The effect of antioxidants on the response of the rabbit urinary bladder to in vitro ischemia/reperfusion

To evaluate the protective effects of two naturally occurring antioxidants, α-Lipoic acid and coenzyme Q10 on the response to in vitro ischemia of the rabbit urinary bladder. We measured free fatty acid (FFA) content, phospholipid (PL) content, malondialdehyde (MDA) levels, and phospholipase A(2) activity (PLA) of subcellular compartments. Twenty New Zealand White male rabbits were separated into four groups of five rabbits each. The in vitro whole bladders from Groups 1 and 2 received a 3 h incubation under normal oxygenated physiological conditions. The bladders were stimulated by field stimulation at 1 and 3 h. The bladders from groups 3 and 4 underwent 1 h incubation time under normal oxygenated physiological conditions. After 1 h, the bladders were stimulated with field stimulation. After a maximal pressure response was recorded, the stimulation was turned off and the bath medium changed to one equilibrated with 95% nitrogen, 5% oxygen without glucose (ischemic medium) and incubated for 1 h with field stimulations occurring at 5 min intervals during this time. At the end of this hour of ischemia with repetitive stimulation, the bath was changed to an oxygenated medium with glucose for a 1-h reperfusion period after which the stimulation was repeated. The rabbits from groups 2 and 4 received α-Lipoic acid (10 mg/kg/day) + Coenzyme Q10 (3 mg/kg/day) by gavage for 4 weeks before the experiment. At the end of the experimental period, each bladder was opened longitudinally, and the muscle and mucosa separated by blunt dissection, frozen under liquid nitrogen, and stored at -80°C for biochemical analyses. Each tissue was fractionated by differential centrifugation into nuclear, mitochondrial, synaptosomal, and cytosol (supernatant) components. PL, FFA, MDA, and PLA were analyzed using standard biochemical techniques. Post-ischemic contractility only returned to 30% of control of the untreated group. However, post-ischemic contractility of the treated group returned to approximately 70% of control. PL loss in the muscle mitochondria and synaptosomes was prevented by antioxidant treatment, while the mucosal layer showed a significant drop in PL with antioxidants treatment. Administration of CoQ + LA significantly decreased MDA levels in both control and ischemic tissues in both the muscle and mucosal bladder layers, especially substantial in the microsomal and mitochondrial components. Treatment had variable effects on PLA(2) activity. Treatment of bladder dysfunction with antioxidants daily can be beneficial in man to prevent or delay the onset of progressive loss of bladder function especially that due to ischemic damage to mitochondrial and microsomal lipids. CoQ10 + LA can provide similar protection of the bladder muscle and mucosa against lipid oxidative stress as they have been shown to protect against protein oxidative damage.