Tanya Bogoslovsky

Cerebral Vascular Injury in Traumatic Brain Injury.

Traumatic cerebral vascular injury (TCVI) is a very frequent, if not universal, feature after traumatic brain injury (TBI). It is likely responsible, at least in part, for functional deficits and TBI-related chronic disability. Because there are multiple pharmacologic and non-pharmacologic therapies that promote vascular health, TCVI is an attractive target for therapeutic intervention after TBI. The cerebral microvasculature is a component of the neurovascular unit (NVU) coupling neuronal metabolism with local cerebral blood flow. The NVU participates in the pathogenesis of TBI, either directly from physical trauma or as part of the cascade of secondary injury that occurs after TBI. Pathologically, there is extensive cerebral microvascular injury in humans and experimental animal, identified with either conventional light microscopy or ultrastructural examination. It is seen in acute and chronic TBI, and even described in chronic traumatic encephalopathy (CTE). Non-invasive, physiologic measures of cerebral microvascular function show dysfunction after TBI in humans and experimental animal models of TBI. These include imaging sequences (MRI-ASL), Transcranial Doppler (TCD), and Near InfraRed Spectroscopy (NIRS). Understanding the pathophysiology of TCVI, a relatively under-studied component of TBI, has promise for the development of novel therapies for TBI.

Fluid Biomarkers of Traumatic Brain Injury and Intended Context of Use

Traumatic brain injury (TBI) is one of the leading causes of death and disability around the world. The lack of validated biomarkers for TBI is a major impediment to developing effective therapies and improving clinical practice, as well as stimulating much work in this area. In this review, we focus on different settings of TBI management where blood or cerebrospinal fluid (CSF) biomarkers could be utilized for predicting clinically-relevant consequences and guiding management decisions. Requirements that the biomarker must fulfill differ based on the intended context of use (CoU). Specifically, we focus on fluid biomarkers in order to: (1) identify patients who may require acute neuroimaging (cranial computerized tomography (CT) or magnetic resonance imaging (MRI); (2) select patients at risk for secondary brain injury processes; (3) aid in counseling patients about their symptoms at discharge; (4) identify patients at risk for developing postconcussive syndrome (PCS), posttraumatic epilepsy (PTE) or chronic traumatic encephalopathy (CTE); (5) predict outcomes with respect to poor or good recovery; (6) inform counseling as to return to work (RTW) or to play. Despite significant advances already made from biomarker-based studies of TBI, there is an immediate need for further large-scale studies focused on identifying and innovating sensitive and reliable TBI biomarkers. These studies should be designed with the intended CoU in mind.

Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials

AIMS Endothelial progenitor cells (EPCs) are markers of vascular repair. Increased numbers of circulating endothelial cells (ECs) are associated with endothelial damage. MATERIALS & METHODS We enumerated EPC-EC by using Enrichment kit with addition of anti-human CD146-PE/Cy7 from peripheral blood mononuclear cell (PBMC) isolated either by red blood cell (RBC) lysing solution or by Ficoll centrifugation, and from fresh and preserved samples. PBMCs were quantified by flow cytometry. RESULTS RBC lysis yielded higher percentage of PBMC (p = 0.0242) and higher numbers of PBMC/ml (p = 0.0039) than Ficoll. Absolute numbers of CD34(+)CD133(+)VEGFR2(+) and CD146(+)CD34(+)VEGFR2(+) were higher (p = 0.0117 for both), when isolated by RBC lysis than by Ficoll, when no difference in other subsets was found. Cryopreservation at -160°C and -80°C and short-term preservation at room temperature decreased EPC-EC. CONCLUSIONS Our data support use of fresh samples and isolation of PBMC from human blood by RBC lysis for enumeration of EPC and EC.

Cryopreservation and Enumeration of Human Endothelial Progenitor and Endothelial Cells for Clinical Trials

Background Endothelial progenitor cells (EPC) are markers of endothelial injury and may serve as a surrogate marker for vascular repair in interventional clinical trials. Objectives of this study were to modify a method of isolation of peripheral blood mononuclear cells (PBMC) and enumeration of EPC and mature endothelial cells (EC) from peripheral blood and to evaluate influence of cryopreservation on viability of PBMC and on numbers of EPC and EC. Patients/Methods EPC and EC were analyzed in healthy volunteers in freshly isolated PBMC collected in CPT (cell preparation tubes) and in PBMC cryopreserved with: 1) Gibco Recovery™ Cell Culture Freezing Medium, 2) custom freezing medium. Viability of PBMC was tested using DAPI. EPC were gated for CD45− CD34+CD133+/−VEGFR2+/− and EC were gated for CD45−CD146+CD34+/−VEGFR2+/−. Results Cryopreservation for 7 days at −80°C decreased viable PBMC from 94 ± 0.5% (fresh) to 84 ± 4% (the custom medium) and to 69 ± 8% (Gibco medium), while cryopreservation at −65°C decreased viability to 60 ± 6% (p<0.001, the custom medium) and 49 ± 5% (p<0.001, Gibco medium). In fresh samples early EPC (CD45− CD34+CD133+VEGFR2+) were enumerated as 0.2 ± 0.06%, late EPC(CD45−CD146+CD34+VEGFR2+) as 0.6 ± 0.1% and mature EC (CD45−CD146+CD34−VEGFR2+) as 0.8 ± 0.3%of live PBMC. Cryopreservation with Gibco and the custom freezing medium at −80°C for 7 days decreased numbers EPC and EC, however, this decrease was not statistically significant. Conclusions Our data indicate that cryopreservation at −80°C for 7 days decreases, although not significantly, viability of PBMC and numbers of subsets of EC and EPC. This method may provide an optimized approach to isolation and short-term cryopreservation of subsets of EPC and of mature EC suitable for multicenter trials.

Dissecting Temporal Profiles of Neuronal and Axonal Damage After Mild Traumatic Brain Injury.

Biomarkers are molecules that can be measured in accessible biological fluids that reflect physiological, pharmacological, or disease processes and can suggest the etiology of, susceptibility to, activity levels of, or progress of a disease. Biomarkers have historically been critical to patient progress in a broad range of clinical conditions. Diagnostic and therapeutic advances in fields as disparate as cardiology and oncology have relied on the ability to measure biomarkers that are reliable indicators of the underlying pathology. The absence of validated biomarkers in the neurotrauma field is a major factor limiting our understanding of the natural history and the long-term effects of traumatic brain injury (TBI), as well as a barrier to drug development in this area. According to the US Food and Drug Administration,1,2 biomarkers fall into the following 4 categories, which are not mutually exclusive: diagnostic, prognostic, predictive, and pharmacodynamic. Diagnostic biomarkers measure disease characteristics that categorize a person by the presence or absence of a specific physiological or pathophysiological state. Prognostic biomarkers are baseline measurements that categorize patients by degree of risk for disease progression and inform about the natural history of the disorder. Predictive biomarkers are baseline characteristics that categorize patients by their likelihood of response to a particular treatment. Finally, pharmacodynamic biomarkers are dynamic measurements that show that biological response has occurred in a patient after a therapeutic intervention. Biomarkers are particularly important for progress in the managementof concussionandmildTBI (mTBI).MildTBI can beachallenge todiagnosebecause thesymptomsareoftenprotean and overlap with other conditions that frequently confound the clinical picture, such as intoxication, delirium, and functional disorders like posttraumatic stress disorder. Mild TBI is defined as a traumatically induced physiological disruptionof brain function resulting from theheadbeing struck or striking an object or the brain undergoing an acceleration or deceleration movement, as manifested by at least 1 of the following: (1) anyperiodof lossof consciousnessup to30minutes; (2)posttraumaticamnesianotexceeding24hours; (3) any periodof confusionor disorientation; (4) transient neurological abnormalities, including focal signs, seizures, and intracranial lesions not requiring surgery; and (5) a score of 13 to 15 on the Glasgow Coma Scale.3 Reliance on recall of the event and subjective report of loss of consciousness, posttraumatic amnesia, and symptoms affects the diagnostic accuracy of mTBI and introduces selection bias,4,5 particularly in cases of unwitnessed traumaorwhen the reportingof symptoms is influenced bypotential secondary gain, such as desire to return to play in athletes or involvement in accident-related litigation.Therefore,while there remainsaneed forprognostic, predictive, andpharmacodynamic biomarkers formTBI, diagnosis remains a common challenge in emergency departments, aswell as at the sitesofunintentional injuries, sportingevents, and military combat zones. In this issue of JAMA Neurology, Papa et al6 report a substantial step toward validation and ultimate clinical usefulness of 2 candidate diagnostic biomarkers of mTBI. The investigators examined the diagnostic accuracy of 2 blood biomarkers (glial fibrillary acidic protein [GFAP] and ubiquitin C-terminal hydrolase L1 [UCH-L1]) separately and in combination in cohorts with mild and moderate TBI within 7 days of the injury with respect to diagnostic precision of TBI, presence of traumatic intracranial lesions detected by computed tomography (CT), and need for neurosurgical interventions. While there has been much work recently investigating TBI biomarkers, GFAP and UCH-L1 are among the most widely studied molecules. The ultimate importance of this single-center prospective study addressing the role of these well-studied blood biomarkers of TBI relates to the following reasons. First, this work is based on a thoughtful study design that included multiple blood sampling (starting from 4 hours up to 180 hours after TBI, with up to 19 time points in total), allowing the authors to carefully track temporal profiles of biomarkers of the TBI population at a level I trauma center. Second, the results and conclusions of this study are derived from the large numbers of patients enrolled and the many blood samples collected (1831 samples from 584 patients), allowing careful examination of temporal profiles of these biomarkers in different subgroups of the TBI cohort (such as mTBI with vs without loss of consciousness and subgroups of patients with TBI requiring surgical intervention vs conservative treatment only). This feature of the data collection allowed the authors to adjust the data for multiple moderators (including age, sex, and Glasgow Coma Scale score), which is the noteworthy advantage of this study. Third, this study is unique in using as controls patients who had experienced orthopedic or other non– central nervous system injuries (with similar mechanisms as for the TBI cohort but with normal mental status and no evidence of TBI). Fourth, the data are collected by meticulous and rigorous methodological assessments of patients with mTBI, ensuring eligibility of the patients by initial screening of emergency department physicians, followed by secondary assessment of the study team, thus confirming the accuracy of classification of the enrolled patients. GFAP is a structural protein expressed almost exclusively in astrocytes and released on disintegration of the cytoskeleton.7 It has been widely studied in TBI, and elevated levels inplasmashowpromiseasadiagnostic andprognostic biomarker.8,9 Inmoderate and severe TBI, GFAP levels are elevated in cerebrospinal fluid and serum, particularly in patients who experienced unfavorable outcome.10 In addiRelated article page 551 Opinion Editorial

Development of a systems‐based in situ multiplex biomarker screening approach for the assessment of immunopathology and neural tissue plasticity in male rats after traumatic brain injury

Traumatic brain injuries (TBIs) pose a massive burden of disease and continue to be a leading cause of morbidity and mortality throughout the world. A major obstacle in developing effective treatments is the lack of comprehensive understanding of the underlying mechanisms that mediate tissue damage and recovery after TBI. As such, our work aims to highlight the development of a novel experimental platform capable of fully characterizing the underlying pathobiology that unfolds after TBI. This platform encompasses an empirically optimized multiplex immunohistochemistry staining and imaging system customized to screen for a myriad of biomarkers required to comprehensively evaluate the extent of neuroinflammation, neural tissue damage, and repair in response to TBI. Herein, we demonstrate that our multiplex biomarker screening platform is capable of evaluating changes in both the topographical location and functional states of resident and infiltrating cell types that play a role in neuropathology after controlled cortical impact injury to the brain in male Sprague–Dawley rats. Our results demonstrate that our multiplex biomarker screening platform lays the groundwork for the comprehensive characterization of changes that occur within the brain after TBI. Such work may ultimately lead to the understanding of the governing pathobiology of TBI, thereby fostering the development of novel therapeutic interventions tailored to produce optimal tissue protection, repair, and/or regeneration with minimal side effects, and may ultimately find utility in a wide variety of other neurological injuries, diseases, and disorders that share components of TBI pathobiology.

Evolution of Traumatic Parenchymal Intracranial Hematomas (ICHs): Comparison of Hematoma and Edema Components

This study seeks to quantitatively assess evolution of traumatic ICHs over the first 24 h and investigate its relationship with functional outcome. Early expansion of traumatic intracranial hematoma (ICH) is common, but previous studies have focused on the high density (blood) component. Hemostatic therapies may increase the risk of peri-hematoma infarction and associated increased cytotoxic edema. Assessing the magnitude and evolution of ICH and edema represented by high and low density components on computerized tomography (CT) may be informative for designing therapies targeted at traumatic ICH. CT scans from participants in the COBRIT (Citicoline Brain Injury Trial) study were analyzed using MIPAV software. CT scans from patients with non-surgical intraparenchymal ICHs at presentation and approximately 24 h later (±12 h) were selected. Regions of high density and low density were quantitatively measured. The relationship between volumes of high and low density were compared to several outcome measures, including Glasgow Outcome Score—Extended (GOSE) and Disability Rating Score (DRS). Paired scans from 84 patients were analyzed. The median time between the first and second scan was 22.79 h (25%ile 20.11 h; 75%ile 27.49 h). Over this time frame, hematoma and edema volumes increased >50% in 34 (40%) and 46 (55%) respectively. The correlation between the two components was low (r = 0.39, p = 0.002). There was a weak correlation between change in edema volume and GOSE at 6 months (r = 0.268, p = 0.037), change in edema volume and DRS at 3 and 6 months (r = −0.248, p = 0.037 and r = 0.358, p = 0.005, respectively), change in edema volume and COWA at 6 months (r = 0.272, p = 0.049), and between final edema volume and COWA at 6 months (r = 0.302, p = 0.028). To conclude, both high density and low density components of traumatic ICHs expand significantly in the first 2 days after TBI. In our study, there does not appear to be a relationship between hematoma volume or hematoma expansion and functional outcome, while there is a weak relationship between edema expansion and functional outcome.

Erythropoietin and Its Derivatives: Mechanisms of Neuroprotection and Challenges in Clinical Translation

Erythropoietin (EPO) was first identified as a regulator of erythropoiesis. Additional studies have demonstrated a myriad of pleotropic effects (eg, an ability to decrease inflammation, oxidative stress, and apoptosis). Further, EPO facilitates both angiogenesis and neurogenesis and modulates core components of primary and secondary brain injury. Animal models have confirmed the therapeutic potential of EPO in both experimental ischemic and traumatic brain injury (TBI). Unfortunately, clinical studies in stroke, TBI, and other related neurological injuries/disorders have thus far failed to demonstrate meaningful improvements after interventions with EPO. The surprising failure of EPO in clinical trials may be linked to inadequate dosing regimens, incorrect timing of treatments, duration of the therapy, and/or to inability (eg, lack of sensitivity) to detect outcomes reflective of clinical improvement. As such, optimized administration of EPO or EPO derivatives may provide a novel therapeutic approach for treatment of currently intractable brain injuries and illnesses.

Reliability of the NINDS common data elements cranial tomography (CT) rating variables for traumatic brain injury (TBI)

ABSTRACT Background: Non-contrast head computer tomography (CT) is widely used to evaluate eligibility of patients after acute traumatic brain injury (TBI) for clinical trials. The NINDS Common Data Elements (CDEs) TBI were developed to standardize collection of CT variables. The objectives of this study were to train research assistants (RAs) to rate CDEs and then to evaluate their performance. The aim was to assess inter-rater reliability (IRR) of CDEs between trained RAs and a neurologist and to evaluate applicability of CDEs in acute and sub-acute TBI to test the feasibility of using CDE CT ratings in future trials and ultimately in clinical practice. The second aim was to confirm that the ratings of CDEs reflect pathophysiological events after TBI. Methods and results: First, a manual was developed for application of the CDEs, which was used to rate brain CTs (n = 100). An excellent agreement was found in combined kappas between RAs on admission and on 24-hour follow-up CTs (Iota = 0.803 and 0.787, respectively). Good IRR (kappa > 0.61) was shown for six CDEs on admissions and for seven CDEs on follow-up CTs. Low IRR (kappa < 0.4) was determined for five CDEs on admission and for four CDEs on follow-up CT. Combined IRR of each assistant with the neurologist were good on admission (Iota = 0.613 and 0.787) and excellent on follow-up CT (Iota = 0.906 and 0.977). Second, Principal Component Analysis (PCA) was applied to cluster the rated CDEs (n = 255) and five major components were found that explain 53% of the variance. Conclusions: CT CDEs are useful in clinical studies of TBI. Trained RAs can reliably collect variables. PCA identifies CDE clusters with clinical and biologic plausibility. Abbreviations: RA, research assistant; CT, Cranial Tomography; TBI, Traumatic Brain Injury; CDE, Common Data Elements; IRR, inter-rater reliability; PCA, Principal Component Analysis; GCS, Glasgow Coma Scale; R, rater; CI, confidence interval; CCC, Concordance correlation coefficient; IVH, Intraventricular haemorrhage; DCA, Discriminant Component analysis; SAH, Subarachnoid Haemorrhage

Biomarkers of Vascular Integrity in Traumatic Brain Injury and Correlation with Cerebrovascular Reactivity

Objective: Several plasma proteins, such as Angiopoietins (Ang), Tumor Necrosis Factor (TNF), Serum Amyloid A (SAA), Soluble Intercellular Adhesion Molecule (sICAM-1), Von Willebrand Factor (vWF), and Tie2, have been implicated as potential biomarkers of vascular integrity and angiogenesis in traumatic brain and vascular injuries (TBI and TVI). Our goal is to test the hypothesis that (1) plasma biomarkers of vascular integrity and angiogenesis can assess TVI during chronic TBI; (2) Cerebrovascular Reactivity (CVR) may be a sensitive biomarker in chronic TBI and TVI and may correlate with the plasma biomarkers. Design: Plasma levels of 18 proteins were measured using high sensitivity electro-chemiluminescent sandwich immunoassays (Meso Scale Discoveries, LLC). Subjects were 23 patients with chronic TBI and persistent post-concussive symptoms (PCS, TBI-Sx), 9 chronic TBI patients without PCS (recovered TBIs or TBI-Rec), and 21 healthy controls (HC). TVI was assessed using MRI by measuring CVR-CO2 by assessing the BOLD response to hypercapnia. All subjects underwent MRI with hypercapnia challenge. Nominal a=0.05 was judged to be statistically significant. Results: Plasma concentration of Ang-1 was decreased in patients with chronic TBI with symptoms (TBI-Sx) compared to HC or TBI-Rec (p<0.05), as was the ratio of Ang-1/Ang-2 (p<0.05). SAA was decreased in TBI-Rec patients compared to the TBI-Sx group (P<0.01). CVR-CO2 was decreased in TBI-Sx group compared to HC (P<0.05). Baseline CVR-CO2 correlated with plasma levels of VEGF, VEGF-C, and P-selectin in all TBI patients, but not in HC group. Conclusions: A panel of plasma biomarkers (Ang-1/Ang-2, SAA, VEGF, P-selectin, and vWF) and assessment of CVR may be useful as predictive biomarkers for therapies designed to improve cerebrovascular function after TBI.