Zhiqun Zhang

Elevated Levels of Serum Glial Fibrillary Acidic Protein Breakdown Products in Mild and Moderate Traumatic Brain Injury Are Associated With Intracranial Lesions and Neurosurgical Intervention

STUDY OBJECTIVE This study examines whether serum levels of glial fibrillary acidic protein breakdown products (GFAP-BDP) are elevated in patients with mild and moderate traumatic brain injury compared with controls and whether they are associated with traumatic intracranial lesions on computed tomography (CT) scan (positive CT result) and with having a neurosurgical intervention. METHODS This prospective cohort study enrolled adult patients presenting to 3 Level I trauma centers after blunt head trauma with loss of consciousness, amnesia, or disorientation and a Glasgow Coma Scale (GCS) score of 9 to 15. Control groups included normal uninjured controls and trauma controls presenting to the emergency department with orthopedic injuries or a motor vehicle crash without traumatic brain injury. Blood samples were obtained in all patients within 4 hours of injury and measured by enzyme-linked immunosorbent assay for GFAP-BDP (nanograms/milliliter). RESULTS Of the 307 patients enrolled, 108 were patients with traumatic brain injury (97 with GCS score 13 to 15 and 11 with GCS score 9 to 12) and 199 were controls (176 normal controls and 16 motor vehicle crash controls and 7 orthopedic controls). Receiver operating characteristic curves demonstrated that early GFAP-BDP levels were able to distinguish patients with traumatic brain injury from uninjured controls with an area under the curve of 0.90 (95% confidence interval [CI] 0.86 to 0.94) and differentiated traumatic brain injury with a GCS score of 15 with an area under the curve of 0.88 (95% CI 0.82 to 0.93). Thirty-two patients with traumatic brain injury (30%) had lesions on CT. The area under these curves for discriminating patients with CT lesions versus those without CT lesions was 0.79 (95% CI 0.69 to 0.89). Moreover, the receiver operating characteristic curve for distinguishing neurosurgical intervention from no neurosurgical intervention yielded an area under the curve of 0.87 (95% CI 0.77 to 0.96). CONCLUSION GFAP-BDP is detectable in serum within an hour of injury and is associated with measures of injury severity, including the GCS score, CT lesions, and neurosurgical intervention. Further study is required to validate these findings before clinical application.

Novel Differential Neuroproteomics Analysis of Traumatic Brain Injury in Rats

Approximately two million traumatic brain injury (TBI) incidents occur annually in the United States, yet there are no specific therapeutic treatments. The absence of brain injury diagnostic endpoints was identified as a significant roadblock to TBI therapeutic development. To this end, our laboratory has studied mechanisms of cellular injury for biomarker discovery and possible therapeutic strategies. In this study, pooled naïve and injured cortical samples (48 h postinjury; rat controlled cortical impact model) were processed and analyzed using a differential neuroproteomics platform. Protein separation was performed using combined cation/anion exchange chromatography-PAGE. Differential proteins were then trypsinized and analyzed with reversed-phase LC-MSMS for protein identification and quantitative confirmation. The results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein 2 (CRMP-2), glyceraldehyde-3-phosphate dehydrogenase, microtubule-associated proteins MAP2A/2B, and hexokinase. Conversely C-reactive protein, transferrin, and breakdown products of CRMP-2, synaptotagmin, and αII-spectrin were found to be elevated after TBI. Differential changes in the above mentioned proteins were confirmed by quantitative immunoblotting. Results from this work provide insight into mechanisms of traumatic brain injury and yield putative biochemical markers to potentially facilitate patient management by monitoring the severity, progression, and treatment of injury.

Human Traumatic Brain Injury Induces Autoantibody Response against Glial Fibrillary Acidic Protein and Its Breakdown Products

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38–50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0–1 days) to late (7–10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype

BackgroundAutophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA).ResultsNMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment.ConclusionsCollectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.

Neuroproteomics and systems biology-based discovery of protein biomarkers for traumatic brain injury and clinical validation

The rapidly growing field of neuroproteomics has expanded to track global proteomic changes underlying various neurological conditions such as traumatic brain injury (TBI), stroke, and Alzheimers disease. TBI remains a major health problem with approximately 2 million incidents occurring annually in the United States, yet no affective treatment is available despite several clinical trials. The absence of brain injury diagnostic biomarkers was identified as a significant road‐block to therapeutic development for brain injury. Recently, the field of neuroproteomics has undertaken major advances in the area of neurotrauma research, where several candidate markers have been identified and are being evaluated for their efficacy as biological biomarkers in the field of TBI. One scope of this review is to evaluate the current status of TBI biomarker discovery using neuroproteomics techniques, and at what stage we are at in their clinical validation. In addition, we will discuss the need for strengthening the role of systems biology and its application to the field of neuroproteomics due to its integral role in establishing a comprehensive understanding of specific brain disorder and brain function in general. Finally, to achieve true clinical input of these neuroproteomic findings, these putative biomarkers should be validated using preclinical and clinical samples and linked to clinical diagnostic assays including ELISA or other high‐throughput assays.

Assessing neuro-systemic & behavioral components in the pathophysiology of blast-related brain injury

Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the “distinct” but shared and partially identified biochemical pathways and neuro-histopathological changes that might be linked to behavioral deficits observed. Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.

Acute Diagnostic Biomarkers for Spinal Cord Injury: Review of the Literature and Preliminary Research Report

OBJECTIVE Many efforts have been made to create new diagnostic technologies for use in the diagnosis of central nervous system injury. However, there is still no consensus for the use of biomarkers in clinical acute spinal cord injury (SCI). The aims of this review are (1) to evaluate the current status of neurochemical biomarkers and (2) to discuss their potential acute diagnostic role in SCI by reviewing the literature. METHODS PubMed (http://www.ncbi.nlm.nih.gov/pubmed) was searched up to 2012 to identify publications concerning diagnostic biomarkers in SCI. To support more knowledge, we also checked secondary references in the primarily retrieved literature. RESULTS Neurofilaments, cleaved-Tau, microtubule-associated protein 2, myelin basic protein, neuron-specific enolase, S100β, and glial fibrillary acidic protein were identified as structural protein biomarkers in SCI by this review process. We could not find reports relating ubiquitin C-terminal hydrolase-L1 and α-II spectrin breakdown products, which are widely researched in other central nervous system injuries. Therefore, we present our preliminary data relating to these two biomarkers. Some of biomarkers showed promising results for SCI diagnosis and outcome prediction; however, there were unresolved issues relating to accuracy and their accessibility. CONCLUSION Currently, there still are not many reports focused on diagnostic biomarkers in SCI. This fact warranted the need for greater efforts to innovate sensitive and reliable biomarkers for SCI.

Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.

Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimers disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser130↓Lys131, Gly157↓Ala158 and Arg380↓Glu381. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-d-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45–42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.

Biomarkers Track Damage after Graded Injury Severity in a Rat Model of Penetrating Brain Injury

The goal of this project was to determine whether biochemical markers of brain damage can be used to diagnose and assess the severity of injury in a rat model of penetrating ballistic-like brain injury (PBBI). To determine the relationship between injury magnitude and biomarker levels, rats underwent three discrete PBBI severity levels defined by the magnitude of the ballistic component of the injury, calibrated to equal 5%, 10%, or 12.5% of total rat brain volume. Cortex, cerebrospinal fluid (CSF), and blood were collected at multiple time points. Levels of three biomarkers (αII-spectrin breakdown product [SBDP150], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1 [UCH-L1]), were measured using quantitative immunoblotting and/or enzyme-linked immunosorbent assays. In injured cortex, SBDP150 and GFAP levels were increased significantly over controls. Cortical SBDP150 was elevated at 1 day but not 7 days, and GFAP at 7 days but not 1 day. At their respective time points, mean levels of SBDP150 and GFAP biomarkers in the cortex rose stepwise as injury magnitude increased. In the CSF, increasing severity of PBBI was associated with increasing concentrations of both neuronal and glial biomarkers acutely at 1 day after injury, but no trends were observed at 7 days. In plasma, SBDP150 was elevated at 5 min after 10% PBBI and at 6 h after 12.5% PBBI. UCH-L1 levels in plasma were elevated acutely at 5 min post-injury reflecting injury severity and rapidly decreased within 2 h. Overall, our results support the conclusion that biomarkers are effective indicators of brain damage after PBBI and may also aid in the assessment of injury magnitude.