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Chronic Cyclic Bladder Over-Distension Upregulates Hypoxia-Dependent Pathways

Saturday, October 20, 2012: 10:09 AM
Grand Ballroom A/B (Hilton Riverside)
Heidi A. Penn, MD1, Stacy T. Tanaka, MD1, Mariana M. Cajaiba, MD2, John C. Thomas, MD1, John C. Pope IV, MD1, Mark C. Adams, MD1, John W. Brock III, MD1 and Douglass B. Clayton, MD1, (1)Division of Pediatric Urology, Monroe Carell Jr. Children's Hospital at Vanderbilt, Nashville, TN, (2)Department of Pathology, Vanderbilt University, Nashville, TN

Purpose: Bladder over-distension secondary to anatomic or functional obstruction can eventually lead to pathologic changes, including decreased elasticity and contractile dysfunction. There has been increasing evidence that ischemia followed by reperfusion and subsequent formation of reactive oxygen species (ROS) may be responsible for the ensuing inflammation and eventual cell death seen from over-distension. We hypothesize that chronic cyclic over-distension in a murine model will activate hypoxia-dependent signaling pathways despite intermittent relief of the distention.

Methods: Female C57/Bl6 mice were ovariectomized at 5-6 weeks and subjected to urethral catheterization and infusion of warmed 0.9% saline at 60cmH20 for two hours while under general anesthesia with isoflourane. At the end of the two hours, the bladders were drained and the mice were allowed to recover.  This process was repeated for a total of 3 consecutive days. Age-matched ovariectomized mice were subjected to 2 hours of general anesthesia each day without bladder distension and acted as controls. Bladder tissue was harvested and used for H&E staining. Immunohistochemical (IHC) staining for hypoxia markers including glucose transporter-1 and the commercially available antibody, Hypoxyprobe™-1 was performed. A single pathologist reviewed all slides. Bladder tissue was snap frozen in liquid nitrogen and used for RNA extraction. Hypoxia PCR Array was performed and statistical significance was calculated based on a Student’s t-test of the replicate 2^(- Delta Ct) values for each gene in the control treatment groups, and p values less than 0.05 were considered significant.

Results: After 3 consecutive over-distensions, bladders exhibited minimal inflammatory changes on H&E staining. IHC was positive for Hypoxyprobe™-1 and glutamine-1. RT-PCR demonstrated up-regulation of hypoxic genes and their fold changes included: connective tissue growth factor (+2.97, p = 0.003), transforming growth factor β (+6.14, p = 0.008), hypoxia inducible factor 1α (+4.57, p = 0.097), vascular endothelial growth factor (+8.3, p = 0.006), and BCL2-associated x protein (BAX) (+2.4, p = 0.02). 

Conclusion: Intermittent periods of cyclic over-distension create a hypoxic environment and over time may result in abnormal bladder function. This bladder injury model might more closely replicate bladder dysfunction in patients with poor bladder emptying due to neurologic disease and particularly in patients who are not compliant with intermittent catheterization.