Human Meninges Research Articles

DNA methylation profile of human dura and leptomeninges.

Healthy meninges are used as control tissue in meningioma studies usually without specification of the exact meningeal layer or macroanatomical origin but the DNA methylation profile of human meninges has not been investigated on a macroanatomical level. We undertook a proof-of-principle analysis to determine whether (1) meningeal tissues show sufficiently homogenous DNA methylation profiles to function as normal control tissue without further specification and (2) if previously described location-specific molecular signatures of meningiomas correspond to region-specific DNA methylation patterns. Dura mater and arachnoid membrane specimens were dissected from 5 anatomical locations in 2 fresh human cadavers and analyzed with the Illumina Infinium MethylationEPIC array. Dura and leptomeninges showed marked differences in global DNA methylation patterns and between rostral and caudal anatomical locations. These differences did not reflect known anatomical predilection of meningioma molecular signatures. The highest numbers of differentially methylated probes were annotated to DIPC2 and FOXP1. Samples from foramen magnum showed hypomethylation of TFAP2B compared to those from remaining locations. Thus, the DNA methylation profiles of human meninges are heterogenous in terms of meningeal layer and anatomical location. The potential variability of DNA methylation data from meningiomas should be considered in studies using meningeal controls.

Potent dual MAGL/FAAH inhibitor AKU-005 engages endocannabinoids to diminish meningeal nociception implicated in migraine pain

BackgroundEngaging the endocannabinoid system through inhibition of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), degrading endocannabinoids (endoCBs) 2-arachidonoylglycerol (2-AG) and anandamide (AEA), was proposed as a promising approach to ameliorate migraine pain. However, the activity of MAGL and FAAH and action of endoCB on spiking activity of meningeal afferents, from which migraine pain originates, has not been explored thus far. Therefore, we here explored the analgesic effects of endoCB enhancement in rat and human meningeal tissues.MethodsBoth MAGL and FAAH activity and local 2-AG and AEA levels were measured by activity-based protein profiling (ABPP) and LC–MS/MS, respectively, in rat meninges obtained from hemiskulls of P38-P40 Wistar rats and human meninges from elderly patients undergoing non-migraine related neurosurgery. The action on endoCBs upon administration of novel dual MAGL/FAAH inhibitor AKU-005 on meningeal afferents excitability was tested by investigating paired KCl-induced spiking and validation with local (co-)application of either AEA or 2-AG. Finally, the specific TRPV1 agonist capsaicin and blocker capsazepine were tested.ResultsThe basal level of 2-AG exceeded that of AEA in rat and human meninges. KCl-induced depolarization doubled the level of AEA. AKU-005 slightly increased spontaneous spiking activity whereas the dual MAGL/FAAH inhibitor significantly decreased excitation of nerve fibres induced by KCl. Similar inhibitory effects on meningeal afferents were observed with local applications of 2-AG or AEA. The action of AKU-005 was reversed by CB1 antagonist AM-251, implying CB1 receptor involvement in the anti-nociceptive effect. The inhibitory action of AEA was also reversed by AM-251, but not with the TRPV1 antagonist capsazepine. Data cluster analysis revealed that both AKU-005 and AEA largely increased long-term depression-like meningeal spiking activity upon paired KCl-induced spiking.ConclusionsIn the meninges, high anti-nociceptive 2-AG levels can tonically counteract meningeal signalling, whereas AEA can be engaged on demand by local depolarization. AEA-mediated anti-nociceptive effects through CB1 receptors have therapeutic potential. Together with previously detected MAGL activity in trigeminal ganglia, dual MAGL/FAAH inhibitor AKU-005 appears promising as migraine treatment.Graphical

CSIG-36. NOTCH3 DRIVES MENINGIOMA TUMORIGENESIS AND RESISTANCE TO RADIOTHERAPY

Abstract There are no approved targeted therapies for meningiomas and the cell types underlying meningeal tumorigenesis are incompletely understood. To address these limitations, we performed single-cell RNA sequencing of 57,114 cells from 8 human meningiomas and 54,607 cells from 3 canine meningiomas. Pseudotime, gene ontology, and copy number variant analyses revealed a population of pericyte-like meningioma cells that were conserved across human and canine tumors and were enriched in expression of Notch3 and other cancer stem cell genes. Deconvolution of cell types from bulk RNA sequencing and DNA methylation profiling of 200 human meningiomas integrated with immunohistochemistry (IHC), immunofluorescence (IF), and RNAScope demonstrated Notch3+ pericytes and Notch3 expression were enriched in high grade or Immune-enriched meningiomas, which were distinguished from other meningioma DNA methylation groups by genes driving vasculature development. IHC and IF of human meninges integrated with lineage tracing approaches using Notch3-CreERT2 ROSAmT/mG alleles in mice demonstrated Notch3 expression was restricted to the perivascular stem cell niche during meningeal development and homeostasis. Mice harboring Notch3-CreERT2 Nf2fl/fl alleles developed meningeal hyperproliferation. Overexpression of constitutively activated Notch3 (Notch3AICD) in Immune-enriched human meningioma cells increased the expression of cancer stem cell genes, driving clonogenic growth in vitro, limiting dilution tumor-initiating capacity in vivo, and resistance to radiotherapy in vivo. A selective Notch3 neutralizing antibody (αNRR3) blocked meningioma cell proliferation and expression of Notch3 target genes, inhibiting meningioma xenograft growth and prolonging overall survival. Single-cell RNA sequencing of 187,366 cells from meningioma xenografts after αNRR3 or radiotherapy treatment ± Notch3AICD overexpression revealed distinct meningioma cell-intrinsic or cell-extrinsic mechanisms driving responses to radiotherapy or αNRR3, respectively. Combined treatment with αNRR3 and radiotherapy additively blocked meningioma xenograft growth and extended survival benefit. In sum, these data shed light on a novel cell type, molecular mechanism, and therapeutic vulnerability in the most common primary intracranial tumor.

Evaluation of an in vivo preclinical model for human middle meningeal artery embolization using the posterior intercostal artery of the swine

BackgroundEmbolization of the middle meningeal artery (MMA) is a promising minimally invasive technique that is gaining traction in the treatment of chronic subdural hematoma. Unfortunately, the human meninges and associated...

ICP-MS Multi-Elemental Analysis of the Human Meninges Collected from Sudden Death Victims in South-Eastern Poland.

Metals perform many important physiological functions in the human body. The distribution of elements in different tissues is not uniform. Moreover, some structures can be the site of an accumulation of essential or toxic metals, leading to multi-directional intracellular damage. In the nervous system, these disorders are especially dangerous. Metals dyshomeostasis has been linked to a variety of neurological disorders which end up leading to permanent injuries. The multi-elemental composition of the human brain is still the subject of numerous investigations and debates. In this study, for the first time, the meninges, i.e., the dura mater and the arachnoid, were examined for their elemental composition by means of inductively coupled plasma mass spectrometry (ICP-MS). Tissue samples were collected post mortem from those who died suddenly as a result of suicide (n = 20) or as a result of injuries after an accident (n = 20). The interactions between 51 elements in both groups showed mainly weak positive correlations, which dominated the arachnoid mater compared to the dura mater. The study showed differences in the distribution of some elements within the meninges in the studied groups. The significant differences concerned mainly metals from the lanthanide family (Ln), macroelements (Na, K, Ca, Mg), a few micronutrients (Co), and toxic cadmium (Cd). The performed evaluation of the elemental distribution in the human meninges sheds new light on the trace metals metabolism in the central nervous system, although we do not yet fully understand the role of the human meninges.

STEM-27. A PERIVASCULAR STEM CELL UNDERLIES VERTEBRATE MENINGEAL TUMORIGENESIS

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

Gut-educated IgA plasma cells defend the meningeal venous sinuses.

The central nervous system has historically been viewed as an immune-privileged site, but recent data have shown that the meninges-the membranes that surround the brain and spinal cord-contain a diverse population of immune cells1. So far, studies have focused on macrophages and T cells, but have not included a detailed analysis of meningeal humoral immunity. Here we show that, during homeostasis, the mouse and human meninges contain IgA-secreting plasma cells. These cells are positioned adjacent to dural venous sinuses: regions of slow blood flow with fenestrations that can potentially permit blood-borne pathogens to access the brain2. Peri-sinus IgA plasma cells increased with age and following a breach of the intestinal barrier. Conversely, they were scarce in germ-free mice, but their presence was restored by gut re-colonization. B cell receptor sequencing confirmed that meningeal IgA+ cells originated in the intestine. Specific depletion of meningeal plasma cells or IgAdeficiency resulted in reduced fungal entrapment in the peri-sinus region and increased spread into the brain following intravenous challenge, showing that meningeal IgA is essential for defending the central nervous system at this vulnerable venous barrier surface.

Single-Cell Transcriptomic Analyses of the Developing Meninges Reveal Meningeal Fibroblast Diversity and Function.

The meninges are a multilayered structure composed of fibroblasts, blood and lymphatic vessels, and immune cells. Meningeal fibroblasts secrete a variety of factors that control CNS development, yet strikingly little is known about their heterogeneity or development. Using single-cell sequencing, we report distinct transcriptional signatures for fibroblasts in the embryonic dura, arachnoid, and pia. We define new markers for meningeal layers and show conservation in human meninges. We find that embryonic meningeal fibroblasts are transcriptionally distinct between brain regions and identify a regionally localized pial subpopulation marked by the expression of μ-crystallin. Developmental analysis reveals a progressive, ventral-to-dorsal maturation of telencephalic meninges. Our studies have generated an unparalleled view of meningeal fibroblasts, providing molecular profiles of embryonic meningeal fibroblasts by layer and yielding insights into the mechanisms of meninges development and function.

A perfusion bioreactor-based 3D model of the subarachnoid space based on a meningeal tissue construct

BackgroundAltered flow of cerebrospinal fluid (CSF) within the subarachnoid space (SAS) is connected to brain, but also optic nerve degenerative diseases. To overcome the lack of suitable in vitro models that faithfully recapitulate the intricate three-dimensional architecture, complex cellular interactions, and fluid dynamics within the SAS, we have developed a perfusion bioreactor-based 3D in vitro model using primary human meningothelial cells (MECs) to generate meningeal tissue constructs. We ultimately employed this model to evaluate the impact of impaired CSF flow as evidenced during optic nerve compartment syndrome on the transcriptomic landscape of MECs.MethodsPrimary human meningothelial cells (phMECs) were seeded and cultured on collagen scaffolds in a perfusion bioreactor to generate engineered meningeal tissue constructs. Engineered constructs were compared to human SAS and assessed for specific cell–cell interaction markers as well as for extracellular matrix proteins found in human meninges. Using the established model, meningeal tissue constructs were exposed to physiological and pathophysiological flow conditions simulating the impaired CSF flow associated with optic nerve compartment syndrome and RNA sequencing was performed.ResultsEngineered constructs displayed similar microarchitecture compared to human SAS with regards to pore size, geometry as well as interconnectivity. They stained positively for specific cell–cell interaction markers indicative of a functional meningeal tissue, as well as extracellular matrix proteins found in human meninges. Analysis by RNA sequencing revealed altered expression of genes associated with extracellular matrix remodeling, endo-lysosomal processing, and mitochondrial energy metabolism under pathophysiological flow conditions.ConclusionsAlterations of these biological processes may not only interfere with critical MEC functions impacting CSF and hence optic nerve homeostasis, but may likely alter SAS structure, thereby further impeding cerebrospinal fluid flow. Future studies based on the established 3D model will lead to new insights into the role of MECs in the pathogenesis of optic nerve but also brain degenerative diseases.

Neurodegenerative Dementias: Improving Brain Health to Decrease Risk.

Neurodegenerative Dementias: Improving Brain Health to Decrease Risk.

Proteomic analysis discovers the differential expression of novel proteins and phosphoproteins in meningioma including NEK9, HK2 and SET and deregulation of RNA metabolism.

BackgroundMeningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is hampered by poor knowledge of their proteomic signature. There is an urgent need to identify new therapeutic targets and biomarkers of meningioma.MethodsWe performed proteomic profiling of grade I, II and III frozen meningioma specimens and three normal healthy human meninges using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry.FindingsWe quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades and normal meninges. Bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation of the total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins identified and validated in meningioma included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, HK2, involved in cellular metabolism.InterpretationOverall, we generated a proteomic thesaurus of meningiomas for the identification of potential biomarkers and therapeutic targets.FundThis study was supported by Brain Tumour Research.

Electron Microscopy of Dural and Arachnoid Disruptions After Subarachnoid Block

(Reg Anesth Pain Med 2017;42:709–718) Postdural puncture headache (PDPH) is commonly explained by intracranial hypotension due to cerebrospinal fluid leakage. An association between larger size dural punctures and an increased incidence of PDPH has been widely accepted. It was generally believed that pencil-point type needles produced smaller dural lesions by allowing separation rather than cutting of the dural fibers, but this has not been supported by morphologic evidence. There has been little investigation on the morphology of dural lesions produced by spinal needles. This study aimed to study human meninges and the morphology of dura-arachnoid lesions produced by 27 G Whitacre and 29 G Quincke spinal needles. The study also sought to determine whether there was a correlation between the diameter of the lesions and the needles used. Finally, authors aimed to determine whether the lesion diameter was altered when the 29 G needle bevel was oriented vertically rather than horizontally.

PO-522 Global proteome and phosphoprotein profiling of meningiomas reveals novel potential therapeutic targets and biomarkers

IntroductionMeningiomas are the most common primary intracranial brain tumour arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate the pathogenic signature of these tumours and identify novel molecular targets by deciphering the global proteome and phosphoprotein profile of different grades of meningiomas.Material and methodsTumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionisation LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data were validated by Western blot and immunohistochemistry. In silico functional annotation of expression data was completed using Perseus 1.5.0.31 software suite, Ingenuity Pathway Analysis (IPA®) and DAVID 6.8.Results and discussionsWe have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis identified 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change ≥1.5; p<0.05) among all grades vs. normal meninges, and further identified differential expression profiles between meningioma grades. Expression data was validated on samples analysed by MS and on an additional cohort of meningiomas for upregulated proteins including SET, EGFR, SRSF1, CKAP4 and HK2; and phosphoproteins including AKT1, AKT2 and RB1. Gene Ontology revealed commonly upregulated proteins to be enriched in terms including RNA helicase activity, whilst phosphoproteins were enriched in the phosphoprotein associated terms of signal complex assembly, Rho guanyl-nucleotide exchange factor activity and EGFR signalling.ConclusionIn summary, we performed a comprehensive quantitative proteomic analysis from meningioma tissue of all WHO grades compared to healthy meninges and have identified several potential candidates that may hold therapeutic potential for targeted treatment of these tumours.

Global proteome and phosphoproteome analysis of meningiomas

Meningiomas are common brain tumours arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate dysregulated pathways in meningioma pathogenesis and identify novel molecular targets by deciphering the proteome and phosphoproteome of different grades of meningiomas. Tumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionization LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data was validated by Western blot and functionally annotated using Ingenuity® Pathway Analysis (IPA®) and DAVID 6.8. We have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis confirmed 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change≥1.5; p<0.05) among all grades vs. normal meninges. We have successfully validated the expression data of several upregulated proteins and phosphoproteins. Gene Ontology revealed biological processes including EFGR and VEGFR signalling to be enriched in the phosphoproteome. Grade-wise comparisons identified 667 proteins and 769 phosphoproteins to be differentially expressed (p<0.05) between grade III meningiomas compared to grade II and I. We have performed a comparative proteomic analysis across all meningioma grades and identified changes in proteomic profiles between these tumours and normal healthy meninges. We will use this data to define novel targets common to all grades and specific to a grade of meningioma for future therapies.

Abstract WP411: Identification and Characterization of Human Meningeal Lymphatics

Late onset Alzheimer’s Disease (AD) is the most common form of dementia, affecting 40 million patients and countless families worldwide. AD is characterized by accumulation of amyloid beta (Aß) in the brain and central nervous system (CNS). A growing body of evidence indicates that while Aß production is unchanged, its clearance from the CNS is attenuated in late onset AD. Therefore, it is critical to determine how Aβ is cleared from the brain and if these processes differ in healthy and AD patients. Recently, two groups independently reported that a network of meningeal lymphatic vasculature participates in the clearance of solutes and macromolecules from the brain and cerebrospinal fluid of mice. However the presence of meningeal lymphatic vessels and their potential role in the clearance of Aß has not yet been defined in humans. Therefore, to determine if human meningeal lymphatic vessels absorb Aβ, we used immunofluorescence to examine superior sagittal sinus-associated dura mater tissue in a cohort of 21 patient samples including 6 subjects with no diagnosed dementia (control), 7 subjects with histopathologically confirmed AD, and 8 with diagnosis of mixed or other dementia. We found podoplanin (PDPN) positive, lumenized vessels in 19/21 patient samples, with 5/6 in control patients, 7/7 in AD patients and 7/8 in other or mixed dementia patients. These vessels were located in the dura mater, lateral to the superior sagittal sinus, and ranged from approximately 10 to 500 microns in diameter. Trace Aβ immunoreactivity was colocalized with podoplanin-positive vessels in 1/5 control, 1/7 AD, and 1/7 other or mixed dementia patients. Aß reactivity was found in meningeal blood vessels of 0/6 control, 1/7 AD, and 0/7 other or mixed dementia patients. To our knowledge, these data are the first to robustly support the existence of lymphatic vasculature in the human meninges. The data further suggest that Aß does not appear to widely deposit along wither these meningeal lymphatic or blood vessels, even among AD subjects.

Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas

The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas.

Comparison of the Inflammatory Responses of Human Meningeal Cells following Challenge withNeisseria lactamicaand withNeisseria meningitidis

The rationale for the present study was to determine how different species of bacteria interact with cells of the human meninges in order to gain information that would have broad relevance to understanding aspects of the innate immune response in the brain. Neisseria lactamica is an occasional cause of meningitis in humans, and in this study we investigated the in vitro interactions between N. lactamica and cells derived from the leptomeninges in comparison with the closely related organism Neisseria meningitidis, a major cause of meningitis worldwide. N. lactamica adhered specifically to meningioma cells, but the levels of adherence were generally lower than those with N. meningitidis. Meningioma cells challenged with N. lactamica and N. meningitidis secreted significant amounts of the proinflammatory cytokine interleukin-6 (IL-6), the C-X-C chemokine IL-8, and the C-C chemokines monocyte chemoattractant protein 1 (MCP-1) and RANTES, but it secreted very low levels of the cytokine growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). Thus, meningeal cells are involved in the innate host response to Neisseria species that are capable of entering the cerebrospinal fluid. The levels of IL-8 and MCP-1 secretion induced by both bacteria were essentially similar. By contrast, N. lactamica induced significantly lower levels of IL-6 than N. meningitidis. Challenge with the highest concentration of N. lactamica (10(8) CFU) induced a small but significant down-regulation of RANTES secretion, which was not observed with lower concentrations of bacteria. N. meningitidis (10(6) to 10(8) CFU) also down-regulated RANTES secretion, but this effect was significantly greater than that observed with N. lactamica. Although both bacteria were unable to invade meningeal cells directly, host cells remained viable on prolonged challenge with N. lactamica, whereas N. meningitidis induced death; the mechanism was overwhelming necrosis with no significant apoptosis. It is likely that differential expression of modulins between N. lactamica and N. meningitidis contributes to these observed differences in pathogenic potential.

Microscopic morphology and histology of the human meninges

The meninges comprise the dura mater and the leptomeninges (arachnoid and pia mater). Dura forms an outer endosteal layer related to the bones of the skull and spine and an inner layer closely applied to the arachnoid mater. Leptomeninges have multiple functions and anatomical relationships. The outer parietal layer of arachnoid is impermeable to CSF due to tight intercellular junctions; elsewhere leptomeningeal cells form demosomes and gap junctions. Trabeculae of leptomeninges compartmentalize the subarachnoid space and join the pia to arachnoid mater. In bacterial meningitis leptomeningeal cells secrete cytokines. Pia mater is reflected from the surface of the brain and spinal cord onto arteries and veins, thus separating the subarachnoid space from the brain and cord. A sheath of leptomeninges accompanies arteries into the brain and is related to the pathways for the drainage of interstitial fluid that play a role in inflammatory responses in the brain and appear to be blocked by amyloid-beta in Alzheimer's disease. Specialised leptomeningeal cells in the stroma of the choroid plexus form collagen whorls that become calcified with age. Leptomeningeal cells also form channels in the core and apical cap of arachnoid granulations for the drainage of CSF into venous sinuses. In the spine, leptomeninges form highly perforated intermediate sheets of arachnoid and delicate ligaments that compartmentalize the subarachnoid space; dentate ligaments anchor subpial collagen to the dura mater and stabilize the spinal cord. Despite the multiple anatomical arrangements and physiological functions, leptomeningeal cells retain many histological features that are similar from site to site.

Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges.

The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.

Electron microscopy and the expansion of regional anesthesia knowledge

We describe the findings obtained with the use of the transmission and scanning electron microscopy in the study of human meninges and peripheral nerves. We show details about morphology and distribution of the fibers inside the dural sac. We provide information about the origin of subdural space and structural organization of the arachnoid trabeculates in sleeves that wrap the nervous roots inside the subarachnoid space. We encountered natural fenestrations in the pia mater and studied ultrastructural components of the peripheral nerves coverings. A brief comment on the history of the electron microscopy and the basics of these microscopes complete the presentation of this work. Copyright 2002, Elsevier Science (USA). All rights reserved.