Metabolic cage

Immediately prior to HEPA‐ and charcoal‐filtered air exposure or CAP exposure, mice were weighed and briefly exposed to D‐glucose/saccharin solution (w/v; 3.0%/0.125%; Sigma) on the mouth (Conklin, Haberzettl, Lesgards, et al., 2009 ; Wood et al., 2001 (link)). After 6h air or CAP exposure, mice were placed singly per metabolic cage (Harvard Apparatus) with D‐glucose/saccharin solution to collect urine (in graduated cylinders inside 4°C water‐jacketed organ baths) for 3 h. After urine collection, mice were placed in home cages overnight with food and water per normal housing arrangements. Urine samples were centrifuged (600 × g, 5 min, 4°C; to pellet any feces and food particles), decanted, and stored at −80°C. The major metabolite of acrolein, 3‐hydroxypropylmercapturic acid (3HPMA), was quantified in urine by mass spectrometry (negative ion mode) as previously described (Conklin et al., 2017 (link)). To account for urinary dilution, values for urinary 3HPMA were normalized to urinary creatinine (mg/dL).

One day prior to injury and at the specified end-points (1 day, 2 days, 7 days, 14 days, and 28 days post-injury), animals were placed in metabolic cages (Harvard Apparatus) in which food (grams of chow consumed over 6 h) and water intake, as well as urine output were recorded. All rats were placed in metabolic cages for about 6 h (started approximately at 08:30 and ended at 14:30). After 6 h was over, rats were removed from the metabolic cages and returned to their home cages. Urine, remaining water, and remaining rat chow were measured and recorded. If rats had not yet urinated after 6 h, they were tickled gently to induce urination. Urine was collected to a chilled collection cup placed on ice and centrifuged at 5000 rpm for 5 min at 4°C, to remove any undissolved material, and supernatant was stored at -80°C, until subsequent processing/analyses.

Adult WKYs and SHRs were adapted in metabolic cages (Harvard Apparatus, Holliston, MA, USA) for 3 days before a seven-day treatment. After rat bladder was emptied by gentle abdominal massage, urine was collected by spontaneous voiding every 24 hours. PU-14 (PU-14 was home synthesized with purity at 99% as determined by HPLC) at 50 mg/kg in 40% (g/ml) 2-hydroxypropyl-β-cyclodextrin was subcutaneously injected every 6 hours (0:30 a.m., 6:30 a.m., 12:30 p.m., and 6:30 p.m.) for one week16 (link). 40% 2-hydroxypropyl-β-cyclodextrin was used as a vehicle control. Two hours after the last administration, blood sample and thoracic aorta were collected under anesthesia with pentobarbital (1%) at 40 mg/kg body weight. The adequacy of anaesthesia was monitored based on the disappearance of the pedal with drawal reflex response to foot pinch. Urinary volume was measured by gravimetry, assuming a density of 1 g/ml. Urinary osmolality was measured by freezing point depression (Micro-osmometer, FISKER ASSOCIATES, Norwood, MA). Urea concentration was measured with QuantiChrom Urea Assay kit (Roche Diagnostics, Indianapolis, IN, USA).

Rats were administered 4 mg/kg MDPV, were placed individually into metabolic cages (Harvard Apparatus, USA) and their 24-h urine fractions were collected. During collection urine was maintained below 4°C throughout. Samples were subsequently stored at −40°C until analysis. The methods used to the screening of MDPV metabolites, and quantification of MDPV and its metabolites has been described more fully in Horsley et al. [73 (link)].