Browsing by Author "Lam, Cornelius Hoktsim"

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    The egress of fluid from the brain via arachnoid transport: foundational work for the tissue engineering of the arachnoid granulation
    (2011-12) Lam, Cornelius Hoktsim
    The arachnoid tissue is a critical component for the removal of cerebrospinal fluid (CSF) and other substances. Failure results in hydrocephalus, increased intracranial pressure, and buildup of toxic materials in the brain. The purpose of this thesis is to establish a foundation for a biomimetic arachnoid construct. First, we characterized arachnoid cell transport in culture and on three-dimensional collagen scaffolds. Arachnoid cells were harvested from rat brainstems and cultured onto bilayered bovine collagen scaffolds. Cells exhibited arachnoid cell phenotype (positive for vimentin, desmoplakin, and cytokeratin), readily penetrated the collagen scaffold, and doubled approximately every 2–3 days. The transepithelial electrical resistance for a monolayer of cells was 160 Ω∙cm2, and permeability of indigo carmine was 6.7+1.1X10- 6 cm/s. Hydraulic conductivity of the collagen construct was 6.39 mL/min/mmHg/cm2. Because of practical limitations of primary culture which include slow growth, early senescence, and poor reproducibility, we created two immortalized rat arachnoid cell lines using retroviral gene transfer of SV40 large T antigen (SV40 LTAg) either with or without human telomerase (hTERT). They stably expressed either SV40 LTAg alone, or SV40 LTAg and hTERT, and demonstrated high proliferative rate, contact inhibition at confluence, and stable expression of protein markers characteristic of native arachnoid cells for more than 160 passages. We subsequently used them to determine arachnoidal barrier properties and paracellular transport. Permeabilities of urea, mannitol, and inulin were 2.9+1.1X10-6, 0.8+.18x10-6, and 1.0+.29x10-6 cm/s respectively. Size differential permeability testing with dextran clarified the arachnoidal blood-CSF-barrier limit and established a rate of intracellular transport to be two orders of magnitude slower than paracellular transport in a polyester membrane diffusion chamber. The theoretical pore size for paracellular space was 11Å and the occupancy to length ratios were 0.8 and 0.72 cm-1 for urea and mannitol respectively. The monolayer permeability was not significantly different from an apical to basal direction or vice versa. Gap junction may have a role in barrier formation. Although up-regulation of claudin by dexamethasone did not significantly alter paracellular transport, increasing intracellular cAMP decreased mannitol permeability. Calcium modulated paracellular transport, but only selectively with the ion chelator, EDTA, and with disruption of intracellular stores. Without the neurovascular unit of the blood-brain-barrier, the blood-CSF-barrier at the arachnoid tissue is anatomically and physiologically different from the vascular based blood-brain-barrier. These studies provide a three dimensional architecture, a stable cellular substrate, and baseline blood-CSF-barrier properties for the establishment of a viable bioartificial arachnoid shunt.