Sodium Fluorescein

To evaluate retinal RPE structure and function in vivo, OCT and FA were performed simultaneously as described previously with some modifications [41 (link)]. Briefly, mice were anesthetized using 2% isoflurane and their pupils were dilated using 1% tropicamide eye drop. Each mouse was then placed on the imaging platform of the Phoenix Micron III retinal imaging microscope supplemented with OCT imaging device (Phoenix Research Laboratories, Pleasanton, CA). Genteal gel was applied liberally to keep the eye moist during imaging. Mice were administered 10 to 20 μL 10% fluorescein sodium (Apollo Ophthalmics, Newport Beach, CA), and rapid acquisition of fluorescent images ensued for ∼5 minutes. Fluorescein leakage manifests as indistinct vascular borders progressing to diffusely hazy fluorescence.

TEER, reflecting the flux of ions through cell layers in culture conditions, was measured by an Epithelial-volt-ohm meter and Endohm-6 chamber electrodes (World Precision Instruments, USA). The TEER of pericyte-layered filters was subtracted from the measured TEER values of the models, shown as Ω×cm². The flux of sodium fluorescein (Na–F) and Evan’s blue-albumin (EBA) across the endothelial cell layers of the in vitro BBB models was determined as previously described [25] (link). Cell culture inserts were transferred to 24-well plates containing 0.6 ml permeability assay buffer (141 mM NaCl, 2.8 mM CaCl₂, 1 mM MgSO₄, 4 mM KCl, 1 mM NaH₂PO₄, 10 mM glucose and 10 mM Hepes, pH 7.4) in the lower or abluminal compartments. In the inserts (luminal compartment), culture medium was replaced by 0.1 ml buffer containing 100 µg/ml Na–F (MW: 376 Da) and 4% bovine serum albumin (Sigma) mixed with 0.67 mg/mL Evan’s blue dye (Sigma) (EBA; MW: 67,000 Da). Samples (400 µL) were removed from each side at 15, 30, 45, 60, 120 and 180 min. To ensure mixing of the layers, we stirred the assay buffer in the receiver chamber, into which test compounds permeate, with a pipette before removing the buffer and immediately replacing with fresh permeability assay buffer. The concentrations of Na-F were determined with a CytoFluor Series 4000 fluorescence multiwall plate reader (PerSeptive Biosystems) using a fluorescein filter pair (Ex(λ) 485±10 nm; Em(λ) 530±10 nm). The EBA concentration in the abluminal chamber was measured by determining the absorbance of samples at 630 nm with an amicroplate reader (Opsys MR, DYNEX Technologies, Chantilly, VA,USA). Flux across the pericyte culture inserts was also measured. The transendothelial permeability coefficient P_trans was calculated as described in Analysis of in vitro permeability data.

Permeability tests using the small molecular marker sodium fluorescein (SF, MW = 376 Da) were carried out on an in-contact type double co-culture BBB model with primary astroglia when high TEER values were recorded. After applying IL-6, IL-10 and IL-6 + IL-10 in combination (both cytokines at 50 ng/ml concentration) for 24 h in the luminal compartment, the test was conducted as described previously [37 (link)]. The concentration of the SF marker molecule in samples from the upper and lower compartments was determined with a microplate reader (excitation at 440 nm, emission at 525 nm; BMG Fluostar Optima; BMG Labtech, Ortenberg, Germany). Flux across coated, cell-free inserts was also measured. Endothelial permeability coefficients (P_e) were calculated from clearance values of tracers as described in detail in our previous publication [32 (link)].

Hesperidin, fluorescein sodium salt, and dimethylsulfoxide (DMSO) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone Laboratories Inc (Logan, UT, USA). Penicillin–Streptomycin, MEM Non‐Essential Amino Acids (NEAA), and 0.25% Trypsin/EDTA were purchased from GIBCO (Gaithersburg, MD, USA). MITO+™ serum extender, basal medium for seeding, and enterocyte differentiation medium for Caco‐2 cell monolayer cultures were purchased from BD Biosciences (San Jose, CA, USA). Other reagents were analytical grade and used without further purification.