High Resolution Strain Analysis Comparing Aorta and Abdominal Aortic Aneurysm with Real Time Three Dimensional Speckle Tracking Ultrasound.

OBJECTIVE/BACKGROUND: Ultrasound measurement of aortic diameter for aneurysm screening allows supervision of aneurysm growth. Additional biomechanical analysis of wall motion and aneurysm deformation can supply information about individual elastic properties and the pathological state of the aortic wall. Local aortic wall motion was analyzed through imaged aortic segments according to age and pathology. METHODS: Sixty-five patients were examined with a commercial four dimensional ultrasound system (4D-US). Three groups were defined: patients with normal aortic diameter and younger than 60 years of age (n = 21); those with normal aortic diameter and older than 60 years of age (n = 25); and those with infrarenal aortic aneurysm (n = 19). A diastolic reference shape of aortic wall segments was obtained and local and temporally resolved wall strain was determined. Indices characterizing the resulting wall strain distribution were determined. RESULTS: The analysis of biomechanical properties displayed increasing heterogeneous and dyssynchronous circumferential strain with increasing patient age. Young patients exhibited higher mean strain amplitude. The distribution of the spatial heterogeneity index and local strain ratio was inversely proportional to age. The maximum local strain amplitude was significantly higher in the young (0.26 ± 0.17) compared with the old (0.16 ± 0.07) or aneurysmal aorta (0.16 ± 0.10). Temporal dyssynchrony significantly differed between young (0.13 ± 0.10) and old (aneurysmal 0.31 ± 0.04, non-aneurysmal 0.29 ± 0.05), regardless of aortic diameter. The spatial heterogeneity index and local strain ratio differentiate non-aneurysmal and aneurysmal aorta, regardless of age. CONCLUSIONS: 4D-US strain imaging enables description of individual wall motion (kinematics) of the infrarenal aorta with high spatial and temporal resolution. Functional differences between young, old, and aneurysmal aorta can be described by mean (circumferential) strain amplitude, the spatial heterogeneity index, and the local strain ratio. Further investigation is required to refine this new perspective of patient individualized characterization of the pathological AAA wall and eventually to rupture risk stratification.

Quantitative reflection contrast microscopy of living cells.

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.

Adhesion induced expression of the serine/threonine kinase Fnk in human macrophages.

Members of the polo subfamily of protein kinases play crucial roles in cell proliferation. To study the function of this family in more detail, we isolated the cDNA of human Fnk (FGF-inducible kinase) which codes for a serine/threonine kinase of 646 aa. Despite the homology to the proliferation-associated polo-like kinase (Plk), tissue distribution of Fnk transcripts and expression kinetics differed clearly. In contrast to Plk no correlation between cell proliferation and Fnk gene expression was found. Instead high levels of Fnk mRNA were detectable in blood cells undergoing adhesion. The transition of monocytes from peripheral blood to matrix bound macrophages was accompanied by increasing levels of Fnk with time in culture. Neither treatment of monocytes with inducers of differentiation nor withdrawal of serum did influence Fnk mRNA levels significantly, suggesting that cell attachment triggers the onset of Fnk gene transcription. The idea that Fnk is part of the signalling network controlling cellular adhesion was supported by the analysis of the cytoplasmic distribution of the Fnk protein and the influence of its overexpression on the cellular architecture. Fnk as fusion protein with GFP localized at the cellular membrane in COS cells. Dysregulated Fnk gene expression disrupted the cellular f-actin network and induced a spherical morphology. Furthermore, Fnk binds to the Ca2+/integrin-binding protein Cib in two-hybrid-analyses and co-immunoprecipitation in assays. Moreover, both proteins were shown to co-localize in mammalian cells. The homology of Cib with calmodulin and with calcineurin B suggests that Cib might be a regulatory subunit of polo-like kinases.

Dimethylaminostyrylmethylpyridiniumiodine (daspmi) as a fluorescent probe for mitochondria in situ.

An investigation has been made on the properties of dimethylaminostyrylmethylpyridiniumiodine (DASPMI), its reaction with isolated pigeon heart mitochondria and its suitability as a vital strain for mitochondria in situ. DASPMI is a low toxicity specific vital stain for mitochondria in living cells. In vitro dye concentrations over 6 nmol/mg protein inhibit fast (state 3) respiration after a preincubation time of more than 5 min in the presence of substrate. No uncoupling was observed. Energization of pigeon heart mitochondria by addition of ATP or various substrated yields an average 8.5-fold increase in fluorescence intensity in relation to DASPMI-stained mitochondria that are under anoxia, substrate deficiency, or under the influence of respiratory inhibitors, or uncouplers. The alterations in fluorescence intensity are not primarily due to ion movements or pH changes. The amount of dye (2.96+/-0.8 nmol) yielding maximal fluorescence response with 1 mg mitochondrial protein remains constant during energization of mitochondria. As indicated by electron microscopic studies the observed changes in emission intensity may be related to changes in the fine structural organisation of cristae. A remarkable difference exists between isolated mitochondria and mitochondria in situ with respect to the reaction to cyanide. According to the reported results DASPMI will be a useful probe for the investigation of mitochondrial activities in living cells.

Mechanics of crawling cells.

Crawling of keratocytes derived from aquatic vertebrates represents a very useful model system for the investigation of cell locomotion because of its ease of handling and the clear structural separation of a thin cytoplasmic layer, the lamella, from the cell body containing the nucleus and other organelles. Spreading of spherical keratocytes results in fried egg shaped cells, which on withdrawing their lamella at one side become polarized and start moving. Hydrostatic pressure, tension at the cortex, traction forces exerted on the adhesion sites and inside the cells along filamentous structures are required to gain a certain shape. Traction forces have been made visible using scanning acoustic microscopy. This method also allowed for the demonstration of cytoplasmic fluxes inside a moving keratocyte and changes of forces while a migrating cell is changing its direction of locomotion. The pros and cons for actin polymerization at the leading front providing the driving force for crawling are discussed on the basis of structural and experimental results: do they stringently identify polymerization of actin as the only driving machinery. Such a mechanism not only should explain the advancement of the leading edge but also the movement of the whole cell, i.e. the material flux taking place from the cell body to the periphery. Even if the lamella periphery itself may be motile by actin turnover this scheme may represent an oversimplification if applied to the whole cell. Considering the complexity of a whole cell simplifying model systems may not lead to adequate descriptions of the mechanisms as they occur within cells with a highly complex structure, although the model might be consistent and sufficient to describe, i.e. crawling in general.

Sequestration of iontophoretically injected calcium by living endothelial cells.

Sequestration of iontophoretically injected Ca2+ by monolayer culture cells (primary Xenopus laevis Tadpole Heart cells, XTH P, and an established cell line, XTH 2) is investigated. Injections are made at different velocities by changing the influx current. On Ca2+ injection the entire ER desintegrates, and near to the tip of the injecting pipette microtubules depolymerize. The time required to attain cell death is taken as the parameter indicating an overload of cellular Ca2+ sequestration capability. Three different Ca2+ transport kinetics are found: at Ca2+ flux rates of up to 20 X 10(-15) mol X s-1 (condition I) cells can tolerate long injection periods before they die; at flux rates from 20 to 40 X 10(-15) mol X s-1 (condition II) the injection time before cell death remains constant. Flux rates exceeding 40 X 10(-15) mol X s-1 decrease cellular Ca2+ sequestration capability to a minimum. These observations support the assumption of two Ca2+ sequestrating mechanisms: one of high affinity, but with low capacity (less than = 5 X 10(-15) mol X s-1) the other with low affinity for Ca2+ and a high capacity (10 to 40 X 10(-15) mol X s-1) for Ca2+ accumulation. Both mechanisms are saturable. As the Ca2+ sequestration velocity remains approximately constant in condition II, the capacity of the second mechanism seems to grow with increasing Ca2+ influx. The highly affin Ca2+ compartment is the ER, mitochondria form the less affin system. XTH 2 differ from primary cells by possessing a 5 to 8 fold higher Ca2+ sequestration capacity, whereas sequestration velocity is equal in both cell types.

The distribution of Tyr- and Glu-microtubules during fish scale regeneration.

Cyclic rearrangements of the microtubules (Mts) occur in the hyposquamal scleroblasts which synthesize the highly ordered three-dimensional collagen network forming the basal plate of the fish scale. The distribution of Mts containing tyrosinated and detyrosinated alpha-tubulin (Tyr-Mts and Glu-Mts, respectively) was analyzed in relation to the frequency of Mt reorganization in the teleostean elasmoid scale during collagen deposition using two specific monoclonal antibodies. In the very flat hyposquamal scleroblasts of fully developed scales (with a very low synthetic activity) two microtubule populations were identified. Most contain Tyr-tubulin while Glu-tubulin is found in some "stable" Mts. In the tall prismatic scleroblasts of regenerating scales (active in collagen synthesis) only Tyr-Mts have been revealed. In late stage of regeneration, when the synthetic activity decreases and the scleroblasts flatten, an increasing centriole and Mt labeling with Glu-tubulin antibody was observed. The intimate relationship of intracellular microtubule arrangement and extracellular collagen fibril pattern reported earlier (Zylberberg et al., Cell Tissue Res. 253, 597-603 (1988], together with the results presented here, support the hypothesis that a changing Mt pattern is involved in the generation of the collagen plywood.

Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.

Signaling of mechanical stretch in human keratinocytes via MAP kinases.

Cells within human skin are permanently exposed to mechanical stretching. Here we present evidence that alterations in cell shape trigger biochemical signaling via MAP kinases in human keratinocytes. In an in vitro attempt we demonstrate a fast but transient activation of extracellular signal-regulated kinases 1/2 in response to cell stretch. This activation is reversed by preincubation with functional blocking antibodies directed towards beta1-integrins. As a second member of MAP kinases, stress-activated protein kinase/c-JUN NH2-terminal kinase was activated in a slower fashion, peaking at 1 h after the initial stimulus. The delay in signal transmission suggests that extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase do not share the same signaling pathway. p38 was not activated by cell stretching. The contribution of cytoskeletal elements in signal perception and transduction was evaluated by selective disruption of either actin filaments, microtubules, or keratin filaments but showed no clear effect on stretch-induced activation of extracellular signal-regulated kinases 1/2 and stress-activated protein kinase/c-JUN NH2-terminal kinase. In conclusion we found evidence of a cell-shape-dependent activation of MAP kinases in human keratinocytes disclosing beta1-integrins as putative mechano-transducers. It is likely that alterations of skin mechanics in vivo underlying pathogenic processes like wound formation and healing trigger physiologic responses via the MAP kinase pathway.

Induction of RANTES, HLA-DR, and intercellular adhesion molecule-1 on highly purified distal tubular cells from human kidney.

BACKGROUND: Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. METHODS: Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. RESULTS: Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. CONCLUSIONS: We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.

In vitro analysis of verapamil-induced immunosuppression: potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells.

BACKGROUND: Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS: A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS: Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS: The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.

Subcellular tension fields and mechanical resistance of the lamella front related to the direction of locomotion.

Keratocytes derived from the epidermis of aquatic vertebrates are now widely used for investigation of the mechanism of cell locomotion. One of the main topics under discussion is the question of driving force development and concomitantly subcellular force distribution. Do cells move by actin polymerization-driven extension of the lamella, or is the lamella edge extended at regions of weakness by a flow of cytoplasm generated by hydrostatic pressure? Thus, elasticity changes were followed and the stiffness of the leading front of the lamella was manipulated by local application of phalloidin and cytochalasin D (CD). In scanning acoustic microscopy (SAM), elasticity is revealed from the propagation velocity of longitudinal sound waves (1 GHz). The lateral resolution of SAM is in the micrometer range. Using this method, subcellular tension fields with different stiffnesses (elasticity) can be determined. A typical pattern of subcellular stiffness distribution is related to the direction of migration. Cells forced to change their direction of movement by exposure to DC electric fields of varying polarity alter their pattern of subcellular stiffness in relationship to the new direction. The cells spread into the direction of low stiffness and retract at zones of high stiffness. The pattern of subcellular stiffness distribution reveals force distribution in migrating cells; i.e., if a cell moves exactly in a direction perpendicular to its long axis, then the contractile forces are largest along the long axis and decrease toward the short axis. Locomotion in any angle oblique to this axis requires an asymmetric stiffness distribution. Inhibition of actomyosin contractions by La3+ (2 mM), which inhibits Ca2+ influx, reduces cytoplasmic stiffness accompanied by an immediate cessation of locomotion and a change of cell shape. Local release of CD in front of a progressing lamella activates a cell to follow the CD gradient: The lamella thickens locally and is extended toward the tip of the microcapillary. Release of phalloidin stops extension of the lamella, and the cell turns away from the releasing microcapillary. The response to CD is assumed to be the result of local weakening of the cytoplasm due to severing of the actin fibrils. Phalloidin is supposed to stabilize the leading front by inhibition of F-actin depolymerization. These observations are in favor of the assumption that migration is due to an extension of the cell into the direction of minimum stiffness, and they are consistent with the hypothesis that local release of hydrostatic pressure provides the driving force for the flux of cytoplasm.

Cell contraction caused by microtubule disruption is accompanied by shape changes and an increased elasticity measured by scanning acoustic microscopy.

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.

Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria.

Mitochondria are semi-autonomous organelles which are endowed with the ability to change their shape (e.g., by elongation, shortening, branching, buckling, swelling) and their location inside a living cell. In addition they may fuse or divide. These dynamics are discussed. Dislocation of mitochondria may result from their interaction with elements of the cytoskeleton, with microtubules in particular, and from processes intrinsic to the mitochondria themselves. Morphological criteria and differences in the fate of some mitochondria argue for the presence of more than one mitochondrial population in some animal cells. Whether these reflect genetic differences remains obscure. Emphasis is laid on the methods for visualizing mitochondria in cells and following their behaviour. Fluorescence methods provide unique possibilities because of their high resolving power and because some of the mitochondria-specific fluorochromes can be used to reveal the membrane potential. Fusion and fission often occur in short time intervals within the same group of mitochondria. At sites of fusion of two mitochondria material of the inner membrane, the matrix compartment seems to accumulate. The original arrangement of the fusion partners is maintained for some minutes. Fission is a dynamic event which, like fusion, in most cases observed in vertebrate cell cultures is not a straight forward process but rather requires several "trials" until the division finally occurs. Regarding fusion and fission hitherto unpublished phase contrast micrographs, and electron micrographs have been included.

A new model of epidermal differentiation: induction by mechanical stimulation.

In vivo, epidermal cells are committed to terminal differentiation in which they undergo a series of morphological and biochemical changes. In vitro, keratinocytes are able to undergo some steps of this differentiation process only. In view of the fact that in vivo skin is continuously subjected to mechanical stress, we investigated the stimulation of differentiation of transformed keratinocytes by mechanical stimulation. The cells, grown in plastic culture dishes, were periodically treated with weights exerting a pressure of 0.015 Ncm-2. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, 3H-thymidine and 14C-amino acid incorporation, SDS polyacrylamide gel electrophoresis, and Western blotting. Following pressure treatment, the previously monolayered keratinocytes locally grew up to several layers, the number of horny scales increased and, after 4 days, the pattern of cytokeratin was modified. The total amount of keratin increased, forming granular accumulations, while the proliferation rate of the cells decreased. Both the 67 kDa and 49.5 kDa keratin subunits increased in stimulated cells. Moreover, a weak keratin band of 44 kDa appeared that was not present in controls. The results demonstrate that cyclic pressure promotes differentiation of cultivated epidermal cells.

Cocultures of fetal and adult cardiomyocytes yield rhythmically beating rod shaped heart cells from adult rats.

Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7-10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior.

An improved method for the isolation of cells from tissues: prevention of cell rounding enhances spreading and locomotion of keratinocytes (epitheliocytes).

Benzamide (10mM) in Ca-free solution anesthetizes small cold blooded animals such as tadpoles of Rana temporaria, Xenopus laevis and an aquarium fish, guppy (Lebistes [Poecilia] reticulatus). Trypsinization of tails of tadpoles in Ca-free, benzamide-supplemented solution permits isolation of keratinocytes (epitheliocytes) maintaining polygonal shapes and prevents cell rounding. Such cells quickly attach to glass, spread and commence locomotion in contrast to those cells which assume spherical shape after standard trypsinization.

Comparative study on effects of cytochalasins B and D on F-actin content in different cell lines and different culture conditions.

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.

The role of mitochondria in apoptosis induced in vitro.

Cell death remains the focus of in vitro toxicology. Xenobiotics are capable of bringing about two types of cell death: apoptosis and necrosis. From our previous study we know that cells treated with xenobiotics showed very dynamic changes in their morphology, particularly vigorous movement of the plasma membrane. Such changes probably depend on adequate energy supply. This observation stands in contradiction with published data showing that generation of ATP in mitochondria is altered very early in apoptosis. In this study we analysed the relationship between mitochondrial activity and cell death induced by Etoposide, a selective inhibitor of topoisomerase II, treatment (10 microg/ml). As a model system we used stabilised cell line Hep2. Several markers of apoptosis, including typical cell morphology and DNA ladder formation were measured. The dynamics of morphological changes was recorded by the time-lapse videomicroscopy. We measured mitochondrial membrane potential with a specific fluorochrome DASPMI, quantification was done by microfluorometric assessment. Our data show that mitochondrial activity was maintained during the first 6 hours after the treatment with Etoposide, at the same time substantial changes in cell morphology as well as typical DNA fragmentation were observed.

Advanced 3D-Sonographic Imaging as a Precise Technique to Evaluate Tumor Volume.

Determination of tumor volume in subcutaneously inoculated xenograft models is a standard procedure for clinical and preclinical evaluation of tumor response to treatment. Practitioners frequently use a hands-on caliper method in conjunction with a simplified formula to assess tumor volume. Non-invasive and more precise techniques as investigation by MR or (µ)CT exist but come with various adverse effects in terms of radiation, complex setup or elevated cost of investigations. Therefore, we propose an advanced three-dimensional sonographic imaging technique to determine small tumor volumes in xenografts with high precision and minimized observer variability. We present a study on xenograft carcinoma tumors from which volumes and shapes were calculated with the standard caliper method as well as with a clinically available three-dimensional ultrasound scanner and subsequent processing software. Statistical analysis reveals the suitability of this non-invasive approach for the purpose of a quick and precise calculation of tumor volume in small rodents.