Development of an In vitro Model to Study Pseudotumor Cerebri

Abstract

Abstract: : Purpose: The objective of this study is to develop an in vitro model to study flow characteristics of CSF across arachnoid granulations. It is hypothesized that arachnoid granulations have a role in the outflow of cerebrospinal fluid (CSF) especially in the diseased state. We are studying the role of arachnoid granulations in the disease pseudotumor cerebri (PTC). PTC is a condition where increased CSF pressure puts pressure on the optic nerve, which can lead to blindness. It is believed that elevated CSF pressure is due to an increased resistance at the pia–arachnoid layer of arachnoid granulations. The first step is the isolation and purification of pia–arachnoid cells. Methods: Primary cell cultures were obtained from human arachnoid granulation explants. These explants were placed in fibronectin coated culture plates with DMEM/F–12 medium supplemented with newborn calf serum, L–glutamine, and antibiotics. Cell migration from the explants was observed 4–7 days after explantation. Media was changed every 3–5 days, and cells passaged every 2–3 weeks. Cell phenotypes were determined by immunostaining. Flow cytometry was used to characterize appropriate immunological markers to identify contaminate cell types (fibroblasts, astrocytes, endothelial cells). Contaminate cell types were labeled with immunomagnetic markers and separated using magnetic cell sorting. Cells are seeded onto filters where the monolayer is grown to confluency for insertion into the flow perfusion system. Results: Staining for vimentin and connexin–43 indicated mixed cell cultures consisting primarily of pia–arachnoid cells and fibroblasts. Negative staining for glial fibrillary acidic protein and DiI labeled acetylated low–density liporotein indicated astrocytes and endothelial cells were not present. Subsequent immunomagnetic cell separation yielded homogenous cultures of pia–arachnoid cells. Conclusions: We have identified a novel technique to study PTC. Pure cultures of pia–arachnoid cells cultured from human arachnoid granulation explants have been isolated using immunomagnetic cell separation. This technique will allow perfusion studies of CSF flow across the arachnoid granulations at normal and elevated CSF pressures such as those seen in individuals with PTC. The outputs from the model include the hydraulic characteristics of pia–arachnoid cells and their response to various drugs used to treat PTC.

Keywords: flow cytometry • immunohistochemistry • visual impairment: neuro–ophthalmological disease