We conducted an 8-week study of Long Evans rats that included 4 treatment groups: 1) control; 2) NNK (2 mg/kg, i.p. on Mondays, Wednesdays, and Fridays from weeks 3 through 8); 3) ethanol (chronic-26% caloric for 8 weeks + binge- 2g/kg intraperitoneal (i.p.), Tuesdays, Thursdays, and Saturdays in weeks 7 and 8); and 4) ethanol+NNK. Control and NNK-treated rats were pair-fed isocaloric liquid diets containing 0% ethanol throughout the 8 weeks of study. Rats not receiving NNK or ethanol on the designated days were administered saline by i.p. injection (see Supplementary (S) Figure 1). Each of the 4 experimental groups had 8 rats. Basal (7 AM) and binge blood alcohol concentrations were measured (STable 1). NNK exposure was verified by detecting O6-Methylguanine adducts in liver (Zabala et al., 2015 (link)). These experiments were approved by the Institutional Animal Care and Use Committee at the Lifespan-Rhode Island Hospital, and the protocols conformed to guidelines established by the National Institutes of Health.
Fresh postmortem brains were sectioned to obtain 3 mm-thick coronal slices including the infundibulum, temporal lobes, and corpus callosum. Cryostat sections (10 µm thick) of frozen tissue blocks were thaw-mounted onto indium tin oxide (ITO)-coated glass slides, vacuum dried, washed with ammonium formate buffer to remove salts and enhance sensitivity for lipid analysis (Angel et al., 2012 (link)), and sublimed with 2,5-dehydroxybenzoic acid (DHB) as matrix (Hankin et al., 2007 (link)). Imaging was performed using a reflectron geometry MALDI-TOF/TOF mass spectrometer (Ultraflextreme, Bruker Daltonics, Bremen, Germany) (Yalcin and de la Monte, 2015 ) and visualized with FlexImaging software v4.0. Data collected in the negative ion mode were processed using FlexAnalysis v3.4. Statistical analyses were performed using ClinProTools v3.0. Adjacent sections stained with Luxol fast blue-Hematoxylin and Eosin (LHE) were used to co-register MALDI-TOF results. Phospholipid and sphingolipids were identified from their mass to charge (m/z) product ion ratios in MS/MS spectra and LIPID MAPS tools (http://www.lipidmaps.org/tools/index.html ).
Fresh postmortem brains were sectioned to obtain 3 mm-thick coronal slices including the infundibulum, temporal lobes, and corpus callosum. Cryostat sections (10 µm thick) of frozen tissue blocks were thaw-mounted onto indium tin oxide (ITO)-coated glass slides, vacuum dried, washed with ammonium formate buffer to remove salts and enhance sensitivity for lipid analysis (Angel et al., 2012 (link)), and sublimed with 2,5-dehydroxybenzoic acid (DHB) as matrix (Hankin et al., 2007 (link)). Imaging was performed using a reflectron geometry MALDI-TOF/TOF mass spectrometer (Ultraflextreme, Bruker Daltonics, Bremen, Germany) (Yalcin and de la Monte, 2015 ) and visualized with FlexImaging software v4.0. Data collected in the negative ion mode were processed using FlexAnalysis v3.4. Statistical analyses were performed using ClinProTools v3.0. Adjacent sections stained with Luxol fast blue-Hematoxylin and Eosin (LHE) were used to co-register MALDI-TOF results. Phospholipid and sphingolipids were identified from their mass to charge (m/z) product ion ratios in MS/MS spectra and LIPID MAPS tools (
