Metabolite transport across central nervous system barriers.

Metabolomic analysis of cerebrospinal fluid (CSF) is used to improve diagnostics and pathophysiological understanding of neurological diseases. Alterations in CSF metabolite levels can partly be attributed to changes in brain metabolism, but relevant transport processes influencing CSF metabolite concentrations should be considered. The entry of molecules including metabolites into the central nervous system (CNS), is tightly controlled by the blood-brain, blood-CSF, and blood-spinal cord barriers, where aquaporins and membrane-bound carrier proteins regulate influx and efflux via passive and active transport processes. This report therefore provides reference for future CSF metabolomic work, by providing a detailed summary of the current knowledge on the location and function of the involved transporters and routing of metabolites from blood to CSF and from CSF to blood.

The influence of fixation and cryopreservation of cerebrospinal fluid on antigen expression and cell percentages by flow cytometric analysis.

The pauci-cellular nature of cerebrospinal (CSF), particularly ventricular CSF, and the rapid cell death following sampling, incumbers the use of flow cytometric analysis of these samples in the investigation of central nervous system (CNS) pathologies. Developing a method that allows long-term storage and batched analysis of CSF samples without compromising cell integrity is highly desirable in clinical research, given that CSF is often sampled after hours creating logistical difficulties for fresh processing. We examined percentages and relative proportion of peripheral and brain-derived immune cells in cryopreserved and transfix-treated CSF, compared to freshly processed CSF. Cell proportions were more comparable between Fresh and Cryopreserved CSF (mean of differences = 3.19), than between fresh and transfix-treated CSF (mean of differences = 14.82). No significant differences in cell percentages were observed in fresh versus cryopreserved CSF; however significantly lower cell percentages were observed in transfix-treated CSF compared to Fresh CSF [(CD11b++ (p = 0.01), CD4+ (p = 0.001), CD8+ (p = 0.007), NK cells (p = 0.04), as well as CD69+ activation marker (p = 0.001)]. Furthermore, loss of marker expression of various lymphocyte sub-populations were observed in transfix-treated CSF. Cryopreservation is a feasible option for long-term storage of ventricular CSF and allows accurate immunophenotyping of peripheral and brain-derived cell populations by flow cytometry.

Comparing the immune abnormalities in MIS-C to healthy children and those with inflammatory disease reveals distinct inflammatory cytokine production and a monofunctional T cell response.

Multisystem inflammatory syndrome in children (MIS-C) is a severe, hyperinflammatory disease that occurs after exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The underlying immune pathology of MIS-C is incompletely understood, with limited data comparing MIS-C to clinically similar paediatric febrile diseases at presentation. SARS-CoV-2-specific T cell responses have not been compared in these groups to assess whether there is a T cell profile unique to MIS-C. In this study, we measured inflammatory cytokine concentration and SARS-CoV-2-specific humoral immunity and T cell responses in children with fever and suspected MIS-C at presentation (n = 83) where MIS-C was ultimately confirmed (n = 58) or another diagnosis was made (n = 25) and healthy children (n = 91). Children with confirmed MIS-C exhibited distinctly elevated serum IL-10, IL-6, and CRP at presentation. No differences were detected in SARS-CoV-2 spike IgG serum concentration, neutralisation capacity, antibody dependant cellular phagocytosis, antibody dependant cellular cytotoxicity or SARS-CoV-2-specific T cell frequency between the groups. Healthy SARS-CoV-2 seropositive children had a higher proportion of polyfunctional SARS-CoV-2-specific CD4+ T cells compared to children with MIS-C and those with other inflammatory or infectious diagnoses, who both presented a largely monofunctional SARS-CoV-2-specific CD4+ T cell profile. Treatment with steroids and/or intravenous immunoglobulins resulted in rapid reduction of inflammatory cytokines but did not affect the SARS-CoV-2-specific IgG or CD4+ T cell responses in MIS-C. In these data, MIS-C had a unique cytokine profile but not a unique SARS-CoV-2 specific humoral or T cell cytokine response.

Rifampicin and protein concentrations in paired spinal versus ventricular cerebrospinal fluid samples of children with tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is the most lethal form of TB. To study the disease, drug concentrations in samples obtained from the spinal CSF are usually used to reflect brain concentrations. Emerging data suggest that transport of substances across capillaries in the brain (ventricular CSF) and spinal cord may differ. METHODS: We examined paired, time-linked samples of ventricular CSF (VCSF) and lumbar CSF (LCSF) of 28 patients with TBM and analysed these for rifampicin and total protein concentrations. Clinically indicated samples from procedures to determine the level of CSF block were collected from children being treated for TBM and hydrocephalus. Total protein concentrations were determined using the bicinchoninic acid (BCA) or turbidimetry assay, and rifampicin concentrations were determined using a validated LC coupled with tandem MS method. A paired Wilcoxon signed-rank test was used to determine significance. RESULTS: TBM was confirmed in 19 cases (68%) using TB culture or GeneXpert Mtb/Rifampicin assay. All other cases were classified as probable. The median total protein concentration in LCSF was 6.0 g/L and in VCSF was 1.3 g/L. The median rifampicin concentration in LCSF was 299 ng/mL and 133 ng/mL in VCSF. The median ratio of LCSF/VSCF for protein was 4.23 and 1.57 for rifampicin. CONCLUSIONS: Total protein and rifampicin concentrations differed significantly between the two compartments, both being higher in LCSF than in VCSF samples (P < 0.0001 for total protein and P = 0.0046 for rifampicin). Further studies are required to explore the causative reasons for the observed differences.

Cell type-specific gene expression dynamics during human brain maturation.

The human brain undergoes protracted post-natal maturation, guided by dynamic changes in gene expression. To date, studies exploring these processes have used bulk tissue analyses, which mask cell type-specific gene expression dynamics. Here, using single nucleus (sn)RNA-Sseq on temporal lobe tissue, including samples of African ancestry, we build a joint paediatric and adult atlas of 54 cell subtypes, which we verify with spatial transcriptomics. We explore the differences in cell states between paediatric and adult cell types, revealing the genes and pathways that change during brain maturation. Our results highlight excitatory neuron subtypes, including the LTK and FREM subtypes, that show elevated expression of genes associated with cognition and synaptic plasticity in paediatric tissue. The new resources we present here improve our understanding of the brain during a critical period of its development and contribute to global efforts to build an inclusive cell map of the brain.

Bacterial meningitis in Africa.

Bacterial meningitis differs globally, and the incidence and case fatality rates vary by region, country, pathogen, and age group; being a life-threatening disease with a high case fatality rate and long-term complications in low-income countries. Africa has the most significant prevalence of bacterial meningitis illness, and the outbreaks typically vary with the season and the geographic location, with a high incidence in the meningitis belt of the sub-Saharan area from Senegal to Ethiopia. Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis (meningococcus) are the main etiological agents of bacterial meningitis in adults and children above the age of one. Streptococcus agalactiae (group B Streptococcus), Escherichia coli, and Staphylococcus aureus are neonatal meningitis's most common causal agents. Despite efforts to vaccinate against the most common causes of bacterial neuro-infections, bacterial meningitis remains a significant cause of mortality and morbidity in Africa, with children below 5 years bearing the heaviest disease burden. The factors attributed to this continued high disease burden include poor infrastructure, continued war, instability, and difficulty in diagnosis of bacterial neuro-infections leading to delay in treatment and hence high morbidity. Despite having the highest disease burden, there is a paucity of African data on bacterial meningitis. In this article, we discuss the common etiologies of bacterial neuroinfectious diseases, diagnosis and the interplay between microorganisms and the immune system, and the value of neuroimmune changes in diagnostics and therapeutics.

Cerebrovascular Pressure Reactivity Has a Strong and Independent Association With Outcome in Children With Severe Traumatic Brain Injury.

OBJECTIVES: To examine cerebrovascular pressure reactivity index (PRx) in a large cohort of children with severe traumatic brain injury (sTBI) in association with physiologic variables and outcome. DESIGN: Retrospective observational cohort study. SETTING: Red Cross War Memorial Children's Hospital in Cape Town, South Africa. PATIENTS: Pediatric (≤ 14 yr old) sTBI patients with intracranial pressure (ICP) monitoring (postresuscitation Glasgow Coma Score [Glasgow Coma Scale (GCS)] of ≤ 8). MEASUREMENTS AND MAIN RESULTS: Data were analyzed from ICM+ files sampled at 100Hz. PRx (a mathematical indicator of pressure reactivity) was calculated as a moving correlation coefficient between ICP and mean arterial pressure (MAP) as previously described. Associations between PRx, age, GCS, ICP, MAP, and cerebral perfusion pressure (CPP) were examined with summary measures and correlation analysis using high-frequency data. Associations between PRx and mortality/outcome were examined with multivariable logistic regression analysis and the prognostic ability of PRx with receiver operating characteristic (ROCs) curves. The dataset included over 1.7 million minutes (28,634 hr) of MAP and ICP data in 196 children. The series mortality was 10.7% (21/196), and unfavorable outcome 29.6% (58/196). PRx had a moderate positive correlation with ICP ( r = 0.44; p < 0.001), a moderate negative correlation with CPP ( r = -0.43; p < 0.001), and a weak negative correlation with MAP ( r = -0.21; p = 0.004). PRx was consistently higher in patients with poor outcome and had a strong, independent association with mortality (ROC area under the curve = 0.91). A PRx threshold of 0.25 showed the best predictive ability for mortality. CONCLUSIONS: This is the largest cohort of children with PRx analysis of cerebrovascular reactivity to date. PRx had a strong association with outcome that was independent of ICP, CPP, GCS, and age. The data suggest that impaired autoregulation is an independent factor associated with poor outcome and may be useful in directing clinical care.

Population Pharmacokinetic Analysis of Rifampicin in Plasma, Cerebrospinal Fluid, and Brain Extracellular Fluid in South African Children with Tuberculous Meningitis.

Limited knowledge is available on the pharmacokinetics of rifampicin in children with tuberculous meningitis (TBM) and its penetration into brain tissue, which is the site of infection. In this analysis, we characterize the distribution of rifampicin in cerebrospinal fluid (CSF), lumbar (LCSF) and ventricular (VCSF), and brain extracellular fluid (ECF). Children with TBM were included in this pharmacokinetic analysis. Sparse plasma, LCSF, and VCSF samples were collected opportunistically, as clinically indicated. Brain ECF was sampled using microdialysis (MD). Rifampicin was quantified with liquid chromatography with tandem mass spectrometry in all samples, and 25-desacetyl rifampicin in the plasma samples. The data were interpreted with nonlinear mixed-effects modeling, with the CSF and brain ECF modeled as "effect compartments." Data were available from 61 children, with median (min-max) age of 2 (0.3 to 10) years and weight of 11.0 (4.8 to 49.0) kg. A one-compartment model for parent and metabolite with first-order absorption and elimination via saturable hepatic clearance described the data well. Allometric scaling, maturation, and auto-induction of clearance were included. The pseudopartition coefficient between plasma and LCSF/VCSF was ~5%, while the value for ECF was only ~0.5%, possibly reflecting low recovery of rifampicin using MD. The equilibration half-life between plasma and LCSF/VCSF was ~4 h and between plasma and ECF ~2 h. Our study confirms previous reports showing that rifampicin concentrations in the LCSF are lower than in plasma and provides novel knowledge about rifampicin in the VCSF and the brain tissue. Despite MD being semiquantitative because the relative recovery cannot be quantified, our study presents a proof-of-concept that rifampicin reaches the brain tissue and that MD is an attractive technique to study site-of-disease pharmacokinetics in TBM.

Biomarkers of Tuberculous Meningitis and Pediatric Human Immunodeficiency Virus on the African Continent.

Biomarkers in body fluids are helpful objective tools in diagnosis, prognosis and monitoring of (therapeutic) responses of many neurological diseases. Cerebrospinal fluid (CSF) biomarkers are part of the diagnostic toolbox for infectious neurological diseases. Tuberculous meningitis (TBM) and Human immunodeficiency virus (HIV), are important burdens of disease in Africa and can negatively affect brain health. Two thirds of the world's population of people living with HIV reside in sub-Saharan Africa and 25% of the global burden of tuberculosis (TB) is carried by the African continent. Neuroinflammation and damage of specific neuronal cell types are key constituents in the pathophysiology of these central nervous system (CNS) diseases, and important potential sources of circulating biomarkers. In this review, we summarize current research in the use of biomarkers in TBM and pediatric HIV as case demonstrations for high prevalence neurological diseases in Africa. Inflammatory molecules, primarily when detected in CSF, appear to have diagnostic value in these diseases, especially when measured as profiles. Brain injury molecules, such as S100, Neuron specific enolase and glial fibrillary acidic protein may have prognostic value in TBM, but more studies are needed. There is a need for more cost-economic and high sensitivity technologies to drive further biomarker discoveries and translate into healthcare improvements for these important healthcare problems in a globally fair way.

Infections in the Developing Brain: The Role of the Neuro-Immune Axis.

Central nervous system (CNS) infections occur more commonly in young children than in adults and pose unique challenges in the developing brain. This review builds on the distinct vulnerabilities in children's peripheral immune system (outlined in part 1 of this review series) and focuses on how the developing brain responds once a CNS infection occurs. Although the protective blood-brain barrier (BBB) matures early, pathogens enter the CNS and initiate a localized innate immune response with release of cytokines and chemokines to recruit peripheral immune cells that contribute to the inflammatory cascade. This immune response is initiated by the resident brain cells, microglia and astrocytes, which are not only integral to fighting the infection but also have important roles during normal brain development. Additionally, cytokines and other immune mediators such as matrix metalloproteinases from neurons, glia, and endothelial cells not only play a role in BBB permeability and peripheral cell recruitment, but also in brain maturation. Consequently, these immune modulators and the activation of microglia and astrocytes during infection adversely impact normal neurodevelopment. Perturbations to normal brain development manifest as neurodevelopmental and neurocognitive impairments common among children who survive CNS infections and are often permanent. In part 2 of the review series, we broadly summarize the unique challenges CNS infections create in a developing brain and explore the interaction of regulators of neurodevelopment and CNS immune response as part of the neuro-immune axis.

A pilot study of inflammatory mediators in brain extracellular fluid in paediatric TBM.

Tuberculous meningitis (TBM) is the most fatal form of tuberculosis and frequently occurs in children. The inflammatory process initiates secondary brain injury processes that lead to death and disability. Much remains unknown about this cerebral inflammatory process, largely because of the difficulty in studying the brain. To date, studies have typically examined samples from sites distal to the site of disease, such as spinal cerebrospinal fluid (CSF) and blood. In this pilot study, we examined the feasibility of using direct brain microdialysis (MD) to detect inflammatory mediators in brain extracellular fluid (ECF) in TBM. MD was used to help guide neurocritical care in 7 comatose children with TBM by monitoring brain chemistry for up to 4 days. Remnant ECF fluid was stored for offline analysis. Samples of ventricular CSF, lumbar CSF and blood were collected at clinically indicated procedures for comparison. Inflammatory mediators were quantified using multiplex technology. All inflammatory markers, with the exception of interleukin (IL)-10 and IL-12p40, were detected in the ECF. Cytokine concentrations were generally lower in ECF than ventricular CSF in time-linked specimens. Individual cases showed ECF cytokine increases coinciding with marked increases in ECF glycerol or decreases in ECF glucose. Cytokine levels and glycerol were generally higher in patients with more severe disease. This is the first report of inflammatory marker analysis from samples derived directly from the brain and in high temporal resolution, demonstrating feasibility of cerebral MD to explore disease progression and possibly therapy response in TBM.

Brain interstitial glycerol correlates with evolving brain injury in paediatric traumatic brain injury.

PURPOSE: A better understanding of the complex pathophysiology of traumatic brain injury (TBI) is needed to improve our current therapies. Cerebral microdialysis (CMD) is an advanced method to monitor the brain, but little is known about its parameters in children. Brain glycerol, one of the CMD variables, is an essential component of the phospholipid bilayer cell membrane and is considered a useful marker of tissue hypoxia in adults. This study examined the time course of glycerol and its associations in paediatric TBI. METHODS: In this retrospective cohort study, we collected data on children (< 13years) with severe TBI who underwent CMD monitoring. The relationship of glycerol was examined with respect to physiological, radiological variables, and clinical outcome. RESULTS: Twenty-eight children underwent CMD monitoring and had evaluable data. Lesion progression on head computed tomography (CT) demonstrated a strong relationship with glycerol (median glycerol, maximum and initial-to-maximum) when lesion size increased by > 30% (p=0.01, p=0.04 and p=0.004). Absolute glycerol values had a weak but statistically significant association with intracranial pressure and brain oxygenation. We did not find an association with clinical outcome. CONCLUSION: This is the first study to provide data on brain interstitial glycerol in children. CMD glycerol, particularly an increase from baseline, is associated with other markers of injury and with a significant increase in lesion size on repeat head CT. As such, it may represent a useful monitorable marker for evolving injury in paediatric TBI.

Infection in the Developing Brain: The Role of Unique Systemic Immune Vulnerabilities.

Central nervous system (CNS) infections remain a major burden of pediatric disease associated with significant long-term morbidity due to injury to the developing brain. Children are susceptible to various etiologies of CNS infection partly because of vulnerabilities in their peripheral immune system. Young children are known to have reduced numbers and functionality of innate and adaptive immune cells, poorer production of immune mediators, impaired responses to inflammatory stimuli and depressed antibody activity in comparison to adults. This has implications not only for their response to pathogen invasion, but also for the development of appropriate vaccines and vaccination strategies. Further, pediatric immune characteristics evolve across the span of childhood into adolescence as their broader physiological and hormonal landscape develop. In addition to intrinsic vulnerabilities, children are subject to external factors that impact their susceptibility to infections, including maternal immunity and exposure, and nutrition. In this review we summarize the current evidence for immune characteristics across childhood that render children at risk for CNS infection and introduce the link with the CNS through the modulatory role that the brain has on the immune response. This manuscript lays the foundation from which we explore the specifics of infection and inflammation within the CNS and the consequences to the maturing brain in part two of this review series.

International Survey Reveals Opportunities to Improve Tuberculous Meningitis Management and the Need for Standardized Guidelines.

BACKGROUND: Tuberculous meningitis (TBM) is a medical emergency, yet there are no standardized treatment guidelines for the medical or neurosurgical management of these patients and little data on neurocritical care. We conducted an international survey to understand current medical and neurosurgical TBM management and resource availability to provide baseline data needed for future multicenter trials addressing unanswered clinical research questions and the establishment of standardized guidelines. METHODS: An online survey of 77 questions covering medical and neurosurgical TBM management aimed at clinicians/nurses treating TBM was distributed as an anonymous link through email invitation, international organizations' membership distribution, and direct links on organizational webpages or social media. The survey remained open for 5 months. Data were summarized with frequencies and percentages. RESULTS: The survey had 222 responses from 43 countries representing 6 continents. Most respondents were from tertiary care facilities, with broad access to medical and neurosurgical resources. There was significant heterogeneity in general supportive care, and TBM-specific management demonstrated considerable divergence from current standard-of-care practices. The lack of standardized guidelines was identified as a major challenge in TBM management. General and neurocritical care were largely absent. Resources for bedside supportive care and noninvasive monitoring were broadly accessible. CONCLUSIONS: These findings suggest that current TBM management could be improved by the establishment of internationally accepted treatment guidelines based on available evidence, and that numerous centers have resources available to participate in future multicenter trials, even for basic interventions, that may further improve patient outcomes globally.

Management of Spasticity After Traumatic Brain Injury in Children.

Traumatic brain injury is a common cause of disability worldwide. In fact, trauma is the second most common cause of death and disability, still today. Traumatic brain injury affects nearly 475 000 children in the United States alone. Globally it is estimated that nearly 2 million people are affected by traumatic brain injuries every year. The mechanism of injury differs between countries in the developing world, where low velocity injuries and interpersonal violence dominates, and high-income countries where high velocity injuries are more common. Traumatic brain injury is not only associated with acute problems, but patients can suffer from longstanding consequences such as seizures, spasticity, cognitive and social issues, often long after the acute injury has resolved. Spasticity is common after traumatic brain injury in children and up to 38% of patients may develop spasticity in the first 12 months after cerebral injury from stroke or trauma. Management of spasticity in children after traumatic brain injury is often overlooked as there are more pressing issues to attend to in the early phase after injury. By the time the spasticity becomes a priority, often it is too late to make meaningful improvements without reverting to major corrective surgical techniques. There is also very little written on the topic of spasticity management after traumatic brain injury, especially in children. Most of the information we have is derived from stroke research. The focus of management strategies are largely medication use, physical therapy, and other physical rehabilitative strategies, with surgical management techniques used for long-term refractory cases only. With this manuscript, the authors aim to review our current understanding of the pathophysiology and management options, as well as prevention, of spasticity after traumatic brain injury in children.

Microglial Metabolism After Pediatric Traumatic Brain Injury - Overlooked Bystanders or Active Participants?

Microglia play an integral role in brain development but are also crucial for repair and recovery after traumatic brain injury (TBI). TBI induces an intense innate immune response in the immature, developing brain that is associated with acute and chronic changes in microglial function. These changes contribute to long-lasting consequences on development, neurologic function, and behavior. Although alterations in glucose metabolism are well-described after TBI, the bulk of the data is focused on metabolic alterations in astrocytes and neurons. To date, the interplay between alterations in intracellular metabolic pathways in microglia and the innate immune response in the brain following an injury is not well-studied. In this review, we broadly discuss the microglial responses after TBI. In addition, we highlight reported metabolic alterations in microglia and macrophages, and provide perspective on how changes in glucose, fatty acid, and amino acid metabolism can influence and modulate the microglial phenotype and response to injury.

Elevated Matrix Metalloproteinase Concentrations Offer Novel Insight Into Their Role in Pediatric Tuberculous Meningitis.

We collected lumbar and ventricular cerebrospinal fluid and serum from 40 children treated for tuberculous meningitis and measured the concentrations of gelatinases and their inhibitors. The concentrations of matrix metalloproteinase 9 (MMP-9), MMP-2, tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2 were significantly elevated in the lumbar CSF samples, and we found interesting dynamics for MMP-9 that offer novel insight into its role in pediatric patients with tuberculous meningitis.

Tuberculous meningitis in children is characterized by compartmentalized immune responses and neural excitotoxicity.

Tuberculous meningitis (TBM) is the most severe form of TB with high rates of mortality and morbidity. Here we conduct RNA-sequencing on whole blood as well as on ventricular and lumbar cerebrospinal fluid (CSF) of pediatric patients treated for TBM. Differential transcript expression of TBM cases are compared with healthy controls in whole blood and with non-TB cerebral infection controls in CSF. Whole blood RNA-Seq analysis demonstrates a distinct immune response pattern in TBM, with significant increase in both canonical and non-canonical inflammasome activation and decrease in T-cell activation. In ventricular CSF, a significant enrichment associated with neuronal excitotoxicity and cerebral damage is detected in TBM. Finally, compartmental comparison in TBM indicates that the ventricular profile represents brain injury whereas the lumbar profile represents protein translation and cytokine signaling. Together, transcriptomic analysis shows that disease processes differ between the periphery and the central nervous system, and within brain compartments.