Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer.

Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.

Locating femoral graft placement from lateral radiographs in anterior cruciate ligament reconstruction: a comparison of 3 methods of measuring radiographic images.

Graft positioning in anterior cruciate ligament (ACL) reconstruction is usually documented from lateral postoperative radiographs. The purpose of this study was to compare 3 measurement methods for femoral graft placement in 50 patients with ACL reconstruction. Intraoperative radiographic images were obtained and divided into 2 groups. The first group showed suboptimal projections, with out-of-plane rotations causing the femoral condyles to not be perfectly overlapped. The second group showed good projection, with optimal rotation and fully overlapped femoral condyles. In our study, only the measurement technique described by Amis produced data with the least measurement error when multiple observers assessed both groups. It is recommended that Amis' method be used to measure femoral ACL graft position so that reliable data are available for comparison between medical centers.

Sagittal plane imaging parameters for computer-assisted fluoroscopic anterior cruciate ligament reconstruction.

Reproducible graft placement in anterior cruciate ligament (ACL) reconstructions is considered to be a critical factor affecting the successful clinical outcome of the procedure. Many current ACL instrument systems rely on intra-articular landmarks to guide the ACL tunnel placement. However, most of these instrument systems use mobile soft tissues as landmarks. We hypothesize that consistently identifiable radiographic contour landmarks can be established that can be used to improve the reproducibility of graft tunnel placement in fluoroscopically and computer-assisted ACL reconstructions. For the tibia, magnetic resonance imaging (MRI) scans showed the average ACL attachment site to be projected at 46% on a line extending from the anterior to the posterior cortices. Intraoperative fluoroscopic images were checked for the reproducibility of this line and its clinical use. For the femur, lateral radiographs demonstrated a consistent relationship between the intercondylar roof line (Blumensaat's line) and the nearly circular profile of the posterior and inferior contour of the lateral femoral condyle. The middle of this circular profile is consistently projected on Blumensaat's line at 66% of its anterior-to-posterior direction. Intraoperative images were used, which showed the aiming drill at the point of entering the lateral femoral condyle. Instead of determining the femoral attachment site relative to Blumensaat's line, we can thus determine its position relative to the center of the circle. Based on intraoperative x-rays, the proposed femoral ACL attachment site can be projected on a line parallel with the Blumensaat's line from the circle center in the posterior direction. Our results indicate that there are consistently identifiable radiographic features on the tibia and femur contours that can be used for fluoroscopic and computer-assisted guidance of ACL graft placement.

Intrinsic cone adaptation modulates feedback efficiency from horizontal cells to cones.

Processing of visual stimuli by the retina changes strongly during light/dark adaptation. These changes are due to both local photoreceptor-based processes and to changes in the retinal network. The feedback pathway from horizontal cells to cones is known to be one of the pathways that is modulated strongly during adaptation. Although this phenomenon is well described, the mechanism for this change is poorly characterized. The aim of this paper is to describe the mechanism for the increase in efficiency of the feedback synapse from horizontal cells to cones. We show that a train of flashes can increase the feedback response from the horizontal cells, as measured in the cones, up to threefold. This process has a time constant of approximately 3 s and can be attributed to processes intrinsic to the cones. It does not require dopamine, is not the result of changes in the kinetics of the cone light response and is not due to changes in horizontal cells themselves. During a flash train, cones adapt to the mean light intensity, resulting in a slight (4 mV) depolarization of the cones. The time constant of this depolarization is approximately 3 s. We will show that at this depolarized membrane potential, a light-induced change of the cone membrane potential induces a larger change in the calcium current than in the unadapted condition. Furthermore, we will show that negative feedback from horizontal cells to cones can modulate the calcium current more efficiently at this depolarized cone membrane potential. The change in horizontal cell response properties during the train of flashes can be fully attributed to these changes in the synaptic efficiency. Since feedback has major consequences for the dynamic, spatial, and spectral processing, the described mechanism might be very important to optimize the retina for ambient light conditions.

Computer assistance in arthroscopic anterior cruciate ligament reconstruction.

Accurate placement of the graft is considered one of the most important factors in anterior cruciate ligament surgery. However, reconstruction with contemporary guiding systems still can result in unacceptable graft placement variability. To improve the reproducibility of graft placement, intraoperative visual feedback was added to the arthroscopic technique. First, fluoroscopic visualization was added to evaluate guidewire placement before tunnel drilling. Second, computer graphic overlays were added to the fluoroscopic view. Three groups of patients were treated: 29 patients with arthroscopy, 53 patients with fluoroscopy added, and 50 patients with computer overlays added. Graft placement variability was reduced significantly with fluoroscopic visualization. Computer overlays resulted in additional significant reductions in graft placement variability. Simple visual enhancements seem to be useful in improving the accuracy of arthroscopic anterior cruciate ligament reconstruction.