Development of Renal Phantoms for the Evaluation of Current and Emerging Ultrasound Technology

The primary aim of this project was to develop novel anatomically realistic renal phantoms for the evaluation of current and emerging ultrasound techniques capable of diagnosing all grades of renal artery stenosis. Renal artery stenosis is considered the most common cause of potentially curable secondary hypertension which if left untreated can lead to renal failure. Its early detection is very important as it offers the possibility of various drug treatments, which are considerably less invasive and poses less risk to the patient. Computer-aided modelling techniques were used to generate a range of anatomically realistic phantoms of the renal artery from medical images of a 64-slice CT scan which was acquired from a healthy volunteer with normal renal vasculature. These phantoms comprised of a normal healthy vessel and vessels with increasing stenosis (30%, 50% 70% and 85%). Using these novel phantoms a comparative study between four of the imaging techniques currently used to detect renal artery stenosis (ultrasound, magnetic resonance imaging, computed tomography and digital subtraction angiography (DSA)) was carried out. A novel kidney perfusion phantom was also developed with the ability to achieve flow velocities comparable with those found in the blood vessels of the renal macrocirculation (renal artery and renal vein) and microcirculation (kidney). By developing an understanding of disease progression in the renal artery and kidney through experimentation, it is possible to improve the outcome of various treatment regimes by early detection of the disease. Recent and ongoing ultrasound technological developments such as ultrasound contrast agents should render accessible the technically more challenging imaging of the renal artery and kidneys and potentially replace invasive intra-arterial DSA technique

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Apports physiopathologiques de l’étude de la perfusion de la moëlle osseuse par IRM

Bone Marrow (BM) microvascular properties are insufficently known in humans. Dynamic-Contrast Enhanced (DCE) MRI allows its non-invasive quantitative assessment. We concentrated on the hip because this joint is frequently affected by debilitating pathologies such as osteoarthritis. Their early diagnosis is a current medical challenge. We implemented a 3D DCE-MRI sequence with isotropic voxels, high spatial resolution and a large coverage. It was used in a study of 60 patients aged 18 to 60, with no previous history of bone disease and with normal-appearing BM on MR images. Semi-quantitative and pharmacokinetic parameters were measured in 15 regions of interest in each patient. All the parameters were different between red and yellow BM. Perfusion was different between acetabular (axial skeleton) and femoral intertrochanteric (appendicular skeleton) BMs. Several parameters were negatively correlated with age. Perfusion was different in men and women. The femoral head perfusion was heterogeneous, likely because of mechanical load exposure. Ktrans, Kep and TTP were correlated with body mass index. This suggests that obesity influences BM metabolism. Smoking and hypercholesterolemia influenced these same parameters in several zones. We hypothesized that these parameters might reflect BM microvascular aletrations. Our results open new research perspectives both in the physiology and pathology of BM.Les propriétés microvasculaires de la moëlle osseuse (MO) sont mal connues chez l’être humain. L’IRM de perfusion en permet une évaluation quantitative non invasive. La hanche a été choisie, car elle est la cible de pathologies fréquentes et handicapantes, qu’il est nécessaire de diagnostiquer plus précocement telle la coxarthrose. Nous avons d’abord implémenté une séquence IRM volumique à voxels isotropiques, avec une couverture large et une résolution spatiale élevée. Celle-ci a ensuite permis l’étude d’une série de 60 patients âgés de 18 à 60 ans, sans antécédent de pathologie osseuse et présentant une MO d’aspect normal en IRM. Les paramètres de perfusion semi-quantitatifs et pharmacocinétiques ont été mesurés dans 15 régions d’intérêt chez chaque patient. Tous les paramètres de perfusion diffèrent entre les zones de MO rouge et de MO jaune. La perfusion est différente entre les MO acétabulaire (squelette axial) et fémorale intertrochantérienne (squelette appendiculaire). Plusieurs paramètres sont corrélés de manière négative à l’âge. Plusieurs paramètres sont différents entre les hommes et les femmes. La perfusion de la tête fémorale est hétérogène, probablement en raison de l’exposition aux contraintes mécaniques. Les paramètres Ktrans, Kep et TTP sont corrélés à l’indice de masse corporelle, ce qui suggère que l’obésité influence le métabolisme de la MO. Enfin, le tabagisme et l’hypercholestérolémie ont une incidence sur ces mêmes paramètres dans certaines zones. Ils pourraient donc être le reflet d’altérations de la microvascularisation osseuse. Ces travaux ouvrent de nouvelles perspectives de recherche sur la physiologie et la pathologie de la MO

Molecular Imaging of Prostate Cancer

Chapter 1 addresses the introduction to the thesis and provides epidemiology, etiology, metastatic spread, current diagnostics and clinical need of new biomarker for risk stratification of prostate cancer. Chapter 2 provides a detailed analysis of the distribution pattern of the three most used choline tracers: 18F-methylcholine, 11C-choline, and 18F-ethylcholine in metabolically and anatomically disease-free patients. The ranges of SUVmax, SUVmean and standard deviations have been presented. Potential pitfalls in evaluation of “non-avid” but clinically significant presentation of different disease entities are also addressed. The chapter provides overview of the variations in choline uptake pattern which is vital for assessment of various organs when imaging is performed for evaluation of metastatic disease. Chapter 3 presents the feasibility of assessing dynamic 18F Ethyl Choline PET with a view to do kinetic modelling in clinical setting of biochemical relapse of Prostate Cancer. This critical piece of work underpins the quantification, tracer kinetics and demonstrates that cancerous tissue shows abnormal perfusion. From these observations I was able to conclude that 18F Choline can act as a biomarker to assess angiogenesis in prostate cancer and introduces 18F Choline as a biomarker for further work presented in chapter 4-8. Chapter 4 addresses the detection of clinically significant and insignificant prostate cancer on 18F-FECH PET/CT and I correlated findings with template guided prostate mapping biopsy (TPM). Sensitivity and Specificity data of 8F-FECH PET/CT has been provided. Chapter 5 addresses the accuracy of 18F Choline PET/MR which is compared to reference standard (template guided prostate mapping biopsy). This work suggests that data obtained from 18F Choline PET/MR can allow detection of clinically significant and insignificant prostate cancer. I noted that multiple previous treatments can give false positive results and 18F Choline PET/MR is the imaging investigation of choice post HIFU. Moreover, false negative results with 18F Choline PET/MR can be due to very small volume (=/<2 mm) disease. Chapter 6 presents the differential diagnosis of abnormal tracer accumulation in the Prostate and periprostatic tissue. Chapter 7 provides spectrum of skeletal findings on dual-phase 18F-fluoroethylcholine (FECH) PET/CT performed during the work-up of patients referred for suspected prostate cancer relapse. I have provided quantification data and explained that SUVmax in isolation cannot be used to characterize these lesions as benign or malignant. Minimal overlap of benign and malignant lesions also exists. Chapter 8 addresses the clinical utility of 18F Choline in the setting of clinical trial in collaboration with Uro-oncology, Nuclear Medicine and Radiology departments. This critical work compares 18F Choline PET-CT and Whole-Body MRI in assessment and decision-making process for salvage treatment of focal radio-recurrent prostate cancer. This chapters concludes that at present WB-MRI cannot be used alone as imaging modality for investigation of biochemical relapse of Prostate Cancer. Chapter 9 is a summary of main findings and discussions from chapters in this thesis. It also highlights the potential applications and future perspectives of novel biomarkers for imaging of prostate cancer

Measuring Chemotherapy Response in Breast Cancer Using Optical and Ultrasound Spectroscopy

Purpose: This study comprises two subprojects. In subproject one, the study purpose was to evaluate response to neoadjuvant chemotherapy (NAC) using quantitative ultrasound (QUS) and diffuse optical spectroscopy imaging (DOS) in locally advanced breast cancer (LABC) during chemotherapy. In subproject two, DOS-based functional maps were analysed with texture-based image features to predict breast cancer response before the start of NAC. Patients and Measurements: The institution’s ethics review board approved this study. For subproject one, subjects (n=22) gave written consent before participating in the study. Participants underwent non-invasive, DOS and QUS imaging. Data were acquired at weeks 0 (i.e. baseline), 1, 4, 8 and before surgical removal of the tumour (mastectomy and/or lumpectomy); corresponding to chemotherapy schedules. QUS parameters including the midband fit (MBF), 0-MHz intercept (SI), and the spectral slope (SS) were determined from tumour ultrasound data using spectral analysis. In the same patients, DOS was used to measure parameters relating to tumour haemoglobin and tissue composition such as %Water and %Lipids. Discriminant analysis and receiver-operating characteristic (ROC) analyses were used to correlate the measured imaging parameters to Miller-Payne pathological response during treatment. Additionally, multivariate analysis was carried out for pairwise DOS and QUS parameter combinations to determine if an increase in the classification accuracy could be obtained using combination DOS and QUS parametric models. For subproject two, 15 additional patients we recruited after first giving their written informed consent. A pooled analysis was completed for all DOS baseline data (subproject 1 and subproject 2; n=37 patients). LABC patients planned for NAC had functional DOS maps and associated textural features generated. A grey-level co-occurrence matrix (texture) analysis was completed for parameters associated with haemoglobin, tissue composition, and optical properties (deoxy-haemoglobin [Hb], oxy-haemoglobin [HbO2], total haemoglobin [HbT]), %Lipids, %Water, and scattering power [SP], scattering amplitude [SA]) prior to treatment. Textural features included contrast (con), vi correlation (cor), energy (ene), and homogeneity (hom). Patients were classified as ‘responders’ or ‘non-responders’ using Miller-Payne pathological response criteria after treatment completion. In order to test if baseline univariate texture features could predict treatment response, a receiver operating characteristic (ROC) analysis was performed, and the optimal sensitivity, specificity and area under the curve (AUC) was calculated using Youden’s index (Q-point) from the ROC. Multivariate analysis was conducted to test 40 DOS-texture features and all possible bivariate combinations using a naïve Bayes model, and k-nearest neighbour (k-NN) model classifiers were included in the analysis. Using these machine-learning algorithms, the pretreatment DOS-texture parameters underwent dataset training, testing, and validation and ROC analysis were performed to find the maximum sensitivity and specificity of bivariate DOS-texture features. Results: For subproject one, individual DOS and QUS parameters, including the spectral intercept (SI), oxy-haemoglobin (HbO2), and total haemoglobin (HbT) were significant markers for response outcome after one week of treatment (p<0.01). Multivariate (pairwise) combinations increased the sensitivity, specificity and AUC at this time; the SI+HbO2 showed a sensitivity/specificity of 100%, and an AUC of 1.0 after one week of treatment. For subproject two, the results indicated that textural characteristics of pre-treatment DOS parametric maps can differentiate treatment response outcomes. The HbO2-homogeneity resulted in the highest accuracy amongst univariate parameters in predicting response to chemotherapy: sensitivity (%Sn) and specificity (%Sp) = 86.5 and 89.0%, respectively and an accuracy of 87.8%. The highest predictors using multivariate (binary) combination features were the Hb-Contrast + HbO2-Homogeneity which resulted in a %Sn = 78.0, a %Sp = 81.0% and an accuracy of 79.5% using the naïve Bayes model. Conclusion: DOS and QUS demonstrated potential as coincident markers for treatment response and may potentially facilitate response-guided therapies. Also, the results of this study demonstrated that DOS-texture analysis can be used to predict breast cancer response groups prior to starting NAC using baseline DOS measurements

Prognostic factors following ischaemic stroke

The work presented for examination in this thesis concerns the investigation and management of patients with stroke. The underlying theme which unites the chapters is that of outcome following stroke, and how it may be influenced or predicted. Chapter one is a broad overview of stroke disease, incorporating a brief summary of the socio-economic burden of stroke in the Western world. Chapter two reports a randomised, double blind placebo-controlled study designed to investigate the effect of the angiotensin converting enzyme inhibitor perindopril upon cerebral and renal perfusion in hypertensive stroke patients with carotid artery disease. Chapter three reports a study to investigate the feasibility of rigorous control of blood glucose in hyperglycaemic patients following stroke. The prognostic significance of triglyceride concentration following ischaemic stroke is investigated in chapter four. In chapter five the prognostic significance of visible infarction on computed tomography in patients with lacunar stroke is examined. In chapter six the development and implementation of a novel diffusion- weighted magnetic resonance sequence is reported. In chapter seven, preliminary experience with the use of recombinant tissue plasminogen activator in a UK stroke centre is reported. Chapter eight contains a synopsis of the work presented, together with a brief discussion of the results in the context of our current understanding of stroke disease Future directions of study arising from the research are identified and discussed. (Abstract shortened by ProQuest.)