Helmut Ahammer

Quantitating the Subtleties of Microglial Morphology with Fractal Analysis

It is well established that microglial form and function are inextricably linked. In recent years, the traditional view that microglial form ranges between “ramified resting” and “activated amoeboid” has been emphasized through advancing imaging techniques that point to microglial form being highly dynamic even within the currently accepted morphological categories. Moreover, microglia adopt meaningful intermediate forms between categories, with considerable crossover in function and varying morphologies as they cycle, migrate, wave, phagocytose, and extend and retract fine and gross processes. From a quantitative perspective, it is problematic to measure such variability using traditional methods, but one way of quantitating such detail is through fractal analysis. The techniques of fractal analysis have been used for quantitating microglial morphology, to categorize gross differences but also to differentiate subtle differences (e.g., amongst ramified cells). Multifractal analysis in particular is one technique of fractal analysis that may be useful for identifying intermediate forms. Here we review current trends and methods of fractal analysis, focusing on box counting analysis, including lacunarity and multifractal analysis, as applied to microglial morphology.

Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7α-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.

Automatically Generated, Anatomically Accurate Meshes for Cardiac Electrophysiology Problems

Significant advancements in imaging technology and the dramatic increase in computer power over the last few years broke the ground for the construction of anatomically realistic models of the heart at an unprecedented level of detail. To effectively make use of high-resolution imaging datasets for modeling purposes, the imaged objects have to be discretized. This procedure is trivial for structured grids. However, to develop generally applicable heart models, unstructured grids are much preferable. In this study, a novel image-based unstructured mesh generation technique is proposed. It uses the dual mesh of an octree applied directly to segmented 3-D image stacks. The method produces conformal, boundary-fitted, and hexahedra-dominant meshes. The algorithm operates fully automatically with no requirements for interactivity and generates accurate volume-preserving representations of arbitrarily complex geometries with smooth surfaces. The method is very well suited for cardiac electrophysiological simulations. In the myocardium, the algorithm minimizes variations in element size, whereas in the surrounding medium, the element size is grown larger with the distance to the myocardial surfaces to reduce the computational burden. The numerical feasibility of the approach is demonstrated by discretizing and solving the monodomain and bidomain equations on the generated grids for two preparations of high experimental relevance, a left ventricular wedge preparation, and a papillary muscle.

Three-Dimensional Models of Individual Cardiac Histoanatomy: Tools and Challenges

Abstract:  There is a need for, and utility in, the acquisition of data sets of cardiac histoanatomy, with the vision of reconstructing individual hearts on the basis of noninvasive imaging, such as MRI, enriched by reference to detailed atlases of serial histology obtained from representative samples. These data sets would be useful not only as a repository of knowledge regarding the specifics of cardiac histoanatomy, but could form the basis for generation of individualized high‐resolution cardiac structure–function models. The current article presents a step in this general direction: it illustrates how whole‐heart noninvasive imaging can be combined with whole‐heart histology in an approach to achieve automated construction of histoanatomically detailed models of cardiac 3D structure and function at hitherto unprecedented resolution and accuracy (based on 26.4 × 26.4 × 24.4 μm MRI voxel size, and enriched by histological detail). It provides an overview of the tools used in this quest and outlines challenges posed by the approach in the light of applications that may benefit from the availability of such data and tools.

Di-4-ANEPPS causes photodynamic damage to isolated cardiomyocytes

Action potential recordings from isolated guinea pig ventricular cells in the whole-cell recording mode were used to study the toxic and photodynamic properties of the voltage-sensitive fluorescent dye di-4-ANEPPS. Staining of the cardiomyocytes with di-4-ANEPPS (30 or 60 μM; 10 min) did not alter the action potential shape. When the stained cells were illuminated (1W/cm2) severe effects on the action potential were observed. There was a prolongation of the action potential duration, occurrence of early afterdepolarizations, reduction of the membrane resting potential and eventually inexcitability. Addition of the antioxidant catalase (100 IU/ml) to the extracellular solution delayed the onset of these effects, suggesting that reactive-oxygen-intermediates take part in di-4-ANEPPS induced photodynamic damage. Since di-4-ANEPPS is a very important tool for optical membrane potential recordings in heart tissue and single cardiomyocytes catalase might be useful in suppressing photodynamic damage during optical potential recordings.

Optical multisite monitoring of cell excitation phenomena in isolated cardiomyocytes

An especially designed setup which consists of an inverted fluorescence microscope, an argon ion laser and a photodiode array system permits membrane potential monitoring in isolated guinea-pig ventricular cardiomyocytes, stained with the voltage-sensitive dye di-4-ANEPPS, which responds linearly with relative fluorescence changes (ΔF/F) ≈ −8% per 100 mV. About a dozen measuring spots covering a single cell were simultaneously monitored with a spatial and temporal resolution of 15 μm and about 20 μs, respectively. In general, the rising phases of the action potentials within a single cell were highly synchronized (i.e. all upstroke velocities peaked within about 20 μs); however, in one cell (out of 25 examined) significant (P < 0.05) time lags exceeding the signal-dependent time resolution were also found. Experiments, simultaneously performed with our optical system and a widely used patch-clamp setup, revealed a slowed and delayed response of the clamp amplifier depending on the cell access resistance. Optical monitoring during whole-cell voltage-clamping demonstrated the influence of graduated series resistance compensation. When field stimulation was used, our results clearly demonstrated the spatially dependent polarization of the cell membrane during the stimulus, as well as a highly synchronized upstroke development. Slight differences in the maximum upstroke velocities within a single cell were also found and were basically in agreement with mathematical models.

Trophoblastic invasion in vitro and in vivo: similarities and differences

BACKGROUND The basic mechanisms of trophoblast invasion are not completely understood. This may be due to the lack of suitable in vitro models which enable experimental modulation of this complex process. In the present study, a three-dimensional co-culture model is used for comparing two factors considered to be implicated in the regulation of trophoblast invasion, the expression of HLA-G and apoptosis, in vitro and in vivo. METHODS Tissue fragments from human first trimester decidua parietalis were put in close contact with spheroids of AC-1M59 trophoblast/choriocarcinoma hybrid cells as a model of the invasive trophoblast. Cryostat sections from these co-cultures were immunohistochemically stained and compared with first trimester placentation sites in vivo. RESULTS Only the invasive trophoblast-derived cells showed an intensive staining for HLA-G, whereas the cells on the periphery of the confrontation culture exhibited only a weak staining. A similar staining pattern was found in vivo. Both in vitro and in vivo CD45(+) apoptotic leukocytes were frequently detected in close proximity to the invasive trophoblastic cells. CONCLUSIONS In this co-culture system, key factors considered to be implicated in trophoblast invasion in vivo can also be demonstrated in vitro. Therefore, it may help in finding strategies for the management of diseases associated with deficient trophoblast invasion.

The influence of edge detection algorithms on the estimation of the fractal dimension of binary digital images

The boundary of a fractal object, represented in a two-dimensional space, is theoretically a line with an infinitely small width. In digital images this boundary or contour is limited to the pixel resolution of the image and the width of the line commonly depends on the edge detection algorithm used. The Minkowski dimension was evaluated by using three different edge detection algorithms (Sobel, Roberts, and Laplace operator). These three operators were investigated because they are very widely used and because their edge detection result is very distinct concerning the line width. Very common fractals (Sierpinski carpet and Koch islands) were investigated as well as the binary images from a cancer invasion assay taken with a confocal laser scanning microscope. The fractal dimension is directly proportional to the width of the contour line and the fact, that in practice very often the investigated objects are fractals only within a limited resolution range is considered too.

Fractal dimension and vessel complexity in patients with cerebral arteriovenous malformations.

The fractal dimension (FD) can be used as a measure for morphological complexity in biological systems. The aim of this study was to test the usefulness of this quantitative parameter in the context of cerebral vascular complexity. Fractal analysis was applied on ten patients with cerebral arteriovenous malformations (AVM) and ten healthy controls. Maximum intensity projections from Time-of-Flight MRI scans were analyzed using different measurements of FD, the Box-counting dimension, the Minkowski dimension and generalized dimensions evaluated by means of multifractal analysis. The physiological significance of this parameter was investigated by comparing values of FD first, with the maximum slope of contrast media transit obtained from dynamic contrast-enhanced MRI data and second, with the nidus size obtained from X-ray angiography data. We found that for all methods, the Box-counting dimension, the Minkowski dimension and the generalized dimensions FD was significantly higher in the hemisphere with AVM compared to the hemisphere without AVM indicating that FD is a sensitive parameter to capture vascular complexity. Furthermore we found a high correlation between FD and the maximum slope of contrast media transit and between FD and the size of the central nidus pointing out the physiological relevance of FD. The proposed method may therefore serve as an additional objective parameter, which can be assessed automatically and might assist in the complex workup of AVMs.

Body composition in sport: a comparison of a novel ultrasound imaging technique to measure subcutaneous fat tissue compared with skinfold measurement

Background Extremely low weight and rapid changes in weight and body composition have become major concerns in many sports, but sufficiently accurate field methods for body composition assessment in athletes are missing. This study aimed to explore the use of ultrasound methods for assessment of body fat content in athletes. Methods 19 female athletes (stature: 1.67(±0.06) m, weight: 59.6(±7.6) kg; age: 19.5(±3.3) years) were investigated by three observers using a novel ultrasound method for thickness measurement of uncompressed subcutaneous adipose tissue and of embedded structures. Two observers also measured skinfold thickness at eight International Society for the Advancement of Kinanthrometry (ISAK) sites; mean skinfold values were compared to mean subcutaneous adipose tissue thicknesses measured by ultrasound. Interobserver reliability of imaging and evaluation obtained by this ultrasound technique: intraclass correlation coefficient ICC=0.968 (95% CI 0.957 to 0.977); evaluation of given images: ICC=0.997 (0.993 to 0.999). Results Skinfold compared to ultrasound thickness showed that compressibility of subcutaneous adipose tissue depends largely on the site and the person: regression slopes ranged from 0.61 (biceps) to 1.59 (thigh) and CIs were large. Limits of agreement ranged from 2.6 to 8.6 mm. Regression lines did not intercept the skinfold axis at zero because of the skin thickness being included in the skinfold. The four ISAK trunk sites caused ultrasound imaging problems in 13 of 152 sites (8 ISAK sites, 19 athletes). Conclusions The ultrasound method allows measurement of uncompressed subcutaneous adipose tissue thickness with an accuracy of 0.1–0.5 mm, depending on the probe frequency. Compressibility of the skinfold depends on the anatomical site, and skin thickness varies by a factor of two. This inevitably limits the skinfold methods for body fat estimation. Ultrasound accuracy for subcutaneous adipose tissue measurement is limited by the plasticity of fat and furrowed tissue borders. Comparative US measurements show that skinfold measurements do not allow accurate assessment of subcutaneous adipose tissue thickness.