Pericytes

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.

In vitro human BBB models were developed by combining three immortalized cell lines [11 (link)]). Briefly, HBPC/ci37 cells were seeded on the bottom side of the collagen IV- and Fibronectin-coated polycarbonate membrane of a transwell insert (Millicell cell culture insert 24-well hanging inserts, 0.4 μm PET; Merck, Darmstadt, Germany) at a density of 1.0 × 10⁴ cells/insert. The cells were then cultured for 1 day to allow them to attach firmly. HASTR/ci35 cells were seeded (5.0 × 10⁴ cells/well) on collagen I-coated 24-well plates (Greiner Bio-one, Frickenhausen, Germany) and maintained in astrocyte culture medium. HBPC/ci37 cells were induced to differentiate by replacing the pericyte medium with pericyte differentiation medium, which was consisted of FBS- and blasticidin S-free pericyte medium; HASTR/ci35 cells were induced to differentiate by replacing the astrocyte culture medium with astrocyte differentiation medium, which was consisted of FBS- and blasticidin S-free astrocyte growth medium supplemented with 1 mM adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate. After the differentiation media were added, both cell lines were cultured at 37 °C for 24 h. To start a coculture, HBMEC/ci18 cells were seeded on the inner side of the HBPC/ci37 cell culture insert at a density of 1.0 × 10⁵ cells. Finally, the transwell inserts with HBMEC/ci18 cells and HBPC/ci37 cells were transferred into 24-well plates containing HASTR/ci35 cells. The cells were re-fed with VEGF- and EGF-free VascuLife complete medium in the inner insert and the Neurobasal medium with N2 supplement in the lower chamber. Day 0 was defined as the day of EC plating on the membrane. The cells were incubated at 33 °C.
On Day 1, the trans-endothelial electrical resistance (TEER) was measured by an EVOM² voltohmmeter (World Precision Instruments, Sarasota, California, USA) with chopstick electrodes. The net resistance value was calculated by subtracting the measured resistance value of the insert membrane from the measured resistance value of the coculture. TEER (Ω × cm²) = the net resistance value (Ω) × surface area (cm²).

The expression levels of TJ proteins and transporter proteins were analyzed by Western blotting [20 ]. After the examination of TEER, Western blots were performed to analyze protein extracts from both endothelial cells and pericytes. These extracts were obtained by lysing the cells with sample buffer (62.5 mM Tris, 2% SDS, 10% glycerin, 0.0125% bromophenol blue, pH 6.8) and homogenizing them on ice. The lysates were resolved by SDS‒PAGE and transferred to PVDF membranes. The membranes were incubated overnight in BlockAce blocking solution at 4 °C. Then, the membranes were incubated with primary antibodies for 1 h at 25 °C. After being washed three times, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (1:5000) for 1 h at 25 °C. The membranes were then washed three times, and signals were visualized with a LAS3000 chemiluminescence detector (Fujifilm Co., Tokyo, Japan). We confirmed that the bands matched the molecular weights of the specific proteins of interest, i.e., CD31 (120 kDa), ZO-1 (225 kDa), Claudin-5Claudin-5 (24 kDa), P-gp (180 kDa), BCRP (65–80 kDa), Glut1 (40–60 kDa), TfR (90 kDa), and b-actin (42 kDa).