Transcytosis

hCMEC/D3 cells, a brain endothelial cell line immortalized by transduction with hTERT and SV40 large T [4] (link) were obtained from Pierre-Olivier Couraud under license (INSERM, Paris, France). Medium and supplements for hCMEC/D3 and primary human astrocytes were obtained from Lonza (Verviers, Belgium). MDCK and rat C6 media and components were obtained from Invitrogen.
Primary human astrocytes (passages 2–4) were obtained from Cell Systems (Troisdorf, Germany) and cultured in ABM medium fully complemented with the AGM SingleQuots kit. Rat C6 Glioma cells from ATCC were cultured in DMEM-F12 containing 5% heat inactivated FBS, 10% horse serum and 2 mM L-Glutamine. MDCK cells from ATCC were cultured in MEM containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate.
hCMEC/D3 cells (passages 26–29) were cultured to confluence on collagen (Sigma, Schnelldorf, Germany) coated coverslips (microscopy) in EBM2 medium containing 2.5% FBS, quarter of the supplied growth factors and fully complemented with supplied hydrocortisone, gentamycin and ascorbic acid.
For all transcytosis assays, high density pore (1×108 pores/cm²) PET membrane filter inserts (0.4 µm, 12 mm diameter; Millipore, Schwalbach, Germany) were used in 12-well cell culture plates (Corning, Amsterdam, Netherlands). Optimum media volumes were calculated to be 400 µl and 1600 µl for apical and basolateral chambers respectively. Apical chambers of Millicell hanging filter inserts were coated with Rat tail Collagen 1 (7.5 µg/cm²; BD Biosciences) followed by Fibronectin (5 µg/ml; Sigma) each incubation lasting for 1 hr at RT. hCMEC/D3 cells were grown to confluent monolayers (∼2×10⁵ cells/cm²) for 10-12 days in EMB2 medium. Empty filters coated with collagen/fibronectin were blocked in PBS containing 1% BSA o/n before the assay and then calibrated for at least 1 h in EBM2 before the assay. For generating endothelial-astrocyte co-cultures, the basolateral side of the filter inserts and TC plastic were coated with 10 µg/ml poly-d-lysine (Sigma) o/n at RT. The solution was aspirated, TC plastic dried for 2 h at RT and the coated surfaces were rinsed 3 × with PBS before astrocytes were seeded at 1×10³ in 200 µl on the bottom side of an inverted filter insert. Cells were allowed to adhere for 15 min at 37°C and the insert was then placed in a 12-well cell culture plate with the respective volumes of medium in apical and basolateral chambers. For culture of astrocytes in a culture plate, 1×10³ cells were plated in 1600 µl. After 48 hours the filters were processed for culturing hCMEC/D3 cells as described above. Experiments with the co-culture model were performed 10–12 days after hCMEC/D3 cells were seeded.

A 96-well ELISA plate was coated with 1/5000 goat antimouse
IgG, F(ab′)₂ fragment-specific antibody (Jackson
ImmunoResearch) diluted in PBS and incubated at 4 °C overnight.
The wells were blocked with 1% bovine serum albumin/phosphate-buffered
saline (BSA/PBS) for 1 h at room temperature on a 500 rpm shaking
platform, followed by washing five times with 0.05% Tween 20/PBS using
a Tecan Hydroflex microplate washer. Diluted and undiluted apical
and basolateral samples from the transcytosis assay, along with known
standard concentrations of monoclonal bivalent antibodies in duplicate
(from 0.5 to 128 pM), were added to the wells and incubated for 2
h at room temperature on a 500 rpm shaking platform. The wells were
washed as previously described and subsequently incubated with 1/5000
goat antimouse HRP (Sigma) diluted in 0.1% BSA/0.05% Tween 20/PBS
for 1 h at room temperature on a 500 rpm shaking platform. Following
a final wash cycle, the wells were developed with K-BlueTMB aqueous
substrate (Neogen) at room temperature according to the manufacturer’s
recommendations using 1 M H₂SO₄ to stop the
reaction (approximately 5–8 min following the addition of TMB).
Absorbance readings at 450 nm were measured immediately using a FLUOstar
Omega ELISA plate reader (BMG Labtech), and the data was analyzed
using Omega Control (BMG Labtech) and Prism 9 for macOS. Using GraphPad
analysis software, interpolation from a standard curve (Sigmoidal,
4PL), based on the concentration of the antibody (ranging from 0.5
to 128 pM), was performed to obtain concentrations of all of the collected
samples. The samples were diluted in such a way that they fell within
the most linear portion of the curve. Statistical analysis between
indicated populations was performed using an unpaired nonparametric
Mann–Whitney test, and the minimal accepted significance level
was P ≤ 0.05.

Another subgroup of SHR-S, SHR-T, Wistar-S, and Wistar-T received, after the functional measurements, an overdose of ketamine + xylazine. Immediately after the respiratory arrest, the thorax was opened and the left ventricle cannulated for sterile saline perfusion (∼30 mL/min, Daigger pump, Vernon Hills IL United States) followed by modified Karnovsky solution (2.5% glutaraldehyde +2% paraformaldehyde in 0.1 M PBS, pH 7.3). Brain was removed and placed on a coronal brain matrix (72–5029, Harvard Apparatus) to obtain hypothalamic and brainstem slices. PVN, NTS, and RVLM nuclei were microdissected with the aid of a magnifying lens, using as anatomic markers the third ventricle and optic chiasma, the central canal and 4th ventricle, and, the nucleus ambiguous, raphe obscurus and inferior olive, respectively. The nuclei were immersed in a 2.5% glutaraldehyde solution for 2 h, washed in PBS and post-fixed in a 2% osmium tetroxide solution for 2 h at 4°C. Tissues were then stained overnight with uranyl acetate, dehydrated in 60% up to 100% ethanol series and immersed in pure resin. Semi-thin slices (400 nm, ultra-microtome Leica EMUC6) were obtained, placed in glass slides and stained with Toluidine Blue in order to select adequate areas for further processing. Ultra-thin slices (60 nm) were obtained with diamond knife, contrasted with 4% uranyl acetate and 0.4% lead acetate and disposed in 200 copper mesh screens.
Transverse sections of PVN, NTS, and RVLM capillaries of the 4 experimental groups were acquired in a transmission electron microscope (FEI Tecnai G20, 200 KV) and analyzed by a blind observer using the ImageJ software. The following parameters were analyzed in 9–11 capillaries/area/rat, 3 rats/experimental group: luminal and abluminal perimeter, lumen diameter, area of the endothelial cell, thickness of the basement membrane, pericytes’ coverage of capillaries, extension of capillary border between adjacent endothelial cells, the occurrence/extension of tight junctions, and, the counting of transcellular vesicles/capillary. To avoid the inclusion of non-transcytotic vesicles such as lysosomes, endosomes, peroxisomes, only the vesicles being formed at the luminal, and abluminal membranes were counted. Vesicle counting was expressed as number/capillary. Using the zoom to expand acquired images, the whole extension of capillaries was analyzed.

Some rats of each experimental group were used to confirm our counting of transcytotic vesicles by means of caveolin-1 expression. Caveolin-1, the main component of transcellular vesicle membranes (Predescu et al., 1997 (link); Zhao et al., 2014 (link)) was used as a marker of transcytosis. After the functional measurements rats received an overdose of ketamine + xylazine. Immediately after the respiratory arrest brains were harvested, post-fixed, cryoprotected and stored as previously described (Fragas et al., 2021 (link)).
For caveolin-1 immunofluorescence assay coronal PVN sections (30 μm) were collected (Fragas et al., 2021 (link)). Briefly, free-floating sections were incubated with a mixture of primary antibodies (rabbit anti-caveolin-1, Cell Signaling 1:100 dilution + mouse-anti-endothelial cell antibody RECA-1, Abcam, 1:800 dilution for 48 h at 4°C) followed by incubation with secondary antibodies (anti-rabbit Alexa Fluor 488 + anti-mouse Alexa Fluor 549, Jackson ImmunoResearch, 1:500 dilution each, at room temperature for 1 h). Sections, mounted in gelatinized slides, were examined by a blind observer in a fluorescence microscope (Axioimager AI, Zeiss, Munchen, Germany attached to a Zeiss Axiocam 512 camera). Caveolin-1 immunofluorescence was normalized by RECA-1 immunoreactivity, both signals being acquired in the same ROI. Images were acquired with identical acquisition settings and analyzed as previously described (Rocha-Santos et al., 2020 (link)). Values of several PVN slices (expressed as integrated density) were averaged to yield a mean value/rat.