Glial fibrillary acidic protein, neurofilament light, matrix metalloprotease 3 and fatty acid binding protein 4 as non-invasive brain tumor biomarkers.

BACKGROUND: Gliomas are aggressive malignant tumors, with poor prognosis. There is an unmet need for the discovery of new, non-invasive biomarkers for differential diagnosis, prognosis, and management of brain tumors. Our objective is to validate four plasma biomarkers - glial fibrillary acidic protein (GFAP), neurofilament light (NEFL), matrix metalloprotease 3 (MMP3) and fatty acid binding protein 4 (FABP4) - and compare them with established brain tumor molecular markers and survival. METHODS: Our cohort consisted of patients with benign and malignant brain tumors (GBM = 77, Astrocytomas = 26, Oligodendrogliomas = 23, Secondary tumors = 35, Meningiomas = 70, Schwannomas = 15, Pituitary adenomas = 15, Normal individuals = 30). For measurements, we used ultrasensitive electrochemiluminescence multiplexed immunoassays. RESULTS: High plasma GFAP concentration was associated with GBM, low GFAP and high FABP4 were associated with meningiomas, and low GFAP and low FABP4 were associated with astrocytomas and oligodendrogliomas. NEFL was associated with progression of disease. Several prognostic genetic alterations were significantly associated with all plasma biomarker levels. We found no independent associations between plasma GFAP, NEFL, FABP4 and MMP3, and overall survival. The candidate biomarkers could not reliably discriminate GBM from primary or secondary CNS lymphomas. CONCLUSIONS: GFAP, NEFL, FABP4 and MMP3 are useful for differential diagnosis and prognosis, and are associated with molecular changes in gliomas.

Plasmatic profiles of cytokines/chemokines, glial fibrillary acidic protein (GFAP) and MRI brain damage in neonates with hypoxic ischemic encephalopathy (HIE).

BACKGROUND: Perinatal hypoxia triggers the release of cytokines and chemokines by neurons, astrocytes and microglia. In response to hypoxia-ischemia resting/ramified microglia proliferate and undergo activation, producing proinflammatory molecules. The brain damage extension seems to be related to both the severity of hypoxia and the balance between pro and anti-inflammatory response and can be explored with neuroimaging. AIMS: The aim of this preliminary study was to explore possible relationships between plasma levels of inflammatory cytokines/chemokines and the severe brain damage detectable by Magnetic Resonance Imaging (MRI), performed during the hospitalization. METHODS: In 10 full terms neonates with hypoxic ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH), divided into cases and controls, according to MRI results, we measured and compared the plasma levels of CCL2/MCP-1, CXCL8, GFAP, IFN y, IL-10, IL-18, IL-6, CCL3, ENOLASE2, GM-CSF, IL-1b, IL-12p70, IL-33, TNFα, collected at four different time points during TH (24, 25-48, 49-72 h of life, and 7-10 days from birth). Five of enrolled babies had pathological brain MRI (cases) and 5 had a normal MRI examination (controls). Cytokines were measured by Magnetic Luminex Assay. MRI images were classified according to Barkovich's score. RESULTS: Mean levels of all cytokines and molecules at time T1 were not significantly different in the two groups. Comparing samples paired by day of collection, the greatest differences between cases and controls were found at times T2 and T3, during TH. At T4, levels tended to get closer again (except for IL-6, IL10 and IL18). Infants with worse MRI showed higher plasmatic GFAP levels than those with normal MRI, while their IL-18 was lower. The mean levels of CCL3MIP1alpha, GMCSF, IL1BETA overlapped throughout the observation period in both groups. CONCLUSION: In a small number of infants with worse brain MRI, we found higher levels of GFAP and of IL-10 at T4 and a trend toward low IL-18 levels than in infants with normal MRI, considered early biomarker of brain damage and a predictor of adverse outcome, respectively. The greatest, although not significant, difference between the levels of molecules was found in cases and controls at time points T2 and T3, during TH.

Pediatric reference intervals for serum neurofilament light and glial fibrillary acidic protein using the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort.

OBJECTIVES: Blood biomarkers have the potential to transform diagnosis and prognosis for multiple neurological indications. Establishing normative data is a critical benchmark in the analytical validation process. Normative data are important in children as little is known about how brain development may impact potential biomarkers. The objective of this study is to generate pediatric reference intervals (RIs) for serum neurofilament light (NfL), an axonal marker, and glial fibrillary acidic protein (GFAP), an astrocytic marker. METHODS: Serum from healthy children and adolescents aged 1 to <19 years were obtained from the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort. Serum NfL (n=300) and GFAP (n=316) were quantified using Simoa technology, and discrete RI (2.5th and 97.5th percentiles) and continuous RI (5th and 95th percentiles) were generated. RESULTS: While there was no association with sex, there was a statistically significant (p<0.0001) negative association between age and serum NfL (Rho -0.400) and GFAP (Rho -0.749). Two statistically significant age partitions were generated for NfL: age 1 to <10 years (lower, upper limit; 3.13, 20.6 pg/mL) and 10 to <19 years (1.82, 7.44 pg/mL). For GFAP, three statistically significant age partitions were generated: age 1 to <3.5 years (80.4, 601 pg/mL); 3.5 to <11 years (50.7, 224 pg/mL); and 11 to <19 years (26.2, 119 pg/mL). CONCLUSIONS: Taken together with the literature on adults, NfL and GFAP display U-shaped curves with high levels in infants, decreasing levels during childhood, a plateau during adolescence and early adulthood and increasing levels in seniors. These normative data are expected to inform future pediatric studies on the importance of age on neurological blood biomarkers.

Minor head injury in anticoagulated patients: performance of biomarkers S100B, NSE, GFAP, UCH-L1 and Alinity TBI in the detection of intracranial injury. A prospective observational study.

OBJECTIVES: Data in literature indicate that in patients suffering a minor head injury (MHI), biomarkers serum levels could be effective to predict the absence of intracranial injury (ICI) on head CT scan. Use of these biomarkers in case of patients taking oral anticoagulants who experience MHI is very limited. We investigated biomarkers as predictors of ICI in anticoagulated patients managed in an ED. METHODS: We conducted a single-cohort, prospective, observational study in an ED. Our structured clinical pathway included a first head CT scan, 24 h observation and a second CT scan. The outcome was delayed ICI (dICI), defined as ICI on the second CT scan after a first negative CT scan. We assessed the sensitivity (SE), specificity (SP), negative predictive value (NNV) and positive predictive value (PPV) of the biomarkers S100B, NSE, GFAP, UCH-L1 and Alinity TBI in order to identify dICI. RESULTS: Our study population was of 234 patients with a negative first CT scan who underwent a second CT scan. The rate of dICI was 4.7 %. The NPV for the detection of dICI were respectively (IC 95 %): S100B 92.7 % (86.0-96.8 %,); ubiquitin C-terminal hydrolase-L1 (UCH-L1) 91.8 % (83.8-96.6 %); glial fibrillary protein (GFP) 100 % (83.2-100 %); TBI 100 % (66.4-100 %). The AUC for the detection of dICI was 0.407 for S100B, 0.563 for neuron-specific enolase (NSE), 0.510 for UCH-L1 and 0.720 for glial fibrillary acidic protein (GFAP), respectively. CONCLUSIONS: The NPV of the analyzed biomarkers were high and they potentially could limit the number of head CT scan for detecting dICI in anticoagulated patients suffering MHI. GFAP and Alinity TBI seem to be effective to rule out a dCI, but future trials are needed.

Astrocyte biomarkers GFAP and YKL-40 mediate early Alzheimer's disease progression.

INTRODUCTION: We studied how biomarkers of reactive astrogliosis mediate the pathogenic cascade in the earliest Alzheimer's disease (AD) stages. METHODS: We performed path analysis on data from 384 cognitively unimpaired individuals from the ALzheimer and FAmilies (ALFA)+ study using structural equation modeling to quantify the relationships between biomarkers of reactive astrogliosis and the AD pathological cascade. RESULTS: Cerebrospinal fluid (CSF) amyloid beta (Aß)42/40 was associated with Aß aggregation on positron emission tomography (PET) and with CSF p-tau181 , which was in turn directly associated with CSF neurofilament light (NfL). Plasma glial fibrillary acidic protein (GFAP) mediated the relationship between CSF Aß42/40 and Aß-PET, and CSF YKL-40 partly explained the association between Aß-PET, p-tau181 , and NfL. DISCUSSION: Our results suggest that reactive astrogliosis, as indicated by different fluid biomarkers, influences the pathogenic cascade during the preclinical stage of AD. While plasma GFAP mediates the early association between soluble and insoluble Aß, CSF YKL-40 mediates the latter association between Aß and downstream Aß-induced tau pathology and tau-induced neuronal injury. HIGHLIGHTS: Lower CSF Aß42/40 was directly linked to higher plasma GFAP concentrations. Plasma GFAP partially explained the relationship between soluble Aß and insoluble Aß. CSF YKL-40 mediated Aß-induced tau phosphorylation and tau-induced neuronal injury.

Pathologic and cognitive correlates of plasma biomarkers in neurodegenerative disease.

INTRODUCTION: We investigate pathological correlates of plasma phosphorylated tau 181 (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) across a clinically diverse spectrum of neurodegenerative disease, including normal cognition (NormCog) and impaired cognition (ImpCog). METHODS: Participants were NormCog (n = 132) and ImpCog (n = 461), with confirmed ß-amyloid (Aß+/-) status (cerebrospinal fluid, positron emission tomography, autopsy) and single molecule array plasma measurements. Logistic regression and receiver operating characteristic (ROC) area under the curve (AUC) tested how combining plasma analytes discriminated Aß+ from Aß-. Survival analyses tested time to clinical dementia rating (global CDR) progression. RESULTS: Multivariable models (p-tau+GFAP+NfL) had the best performance to detect Aß+ in NormCog (ROCAUC = 0.87) and ImpCog (ROCAUC = 0.87). Survival analyses demonstrated that higher NfL best predicted faster CDR progression for both Aß+ (hazard ratio [HR] = 2.94; p = 8.1e-06) and Aß- individuals (HR = 3.11; p = 2.6e-09). DISCUSSION: Combining plasma biomarkers can optimize detection of Alzheimer's disease (AD) pathology across cognitively normal and clinically diverse neurodegenerative disease. HIGHLIGHTS: Participants were clinically heterogeneous, with autopsy- or biomarker-confirmed Aß. Combining plasma p-tau181, GFAP, and NfL improved diagnostic accuracy for Aß status. Diagnosis by plasma biomarkers is more accurate in amnestic AD than nonamnestic AD. Plasma analytes show independent associations with tau PET and post mortem Aß/tau. Plasma NfL predicted longitudinal cognitive decline in both Aß+ and Aß- individuals.

Tau, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain as Brain Protein Biomarkers in Cerebrospinal Fluid and Blood for Diagnosis of Neurobiological Diseases.

Neurological damage is the pathological substrate of permanent disability in various neurodegenerative disorders. Early detection of this damage, including its identification and quantification, is critical to preventing the disease's progression in the brain. Tau, glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL), as brain protein biomarkers, have the potential to improve diagnostic accuracy, disease monitoring, prognostic assessment, and treatment efficacy. These biomarkers are released into the cerebrospinal fluid (CSF) and blood proportionally to the degree of neuron and astrocyte damage in different neurological disorders, including stroke, traumatic brain injury, multiple sclerosis, neurodegenerative dementia, and Parkinson's disease. Here, we review how Tau, GFAP, and NfL biomarkers are detected in CSF and blood as crucial diagnostic tools, as well as the levels of these biomarkers used for differentiating a range of neurological diseases and monitoring disease progression. We also discuss a biosensor approach that allows for the real-time detection of multiple biomarkers in various neurodegenerative diseases. This combined detection system of brain protein biomarkers holds significant promise for developing more specific and accurate clinical tools that can identify the type and stage of human neurological diseases with greater precision.

Impact of pre-analytical sample handling factors on plasma biomarkers of Alzheimer's disease.

An unmet need exists for reliable plasma biomarkers of amyloid pathology, in the clinical laboratory setting, to streamline diagnosis of Alzheimer's disease (AD). For routine clinical use, a biomarker must provide robust and reliable results under pre-analytical sample handling conditions. We investigated the impact of different pre-analytical sample handling procedures on the levels of seven plasma biomarkers in development for potential routine use in AD. Using (1) fresh (never frozen) and (2) previously frozen plasma, we evaluated the effects of (A) storage time and temperature, (B) freeze/thaw (F/T) cycles, (C) anticoagulants, (D) tube transfer, and (E) plastic tube types. Blood samples were prospectively collected from patients with cognitive impairment undergoing investigation in a memory clinic. ß-amyloid 1-40 (Aß40), ß-amyloid 1-42 (Aß42), apolipoprotein E4, glial fibrillary acidic protein, neurofilament light chain, phosphorylated-tau (phospho-tau) 181, and phospho-tau-217 were measured using Elecsys® plasma prototype immunoassays. Recovery signals for each plasma biomarker and sample handling parameter were calculated. For all plasma biomarkers measured, pre-analytical effects were comparable between fresh (never frozen) and previously frozen samples. All plasma biomarkers tested were stable for ≤24 h at 4°C when stored as whole blood and ethylenediaminetetraacetic acid (EDTA) plasma. Recovery signals were acceptable for up to five tube transfers, or two F/T cycles, and in both polypropylene and low-density polyethylene tubes. For all plasma biomarkers except Aß42 and Aß40, analyte levels were largely comparable between EDTA, lithium heparin, and sodium citrate tubes. Aß42 and Aß40 were most sensitive to pre-analytical handling, and the effects could only be partially compensated by the Aß42/Aß40 ratio. We provide recommendations for an optimal sample handling protocol for analysis of plasma biomarkers for amyloid pathology AD, to improve the reproducibility of future studies on plasma biomarkers assays and for potential use in routine clinical practice.

Autoimmune glial fibrillary acidic protein astrocytopathy associated with breast cancer: a case report.

BACKGROUND: Autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy (GFAP-A) is an autoimmune inflammatory central nervous system disorder characterized by the detection of autoantibodies that recognize GFAP in CSF. The pathogenesis of GFAP-A is poorly understood. Some patients had a neoplasm detected and GFAP expressed by neoplasms is plausible as immunogen triggering paraneoplastic neurological autoimmunity. CASE PRESENTATION: We report a case of 76-year-old female patient with GFAP-A complicated with breast cancer. She presented with altered consciousness, nuchal rigidity, speech disturbances, and weakness. Her clinical symptoms were improved by immunotherapy and cancer treatments. Immunohistochemical analysis showed that the restricted tumor expressed GFAP. The infiltration of CD3 + T cells were observed in the peritumoral and intratumoral areas. The most common infiltrating lymphocytes were CD8 + T cells. CD4 + T cells and CD20 + B cells were also observed in the predominant peritumoral area. CONCLUSIONS: These results suggest that GFAP-A may occur in a paraneoplastic neurological syndrome associated with breast cancer.

Serum GFAP - pediatric reference interval in a cohort of Danish children.

OBJECTIVES: Glial fibrillary acidic protein (GFAP) in blood is an emerging biomarker of brain injury and neurological disease. Its clinical use in children is limited by the lack of a reference interval (RI). Thus, the aim of the present study was to establish an age-dependent continuous RI for serum GFAP in children. METHODS: Excess serum from routine allergy testing of 391 children, 0.4-17.9 years of age, was measured by a single-molecule array (Simoa) assay. A continuous RI was modelled using non-parametric quantile regression and presented both graphically and tabulated as discrete one-year RIs based on point estimates from the model. RESULTS: Serum GFAP showed a strong age-dependency with declining levels and variability from infants to adolescents. The estimated median level decreased 66 % from four months to five years of age and another 65 % from five years to 17.9 years of age. No gender difference was observed. CONCLUSIONS: The study establishes an age-dependent RI for serum GFAP in children showing high levels and variability in the first years of life.

Atretic cephalocele and encephalocele: A single-institution clinicopathological study.

BACKGROUND: Encephaloceles are neural tube defects characterized by herniation of meninges, neural tissue and cerebrospinal fluid, while atretic cephaloceles denote a rudimentary connection to the intracranial space with absence of herniated neural tissue and represent an infrequent dermatopathologic diagnosis. Limited reports of these entities confound the challenge in their histopathologic distinction. Accurate classification is important given associated anomalies and neurologic manifestations that impact prognosis. METHODS: We describe the clinicopathological and immunohistochemical [glial fibrillary acidic protein (GFAP), S100, epithelial membrane antigen (EMA), and somatostatin receptor subtype 2A (SSTR2A)] features in a retrospective series encountered at a single institution between 1994 and 2020. RESULTS: We identified 13 cases classified as atretic cephalocele (n = 11) and encephalocele (n = 2). Hamartomatous changes and multinucleated cells were unique to atretic cephaloceles while myxoid areas were unique to encephaloceles. At least focal staining for SSTRA was seen in all atretic cephaloceles with the majority (87.5%) staining for EMA; negative staining for GFAP and S100 confirmed absence of neural tissue. Encephaloceles were GFAP and S100 positive, and negative for SSTR2 and EMA. Atretic cephaloceles had a favorable prognosis compared to encephaloceles, with severe morbidity present in both encephalocele cases. CONCLUSION: Our study raises awareness of atretic cephalocele and encephalocele among dermatopathologists and reveals a mutually exclusive immunophenotype that facilitates their distinction for prognostication and management.

Clinical characteristics of patient with GFAP-IgG: a review of 31 patients from two tertiary referral centers in China.

OBJECTIVE: This study presents a comprehensive analysis of the clinical characteristics of 31 patients exhibiting cerebrospinal fluid (CSF) and/or serum positivity for GFAP-IgG, with a specific emphasis on 24 cases demonstrating only GFAP-IgG positivity. The investigation thoroughly evaluates their clinical, radiological, and laboratory features, as well as treatment responses, with the objective of offering clinicians potential diagnostic and therapeutic approaches. METHODS: A total of 31 patients with GFAP-IgG in the CSF and/or serum were registered between August 2016 and August 2021 at the General Hospital of Ningxia Medical University and Huashan Hospital of Fudan University. We retrospectively reviewed their clinical records. RESULTS: Overall, the patients were positive with GFAP-IgG in their CSF (15/31), in serum (6/31), and both CSF and serum (10/31). Among them, two were eventually diagnosed with astroglioma and primary central nervous system lymphoma, respectively; one patient had typical multiple sclerosis; three exhibited overlapping GFAP-IgG and aquaporin-4-IgG (AQP4-IgG); and one patient was coexisting N-methyl-D-aspartate receptor IgG. The remaining 24 patients were only GFAP-IgG positive. In total, 22 out of the 24 patients had abnormal MRI outcomes, involving the brain, meninges, and spinal cord. Besides, seven of the 24 patients developed optic neuritis. The CSF protein levels positively correlated with the Expanded Disability Status Scale score (EDSSs). Significantly decreased EDSSs, modified Rankin Scale score, GFAP-IgG titer, CSF protein level, and CSF white blood cell counts were observed after immunomodulatory therapy. CONCLUSION: The clinical manifestations of GFAP-IgG exhibit a wide range of phenotypes that lack specificity. These findings emphasize the significance of not exclusively relying on the presence of antibodies to diagnose GFAP-A, but rather integrating them with the clinical phenotypes. GFAP-IgG testing enables the diagnosis of autoimmune GFAP astrocytopathy, a treatable autoimmune disease affecting the central nervous system. This condition provides opportunities for investigating innovative mechanisms of CNS autoimmunity and inflammation.

A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images.

Immunohistochemical staining of cell and molecular targets in brain samples is a powerful tool that can provide valuable information on neurological mechanisms. However, post-processing of photomicrographs acquired after 3,3'-Diaminobenzidine (DAB) staining is particularly challenging due to the complexity associated with the size, samples number, analyzed targets, image quality, and even the subjectivity inherent to the analysis by different users. Conventionally, this analysis relies on the manual quantification of distinct parameters (e.g., the number and size of cells and the number and length of cell branching) in a large set of images. These represent extremely time-consuming and complex tasks, defaulting the processing of high amounts of information. Here we describe an improved semi-automatic method to quantify glial fibrillary acidic protein (GFAP)-labelled astrocytes in immunohistochemistry images of rat brains, at magnifications as low as 20×. This method is a straightforward adaptation of the Young & Morrison method, using ImageJ's plugin Skeletonize, coupled with intuitive data processing in datasheet-based software. It allows swifter and more efficient post-processing of brain tissue samples, regarding astrocyte size and number quantification, the total area occupied, as well as astrocyte branching and branch length (indicators of astrocyte activation), thus contributing to better understand the possible inflammatory response developed by astrocytes.

Microglia and Astroglia-The Potential Role in Neuroinflammation Induced by Pre- and Neonatal Exposure to Lead (Pb).

Neuroinflammation is one of the postulated mechanisms for Pb neurotoxicity. However, the exact molecular mechanisms responsible for its pro-inflammatory effect are not fully elucidated. In this study, we examined the role of glial cells in neuroinflammation induced by Pb exposure. We investigated how microglia, a type of glial cell, responded to the changes caused by perinatal exposure to Pb by measuring the expression of Iba1 at the mRNA and protein levels. To assess the state of microglia, we analyzed the mRNA levels of specific markers associated with the cytotoxic M1 phenotype (Il1b, Il6, and Tnfa) and the cytoprotective M2 phenotype (Arg1, Chi3l1, Mrc1, Fcgr1a, Sphk1, and Tgfb1). Additionally, we measured the concentration of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α). To assess the reactivity and functionality status of astrocytes, we analyzed the GFAP (mRNA expression and protein concentration) as well as glutamine synthase (GS) protein level and activity. Using an electron microscope, we assessed ultrastructural abnormalities in the examined brain structures (forebrain cortex, cerebellum, and hippocampus). In addition, we measured the mRNA levels of Cxcl1 and Cxcl2, and their receptor, Cxcr2. Our data showed that perinatal exposure to Pb at low doses affected both microglia and astrocyte cells' status (their mobilization, activation, function, and changes in gene expression profile) in a brain-structure-specific manner. The results suggest that both microglia and astrocytes represent a potential target for Pb neurotoxicity, thus being key mediators of neuroinflammation and further neuropathology evoked by Pb poisoning during perinatal brain development.

[Effect of Xiaoxuming Decoction on activation of astrocytes in acute cerebral ischemia/reperfusion injury].

This study investigated the effect of Xiaoxuming Decoction(XXMD) on the activation of astrocytes after cerebral ischemia/reperfusion(I/R) injury. The model of cerebral IR injury was established using the middle cerebral artery occlusion method. Fluorocitrate(FC), an inhibitor of astrocyte activation, was applied to inhibit astrocyte activation. Rats were randomly divided into a sham group, a model group, a XXMD group, a XXMD+FC group, and a XXMD+Vehicle group. Neurobehavioral changes at 24 hours after cerebral IR injury, cerebral infarction, histopathological changes observed through HE staining, submicroscopic structure of astrocytes observed through transmission electron microscopy, fluorescence intensity of glial fibrillary acidic protein(GFAP) and thrombospondin 1(TSP1) measured through immunofluorescence, and expression of GFAP and TSP1 in brain tissue measured through Western blot were evaluated in rats from each group. The experimental results showed that neurobehavioral scores and cerebral infarct area significantly increased in the model group. The XXMD group, the XXMD+FC group, and the XXMD+Vehicle group all alleviated neurobehavioral changes in rats. The pathological changes in the brain were evident in the model group, while the XXMD group, the XXMD+FC group, and the XXMD+Vehicle group exhibited milder cerebral IR injury in rats. The submicroscopic structure of astrocytes in the model group showed significant swelling, whereas the XXMD group, the XXMD+FC group, and XXMD+Vehicle group protected the submicroscopic structure of astrocytes. The fluorescence intensity and protein expression of GFAP and TSP1 increased in the model group compared with those in the sham group. However, the XXMD group, the XXMD+FC group, and XXMD+Vehicle group all down-regulated the expression of GFAP and TSP1. The combination of XXMD and FC showed a more pronounced effect. These results indicate that XXMD can improve cerebral IR injury, possibly by inhibiting astrocyte activation and down-regulating the expression of GFAP and TSP1.

The innate immune system stimulating cytokine GM-CSF improves learning/memory and interneuron and astrocyte brain pathology in Dp16 Down syndrome mice and improves learning/memory in wild-type mice.

Down syndrome (DS) is characterized by chronic neuroinflammation, peripheral inflammation, astrogliosis, imbalanced excitatory/inhibitory neuronal function, and cognitive deficits in both humans and mouse models. Suppression of inflammation has been proposed as a therapeutic approach to treating DS co-morbidities, including intellectual disability (DS/ID). Conversely, we discovered previously that treatment with the innate immune system stimulating cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which has both pro- and anti-inflammatory activities, improved cognition and reduced brain pathology in a mouse model of Alzheimer's disease (AD), another inflammatory disorder, and improved cognition and reduced biomarkers of brain pathology in a phase II trial of humans with mild-to-moderate AD. To investigate the effects of GM-CSF treatment on DS/ID in the absence of AD, we assessed behavior and brain pathology in 12-14 month-old DS mice (Dp[16]1Yey) and their wild-type (WT) littermates, neither of which develop amyloid, and found that subcutaneous GM-CSF treatment (5 µg/day, five days/week, for five weeks) improved performance in the radial arm water maze in both Dp16 and WT mice compared to placebo. Dp16 mice also showed abnormal astrocyte morphology, increased percent area of GFAP staining in the hippocampus, clustering of astrocytes in the hippocampus, and reduced numbers of calretinin-positive interneurons in the entorhinal cortex and subiculum, and all of these brain pathologies were improved by GM-CSF treatment. These findings suggest that stimulating and/or modulating inflammation and the innate immune system with GM-CSF treatment may enhance cognition in both people with DS/ID and in the typical aging population.

The GFAP Monoclonal Antibody GA-5 Identifies Astrocyte Remodeling and Glio-Vascular Uncoupling During the Evolution of EAE.

To examine how astrocyte activation is regulated at different phases of relapsing-remitting EAE, we performed an immunofluorescent analysis of the spinal cord using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody GA-5. In keeping with previous studies, gray matter astrocytes showed strongly increased GFAP expression during the peak phase of disease (14 days post-immunization), which remained elevated during the remission phase (21-28 days post-immunization). In sharp contrast, during the peak phase of disease, the GA-5 signal in sub-meningeal white matter transiently disappeared in areas containing high levels of infiltrating leukocytes, but during the remission phase, the GFAP signal was fully restored. Parallel staining of the same sections with a polyclonal GFAP antibody confirmed elevated GFAP expression in the gray matter but no loss of signal in white matter. Interestingly, loss of GA-5 signal in sub-meningeal white matter was strongly associated with vascular disruption as defined by extravascular fibrinogen leak and by glio-vascular uncoupling, as defined by dissociation of AQP4-positive astrocyte endfeet and CD31-positive blood vessels. GA-5-negative areas were also associated with demyelination. These findings demonstrate a novel staining pattern of a GFAP antibody during EAE progression and suggest that the GFAP epitope recognized by the GA-5 monoclonal antibody transiently disappears as white matter astrocytes undergo remodeling during the peak phase of EAE. They also suggest that the GA-5 antibody provides a novel tool to identify astrocyte remodeling in other neurological conditions.

Serum GFAP - reference interval and preanalytical properties in Danish adults.

OBJECTIVES: Glial fibrillary acidic protein (GFAP) is a promising biomarker that could potentially contribute to diagnosis and prognosis in neurological diseases. The biomarker is approaching clinical use but the reference interval for serum GFAP remains to be established, and knowledge about the effect of preanalytical factors is also limited. METHODS: Serum samples from 371 apparently healthy reference subjects, 21-90 years of age, were measured by a single-molecule array (Simoa) assay. Continuous reference intervals were modelled using non-parametric quantile regression and compared with traditional age-partitioned non-parametric reference intervals established according to the Clinical and Laboratory Standards Institute (CLSI) guideline C28-A3. The following preanalytical conditions were also examined: stability in whole blood at room temperature (RT), stability in serum at RT and -20 °C, repeated freeze-thaw cycles, and haemolysis. RESULTS: The continuous reference interval showed good overall agreement with the traditional age-partitioned reference intervals of 25-136 ng/L, 34-242 ng/L, and 5-438 ng/L for the age groups 20-39, 40-64, and 65-90 years, respectively. Both types of reference intervals showed increasing levels and variability of serum GFAP with age. In the preanalytical tests, the mean changes from baseline were 2.3% (95% CI: -2.4%, 6.9%) in whole blood after 9 h at RT, 3.1% (95% CI: -4.5%, 10.7%) in serum after 7 days at RT, 10.4% (95% CI: -6.0%, 26.8%) in serum after 133 days at -20 °C, and 10.4% (95% CI: 9.5%, 11.4%) after three freeze-thaw cycles. CONCLUSIONS: The study establishes age-dependent reference ranges for serum GFAP in adults and demonstrates overall good stability of the biomarker.

Serum biomarkers identify critically ill traumatic brain injury patients for MRI.

BACKGROUND: Magnetic resonance imaging (MRI) carries prognostic importance after traumatic brain injury (TBI), especially when computed tomography (CT) fails to fully explain the level of unconsciousness. However, in critically ill patients, the risk of deterioration during transfer needs to be balanced against the benefit of detecting prognostically relevant information on MRI. We therefore aimed to assess if day of injury serum protein biomarkers could identify critically ill TBI patients in whom the risks of transfer are compensated by the likelihood of detecting management-altering neuroimaging findings. METHODS: Data were obtained from the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. Eligibility criteria included: TBI patients aged ≥ 16 years, Glasgow Coma Score (GCS) < 13 or patient intubated with unrecorded pre-intubation GCS, CT with Marshall score < 3, serum biomarkers (GFAP, NFL, NSE, S100B, Tau, UCH-L1) sampled ≤ 24 h of injury, MRI < 30 days of injury. The degree of axonal injury on MRI was graded using the Adams-Gentry classification. The association between serum concentrations of biomarkers and Adams-Gentry stage was assessed and the optimum threshold concentration identified, assuming different minimum sensitivities for the detection of brainstem injury (Adams-Gentry stage 3). A cost-benefit analysis for the USA and UK health care settings was also performed. RESULTS: Among 65 included patients (30 moderate-severe, 35 unrecorded) axonal injury was detected in 54 (83%) and brainstem involvement in 33 (51%). In patients with moderate-severe TBI, brainstem injury was associated with higher concentrations of NSE, Tau, UCH-L1 and GFAP. If the clinician did not want to miss any brainstem injury, NSE could have avoided MRI transfers in up to 20% of patients. If a 94% sensitivity was accepted considering potential transfer-related complications, GFAP could have avoided 30% of transfers. There was no added net cost, with savings up to £99 (UK) or $612 (US). No associations between proteins and axonal injury were found in intubated patients without a recorded pre-intubation GCS. CONCLUSIONS: Serum protein biomarkers show potential to safely reduce the number of transfers to MRI in critically ill patients with moderate-severe TBI at no added cost.

Confocal Laser and Electron Microscopic Investigation of Gap Junctions in Anaplastic Astrocytomas.

Connexin 43 (Cx43) is a multifunction protein that forms gap junction channels and hemichannels and is suggested to play an essential role in oxygen-glucose deprivation, induced via neuroinflammation during astrocytoma expansion into healthy tissue. To prove this assumption we studied connexin 43 localisation and ultrastructure of gap junctions in samples of malignant brain tumour (anaplastic astrocytomas grade III). For confocal laser microscopy, vibratome sections of tumour fragments were incubated in a mixture of primary antibodies to connexin 43 and glial fibrillary acidic protein (GFAP), then in a mixture of secondary antibodies conjugated with a fluorescent label. After the immunofluorescence study, sections were washed in phosphate buffer, additionally postfixed with 1% OsO4 solution, dehydrated and embedded in epoxy resin by a plane-parallel method. Ultra-thin sections obtained from these samples were contrasted with uranyl acetate and lead citrate and viewed under a Jem 1011 electron microscope. Confocal laser examination detected a positive reaction to Cx43 in the form of point fluorescence. These points were of various sizes. Most of them were localised around or at the intersection of small processes containing GFAP. Electron microscopy of the tumour samples containing the most significant number of Cx43 revealed single and closely spaced gap junctions with a typical ultrastructure on the processes and bodies of tumour cells. Sequential analysis in the fields of view revealed 62 gap junctions in the area of 100 µm2. Numerous gap junctions in anaplastic astrocytomas revealed in our study may indicate electrotonic and metabolic transmission between glioma cells, possibly promoting its progression.