Occludin deficiency with BACE1 elevation in cerebral amyloid angiopathy.

OBJECTIVE: A significant cause of spontaneous hemorrhages in the elderly is cerebral amyloid angiopathy (CAA), which causes degeneration of cerebral vessels, but the mechanisms are unclear. METHODS: We isolated leptomeningeal vessels from rapidly autopsied brains (the average of postmortem intervals was 3.28 hours) from 9 patients with CAA and 10 age-matched controls, and used molecular, cell biology, and immunohistochemical approaches to examine ß-site APP-cleaving enzyme 1 (BACE1) protein expression and enzymatic activities as well as tight junction molecular components in small- and medium-sized arteries of the cerebral cortex and leptomeninges. RESULTS: We not only identified that the cerebral vessels, including leptomeningeal and cortical vessels, synthesize and express BACE1, but also found a significant elevation of both BACE1 protein levels and enzymatic activities in leptomeningeal vessels from patients with CAA. Moreover, overexpression of BACE1 in endothelial cells resulted in a significant reduction of occludin, a tight junction protein in blood vessels. CONCLUSION: These findings suggest that in addition to neurons, cerebral vascular cells express functional BACE1. Moreover, elevated vascular BACE1 may contribute to deficiency of occludin in cerebral vessels, which ultimately has a critical role in pathogenesis of CAA and its related hemorrhage.

St. John's wort reversal of meningeal nociception: a natural therapeutic perspective for migraine pain.

Despite a number of antimigraine drugs belonging to different pharmacological classes are available, there is a huge unmet need for better migraine pharmacotherapy. We here demonstrated the capability of Hypericum perforatum, popularly called St. John's wort (SJW), to relieve meningeal nociception in an animal model induced by administration of the nitric oxide (NO) donors glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). GTN and SNP produced a delayed meningeal inflammation, as showed by the upregulation of interleukin (IL)-1ß and inducible NO synthase (iNOS), and a prolonged cold allodynia and heat hyperalgesia with a time-course consistent with NO-induced migraine attacks. A single oral administration of a SJW dried extract (5mg/kg p.o.) counteracted the nociceptive behaviour and the overexpression of IL-1ß and iNOS. To clarify the cellular pathways involved, the expression of protein kinase C (PKC) and downstream effectors was detected. NO donors increased expression and phosphorylation of PKCγ, PKCɛ and transcription factors, such as nuclear factor (NF)-κB, cyclic AMP response element binding protein (CREB), Signal Transducer and Activator of Transcription (STAT)-1. All these molecular events were prevented by SJW and hypericin, a SJW main component. In conclusion, SJW counteracted the NO donor-induced pain hypersensitivity and meningeal activation by blocking PKC-mediated pathways involving NF-κB, CREB, STAT1. These results might suggest SJW as an innovative and safe perspective for migraine pain.

Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors.

OBJECTIVE: To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG). METHODS: Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO. RESULTS: Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization. INTERPRETATION: The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

Activation of PI3 kinase/Akt signaling in chronic subdural hematoma outer membranes.

Chronic subdural hematoma (CSDH) is an angiogenic disease that is recognized as a cause of treatable dementia with unknown pathogenesis. Vascular endothelial growth factor (VEGF), a potent growth factor regulating angiogenesis through the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, has been implicated in its etiology. The status of this signaling pathway in CSDH outer membranes was examined in the present study, using outer membranes obtained during trepanation surgery. Expressions of PI3-kinase, PKB-kinase, Akt, phosphorylated Akt at Ser(473) (p-Akt), endothelial nitric oxide synthase (eNOS), vascular endothelial-cadherin (VE-cadherin), and actin were examined by Western blot analysis, together with their immunohistochemistry. PI3-kinase, Akt, eNOS, and VE-cadherin were detected in all cases. The magnitude of the expression of p-Akt varied among cases; however, the localization was revealed to be present in endothelial cells of vessels in CSDH outer membranes, together with VEGF and VE-cadherin detected in endothelial cells of vessels. These findings suggest that the PI3-kinase/Akt signaling is activated in CSDH outer membranes, and indicate the possibility that the PI3 kinase/Akt pathway might be activated by VEGF and play a critical role in the angiogenesis of CSDH.

Transforming growth factor-beta1 induces matrix metalloproteinase-9 expression in human meningeal cells via ERK and Smad pathways.

Transforming growth factor (TGF)-beta1, a cytokine released into the cerebrospinal fluid (CSF) after intraventricular hemorrhage (IVH), stimulates the expression of the components of the extracellular matrix (ECM), which causes progressive ventricular dilatation by impaired CSF absorption. Matrix metalloproteinase-9 (MMP-9), a proteinase involved in the removal of ECM proteins, has been shown to contribute to the resolution of progressive ventricular dilation after IVH. The aim of this study is to clarify the mechanism by which MMP-9 is expressed following IVH. Cultured human meningeal cells were treated with human recombinant TGF-beta1. RT-PCR demonstrated that TGF-beta1 induced MMP-9 expression in the meningeal cells in a dose-dependent manner. The TGF-beta1-induced MMP-9 expression was attenuated in the presence of either MEK or Smad 3 inhibitor. Our data indicated that MMP-9 is released into the CSF from meningeal cells in response to TGF-beta1, most probably through the activation of ERK and Smad pathways.

Immunohistochemical analysis of MMP-9, MMP-2 and TIMP-1, TIMP-2 expression in the central nervous system following infection with viral and bacterial meningitis.

Matrix metalloproteinases (MMPs) are capable of degrading components of the basal lamina of cerebral vessels, thereby disrupting the blood-brain barrier and inducing leukocyte recruitment. This study provides comprehensive information regarding the cell specificity of matrix metalloproteinases (MMP-2, MMP-9) and their binding tissue inhibitors (TIMP-1, TIMP-2) in the central nervous system during viral and bacterial meningitis. Specifically, we evaluated the immunoreactivity of MMPs and TIMPs in various cell types in brain parenchyma and meninges obtained from autopsy tissues. We found that a higher proportion of endothelial cells were positive for MMP-9 during meningitis when compared to controls. In addition, the immunoreactivity of MMP-9 decreased and the immunoreactivity of TIMP-1 increased in astrocytes upon infection. Furthermore, the results of this study revealed that mononuclear cells were highly immunoreactive for TIMP-1, TIMP-2 and MMP-9 during viral meningitis and that the expression of TIMPs in polymorphonuclear cells was even higher during bacterial meningitis. Taken together the results of this study indicated that the central nervous system resident cells and inflammatory infiltrates contribute to MMPs activity and that the expression patterns vary between cell types and in response to viral and bacterial meningitis.

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes.

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as beta-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.

Effects of interleukin-1beta and prostaglandin E2 on prostaglandin D synthase production in cultivated rat leptomeningeal cells.

Although the interleukin (IL)-1 receptor is densely distributed in the leptomeninges constituting the blood/cerebrospinal fluid barrier, its physiologic significance has remained unclear. In the present study, we show that in cultured leptomeningeal cells, IL-1beta, tumor necrosis factors, or lipopolysaccharide causes a prominent increase in the synthesis and release of prostaglandin (PG) D synthase, which catalyzes the final step in the biosynthesis of PGD2. Although significant increases in the amount of PGD synthase were also observed with cells exposed to somatostatin, thrombin, or ciliary neurotrophic factor, these were much smaller than were those induced by the proinflammatory cytokines. Other agents tested including IGF-I had no effect upon the enzyme levels in the culture media. Furthermore, we found that the increased secretion of PGD synthase by IL-1beta was completely inhibited by 10(-7) M PGE2. The same dose of PGD2 or 15-deoxy-Delta(12-14)PGJ2 had no effect upon the IL-1beta action. In addition, PGE2 increased the level of fibronectin and eliminated the expression of zonula occludentes-1, a tight junction-associated protein from cultured cells, effects likely reflecting a loss of barrier integrity. These results demonstrate the importance of inflammatory stimuli as a physiologic regulator of the leptomeningeal cell function.

Dominant localization of adenosine deaminase in leptomeninges and involvement of the enzyme in sleep.

Adenosine is an endogenous hypnotic molecule. However, the mechanism by which the level of extracellular adenosine is regulated remains to be elucidated. We found by Northern hybridization and enzyme assay that ecto-5(')-nucleotidase and adenosine deaminase (ADA), major enzymes responsible for the production and degradation of adenosine, respectively, were localized most abundantly in the leptomeninges within the rat brain. Immunohistochemical study showed that ADA was dominantly localized in arachnoid barrier and trabecular cells of the leptomeninges. In vivo microdialysis demonstrated that externally applied adenosine was rapidly metabolized by ADA to inosine in the subarachnoid space. Perfusion of an ADA inhibitor, coformycin, increased the extracellular adenosine level in the subarachnoid space under the rostral basal forebrain. When coformycin was continuously infused into the subarachnoid space, non-rapid eye movement sleep was increased with prolonged duration of the sleep episode. These results demonstrate that the leptomeninges control the extracellular level of adenosine in the subarachnoid space by their high 5(')-nucleotidase and ADA activities and regulate non-rapid eye movement sleep.

The meninges is a source of retinoic acid for the late-developing hindbrain.

One general function for retinoic acid (RA) is pattern organization in the CNS. This regulatory factor has an essential role in spinal cord motor neuron and early posterior hindbrain development. In the anterior CNS, however, there is only a limited number of foci of RA synthesis, and less attention has been placed on regions such as the anterior hindbrain where RA synthesizing enzymes are absent. This study shows that a rich source of RA lies around the hindbrain from the RA synthetic enzyme retinaldehyde dehydrogenase-2 (RALDH2) present in the surrounding meninges and mesenchyme by embryonic day 13. RALDH2 is not distributed uniformly throughout the meninges but is restricted to territories over the developing hindbrain, suggesting that RA signaling may be localized to those regions. Further regulation of RA signaling is provided by the presence of a RA sink in the form of the CYP26B1 RA catabolic enzyme expressed in deeper regions of the brain. As a guide to the neural anatomy of hindbrain RA signaling, we used a mouse transgenic for a lacZ reporter gene driven by a RA response element (RAREhsplacZ) to identify regions of RA signaling. This reporter mouse provides evidence that RA signaling in the hindbrain after embryonic day 13 occurs in the regions of the cerebellum and precerebellar system adjacent to sources of RA, including the inferior olive and the pontine nuclei.

Nitric oxide synthase inhibition lowers activity of neurons with meningeal input in the rat spinal trigeminal nucleus.

Nitric oxide is thought to control transmitter release and neuronal activity in the spinal dorsal horn and the spinal trigeminal nucleus, where nociceptive information from extra- and intracranial tissues is processed. Extracellular impulse activity was recorded from neurons in the rat spinal trigeminal nucleus with afferent input from the cranial dura mater. In contrast to the inactive isomer D-NAME, infusion of the nitric oxide synthase inhibitor L-NAME (20 mg/kg) significantly reduced neuronal activity and increased systemic blood pressure. It is concluded that nitric oxide production contributes to the ongoing activity of sensitized neurons in the spinal trigeminal nucleus. The results suggest that nitric oxide may be involved in the generation and maintenance of primary headaches such as migraine.

Expression of a membrane-bound form of the ferroxidase ceruloplasmin by leptomeningeal cells.

Ceruloplasmin is a key enzyme involved in detoxifying ferrous iron, which can generate free radicals. The secreted form of ceruloplasmin is produced by the liver and is abundant in serum. We have previously identified a membrane-bound glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin (GPI-Cp) that is expressed by astrocytes in the central nervous system (CNS) (Patel and David. 1997. J Biol Chem 272:20185-20190). We now provide direct evidence that rat leptomeningeal cells, which cover the surface of the brain, also express GPI-Cp. The expression of GPI-Cp on the surface of these cells increases with postnatal development and is regulated in vitro by cell density, time in culture, and various extracellular matrix molecules. The expression of GPI-Cp also appears to be regulated differently in astrocytes and leptomeningeal cells in vitro. The abundant expression of GPI-Cp on the surface of leptomeningeal cells suggests that these cells play a role in antioxidant defense along the surface of the postnatal CNS possibly by detoxifying the cerebrospinal fluid.

Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells.

The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (-1250 to +77) showed that an AP-2 element at -109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1 beta to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-kappa B elements at -1106 and -291 were essential for this up-regulation. Binding of two NF-kappa B subunits, p65 and c-Rel, to these two NF-kappa B elements occurred after the interleukin-1 beta treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-kappa B system.

Increase in meningeal blood flow by nitric oxide--interaction with calcitonin gene-related peptide receptor and prostaglandin synthesis inhibition.

This study addresses possible interactions of the vasodilators nitric oxide (NO), calcitonin gene-related peptide (CGRP) and prostaglandins, which may be implicated in the generation of vascular headaches. Local application of the NO donator diethylamine-NONOate (NONOate) to the exposed dura mater encephali of the rat caused dose-dependent increases in meningeal blood flow recorded by laser Doppler flowmetry. Pre-application of the CGRP receptor antagonist CGRP8-37 significantly attenuated the evoked blood flow increases, while the cyclooxygenase inhibitors acetylsalicylic acid and metamizol were only marginally effective. Stimulation of rat dura mater with NONOate in vitro caused increases in CGRP release. NADPH-diaphorase activity indicating NO production was restricted to the endothelium of dural arterial vessels. We conclude that increases in meningeal blood flow caused by NO depend partly on the release and vasodilatory action of CGRP from dural afferents, while prostaglandins are not significantly involved.

Retinoic acid synthesis for the developing telencephalon.

The small lipid retinoic acid is known to promote neuronal differentiation in vitro and to act as a teratogen in the embryonic brain, but very little is known about the natural role of endogenously synthesized retinoic acid in forebrain development. Retinoic acid is synthesized mainly by three retinaldehyde dehydrogenases. We show here where the retinaldehyde dehydrogenases for the developing telencephalon are expressed and how their expression patterns change over developmental time. Retinoic acid diffusing from the retinaldehyde dehydrogenase sites is likely to influence the early telencephalon before the beginning of neurogenesis, as well as differentiation and radial migration of neurons into the cerebral cortex. Because of its diffusible character, retinoic acid represents a unique tool for the coordination of growth processes over an intermediate distance range in the developing telencephalon.

Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid.

BACKGROUND AND PURPOSE: Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF. METHODS: We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or beta-galactosidase (Adbeta-gal) into the cisterna magna of rabbits. RESULTS: Total SOD activity in CSF was 39+/-11 U/mL (mean+/-SE) before virus injection. Three days later, total SOD activity in CSF increased to 148+/-22 U/mL after AdEC-SOD and 92+/-10 U/mL after AdCuZnSOD (P:<0.05 versus AdEC-SOD), with no change after Adbeta-gal (49+/-5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adbeta-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adbeta-gal. CONCLUSIONS: -We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Cyclooxygenase-2 immunoreactivity in the ischemic neonatal human brain. An autopsy study.

Cyclooxygenase-2 (COX-2) is known to be expressed in rat brain and up-regulated by ischemia. The administration of COX inhibitors before as well as soon after the ischemic insult reduces the extension of cerebral damage in rats. Overexpression of COX-2 has also been shown in the ischemic brain of adult human patients, while no information concerning COX-2 expression in neonatal ischemia is available. Intrapartum asphyxia and perinatal brain injury may result in cerebral palsy, mental retardation or epilepsy. COX-2 expression in the brain of neonates delivered after severe birth asphyxia was investigated using immunohistochemistry. Meningeal vessel walls of term and preterm babies widely expressed COX-2 immunoreactivity, as did periventricular large vessels in preterms. A number of brain cells (mature and immature cortical, periventricular and basal ganglia neurons, and oligodendrocytes of the cerebral white matter in brains from term neonates) also expressed COX-2. The present findings suggest that COX-2 may take part in enhancing neonatal brain damage via different mechanisms, such as those involving excitotoxicity and production of reactive oxygen species.

Cellular localization of lipocalin-type prostaglandin D synthase (beta-trace) in the central nervous system of the adult rat.

We applied high-resolution laser-scanning microscopy, electron microscopy, and non-radioactive in situ hybridization histochemistry to determine the cellular and intracellular localization of lipocalin-type prostaglandin D synthase, the major brain-derived protein component of cerebrospinal fluid, and its mRNA in leptomeninges, choroid plexus, and parenchyma of the adult rat brain. Both immunoreactivity and mRNA for prostaglandin D synthase were located in arachnoid barrier cells, arachnoid trabecular cells, and arachnoid pia mater cells. Furthermore, meningeal macrophages and perivascular microglial cells, identified by use of ED2 antibody, were immunopositive for prostaglandin D synthase. In the arachnoid trabecular cells, the immunoreactivity for prostaglandin D synthase was located in the nuclear envelope, Golgi apparatus, and secretory vesicles, indicating the active production and secretion of prostaglandin D synthase. In the meningeal macrophages, prostaglandin D synthase was not found around the nucleus but in lysosomes in the cytoplasm, pointing to an uptake of the protein from the cerebrospinal fluid. Furthermore, the existence of meningeal cyclooxygenase (COX) -1 and COX-2 was investigated by Western blot, Northern blot, and reverse transcriptase-polymerase chain reaction (RT-PCR), and the colocalization of COX-2 and prostaglandin D synthase was demonstrated in virtually all cells of the leptomeninges, choroid plexus epithelial cells, and perivascular microglial cells, suggesting that these cells synthesize prostaglandin D(2) actively. Alternatively, oligodendrocytes showed prostaglandin D synthase immunoreactivity without detectable COX-2. The localization of lipocalin-type prostaglandin D synthase in meningeal cells and its colocalization with COX-2 provide evidence for its function as a prostaglandin D(2)-producing enzyme.

Acetylcholinesterase and butyrylcholinesterase are expressed in the spinal meninges of monkeys and pigs.

BACKGROUND: Acetylcholinesterase inhibition at the spinal level has been shown to produce a potent antinociceptive effect. However, the site of cholinesterase inhibition is unknown. To determine whether the spinal meninges participate in acetylcholine metabolism, the spinal meninges of monkeys and pigs were assayed for cholinesterase activity. METHODS: Spinal cord, dura mater, and arachnoid mater specimens from anesthetized pigs and monkeys were mechanically homogenized and cholinesterase activity was determined quantitatively using a commercially available colorimetric assay. The ability of neostigmine to inhibit cholinesterase activity in vitro was also measured. Finally, the reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the cholinesterase metabolizing enzymes expressed by the spinal meninges. RESULTS: All spinal cord and meningeal specimens showed cholinesterase activity. In pigs, the dura mater showed less enzyme activity (36 +/- 17.7 U/mg protein) than the arachnoid mater (73.4 +/- 30.3 U/mg protein; P < 0.05), and the arachnoid mater showed less activity than the spinal cord (131.3 +/- 55.2 U/mg protein; P < 0.05). In monkeys, the dura mater again showed less cholinesterase activity (45.8 +/- 20.1 U/mg protein; P < 0.05), whereas cholinesterase activity in the arachnoid mater (90.3 +/- 45.9 U/mg protein) and spinal cord specimens (101.9 +/- 37.5 U/mg protein) were not significantly different. There were no significant species-related differences in cholinesterase activity. Neostigmine inhibited cholinesterase activity in a log-dose-dependent manner. The RT-PCR identified mRNA for acetylcholinesterase and butyrylcholinesterase in monkey pia-arachnoid mater. CONCLUSIONS: These data show that the spinal meninges express acetylcholinesterase and butyrylcholinesterase; for monkeys, although not pigs, the level of cholinesterase activity is comparable with that found in the spinal cord. This finding suggests that the meninges may be an important site for acetylcholine metabolism and may play a role in the analgesic effect produced by intrathecally administered cholinesterase inhibitors.