Introduction: Oxidative inflammatory damage to the brain may lead to dysfunction of peripheral organs including bladder disorders. However, the source of reactive oxygen species (ROS) in specific brain regions that regulate the function of peripheral systems is poorly understood. Of all ROS-generating enzymes, the NADPH oxidase (Nox) family produces ROS as its sole function and offers an advantage over other enzymes as a drug targetable molecule to selectively control excessive ROS. Our pilot study demonstrated such system in the bladder. This study tested the hypothesis that the major Nox subtype, Nox 2 is expressed in the micturition regulatory regions - Pontine micturition centre (PMC) and periaqueductal gray (PAG), and such system is able to produce substantial superoxide.
Methods: Male adult C57BL/6J mice (2-5 months) were used. PAG, PMC were dissected according to a mouse stereotaxic atlas. Cardiac and bladder tissues were isolated under microscopic guidance. Western blot determined Nox 2 expression using Nox 2 primary antibody and Infra-red dye-conjugated secondary antibodies. Lucigenin enhanced chemiluminescence quantified real-time superoxide production in live tissues, verified by the superoxide scavenger Tiron. Paired and non-paired t-test tested the difference in two group means and ANOVA with pairwise comparisons tested multiple means.
Results: Western blot experiments demonstrated the presence of Nox 2 in PAG and PMC tissues, with a density comparable to that of positive control (n=6). There was a significant level of NADPH dependent superoxide production in both brain tissues (RLU/mg, PMC: 127±17; PAG102±13, N=11, higher than that from cardiac tissue (32±3, P<0.01); similar to that from bladder smooth muscle (91±19, p>0.05) and lower than that in bladder mucosa (262±55, p<0.01). The time course analysis shows that the rise of superoxide production in these two brain tissues was more sustained than in bladder mucosa and smooth muscle (Half peak duration PD50, minutes: PMC, 101±2; PAG, 99±2; bladder mucosa, 48±3, p<0.01; smooth muscle, 67±9, p<0.01). The superoxide generation from these brain tissues was significantly suppressed by the Nox inhibitor diphenyleneiodonium (DPI).
Conclusions: These data provide the first evidence for the presence of Nox 2 in brain micturition centres and its high capacity to produce superoxide. The study has also identified the sustained nature of the superoxide rise from this ROS-generating pathway. The predominance of this system and its characteristics revealed by this study provide mechanistic basis for the pathology of aging and inflammation associated neurogenic bladder disorders.
Source of Funding: BBSRC (BB/P004695/1)
NIA (NIA1R01AG049321-01A1)
QW: Jiangsu Overseas Visiting Scholar Program for University Prominent Young and Middle-aged Teachers and Presidents.
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