Multi-Foci Beamforming Using Curved Linear Array Transducer for Qualitative Identification of Lipids in Human Liver

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of liver chronic diseases in the U.S. and its prevalence is growing in the world. In the United States, it affects an estimate of 80 to 100 million people. In less than a decade, NAFLD will likely become the number one cause of liver transplants in the country. NAFLD cases have risen rapidly over the last three decades and is the most common liver disease in children. NAFLD encompasses a disease spectrum of a variety of liver conditions ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). SS is a benign form of the disease, characterized by the accumulation of lipid in the liver. On the other hand, NASH is defined by hepatic steatosis with cell injury, hepatic ballooning and various degrees of fibrosis. NASH may further develop into cirrhosis, liver failure and hepatocellular carcinoma (HCC). A non-invasive, early detection and accurate staging of NAFLD may allow for a timely intervention and treatment to prevent the progression of the disease to cirrhosis and HCC. We hypothesized a new dual-modality ultrasound imaging combining acoustic radiation force impulse (ARFI) imaging and thermal strain imaging (TSI) implemented on a clinical ultrasound probe. ARFI imaging utilizes high intensity focused ultrasound to generate a push in a region of interest (ROI). The response of the tissue inside the region of excitation due to the acoustic radiation push is determined by estimating the displacement between the pre-push reference frames and the post-push tracking frames. TSI has been used in the field of medical imaging for detecting lipids in atherosclerotic plaques and quantification of liver fat in ob/ob mice. TSI is based on the fact that the speed of sound changes differently in respect to the increase in temperature for different tissue composition. Lipids register a decreasing sound speed with increasing temperature, whereas water-bearing tissue exhibit an increasing sound speed with increasing temperature. Development of the proposed multi-modality system will be a step towards a novel clinical system which would permit the creation of a single co-registered image featuring information regarding lipid content and liver stiffness

NASH and Systemic Complications

Nonalcoholic fatty liver disease (NAFLD) is known as the hepatic manifestation of the metabolic syndrome, and while most patients develop simple steatosis, up to one-third can develop nonalcoholic steatohepatitis (NASH). NASH is a chronic inflammatory condition of the liver that can further progress to fibrosis and cirrhosis, which may eventually lead to liver failure and death. While we have increased our mechanistic knowledge regarding the pathogenesis of NASH within the last decade, treatment options are still limited and liver biopsies have remained the gold standard for diagnosis. To achieve major clinical breakthroughs for NASH patients, it is not sufficient to use a single animal model, since each model has specific limitations. Furthermore, we should rely more on alternative models such as organ-on-a-chip, which will enable us to explore unknown aspects of disease pathogenesis much faster and serve as clinically relevant surrogates for murine models. Another important direction for the improvement of patient health is to pay more attention to extrahepatic, organ-specific and systemic effects, which are associated with NASH. The articles in this Special Issue include an up-to-date overview of the rapidly developing technologies, novel targets for intervention and insights in the field in NASH. Additionally, these articles describe the major challenges in the field, strategies to overcome them and suggestions for future directions. To improve patient’s outcome, clinicians, as well as scientists with biomedical, nutrition, physics and mathematics backgrounds, should join forces. Although challenges remain, the future of the field seems promising as these novel technologies and developments are expected to lead to progress in NASH

Novel interventional therapies to improve cardiovascular risk in women with polycystic ovary syndrome

Introduction:Polycystic ovary syndrome (PCOS) is associated with a diverse range of endocrine, metabolic and cardiovascular risk factors. Low vitamin D in PCOS is associated with multiple cardiovascular risk factors and vitamin D replacement therapy has been suggested as a promising alternative for the prevention and treatment of PCOS. Empagliflozin; a sodium-glucose cotransporter 2 (SGLT2) inhibitor, has been shown to have several favourable effects on inflammatory and cardiovascular risk factors in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients and could potentially be a treatment option in women with PCOS.Methods:The first study was a randomized, double-blind, placebo-controlled trial with an objective to determine the effect of vitamin D supplementation on liver fibrosis markers. Forty overweight and obese women with PCOS were randomization to either vitamin D 3200 IU daily or placebo for 3 months. The second trial was a randomised open-label parallel study to look at the effects of empagliflozin versus metformin on hormonal, metabolic and cardiovascular risk factors in women with PCOS. Forty overweight and obese women with PCOS were randomization to either empagliflozin 25mg or metformin 1500mg daily for 3 months.Results:For vitamin D treatment, there were significant reductions in individual liver fibrosis markers [hyaluronic acid (HA), N-terminal pro-peptide type-III pro-collagen (PIIINP), tissue inhibitor of metalloproteinase-1 (TIMP-1)] and their cumulative enhanced liver fibrosis (ELF) score was associated with a significant improvement in alanine aminotransferase (ALT) levels in patients randomized to vitamin D, whereas there were no changes in any of these parameters in the placebo group. There were no changes in free androgen index (FAI), insulin resistance (Homeostasis model assessment-insulin resistance; HOMA-IR), other anthropometric, inflammatory and body composition parameters in either group. There were no significant changes in endothelial microparticles (EMPs) in the vitamin D group as compared to the placebo group. In the second study, there were significant reductions in anthropometric and body composition parameters in patients randomized to empagliflozin while patients on metformin had significant increases in these parameters as compared to baseline. Between groups comparisons at the end of the study showed that the percentage reductions in anthropometric and body composition parameters were statistically significant in the empagliflozin group as compared to the metformin group. There was significant reduction in total testosterone levels in the metformin group only but not in the empagliflozin group. There were no significant changes in FAI, HOMA-IR, reactive hyperaemic index (RHI), fasting lipids and other hormonal and metabolic markers in both the groups. However, there were significant increases in cluster of differentiation 54 (CD54) and cluster of differentiation 62 (CD62) microparticles in the empagliflozin group and cluster of differentiation (CD106) microparticles in both the empagliflozin and metformin groups.Conclusions:Direct beneficial effects of vitamin D supplementation on markers of hepatic fibrosis were seen in overweight and obese women with PCOS shown by a reduction in the ELF score and its constituent liver fibrosis markers (HA, PIIINP, TIMP-1). However, vitamin D supplementation did not improve endothelial function as suggested by no significant changes in EMPs in the vitamin D group as compared to the placebo group. In the second trial, empagliflozin improved anthropometric and body composition indices after three months of treatment. However, there was significant increase in CD54 and CD62 microparticles after empagliflozin and CD106 microparticles after both empagliflozin and metformin treatments suggesting that the effects of empagliflozin and metformin could be partly mediated through modulation of endothelial function. This research work suggests both vitamin D and SGLT2 inhibition (empagliflozin) as potential treatment options in women with PCOS for improving future cardiovascular risk

Liver Biopsy

Liver biopsy is recommended as the gold standard method to determine diagnosis, fibrosis staging, prognosis and therapeutic indications in patients with chronic liver disease. However, liver biopsy is an invasive procedure with a risk of complications which can be serious. This book provides the management of the complications in liver biopsy. Additionally, this book provides also the references for the new technology of liver biopsy including the non-invasive elastography, imaging methods and blood panels which could be the alternatives to liver biopsy. The non-invasive methods, especially the elastography, which is the new procedure in hot topics, which were frequently reported in these years. In this book, the professionals of elastography show the mechanism, availability and how to use this technology in a clinical field of elastography. The comprehension of elastography could be a great help for better dealing and for understanding of liver biopsy

Fluorescence and Diffuse Reflectance Spectroscopy and Endoscopy for Tissue Analysis

Biophotonics techniques are showing great potential for practical tissue diagnosis, capable of localised optical spectroscopy as well as wide field imaging. Many of those are generally based on the same concept: the spectral information they enable to acquire encloses clues on the tissue biochemistry and biostructure and these clues carry diagnostic information. Biophotonics techniques present the added advantage to incorporate easily miniaturisable hardware allowing several modalities to be set up on the same systems and authorizing their use during minimally invasive surgery (MIS) procedures. The work presented in this thesis aims to build on these advantages to design biophotonics instruments for tissue diagnosis. Fluorescence and diffuse reflectance, the two modalities of interest in this work, were implemented in their single point spectroscopic and imaging declinations. Two “platforms”, a spectroscopic probe setup and an optical imaging laparoscope, were built; they included either one of the two aforementioned modalities or the two of them together. The spectroscopic probe system was assembled to detect lesions in the digestive tract. In its first version, the setup included a dual laser illumination system to carry out an ex vivo fluorescence study of non-alcoholic fatty liver diseases (NAFLD) in the mouse model. Outcomes of the study demonstrated that healthy livers could be distinguished from NAFLD livers with high classification accuracy. Then, the same fluorescence probe inserted in a force adaptive robotic endoscope was applied on a fluorescence phantom and a liver specimen to prove the feasibility of recording spectra at multiple points with controlled scanning pattern and probe/sample pressure (known to affect the spectra shape). This approach proposed therefore a convincing method to perform intraoperative fluorescence measurements. The fluorescence setup was subsequently modified into a combined fluorescence/diffuse reflectance spectroscopic probe and demonstrated as an efficient method to separate normal and diseased tissue samples from the human gastrointestinal tract. Following the single point spectroscopy work, imaging studies were conducted with a spectrally resolved laparoscope. The system, featuring a CCD/filter wheel unit clipped on a traditional laparoscope was validated on fluorescence phantoms and employed in two experiments. The first one, building on the spectroscopy study of the gastrointestinal tract, was originally aimed at locating tumour in the oesophagus but a lack of tissue availability prevented us from doing so. The system design and validation on fluorophores phantoms were nevertheless described. In the second one, the underarm of a pig was imaged after injection of a nerve contrast agent in order to test the feasibility of in vivo nerve delineation. Fluorescence was detected from the region of interest but no clear contrast between the nerve and the surrounding muscle tissue could be detected. Finally, the fluorescence imaging laparoscope was modified into a hyperspectral reflectance imaging laparoscope to perform tissue vasculature studies. It was first characterized and tested on haemoglobin phantoms with varying concentrations and oxygen saturations and then employed in vivo to follow the haemoglobin concentration and oxygen saturation temporal evolutions of a porcine intestine subsequently to the pig’s termination. A decrease in oxygen saturation was observed. The last experiment consisted in monitoring the tissue re-oxygenation of a rabbit uterus transplant on the recipient animal, a successful tissue re-perfusion after the graft was highlighted

Nutritional modulation of hepatic lipid metabolism in health and disease

The objective of this thesis was to assess the impact of altering macronutrient intakes on hepatic lipid metabolism. Two separate studies were performed, with liver triglyceride content being the principal outcome of both. In the first study 32 healthy and centrally overweight males were randomised to 2 periods, each of 2 weeks, of either a high fructose or glucose intake in a non-crossover fashion. Isoenergetic status was maintained by providing foodstuffs during the first period, followed by a 6 week washout and then a second period of ad libitum overfeeding. In the second study 55 patients with biopsy proven non-alcoholic fatty liver disease were randomised to 3 months 5g a day of capsules containing either n-3 polyunsaturated fatty acid or oleic enriched sunflower oil. The main findings are summarised. High intakes of fructose and glucose in the isoenergetic period resulted in a stable weight, and no change in hepatic, serum and ectopic triglyceride content. There was a raised serum uric acid with fructose. During the hyperenergetic period there was a tendency for greater uric acid with fructose, whilst both groups had a matched weight gain, elevation of liver biochemistry and an increase in hepatic, serum and muscle triglycerides. Changes in liver biochemistry and triglycerides were associated with changes in weight. During both periods there was calorimetric evidence for a shift in whole body metabolism towards that reflective of a high carbohydrate intake. There was no alteration in renal function or cardiovascular haemodynamic parameters or consistent change in insulin resistance. The n-3 polyunsaturated versus oleic acid study resulted in significant alterations of serum fatty acid profiles between the groups, which were in line with the capsules’ contents. These changes however failed to translate, in the whole group, to any detected metabolic or hepatic changes beyond a reduction in serum triglyceride with n-3 polyunsaturated fatty acids. Only 43 of the 55 patients had elevated liver triglycerides on baseline MRI. Amongst this 43 there was a reduction in liver triglyceride with n-3 polyunsaturated fatty acids, but no other associated metabolic changes. The uric acid findings support the notion of fructose and glucose differing in their pre triose metabolism. There was however no differing outcomes in terms of lipid synthesis or storage. There was a suggestion of reduced liver triglycerides with n-3 polyunsaturated fatty acids though this was an isolated result only found amongst those with a steatotic liver at baseline. Ultimately the exquisite sensitivity of the liver to nutrient intakes was highlighted by the 0.8% gain in weight in the fructose / glucose study resulting in a 24% increase in liver lipid. This affirms the notion that dietary energy intakes have a profound influence on hepatic metabolism, but there is no evidence from this thesis that this influence is macronutrient specific. In the future macronutrient comparisons need to be made

Role of extracellular vesicles in non-alcoholic fatty liver disease

Once thought to be a benign accumulation of hepatic fat, non-alcoholic fatty liver disease (NAFLD) is now recognised as a prevalent complication of obesity with serious consequences if left untreated. This includes liver inflammation and impaired glucose metabolism, and with time may result in cirrhosis and organ failure. While basic diagnostic tools exist, such as ultrasound and serum protein markers, these are often unreliable and warrant the use of more invasive techniques such as tissue biopsy. Given most patients only become symptomatic once the disease is advanced, there is an urgent need to develop a better diagnostic strategy for NAFLD. This project aims to address the issue by exploring a novel biomarker candidate: extracellular vesicles (EVs). These submicron vesicles can be isolated from the circulation and tracked back to their parent cells by nature of their surface markers. EVs are constitutively released by all cell types, however, their abundance and molecular cargo are shown to be changed in various conditions and disease states, which also affects the functions they mediate as “intercellular messengers”. Using rodent models of diet-induced NAFLD, we aim to determine whether plasma or liver-derived EVs could be effectively used as a marker for the progression of NAFLD, and whether suitable interventions exist to slow or even reverse the disease. Finally, using in vitro models of hepatocyte EV uptake, the project will also investigate how EVs impart signals within the liver, in a paracrine fashion and from extraheptic tissue