Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease

International audience

Animal Models of Friedreich Ataxia

Friedreich ataxia (FRDA) is the most common form of autosomal-recessive ataxia. Common nonmotor features include cardiomyopathy and diabetes mellitus. At present, no effective treatments are available to prevent disease progression. Age of onset varies from infancy to adulthood. In the majority of patients, FRDA is caused by intronic GAA expansions in FXN, which encodes a highly-conserved small mitochondrial matrix protein, frataxin. A mouse model of FRDA has been difficult to generate because complete loss of frataxin causes early embryonic lethality. Although there are some controversies about the function of frataxin, recent biochemical and structural studies have confirmed that it is a component of the multiprotein complex that assembles iron-sulfur clusters in the mitochondrial matrix. The main consequences of frataxin deficiency are energy deficit, altered iron metabolism, and oxidative damage.

Good laboratory practice: preventing introduction of bias at the bench

Background and Purpose—As a research community, we have failed to demonstrate that drugs which show substantial efficacy in animal models of cerebral ischemia can also improve outcome in human stroke. Summary of Review—Accumulating evidence suggests this may be due, at least in part, to problems in the design, conduct and reporting of animal experiments which create a systematic bias resulting in the overstatement of neuroprotective efficacy. Conclusions—Here, we set out a series of measures to reduce bias in the design, conduct and reporting of animal experiments modeling human stroke.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Characterization of bone pathologies in the ins2+/akita mouse, a new model for diabetes insulindependent: contribution for a better understanding of the disease in humans

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Bioética animal: antropocentrismo y otras reflexiones

En este artículo se hace un recorrido histórico-filosófico acerca de las diferentes concepciones que el ser humano se ha planteado en torno a los animales, y su relación con éstos. Finalmente, se elabora un análisis crítico en relación con el antropocentrismo y el biocentrismo, además se asume una postura moderada con respecto a ambas tesis.

A real time electromagnetic analysis of electric machines for educational purposes and laboratory implementation

This thesis presents a system for visually analyzing the electromagnetic fields of the electrical machines in the energy conversion laboratory. The system basically utilizes the finite element method to achieve a real-time effect in the analysis of electrical machines during hands-on experimentation. The system developed is a tool to support the student's understanding of the electromagnetic field by calculating performance measures and operational concepts pertaining to the practical study of electrical machines. Energy conversion courses are fundamental in electrical engineering. The laboratory is conducted oriented to facilitate the practical application of the theory presented in class, enabling the student to use electromagnetic field solutions obtained numerically to calculate performance measures and operating characteristics. Laboratory experiments are utilized to help the students understand the electromagnetic concepts by the use of this visual and interactive analysis system. In this system, this understanding is accomplished while hands-on experimentation takes place in real-time.

Lattice Boltzmann Modeling and Specialized Laboratory Techniques to Determine the Permeability of Megaporous Karst Rock

The Pleistocene carbonate rock Biscayne Aquifer of south Florida contains laterally-extensive bioturbated ooltic zones characterized by interconnected touching-vug megapores that channelize most flow and make the aquifer extremely permeable. Standard petrophysical laboratory techniques may not be capable of accurately measuring such high permeabilities. Instead, innovative procedures that can measure high permeabilities were applied. These fragile rocks cannot easily be cored or cut to shapes convenient for conducting permeability measurements. For the laboratory measurement, a 3D epoxy-resin printed rock core was produced from computed tomography data obtained from an outcrop sample. Permeability measurements were conducted using a viscous fluid to permit easily observable head gradients (~2 cm over 1 m) simultaneously with low Reynolds number flow. For a second permeability measurement, Lattice Boltzmann Method flow simulations were computed on the 3D core renderings. Agreement between the two estimates indicates an accurate permeability was obtained that can be applied to future studies.

Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas

Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies.

Dairy cattle-slurry treatment technologies: towards an integrating approach of crop production, animal feeding and environmental impacts

Dairy cattle farms have a well-known environmental impact that affects all ecological compartments: air, soil, water and biosphere [1]. Dairy cattle farming are a significant source of anthropogenic gases from enteric fermentation, manure storage and land application, mainly ammonia (NH3), nitric oxide (NO), nitrous oxide (N2O), carbon dioxide (CO2) and methane (CH4). The emission of such gases represents not only an environmental problem but also leads to energy and nitrogen (N) losses in ruminant production systems [2-5]. Several efforts are required on the development of new technologies and strategies that mitigate gaseous emissions, N losses and improve the efficiency of the energy and N cycles [6, 7]. In the Northwest of Portugal, dairy cattle production has a major impact on the economy, with strong repercussions at national scale. Therefore, our Ph.D. thesis project aims to: a) Study natural supplements as additives in the dairy cattle diet towards a decrease in GHG emissions from feeding operations; b) Compare commercial dairy cattle diets with and without additives on gaseous emissions from manure deposited in a simulated concrete floor; c) Assess the concentrations and emissions of NH3 and greenhouse gases from commercial dairy cattle facilities; d) Evaluate the effects of different additives on lowering gaseous emissions from dairy cattle excreta, using a laboratory system simulating a dairy house concrete floor.

Cold acclimation of chill coma mechanisms in the locust CNS

Temperature has profound effects on the neural function and behaviour of insects. When exposed to low temperature, migratory locusts (Locusta migratoria) enter chill coma (neuromuscular paralysis) and can resume normal body functions after returning to normal temperature. Our laboratory has studied phenomena underlying environmental stress-induced comas in locusts and found that they are associated with a sudden loss of K+ homeostasis and also a temporary electrical silence in the central nervous system (CNS). However, the mechanisms underlying chill coma entry and recovery are not well understood, particularly the role of the CNS has not been determined. Here, I investigated neural function during chill coma in the locust by measuring electrical activity in the CNS. As pre-exposure to moderately low temperatures, either chronically (cold acclimation) or acutely (rapid cold hardening; RCH), has been found to improve the insect’s cold tolerance, I also determined cold acclimation and RCH protocols that will improve the locust's cold tolerance and whether these protocols affect neural shutdown during chill coma in the locust. With an implanted thermocouple in the thorax, I determined the temperature associated with a loss of responsiveness (CTmin) in intact male adult locusts. In parallel experiments, I recorded field potential (FP) in the metathoracic ganglion (MTG) in semi-intact preparations to determine the temperature that would induce neural shutdown. I found that acclimation at 10 ˚C and RCH at 4 ˚C reduced chill coma recovery time (CCRT) in intact animal preparations and RCH at 4 ˚C for 4 hours reduced the temperature at neural shutdown in semi-intact preparations. These results suggest that pre-exposure to cold can improve the locust's resistance to chill coma and support the notion that the CNS has a role in determining entry into and exit from chill coma in locusts.

Salmonella Enterica serovar Typhimurium Infection in Mouse Pregnancy

Salmonella are Gram-negative, intracellular food-borne pathogens that cause pregnancy complications. In pregnant mice, Salmonella enterica serovar Typhimurium (S.Tm) infection results in placental bacterial replication, inflammation, necrosis, and fetal loss by unknown mechanisms. Necroptosis, or programmed necrosis mediated by RIPK3 (receptor-interacting protein kinase 3), an inflammatory cell death pathway, is implicated in the pathogenesis of S.Tm in non-pregnant mice. This goal of this thesis was to investigate the role of necroptosis in the pathogenesis of S.Tm infection during mouse pregnancy. I hypothesized that elimination of the key necroptotic cell death protein RIPK3 would decrease placental inflammation and trophoblast cell death, and increase conceptus survival compared to controls. Mice expressing a functional Slc11a1 (encodes the natural resistance-associated macrophage protein 1, NRAMP1) gene with or without RIPK3 function (Ripk3-/-Slc11a1+/+ compared to Slc11a1+/+) were infected with 103 S.Tm by tail vein injection on gestational day (GD) 12. Mice were euthanized on GD 14 (48h post-infection) or GD 15 (72h post-infection) and implantation sites (IS) and maternal serum were harvested for analyses. In nearly all challenged mice (except one outlier), S.Tm were detected in most IS within a litter but there was limited immune cell infiltration, placental damage or cell death in Slc11a1 competent mice regardless of Ripk3 gene deletion. Maternal serum cytokine analyses confirmed lack of maternal immune responses to S.Tm infection. IS amongst the litter of a single dam (Ripk3-/-Slc11a1+/+ at 72h postinfection) displayed heavy but not universal placental S.Tm infection of decidual tissues and spongiotrophoblast, associated with elevated maternal serum pro-inflammatory cytokines. S.Tm infection of the fetal yolk sac (YS) was observed in 54.5% of IS from this dam. YS infection was confirmed in archival samples in mice expressing Ripk3 with intact Slc11a1 and in mice lacking functional Slc11a1. In Slc11a1 incompetent mice, S.Tm were detected in placental labyrinthine trophoblast. Based on the available data, this thesis suggests that Ripk3 and necroptosis have no significant roles in either promotion or prevention of progressive Salmonella infection during mouse pregnancy. It also provides pilot data that NRAMP1 controls placental localization and lethality due to YS infection.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

New prophylactic and therapeutic treatments to combat pathogenic Enterohaemorrhagic Escherichia coli

Bacterial diarrhoeal diseases have significant influence on global human health, and are a leading cause of preventable death in the developing world. Enterohaemorrhagic Escherichia coli (EHEC), pathogenic strains of E. coli that carry potent toxins, have been associated with a high number of large-scale outbreaks caused by contaminated food and water sources. This pathotype produces diarrhoea and haemorrhagic colitis in infected humans, and in some patients leads to the development of haemolytic uremic syndrome (HUS), which can result in mortality and chronic kidney disease. A major obstacle to the treatment of EHEC infections is the increased risk of HUS development that is associated with antibiotic treatment, and rehydration and renal support are often the only options available. New treatments designed to prevent or clear E. coli infections and reduce symptoms of illness would therefore have large public health and economic impacts. The three main aims of this thesis were: to explore mouse models for pre-clinical evaluation in vivo of small compounds that inhibit a major EHEC colonisation factor, to assess the production and role of two proteins considered promising candidates for a broad-spectrum vaccine against pathogenic E. coli, and to investigate a novel compound that has recently been identified as a potential inhibitor of EHEC toxin production. As EHEC cannot be safely tested in humans due to the risk of HUS development, appropriate small animal models are required for in vivo testing of new drugs. A number of different mouse models have been developed to replicate different features of EHEC pathogenesis, several of which we investigated with a focus on colonisation mediated by the Type III Secretion System (T3SS), a needle-like structure that translocates bacterial proteins into host cells, resulting in a tight, intimate attachment between pathogen and host, aiding colonisation of the gastrointestinal tract. As E. coli models were found not to depend significantly on the T3SS for colonisation, the Citrobacter rodentium model, a natural mouse pathogen closely related to E. coli, was deemed the most suitable mouse model currently available for in vivo testing of T3SS-targeting compounds. Two bacterial proteins, EaeH (an outer membrane adhesin) and YghJ (a putative secreted lipoprotein), highly conserved surface-associated proteins recently identified as III protective antigens against E. coli infection of mice, were explored in order to determine their suitability as candidates for a human vaccine against pathogenic E. coli. We focused on the expression and function of these proteins in the EHEC O157:H7 EDL933 strain and the adherent-invasive E. coli (AIEC) LF82 strain. Although expression of EaeH by other E. coli pathotypes has recently been shown to be upregulated upon contact with host intestinal cells, no evidence of this upregulation could be demonstrated in our strains. Additionally, while YghJ was produced by the AIEC strain, it was not secreted by bacteria under conditions that other YghJ-expressing E. coli pathotypes do, despite the AIEC strain carrying all the genes required to encode the secretion system it is associated with. While our findings indicate that a vaccine that raises antibodies against EaeH and YghJ may have limited effect on the EHEC and AIEC strains we used, recent studies into these proteins in different E. coli pathogens have suggested they are still excellent candidates for a broadly effective vaccine against E. coli. Finally, we characterised a small lead compound, identified by high-throughput screening as a possible inhibitor of Shiga toxin expression. Shiga toxin production causes both the symptoms of illness and development of HUS, and thus reduction of toxin production, release, or binding to host receptors could therefore be an effective way to treat infections and decrease the risk of HUS. Inhibition of Shiga toxin production by this compound was confirmed, and was shown to be caused by an inhibitory effect on activation of the bacterial SOS response rather than on the Shiga toxin genes themselves. The bacterial target of this compound was identified as RecA, a major regulator of the SOS response, and we hypothesise that the compound binds covalently to its target, preventing oligomerisation of RecA into an activated filament. Altogether, the results presented here provide an improved understanding of these different approaches to combating EHEC infection, which will aid the development of safe and effective vaccines and anti-virulence treatments against EHEC.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.