Hydraulic Conductivity of Human Arachnoid Granulation Cap Cells in an in vitro Model of Cerebrospinal Fluid Outflow

Abstract

Abstract: : Purpose:Pseudotumor cerebri (PTC) is a clinical condition of increased intracranial pressure (ICP) that can damage the optic nerves and result in blindness. A suggested mechanism for PTC is a decreased outflow of cerebrospinal fluid (CSF) across the arachnoid granulations (AGs). Due to the difficulty in visualizing CSF outflow in humans in vivo, we have utilized an in vitro model that simulates the CSF outflow pathway. AG cap cells are grown on filter membranes as a model of CSF egress through the AGs. This study describes AG cap cells on filter supports that have been perfused at conditions that mimic normal ICP, in order to establish the baseline hydraulic conductivity (L_p) of these cells. Methods:Primary cell cultures were obtained from human AG explants and tissue digestions. Using fluorescent microscopy AG cap cells were identified in culture by their expression of epithelial markers and junctional proteins. Cells were seeded onto filter membranes at a density of 5*10⁵ cells/cm². AG cap cells were grown to confluence, and the integrity of the monolayer was confirmed by testing a representative filter for the tight junction protein ZO–1. Confluent filter membranes were inserted into our perfusion system and perfused for 3 hrs at a pressure of 3mmHg (normal ICP). Using laser scanning confocal microscopy and electron microscopy (EM), the intracellular structures and cytoskeleton of the monolayer were analyzed for viability and cell attachment. Results:Human AG cells in culture expressed cytokeratin, EMA, and connexin43. Confluent monolayers of AG cap cells expressed ZO–1. The average volumetric flow rate across the monolayer perfused for 3 hrs at 3mmHg was 6.3 µl/min and the average L_p was 0.533 µl/min/mmHg/cm². Confocal microscopic analysis of the perfused monolayer indicated viable cells. EM showed that the cells were anchored to the filter membrane by focal adhesions. Conclusions:We have perfused human AG cap cells on filter membranes under conditions that replicate normal ICP. Future studies will examine the effect of increased pressure on these cells in order to approximate the conditions seen in patients with PTC. Additionally we will treat our cell cultures with factors that are known to be associated with secondary PTC, most notably vitamin A. Further perfusion studies will focus on the effects of vitamin A on the hydraulic conductivity of AG cap cells in order to create an in vitro model of PTC.

Keywords: visual impairment: neuro-ophthalmological disease • pump/barrier function • microscopy: light/fluorescence/immunohistochemistry