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Chronic Social Defeat, But Not Restraint Stress, Alters Bladder Function in Mice
Elizabeth A. Mann, Ph.D., Zaheer Alam, MD, Jillian R. Hufgard, BA, Melissa Mogle, BA, Michael T. Williams, Ph.D., Charles V. Vorhees, Ph.D., Pramod Reddy, MD.
Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

BACKGROUND: Voiding disorders are associated with increased incidence of behavioral issues as well as past history of childhood abuse. We hypothesized that both chronic social defeat (SD) stress and restraint stress in shallow water (RSSW) in mice would engender changes in bladder function, morphology, and behavior.
METHODS: For SD stress (14 days), C57Bl/6 male mice were exposed daily to a larger aggressive CD-1 male for 10 minutes, followed by sensory exposure in a barrier cage for 24 hours. Control mice were similarly housed with no exposure. For RSSW (21 days), C57Bl/6 mice were put in a perforated conical tube with feet immersed in water daily for 4 hours, then returned to single housing cages. Control mice were also in single housing. After the stress period, voiding patterns were obtained on filter paper, followed by behavioral tests. At necropsy, blood was taken for corticosterone analysis, and bladder and body weights measured. Bladder cryosections were stained with H&E for morphological assessment. Sequential sections were immunostained with antibodies to Ki-67 as a proliferation marker, CD31 (endothelial cell marker), and Uroplakin-II. Image J software was used to measure bladder wall thickness on blinded H&E photomicrographs as well as quantitate CD31 staining. Both Ki-67 -positive and -negative nuclei were counted with Imaris software to obtain a proliferation index.
RESULTS: Only SD mice had a single large void pattern. Bladder- to- body weight ratios increased in SD mice (p≤ 0.02) but not in RSSW mice. Plasma corticosterone levels were elevated in all stressed mice. SD mice exhibited lower levels of locomotor activity compared with controls; RSSW mice were hyperactive. In SD mice, bladder wall thickness was increased (p ≤ 0.002), but no change was seen in Ki-67 proliferation index, consistent with hypertrophy. No difference with control mice was seen in vascularity as visualized by CD31 staining. Uniform Uroplakin-II staining lined the urothelium of both SD and control mice.
CONCLUSIONS: Mice exposed to repeated SD (14 days) respond with altered voiding indicative of urine retention, and exhibit bladder wall changes consistent with hypertrophy while the urothelial barrier is maintained. These changes were not observed with repeated RSSW. SD, in contrast to RSSW, provides a model of psychological stress to further study the interplay of behavior and bladder dysfunction, enabling an improved therapeutic care plan in the future for children presenting with voiding dysfunction.

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