Glial Fibrillary Acidic Protein (GFAP) as a TBI Biomarker
Puffer and team (2021) isolated extracellular vesicles (EVs) from blood plasma of TBI patients and controls to identify novel biomarkers assisting in the rapid diagnosis and classification of brain injury. On isolating biomarkers, researchers found an increased concentration of Glial Fibrillary Acidic Protein (GFAP) in traumatic brain injury (TBI) patients with altered consciousness. They also identified the differential expression of multiple miRNAs targeting pathways relevant to TBIs.
What are Extracellular Vesicles (EVs)
EVs contain DNA, mRNA, protein, and noncoding RNA released into the microenvironment. EVs participate in cell-to-cell communication between glia, neurons, and endothelial cells. They accomplish complex functions such as moderation of the blood-brain barrier. EVs are heterogenous and consist of smaller exosomes and micro-vesicles generated through distinct cellular pathways, resulting in distinct cargo.
Common TBI Biomarkers
Severe TBI is diagnosed in 200,000 individuals in the United States every year and is a major cause of mortality among men under 35 years. mTBI is underreported although it could be ten times more common when compared to severe TBI. mTBI may contribute to chronic neurologic dysfunction. Plasma biomarkers of TBI discovered recently include glial fibrillary acidic protein (GFAP), neurofilament light, tau, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1). These biomarkers play an integral role in diagnosis and prognosis of TBI, specifically mTBI.
Extracellular Vesicles (EVs) in TBI
Previous studies have identified a higher number of EVs in cerebrospinal fluid (CSF) of patients with severe TBI i.e. with a Glasgow Coma Scale (GCS) score of 3–8. Higher quantities of UCH-L1 and GFAP were isolated from the patients. It must be noted that plasma is more readily available when compared to CSF. Brain-specific biomarkers can be isolated from the EVs of peripheral blood. The lipid membranes of EVs in peripheral blood contain RNA, DNA, and proteins that are protected from circulating degradation enzymes, and provide insight into the cellular microenvironment during initial brain injury phase and short- and long-term recovery.
In their present study, researchers obtained 20 plasma samples (15 TBI and 5 healthy controls). Samples consisted of the following:
- Mild TBI (mTBI) i.e. GCS score 13–15 with evidence of injury on CT scan.
- Moderate TBI i.e. GCS score of 9–12.
- Severe TBI i.e. GCS score 3–8.
- Healthy controls were uninjured.
EV Isolation and Analysis
EVs were isolated from plasma using ultracentrifugation, analyzed using nanotracker particle analysis to characterize concentration and size of EVs. Common EV proteins were identified with Western Blot analysis and proteins were separated with electrophoresis. Subsequent to membrane transfer, proteins were probed with antibodies. Enhanced chemiluminescence was used for detection. To break membranes, EVs were sonicated for performing GFAP analysis. Expression of GFAP was tested in all samples with ELISA.
Short, noncoding RNA was isolated from samples, quality assessment was performed, and samples were subject to next-generation sequencing. Short noncoding RNA sequences were analyzed and differential expression analysis was performed to identify differences in miRNA expression between groups. Subsequently, descriptive statistical analysis was performed — interquartile range, median, and proportions were used to describe continuous and categorical variables. Levels of biomarkers were regarded as continuous data. Between-group differences for continuous variables were analyzed with Mann-Whitney U-Test.
Higher GFAP Concentration in TBI Patients
EV size and concentration, RNA yield, and GFAP concentration was determined for all samples. No significant difference was found in EV size, concentration, and RNA yield between controls and TBI patients with normal consciousness (GCS score = 15) or altered consciousness (GCS score ≤14). However, analysis of GFAP yield with ELISA technique showed higher GFAP concentration in TBI patients with altered consciousness (GCS score ≤14) compared to normal controls.
Disease Pathways Relevant to Trauma Identified
Short noncoding RNA species were identified beyond miRNAs in EVs from TBI patients. Differential expression analysis was performed for miRNA sequences beyond a threshold value. In TBI patients with altered consciousness, 9 highly upregulated miRNAs and 2 highly downregulated miRNAs were identified.
Ingenuity pathway analysis of highly differentially expressed miRNAs among TBI patients identified top disorder and disease pathways — gastrointestinal disease, cancer, and organismal abnormalities or injuries. Molecular and cellular pathways identified were related to cell-to-cell signaling, cell death or survival, and cellular organization or assembly.
Researchers also found several species of short noncoding RNA such as snRNA, snoRNA, rRNA, and long intervening noncoding RNA (lincRNA). GFAP in plasma EV indicates the presence of proteomic, transcriptomic, and metabolic signatures that are unique to TBI diagnosis and can be isolated and analyzed. The pathways indicated in analysis represent logically relevant pathways in trauma.
Conclusion
Findings from the study indicate that brain-specific EVs are released shortly after TBI, that leads to an altered level of consciousness. EV isolation and analysis can make it possible to identify potential biomarkers related to severity, prognosis, and therapeutic response of injury. It may be possible that not just the CNS, but also multiple organ systems change EV release patterns following trauma. Samples studied are snapshots of time and their signatures change in response to insult (decreased cerebral blood flow, persistently raised intracranial pressure, and poor oxygen delivery), or therapeutic and surgical interventions. The signatures obtained could be used to monitor function and recovery of CNS at the cellular level. The technique can also be used to study EVs specific to organ systems. Novel biomarkers identified must be correlated with disease states, clinical presentations, and outcomes, to ensure that signatures are specific to disease process and treatment.
Reference
Puffer, R. C., Milbeth, L., Himes, B., Jung, M., Meyer, F. B., Okonkwo, D. O., & Parney, I. F. (2021). Plasma extracellular vesicles as a source of biomarkers in traumatic brain injury. Journal of Neurosurgery, 134(6), 1921–1928. https://doi.org/10.3171/2020.4.jns20305
