volunteers, a cut-off of >128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained ≤20 mm Hg throughout the study period. In contrast, the marker had no prognostic value in predicting elevated ICP in polytrauma patients with TBI. When the levels of serum IL-6 were assessed in patients with orthopedic injury (n = 7) in the absence of TBI, a significant increase was found in these patients compared to healthy volunteers, albeit lower than that observed in TBI patients.ConclusionsOur results suggest that serum IL-6 can be used for the differential diagnosis of elevated ICP in isolated TBI.

Biomarkers in the clinical diagnosis and management of traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability among young adults. Numerous safety improvements in the workplace, the addition of airbags to vehicles, and the enforcement of speed limits have all helped to reduce the incidence and severity of head trauma. While improvements in emergency response times and acute care have increased TBI survivability, this has heightened the necessity for developing reliable methods to identify patients at risk of developing secondary pathologies. At present, the primary clinical indicators for the presence of brain injury are the Glasgow Coma Scale (GCS), pupil reactivity, and head computed tomography (CT). While these indices have proven useful for stratifying the magnitude and extent of brain damage, they have limited utility for predicting adverse secondary events or detecting subtle damage. Biomarkers, reflecting a biological response to injury or disease, have proven useful for the diagnosis of many pathological conditions including cancer, heart failure, infection, and genetic disorders. For TBI, several proteins synthesized in astroglial cells or neurons have been proposed as potential biomarkers. These proteins include the BB isozyme of creatine kinase (CK-BB, predominant in brain), glial fibrilary acidic protein (GFAP), myelin basic protein (MBP), neuron-specific enolase (NSE), and S100B.The presence of these biomarkers in the cerebrospinal fluid and serum of patients with moderate-to-severe TBI, and their correlation with outcome, suggest that they may have utility as surrogate markers in clinical trials. In addition, many of these markers have been found to be sensitive indicators of injury, and therefore may have the potential to diagnose persons with mild TBI. In addition to biomarkers that correlate with long-term outcome, a few studies have identified prognostic biomarkers for secondary injury that may be useful in individualizing patient management.

Increased plasma interleukin-6 in donors is associated with lower recipient hospital-free survival after cadaveric organ transplantation

Objectives:Brain death induces a massive inflammatory response. However, the influence of this inflammatory response on organ procurement, transplantation, and recipient outcome is unknown. We describe the inflammatory response characteristics in brain-dead organ donors and examine associations with organ transplantation and recipient survival. We test the hypothesis that increased inflammatory response is associated with fewer organs transplanted and decreased recipient survival. Design:Prospective, observational, cohort study. Setting:Two large intensive care units of tertiary care university hospitals in the United States. Patients:We recruited 30 consecutive brain-dead organ donors and 78 recipients between April 11, 2004, and November 23, 2004; recipients were followed through May 2005. Following declaration of brain death, we collected data on donor demographics, mechanism of brain death, number of organs procured and transplanted, and recipient characteristics. Plasma cytokines (tumor necrosis factor, interleukin-6, interleukin-10) were measured in donors at baseline following study enrollment, every hour for the first 4 hrs, and immediately before organ procurement for transplantation. Interventions:None. Measurements and Main Results:We examined the relationships among clinical characteristics, demographics, and cytokine response in donors and their influence on organ procurement and transplantation using multivariable regression and recipient’s 6-month hospital-free survival using a Cox proportional hazards regression. One hundred-eighteen organs were procured from 30 donors, and 91 (77%) were transplanted (mean of three organs transplanted per donor). All cytokines were increased following brain death. Older age in donors was significantly associated with lower number of organs transplanted (p < .001). Higher plasma interleukin-6 concentrations in donors before organ procurement was significantly associated with lower 6-month hospital-free survival in recipients (hazard ratio 1.77; 95% confidence interval, 1.17–2.69, p < .007). Conclusions:Among brain-dead organ donors, older age donors contribute fewer organs for transplantation, and increased donor interleukin-6 level before organ procurement is associated with lower recipient six-month hospital-free survival.

Feasibility study of cytokine removal by hemoadsorption in brain-dead humans.

Background:Inflammatory cytokines occur in the circulation and in the tissues after brain death and have been associated with dysfunction of donor organs before and after transplantation. Objective:To determine the feasibility of removing cytokines using a hemoadsorption device. Design:Two-center, randomized, open-label, feasibility study in which brain-dead subjects were randomized to two treatment groups. Setting:Two U.S. academic hospitals. Participants:Eight brain-dead subjects deemed unsuitable for organ donation by respective organ procurement organizations. Main Outcome Measures:After obtaining consent from families, subjects were treated with hemoadsorption for 4 hrs using CytoSorb. Effects on cytokines (tumor necrosis factor, interleukin [IL]-6, and IL-10) were assessed both across the device and in the plasma over time. Feasibility for cytokine removal was assessed using objective criteria. Results:Cytokine removal across the CytoSorb device ranged from 4% to 30% and was not significantly different from 1 hr to 4 hrs. Overall removal was greatest for IL-6, 28% (p = .006), and least for tumor necrosis factor, 8.5% (p = .13). Plasma concentrations of both IL-6 and tumor necrosis factor, but not IL-10, were significantly reduced after the first hour of therapy; mean differences were −13% ± 7% for IL-6 (p = .039), −23% ± 9% for tumor necrosis factor (p = .02), and −2% ± 7% of IL-10 (p = 23). However, plasma concentrations for all three cytokines increased over time and were above baseline by the end of the intervention. No adverse effects of therapy were observed. However, removal of cortisol and triiodothyronine was similar to removal of cytokines. Conclusions:Hemoadsorption for removal of cytokines in brain-dead subjects is feasible. Evaluation of possible clinical benefit will require controlled trials in actual donors. However, the significant capacity for cytokine removal and absence of adverse events suggest that such trials are warranted.

Human Mild Traumatic Brain Injury Decreases Circulating Branched-Chain Amino Acids and Their Metabolite Levels

The pathophysiology of traumatic brain injury (TBI) is complex and not well understood. Because pathophysiology has ramifications for injury progression and outcome, we sought to identify metabolic cascades that are altered after acute human mild and severe TBI. Because catabolism of branched-chain amino acids (BCAAs; i.e., valine, isoleucine, and leucine) leads to glucose and energy metabolism, and neurotransmitter synthesis and availability, we investigated BCAA metabolites in plasma samples collected within 24 h of injury from mild TBI (Glasgow Coma Scale [GCS] score >12), severe TBI (GCS ≤8), orthopedic injury, and healthy volunteers. We report decreased levels of all three BCAAs in patients with mild TBI relative to healthy volunteers, while these BCAAs levels in patients with severe TBI were further reduced compared with all groups. Orthopedic patients exhibited reductions in BCAA comparable to those in patients with mild TBI. The decrease in patients with mild and severe TBI persisted for derivatives of BCAA catabolic intermediates. Only plasma levels of methylglutarylcarnitine, a derivative of a leucine metabolite, were increased in patients with severe TBI compared with all other groups. Notably, logistic regression combination of three BCAA metabolites whose levels were changed by 24 h post-injury provided prognostic value (area under the curve=0.92) in identifying patients with severe TBI in whom elevated intracranial pressure (≥25 mm Hg) developed. These changes suggest alteration of BCAA metabolism after TBI may contribute to decreased energy production and neurotransmitter synthesis and may contribute to TBI pathophysiology. Supplementation of BCAAs and/or their metabolites may reduce TBI pathology and improve outcome.

ZIP4 is a novel molecular marker for glioma

BACKGROUND Dysregulated zinc transport has been observed in many cancers. However, the status of zinc homeostasis and the expression profile of zinc transporters in brain and brain tumors have not been reported. METHODS The gene profiles of 14 zinc importers (ZIPs) and 10 zinc exporters (ZnTs) in patients with glioma were studied by investigating the association between the zinc transporters and brain tumor characteristics (tumor grade and overall survival time). Three independent cohorts were analyzed to cross-validate the findings: the Chinese Glioma Genome Atlas (CGCA) cohort (n = 186), the US National Cancer Institute Repository for Molecular Brain Neoplasia Data (REMBRANDT) cohort (n = 335), and The University of Texas (UT) cohort (n = 34). RESULTS The expression of ZIP3, 4, 8, 14, ZnT5, 6, and 7 were increased, and the expression of ZnT10 was decreased in grade IV gliomas, compared with grade II gliomas. Among all 24 zinc transporters, ZIP4 is most significantly associated with tumor grade and overall survival; this finding is consistent across 2 independent cohorts (CGCA and REMBRANDT) and is partially validated by the third cohort (UT). High ZIP4 expression was significantly associated with higher grade of gliomas and shorter overall survival (hazard ratio = 1.61, 95% confidence interval = 1.02-2.53, P = .040 in CGCA cohort; hazard ratio = 1.32, 95% confidence interval = 1.08-1.61, P = .007 in REMBRANDT cohort). CONCLUSIONS Dysregulated expression of zinc transporters is involved in the progression of gliomas. Our results suggest that ZIP4 may serve as a potential diagnostic and prognostic marker for gliomas.