To test this hypothesis, we used a preclinical murine model to investigate whether 4 weeks of dietary supplementation was sufficient to decrease markers of inflammation and reduce sickness behavior in adult and aged

mice challenged with LPS. Sickness behavior and molecular inflammatory response have been well characterized in our model of LPS-challenged aged mice, and these measurements will provide useful information for determining whether broccoli supplementation attenuates behavioral complications of inflammation. A reduction in LPS-induced proinflammatory markers in the broccoli-supplemented mice would indicate that broccoli is a suitable dietary addition to temper inflammation. Adult (4-month-old) and aged (18-month-old) BALB/c mice reared in-house were individually housed in a temperature-controlled environment with a reversed-phase light/dark cycle (lights on 8:00 pm). Ibrutinib During the 28-day experimental period, mice were given ad libitum access to water and diet consisting of AIN-93M or AIN-93M + 10% freeze-dried broccoli (Table). Soy oil was replaced with corn oil to mitigate any potential anti-inflammatory effects derived from increased omega-3 fatty acid content of soy oil. The

broccoli used in the diet provided 5.22 μmol SFN/g as determined by laboratory hydrolysis using the methods described by Dosz and Jeffery [22]. Therefore, it is estimated that mice fed the 10% broccoli diet were exposed to 0.5 μmol glucoraphanin per gram of diet consumed, E7080 providing up to 0.5

μmol SFN/g, depending on the extent of glucoraphanin hydrolysis. To diminish the potential for degradation of glucosinolates from the broccoli-containing diet, we replaced both diets every other day. Body weight was recorded weekly. Mice were handled 1 to 2 minutes per day for 1 week before behavior testing. All studies were carried out in accordance with United States National Institutes of Health guidelines and were approved by the University of Illinois Institutional Animal Care and Use Committee. Escherichia coli LPS (serotype 0127:B8, Sigma, St. Louis, Missouri) was dissolved DOCK10 in sterile saline before experimentation. On day 29 of dietary intervention, mice from each diet group (n = 7) were given LPS (0.33 mg/kg body weight) or saline intraperitoneally. Treatments were administered during the first hour after onset of the dark phase of the light/dark cycle. To determine whether broccoli diet reduced sickness behavior, we assessed social exploratory behavior in all mice 2, 4, 8, and 24 hours after treatment, as previously described in detail [23]. Baseline social exploratory behavior was determined 24 hours before treatment and was used as a basis of comparison for calculating percent baseline time spent investigating a novel juvenile. A novel juvenile conspecific mouse was placed inside a protective cage before being placed in the home cage of the experimental mouse.