After hemodynamic recordings a subgroup of SHR-S, SHR-T, Wistar-S, and Wistar-T was again anesthetized for catheterization of right carotid artery. A mixture of dyes [rhodamine isothiocyanate dextran, 70 kDa (RHO) and fluorescein isothiocyanate dextran 10 kDa (FITC], Sigma-Aldrich) was slowly administered and allowed to recirculate as previously described (Biancardi et al., 2014 (link)). Rats received then and overdose of anesthesia (300 mg/kg ketamine +60 mg/kg xylazine ip) for brain harvesting immediately after the respiratory arrest. Brains were post-fixed (4% phosphate-buffered paraformaldehyde, 48 h), cryoprotected (30% sucrose in PBS for 72 h) and stored until processing (Buttler et al., 2017 (link); Rocha-Santos et al., 2020 (link)).
Sequential coronal PVN, NTS and RVLM slices (30 μm, Leica CM1850 cryostat, Germany) were collected and mounted in gelatinized slides as previously described (Buttler et al., 2017 (link)). The BBB permeability was analyzed by the quantitative assessment of intravascular and extravascular dyes according the technique developed by Biancardi et al. (2014) (link). With an intact BBB both dyes are colocalized within brain capillaries; in the presence of compromised barrier integrity the large-size dye are still contained by the capillaries whereas the small-size dye partially leaks into the brain parenchyma (Biancardi et al., 2014 (link)). Tissues were examined by a blind observer on a fluorescent microscope (Leica BMLB, Nussloch, Germany) attached to an Exiblue camera (Imaging, Canada). Selected images were acquired by Image-Pro Plus software (Media Cybernetics, United States) and quantified by the ImageJ software (NIH, United States).
Sequential coronal PVN, NTS and RVLM slices (30 μm, Leica CM1850 cryostat, Germany) were collected and mounted in gelatinized slides as previously described (Buttler et al., 2017 (link)). The BBB permeability was analyzed by the quantitative assessment of intravascular and extravascular dyes according the technique developed by Biancardi et al. (2014) (link). With an intact BBB both dyes are colocalized within brain capillaries; in the presence of compromised barrier integrity the large-size dye are still contained by the capillaries whereas the small-size dye partially leaks into the brain parenchyma (Biancardi et al., 2014 (link)). Tissues were examined by a blind observer on a fluorescent microscope (Leica BMLB, Nussloch, Germany) attached to an Exiblue camera (Imaging, Canada). Selected images were acquired by Image-Pro Plus software (Media Cybernetics, United States) and quantified by the ImageJ software (NIH, United States).
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