No description available.

Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1 to 100 ng/ml) and cultured at 5% and 21% oxygen tension for 48 hours. In separate experiments, constructs were subjected to dynamic compression (15%) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5% and 21% oxygen tension using an ex-vivo bioreactor for 48 hours. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (p < 0.01) with values greater at 5% oxygen tension than 21%. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21%. The stimulation of GAG synthesis by dynamic compression was greater at 21% than 5% oxygen tension and this response was reduced with TNFα or reversed with L-NIO. The present findings revealed that TNFα has dose-dependent catabolic activities and increased production of inflammatory mediators at low oxygen tension. Dynamic compression or the NOS inhibitor downregulated the inflammatory effects induced by TNFα, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα induced pathways.

The migration of keratinocytes across wound beds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease, and reactive oxygen species damage accumulation increases, but it is unclear if these factors relate specifically to migration of human skin keratinocytes (HSKs). In this study, two concentrations of oxygen (4% and 21%) were used to model low and high oxidative stress to produce varying levels of reactive oxygen species. When migration of HSKs from young and old primary skin were compared by scratch assay, those from old skin migrated faster in high oxygen tension than did young HSKs, which was an opposite trend from that seen in young skin. An intense increase in reactive oxygen species at margins immediately after scratching was seen in both young and old HSKs, but reactive oxygen species disappeared from young skin at 21% oxygen most quickly. These cells also had the slowest migration. These findings suggest that old and young keratinocytes respond differently to oxidative stress, and that migration of keratinocytes--a key step in re-epithelialization of wounds, is effected by the efficacy of reactive oxygen species removal.

Blood flow and its payload of molecular oxygen are two parameters of most physiological interest. Systemic tissue health is routinely gauged through measurements of vitals and oxygen saturation to estimate the state of these physiological parameters. We design, develop, and deploy optical imaging systems for examining perfusion and oxygenation at the local tissue level and apply these techniques for elucidating the normal and pathological processes associated with neurovascular disease. Specifically, we develop and validate the ability to use Multi-Exposure Speckle Imaging (MESI) to estimate microvascular flow dynamics in rodents over acute and chronic periods. Next, we pose significant optimizations to improve the efficacy of the widefield imaging technique for adoption by bench-side and clinical perfusion studies. We also introduce re-interpretations of the underlying physics to advance the theory that quantifies motion from the imaged speckle patterns. Finally, the technique is deployed for chronic monitoring of cortical flow dynamics before after focal ischemia of the motor cortex as part of a behavioral study in rodents. At the microscale, we develop and validate Two Photon Phosphorescence Lifetime Microscopy (2PLM) to examine dissolved oxygen concentration in microvasculature in three dimensions. We examine the technique’s ability for functional mapping of the rodent cortical microvascular network by quantifying the partial pressure of oxygen (pO₂) before and after occlusion of critical arterioles. Automation of acquisitions and processing for robust oxygen mapping within the micro-vascular network are developed and evaluated. The in vivo results are presented in light of those from studies utilizing more invasive mapping electrodes to provide independent corroboration of the observed neurovascular oxygen distributions. The technique is deployed for examining high resolution functional and structural remodeling after focal cerebral ischemia. / text

There is now ample evidence to support the notion that a lack of oxygen (hypoxia) within the tumour adversely affects the outcome of radiotherapy and whether a patient is able to remain disease free. Thus, there is increasing interest in accurately determining oxygen concentration levels within a tumour. Hypoxic regions arise naturally in cancerous tumours because of their abnormal vasculature and it is believed that oxygen is necessary in order for radiation to be effective in killing cancer cells. One method of measuring oxygen concentration within a tumour is the Eppendorf polarographic needle electrode; a method that is favored by many clinical researchers because it is the only device that is inserted directly into the tumour, and reports its findings in terms of oxygen partial pressure (PO2). Unfortunately, there are often anomalous readings in the Eppendorf measurements (negative and extremely high values) and there is little consensus as to how best to interpret the data. In this thesis, Bayesian methods are applied to estimate two measures commonly used to quantify oxygen content within a tumour in the current literature: the median PO2, and Hypoxic Proportion (HP5), the percentage of readings less than 5mmHg. The results will show that Bayesian methods of parameter estimation are able to reproduce the standard estimate for HP5 while providing an additional piece of information, the error bar, that quantifies how uncertain we believe our estimate to be. Furthermore, using the principle of Maximum Entropy, we will estimate the true median PO2 of the distribution instead of simply relying on the sample median, a value which may or may not be an accurate indication of the actual median PO2 inside the tumour. The advantage of the Bayesian method is that it takes advantage of probability theory and presents its results in the form of probability density functions. These probability density functions provide us with more information about the desired quantity than the single number that is produced in the current literature and allows us to make more accurate and informative statements about the measure of hypoxia that we are trying to estimate.

There is now ample evidence to support the notion that a lack of oxygen (hypoxia) within the tumour adversely affects the outcome of radiotherapy and whether a patient is able to remain disease free. Thus, there is increasing interest in accurately determining oxygen concentration levels within a tumour. Hypoxic regions arise naturally in cancerous tumours because of their abnormal vasculature and it is believed that oxygen is necessary in order for radiation to be effective in killing cancer cells. One method of measuring oxygen concentration within a tumour is the Eppendorf polarographic needle electrode; a method that is favored by many clinical researchers because it is the only device that is inserted directly into the tumour, and reports its findings in terms of oxygen partial pressure (PO2). Unfortunately, there are often anomalous readings in the Eppendorf measurements (negative and extremely high values) and there is little consensus as to how best to interpret the data. In this thesis, Bayesian methods are applied to estimate two measures commonly used to quantify oxygen content within a tumour in the current literature: the median PO2, and Hypoxic Proportion (HP5), the percentage of readings less than 5mmHg. The results will show that Bayesian methods of parameter estimation are able to reproduce the standard estimate for HP5 while providing an additional piece of information, the error bar, that quantifies how uncertain we believe our estimate to be. Furthermore, using the principle of Maximum Entropy, we will estimate the true median PO2 of the distribution instead of simply relying on the sample median, a value which may or may not be an accurate indication of the actual median PO2 inside the tumour. The advantage of the Bayesian method is that it takes advantage of probability theory and presents its results in the form of probability density functions. These probability density functions provide us with more information about the desired quantity than the single number that is produced in the current literature and allows us to make more accurate and informative statements about the measure of hypoxia that we are trying to estimate.

Noninvasive measurements of myocardial carboxymyoglobin levels (%MbCO) and oxygen tensions (PtO2) are difficult to obtain experimentally. We have developed a compartmental model which allows prediction of myocardial %MbCO levels and PtO2 for varied carbon monoxide (CO) exposures. The cardiac compartment in the model consists of vascular subcompartments which contain two tissue subcompartments varying in capillary density. Mass-balance equations for oxygen (O2) and CO are applied for all compartments. Myocardial oxygen consumption and blood flow are quantified from predictive formulas based on heart rate. Model predictions are validated with experimental data at normoxia, hypoxia, exercise and hyperoxia. CO exposures of varying concentration and time (short-high, long-low), CO rebreathing during 100% O2, and exposure during exercise is simulated. Results of the simulations demonstrate that during CO exposures and subsequent therapies, the temporal changes of %MbCO in the heart differ from those of carboxyhemoglobin levels (%HbCO). Analysis of correlation between %HbCO, %MbCO and PtO2 was done to understand myocardial injury due to CO hypoxia. This thesis demonstrates that the model is able to anticipate the uptake and distribution of CO in the human myocardium and thus can be used to estimate the extravascular burden (MbCO, PtO2 ) of CO on the human heart.

Most in vitro systems employ the standard cell culture maintenance conditions of 95 % air with 5 % CO2 to balance medium pH, which translates to culture oxygen tensions of approximately 20 % - above the typical ≤ 6 % found in most tissues. The current investigation, therefore, aims to characterize the effect of maintenance and toxicant exposure with a particular focus on the α,β-unsaturated aldehyde, acrolein, in the presence of physiologically relevant oxygen tension using a differentiated H9c2 cardiomyoblast subclone. H9c2 cells were maintained separately in 20.1 and 5 % oxygen, after which cells were differentiated for five days, and then exposed to acrolein in media containing varying concentrations of tricarboxylic acid and glycolytic substrates. Cells were then assessed for viability and metabolism via the MTT conversion assay. H9c2 cells were assessed for mechanistic elucidation to characterize contributors to cellular death, including mitochondrial membrane potential (ΔΨm) reductions (JC-1), intracellular calcium influx (Fluo-4), and PARP activation. Exposure to acrolein in differing oxygen tensions revealed that standard culture cells are particularly sensitive to acrolein, but cells cultured in 5 % oxygen, depending on the medium pyruvate concentration, can be rescued significantly. Further, reductions in ΔΨm were reversed by co-exposure of 5-10 mM EGTA for both culture conditions, while intracellular calcium transients were noted only for standard cultures. The results demonstrate significant metabolic reprogramming which desensitizes differentiated H9c2 to acrolein-induced cytotoxicity. Further, PARP and extracellular calcium contribute to the fate of these cells exposed to acrolein, though clotrimazole-associated TRPM2 channels may not be significantly involved. Conclusively, significant alteration of toxicogenic response was noted in this cell line when cultured under physiologically relevant conditions, and may have a substantial impact on the reliability and predictive power and interpretive application of in vitro-based toxicity models cultured under standard culture conditions, depending on the parent tissue.

No description available.

This study is concerned with certain factors affecting the content of lactobacillic acid in Lactobacillus plantarum. Three main areas of investigation are reported herein. The effects of both the oxygen tension and the pH of the culture medium on the accumulation of lactobacillic acid were determined. In addition, monolayer studies were conducted to determine the influence of cyclopropane fatty acid content on the molecular packing of membrane lipids.