Generation and Study of Antibodies against Two Triangular Trimers Derived from Aß.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aß) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aß assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aß. These antibodies are the first mAbs generated against Aß oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aß42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aß enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Chikungunya virus cell-to-cell transmission is mediated by intercellular extensions in vitro and in vivo.

Chikungunya virus (CHIKV) has recently emerged to cause millions of human infections worldwide. Infection can induce the formation of long intercellular extensions that project from infected cells and form stable non-continuous membrane bridges with neighbouring cells. The mechanistic role of these intercellular extensions in CHIKV infection was unclear. Here we developed a co-culture system and flow cytometry methods to quantitatively evaluate transmission of CHIKV from infected to uninfected cells in the presence of neutralizing antibody. Endocytosis and endosomal acidification were critical for virus cell-to-cell transmission, while the CHIKV receptor MXRA8 was not. By using distinct antibodies to block formation of extensions and by evaluation of transmission in HeLa cells that did not form extensions, we showed that intercellular extensions mediate CHIKV cell-to-cell transmission. In vivo, pre-treatment of mice with a neutralizing antibody blocked infection by direct virus inoculation, while adoptive transfer of infected cells produced antibody-resistant host infection. Together our data suggest a model in which the contact sites of intercellular extensions on target cells shield CHIKV from neutralizing antibodies and promote efficient intercellular virus transmission both in vitro and in vivo.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Spontaneous Resolution of Diffuse Idiopathic Slit-Like "Syrinx" in a Pediatric Patient.

It is important to recognize that a prominent central canal of the spinal cord can be a normal variant and can spontaneously regress. A five-year-old male presented for evaluation of abnormal gait. Prior brain magnetic resonance imaging showed no hindbrain malformation, and the patient had no history of trauma. Full spine magnetic resonance imaging showed a vertical slit-like linear cavity within the center of the spinal cord, from C6-7 to the conus medullaris with a diameter ranging from 0.5 to 2 mm. This was initially reported as a syrinx. The patient's symptoms remained stable. Three years later, follow-up magnetic resonance imaging showed spontaneous resolution of the slit-like cavity. This case likely represented a prominent central canal (a normal variant) that underwent normal closure.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Evolving White Matter Injury following Pediatric Traumatic Brain Injury.

This study is unique in that it examines the evolution of white matter injury very early and at 12 months post-injury in pediatric patients following traumatic brain injury (TBI). Diffusion tensor imaging (DTI) was acquired at two time-points: acutely at 6-17 days and 12 months following a complicated mild (cMild)/moderate (mod) or severe TBI. Regional measures of anisotropy and diffusivity were compared between TBI groups and against a group of age-matched healthy controls and used to predict performance on measures of attention, memory, and intellectual functioning at 12-months post-injury. Analysis of the acute DTI data using tract based spatial statistics revealed a small number of regional decreases in fractional anisotropy (FA) in both the cMild/mod and severe TBI groups compared with controls. These changes were observed in the occipital white matter, anterior limb of the internal capsule (ALIC)/basal ganglia, and corpus callosum. The severe TBI group showed regional differences in axial diffusivity (AD) in the brainstem and corpus callosum that were not seen in the cMild/mod TBI group. By 12-months, widespread decreases in FA and increases in apparent diffusion coefficient (ADC) and radial diffusivity (RD) were observed in both TBI groups compared with controls, with the overall number of regions with abnormal DTI metrics increasing over time. The early changes in regional DTI metrics were associated with 12-month performance IQ scores. These findings suggest that there may be regional differences in the brain's reparative processes or that mechanisms associated with the brain's plasticity to recover may also be region based.

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer ≥1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 ( COVID-19 ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( S ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F ™ and ExpiCHO-S ™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Longitudinal Metabolite Changes after Traumatic Brain Injury: A Prospective Pediatric Magnetic Resonance Spectroscopic Imaging Study.

The aims of this study were to evaluate longitudinal metabolite changes in traumatic brain injury (TBI) subjects and determine whether early magnetic resonance spectroscopic imaging (MRSI) changes in discrete brain regions predict 1-year neuropsychological outcomes. Three-dimensional (3D) proton MRSI was performed in pediatric subjects with complicated mild (cMild), moderate, and severe injury, acutely (6-17 days) and 1-year post-injury along with neurological and cognitive testing. Longitudinal analysis found that in the cMild/Moderate group, all MRSI ratios from 12 regions returned to control levels at 1 year. In the severe group, only cortical gray matter regions fully recovered to control levels whereas N-acetylaspartate (NAA) ratios from the hemispheric white matter and subcortical regions remained statistically different from controls. A factor analysis reduced the data to two loading factors that significantly differentiated between TBI groups; one included acute regional NAA variables and another consisted of clinically observed variables (e.g., days in coma). Using scores calculated from the two loading factors in a logistic regression model, we found that the percent accuracy for classification of TBI groups was greatest for the dichotomized attention measure (93%), followed by Full Scale Intelligence Quotient at 91%, and the combined memory Z-score measure (90%). Using the acute basal ganglia NAA/creatine (Cr) ratio alone achieved a higher percent accuracy of 94.7% for the attention measure whereas the acute thalamic NAA/Cr ratio alone achieved a higher percent accuracy of 91.9% for the memory measure. These results support the conclusions that reduced NAA is an early indicator of tissue injury and that measurements from subcortical brain regions are more predictive of long-term cognitive outcome.

Combined Diffusion Tensor and Magnetic Resonance Spectroscopic Imaging Methodology for Automated Regional Brain Analysis: Application in a Normal Pediatric Population.

During human brain development, anatomic regions mature at different rates. Quantitative anatomy-specific analysis of longitudinal diffusion tensor imaging (DTI) and magnetic resonance spectroscopic imaging (MRSI) data may improve our ability to quantify and categorize these maturational changes. Computational tools designed to quickly fuse and analyze imaging information from multiple, technically different datasets would facilitate research on changes during normal brain maturation and for comparison to disease states. In the current study, we developed a complete battery of computational tools to execute such data analyses that include data preprocessing, tract-based statistical analysis from DTI data, automated brain anatomy parsing from T1-weighted MR images, assignment of metabolite information from MRSI data, and co-alignment of these multimodality data streams for reporting of region-specific indices. We present statistical analyses of regional DTI and MRSI data in a cohort of normal pediatric subjects (n = 72; age range: 5-18 years; mean 12.7 ± 3.3 years) to establish normative data and evaluate maturational trends. Several regions showed significant maturational changes for several DTI parameters and MRSI ratios, but the percent change over the age range tended to be small. In the subcortical region (combined basal ganglia [BG], thalami [TH], and corpus callosum [CC]), the largest combined percent change was a 10% increase in fractional anisotropy (FA) primarily due to increases in the BG (12.7%) and TH (9%). The largest significant percent increase in N-acetylaspartate (NAA)/creatine (Cr) ratio was seen in the brain stem (BS) (18.8%) followed by the subcortical regions in the BG (11.9%), CC (8.9%), and TH (6.0%). We found consistent, significant (p < 0.01), but weakly positive correlations (r = 0.228-0.329) between NAA/Cr ratios and mean FA in the BS, BG, and CC regions. Age- and region-specific normative MR diffusion and spectroscopic metabolite ranges show brain maturation changes and are requisite for detecting abnormalities in an injured or diseased population.

Jugular foramen schwannoma mimicking paraganglioma: case report and review of imaging findings.

We present the case of a 30-year-old woman who presented with an 11-year history of chronic occipital headaches and a 12-month history of worsening difficulty speaking and/or swallowing, facial spasms, hearing loss, and dizziness. A large lytic mass was found centered in the left jugular foramen (JF) on computed tomography examination; follow-up magnetic resonance imaging showed an avidly enhancing mass with prominent central flow voids. Histopathologic examination after surgical resection revealed the mass to be a schwannoma. Prominent central vascularity is an unusual presentation for JF schwannomas. Our report provides a review of magnetic resonance imaging features of intrinsic JF lesions relevant to our case.

Susceptibility-weighted imaging and quantitative susceptibility mapping in the brain.

Susceptibility-weighted imaging (SWI) is a magnetic resonance imaging (MRI) technique that enhances image contrast by using the susceptibility differences between tissues. It is created by combining both magnitude and phase in the gradient echo data. SWI is sensitive to both paramagnetic and diamagnetic substances which generate different phase shift in MRI data. SWI images can be displayed as a minimum intensity projection that provides high resolution delineation of the cerebral venous architecture, a feature that is not available in other MRI techniques. As such, SWI has been widely applied to diagnose various venous abnormalities. SWI is especially sensitive to deoxygenated blood and intracranial mineral deposition and, for that reason, has been applied to image various pathologies including intracranial hemorrhage, traumatic brain injury, stroke, neoplasm, and multiple sclerosis. SWI, however, does not provide quantitative measures of magnetic susceptibility. This limitation is currently being addressed with the development of quantitative susceptibility mapping (QSM) and susceptibility tensor imaging (STI). While QSM treats susceptibility as isotropic, STI treats susceptibility as generally anisotropic characterized by a tensor quantity. This article reviews the basic principles of SWI, its clinical and research applications, the mechanisms governing brain susceptibility properties, and its practical implementation, with a focus on brain imaging.

Application of advanced neuroimaging modalities in pediatric traumatic brain injury.

Neuroimaging is commonly used for the assessment of children with traumatic brain injury and has greatly advanced how children are acutely evaluated. More recently, emphasis has focused on how advanced magnetic resonance imaging methods can detect subtler injuries that could relate to the structural underpinnings of the neuropsychological and behavioral alterations that frequently occur. We examine several methods used for the assessment of pediatric brain injury. Susceptibility-weighted imaging is a sensitive 3-dimensional high-resolution technique in detecting hemorrhagic lesions associated with diffuse axonal injury. Magnetic resonance spectroscopy acquires metabolite information, which serves as a proxy for neuronal (and glial, lipid, etc) structural integrity and provides sensitive assessment of neurochemical alterations. Diffusion-weighted imaging is useful for the early detection of ischemic and shearing injury. Diffusion tensor imaging allows better structural evaluation of white matter tracts. These methods are more sensitive than conventional imaging in demonstrating subtle injury that underlies a child's clinical symptoms. There also is an increasing desire to develop computational methods to fuse imaging data to provide a more integrated analysis of the extent to which components of the neurovascular unit are affected. The future of traumatic brain injury neuroimaging research is promising and will lead to novel approaches to predict and improve outcomes.

Impaired neurovascular unit function contributes to persistent symptoms after concussion: a pilot study.

Research shows that approximately 14% of school age children with mild traumatic brain injury (TBI) including sports-related concussions (SRCs) remain symptomatic three months after injury. Advanced imaging studies early after injury have shown evidence of axonal damage, reduced N-acetyl aspartate (NAA) and impaired cerebral blood flow (CBF) in individuals with mild TBI. This study was undertaken to determine whether these techniques can provide valuable information in pediatric SRC patients with persistent post-concussive symptoms. Fifteen pediatric subjects ages 8 to 17 years with persistent post-concussive symptoms were evaluated using perfusion-weighted imaging (PWI), three-dimensional (3D) magnetic resonance spectroscopic imaging, and diffusion tensor imaging (DTI) three to 12 months post-SRC. Data were compared with 15 demographically similar (age, gender, and body mass index) controls. In the bilateral thalami, SRC patients showed reduced CBF (p=0.02 and p=0.02) and relative cerebral blood volume (CBV; p=0.05 and p=0.03), compared with controls. NAA/creatine (Cr) and NAA/choline (Cho) ratios were reduced in the corpus callosum (p=0.003; p=0.05) and parietal white matter (p<0.001; p=0.006) of SRC subjects, compared with controls. Significant differences in DTI metrics differentiated patients with cognitive symptoms, compared with those without cognitive symptoms and controls. Advanced imaging methods detect a spectrum of injury including impaired axonal function, neuronal metabolism and perfusion, suggesting involvement of the neurovascular unit in the presence of persistent symptoms in pediatric SRC patients.