Determination of regional bone blood flow by means of fluorescent microspheres using an automated sample-processing procedure

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 x 10(6) spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 +/- 1.1 and 8.5 +/- 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 +/- 1.1 to 6.0 +/- 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 +/- 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF. Copyright (C) 2003 S. Karger AG, Basel

Autoinducers act as biological timers in Vibrio harveyi

Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations

Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri

The marine bacterium Vibrio fischeri regulates its bioluminescence through a quorum sensing mechanism: the bacterium releases diffusible small molecules (autoinducers) that accumulate in the environment as the population density increases. This accumulation of autoinducer (AI) eventually activates transcriptional regulators for bioluminescence as well as host colonization behaviors. Although V.fischeri quorum sensing has been extensively characterized in bulk populations, far less is known about how it performs at the level of the individual cell, where biochemical noise is likely to limit the precision of luminescence regulation. We have measured the time-dependence and AI-dependence of light production by individual V.fischeri cells that are immobilized in a perfusion chamber and supplied with a defined concentration of exogenous AI. We use low-light level microscopy to record and quantify the photon emission from the cells over periods of several hours as they respond to the introduction of AI. We observe an extremely heterogeneous response to the AI signal. Individual cells differ widely in the onset time for their luminescence and in their resulting brightness, even in the presence of high AI concentrations that saturate the light output from a bulk population. The observed heterogeneity shows that although a given concentration of quorum signal may determine the average light output from a population of cells, it provides far weaker control over the luminescence output of each individual cell

Identification and characterization of a direct activator of a gene transfer agent

Gene transfer agents (GTAs) are thought to be ancient bacteriophages that have been co-opted into serving their host and can now transfer any gene between bacteria. Production of GTAs is controlled by several global regulators through unclear mechanisms. In Rhodobacter capsulatus, gene rcc01865 encodes a putative regulatory protein that is essential for GTA production. Here, I show that rcc01865 (hereafter gafA) encodes a transcriptional regulator that binds to the GTA promoter to initiate production of structural and DNA packaging components. Expression of gafA is in turn controlled by the pleiotropic regulator protein CtrA and the quorum-sensing regulator GtaR. GafA and CtrA work together to promote GTA maturation and eventual release through cell lysis. Identification of GafA as a direct GTA regulator allows the first integrated regulatory model to be proposed and paves the way for discovery of GTAs in other species that possess gafA homologues

[The architecture of the subacromial space after full thickness supraspinatus tears]

AIM: The etiology of rotator cuff tears is multifactorial. An important factor is the damage of the rotator cuff by narrowing of the subacromial space. The purpose of this investigation was to estimate the influence of various metrical parameters on the size of the subacromial space. METHOD: We investigated 161 human macerated scapulae, 36 of them had a known tear of the supraspinatus tendon. All scapulae were photographed in two standard positions from the front and the lateral side. Defined distances and angles were measured using an image analyzing system followed by statistical analysis. RESULTS: Shoulders with a tear of the supraspinatus tendon showed a trend towards higher incidences of hooked acromions. Furthermore we found a significant higher incidence of elongated oval shaped glenoids in the group with supraspinatus tendon tear. In comparison to normal shoulders a significantly smaller distance from the top of the glenoid to the tip of the acromion and a greater distance from the top of the glenoid to the tip of the coracoid process was measured. In addition there was a significantly smaller coracoid angle and a smaller glenoid-spinal angle in this group. CONCLUSION: The width of the subacromial space depends on various parameters. Our data suggest that besides the acromion type the shape of the coracoid, the acromial angle, the spine-scapula angle and the cavitas-spine angle should be taken into account for diagnostic and therapeutic decisions

Determination of regional bone blood flow by means of fluorescent microspheres using an automated sample-processing procedure.

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 x 10(6) spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 +/- 1.1 and 8.5 +/- 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 +/- 1.1 to 6.0 +/- 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 +/- 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF

Fluorescent microspheres are reliable for serial bone blood flow measurements.

The fluorescent microsphere method is one of the current techniques to determine regional blood flow in various organs. The purpose of this study was to examine the suitability of fluorescent microspheres for serial measurement of regional bone blood flow. Six anesthetized female New Zealand rabbits received five left ventricular injections of fluorescent microspheres in 20-minute intervals. To test the precision of the measurement two types of fluorescent microspheres were injected simultaneously at the first and last injections. Blood flow was calculated in the kidneys, lungs, brain, femurs, and tibias after measuring the fluorescence intensity in each reference blood and tissue sample. Comparison of blood-flow values obtained by simultaneously injected microspheres showed an excellent correlation and a minimal percentage difference at the first and last injections, indicating valid measurements of regional bone blood flow. No significant differences were observed when comparing blood flow in the corresponding regions of bones on the right side and left side. Mean blood flow in the femur and tibia significantly increased at the fourth injection whereas flow distribution within the femur and tibia essentially remained unchanged throughout the experiment. Comparison of blood flow values obtained by simultaneously injected microspheres showed moderate agreement for the kidneys and lungs at the last injections. Because this finding might be attributable to disturbances of microcirculation caused by accumulation of spheres in high-flow organs, the increase in regional bone blood flow observed in our experiments has to be interpreted carefully. This study showed that bone blood flow can be determined reliably in anesthetized rabbits by as many as three serial injections of fluorescent microspheres

Microspheres accurately predict regional bone blood flow.

Even though the microsphere method frequently is used to determinate bone blood flow, validation of this technique for bone blood flow measurement is incomplete. The method is based on the principle that injected microspheres are distributed with the arterial blood and trapped in the capillaries because of their diameter (15 microm). The number of spheres lodged in an organ is proportional to its blood flow. The number of radioactive or fluorescent microspheres in a specific organ is determined indirectly by measuring radioactivity or fluorescence intensity in the organ. In this study the reliability and precision of the microsphere method for determining bone blood flow was established using radioactive and fluorescent microspheres. Six female, anesthetized New Zealand rabbits received left ventricular injections of pairs of fluorescent and/or radioactive microspheres. The humerus, femur, and tibia were dissected in a standardized manner and blood flow was determined in each sample. Comparison of relative blood flow values showed an excellent correlation between radioactive and fluorescent microspheres. The percentage difference and variation between two simultaneously injected sets of microspheres was minimal for radioactive microspheres (0.8% +/- 9.6%) and for fluorescent microspheres (0.2% +/- 11.4%). Regional bone blood flow in different regions of the femur, tibia, or humerus ranged from 2.2-28.1 mL/minute/100 g, but there was no significant difference between right and left bone samples of the same region after repeated measurement. Radioactive and fluorescent microspheres allow precise determination of regional bone blood flow