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

Computed tomography osteoabsorptiometry is reliable for the determination of the subchondral bone mineralization distribution in the rabbit knee

Rabbits are among the most frequently used animals in osteoarthritis research. It is meanwhile accepted that the subchondral bone plate (SBP) plays a key role in the development of osteoarthritis. The most suitable technique for analyzing subchondral bone mineralization is computed tomography osteoabsorptiometry (CT-OAM). Because CT-OAM has not yet been applied to smaller animals, the purpose of this study is to test the reliability of CT-OAM in the rabbit knee. Another important task in animal experiments is the intra- and interindividual difference of the measurement parameters. Our hypothesis is that there is no difference regarding both the position of the density maxima and the bone mineral density (BMD) of the SBP comparing right and left tibial plateaus of rabbits. For evaluating the reliability, a rabbit knee was examined by computed tomography 6 times at weekly intervals. The subchondral mineralization distribution was measured by means of CT-OAM. Positions of the density maxima and BMD of the SBP were determined in a standardized procedure. Furthermore, both parameters were evaluated in 6 female White New Zealand rabbits. Positions of density maxima and BMD in the SBP in left tibial plateaus were compared with right tibial plateaus. The relative coefficient of variation as a parameter for reproducibility was 1.6% for determining the position of the density maxima and 1.2% for measuring the BMD. The positions of density maxima and relative BMD between right and left tibial plateaus varied only about 2% intraindividually, whereas interindividual variance was about 10%. In conclusion, determination of the position of density maxima as well as BMD of the SBP by means of CT-OAM is reliable and reproducible in the rabbit knee. We recommend using the contralateral limb as control, because intraindividual accordance of the mineralization patterns and of the BMD of the SBP was higher than interindividual accordance

Meniscectomy: indications, procedure, outcomes, and rehabilitation

Hermann Anetzberger,1 Christof Birkenmaier,2 Stephan Lorenz3 1Orthopädische Gemeinschaftspraxis am OEZ, Munich, Germany; 2Department of Orthopedics, Ludwig-Maximilian-University Munich, Munich, Germany; 3Department of Orthopedic Sports Medicine, Technical University of Munich, Klinikum rechts der Isar, Munich, Germany Abstract: Meniscal injuries are among the most frequent reasons for knee problems. The goal of this manuscript is to review the biomechanical relevance of the human knee's menisci in relation to surgical indications, surgical techniques, rehabilitation, and outcomes. In order to identify the relevant literature, we performed a PubMed search for the years ranging from 1980–2013 using the following search terms: meniscus; biomechanical function; meniscectomy; meniscal repair; and clinical outcome. The meniscus helps to distribute the forces between the tibial and femoral articular cartilage layers in a load-sharing capacity. Meniscus damage or meniscectomy intuitively leads to an overloading of the cartilage and, hence, to the development of osteoarthrosis. Precise knowledge of meniscal shape and function, of the type of injury, of surgical techniques, as well as of postsurgical rehabilitative care are of decisive importance for an individually-adjusted treatment strategy. Other underlying coexisting knee pathologies also need to be considered. The diagnosis of a meniscal injury is based upon clinical history, physical examination, and imaging studies. The treatment of a meniscal lesion includes conservative, as well as operative, procedures. The goals of surgery are to reduce pain and disability, as well as to preserve meniscal function without causing additional cartilage damage. The resection of meniscal tissue should be restricted to as much as is necessary, and as little as is reasonably possible. Postoperative rehabilitation serves the purpose of improving functional deficits and pain, as well as of restoring a good range of motion and preventing secondary damage. It is the surgical therapy that dictates the aftercare, and with regards to the latter, there are clear differences between meniscectomy and meniscal repair. Keywords: meniscectomy, osteoarthritis, meniscus pathology, meniscus functio