Apocynin 4 hydroxy 3 methoxyacetophenone

The murine MS1 (Mile Sven 1) pancreatic islet endothelial cell line, obtained from American Type Culture Collection (ATCC®, CRL-2279^TM), is a commonly used model of microvascular endothelium. MS1 cells were cultured in high glucose (4.5 g/l) Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% decomplemented (heat inactivated) fetal bovine serum (FBS), 120 U/mL streptomycin/penicillin and 4 mM L-Glutamine, in 5% CO₂ at 37 °C. MOVAS (ATCC®, CRL-2797^TM) is an established VSMC line from C57BL/6 mice. Cells were grown in DMEM supplemented with 10% FBS, 2 mM L-Glutamine and 0.2 mg/ml G-418 (all reagents obtained from Lonza). For experiments, cells at 80% confluence were growth-arrested by serum starvation for 24 h. Endotoxin-free CsA (Calbiochem, Merck Chemicals) and tacrolimus (USBiological) stock solutions (10 mg/ml) were dissolved in ethanol. CLI-095 (InvivoGen) was used according to manufacturer’s time and dose recommendations. The NF-κB inhibitor parthenolide, the TRIF inhibitor resveratrol and the antioxidants 4′-hydroxy-3′methoxyacetophenone (apocynin) and diphenyleneiodonium chloride (DPI) were from Sigma-Aldrich. These reagents were used at concentration derived from prior dose-response studies in our laboratory or from bibliographic data.

RPMI-1640 culture medium and Hanks' balanced salt solution (HBSS) were obtained from Gibco (Inchinnan, Scotland). Fetal bovine serum (FBS) and human type AB serum were purchased from PAA The Cell Culture Company (Pasching, Austria). Middlebrook 7H10 agar, Middlebrook 7H9 broth, and Middlebrook OADC enrichment were acquired from Becton Dickinson (Franklin Lakes, NJ, USA). Phorbol-12-myristate-13-acetate (PMA), diethylenetriamine/nitric oxide adduct (DETA/NO), menadione, 4′-hydroxy-3′-methoxyacetophenone (apocynin), L-N⁶-(1-iminoethyl)lysine dihydrochloride (L-NIL), Triton X-100, β-mercaptoethanol, penicillin (10,000 U/ml)/streptomycin (10 mg/ml) solution (P/S), fluorescein isothiocyanate (FITC), Tween-80, 37% formaldehyde (FA) solution, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Sprague-Dawley rats were anesthetized with a mixture of air and isoflurane and placed in a stereotaxic frame. Cannula (Plastic One) was implanted unilaterally into VTA [coordinates: AP +3.4 (Lambda); L -2.1; DV-7.2, 12 degrees angle] (Rivera-Meza et al., 2014 (link)) according to the atlas of Paxinos and Watson (1998 ) (Paxinos and Watson, 1998 ). After recovery, animals were transferred to individual cages at the animal station, with access to water and food ad libitum. One week after surgery, the rats were infused each day with 100 ng of gp91 ds–tat (Anaspec) 20 min before exposure to CPP assay.
Apocynin (4′-Hydroxy-3′-methoxyacetophenone, Sigma) was added to drinking water at 5 mM and treatment was performed for 5 weeks (P30–P70) (Furukawa et al., 2004 (link); Harraz et al., 2008 (link); Espinosa et al., 2013 (link)).