Improved Tissue Corrosion of Vascular Casts: A Quantitative Filtration Method Used to Compare Tissue Corrosion in Various Concentrations of Sodium and Potassium Hydroxide

In this study, we compare weights of filter retained material (11 μm particle retention) after solubilization and filtration of unfixed, un-perfused tissue (fat, liver and trachea) in sodium and potassium hydroxide (1% , 5%, 10% and 20% weight/volume, w/v) at 8, 24 and 48 hour time points at 45°C. Three detergents [1% Triton-X-100 (volume/volume, v/v), 1% 7X (v/v), 1% Terg-A-Zyme (w/v)] used in combination with hydroxide were evaluated for use in solubilizing fat. Additionally, vascular casts from mouse kidneys were corroded to test the practical effectiveness of corroding solutions on resin infused tissue. Five percent KOH for eight hours proved to be the most effective concentration and time required to corrode fatty tissue. Liver tissue was corroded most rapidly in 1% to 5% NaOH or in 1% to 20% KOH. Corrosion of trachea tissue showed that 5, 10 and 20% hydroxide (NaOH or KOH) are equally effective after 8 hours of corrosion. Use of detergents improved solubilization of fat when combined with 2% , 3% or 5% NaOH. However, fatty tissue was solubilized more rapidly without the use of detergents in 1% NaOH. Scanning electron microscopy of vascular casts shows that corrosion in 1% NaOH appears equally as effective as corrosion in 15% KOH

Correlative Light and Electron Microscopy of Platelet Adhesion and Fibrinogen Receptor Expression using Colloidal-Gold Labeling

Differences in the shape change responses of platelets to various polymers may determine the thrombotic potential of these materials. Substrate-dependent variation in the expression and motility of the platelet fibrinogen receptor may underlie these differences due to this ligand\u27s essential role in platelet aggregation. In this study we examine platelet activation on polyetherurethaneureas (PEUUs) presently being evaulated for vascular prosthetic applications. These polymers are prepared as 50-100nm thin films suitable as substrates for consecutive light microscopy, high voltage electron microscopy (HVEM), and SEM. 18nm colloidal gold coupled to fibrinogen permits visualization of that receptor\u27s motility in living cells by video-enhanced light microscopy. Subsequent HVEM and SEM of identified cells provides correlative ultrastructure and surface morphology. The use of these novel support films coupled with the multiple modes of microscopy and colloidal gold labeled ligands permits in depth study of the molecular biology of cell adhesion to materials with varied, and known, surface properties. The motility of the platelet fibrinogen receptor was related to the extent of cytoskeletal reorganization, which, in turn, was influenced by polymer surface energetics. Platelets adherent to more hydrophobic PEUUs had greater receptor mobility and receptor redistribution than platelets adherent to more hydrophilic PEUUs. The most extensive receptor motility and redistribution was observed on Formvar, a non-PEUU with low surface-water energy, suggesting that additional surface properties are of importance in determining platelet spreading and fibrinogen receptor motility

Expression of Major Histocompatibility Complex Antigens on Macrophages: Correlative Study Using Flow Cytometry, Radioimmunoassay, and Colloidal Gold Immunolabeling

Correlative scanning electron microscopy (SEM), radioimmunoassay (RIA), and flow cytometric analysis were used to characterize levels of class I and class II major histocompatibility complex-encoded (MHC) antigen expression on peritoneal exudate cells of mice chronically infected with Chlamydia psittaci. Analysis of peritoneal macrophages by all three techniques revealed a marked induction of H-2 K,D (class I) and I-A, l-E (class II) antigens on cells from infected C3H mice when compared to uninfected controls. Scanning electron micrographs further document that the increases in class I and II MHC antigens are due to an increase in la/H-2 bearing cells as well as an increase in MHC molecules/cell. These immune macrophages have a flattened morphology, almost completely devoid of the membrane ruffles and villi which are characteristic of control peritoneal macrophages. These studies suggest that while both flow cytometry and RIA can provide an accurate quantitative estimate of antigen expression in a cell population, the immunogold labeling technique can allow visualization of individual cells and additional analysis of the topographical distribution of cell surface antigens

Surface Characterization of Biomaterials by Immunogold Staining - Quantitative Analysis

The labeling of target proteins by immunogold particles has been analyzed based on Einstein\u27s law of Brownian motion. The theory was confirmed from the experiments which employed antifibrinogen gold markers to label fibrinogen molecules adsorbed on the polyethylene surface. The theory predicts that the degree of labeling depends on the concentration of gold markers, temperature, medium viscosity, size of gold markers, and staining time. Of these factors most important is the concentration of immunogold particles. Small change in the marker concentration results in a significant variation in the staining efficiency when other variables are kept constant. The effect of temperature is always accompanied with that of the medium viscosity. There is a linear relationship between the degree of labeling and the temperature when the viscosity effect is combined. The staining of fibrinogen molecules adsorbed on the polyethylene surface at three different temperatures shows a temperature dependence which is in close agreement with the theory. The degree of labeling is inversely related to a square root of the size of gold markers. This analysis makes it possible to maximize the staining sensitivity and to improve the reproducibility of the labeling. Thus, the immunogold staining under a well defined condition allows quantification as well as positive identification and localization of target proteins. This technique has been used to study protein adsorption on biomaterials

Vascular Changes in Popliteal Lymph Nodes due to Antigen Challenge in Normal and Lethally Irradiated Mice

The microvascular system of the murine popliteal lymph node was investigated using scanning electron microscopy of microcorrosion casts. Time-dependent changes in the microvasculature following regional antigen challenge in normal and lymphocyte-depleted mice were studied. Normal lymph node microvasculature exhibited a significant increase in both the vascular bed and post-capillary venules containing high-endothelium in response to antigen challenge. Lymph nodes of lymphocyte-depleted mice showed no microvascular size increase following antigen challenge and a reduction in the amount of high-endothelium was observed

Labeling of Sweet Taste Binding Sites using a Colloidal Gold-Labeled Sweet Protein, Thaumatin

Thaumatin, an intensely sweet tasting protein, was bound to colloidal gold and applied to the taste bud-bearing foliate papillae of Rhesus monkeys. Examination of thin sections of taste pores showed that gold particles were bound to merocrine secretions of Type I taste bud cells, to some cell remnants of lysed cells, and, most importantly, to small, membrane bounded blebs of cytoplasm. These blebs are thought to be shed into the pore from the tips of taste bud cell microvilli, particularly those arising from Type II cells. The binding of gold particles to microvillus tips and to the blebs suggest that this may be an important means by which taste bud cells rid themselves of taste stimulus-receptor complexes

Ex vivo Platelet Deposition on Fibronectin-Preadsorbed Surfaces

Temporal platelet deposition profiles of canine plasma fibronectin (CPFN) adsorbed to different polymers ex vivo and the in vitro characteristics of CPFN adsorption were studied in an attempt to correlate the two. The maximum platelet deposition (pltmax) obtained at a protein preadsorption time of 30 min was greater than that obtained using an adsorption time of 120 min for all surfaces studied. At 30 min of preadsorption, pltmax was 520, 560 and 1230 platelets/1000 μm2 on Biomer, polyethylene (PE) and oxidized PE (OXPE), respectively. In contrast, the platelet deposition at 120 min. of fibronectin preadsorption was about 60-90 platelets/1000 μm2 on all polymers studied. The surface concentrations of adsorbed CPFN measured using 125I-CPFN, were in the order PE\u3e OXPE \u3e Biomer. The adsorbed protein concentration increased with increasing adsorption time. The surface distribution of adsorbed CPFN was visualized with antibody-labelled colloidal gold and scanning electron microscopy. The extent of staining was lowest on PE, greater on Biomer, and highest on OXPE, roughly similar to the order of platelet deposition. Platelet deposition ex vivo appears to correlate with the irnmunogold-stainable-adsorbed protein rather than with the total amount of adsorbed protein

Nanometer Scale Dielectric Fluctuations at the Glass Transition

Using non-contact scanning probe microscopy (SPM) techniques, dielectric properties were studied on 50 nanometer length scales in poly-vinyl-acetate (PVAc) films in the vicinity of the glass transition. Low frequency (1/f) noise observed in the measurements, was shown to arise from thermal fluctuations of the electric polarization. Anomalous variations observed in the noise spectrum provide direct evidence for cooperative nano-regions with heterogeneous kinetics. The cooperative length scale was determined. Heterogeneity was long-lived only well below the glass transition for faster than average processes.Comment: 4 pages, 4 embedded PS figures, RevTeX - To appear in Phys. Rev. Let

High Amplitude Phase Resetting in Rev-Erbα/Per1 Double Mutant Mice

Over time, organisms developed various strategies to adapt to their environment. Circadian clocks are thought to have evolved to adjust to the predictable rhythms of the light-dark cycle caused by the rotation of the Earth around its own axis. The rhythms these clocks generate persist even in the absence of environmental cues with a period of about 24 hours. To tick in time, they continuously synchronize themselves to the prevailing photoperiod by appropriate phase shifts. In this study, we disrupted two molecular components of the mammalian circadian oscillator, Rev-Erbα and Period1 (Per1). We found that mice lacking these genes displayed robust circadian rhythms with significantly shorter periods under constant darkness conditions. Strikingly, they showed high amplitude resetting in response to a brief light pulse at the end of their subjective night phase, which is rare in mammals. Surprisingly, Cry1, a clock component not inducible by light in mammals, became slightly inducible in these mice. Taken together, Rev-Erbα and Per1 may be part of a mechanism preventing drastic phase shifts in mammals

Interactions of polymorphisms in different clock genes associated with circadian phenotypes in humans

Several studies have shown that mutations and polymorphisms in clock genes are associated with abnormal circadian parameters in humans and also with more subtle non-pathological phenotypes like chronotypes. However, there have been conflicting results, and none of these studies analyzed the combined effects of more than one clock gene. Up to date, association studies in humans have focused on the analysis of only one clock gene per study. Since these genes encode proteins that physically interact with each other, combinations of polymorphisms in different clock genes could have a synergistic or an inhibitory effect upon circadian phenotypes. In the present study, we analyzed the combined effects of four polymorphisms in four clock genes (Per2, Per3, Clock and Bmal1) in people with extreme diurnal preferences (morning or evening). We found that a specific combination of polymorphisms in these genes is more frequent in people who have a morning preference for activity and there is a different combination in individuals with an evening preference for activity. Taken together, these results show that it is possible to detect clock gene interactions associated with human circadian phenotypes and bring an innovative idea of building a clock gene variation map that may be applied to human circadian biology