Oxidative inflammatory damage to the specialised brain centres may lead to aging and the dysfunction of their associated peripheral organs such as bladder disorders (1). However, the source of reactive oxygen species (ROS) in specific brain regions that regulate bladder function is poorly understood. Of all ROS-generating enzymes, NADPH oxidase (Nox) family produces ROS as its sole function and offers advantage over other enzymes as a drug targetable molecule to selectively control excessive ROS generation without compromising physiological oxidation. Our pilot studies have identified the Nox system in the bladder tissue and demonstrated its functional and pathological significance (2). However whether Nox system exists in the brain micturition centre and hence affects the micturition process has not been examined. We have investigated whether the major pathological Nox subtype, Nox 2,  is expressed in micturition regulatory Periaqueductal gray (PAG) and Barrington’s nucleus (pontine micturition centre, PMC) and examined the Nox-derived ROS production in these structures.

C57BL/6J mice (male, 2-5 months) were used as the experimental model in compliance with the UK regulations. PAG and PMC were obtained by stereotaxic dissection using a rodent brain slicer. Cardiac tissue, bladder tissue and aorta were micro-dissected under the microscopic guidance. The Nox 2 expression was determined by Western blot with primary antibodies for NOX2/gp91phox and the reference protein beta-actin, and Infra-red dye conjugated secondary antibodies. Lucigenin enhanced chemiluminescence quantified real-time superoxide production in live tissues, incubated with an artificial cerebral spinal fluid (ACSF). ACSF contained 128 mM NaCl, 3 mM KCl, 1 mM MgCl2.6H2O, 24 mM NaHCO3, 0.5 mM NaH2PO4.2 H2O, 30 mM glucose, and 1.5 mM CaCl2. The NADPH-dependent superoxide production was stimulated by 100µM NADPH. The specificity was verified by superoxide scavenger Tiron. The quantity of photons collected was expressed as relative light units (RLU) per unit tissue weight (RLU/mg). Data are expressed as mean ± SEM. For data with a normal distribution, the difference between two group means was tested with Student’s t-test, paired or unpaired as appropriate. For data which did not follow a normal distribution or had an unknown distribution, the difference between two group means was tested with equivalent non-parametric tests. The difference among multiple means was tested with ANOVA followed by pair-wise comparisons or non-parametric equivalents. The null hypothesis was rejected at p<0.05.

Western blot results show a clear protein band with the expected molecular weight of Nox 2 from PAG and PMC extracts (N=6). This was validated by Nox 2 positive HEK 293 cells with the same molecular weight and comparable intensity confirming significant expression of Nox 2 in the PAG and PMC. There was a significant level of NADPH dependent superoxide production in both brain tissues (RLU/mg, PMC: 127±17; PAG102±13, N=110). The magnitude was 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 that 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 broad spectrum Nox inhibitor diphenyleneiodonium (DPI, 20µM). Further experiment using Nox2 specific inhibitor GSK2795039 (25µM) also attenuated the superoxide production in both brain tissues with 2/3 of the inhibition observed in the aortic tissue.

Western blots results with Nox2 protein band with specific molecular weight proves the existence of Nox2 molecules in PAG and PMC. The comparable density of Nox2 proteins in these brain regions to that of positive control cells suggests the abundance of Nox2 expression in these brain tissues. These Nox 2 proteins serve as a molecular basis for Nox-2 derived superoxide production. Measurement of NADPH-stimulated superoxide production, verified by superoxide scavenger Tiron, shows that PAG and PMC produce significant amount of NADPH-dependent superoxide, hence Nox-derived superoxide production. The magnitude of superoxide production, greater than that from the cardiac tissue, known to produce significant amount of reactive oxygen species and subject to oxidative damage, demonstrates the ability of the PAG and PMC to produce sufficient superoxide and hence cause oxidative damage. The significant inhibition of the superoxide production by the broad spectrum Nox enzyme inhibitor DPI proves that the source of superoxide is mainly from from the Nox enzymes in these brain tissues. The inhibition of the superoxide production by Nox2 specific inhibitor GSK2795039, comparable to that of aortic tissue, known to express mainly Nox2 subtype (3), supports the contribution from Nox 2 subtype in the PAG and PMC. The sustained superoxide production in both brain tissues compared with other peripheral tissues implies that Nox derived ROS in these brain regions, once initiated, produces long lasting oxidative damage to the tissue. This brain region specific mode of action of Nox driven superoxide production may serve as a molecular mechanism for chronic damage to these brain regions. As Nox 2 is a major pathologically important Nox subtype in the central nervous system, the findings from this work have pathological implications for neurogenic damage to micturition and bladder function.

Nox 2 subtype is expressed in the PAG and PMC with relatively high density. These brain regions have the ability to produce significant amount of NADPH-dependent superoxide, comparable to that of the peripheral tissues known to subject to oxidative stress. The source of superoxide is mainly from Nox enzymes with contributions from the Nox 2 subtype. The Nox-derived superoxide production in the PAG and PMC has sustained kinetics favouring chronic oxidative damage. The study  provides the first evidence for Nox 2 subtype expression and Nox-derived superoxide production in these micturition regulatory structures in the central nervous system, with functional and pathological implications.

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Continence 2S2 (2022) 100320
DOI: 10.1016/j.cont.2022.100320