Protection of cerebral microcirculation, mitochondrial function, and electrocortical activity by small-volume resuscitation with terlipressin in a rat model of haemorrhagic shock.

BACKGROUND: During early treatment of haemorrhagic shock, cerebral perfusion pressure can be restored by small-volume resuscitation with vasopressors. Whether this therapy is improved with additional fluid remains unknown. We assessed the value of terlipressin and lactated Ringer's solution (LR) on early recovery of microcirculation, tissue oxygenation, and mitochondrial and electrophysiological function in the rat cerebral cortex. METHODS: Animals treated with LR replacing three times (3LR) the volume bled (n=26), terlipressin (n=27), terlipressin plus 1LR (n=26), 2LR (n=16), or 3LR (n=15) were compared with untreated (n=36) and sham-operated rats (n=17). In vivo confocal microscopy was used to assess cortical capillary perfusion, changes in tissue oxygen concentration, and mitochondrial membrane potential and redox state. Electrophysiological function was assessed by cortical somatosensory evoked potentials, spinal cord dorsum potential, and peripheral electromyography. RESULTS: Compared with sham treatment, haemorrhagic shock reduced the mean (SD) area of perfused vessels [82% (sd 10%) vs 38% (12%); P<0.001] and impaired oxygen concentration, mitochondrial redox state [99% (4%) vs 59% (15%) of baseline; P<0.001], and somatosensory evoked potentials [97% (13%) vs 27% (19%) of baseline]. Administration of terlipressin plus 1LR or 2LR was able to recover these measures, but terlipressin plus 3LR or 3LR alone were not as effective. Spinal cord dorsum potential was preserved in all groups, but no therapy protected electromyographic function. CONCLUSIONS: Resuscitation from haemorrhagic shock using terlipressin with small-volume LR was superior to high-volume LR, with regard to cerebral microcirculation, and mitochondrial and electrophysiological functions.

In Vivo Imaging of Flavoprotein Fluorescence During Hypoxia Reveals the Importance of Direct Arterial Oxygen Supply to Cerebral Cortex Tissue.

Live imaging of mitochondrial function is crucial to understand the important role played by these organelles in a wide range of diseases. The mitochondrial redox potential is a particularly informative measure of mitochondrial function, and can be monitored using the endogenous green fluorescence of oxidized mitochondrial flavoproteins. Here, we have observed flavoprotein fluorescence in the exposed murine cerebral cortex in vivo using confocal imaging; the mitochondrial origin of the signal was confirmed using agents known to manipulate mitochondrial redox potential. The effects of cerebral oxygenation on flavoprotein fluorescence were determined by manipulating the inspired oxygen concentration. We report that flavoprotein fluorescence is sensitive to reductions in cortical oxygenation, such that reductions in inspired oxygen resulted in loss of flavoprotein fluorescence with the exception of a preserved 'halo' of signal in periarterial regions. The findings are consistent with reports that arteries play an important role in supplying oxygen directly to tissue in the cerebral cortex, maintaining mitochondrial function.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-µm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 µM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 µM across the cell, or ∼0.6 µM/µm. Deeply deoxygenated (5-15 µM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues.

pH-sensitive perylene bisimide probes for live cell fluorescence lifetime imaging.

Several new perylene bisimide (PBI) probes comprising oligo-guanidine conjugates and cationic hydrogel nanoparticle structures were designed for sensing intracellular pH in live cell fluorescence lifetime imaging microscopy (FLIM). Using adherent mammalian cells (2D) and neurosphere (3D) cell models, we evaluated their performance by confocal FLIM-TCSPC. The nanoparticle PBI probe showed stable pH calibration and lifetime changes from 4.7 to 3.7 ns between pH 4.4 and 8 attributed to photo-induced electron transfer (PET). The molecular oligo-guanidine probe showed fast cell penetration and bright staining, but its calibration is affected by the microenvironment being unreliable for quantitative FLIM. Thus, nanoparticle structures are preferred for the design of quantitative pH measurement by FLIM. High brightness and photostability, efficient staining of different cell types and positive optical response to acidification in fluorescence intensity and lifetime modalities are the advantages of the nanoparticle PBI probes compared to conventional pH probes such as BCECF (2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein). Other PBI derivatives with stronger PET can be developed for future high-resolution FLIM of intracellular pH.

Nondestructive and continuous monitoring of oxygen levels in modified atmosphere packaged ready-to-eat mixed salad products using optical oxygen sensors, and its effects on sensory and microbiological counts during storage.

The objective of this study was to determine the percentage oxygen consumption of fresh, respiring ready-to-eat (RTE) mixed leaf salad products (Iceberg salad leaf, Caesar salad leaf, and Italian salad leaf). These were held under different modified atmosphere packaging (MAP) conditions (5% O2 , 5% CO2 , 90% N2 (MAPC-commercial control), 21% O2 , 5% CO2 , 74% N2 (MAP 1), 45% O2 , 5% CO2 , 50% N2 (MAP 2), and 60% O2 , 5% CO2 , 35% N2 (MAP 3)) and 4 °C for up to 10 d. The quality and shelf-life stability of all packaged salad products were evaluated using sensory, physiochemical, and microbial assessment. Oxygen levels in all MAP packs were measured on each day of analysis using optical oxygen sensors allowing for nondestructive assessment of packs. Analysis showed that with the exception of control packs, oxygen levels for all MAP treatments decreased by approximately 10% after 7 d of storage. Oxygen levels in control packs were depleted after 7 d of storage. This appears to have had no detrimental effect on either the sensory quality or shelf-life stability of any of the salad products investigated. Additionally, the presence of higher levels of oxygen in modified atmosphere packs did not significantly improve product quality or shelf-life stability; however, these additional levels of oxygen were freely available to fresh respiring produce if required. This study shows that the application of optical sensors in MAP packs was successful in nondestructively monitoring oxygen level, or changes in oxygen level, during refrigerated storage of RTE salad products.

Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Analysis of total aerobic viable counts in raw fish by high-throughput optical oxygen respirometry.

A simple, miniaturized, and automated screening assay for the determination of total aerobic viable counts in fish samples is presented here. Fish tissue homogenates were prepared in peptone buffered water medium, according to standard method, and aliquots were dispensed into wells of a 96-well plate with the phosphorescent, oxygen-sensing probe GreenLight. Sample wells were covered with mineral oil (barrier for ambient oxygen), and the plate was monitored on a standard fluorescent reader at 30°C. The samples produced characteristic profiles, with a sharp increase in fluorescence above the baseline level at a certain threshold time, which could be correlated with initial microbial load. Five different fish species were analyzed: salmon, cod, plaice, mackerel, and whiting. Using a conventional agar plating method, the relationship between the threshold time and total aerobic viable counts load (in CFU per gram) was established, calibration curve generated, and the test was validated with 169 unknown fish samples. It showed a dynamic range of 10(4) to 10(7) CFU/g, accuracy of ± 1 log(CFU/g), assay time of 2 to 12 h (depending on the level of contamination), ruggedness with respect to the key assay parameters, simplicity (three pipetting steps, no serial dilutions), real-time data output, high sample throughput, and automation. With this test, quality of fish samples, CFU-per-gram levels, and their respective time profiles were determined.

Analysis of close proximity quenching of phosphorescent metalloporphyrin labels in oligonucleotide structures.

Quenching of phosphorescent platinum(II) and palladium(II) coproporphyrin (MeCP) labelled oligonucleotides was investigated. Strong hybridization-specific quenching was observed in duplex DNA structures with a variety of quenchers and with two identical porphyrin labels when in close proximity. Classical resonance energy transfer mechanism was ruled out, since quenching did not correlate with spectral overlaps and lifetime changes were insignificant. Quenching of MeCP by the free quenchers in solution revealed that porphyrin-porphyrin quenching is predominantly static while other dyes quench dynamically. The results suggest that the quenching in DNA duplex proceeds via direct contact.

Use of oxygen sensors to non-destructively measure the oxygen content in modified atmosphere and vacuum packed beef: impact of oxygen content on lipid oxidation.

The ability of optical oxygen sensors to monitor the levels of oxygen in raw and cooked beef was investigated. Raw and cooked beef slices were vacuum packaged and cooked beef slices were modified atmosphere packaged MAP, (60% N(2): 40% CO(2)) and held under refrigerated display (4 °C) for 15 or 35 days for MAP and vacuum packed samples, respectively. Oxygen sensors attached to the inside of the lidding material in modified atmosphere packages, or inserted into vacuum packages, were capable of monitoring changes in oxygen levels in all packaged samples. Lipid oxidation of samples was measured at regular intervals. Oxygen contents detected, ranged from 1.15 to 1.26% and 0.07-0.55% in MAP and vacuum packed samples, respectively. Samples containing greatest levels of oxygen were most oxidised and cooked samples were significantly (P<0.05) more oxidised than raw samples.

Spectral-luminescent study of the porphyrin-diketones and their complexes.

Syntheses of octaethylporphine-diketone (OEPDK) and its platinum(II) and palladium(II) complexes (PtOEPDK, PdOEPDK) were optimized, and the dyes were isolated in a pure form in preparative quantities. They were characterized by the NMR, UV-VIS absorption and emission spectroscopy. Electronic spectra of these dyes (absorption and luminescence) were investigated in detail, and compared to corresponding porphyrins and porphyrin-monoketones. OEPDK showed a strong fluorescence at about 700 nm, while PtOEPDK and PdOEPDK showed very weak room-temperature phosphorescence in the region of 850-1100 nm and practically no fluorescence. Protonation mechanisms were studied for these dyes. Protonation at sites other than pyrrole nitrogen atoms was shown to occur, corresponding protomeric spectral forms are presented. The possibilities of the use of porphyrin-diketones as longwave fluorescent and phosphorescent probes are discussed.

Monofunctional derivatives of coproporphyrins for phosphorescent labeling of proteins and binding assays.

p-Isothiocyanatophenyl derivatives of Pt(II)- and Pd(II)-coproporphyrin I are described as stable monofunctional reagents which enable simple covalent labeling of proteins and other biomolecules under mild conditions in aqueous solutions. Labeling procedure was optimized for antibodies, avidin, and neutravidin. Photophysical properties of resulting conjugates important for their use in binding assays based on time-resolved phosphorescence detection were studied. The functional activity and long-term storage stability of antibody conjugates were assessed in comparison with unmodified proteins. The new labels and their conjugates were evaluated in the solid-phase immunoassays using commercial time-resolved phosphorescence readers Victor(2) and Arcus-1230 (Wallac). Potential applications of these reagents in in vitro diagnostics are discussed.

Phosphorescent porphyrin probes in biosensors and sensitive bioassays.

Platinum(II) and palladium(II) complexes of porphyrins and related tetrapyrrolic pigments emit strong phosphorescence at room temperatures, which is characterized by long lifetimes falling into the sub-millisecond range and long-wave spectral characteristics. These features make the dyes useful as probes for a number of bioanalytical applications, particularly those employing time-resolved fluorescent detection. They can provide high sensitivity and selectivity, together with rather simple instrumental set-up. A number of analytical systems are now under development that are based on the use of phosphorescent porphyrin probes. Experimental results are presented on the following systems: (i) fibre-optic phosphorescence lifetime-based oxygen sensor on the basis of hydrophobic platinum-porphyrins and development of advanced sensing materials and prototype instrumentation; (ii) practical applications of the optical oxygen sensor, including a sensitive immunosensor that employs glucose oxidase labels, a rapid screening method for cell viability in microtitre-plate format, non-destructive measurement of oxygen in packaged foods and reagentless biosensors for metabolites (glucose, lactate); and (iii) the use of water-soluble platinum- and palladium-porphyrins as labels for ultra-sensitive time-resolved phosphorescence immunoassays.

A cell viability assay based on monitoring respiration by optical oxygen sensing.

A cell viability assay based on monitoring of the metabolic activity of living cells via their consumption of dissolved oxygen has been developed. It uses a microwell plate format and disposable phosphorescent sensor inserts incorporated into each sample. The wells are subsequently sealed from ambient oxygen using a layer of mineral oil, and periodically scanned from underneath with a simple fiber-optic phosphorescent phase detector. Thus, dissolved oxygen levels and time profiles of cell respiration can be determined noninvasively and compared to each other. The system was tested by monitoring the viability of the fission yeast Schizosaccharomyces pombe. In comparison with the conventional cell densitometry assay, the optical oxygen sensor method could reliably monitor lower numbers of cells (10(4)-10(5) vs 10(6)-10(7) cells/ml for densitometry), and accurately determine culture viability within 1 h. The assay was then applied to determine the viability of samples treated with toxic agents such as azide and in response to expression of a physiological inducer of cell death, the Bcl-2 family member Bak. The results obtained confirm that measurement of cell respiration by this assay can serve as a predictable, reliable, and fast method for high-throughput determination of cell viability and growth.

An immunosensor based on the glucose oxidase label and optical oxygen detection.

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10(-11)-10(-12) M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of approximately 1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility.