Journal: Journal of neurochemistry

Article Title: Trafficking of AAV vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells

doi: 10.1111/jnc.13861

Figure Lengend Snippet: (A) BMVEC cultures from a single donor were prepared on collagen-coated electrode arrays designed for use with an Electric Cell-substrate Impedance System. Transendothelial electrical resistance (TEER) readings were acquired continuously in 30-minute intervals for one week. After BMVEC cultures established stable TEER readings, cells were exposed to either AAV2 or AAV9 at three different concentrations (1×104 gc/cell, 1×105 gc/cell, and 1×106 gc/cell) for 48 hours. All treatments were performed in triplicate. Cultures never exposed to virus were used as stable baseline controls. EDTA was used as a control for the reduction of baseline TEER values. Data are presented as the normalized change in resistance from baseline TEER + SEM over a 45-hour period. Right-hand y-axis shows absolute TEER values (Ohms • cm2). Arrow indicates addition of AAV vectors to BMVEC cultures with stable TEER readings (last 10 hours shown). Statistical analysis revealed no significant changes in TEER between AAV treated cultures and controls. (B) BMVECs exhibiting monolayer formation indicative of barrier properties were prepared on collagen-coated cell culture inserts and incubated with AAV9 vectors (2×105 gc/cell) for 0, 2, 4, 6, 24 and 48 hours. At the indicated times, permeability measures were determined using two tetramethylrhodamine (TMR)-labeled dextrans (3 kDa and 40 kDa) applied to the upper compartment for 20 minutes. Media from the lower compartment was then analyzed for the presence of dextran-TMR tracers. Data are presented as mean fluorescence (RU) + SEM for treatments performed in quadruplicate. Statistical analyses revealed no significant difference between cultures incubated with AAV9 and 0-hour controls. NS denotes p > 0.05. (C-E) Additional BMVEC cultures were incubated with AAV9 (2.5×105 gc/cell) for 24 hours in order to determine the cellular localization and relative expression of key tight junction (TJ) proteins that help to stabilize BBB integrity. BMVEC cultures not exposed to AAV were used as controls. (C) Immunocytochemistry reveals cellular localization of occludin (Occ) at intercellular borders consistent with TJ formation both in control cultures and in the presence of AAV9. (D) The histogram represent inserts values from intensity/pixel from intensity line profile drawn across the images. (E) On the basis of the intensity line profiles from sets of N=5, the graph of the average ± SEM shows no statistical significance observed between no virus and AAV9 exposed cultures. (F) Western blots depict similar expression levels of ZO-1, Occ, and claudin-5 (CLD-5) in BMVEC control cultures and those incubated with AAV9. β-actin is presented as a loading control for equal protein.

Article Snippet: Transendothelial electrical resistance An Electrical Cell-substrate Impedance Sensor (ECIS) Zθ system produced by Applied BioPhysics (Troy, NY) was used to measure BMVEC transendothelial electrical resistance (TEER).

Techniques: Cell Culture, Incubation, Permeability, Labeling, Fluorescence, Expressing, Immunocytochemistry, Western Blot