The ultrastructure of spinal cord perivascular spaces: Implications for the circulation of cerebrospinal fluid.

Perivascular spaces play a pivotal role in the exchange between cerebrospinal and interstitial fluids, and in the clearance of waste in the CNS, yet their precise anatomical components are not well described. The aim of this study was to characterise the ultrastructure of perivascular spaces and their role in the transport of fluid, in the spinal cord of healthy rats, using transmission electron microscopy. The distribution of cerebrospinal fluid tracers injected into the subarachnoid space was studied using light, confocal and electron microscopy. Perivascular spaces were found around arterioles and venules, but not capillaries, throughout the spinal cord white and grey matter. They contained fibroblasts and collagen fibres, and were continuous with the extracellular spaces of the surrounding tissue. At 5 min post injection, tracers were seen in the subarachnoid space, the peripheral white matter, the perivascular spaces, basement membranes, extracellular spaces of the surrounding tissue, and surprisingly, in the lumen of blood vessels, suggesting trans-vascular clearance. These findings point out an unrecognised outflow pathway for CNS fluids, with potential implications for volume regulation in health and disease states, but also clinically for the detection of CNS-derived biomarkers in plasma, the immune response and drug pharmacokinetics.

Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma.

Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed 'glymphatic' clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.

[The cerebral perivascular spaces: review of the literature on diffuse or focal expansion]. / Gli spazi perivascolari encefalici: revisione della letteratura sulle dilatazioni focali o diffuse.

Virchow-Robin spaces (VRS) are pial-lined, interstitial fluid-filled structures that do not directly communicate with the subarachnoid space, accompany penetrating arteries and veins and can be visualized on magnetic resonance imaging. This article reviews the imageology characteristics, the functions, the causes and the relation with neurological disorders of VRS.

[Enlargment of Virchow-Robin spaces in cranial trauma: literature review]. / La dilatazione degli spazi di Virchow-Robin nel trauma cranico. Aspetti caratteristici in RM e revisione della letteratura.

In this review it is discussed the role of mild traumatic brain injury as a cause of Virchow-Robin spaces (VRS) pathological enlargement. Anatomy and physiology of normal VRS, and their immunological role are described. Special attention is given to magnetic resonance imaging findings of both normal and enlarged perivascular spaces.

Comparative pharmacokinetics of continuous and conventional intrathecal amphotericin B in rabbits.

We previously reported a new effective therapy, continuous intrathecal amphotericin B (AMB), for the treatment of cryptococcal meningitis, which had fewer side effects and complications than conventional intrathecal AMB. In this study, the pharmacokinetics of continuous intrathecal administration and conventional intrathecal AMB were compared in rabbits, providing a pharmacokinetic basis for the use of continuous intrathecal AMB therapy. The AMB concentration in the cerebrospinal fluid (CSF), sampled via an inserted cisterna magna catheter, was determined by a liquid chromatography-tandem mass spectrometry assay. The results revealed significant pharmacokinetic differences between the two groups. In the continuous intrathecal group (0.15 mg/kg/24 h), the concentration of AMB peaked 7.01 µg/ml at 4 h and then decreased to a stable level of 1.0 to 1.34 µg/ml, with no neurological impairments, while in the conventional intrathecal group (0.015 mg/kg), the drug concentration reached a peak of 3.41 µg/ml at 1 h and then decreased progressively, with fever and neurological impairments, including convulsion and paralysis. The pharmacokinetic results indicated that the continuous intrathecal AMB is a more effective and safe therapy than the conventional intrathecal AMB, with comparatively rational pharmacokinetics and fewer neurological impairments.

Morfología de la lámina aracnoidea espinal humana. Barrera que limita la permeabilidad del saco dural / Structure of the arachnoid layer of the human spinal meninges: a barrier that regulates dural sac permeability

OBJETIVOS: Se ha demostrado que las moléculas inyectadas en el espacio epidural pasan desde éste al espacio subaracnoideo por difusión simple a través de la pared del saco dural. Nuestro objetivo fue estudiar la ultraestructura de células de la lámina aracnoidea y tipo de uniones especializadas responsables del efecto barrera que gobierna el tránsito de moléculas a través del saco dural humano. MATERIAL Y MÉTODO: Se estudiaron catorce muestras de la lámina aracnoidea obtenidas de dos pacientes durante intervenciones con apertura del saco dural lumbar. Las muestras se trataron con glutaraldehido, tetróxido de osmio, ferrocianuro, acetona, e incluyeron en resina. Los cortes ultrafinos se contrastaron con citrato de plomo, para poder ser observados con un microscopio electrónico de transmisión. RESULTADOS: La lámina aracnoidea posee un espesor de 35-40 μm. En su porción externa se hallan células neuroteliales del compartimento subdural, mientras que su porción interna está formada por un plano celular de 5-8 μm de espesor, constituido por la superposición de 4-5 células aracnoideas que forman la capa barrera. El espacio intercelular de este plano fue de 0,02-0,03 μm. Entre las células aracnoideas se encontraron uniones especializadas de membrana de tipo desmosomas y uniones estrechas. CONCLUSIONES: Las células aracnoideas poseen características estructurales que aseguran la función barrera del saco dural humano y no ocupan todo el espesor de la lámina aracnoidea, sólo su porción interna. La presencia de uniones especializadas de membrana entre sus células justifica la permeabilidad selectiva de esta lámina(AU)

OBJETIVES: Drugs injected into the epidural space are known to penetrate the subarachnoid space by simple diffusion through the dural sac. We aimed to study the cellular ultrastructure of the arachnoid membrane and the type of intercellular junctions responsible for creating the barrier that regulates the passage of drugs through the dural sac in humans. MATERIAL AND METHODS: Fourteen tissue samples of arachnoid membrane were taken from 2 patients during procedures that required opening the lumbar dural sac. The samples were treated with glutaraldehyde, osmium tetroxide, ferrocyanide and acetone, and then embedded in resin. Ultrathin sections were stained with lead citrate for examination by transmission electron microscopy. RESULTS: The arachnoid membrane was 35 to 40 μm thick. The outer surface contained neurothelial cells (dural border cells) along the subdural compartment, while the internal portion was made up of a plane 5 to 8 μm thick with 4 to 5 arachnoid cells overlapping to form a barrier layer. The intercellular spaces on this plane were 0.02 to 0.03 μm wide; the arachnoid cells were bridged by specialized junctions (desmosomes and other tight junctions). CONCLUSIONS: Structural features of the arachnoid cells provide a barrier within the human dural sac. They occupy only the internal portion of the arachnoid membrane. Specialized intercellular junctions explain the selective permeability of this membrane(AU)

Primary cell culture of meningothelial cells--a new model to study the arachnoid in glaucomatous optic neuropathy.

BACKGROUND: In a previous report, we found that the occurrence and amount of meningothelial cell nests in the subarachnoid space are significantly increased in glaucomatous optic nerves compared to normals. In order to allow research into the role of meningothelial cells during diseases of the optic nerve, an in vitro model is necessary. For this purpose, we developed a culture method for porcine meningothelial cells from the arachnoid layer covering the optic nerve. METHODS: Meningothelial cells were scraped from the arachnoid layer of porcine optic nerves and cultured for 2-3 weeks until the cells formed a monolayer. To eliminate contaminating fibroblasts from the culture, cells were negatively selected using magnetic anti-fibroblast beads after the first passage. Cells were detached using 0.05% Trypsin-EDTA, incubated with anti-fibroblast beads, separated using a magnetic column and the flow-through was collected. The purified primary meningothelial cells were characterized by electron microscopy and immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) and anti-keratan sulfate antibodies. RESULTS: Primary cells grew out after dissection and formed a monolayer within 2-3 weeks, which was composed of two morphologically different cell types, flattened cells with round nuclei and fibroblast-like cells with long processes. The fibroblast-like cells in the culture could be labelled and selected using anti-fibroblast microbeads. The second cell type did not bind to the anti-fibroblast beads, and upon immunocytochemistry showed a marked expression of both GFAP and keratan sulphate. In addition, examination of these cells by electron microscopy revealed morphological characteristics of meningothelial cells, including hemidesmosomes and cytoplasmatic filaments. CONCLUSIONS: The technique described in this paper for the primary culture of meningothelial cells from the subarachnoid space of the optic nerve and using magnetic beads for the removal of fibroblasts is effective in obtaining a highly enriched meningothelial cell culture.

Effect of anatomical fine structure on the dispersion of solutes in the spinal subarachnoid space.

The dispersion of a solute bolus is calculated for cerebrospinal fluid undergoing oscillatory flow in the subarachnoid space of the spine. The fine structure of the subarachnoid space (nerves and trabeculae) enhances both longitudinal and transverse dispersions five to ten times over a simple model with an open annular space. Overall, dispersion is >10(3) times simple molecular diffusion. The result of enhanced dispersion is rapid spread and dilution of the bolus, effectively stirred by fluid movement around the fine structure.

The olfactory route for cerebrospinal fluid drainage into the peripheral lymphatic system.

Drainage of the cerebrospinal fluid through the olfactory nerves into the nasal lymphatics has been suggested repeatedly. To investigate precisely the morphology of this pathway, India ink was injected into the subarachnoidal space of the rat brain, and samples including the olfactory bulbs, olfactory tracts and the nasal mucosa were observed by light and electron microscopy. Under the dissecting microscope, ink particles were found within the subarachnoid space and along the olfactory nerves. At the nasal mucosa, a lymphatic network stained in black was identified near the olfactory nerves, which finally emptied into the superficial and deep cervical lymph nodes. Light microscopically, ink particles were found in the subarachnoid space, partially distributed around the olfactory nerves and within the lymphatic vessels. By electron microscopy, the subarachnoid space often formed a pocket-like space in the entrance of the fila olfactoria. The olfactory nerves were partially surrounded by ink particles within the space between perineurial cells and epineurial fibroblasts. At the nasal mucosa, the lymphatics were frequently located close to the nerves. These results indicate that the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics, which in turn, empties into the cervical lymph nodes. This anatomical communication, thus, allows the central nervous system to connect with the lymphatic system. The presence of this route may play an important role in the movement of antigens from the subarachnoidal space to the extracranial lymphatic vessels, resulting in inducement of an immune response of the central nervous system.

Effect of anatomical fine structure on the flow of cerebrospinal fluid in the spinal subarachnoid space.

The lattice Boltzmann method is used to model oscillatory flow in the spinal subarachnoid space. The effect of obstacles such as trabeculae, nerve bundles, and ligaments on fluid velocity profiles appears to be small, when the flow is averaged over the length of a vertebra. Averaged fluid flow in complex models is little different from flow in corresponding elliptical annular cavities. However, the obstacles stir the flow locally and may be more significant in studies of tracer dispersion.

The optic nerve: a new window into cerebrospinal fluid composition?

Cerebrospinal fluid (CSF) pressure and composition are generally thought to be homogeneous within small limits throughout all CSF compartments. CSF sampled during lumbar puncture therefore should be representative for all CSF compartments. On the basis of clinical findings, histology and biochemical markers, we present for the first time strong evidence that the subarachnoid spaces (SAS) of the optic nerve (ON) can become separated from other CSF compartments in certain ON disorders, thus leading to an ON sheath compartment syndrome. This may result in an abnormal concentration gradient of CSF molecular markers determined in locally sampled CSF compared with CSF taken during lumbar puncture.

Functional effectiveness of the blood-brain barrier to small water-soluble molecules in developing and adult opossum (Monodelphis domestica).

We have evaluated a small water-soluble molecule, biotin ethylenediamine (BED, 286 Da), as a permeability tracer across the blood-brain barrier. This molecule was found to have suitable characteristics in that it is stable in plasma, has low plasma protein binding, and appears to behave in a similar manner across brain barriers as established by permeability markers such as sucrose. BED, together with a 3000-Da biotin-dextran (BDA3000), was used to investigate the effectiveness of tight junctions in cortical vessels during development and adulthood of a marsupial opossum (Monodelphis domestica). Marsupial species are born at an early stage of brain development when cortical vessels are just beginning to appear. The tracers were administered systemically to opossums at various ages and localized in brains with light and electron microscopy. In adults, the tight junctions restricted the movement of both tracers. In neonates, as soon as vessels grow into the neocortex, their tight junctions are functionally restrictive, a finding supported by the presence of claudin-5 in endothelial cells. However, both tracers are also found within brain extracellular space soon after intraperitoneal administration. The main route of entry for the tracers into immature neocortex appears to be via the cerebrospinal fluid over the outer (subarachnoid) and inner (ventricular) surfaces of the brain. These experiments demonstrate that the previously described higher permeability of barriers to small molecules in the developing brain does not seem to be due to leakiness of cerebral endothelial tight junctions, but to a route of entry probably via the choroid plexuses and cerebrospinal fluid.

Architecture of arachnoid trabeculae, pillars, and septa in the subarachnoid space of the human optic nerve: anatomy and clinical considerations.

AIMS: To describe the anatomy and the arrangement of the arachnoid trabeculae, pillars, and septa in the subarachnoid space of the human optic nerve and to consider their possible clinical relevance for cerebrospinal fluid dynamics and fluid pressure in the subarachnoid space of the human optic nerve. METHODS: Postmortem study with a total of 12 optic nerves harvested from nine subjects without ocular disease. All optic nerves used in this study were obtained no later than 7 hours after death, following qualified consent for necropsy. The study was performed with transmission (TEM) and scanning electron microscopy (SEM). RESULTS: The subarachnoid space of the human optic nerve contains a variety of trabeculae, septa, and stout pillars that are arranged between the arachnoid and the pia layers of the meninges of the nerve. They display a considerable numeric and structural variability depending on their location within the different portions of the optic nerve. In the bulbar segment (ampulla), adjacent to the globe, a dense and highly ramified meshwork of delicate trabeculae is arranged in a reticular fashion. Between the arachnoid trabeculae, interconnecting velum-like processes are observed. In the mid-orbital segment of the orbital portion, the subarachnoid space is subdivided, and can appear even loosely chambered by broad trabeculae and velum-like septa at some locations. In the intracanalicular segment additionally, few stout pillars and single round trabeculae are observed. CONCLUSION: The subarachnoid space of the human optic nerve is not a homogeneous and anatomically empty chamber filled with cerebrospinal fluid, but it contains a complex system of arachnoid trabeculae and septa that divide the subarachnoid space. The trabeculae, septa, and pillars, as well as their arrangement described in this study, may have a role in the cerebrospinal fluid dynamics between the subarachnoid space of the optic nerve and the chiasmal cistern and may contribute to the understanding of the pathophysiology of asymmetric and unilateral papilloedema. All the structures described are of such delicate character that they can not even be visualised with high resolution magnetic resonance imaging (MRI).

Microsurgical anatomy of the Liliequist's membrane and surrounding neurovascular territories.

Since the original description by Liliequist only a few microanatomical studies of the Liliequist membrane have been performed. These studies contain some discrepancies in the description, boundaries, and attachments of the membrane. Using a surgical microscope the authors examined the microsurgical anatomy of Liliequist's membrane and surrounding neurovascular structures in twenty adult brains injected with silicone rubber, with special emphasis given to the analysis of controversial details. This description is intended as an aid for neurosurgeons performing neuroendoscopic procedures.

Fenestrated capillaries in subarachnoid space in the caudal spinal cord of the premature rat: an electron microscopic observation.

The fine structure of the conus medullaris of the spinal cord and surrounding structures were studied in postnatal developing rats, with special attention being paid to the vasculature. The most striking finding was the presence of fenestrae in the capillary endothelium of the subarachnoid space. These structures were not obvious in adult rats. The fenestrated capillaries may influence fluid dynamics in the subarachnoid space of the spinal cord at the level of the conus medullaris, during early stage of postnatal development.

The time course of intracranial pathophysiological changes following experimental subarachnoid haemorrhage in the rat.

The rat subarachnoid haemorrhage (SAH) model was further studied to establish the precise time course of the globally reduced CBF that follows and to ascertain whether temporally related changes in cerebral perfusion pressure (CPP) and intracranial pressure (ICP) take place. Parallel ultrastructural studies were performed upon cerebral arteries and their adjacent perivascular subarachnoid spaces. SAH was induced by a single intracisternal injection of autologous arterial blood. Serial measurements of regional cortical CBF by hydrogen clearance revealed that experimental SAH resulted in an immediate 50% global reduction in cortical flows that persisted for up to 3 h post SAH. At 24 h, flows were still significantly reduced at 85% of control values (p less than 0.05), but by 48 h had regained normal values and were maintained up to 5 days post SAH. ICP rose acutely after haemorrhage to nearly 50 mm Hg with C-type pressure waves being present. ICP then fell slowly, only fully returning to control levels at 72 h. Acute hydrocephalus was observed on autopsy examination of SAH animals but not in controls. Reductions in CPP occurred post SAH, but only in the order of 15%, which could not alone account for the fall in CBF that took place. At 48 and, to a lesser extent, 24 h post SAH, myonecrosis confined largely to smooth muscle cells of the immediately subintimal media was observed. No significant changes in the intima or perivascular nerve plexus were seen. Within 24 h of haemorrhage, a limited degree of phagocytosis of erythrocytes by pial lining cells took place. However, early on the second day post SAH, a dramatic increase in the numbers of subarachnoid macrophages arose from a transformation of cells of the pia-arachnoid. This period was characterised by intense phagocytic activity, erythrocytes, fibrin, and other debris being largely cleared over the next 24 h. At 5 days post SAH the subarachnoid macrophage population declined, cells losing their mobile active features to assume a more typical pia-arachnoid cell appearance once more. Our studies indicate that this increasingly utilised small animal model of SAH develops global cortical flow changes only acutely, and it is likely that early vasospasm, secondary to released blood products rather than pressure changes per se, is responsible for the initial cerebral ischaemia that develops. Interestingly, both cerebral arterial vasculopathy and perivascular macrophage phagocytic activity are most marked at approximately 48 h following SAH in the rat, a time at which a phase of delayed cerebral arterial narrowing has previously been documented.

Ultrastructure of the orbital pathway for cerebrospinal fluid drainage in rabbits.

An increasing number of physiological and morphological studies indicate that cerebrospinal fluid (CSF) drains via nonarachnoidal pathways in several mammalian species. Ultrastructural tracer studies were undertaken to examine the orbital route for CSF absorption in the rabbit. At the termination of the optic nerve subarachnoid space, an area of connective tissue containing numerous small tortuous channels is present. Ferritin (molecular weight 400,000) infused into the ventricles at normal and increased intraventricular pressure was present in these channels by 15 minutes postinfusion, and subsequently reached the intraorbital connective tissue. Elevating the intraventricular pressure did not noticeably alter the morphological appearance of this region or change the gross distribution pattern of the ferritin. Ferritin did not penetrate the scleral barrier to reach the choriocapillaris, nor did it breach the arachnoid barrier layer proximal to the transitional zone at the optic subarachnoid space to reach the dura mater. These results are very similar to those described for the hamster orbital region and the rabbit cribriform region. These experiments support the concept that macromolecules exit the subarachnoid space at the termination of the optic nerve via open channels, and that no significant barrier to drainage of macromolecules in CSF is present at this location.

Blood-brain barrier damage during the acute stage of subarachnoid hemorrhage, as exemplified by a new animal model.

Models have been devised and characterized in the laboratory rat for studying the neuropathology of subarachnoid hemorrhage. Several ways of injecting blood via different routes have been tried; cortical subarachnoid administration is the most reproducible suitable model. The location of injected blood was detected in histological sections. In this rat model for subarachnoid hemorrhage, the arterial blood pressure and the intracranial pressure did not elevate significantly, and the influence of major ischemic components in the development of brain edema could also be ruled out. Measurements performed on the water, electrolyte, and albumin contents of brain tissue have clearly indicated that the brain edema developing in the acute stage of rat experimental subarachnoid hemorrhage could be classified as having a primarily vasogenic component as well. These findings may have implications in the treatment of subarachnoid hemorrhage.

Scanning electron microscopy of the subarachnoid space in the dog: inflammatory response after injection of defibrinated chicken erythrocytes.

The leptomeningeal reaction and the cerebrospinal fluid reaction of the canine inflammatory response were investigated concurrently. One-half milliliter cerebrospinal fluid (CSF) was withdrawn from the cisterna magna of 17 anesthetized mongrel dogs and analyzed. Using this same spinal tap, control and experimental animals were injected with 0.5 ml sterile saline and 0.5 ml defibrinated chicken erythrocytes, respectively. A second spinal tap was performed 2 to 168 hr later. The CSF from the first spinal tap contained less than 1 WBC/mm3. The cell population was unchanged in the second spinal tap of control animals. In experimental animals, the WBC population increased more than 100-fold by 24 hr. Polymorphonuclear cells (PMNs) appeared in the CSF first, followed by lymphocytes and monocytes. Injected erythrocytes seemed trapped in the subarachnoid space (SAS), especially in the inner sheet of the arachnoid mater. The leptomeninges had a substantial increase in free cells without fibrosis. Pial and leptomeningeal cells of the arachnoid trabeculae appeared swollen. Two hours after injection, chicken erythrocytes were phagocytosed by pial cells, macrophages, and free cells adherent to the leptomeninges. The epiplexus cell populations for saline-control and erythrocyte-experimental animals were similar, suggesting that the choroid plexuses were not a gateway for PMN, lymphocyte, or monocyte infusion into the SAS.

Meningothelial rosettes in the canine subarachnoid space.

A previously unrecognized type of rosette consisting of meningothelial cells is reported. Meningothelial clusters with a formation of numerous rosettes were incidentally observed in the canine subarachnoid space after single subarachnoid injection of 0.2 mg/kg of epinephrine. Light microscopy revealed that the cell clusters with distinct rosettes occurred in the extensive cords and nests, surrounding the small subarachnoid arteries or adhering to the arachnoid epithelium. There were scattered mast cells adjacent to the cell clusters. Electron microscopy revealed that the rosettes usually consisted of four to 12 tightly packed cells which were arranged like spokes and contained concentrations of tonofilaments. The rosette cells were knitted together by numerous junctional devices, such as desmosomes and desmosome-like junctions as well as a small number of interdigitations at the lateral surfaces toward the central core. The latter measured approximately 0.77-12.72 micron in long diameter, containing electron-dense, coarsely granular material. Both intracytoplasmic and extracellular inclusions with projecting microvilli were reminiscent of hyaline inclusions in certain meningiomas.