3D Reconstruction of Neural Circuits from Serial EM Images

A basic requirement for reconstructing and understanding complete circuit diagrams of neuronal processing units is the availability of electron microscopic 3D data sets of large ensembles of neurons. A recently developed technique, "Serial Block Face Scanning Electron Microscopy" (SBFSEM, Denk and Horstmann 2004) allows automatic sectioning and imaging of biological tissue inside the vacuum chamber of a scanning electron microscope. Image stacks generated with this technology have a resolution sucient to distinguish different cellular compartments, including synaptic structures. Such an image stack contains thousands of images and is recorded with a voxel size of 23 nm in the x- and y-directions and 30 nm in the z-direction. Consequently a tissue block of 1 mm3 produces 63 terabytes of data. Therefore new concepts for managing large data sets and automated image processing are required. I developed an image segmentation and 3D reconstruction software, which allows precise contour tracing of cell membranes and simultaneously displays the resulting 3D structure. The software contains two stand-alone packages: Neuron2D and Neuron3D, both oering an easy-to-operate graphical user interface (GUI). The software package Neuron2D provides the following image processing functions: • Image Registration: Combination of multiple SBFSEM image tiles. • Image Preprocessing: Filtering of image stacks. Implemented are Gaussian and Non-Linear-Diusion lters in 2D and 3D. This step enhances the contrast between contour lines and image background, leading to a higher signal-to-noise ratio, thus further improving detection of membrane borders. • Image Segmentation: The implemented algorithms extract contour lines from the preceding image and automatically trace the contour lines in the following images (z-direction), taking into account the previous image segmentation. They also permit image segmentation starting at any position in the image stack. In addition, manual interaction is possible. To visualize 3D structures of neuronal circuits the additional software Neuron3D was developed. The program relies on the contour line information provided by Neuron2D to implement a surface reconstruction algorithm based on dynamic time warping. Additional rendering techniques, such as shading and texture mapping, are provided. The detailed anatomical reconstruction provides a framework for computational models of neuronal circuits. For example in ies, where moving retinal images lead to appropriate course control signals, the circuit reconstruction of motion-sensitive neurons can help to further understand the neural processing of visual motion in ies

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Contour-propagation Algorithms For Semi-automated Reconstruction of Neural Processes

A new technique, "serial block face scanning electron microscopy" (SBFSEM), allows for automatic sectioning and imaging of biological tissue with a scanning electron microscope. Image stacks generated with this technology have a resolution sufficient to distinguish different cellular compartments, including synaptic structures, which should make it possible to obtain detailed anatomical knowledge of complete neuronal circuits. Such an image stack contains several thousands of images and is recorded with a minimal voxel size of 10-20 nm in the x- and y-direction and 30 mu in Z-direction. Consequently, a tissue block of 1 mm(3) (the approximate volume of the Calliphora vicina brain) will produce several hundred terabytes of data. Therefore, highly automated 3D reconstruction algorithms are needed. As a first step in this direction we have developed semi-automated segmentation algorithms for a precise contour tracing of cell membranes. These algorithms were embedded into an easy-to-operate user interface, which allows direct 3D observation of the extracted objects during the segmentation of image stacks. Compared to purely manual tracing, processing time is greatly accelerated. (c) 2007 Elsevier B.V. All rights reserved

Mitochondrial ROS and mitochondria-targeted antioxidants in the aged heart.

Excessive mitochondrial ROS production has been causally linked to the pathophysiology of aging in the heart and other organs, and plays a deleterious role in several age-related cardiac pathologies, including myocardial ischemia-reperfusion injury and heart failure, the two worldwide leading causes of death and disability in the elderly. However, ROS generation is also a fundamental mitochondrial function that orchestrates several signaling pathways, some of them exerting cardioprotective effects. In cardiac myocytes, mitochondria are particularly abundant and are specialized in subcellular populations, in part determined by their relationships with other organelles and their cyclic calcium handling activity necessary for adequate myocardial contraction/relaxation and redox balance. Depending on their subcellular location, mitochondria can themselves be differentially targeted by ROS and display distinct age-dependent functional decline. Thus, precise mitochondria-targeted therapies aimed at counteracting unregulated ROS production are expected to have therapeutic benefits in certain aging-related heart conditions. However, for an adequate design of such therapies, it is necessary to unravel the complex and dynamic interactions between mitochondria and other cellular processes

HMGB1 is associated with atherosclerotic plaque composition and burden in patients with stable coronary artery disease.

OBJECTIVES: The role of inflammation in atherosclerosis is widely appreciated. High mobility group box 1 (HMGB1), an injury-associated molecular pattern molecule acting as a mediator of inflammation, has recently been implicated in the development of atherosclerosis. In this study, we sought to investigate the association of plasma HMGB1 with coronary plaque composition in patients with suspected or known coronary artery disease (CAD). DESIGN: HMGB1, high sensitive troponin T (hsTnT) and high sensitive C-reactive protein (hsCRP) were determined in 152 consecutive patients with suspected or known stable CAD who underwent clinically indicated 256-slice coronary computed tomography angiography (CCTA). Using CCTA, we assessed 1) coronary calcification, 2) non-calcified plaque burden and 3) the presence of vascular remodeling in areas of non-calcified plaques. RESULTS: Using univariate analysis, hsCRP, hsTnT and HMGB1 as well as age, and atherogenic risk factors were associated with non-calcified plaque burden (r = 0.21, p = 0.009; r = 0.48, p<0.001 and r = 0.34, p<0.001, respectively). By multivariate analysis, hsTnT and HMGB1 remained independent predictors of the non-calcified plaque burden (r = 0.48, p<0.01 and r = 0.34, p<0.001, respectively), whereas a non-significant trend was noticed for hs-CRP (r = 0.21, p = 0.07). By combining hsTnT and HMGB1, a high positive predictive value for the presence of non-calcified and remodeled plaque (96% and 77%, respectively) was noted in patients within the upper tertiles for both biomarkers, which surpassed the positive predictive value of each marker separately. CONCLUSIONS: In addition to hs-TnT, a well-established cardiovascular risk marker, HMGB1 is independently associated with non-calcified plaque burden in patients with stable CAD, while the predictive value of hs-CRP is lower. Complementary value was observed for hs-TnT and HMGB1 for the prediction of complex coronary plaque