Era32rb

To determine the Iba1, CD86, IL-6, and IL-1β protein concentrations within the hippocampus, an enzyme-linked immunosorbent assay (ELISA) was used. Rats were anesthetized with isoflurane using rodent anesthesia vaporizer (VetFlo, Kent Scientific Corporation, Torrington, CT, USA) and each hippocampus was quickly extracted, frozen in liquid nitrogen, and stored at a temperature of −70 °C. The following ELISA kits were used for quantification of Iba1 (NBP2-66675, NovusBio), CD86 (RAB0887-1KT, Sigma-Aldrich, St. Louis, MO, USA), IL-6 (ERA32RB, Invitrogen), and IL-1beta (BMS630, Invitrogen) according to the manufacturer’s instructions. For analysis, we used both right and left hippocampi. The homogenization buffer consisted of 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate, with a protease inhibitor cocktail (cOmplete, Sigma-Aldrich, St. Louis, MO, USA). The BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used for determining protein concentration. The optical density was measured in an iMark plate spectrophotometer (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm.

Atrial tissue was minced and homogenized using a tissue homogenizer (TissueRuptor, QIAGEN) according to the manufacturer protocol. Inflammatory cytokines and chemokines were measured using a multiplex Luminex-based assay (LXSARM, R&D Systems). Each sample was run in duplicate in a 96-well plate. IL-1β, IL-2, IL-18, and TNF-α were measured using a MAGPIX system (C4447b, Luminex). Acquired mean fluorescence data were analyzed and calculated by xPONENT software. IL-6 (ERA32RB, Invitrogen), TGF-β1 (ab119558, Abcam), PDGF-AB (ab213906, Abcam), and MCP-1 (ab100778, Abcam) were quantified using commercially available ELISAs according to the manufacturers’ protocols.

Arterial blood samples were withdrawn at different time points as designed. Plasma samples were then obtained after centrifugation (1,000 rcf, 20 min, 20°C) and stored at −80°C. Parietal cortex and basal cortex samples of each left hemisphere were collected as previously described (Bach et al., 2019 (link)). Briefly, after adding RIPA buffer, tissue samples were homogenized using a shredder column (QIA Shredder; Qiagen Inc., Hilden; Germany). Supernatant was collected after centrifugation (4,000 rpm, 2 min at 4°C) and stored at −80°C until measurement.
IL-6 and TNF-α concentrations in plasma and brain cortex tissues were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Plasma samples: R6000B for IL-6, RTA00 for TNF-α, R&D, Minneapolis, USA; Brain cortex samples: ERA32RB for IL-6, ERA57RB for TNF-α, ThermoFisher, Carlsbad, USA) in line with manufacturer’s manual. At the end, following the addition of stop solution to terminate the reaction, optical density was immediately analyzed at 450 nm using a microplate reader (Synergy HT Multi-Mode Microplate Reader; BioTek, Winooski, USA) with a second reading at 540 nm for wavelength correction.