David W. Holman

Ex Vivo Model of Cerebrospinal Fluid Outflow across Human Arachnoid Granulations

PURPOSE The brains arachnoid membrane with granulations is an important biological barrier whose responsibilities include the transmission of cerebrospinal fluid (CSF) and the regulation of pressure. Membrane disturbance may cause changes that are difficult to replicate with animal models, suggesting the need for a model using human arachnoid membrane with granulations for the study of conditions such as Alzheimer disease, hydrocephalus, and pseudotumor cerebri. The authors detail the development and validation of an ex vivo model of CSF outflow across human arachnoid granulations (AGs) as an approximation of in vivo conditions. METHODS Human AGs were perfused at normal physiological pressure in physiological and nonphysiological directions for permeability data. Fluorescent particle perfusion with electron microscopy identified outflow pathways through the AGs. RESULTS This human ex vivo model demonstrated in vivo properties of unidirectionality, particle transport, and ultrastructure, similar to our 2005 in vitro model. The average baseline hydraulic conductivity in the physiological direction (n = 20) was 1.05 +/- 0.15 microL/min/mm Hg/cm(2) compared with 0.11 +/- 0.03 microL/min/mm Hg/cm(2) in the nonphysiological direction (n = 3) under statistically equivalent (P = 0.46) average normal physiological pressures (5.88 +/- 0.22 mm Hg and 6.14 +/- 0.23 mm Hg, respectively). CONCLUSIONS The ex vivo model is feasible and herein demonstrated. These findings agree with in vivo CSF outflow. This model increases understanding of the clearance not only of CSF but also of metabolites through the arachnoid membrane. Additional evidence suggests, but does not yet prove, that CSF outflow may occur in a similar manner in the arachnoid membrane adjacent to the granulations, in addition to the flow through the AGs. This is a topic for further investigation.

Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

BackgroundThe arachnoid granulations (AGs) are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF) to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow.MethodsHuman AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144) and their expression was quantified using flow cytometry analysis.ResultsConfluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall.ConclusionTo our knowledge, this is the first report of the in vitro culture of arachnoidal cells grown from human AG tissue. We demonstrated that these cells in vitro continue to express some of the cytoskeletal and junctional proteins characterized previously in human AG tissue, such as proteins involved in the formation of gap junctions, desmosomes, epithelial specific adherens junctions, as well as tight junctions. These junctional proteins in particular may be important in allowing these arachnoidal cells to regulate CSF outflow.

Cerebrospinal fluid dynamics in the human cranial subarachnoid space: an overlooked mediator of cerebral disease. II. In vitro arachnoid outflow model

The arachnoid membrane (AM) and granulations (AGs) are important in cerebrospinal fluid (CSF) homeostasis, regulating intracranial pressure in health and disease. We offer a functional perspective of the human AMs transport mechanism to clarify the role of AM in the movement of CSF and metabolites. Using cultures of human AG cells and a specialized perfusion system, we have shown that this in vitro model mimics the in vivo characteristics of unidirectional fluid transport and we present the first report of serum-free permeability values (92.5 µl min−1 mm Hg−1 cm−2), which in turn are in agreement with the CSF outflow rates derived from a dynamic, in vivo magnetic resonance imaging-based computational model of the subarachnoid cranial space (130.9 µl min−1 mm Hg−1 cm−2). Lucifer yellow permeability experiments have verified the maintenance of tight junctions by the arachnoidal cells with a peak occurring around 21 days post-seeding, which is when all perfusion experiments were conducted. Addition of ruthenium red to the perfusate, and subsequent analysis of its distribution post-perfusion, has verified the passage of perfusate via both paracellular and transcellular mechanisms with intracellular vacuoles of approximately 1 µm in diameter being the predominant transport mechanism. The comparison of the computational and in vitro models is the first report to measure human CSF dynamics functionally and structurally, enabling the development of innovative approaches to modify CSF outflow and will change concepts and management of neurodegenerative diseases resulting from CSF stagnation.

Retinol effects on CSF outflow through cultured human arachnoid granulation cells: implications for PTC

Background The underlying etiology of pseudotumor cerebri (PTC) remains unknown, however it has been estimated that an identifiable secondary cause is present in up to 10% of PTC cases. Of these secondary associations, vitamin A is of particular interest, as vitamin A toxicity is associated with an increased intracranial pressure similar to that seen in PTC. Recently, several clinical studies have suggested that serum and CSF retinol levels may be altered in PTC. We hypothesize that increased concentrations of retinoids in the CSF might increase resistance to CSF outflow at the arachnoid granulations (AGs).

Human arachnoid membrane: active transport of amyloid-beta

Address: 1Department of Ophthalmology, The Ohio State University, College of Medicine, Columbus, OH 43210, USA, 2Department of Biomedical Engineering Center, The Ohio State University, College of Medicine, Columbus, OH 43210, USA, 3Division of Neuropathology, Dept. of Pathology, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI 02903, USA and 4Clinical Neuroscience, Brown Medical School, Providence, RI 02903, USA

Ultrastructural study of the permeability of in-vitro and ex-vivo human models of human arachnoid granulation CSF outflow pathway

Background In communicating hydrocephalus and also idiopathic intracranial hypertension, disturbed CSF dynamics may result from an increased resistance to CSF outflow at the arachnoid granulations (AGs). To better understand the mechanism of CSF egress, we modelled the outflow of CSF through human AGs using both cell culture (in-vitro) and whole tissue (ex-vivo) perfusion models. Ultrastructural studies were done using microparticles, ruthenium red, and TEM and to elucidate the mechanism of fluid flow.

Human arachnoid granulation probability of occurrence and surface area quantification

Meeting abstracts - A single PDF containing all abstracts in this supplement is available here .

Mechanism of CSF outflow through human arachnoid granulations using in-vitro and ex-vivo perfusion models

Bifida Meeting abstracts - A single PDF containing all abstracts in this supplement is available here .