Male C57BL/6J mice received a single injection of DSP-4 (50 mg/kg, i.p.), followed by LPS (15×106 EU/kg, i.p.) treatment one week later. (A) “two-hit” mice exhibited decreased defecation frequency compared with age-matched control animals at 6 and 10 months, [effect of treatment, F(3,127)=11.76, p<0.0001]. (B) The ratios of dry weight and wet weight of feces were measured as an indication of constipation, [effect of treatment, F(3,138)=6.179, p=0.0006]. Results represent average defecation frequency for two trials ± SEM for 9 animals per group. *p< 0.05, **p< 0.01 compare with age-matched vehicle controls. #p< 0.05 compare with LPS-alone group.