The urothelium and lamina propria as an alternative target for clinical antimuscarinics

Introduction: Overactive bladder is the most common type of bladder dysfunction and involves spontaneous contractions of the urinary bladder during the filling phase. The first-line pharmaceutical therapies for managing this disorder are antimuscarinics (Moro et al., 2011), which have a primary action of blocking the action of acetylcholine in the urothelium and lamina propria (Nardulli et al., 2012). However, more than 70% of patients who are administered these drugs cease their treatment regimen due to lower than expected treatment benefits or adverse side effects (Vouri et al., 2019). The reason for this is unclear, although this does suggest a varied effectiveness or selectivity of antimuscarinics on urinary bladder tissue. Aim: This study aims to find the differences in the abilities to inhibit contractions of the U&LP for commonly prescribed clinical antimuscarinics. Methods: Strips of porcine U&LP were mounted in carbogen-gassed Krebs-bicarbonate solution at 37°C. The tissues were paired with carbachol concentration-response curves performed in the absence or presence of clinically used antimuscarinics. The concentration for each antagonist was chosen at which the inhibited contractions reached a significant, but sub-maximal, extent. pEC50 values for each curve were analysed and estimated affinities calculated. Ethical approval was not required for this study as tissues were sourced from the local abattoir after slaughter for the routine commercial provision of food. Results: The clinical antimuscarinics producing right parallel shifts from the control in the U&LP (concentration; n value; estimated affinity or pkD; paired Student’s two-tailed t-test) included oxybutynin (1µM; 18; 7.08; p<0.001), solifenacin (1µM; 11; 6.88; p<0.001), darifenacin (100nM; 10; 6.48; p<0.001), tolterodine (1µM; 10; 8.00; p<0.001), trospium (100nM; 10; 7.63; p<0.001) and fesoterodine (100nM; 11; 7.40; p<0.001). Propiverine (concentration; n value; paired Student’s two-tailed t-test) did not produce a shift (1µM; 11; p=0.50). Conclusion: The data highlights a variance in the effectiveness of each clinically used antimuscarinic to antagonise the response to muscarinic receptor activation of the U&LP

Development of novel magnetic resonance methods for advanced parametric mapping of the right ventricle

The detection of diffuse fibrosis is of particular interest in congenital heart disease patients, including repaired Tetralogy of Fallot (rTOF), as clinical outcome is linked to the accurate identification of diffuse fibrosis. In the Left Ventricular (LV) myocardium native T1 mapping and Diffusion Tensor Cardiac Magnetic Resonance (DT-CMR) are promising approaches for detection of diffuse fibrosis. In the Right Ventricle (RV) current techniques are limited due to the thinner, mobile and complex shaped compact myocardium. This thesis describes technical development of RV tissue characterisation methods. An interleaved variable density spiral DT-CMR method was implemented on a clinical 3T scanner allowing both ex and in vivo imaging. A range of artefact corrections were implemented and tested (gradient timing delays, off-resonance and T2* corrections). The off- resonance and T2* corrections were evaluated using computational simulation demonstrating that for in vivo acquisitions, off-resonance correction is essential. For the first-time high-resolution Stimulated Echo Acquisition Mode (STEAM) DT-CMR data was acquired in both healthy and rTOF ex-vivo hearts using an interleaved spiral trajectory and was shown to outperform single-shot EPI methods. In vivo the first DT-CMR data was shown from the RV using both an EPI and an interleaved spiral sequence. Both sequences provided were reproducible in healthy volunteers. Results suggest that the RV conformation of cardiomyocytes differs from the known structure in the LV. A novel STEAM-SAturation-recovery Single-sHot Acquisition (SASHA) sequence allowed the acquisition of native T1 data in the RV. The excellent blood and fat suppression provided by STEAM is leveraged to eliminate partial fat and blood signal more effectively than Modified Look-Locker Imaging (MOLLI) sequences. STEAM-SASHA T1 was validated in a phantom showing more accurate results in the native myocardial T1 range than MOLLI. STEAM-SASHA demonstrated good reproducibility in healthy volunteers and initial promising results in a single rTOF patient.Open Acces

Micro-computed tomography for high resolution soft tissue imaging; applications in the normal and failing heart

The normal structure and function of the heart, the common pathological changes that cause abnormal function and the interventions proposed to improve or restore its function are fundamentally based on cardiac anatomy. Therefore in all these areas a detailed and accurate understanding of 3D structure is essential. However there is still disparity over some aspects of the form and function of the healthy heart. Furthermore, in heart failure (HF) the transition from compensated to decompensated HF is poorly understood, and details of ventricular, and particularly atrial, remodelling and their effects on cardiac function are yet to be fully elucidated. In addition little is known on how the 3D morphology of the cardiac conduction system is affected in disease, and further knowledge is required on the structural substrates for arrhythmogenesis associated with HF. Here we have developed contrast enhanced micro-CT for soft tissue imaging, allowing non-invasive high resolution (~5 µm attainable) differentiation of multiple soft tissue types including; muscle, connective tissue and fat. Micro-CT was optimised for imaging of whole intact mammalian hearts and from these data we reveal novel morphological and anatomical detail in healthy hearts and in hearts after experimental HF (volume and pressure overload). Remodelling of the myocardium in HF was dramatic with significant hypertrophy and dilatation observed in both atria and ventricles. The atria showed a 67% increase in myocardial volume, with the left atrium showing a 93% increase. The pectinate muscle: wall thickness ratio was significantly increased in both atria (p

Molecular Mechanism of Congenital Heart Disease and Pulmonary Hypertension

This open access book focuses on the molecular mechanism of congenital heart disease and pulmonary hypertension, offering new insights into the development of pulmonary circulation and the ductus arteriosus. It describes in detail the molecular mechanisms involved in the development and morphogenesis of the heart, lungs and ductus arteriosus, covering a range of topics such as gene functions, growth factors, transcription factors and cellular interactions, as well as stem cell engineering technologies. The book also presents recent advances in our understanding of the molecular mechanism of lung development, pulmonary hypertension and molecular regulation of the ductus arteriosus. As such, it is an ideal resource for physicians, scientists and investigators interested in the latest findings on the origins of congenital heart disease and potential future therapies involving pulmonary circulation/hypertension and the ductus arteriosus

Imaging fascicular organisation in mammalian vagus nerve for selective VNS

Nerves contain a large number of nerve fibres, or axons, organised into bundles known as fascicles. Despite the somatic nervous system being well understood, the organisation of the fascicles within the nerves of the autonomic nervous system remains almost completely unknown. The new field of bioelectronics medicine, Electroceuticals, involves the electrical stimulation of nerves to treat diseases instead of administering drugs or performing complex surgical procedures. Of particular interest is the vagus nerve, a prime target for intervention due to its afferent and efferent innervation to the heart, lungs and majority of the visceral organs. Vagus nerve stimulation (VNS) is a promising therapy for treatment of various conditions resistant to standard therapeutics. However, due to the unknown anatomy, the whole nerve is stimulated which leads to unwanted off-target effects. Electrical Impedance Tomography (EIT) is a non-invasive medical imaging technique in which the impedance of a part of the body is inferred from electrode measurements and used to form a tomographic image of that part. Micro-computed tomography (microCT) is an ex vivo method that has the potential to allow for imaging and tracing of fascicles within experimental models and facilitate the development of a fascicular map. Additionally, it could validate the in vivo technique of EIT. The aim of this thesis was to develop and optimise the microCT imaging method for imaging the fascicles within the nerve and to determine the fascicular organisation of the vagus nerve, ultimately allowing for selective VNS. Understanding and imaging the fascicular anatomy of nerves will not only allow for selective VNS and the improvement of its therapeutic efficacy but could also be integrated into the study on all peripheral nerves for peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. Chapter 1 provides an introduction to vagus nerve anatomy and the principles of microCT, neuronal tracing and EIT. Chapter 2 describes the optimisation of microCT for imaging the fascicular anatomy of peripheral nerves in the experimental rat sciatic and pig vagus nerve models, including the development of pre-processing methods and scanning parameters. Cross-validation of this optimised microCT method, neuronal tracing and EIT in the rat sciatic nerve was detailed in Chapter 3. Chapter 4 describes the study with microCT with tracing, EIT and selective stimulation in pigs, a model for human nerves. The microCT tracing approach was then extended into the subdiaphragmatic branches of the vagus nerves, detailed in Chapter 5. The ultimate goal of human vagus nerve tracing was preliminarily performed and described in Chapter 6. Chapter 7 concludes the work and describes future work. Lastly, Appendix 1 (Chapter 8) is a mini review on the application of selective vagus nerve stimulation to treat acute respiratory distress syndrome and Appendix 2 is morphological data corresponding to Chapter 4

Recent Developments in Atomic Force Microscopy and Raman Spectroscopy for Materials Characterization

This book contains chapters that describe advanced atomic force microscopy (AFM) modes and Raman spectroscopy. It also provides an in-depth understanding of advanced AFM modes and Raman spectroscopy for characterizing various materials. This volume is a useful resource for a wide range of readers, including scientists, engineers, graduate students, postdoctoral fellows, and scientific professionals working in specialized fields such as AFM, photovoltaics, 2D materials, carbon nanotubes, nanomaterials, and Raman spectroscopy

Nutrient supply impacts osteocytic specification by regulating a nuclear transcription program

[spa] El hueso es un órgano con múltiples funciones. No sólo actúa como elemento de soporte, protección y locomoción, sino que también resulta indispensable en el mantenimiento del equilibrio mineral y ácido/base, conforma un nicho adecuado para el desarrollo de la hematopoyesis, y mantiene la homeostasis energética del organismo. En estos procesos, los osteocitos tienen un papel especialmente relevante, ya que actúan transduciendo estímulos mecánicos en señales bioquímicas. Los osteocitos constituyen el principal componente celular óseo. Derivan de osteoblastos, los cuales a su vez proceden de células madre mensenquimales (MSC). Los osteoblastos pueden seguir tres destinos alternativos: entrar en apoptosis, originar células de revestimiento óseo o progresar en la diferenciación hacia osteocitos. Actualmente, los estímulos y vías de señalización que regulan cada uno de estos procesos son desconocidos. Por ello, y teniendo en cuenta la importancia de los osteocitos en la homeostasis del organismo, consideramos necesario profundizar en su investigación. Durante el proceso de diferenciación ósea, los osteoblastos quedan embebidos en una matriz mineralizada que limita su disponibilidad de nutrientes, estando expuestos a un ambiente hipoglucémico al cual deben adaptarse. En este contexto Wei et al. demostraron que la glucosa juega un papel importante en la regulación de la diferenciación osteoblástica. Por otro lado, se ha observado que, en ambientes hiperglucémicos, típicos de pacientes diabéticos, se produce una reducción del número y función osteoblástica, así como una disminución de la densidad mineral ósea y alteración de la microarquitectura ósea. Teniendo en cuenta todo lo expuesto, estudiamos la capacidad de diferenciación de las IDG-SW3 en condiciones de hipoglucemia (1mM glucosa), normoglucemia (5mM glucosa) o hiperglucemia (25mM glucosa). Las condiciones de hipoglucemia promueven la diferenciación osteocitica, mientras que altas concentraciones de glucosa dificultan este proceso. A nivel metabólico, las condiciones de baja glucosa aumentan la cantidad de mitocondrias y su agrupación en forma de redes. Por otro lado, los ambientes hipoglucémicos, promueven los eventos de fisión mitocondrial. En este contexto PGC1α podría desempeñar un papel crucial como nexo entre el estrés metabólico y la reprogramación génica de los osteoblastos. PGC1α es un coactivador transcripcional que responde a diferentes tipos de estrés. Aunque sus dianas son múltiples, afectan principalmente a la expresión de genes implicados en el metabolismo, así como la biogénesis y función mitocondrial. PGC1 se activa a través de fosforilación y acetilación mediadas por AMPK y SIRT1. La activación de PGC1α, podría iniciar una reprogramación metabólica y génica que culminaría en una inducción de la diferenciación osteocítica

Life Sciences Program Tasks and Bibliography for FY 1997

This document includes information on all peer reviewed projects funded by the Office of Life and Microgravity Sciences and Applications, Life Sciences Division during fiscal year 1997. This document will be published annually and made available to scientists in the space life sciences field both as a hard copy and as an interactive internet web page