4.2. Animal Studies

Eight-week-old male and female C57BL/6J mice (Jackson’s Laboratories, Bar Harbor, ME, USA) were housed 4 mice to a cage (separated by sex) and maintained on 12-h light, 12-h dark cycles (lights on at 0700 h) in the animal facility at the Department of Animal Sciences, Rutgers University. Following 1-week acclimation, animals were given ad libitum access to water and a polyphenol-free diet (PFD; 3.82 kcal/g, 10% fat, 20% protein, 70% carbohydrate; D12450H; Research Diets, Inc., New Brunswick, NJ, USA) for at least 10 days. For PK studies, after an overnight fast, mice were administered 50 mg/kg DHM (dissolved in 0.9% saline with 10% DMSO) by either IP injection or oral gavage. Oral gavage was performed using single-use, sterile plastic feeding tubes for mice (20 ga × 30 mm; cat# FTP-20-30, Instech Laboratories, (Plymouth Meeting, PA, USA). Mice were deeply anesthetized with 5% isoflurane mixed with oxygen, and blood was collected by cardiac puncture. Following cardiac puncture, animals were exsanguinated and transcardial perfusion with 0.9% saline was performed to ensure blood removal from the brain tissue. Blood and brain samples were collected from a total of 85 mice at 0.25, 0.75, 1.5, 3, 6, 12, 24 h (3–4 mice for each sex and timepoint). Tissues were immediately homogenized with 0.2% formic acid at 1:2 (w/v), and frozen at −80 °C until analysis. Blood was centrifuged at 3000× g for 10 min at 4 °C to isolate serum. For efficacy studies, mice (n = 8/sex) were fasted at 5 h prior to Ethanol gavage 5 g/kg (37% Ethanol in water). DHM 50 mg/kg IP (dissolved in 0.9% saline with 10% DMSO) was administered at 0, 15, 45, 90 and 180 min prior to Ethanol oral gavage. Each mouse was tested at each time point 7 days apart over 6-week period. At the time of Ethanol gavage, mice were placed in oversized conical holders (L: 152 mm X W: 37 mm) and observed for ability to return upright upon being placed in a supine position. Duration of LORR was determined by ability to return upright 3 times within 30 s [48]. To determine whether ethanol-induced tolerance developed, one week (Week 1; Baseline) prior to the 6-week repeated DHM dosing study all mice were oral dose with ethanol (5 g/kg) without any IP injection and LORR was measured. Mice were then re-tested in a similar fashion with oral ethanol 1 week after the DHM 6-week period (Week 8; Post). Mice that demonstrated a significant reduction in LORR from Week 1 to Week 8 were considered “tolerant”, whereas mice that did not demonstrate a significant reduction in LORR from Baseline (Week 1) to Post (Week 8) were considered “responsive”. All animal studies were approved by the Institutional Animal Care and Use Committee of Rutgers University (Bello; PROTO999900014, OLAW #A3262-01, 15 March 2017) and complied with NIH Guide for the Care and Use of Laboratory Animals.