Abstract
The world’s population is rapidly ageing, and with age the risk of chronic diseases increases. The bladder is particularly susceptible to ageing, as bladder conditions affect one in three adults over the age of 65. Common symptoms observed are urinary incontinence, urgency, nocturia and increased urinary frequency. These symptoms are highly stigmatised, impair the patients’ quality of life and have a substantial economic and human cost. Age-related bladder conditions are caused by physiological changes in the function and control of this organ, which are regulated by multiple signalling molecules released by the urothelium (inner lining of the bladder). Amongst the transmitters released by the urothelium, nitric oxide (NO) is a key relaxing mediator in the bladder. Although the importance of NO in the physiology and pathology of the lower urinary tract has been recognised, its role in age-related bladder conditions is largely unknown. The aim of this thesis was to develop electrochemical sensors to study how nitrergic species (nitric oxide and nitrite (NO2 - )) released by the bladder urothelium alter with age.Compared to other techniques, electrochemical methods provide the only means to detect NO directly and in situ from the bladder urothelium. MWCNT composite microelectrodes were made and chemically modified with different concentrations of Pt black for detection of nitrergic species. These microelectrodes were characterised through electrochemical techniques and imaging to identify which composition provides the most sensitive, selective and stable detection of nitrergic species. After optimisation of the nitrergic sensors, using 20% Pt black MWCNT composite microelectrodes, a robust methodology was developed to detect NO and NO2 - in the bladder by removing all factors that could introduce bias into the measurements. The refined protocol for robust bioanalytical measurements from bladder strips was achieved by conducting measurements inside a CO2 incubator using multi-step amperometry (amperometric technique used to monitor multiple analytes in a single measurement) at 0.4, 0.65 and 0.85V vs non-leak Ag/AgCl. The difference in the voltage applied provided the ability to detect both NO and NO2 - . Using this protocol, recordings were conducted to measure NO and NO2 - levels from bladder strips from 3-4-month (young) and 24-month-old (aged) C57BL/6J mice. Nitrergic levels were measured when the bladder samples were placed under different conditions of mechanical stretch and the contribution of the different isoforms of nitric oxide synthase (NOS) to the synthesis of NO was investigated.
Interestingly, the level of nitrergic species was lower in the aged than in the young bladder. Although the reduction in nitrergic release with ageing was not significant in the stretched group, it was clearly lower in aged bladder samples. In contrast, the reduction in nitrergic species with age was significant for the samples under no stretch, where in some cases nitrergic levels were undetectable in the aged group. These findings highlight that in the young bladder NO is released to maintain bladder relaxation, playing an important role during bladder filling where its concentration is reduced when the bladder is fully stretched to allow micturition to occur. However, with age, the bladder would be in a more contractile state as the decline in nitrergic release makes it difficult for the bladder to maintain relaxation. The inhibition of iNOS on bladder tissues showed a greater reduction in nitrergic release in the aged bladder, whilst in the young bladder the majority of NO is synthesised by eNOS. This highlights that changes in NO production with age are due to alterations in the NOS isoforms responsible for the synthesis of NO.
The findings clearly show that the bladder’s nitrergic signalling mechanisms are altered with age, both in the NOS isoforms responsible for NO production and in the physiological processes through which NO may regulate bladder function. These changes could help explain why the prevalence of bladder symptoms increases with age, and also provide potential targets for therapeutic treatment of age-related bladder conditions.
| Date of Award | Jun 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Bhavik Patel (Supervisor), Dr John S. Young (Supervisor) & Jon Mabley (Supervisor) |