Application of a 3D volume 19F MR imaging protocol for mapping oxygen tension (pO2) in perfluorocarbons at low field

Achalasia is a motility disorder of the esophagus characterized by the loss of inhibitory neurons in the distal esophagus. Although idiopathic in nature, autoimmune mechanisms have been proposed, and we set out to determine the presence of myenteric neuronal antibodies. We prospectively studied 18 patients with well-characterized achalasia (by clinical, x-ray, and manometric evidence), nine with gastroesophageal reflux disease, and analyzed the sera from 22 disease-free controls. Using double-label, indirect immunofluorescence techniques, rat esophageal and intestinal sections were double-labeled with sera (dilutions of 1:50 to 1:400) from the three groups and with neurofilament antibody to localize neurons. Seven of 18 achalasia patients had sera that stained the majority of neurons within plexi in the esophageal and intestinal sections, including both NADPH diaphorase (nitric oxide synthase) -positive and -negative neurons. None of the gastroesophageal reflux patients or the controls showed staining. Neuronal antibodies in achalasia provide an attractive hypothesis to explain this diffuse, possibly immune-based disorder.     

Multiple respiratory gas monitoring causes changes of inspired oxygen concentration in closed anesthesia system

This case report describes an accessory hepatic duct (AHD) identified by intraoperative cholangiography during excisional surgery of a choledochal cyst (CC). The accessory duct was divided and reconstructed successfully to the Roux-en-Y jejunal loop. The postoperative course was uneventful, and follow-up abdominal sonography revealed neither evidence of biliary tract obstruction nor atrophic changes of the liver. It is advocated that an AHD should be meticulously reconstructed if it is divided during excisional surgery of a CC.     

Water diffusion in biological porous systems: a NMR approach

The spin-echo nuclear magnetic resonance (NMR) technique together with paramagnetic ion doping are used to study structural parameters of plant samples, such as restricted dimensions, and cell interconnection both through membranes and by cell contact by studying simultaneous restricted diffusion and intercellular water transfer via various pathways. Also, peculiarities of water diffusion on the surface of cell-wall cellulose are studied over a wide range of water content.     

NMR spectroscopic study of cell cultures of astrocytes and neurons exposed to hypoxia: compartmentation of astrocyte metabolism

Primary cultures of murine cerebral cortical astrocytes or cerebellar granule neurons were exposed to 7 h of hypoxia (3 h in some cases). The culture medium was analyzed at the end of the hypoxic or normoxic period by 1H NMR spectroscopy and intracellular components were analyzed as perchloric acid extracts by 31P and 1H NMR spectroscopy. Lactate production in astrocytes increased only marginally, whereas high energy phosphate concentrations were reduced, during 7 h of hypoxia and after 17 h of reoxygenation. After 3 h of hypoxia full recovery was possible during reoxygenation. Citrate and glutamine secretion was reduced or unchanged, respectively, during 7 h of hypoxia. Succinate secretion was only observed during normoxia, whereas pyruvate was secreted during hypoxia. Cerebellar granule neurons were more efficient in increasing glycolysis and were, therefore, more resistant to the effects of hypoxia than astrocytes. In the neurons lactate production was doubled and no effects on levels of high energy phosphates were seen after 7 h of hypoxia. Astrocytes were reoxygenated for 17 h after hypoxia or normoxia in a medium containing [2-13C]acetate in order to access if astrocytes were still capable of supplying neurons with essential precursors. The media were subsequently analyzed by 13C NMR spectroscopy. After shorter periods of hypoxia (3 h) full recovery was possible. Citrate and glutamine production remained however decreased during reoxygenation after 7 h of hypoxia. 13C incorporation into glutamine was greatly reduced but that into citrate was unchanged. These results suggest that under the present conditions, neurons are more efficient than astrocytes in switching the energy metabolism from aerobic to anaerobic glycolysis and that astrocytes may suffer long term damage to mitochondria from longer periods of hypoxia. Furthermore, evidence is presented for the existence of several TCA cycles within astrocytes based on labeling ratios. During normoxia the labeling ratios in the C-2/C-4 positions in glutamine and in the equivalent positions in citrate were 0.27 and 0.11, respectively.     

Blood microdialysis in humans: a new method for monitoring plasma compounds

A new venous microdialysis probe for humans was developed. The active area was made with cellulose membrane (220 mm outside diameter, length 20 mm, and 6000 molecular weight cut off). Glucose measurements were used to test it. The relative recovery was 50% in dialysates obtained in vitro, the step response was 91.8% in the first 2 min. The in vivo recovery was 65.8% obtained by the no net flux method, with the probe placed in the cubital vein of the subjects arms. Corrected glucose in dialysates was non significantly different from plasma glucose simultaneously collected. In healthy male volunteers from 18 to 37 years of age, mean 22.1 years, the glucose levels in the blood dialysates were measured every 30 min, 60 min before and 150 min after an oral load of 75 g of glucose or vehicle. Glucose increased significantly after the load. The differences with the control group were significant at 60 and 90 min after the treatment. The results showed the easy and safe application of this technique to monitor endogenous and exogenous compounds in the extracellular compartment of blood in out patients.     

Transport in a grooved perfusion flat-bed bioreactor for cell therapy applications

We retrospectively reviewed the cases of seventy-two consecutive patients who had a lumbar discectomy, between 1950 and 1983, when they were sixteen years of age or younger. There were forty boys and thirty-two girls. At the time of the lumbar discectomy, twelve patients (17 per cent) also had a spinal arthrodesis. The mean duration of follow-up was 27.8 years (range, twelve to forty-five years). Twenty patients (28 per cent) had one reoperation or more, with the first reoperation performed at a mean of 9.7 years after the initial discectomy. Fourteen patients had one reoperation, four had two reoperations, one had three, and one had five. Fifty-two patients (72 per cent) did not need a reoperation. At the time of the latest follow-up, forty-eight (92 per cent) of the fifty-two patients either had no pain or had occasional pain related to strenuous activity and fifty-one (98 per cent) could participate in daily activities with no or mild limitations. Survivorship analysis showed that the overall probability that a patient would not need a reoperation was 80 per cent at ten years and 74 per cent at twenty years after the initial operation. With the numbers available for study, we could not show that age, gender, or an arthrodesis performed at the time of the initial operation were risk factors for a reoperation. We could not detect a difference, with respect to pain or the level of activity, between the patients who had had an arthrodesis at the initial operation and those who had not or between those who had a coexisting structural abnormality of the lumbar spine and those who did not.     

Rhodamine 123: a useful probe for monitoring T cell activation

The effect of nine factors on the outcome of classic in vitro screens testing the antagonistic action of endophytic bacterial isolates from grape vines against virulent Agrobacterium vitis has been examined. These factors were (i) the strain of A. vitis, (ii) the strain of endophyte, (iii) the growth medium of the pathogen, (iv) the growth medium of the endophyte, (v) the temperature of growth of the pathogen, (vi) the temperature of growth of the endophyte, (vii) the pH of growth of the pathogen, (viii) the pH of growth of the endophyte, and (ix) the medium of the assay plate. Analyses of variance of the full factorial design incorporating main effects and two- and three-way interactions accounted for 66% of the variance. All nine factors had a significant effect on the diameter of inhibition zones (p < 0.001). An examination of the three-way interactions revealed that generalizations were difficult to draw; each target agrobacterium had a specific response to a given antagonistic isolate. It was possible to determine that the growth history of bacterial strains, before they were administered to an assay plate to test for antagonism (especially the composition of the growth medium and the temperature of growth), had a profound effect on the outcome of the test. Generally the more chemically defined media produced less inhibition whereas the lower growth temperature of 15 degrees C produced more inhibition. These findings could be relevant to in situ inhibitory activity. The method used to conduct the inhibitory screen (order of strain application and the medium of the assay plate) had a profound influence on the results. These influences add to the caution necessary in the use of in vitro antagonistic screens for finding successful biocontrol agents.     

Regional myocardial perfusion and mechanics: a model-based method of analysis

A new parametric model-based method has been developed that allows epicardial strain distributions to be computed on the left ventricular free wall in normal and ischemic myocardium and integrated with the regional distributions of anatomic and physiological measurements so that underlying relationships can be explored. An array of radiopaque markers was sewn on the anterior wall of the left ventricle (LV) in three anesthetized open-chest canines, and their positions were recorded using biplane video fluoroscopy before and 2 min after occlusion of the left anterior descending coronary artery. The three-dimensional (3D) anatomy of the LV and epicardial fiber angles were measured post-mortem using a 3D probe. A prolate spheroidal finite element model was fitted to the epicardial surface points (with <0.2 mm accuracy) and fiber angles (<5 degrees error). Regional myocardial blood flows (MBFs) were measured using fluorescent microspheres and fitted into the model (<0.3 ml min(-1) g(-1) error). Epicardial fiber and cross-fiber strain distributions were computed by allowing the model to deform from end-diastole to end-systole according to the recorded motion of the surface markers. Systolic fiber strain varied from -0.05 to 0.01 within the region of the markers during baseline, and regional MBF varied from 1.5 to 2.0 ml min(-1) g(-1). During 2 min ischemia, regional MBF was less than 0.3 ml min(-1) g(-1) in the ischemic region and 1.0 ml min(-1) g(-1) in the nonischemic region, and fiber strain ranged from 0.05 in the central ischemic zone to -0.025 in the remote nonischemic tissue. This analysis revealed a zone of impaired fiber shortening extending into the normally perfused myocardium that was significantly wider at the base than the apex. A validation analysis showed that a regularizing function can be optimized to minimize both fitting errors and numerical oscillations in the computed strain fields.     

Calibration of Pd-porphyrin phosphorescence for oxygen concentration measurements in vivo

Quantitative measurement of oxygen concentrations in the microvasculature is of prime importance in issues related to oxygen transport to tissue. The introduction of the quenching of the Pd-porphyrin phosphorescence as oxygen sensor in vivo by Wilson et al. (J. Appl. Physiol. 74: 580-589, 1993) has provided in this context a major advance in this area of research. For in vivo application, the dye is coupled to albumin to restrict the dye to the circulation and to measure oxygen in the physiological range. In this study a phosphorimeter with a gated photomultiplier is presented and validated. Furthermore, a nonlinear-fit method using the Marquardt-Levenberg algorithm is used to calculate the decay time. With this new phosphorimeter, calibration measurements were performed to investigate the effects of pH, temperature, and diffusivity. The results present a preparation method for albumin coupling of the dye that eliminates the pH dependency of the quenching kinetics. Furthermore, the decreased oxygen diffusivity of serum was compared with that of water, and it was shown that calibration constants measured in water can be extrapolated to serum.     

Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo

Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.     

Evaluation of a regional oxygen saturation catheter for monitoring SjvO2 in head injured patients

Exercise in the water offers several physiological advantages to the pregnant woman. The hydrostatic force of water pushes extravascular fluid into the vascular spaces, producing an increase in central blood volume that may lead to increased uterine blood flow. This force is proportional to the depth of immersion. The increase in blood volume is proportional to the woman's edema. A marked diuresis and natriuresis accompanies the fluid shifts. The buoyancy of water supports the pregnant women. Water is thermoregulating. Studies of pregnant women exercising in the water have shown less fetal heart rate changes in the water than on land in response to exertion. Pregnant women's heart rates and blood pressures during water exercise are lower than on land exercise, reflecting the immersion-induced increase in circulating blood volume. The physiology of water exercise offers some compensation for the physiological changes of exercise on land that may beneficially affect pregnancy.     

Quantitative pO2 imaging in vivo with perfluorocarbon F-19 NMR: tracking oxygen from the airway through the blood to organ tissues

The physiological redistribution of perfluorocarbon (PFC) compounds to liver, spleen, bone marrow, and lung after intravenous (i.v.) or intraperitoneal (IP) administration of PFC emulsions affords the unique opportunity for non-invasive monitoring of oxygenation status of these organs and tissues utilizing fluorine (F-19) nuclear magnetic resonance (NMR) imaging techniques. PFCs also may be introduced directly into the pulmonary airways by procedures such as liquid ventilation, intratracheal instillation, or aerosol inhalation. Considerations of importance when establishing methodology for accurate quantitation of oxygen partial pressure (pO2) in vivo using F-19 NMR include: 1.) error analysis of the calibration curves which relate pO2 to the measured PFC F-19 relaxation rate, 2.) optimization of the NMR pulse sequence for efficient oxygen sensitive data acquisition and, 3.) fluorine signal independence from emulsion aqueous phase bioconstituents. The porcine model was investigated at 0.14T following i.v. or IP administration of the PFC emulsion containing perfluorotributylamine (FC-43) to demonstrate the capability for tracking oxygen with F-19 NMR from the lung through the blood to selected organ tissues. Quantitative pO2 projection images and isobaric contour graphs were derived for the liver, spleen, and lungs as a function of inspired oxygen. Blood pO2 levels in aorta, pulmonary artery, and hepatic vein were monitored simultaneously with NMR imaging for correlative analysis.     

The modified bitegard: a method for administering supplemental oxygen and measuring carbon dioxide

HUGE is a database for human large proteins newly identified by Kazusa cDNA project, which aims to predict protein primary structures from sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are current targets of the project. More than 700 sequences of human cDNAs (average size, 5.1 kb) have been determined to date and deposited in the public databases. Notable information implied from the cDNAs and the predicted protein sequences can be obtained through HUGE via the World Wide Web at URL http://www.kazusa.or.jp/huge     

C19 steroids estrogenic activity in human breast cancer cell lines: importance of dehydroepiandrosterone sulfate at physiological plasma concentration

The estrogenic action of C19 steroids on breast cancer cells was measured by bioluminescence in stably transfected human breast cancer MCF-7 and T47D cell lines with a reporter gene that allows expression of the firefly luciferase enzyme under control of an estrogen regulatory element. The "estrogenic activity" of C19 steroids, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), androst-5-en-3 beta,17 beta-diol, androst-4-en-3,17-dione, dihydrotestosterone, testosterone, and 5 alpha-androstan-3 beta,17 beta-diol was studied. This showed that DHEAS, at concentration observed in physiological conditions (10(-6) M), had a high "estrogen-like effect" in MCF-7 and T47D cell lines. Other C19 steroids, at physiological plasma concentration, alone or together did not have any significant effect on the luciferase activity. Moreover aminoglutethimide, an inhibitor of the aromatase enzyme, in the presence of C19 steroids, partially decreased the luciferase activity. These results suggest that MCF-7 and T47D cell lines could convert DHEAS to estrogen-like compounds by different enzymatic systems.     

Automated static perimetry as a screening method for evaluation of retinal perfusion in diabetic retinopathy

Differential diagnosis of syncope and seizures frequently imposes a major problem, particularly if interictal examinations are normal. We performed orthostatic testing combined with surface electroencephalography (EEG) and non-invasive measurements of cerebral blood flow velocity. Ten healthy controls, ten patients with confirmed diagnosis of epilepsy and 25 patients with history of syncope of unknown origin were examined. The following parameters were evaluated continuously and simultaneously during orthostatic challenge: computerized EEG with synchronous video-monitoring, transcranial Doppler sonography (TCD), heart rate and blood pressure. Isolated cerebrovascular dysregulation (i.e. a drop in cerebral perfusion despite the absence of a significant drop in peripheral blood pressure) occurred in 2/10 controls, 3/10 patients with epilepsy and 11/25 patients with syncope of unknown origin. The combined EEG and TCD measurements represent a new approach to the work-up of patients with otherwise unexplained syncope, helping us to understand the interdependence of neuronal activity and peripheral/cerebrovascular autoregulation under postural stress.     

A perfluorochemical oxygen carrier (fluosol-43) in a synthetic medium used for perfusion of isolated rat liver

The action of primaquine was investigated on male Wistar rats depleted on the vitamin B complex (approximately 50% of their requirement for optimal growth), on thiamine (approximately 50% of their requirement for optimal growth), and pair-fed control animals. There was only a marginal increase in adverse primaquine reactions in the malnourished, especially in the thiamine deficient rats.     

Solid-state NMR for the study of membrane systems: the use of anisotropic interactions

The use of solid-state nuclear magnetic resonance (NMR) as a tool to determine the structure of membrane molecules is reviewed with a particular emphasis on techniques that provide information on orientation or order. Experiments reported here have been performed in membranes, rather than in micelles or organic solvents. Several ways to prepare and handle the samples are discussed, like sample orientation and magic-angle spinning (MAS). Results concerning lipids, membrane peptides and proteins are included, as well as a discussion regarding the potential of such methods and their pitfalls.     

Selective coronary artery perfusion in vitro: a method to study cardiac effects of contrast media

RATIONALE AND OBJECTIVES: The authors evaluated selective perfusion of the coronary arteries in the isolated rat heart as a model for studying contrast medium-induced cardiac effects and compared the effects of iodixanol, iotrolan, and ioxaglate with this model. MATERIALS AND METHODS: Isolated, spontaneously beating rat hearts were used. Control hearts were perfused in the Langendorff or the selective perfusion mode receiving Krebs Henseleit buffer. Contrast media were injected selectively into the left coronary artery. Left ventricular pressure and electrocardiographic parameters were monitored continuously throughout the experiments. RESULTS: The stability of the selective perfusion preparation was similar to that of the conventional Langendorff preparation. Ioxaglate (0.3 g iodine per kilogram body weight) significantly (P < .05) depressed left ventricular contractility and decreased (P < .05) left ventricular pressure. Iodixanol and iotrolan had minor cardiac effects. CONCLUSION: Selective coronary artery perfusion seems to be a suitable model for studying direct cardiac effects of contrast media. The nonionic dimers, iodixanol and iotrolan, induce only minor changes in cardiac function.