Animals
Male apolipoprotein E–deficient (ApoE−/−) on the C57BL6/J strain background were purchased from Jackson Laboratory (Bar Harbor, Maine) at 8 weeks of age. Mice were housed under specific pathogen-free conditions in static microisolator cages with tap water ad libitum in a temperature-controlled room with a 12:12-hour light/dark cycle. At 10 weeks of age mice were started on a Western diet (TD88137, Harlan, WI) and at 14 weeks of age, mice were randomly allocated to the CCI or sham procedures, with or without metoprolol. Mice receiving metoprolol were grouped in same cage as drug was supplied in water source. Metoprolol or vehicle control was administered via the drinking water at a concentration of 2 mg/mL for 6 weeks following TBI. Jintao Wang was aware of cage allocation following TBI procedure.
Ethics Statement
All animal use protocols complied with the Principle of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use and Care of Animals. This study is reported in accordance with ARRIVE guidelines as set by the National Centre for the Replacement Refinement and Reduction of Animals in Research [26].
Model of TBI
To induce TBI, male ApoE−/− mice were anesthetized with 2% isoflurane and placed in a stereotactic frame (Kopf,Tujunga,CA, USA) as previously described [27,28]. Briefly, a 5 mm circular craniotomy, centered between the bregma and lambda, was made and then a controlled cortical impact (CCI) was delivered to the midline at an impact speed of 3.00 m/s, tissue displacement of 1.1 mm, and impact duration of 50 ms. Following impact, the circular bone fragment from the craniotomy was glued back to the cranial window. The sham procedure was identical except for craniotomy and delivery of the CCI.
Blood Pressure Measurement
Blood pressure and pulse rate were measured 3 weeks after CCI or sham operation in non-anesthetized, trained mice by tail plethysmography using the BP-2000 Blood Pressure Analysis System (Visitech System, Apex, NC) as previously described [23].
Histological Analysis
Quantification of atherosclerosis, the primary outcome, was performed as previously described [23]. Briefly, mice were euthanized under pentobarbital anesthesia (i.p., 100 mg/kg), and arterial trees were perfused at physiological pressure and fixed in 10% zinc formalin. Paraffin-embedded hearts, which included aortic valves, were sectioned for lesion analysis. A series of 5 μm sections were obtained at the level of the aortic sinus and 4 cross sections were analyzed from each mouse. Sections were stained with hematoxylin and eosin for quantification of lesion area normalized by adjacent medial area of aorta to control for possible tangential sectioning [23,29]. The lesion area was defined as the area between the endothelial cell layer and internal elastic lamina.
For plaque composition analysis, macrophages were quantified with an antibody to MAC3 (1:100, BD Biosciences, San Jose, CA). Negative controls consisted of tissues handled identically to experimental samples except that the primary antibody was omitted. The detection system was streptavidin-HRP and endogenous peroxidase was quenched with hydrogen peroxide. Sections were counterstained with hematoxylin. Positive staining area was analyzed from three fields in each section and expressed as percentage of the total area. All images were analyzed by automated detection of positive stained area using Nikon MetaMorph software with observer blinded to treatment allocation.
Measurements of Plasma Samples
Plasma samples were collected via terminal heart puncture bleeding 6 weeks after TBI or sham operation. Total cholesterol was measured using Infinity cholesterol enzymatic-colorimetric kit (Thermo Fisher, #TR13421).
Statistical analysis
All data are presented as mean ± standard deviation. Statistical analysis was carried out using GraphPad Prism. Shapiro-Wilk normality test was used for normal distribution testing. Results were analyzed using 2-tailed t-tests for comparison between two groups. Sample size was determined by power calculation based on variability of atherosclerosis previously established in this model [6]. No animals were excluded from analysis.