In vivo measurement of glucose concentration with lasers.

Common polarimeters defined the concentration of an optically active sample by measuring the intensity of the light beam after it has passed through the analyser. Consequently, it can be stated that up to now, there have been the disadvantages of having a signal dependent on absolute light intensity. The new method that is described has a sensitivity that is in great measure independent of absolute light intensity, whereby only one light trace is necessary. The new principle uses no mechanical rotations. Instead, an electrical signal indicates the amount of optical rotation of the sample. The high sensitivity that can be reached is theoretically only limited by polarization noise. By going to the uttermost physical and electronic lengths, sensitivity values of more than 10(-5) degrees can be reached. Furthermore, the mechanical dimensions of the apparatus can be made very small by the application of a solid-state laser.     

In vivo oxygen imaging using green fluorescent protein

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs. heart; ischemia; hypoxia; molecular imaging     

Protein disulphide isomerase-induced refolding of sonochemically prepared Ribonuclease A microspheres

The present communication describes for the first time the development of Ribonuclease A (RNase A) microspheres using the sonochemical method followed by an enzymatic treatment with protein disulphide isomerase (PDI). Ultrasound application induced changes on the protein physicochemical and biological properties: the enzymatic activity of RNase A was decreased in 35% and the free thiol groups content was significantly increased, probably due to the breakage of protein disulphide bonds and assembly of RNase A monomers. The deconvolution of amide I band, from Fourier Transform Infrared Spectroscopy, showed that the secondary structure of RNase A was slightly changed after microspherization. The PDI application on microspheres promoted the recovery of RNase A biological activity and induced the release of active protein into solution in its native state. These results were promoted by different states of PDI active site: oxidized and reduced, respectively. The PDI aptitude to catalyze the refolding of a protein substrate in the form of spheres is here reported.     

In vivo measurement of variations in the oxygen 18 content of water in rat brain.

A method of analysis for oxygen 18 is described, based on the anodic oxidation of specially prepared tantalum wires implanted through cannulas into the tissue of living animals. The thin anodic oxide layer formed on these wires is then analyzed by the 18O(p,alpha)15N reaction. The isotopic oxygen concentration of water in the brain obtained by this method compares very well with the values obtained by conventional analysis using mass spectrometry. This in vivo method of nuclear microanalysis has been used in both metabolic and equilibration experiments involving oxygen 18. The half-life for the turnover of 18O in body water was found to be about 3 days. A simple three-pool model is presented which can account for the experimental results obtained from the dilution by body water of interperitoneally injected water highly enriched in 18O.     

In vivo measurement of tumor redox environment using EPR spectroscopy

Solid tumors are characterized by a number of physiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements of these parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-1 murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement of redox status and its manipulation may have important implications in the understanding of tumor growth and therapy.     

In vivo measurement of tumor redox environment using EPR spectroscopy

Solid tumors are characterized by a number of physiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements of these parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-1 murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement of redox status and its manipulation may have important implications in the understanding of tumor growth and therapy.     

In vivo measurement of intramolecular distances using genetically encoded reporters

The function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to Shaker potassium channels expressed in live Xenopus oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.     

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.     

In vivo measurement of leukocyte viscosity during capillary plugging.

Leukocyte plugging of capillaries in vivo was measured in rat spinotrapezius muscle. The plug durations, leukocyte and capillary dimensions, and arteriolar pressure at the plug sites were applied to the mechanical model of Needham and Hochmuth (1990) to estimate the leukocyte viscosities. The viscosity distribution of 389 cells was lognormal with a median value of 232 Poise. 3.1 percent of the cells were apparently activated and displayed viscosities greater than 3000 Poise. The median viscosity suggests that inactivated leukocytes have a minimal effect on blood flow, but that leukocyte activation may result in significant increases in microvascular flow resistance.     

In vivo skin dose measurement using MOSkin detectors in tangential breast radiotherapy

The purpose of this study is to measure patient skin dose in tangential breast radiotherapy. Treatment planning dose calculation algorithm such as Pencil Beam Convolution (PBC) and in vivo dosimetry techniques such as radiochromic film can be used to accurately monitor radiation doses at tissue depths, but they are inaccurate for skin dose measurement. A MOSFET-based (MOSkin) detector was used to measure skin dose in this study. Tangential breast radiotherapies (“bolus” and “no bolus”) were simulated on an anthropomorphic phantom and the skin doses were measured. Skin doses were also measured in 13 patients undergoing each of the techniques. In the patient study, the EBT2 measurements and PBC calculation tended to over-estimate the skin dose compared with the MOSkin detector (p < 0.05) in the “no bolus radiotherapy”. No significant differences were observed in the “bolus radiotherapy” (p > 0.05). The results from patients were similar to that of the phantom study. This shows that the EBT2 measurement and PBC calculation, while able to predict accurate doses at tissue depths, are inaccurate in predicting doses at build-up regions. The clinical application of the MOSkin detectors showed that the average total skin doses received by patients were 1662 ± 129 cGy (medial) and 1893 ± 199 cGy (lateral) during “no bolus radiotherapy”. The average total skin doses were 4030 ± 72 cGy (medial) and 4004 ± 91 cGy (lateral) for “bolus radiotherapy”. In some cases, patient skin doses were shown to exceed the dose toxicity level for skin erythema. Hence, a suitable device for in vivo dosimetry is necessary to accurately determine skin dose.     

In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

A methodinvolving surgical exposure of the colonic mucosa, fluorescent dyeaddition, and confocal microscopy has been developed for monitoringcolonic crypt function in vivo in mice. Na⁺ concentrationin the extracellular pericryptal space of descending colon was measuredusing a low-affinity Na⁺-sensitive fluorescent indicatorconsisting of an Na⁺-sensitive chromophore (sodium red) andan Na⁺-insensitive chromophore (Bodipy-fl) immobilized on200-nm-diameter polystyrene beads. The Na⁺ indicator beadsaccumulated in the pericryptal spaces surrounding the colonic cryptsafter a 1-h exposure of the colonic luminal surface to the beadsuspension. Na⁺ concentration ([Na⁺]) in thepericryptal space was 491 ± 62 mM (n = 4). Aftera 70-min exposure to amiloride (0.25 mM), pericryptal[Na⁺] was reduced to 152 ± 21 mM. Blockage of thecrypt lumen with mineral oil droplets reduced pericryptal[Na⁺] to 204 ± 44 mM. Exposure of the colonicmucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dyeinto the crypt lumen with a half time of 19.8 ± 1.0 s, whichwas increased to 77.9 ± 6.0 s after amiloride treatment.These results establish an in vivo fluorescence method to measurecolonic crypt function and provide direct evidence for accumulation ofa hypertonic absorbate in the pericryptal space of descending colon.The pericryptal space represents the first example of a hypertonicextracellular compartment in mammals that is not created by acountercurrent amplification mechanism.

     

In vivo measurement of protein functional changes

Conformational changes in proteins are fundamental to all biological functions. In protein science, the concept of protein flexibility is widely used to describe protein dynamics and thermodynamic properties that control protein conformational changes. In this study, we show that urea, which has strong sedative potency, can be administered to fish at high concentrations, and that protein functional changes related to anesthesia induction can be measured in vivo. Ctenopharyngodon idellus (the grass carp) has two different types of N-methyl d-aspartate (NMDA) receptors, urea-insensitive and urea-sensitive, which are responsible for the heat endurance of fish. The urea-sensitive NMDA receptor showed high protein flexibility, the gamma aminobutyric acid (GABA) receptor showed less flexibility, and the protein that is responsible for ethanol anesthesia showed the lowest flexibility. The results suggest that an increase in protein flexibility underlies the fundamental biophysical mechanisms of volatile general anesthetics.     

In vitro and in vivo anticandidal efficacy of green synthesized gold nanoparticles using Spirulina maxima polysaccharide

This study, for the first time, demonstrated an unprecedented approach for the green synthesis of gold (Au) nanoparticles (NPs) using the polysaccharide of Spirulina maxima as a reducing agent. Time-kill kinetic analysis was used to evaluate the antifungal activity of the green synthesized Au NPs against the pathogenic Candida albicans (C. albicans). The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were found to be 32 μg/mL and 64 μg/mL, respectively. Ultra-structural analysis indicated prominent damage on cell wall of the C. albicans after Au NPs treatment, and suggested that the treatment could increase the membrane permeability and disintegration of cells leading to cellular death. The results of propidium iodide (PI) uptake assay showed the higher level of cell death in Au NPs treated C. albicans cells, further confirming the loss of plasma membrane integrity. Cytotoxicity analysis of Au NPs on HEK293T and A549 cells showed no cytotoxic effect up to 64 μg/mL of Au NPs concentration, indicating the potential use in in vivo studies. Also, the recovery of C. albicans infected zebrafish after Au NPs therapy suggest green synthesized Au NPs from S. maxima polysaccharide as a prospective anticandidal agent.     

In vivo measurement of microtubule dynamics using stable isotope labeling with heavy water. Effect of taxanes

Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium ((2)H(2)) from heavy water ((2)H(2)O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of (2)H(2)O labeling in vivo reduced (2)H labeling in polymers while increasing (2)H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.     

Detection of microspheres in vivo using multispectral optoacoustic tomography

We introduce a new approach to detect individual microparticles that contain NIR fluorescent dye by multispectral optoacoustic tomography in the context of the hemoglobin-rich environment within murine liver. We encapsulated a near infrared (NIR) fluorescent dye within polystyrene microspheres, then injected them into the ileocolic vein, which drains to the liver. NIR absorption was determined using multispectral optoacoustic tomography. To quantitate the minimum diameter of microspheres, we used both colorimetric and spatial information to segment the regions in which the microspheres appear. Regional diameter was estimated by doubling the maximum regional distance. We found that the minimum microsphere size threshold for detection by multispectral optoacoustic tomography images is 78.9 µm.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.