Astrocytes regulate α-secretase-cleaved soluble amyloid precursor protein secretion in neuronal cells: Involvement of group IIA secretory phospholipase A2.

Astrocytes are major supportive cells in brains with important functions including providing nutrients and regulating neuronal activities. In this study, we demonstrated that astrocytes regulate amyloid precursor protein (APP) processing in neuronal cells through secretion of group IIA secretory phospholipase A2 (sPLA2-IIA). When astrocytic cells (DITNC) were mildly stimulated with the pro-inflammatory cytokines, such as TNF α and IL-1ß, sPLA2-IIA was secreted into the medium. When conditioned medium containing sPLA2-IIA was applied to human neuroblastoma (SH-SY5Y) cells, there was an increase in both cell membrane fluidity and secretion of α-secretase-cleaved soluble amyloid precursor protein (sAPPα). These changes were abrogated by KH064, a selective inhibitor of sPLA2-IIA. In addition, exposing SH-SY5Y cells to recombinant human sPLA2-IIA also increased membrane fluidity, accumulation of APP at the cell surface, and secretion of sAPPα, but without altering total expressions of APP, α-secretases and ß-site APP cleaving enzyme (BACE1). Taken together, our results provide novel information regarding a functional role of sPLA2-IIA in astrocytes for regulating APP processing in neuronal cells.

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes.

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.

A molecular rotor as viscosity sensor in aqueous colloid solutions.

BACKGROUND: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. METHOD OF APPROACH: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. RESULTS: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of -5.5% to 5.1%). CONCLUSIONS: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.

Venous ligation-mediated bone adaptation is NOS 3 dependent.

Interstitial fluid flow (IFF) in bone has been hypothesized to mediate bone modeling in the absence of mechanical strain. The mechanism of this effect has not been clearly defined, though previous studies indicate that nitric oxide (NO) may play an important role in mediating IFF. In the current study, mice with a targeted disruption of the NOS 3 gene were used according to a previously established model of altered interstitial fluid flow in bone. Femoral vein ligation was performed in one limb to increase intramedullary pressure and consequently its IFF; a sham operation was performed on the contralateral limb. The mice were then hindlimb suspended to uncouple the effects of altered flow in the limb from mechanical loading. Differences in radiographic bone density and bone strength were compared for the sham and venous-ligated femurs in wild-type (WT) mice and NOS 3 knockout (KO) mice. Suspension-induced bone loss in the femurs, as evidenced by a loss in radiographic bone mineral density (BMD), was seen in both groups. Differences between sham and venous-ligated femurs were significant only for the WT mice, in which there appeared to be a protective effect of venous ligation against bone loss [-6.69% (ligated) vs. -12.36% (sham), P<0.05]. Furthermore, the difference in bone density between sham and venous-ligated femurs was muted by NOS 3 knockout, suggesting that the protective effect of venous ligation against bone loss observed in the WT group was NO dependent. The differences in relative BMD were mirrored in the mechanical testing experiments, where maximum load to fracture was significantly higher in the venous-ligated limbs relative to the sham limbs of the WT mice, but not in the NOS 3 group. Taken together, these data further support the hypothesis that fluid flow can modulate bone modeling and suggest that IFF-mediated bone adaptation is NOS 3 dependent.

Evidence for shear-induced increase in membrane fluidity in the dinoflagellate Lingulodinium polyedrum.

Fluid shear stress has been demonstrated to affect the structure and function of various cell types. In mammalian cells, it was hypothesized that shear-induced membrane fluidization leads to activation of heterotrimetric G-proteins. The purpose of this study was to determine if a similar mechanism exists in the dinoflagellate Lingulodinium polyedrum, a single-celled eukaryotic aquatic organism that bioluminesces under shear stress. Membrane fluidity changes in L. polyedrum were monitored using the molecular rotor 9-(dicyanovinyl)-julolidine, whose fluorescence intensity changes inversely with membrane fluidity. Dual-staining with 9-(dicyanovinyl)-julolidine and the membrane dye 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate indicates membrane localization. Subjecting L. polyedrum cells to increasing shear stress reversibly decreased 9-(dicyanovinyl)-julolidine fluorescence, while autofluorescence of the cytoplasmic chlorophyll did not change. The relationship between shear stress (0.63 Pa, 1.25 Pa, 1.88 Pa, and 2.5 Pa) and membrane fluidity changes was linear and dose-dependent with a 12% increase in fluidity at 2.5 Pa. To further explore this mechanism a membrane fluidizing agent, dimethyl sulfoxide was added. Dimethyl sulfoxide decreased 9-(dicyanovinyl)-julolidine emission by 41+/-15% and elicited a dose-dependent bioluminescent response at concentrations of 0.2%, 0.5%, 1.0%, and 1.25%. This study demonstrates a link between fluid shear stress and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of dinoflagellates.

Analysis of temporal shear stress gradients during the onset phase of flow over a backward-facing step.

Endothelial cells in blood vessels are exposed to bloodflow and thus fluid shear stress. In arterial bifurcations and stenoses, disturbed flow causes zones of recirculation and stagnation, which are associated with both spatial and temporal gradients of shear stress. Such gradients have been linked to the generation of atherosclerotic plaques. For in-vitro studies of endothelial cell responses, the sudden-expansion flow chamber has been widely used and described. A two-dimensional numerical simulation of the onset phase of flow through the chamber was performed. The wall shear stress action on the bottom plate was computed as a function of time and distance from the sudden expansion. The results showed that depending on the time for the flow to be established, significant temporal gradients occurred close to the second stagnation point of flow. Slowly ramping the flow over 15 s instead of 200 ms reduces the temporal gradients by a factor of 300, while spatial gradients are reduced by 23 percent. Thus, the effects of spatial and temporal gradients can be observed separately. In experiments on endothelial cells, disturbed flow stimulated cell proliferation only when flow onset was sudden. The spatial patterns of proliferation rate match the exposure to temporal gradients. This study provides information on the dynamics of spatial and temporal gradients to which the cells are exposed in a sudden-expansion flow chamber and relates them to changes in the onset phase of flow.

New fluorescent probes for the measurement of cell membrane viscosity.

BACKGROUND: Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS: Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS: The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence.

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.

Issues of threshold selection when determining the fractal dimension in HRCT slices of lumbar vertebrae.

The box counting dimension is a frequently applied tool for the classification of trabecular bone structure. The algorithm requires a binarization of the gray value data, for example that acquired by high resolution CT (HRCT). We recently proposed a method to eliminate bone mineral density (BMD) by applying a linear normalization scheme. Further consideration has shown that full BMD independence has not been achieved, and the structural parameter proposed was therefore difficult to interpret. In this study we present an alternative approach to obtain a structural parameter that is independent of BMD. HRCT volume data was acquired on 21 lumbar vertebrae from five cadavers. In the segmented spongiosa, thresholding was based on different quantiles of the gray value histogram, yielding invariance over linear and non-linear transformations. Thresholding at high gray value levels (80% quantile) shows the highest level of significance when discriminating between osteoporotic and non-osteoporotic cases. As an addition to the measurement of BMD alone, the determination of structural properties allows an improvement of the assessment of the individual fracture risk.

CT determination of bone mineral density and structural investigations on the axial skeleton for estimating the osteoporosis-related fracture risk by means of a risk score.

In addition to the pure measurement of bone mineral density (BMD) in osteodensitometry, the investigation of bone structure is becoming increasingly important for estimating fracture risk. In a clinical study, a risk score was proposed which separately assesses BMD and structural parameters for spongious and cortical bone and aggregates them into a single diagnostic parameter. In 120 lumbar vertebrae from 40 patients, BMD was determined separately for spongious and cortical bone by means of quantitative CT using a single energy procedure (SE-QCT/85 kV). In addition, structural parameters based on high resolution CT were calculated for the spongiosa and cortical bone. For all patients the number of osteoporosis-related fractures was determined on the entire skeletal system. According to WHO criteria, the patients were subdivided into four groups: 1, normal; 2, osteopenic; 3, osteoporotic without fractures; 4, severely osteoporotic. Weighting factors were determined by means of multivariate least-squares analysis and used to calculate a risk score of all parameters. The ability of the individual parameters and of the sum of discriminate between the individual groups was tested. If one considers the individual parameters (BMD and the fractal structural values for spongious and cortical bone), they allow a statistically significant separation of the four groups, although there is overlapping in the value ranges. In patients with fractures, there was a significant reduction in the cortical mineral density, accompanied by a deterioration in structural properties. The following individual values were obtained (minimum-mean-maximum): spongiosa BMD (mg ml-1), unfractured: 62-112-163, fractured: 9-48-77; cortical BMD (mg ml-1), unfractured: 190-287-405, fractured: 133-191-269; spongiosa structural parameter, unfractured: 0.35-0.73-1.01, fractured: 0.95-1.24-1.58; cortical structural parameter, unfractured: 18-31-65, fractured: 21-44-66. Above 77 mg ml-1 CaHA in the spongiosa and 270 mg ml-1 CaHA in cortical bone, no fractures were observed. By appropriately selecting the weighting factors, the score is free of overlapping between the groups with and without fractures (values: unfractured 1-9-15, fractured 16-21-29). With higher score values, the fracture risk is increasing.

Relationship between structural parameters, bone mineral density and fracture load in lumbar vertebrae, based on high-resolution computed tomography, quantitative computed tomography and compression tests.

Different noninvasive techniques for the assessment of the individual fracture risk in osteoporosis are introduced, and the relation between structural properties of high-resolution computed tomography (HR-CT) images of vertebral bodies, their bone mineral density (BMD) and the fracture load is analyzed. In 24 unfractured lumbar vertebrae with different degrees of demineralization from six specimens, the trabecular and cortical BMD was determined using quantitative CT. A lateral X-ray image revealed the number of fractures in the entire spine. A structural analysis of spongy and cortical bone was performed based on the HR-CT images. In the spongiosa, the fractal dimension was calculated as a function of the threshold value. In the cortical shell, the maximum number of clusters of low BMD was determined at varying threshold values. After the CT measurements the vertebrae were excised and compressed until fractured. On the basis of the spongiosa BMD and the number of fractures, 3 cases were found to be severely osteoporotic; the other 3 cases showed osteopenia. The average fracture loads were determined as 3533 N for the non-osteoporotic cases (range 2602-5802 N) and 1725 N for the osteoporotic cases (range 1311-2490 N). The parameters were determined as follows: average spongiosa BMD 115.2 mg/ml (101.8-135.3 mg/ml) for the non-osteoporotic cases, 46.2 mg/ml (34.8-57.6 mg/ml) for the osteoporotic cases; average cortical BMD 285.1 mg/ml (216.4-361.9 mg/ml) for the non-osteoporotic cases, 136. 1 mg/ml (142.5-215.2 mg/ml) for the osteoporotic cases; spongiosa structure: average 0.5 (range 0.32-0.75) for the non-osteoporotic cases, average 1.05 (range 0.87-1.24) for the osteoporotic cases; cortical structure: average 81 (range 55-104) for the non-osteoporotic cases), average 136 (range 102-159) for the osteoporotic cases. Single parameters (BMD and structure) and weighted sums of these parameters were correlated with the fracture load, resulting in correlation coefficients of r(sBMD) = 0.82 (spongiosa BMD), r(cBMD) = 0.82 (cortical BMD), r(sStr) = -0.75 (spongiosa structure) and r(cStr) = -0.86 (cortical structure). The weighted sum of cortical and spongiosa BMD resulted in r(BMD) = 0.86, of cortical and spongiosa structure in r(Str) = -0.86. A weighted combination of all four parameters correlates with the fracture load at r(4) = 0.89, all correlations being statistically significant (p<0.0001). The four individual parameters show only a slight overlap between non-osteoporotic and osteoporotic subjects. The high correlation of the cortical BMD and the structural parameter in cortical bone indicates the important contribution of the cortical shell to vertebral stability. A weighted sum of multiple parameters results in a higher correlation with the fracture load and does not show an overlap between the two groups. It is best suited to estimate the individual fracture risk. The presented methods are generally applicable in vivo; and allow an improvement of the diagnosis of osteoporosis compared with the measurement of the BMD alone.

Computerized analysis of gray-value profiles in spongy and cortical bone. Clinical experience.

RATIONALE AND OBJECTIVES: Osteoporosis is characterized by a loss of bone mineral density and deterioration of structure. The authors present a structural parameter for the quantitative assessment of osteoporotic changes in vertebral bone. METHODS: In 40 patients without or with known osteoporotic fractures, spongiosa and cortical bone mineral density was measured in lumbar vertebrae 1 to 3 by quantitative CT. Additional axial high-resolution CT slices were obtained for the structural analysis. In the spongiosa, the gray-value profile along a horizontal line in the CT slice was used, whereas in the cortical shell a profile was obtained from the cortical ridge. Both profiles were intersected with a horizontal line of variable position, and the maximum number of intersections was determined. RESULTS: The maximum number of intersections is significantly higher in cases with fractures (spongiosa 48.6, cortical shell 77.3) than in cases without fractures (spongiosa 42.1, cortical shell 62.4). It also correlates with bone mineral density and age. CONCLUSIONS: The presented method shows significantly different numeric results for patients with and without osteoporotic fractures. The analysis is easy to perform and provides additional information on the bone structure that may be used in combination with bone mineral density measurements.

Projecting the sulcal pattern of human brains onto a 2D plane--a new approach using potential theory and MRI.

A new method is introduced to project the sulcal pattern of the brain surface onto a 2D plane. Twin brains are compared against each other using the planar representation. We obtained T1-weighted Flash-3D MRI volumes from 14 male twins (seven monozygotic, seven dizygotic) with 3 mm-thick coronal slices. The projection is based on potential theory: A virtual electrostatic field is calculated between the area of the segmented brain and a surrounding spherical electrode. Field lines starting from each border point of the segmented brain follow the gradient towards the sphere, leading to field line concentrations due to the underlying sulci. The unwrapped sphere surface with the number of field lines per area unit is used as the 2D representation of the sulcal pattern. The resulting brain projections show a distinctive pattern, and a visual assignment of the twin pairs from the unsorted set is possible because of a high similarity of the patterns between twin pairs. Global correlation coefficients for each pair of maps yield significantly higher values for matching monozygotic twin pairs (mean = 20.2, range 12.3-25.6) than for unmatched pairs (mean = 13.0, range 1.1-28.5). As a conclusion, our method allows us to map the location and depth of the sulci on a 2D plane. The resulting maps allow quantitative inter-individual comparisons on the entire brain or parts of the brain surface.

Assessing the degree of osteoporosis in the axial skeleton using the dependence of the fractal dimension on the grey level threshold.

Combining the measurement of bone mineral density (BMD) and the classification of the trabecular structure in cancellous bone improves the estimation of the degree of osteoporosis. A fractal method for the automatic quantitative classification of the trabecular structure in midvertebral slices of lumbar vertebrae is introduced. This method is based on the computation of the fractal dimension (box counting method) for varying binarization thresholds. Radiographic images from 30 lumbar vertebrae and CT images from an additional 16 lumbar vertebrae were analysed by calculating the dimension D in dependency of the threshold value T. The function D(T) was normalized by the average image grey value, eliminating the bone mineral density from the computations. The results show that the images of the lumbar vertebrae have fractal properties, and the function D(T) has a typical behaviour that allows the discrimination of the degree of osteoporosis. With two parameters extracted from the function D(T) the correlation coefficients with BMD were both -79% for the radiographic images, and -93% and -91% for the CT data, respectively.

Evaluation of the cortical structure in high resolution CT images of lumbar vertebrae by analysing low bone mineral density clusters and cortical profiles.

The structural classification of trabecular bone is of considerable clinical importance for the diagnosis of osteoporosis. Assessment of the cortical bone mineral density (BMD) and analysis of cortical structure is necessary because the cortex is also affected by osteoporosis. We describe a method for the automatic classification of the cortex from its structure on high resolution (HR) CT images. The method is based on the distribution of areas with low BMD in the cortex. Two different approaches are presented; one uses the grey scale profile of the cortical ridge, and the other one evaluates the distribution of connected regions (clusters) of low BMD. i.e. areas that lie below a certain threshold value. In HRCT images from 22 lumbar vertebrae, the number of intersections of the cortical intensity profile with a horizontal line was determined at various positions of this threshold line. At a certain position, there was a local maximum in the number of intersections which was 38% higher in the osteoporotic cases. The maximum shows a moderate correlation with the cortical BMD of rni = -0.72 (p < 0.0001). The number nc of connected regions (clusters) with pixel values below a certain threshold value was determined in relation to the threshold value T. The resulting function nc(T) shows a relative maximum slightly below the average grey scale value of the respective image. The curve depends on the degree of osteoporosis: the height of the maximum (i.e. the maximal number of clusters ncmax) allows distinction to be made between different degrees of osteoporosis, and ncmax shows a correlation with the cortical BMD of rnc = -0.84 (p < 0.0001).