Oxidative Stress on Human Cells in the Presence of Nano-Sized Titanium Dioxide

Nanosized TiO2 (nanoTiO2) is one of the most widely used nanomaterials, with applications ranging from paints, self-cleaning coatings, pharmaceuticals, to food and cosmetics. In spite of this massive use of nanoTiO2, its biological activity and toxicity remains subject of the intense debate. In particular, there is still considerable uncertainty in the current understanding of the relationship between physico-chemical parameters of nanoTiO2, such as crystalline phase, size, aspect ratio, surface properties, surface defects and surface chemistry and its potential toxicological effects. Motivated by this general problem, this thesis provides a multidisciplinary experimental insight into to the toxicity and photo-toxicity of various forms of nanoTiO2. Firstly, since all of the widely accepted models of nanoTiO2 toxicity involve reactive oxygen species (ROS), electron spin resonance (ESR) in combination with spintrapping was used to measure ROS formation efficiency for the two most industrially important polymorphs of TiO2, anatase and rutile. The study was performed in an unprecedentedly broad range of particle sizes: 3.8 nm to 150 nm and 5 nm to 215 nm, for anatase and rutile nanoTiO2, respectively. Moreover, the photocatalytic and toxic properties of custom-made anatase-based TiO2 nanowires (with a diameter of 35 nm and a length of 0.5-1 µm) were characterized for the first time. For pure anatase nanoTiO2, the maximum ROS generation efficacy was found for nanoparticle sizes in the range of 25 – 30 nm. The ROS generation efficacy of the custom-made TiO2 nanowires was ca. 30% lower, being close to that of the commercial anatase nanoTiO2 with primary grain sizes of 5.3 nm. Secondly, this thesis addressed challenging, complex and still poorly understood processes occurring when nanoTiO2 particles are brought into contact with living cells. In particular, the comparative nanotoxicity study towards human melanoma Lu1205 and WM793 cell lines was performed for three selected nanomaterials: the custom-made anatase-based TiO2 nanowires, the commercial anatase nanoTiO2 with a primary particle size of 5.3 nm and similar in vitro ROS formation efficacy, and the industrial photocatalytic standard, P25 Degussa (a formulation consisting of 80% anatase and 20% rutile, with primary grain sizes of 25 nm). This comparative nanotoxicity study was performed using very low concentrations of nanoTiO2 (2 – 2.5 µg/mL), considerably lower than that applied in the majority of previous ex vivo cell culture studies. A wide palette of spectroscopic and microscopic methods was applied to verify the cellular response to the presence of selected nanoTiO2-based particles, both in the dark and under illumination with UV-A light of low intensity (λ = 365 nm, 1 mW/cm2). In particular, to detect intracellular ROS in cells incubated with the selected nanoTiO2, ESR spin-trapping with an intracellular spin-trap (ACP) and histochemical detection with a ROS-sensitive dye (NBT) were used. These studies were completed by optical microscopy visualization of ROS-induced changes in cell morphology and actin filament organization (FITC-phalloidin staining). Moreover, atomic force microscopy (AFM) was used to follow the changes in the cell topology and elastic properties (AFM force spectroscopy) of cells incubated with nanoTiO2 and exposed to photo-oxidative stress under UV-A illumination. All of these techniques revealed, either directly or indirectly, deleterious effects induced by incubation of living cells with nanoTiO2 and by photo-oxidative stress under illumination with UV-A. Primarily, optical microscopy visualization of selectively stained actin stress fibers and AFM topography images pointed to a rapid disorganization of the actin cortex for cells exposed to photo-oxidative stress in the presence of nanoTiO2. These results corroborated the changes in the cell shape and elasticity measured by AFM. The cell elasticity measurements, quantitatively described in terms of local Young's modulus values, pointed to an internal remodeling of the cytoskeleton in cells exposed to the presence of nanoTiO2 and photo-oxidative stress. The custom made anatase-based TiO2 nanowires revealed generally stronger cytotoxic effects than the anatase nanoTiO2 with a particle size of 5.3 nm, despite having similar photocatalytic properties in vitro. These observations were confirmed by AFM measurements of the cellular elasticity, histochemical detection with a ROS-sensitive dye (NBT) and by intracellular detection of ROS with ESR spin-trapping. The latter technique provided also a new important observation pointing to the enhanced ROS formation in cells incubated with nanoTiO2 in the dark. Overall, the findings stemming from cell toxicity studies were much more complex than those obtained from measurements of ROS generation efficacy in the presence of nanoTiO2 in vitro. In contrast to a marked evolution of ROS generation efficacy as a function of the primary grain size in vitro, the cellular work pointed to the lack of direct and simple correlations between the physical parameters of nanoTiO2, such as particles size, their aspect ratio, and aggregation state, and cell toxicity

Improvement of quality of life after therapeutic plasma exchange in patients with myasthenic crisis

Introduction We sought to evaluate quality of life patients with myasthenic crisis before and after therapeutic plasma exchange. Materials and methods In our study we conducted an assessment of the quality of life with the use of the questionnaire SF-36, when executed eleven therapeutic plasma exchange. The assessment was made on baseline and after 4 weeks. We also did neurological clinical evaluation before and after TPE. Results Patients in the study showed significant improvement in quality of life after performed therapeutic plasma exchange. The changes were observed in physical functioning, which confirmed the results of the statistical significance of p<0.05. In the analysis, the assessment of mental functioning not obtained the results of statistical significance, but the results also showed improvement in self-assessment. We observed high correlation between general health and physical mental functioning, between the role limitations due to physical health problems and role limitations due to emotional problems, and general health perception and bodily pain. Conclusions Therapeutic plasma exchange significantly improves the quality of life of patients with myasthenia gravis during the crisis

Fat fraction mapping using bSSFP Signal Profile Asymmetries for Robust multi-Compartment Quantification (SPARCQ)

Purpose: To develop a novel quantitative method for detection of different tissue compartments based on bSSFP signal profile asymmetries (SPARCQ) and to provide a validation and proof-of-concept for voxel-wise water-fat separation and fat fraction mapping. Methods: The SPARCQ framework uses phase-cycled bSSFP acquisitions to obtain bSSFP signal profiles. For each voxel, the profile is decomposed into a weighted sum of simulated profiles with specific off-resonance and relaxation time ratios. From the obtained set of weights, voxel-wise estimations of the fractions of the different components and their equilibrium magnetization are extracted. For the entire image volume, component-specific quantitative maps as well as banding-artifact-free images are generated. A SPARCQ proof-of-concept was provided for water-fat separation and fat fraction mapping. Noise robustness was assessed using simulations. A dedicated water-fat phantom was used to validate fat fractions estimated with SPARCQ against gold-standard 1H MRS. Quantitative maps were obtained in knees of six healthy volunteers, and SPARCQ repeatability was evaluated in scan rescan experiments. Results: Simulations showed that fat fraction estimations are accurate and robust for signal-to-noise ratios above 20. Phantom experiments showed good agreement between SPARCQ and gold-standard (GS) fat fractions (fF(SPARCQ) = 1.02*fF(GS) + 0.00235). In volunteers, quantitative maps and banding-artifact-free water-fat-separated images obtained with SPARCQ demonstrated the expected contrast between fatty and non-fatty tissues. The coefficient of repeatability of SPARCQ fat fraction was 0.0512. Conclusion: The SPARCQ framework was proposed as a novel quantitative mapping technique for detecting different tissue compartments, and its potential was demonstrated for quantitative water-fat separation.Comment: 20 pages, 7 figures, submitted to Magnetic Resonance in Medicin

Lessons on brain edema in HE : from cellular to animal models and clinical studies

Brain edema is considered as a common feature associated with hepatic encephalopathy (HE). However, its central role as cause or consequence of HE and its implication in the development of the neurological alterations linked to HE are still under debate. It is now well accepted that type A and type C HE are biologically and clinically different, leading to different manifestations of brain edema. As a result, the findings on brain edema/swelling in type C HE are variable and sometimes controversial. In the light of the changing natural history of liver disease, better description of the clinical trajectory of cirrhosis and understanding of molecular mechanisms of HE, and the role of brain edema as a central component in the pathogenesis of HE is revisited in the current review. Furthermore, this review highlights the main techniques to measure brain edema and their advantages/disadvantages together with an in-depth description of the main ex-vivo/in-vivo findings using cell cultures, animal models and humans with HE. These findings are instrumental in elucidating the role of brain edema in HE and also in designing new multimodal studies by performing in-vivo combined with ex-vivo experiments for a better characterization of brain edema longitudinally and of its role in HE, especially in type C HE where water content changes are small

Lessons on brain edema in HE: from cellular to animal models and clinical studies

Brain edema is considered as a common feature associated with hepatic encephalopathy (HE). However, its central role as cause or consequence of HE and its implication in the development of the neurological alterations linked to HE are still under debate. It is now well accepted that type A and type C HE are biologically and clinically different, leading to different manifestations of brain edema. As a result, the findings on brain edema/swelling in type C HE are variable and sometimes controversial. In the light of the changing natural history of liver disease, better description of the clinical trajectory of cirrhosis and understanding of molecular mechanisms of HE, and the role of brain edema as a central component in the pathogenesis of HE is revisited in the current review. Furthermore, this review highlights the main techniques to measure brain edema and their advantages/disadvantages together with an in-depth description of the main ex-vivo/in-vivo findings using cell cultures, animal models and humans with HE. These findings are instrumental in elucidating the role of brain edema in HE and also in designing new multimodal studies by performing in-vivo combined with ex-vivo experiments for a better characterization of brain edema longitudinally and of its role in HE, especially in type C HE where water content changes are small

Diffusion of brain metabolites highlights altered brain microstructure in type C hepatic encephalopathy: a 9.4 T preliminary study

IntroductionType C hepatic encephalopathy (HE) is a decompensating event of chronic liver disease leading to severe motor and cognitive impairment. The progression of type C HE is associated with changes in brain metabolite concentrations measured by 1H magnetic resonance spectroscopy (MRS), most noticeably a strong increase in glutamine to detoxify brain ammonia. In addition, alterations of brain cellular architecture have been measured ex vivo by histology in a rat model of type C HE. The aim of this study was to assess the potential of diffusion-weighted MRS (dMRS) for probing these cellular shape alterations in vivo by monitoring the diffusion properties of the major brain metabolites.MethodsThe bile duct-ligated (BDL) rat model of type C HE was used. Five animals were scanned before surgery and 6- to 7-week post-BDL surgery, with each animal being used as its own control. 1H-MRS was performed in the hippocampus (SPECIAL, TE = 2.8 ms) and dMRS in a voxel encompassing the entire brain (DW-STEAM, TE = 15 ms, diffusion time = 120 ms, maximum b-value = 25 ms/μm2) on a 9.4 T scanner. The in vivo MRS acquisitions were further validated with histological measures (immunohistochemistry, Golgi-Cox, electron microscopy).ResultsThe characteristic 1H-MRS pattern of type C HE, i.e., a gradual increase of brain glutamine and a decrease of the main organic osmolytes, was observed in the hippocampus of BDL rats. Overall increased metabolite diffusivities (apparent diffusion coefficient and intra-stick diffusivity—Callaghan’s model, significant for glutamine, myo-inositol, and taurine) and decreased kurtosis coefficients were observed in BDL rats compared to control, highlighting the presence of osmotic stress and possibly of astrocytic and neuronal alterations. These results were consistent with the microstructure depicted by histology and represented by a decline in dendritic spines density in neurons, a shortening and decreased number of astrocytic processes, and extracellular edema.DiscussiondMRS enables non-invasive and longitudinal monitoring of the diffusion behavior of brain metabolites, reflecting in the present study the globally altered brain microstructure in BDL rats, as confirmed ex vivo by histology. These findings give new insights into metabolic and microstructural abnormalities associated with high brain glutamine and its consequences in type C HE

Abnormal brain oxygen homeostasis in an animal model of liver disease

Background & Aims: Increased plasma ammonia concentration and consequent disruption of brain energy metabolism could underpin the pathogenesis of hepatic encephalopathy (HE). Brain energy homeostasis relies on effective maintenance of brain oxygenation, and dysregulation impairs neuronal function leading to cognitive dysfunction. We hypothesise that HE is associated with reduced brain oxygenation and explored the potential role of ammonia as an underlying pathophysiological factor. / Methods: In a rat model of chronic liver disease with minimal HE (mHE; bile duct ligation [BDL]), brain tissue oxygen measurement and proton magnetic resonance spectroscopy were used to investigate how hyperammonemia impacts oxygenation and metabolic substrate availability in the CNS. Ornithine phenylacetate (OP, OCR-002; Ocera Therapeutics, USA) was used as an experimental treatment to reduce ammonia concentration in plasma. / Results: In BDL animals, glucose, lactate and tissue oxygen concentrations in the cerebral cortex were significantly lower compared to sham-operated controls. OP treatment corrected the hyperammonemia and restored brain tissue oxygen. While BDL animals were hypotensive, cortical tissue oxygen was significantly improved by treatments which increased arterial blood pressure. Cerebrovascular reactivity to exogenously applied CO2 was found to be normal in BDL animals. / Conclusions: These data suggest that hyperammonemia significantly decreases cortical oxygenation, potentially compromising brain energy metabolism. These findings have potential clinical implications for the treatment of patients with mHE aiming to restore normal brain blood flow alongside ammonia-reducing strategies

Influence of anatomical variations on lumbar foraminal stenosis pathogenesis

Introduction: Symptomatic foraminal stenosis has been observed in patients with degenerative disc disease, scoliosis, asymmetrical disc degeneration and spondylolisthesis. Nevertheless not all patients with the above pathologies will develop symptomatic foraminal stenosis. We hypothesised that symptomatic patients have anatomical predisposition to foraminal stenosis, namely a larger pedicle height (PH) to vertebral body height (VH) ratio, leaving less room below the pedicle for the exiting nerve root compared to asymptomatic patients. Patient sample: 66 Patients were divided in two groups. The surgical group consisted of 37 patients (average age of 61years) who presented with severe radicular symptoms resisting to conservative measures and requiring decompression and transforaminal lumbar interbody fusion (TLIF). The control group consisted of 29 patients (average age of 51years) presenting with low back pain (LBP) but with no radicular symptoms and who were treated conservatively. Methods: We measured VH at the level of the posterior wall as well as PH on parasagittal images (CT or MRI) on all lumbar levels (L1 to L5). Statistical analysis was performed using Student's t test. Results: No difference in PH was found between the two groups for L1 to L4 levels. By contrast, there was a highly statistically significant difference in VH between the two groups from L1 to L4 level. In the surgical group, the VH was smaller (p<0.001). Conclusions: Symptomatic patients with foraminal stenosis have smaller VH leading to lesser space beneath the pedicle and putting the exiting nerve root at risk in cases of spondylolisthesis or disc degeneration