Biomedical subjects

R M Albrecht

Publications and source records attributed to R M Albrecht.

At least 55 records · Page 3Linked to original sources

PubMed1990

Advances in corrosion casting methods.

This paper briefly discusses the concept of corrosion cast preparation (primarily of blood vessels), the use of the scanning electron microscope (SEM) to study these casts and the observations which can be made, together with the merits and the limitations in various applications. A number of reviews and surveys are quoted in which the different injection media, injection methods, animal preparations and corrosion procedures are described. A new procedure of cleaning the corrosion casts with sodium hydroxide and Triton X-100 is described. The observations which can be made are listed and illustrated both on the cellular level as well as in organ systems as a whole. The discussion centers around some common misconceptions, the feasibility in various applications and the limitations of the method. The conclusion is that the method has proven to be useful especially in conjunction with other methods. Moreover, while the concept of the method may be very straight-forward the approach and the interpretation often need careful consideration and might not be as straight-forward as one tends to expect from the simple sounding principle.

Animals↗

PubMed1989

Platelet shape change and cytoskeletal reorganization on polyurethaneureas.

Understanding how platelet activation responses are affected by polymers having varied surface physicochemical properties can lead to improved materials for vascular applications. The in vitro responses of human platelets were studied upon adherence to four polyurethaneureas with different soft segments, as well as to Biomer, and to Formvar. Platelets were observed by video-enhanced light microscopy (VLM) as they adhered to polymer films. Platelets were subsequently prepared for high-voltage transmission electron microscopy (HVEM) to view the cytoskeleton and other ultrastructural features. Scanning electron microscopy (SEM) was then used to characterize cell surface morphology and to survey platelet populations. Shape change and cytoskeletal reorganization differed on the various surfaces. The extent of shape change and cytoskeletal reorganization was related to polyurethane surface energetic properties. While the most extensive shape change was observed on the hydrophilic and polar Formvar surface, the least shape change was observed on a polyethylene oxide soft segment polyurethane with similar surface-water energetic properties. Therefore properties other than surface-water energetics must be involved in determining platelet responses to different classes of polymers. HVEM also showed that cytoskeletal reorganization proceeded to completion only on Formvar. Polyurethane adherent platelets, although appearing fully spread by SEM or VLM, never exhibited complete cytoskeletal reorganization.

Biocompatible Materials↗

PubMed1989

Carnitine femoral arterial-venous differences in the stressed critically ill.

Femoral arterial and venous carnitine concentrations from critically ill patients were measured in order to determine if the large urinary carnitine excretions seen in these patients was associated with a net loss of carnitine from skeletal muscle. Bloods were drawn two or three times during the 7-day study period. A 24-hr urine sample was obtained on the same day. The arterial-venous difference for free carnitine plus short chain acylcarnitine was -2.8 +/- 0.9 microM (means +/- SEM), and -2.7 +/- 1.0 microM for total carnitine. Both values were significantly less than zero (p less than 0.05). Median urinary free carnitine excretion was 1237 mumol/day while the median acylcarnitine excretion was 544 mumol/day. We conclude that skeletal muscle in these patients is in negative carnitine balance, and is at least one source of the increase in carnitine excretion seen in critically ill patients.

Adult↗

PubMed1989

Immunoelectron microscopic localization of fibronectin in adherence of Staphylococcus aureus to cultured bovine endothelial cells.

Fibronectin binds to Staphylococcus aureus and may have a role in mediating its adherence to host tissue. Fibronectin was localized ultrastructurally on S. aureus in suspension and in interactions with cultured bovine endothelial cells. Probes were constructed by adsorbing fibronectin or its antibodies to colloidal gold beads (FN-Au or aFN-Au, respectively). Sites of fibronectin binding to S. aureus were demonstrated by reacting FN-Au with S. aureus in suspension. Transmission electron microscopy showed that FN-Au localized uniformly over the surface of S. aureus in suspension; most localized within 65 nm of the cell wall. The distribution of aFN-Au on S. aureus adherent to endothelial cells was concentrated in areas between S. aureus and endothelial cells. Areas of S. aureus not facing endothelial cells bound few aFN-Au. This suggests that the fibronectin labeled by aFN-Au in areas between S. aureus and endothelial cells was involved in adherence of the S. aureus, consistent with a role for fibronectin in endocarditis.

Animals↗

PubMed1989

Probe size and bound label conformation in colloidal gold-ligand labels and gold-immunolabels.

Colloidal gold can be produced in sizes ranging from 1.0nm to 150nm. All sizes of gold can be conjugated, principally by hydrophobic bonding, to a variety of molecules including ligands, enzymes and antibodies, as well as lectins and polysaccharides. The activity of most of these biological molecules is retained on conjugation with gold particles irregardless of size range, although the ratio of protein surface area to gold particle surface area varies widely depending on particle and protein size. We have employed low voltage high resolution scanning electron microscopy to compare, microscopically, the shapes of biological molecules unbound, bound to very small (3nm) gold particles, and bound to larger (18nm-30nm) gold particles. When very small gold particles are conjugated to large protein molecules, several particles bind along the length of each molecule, while smaller protein molecules often wrap around a single small gold particle. With larger gold particles, several biological molecules bind to a single gold particle. In addition, the shape of protein molecules bound to larger gold particles differs from that of molecules bound to small gold particles.

Animals↗

PubMed1989

Polyurethane support films: structure and cellular adhesion.

It is desirable to examine the cytobiology of cell adhesion to the same materials which are contemplated for use in biomedical and biotechnological devices. It is also of fundamental interest to examine adhesion to substrates with properties which are likely to influence adhesion in controlled ways. In many of these applications the materials of choice are polyurethane elastomers due to their physical properties and resistance to biodegradation. Polyurethanes have a two phase microstructure consisting of hydrophilic hard segments and hydrophobic soft segment domains. Variations of both the chemistry and the morphology of these microdomains may be produced. It is well understood that the hydrophilic/hydrophobic nature of surfaces affects cellular adhesion and the adsorption of extracellular proteins. Since polyurethane microdomains have dimensions in the range of 10-100 nm, hence the size of proteins and cell-surface receptors, polyurethane microdomain structure could influence order at the cell-material interface. Polyurethanes may be prepared as thin films with excellent properties for use as specimen supports in High Voltage transmission Electron Microscopy (HVEM) at 1 MeV. This permits the imaging of the cytoskeleton and other internal features of whole mounts of adherent cells, rather than tedious thin sectioning required for conventional TEM. Subsequently the surface morphology of these preparations may be imaged with high resolution SEM. Finally, the polyurethane itself may be stained and imaged by either HVEM or high resolution SEM in order to relate polyurethane micro-morphology to cellular features.

Biocompatible Materials↗

PubMed1989

The evolution of correlative techniques for electron microscopy--an overview.

The papers presented at this Pfefferkorn Conference demonstrate the dramatic recent progress in the science of biological specimen preparation for electron microscopy. This progress results largely from increased use of more diverse, critical, correlative scientific methods. This paper outlines several strategies that tend to promote this type of scientific approach, and that have proven generally useful in biological research. The strategy most commonly chosen to augment both the empirical and the cross-disciplinary components of structural studies is the correlative use of diverse experimental techniques on samples which are parallel to those prepared for microscopy. This type of approach tends to advance our understanding of biological structure and function and also of the scientific methodology. Such approaches redirect attention to the biological problem under study and tend to open new areas of investigation. A second strategy which promotes more rigorous scientific approaches is the application of correlative techniques to identical structures. In contrast with parallel studies, data from identical structures document directly the coincidence of different features within each individual structure, and these data establish the distributions of these features in the study population based on relatively few observations. A third strategy to promote more critical science is to utilize the effects of the specimen preparation as experimental parameters by varying the preparative methods with appropriate controls. This approach is especially valuable in studies of biological specimen preparation, where the potential impact of systematic errors warrants especially rigorous scientific practice.

Animals↗

PubMed1989

Observations of colloidal gold labelled platelet microtubules: high voltage electron microscopy and low voltage-high resolution scanning electron microscopy.

18 nm colloidal gold-antitubulin and 4 nm colloidal gold-antitubulin were used to label microtubules in adherent, fully spread platelets. Both sizes of marker effectively labelled microtubules in the partially extracted platelets. However only the 4 nm gold penetrated the dense microfilament matrix of the inner filamentous zone so that portions of microtubules within this cytoskeletal zone could be tracked. The gold marker could be visualized well with 1 MeV high voltage transmission EM and with 5 kV or greater secondary imaging or 20 kV backscattered imaging of carbon only coated samples. 1 kV secondary imaging permitted high resolution imaging of the surface of tubules and the microfilaments with their respective associated material. Individual gold-antibody complexes were difficult to identify by shape alone due to the tendency of the antibody coats to blend together when in very close approximation and due to the presence of other molecules or molecular aggregates similar in size to the gold-antibody labels. Microtubules were seen to wind in and out of the inner and outer filamentous zones as they encircled the granulomere. Some tubules were seen to "dead end" at the peripheral web. Numerous smaller microtubule loops were present principally in the outer filamentous zone and tubules could be followed as they went from the outer filamentous zone through the inner filamentous zone and into the granulomere.

Blood Platelets↗

PubMed1988

Increased plasma carnitine in trauma patients given lipid-supplemented total parenteral nutrition.

The purpose of this study was to determine the effects of altering the fuel substrate mix of total parenteral nutrition (TPN) on plasma and urinary carnitine in trauma patients. TPN solutions were either 100% carbohydrate (CHO) based or lipid based (70% CHO, 30% lipid). There were statistically significant (p less than 0.05) increases in plasma levels of free carnitine, short-chain acyl carnitine, and total carnitine in trauma patients receiving lipid-based TPN. No significant differences in urinary carnitine excretion were noted between groups. We conclude that the use of lipids in the TPN of trauma patients leads to an alteration in plasma carnitine metabolism.

Adult↗

PubMed1987

Scanning microscopy of colonic mucin during carcinogenesis: is it clinically applicable?

The integrity of the colonic mucin layer has been reported to be altered during carcinogenesis in both humans and rodents. Prior to attempting scanning microscopic techniques on colonic mucosa of patients at high risk to develop colorectal cancer, these procedures were performed on colonic mucosa from rats with chemically induced colon cancers. Substantial technical difficulties in preparation and serious subjectivity in interpretation of the scanning micrographs were encountered. The major technical problem was the unpredictable retention of the mucin layer upon both normal and cancerous mucosae. Visual interpretation of the integrity or disruption of the mucin layer with the scanning electron microscope revealed variable fenestration and fraying of the mucin in both normal and cancerous colons. Our findings suggest that scanning electron microscopy of colonic mucin may not be a reliable screening procedure for (pre)cancerous changes in human colonic mucosae.

Animals↗

PubMed1987

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 (gamma 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, gamma pltmax was 520,560 and 1230 platelets/1000 micron2 on Biomer, polyethylene (PE) and oxidized PE (OXPE), respectively. In contrast, the platelet deposition at 120 min. of fibronectin preadsorption was about 60 approximately 90 platelets/1000 micron2 on all polymers studied. The surface concentrations of adsorbed CPFN measured using 125I-CPFN, were in the order PE greater than OXPE greater than 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 immunogold-stainable-adsorbed protein rather than with the total amount of adsorbed protein.

Adsorption↗

PubMed1987

Surface characterization of biomaterials by immunogold staining--quantitative analysis.

The labeling of target proteins by immunogold particles has been analyzed based on Einstein's 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.

Adsorption↗

PubMed1987

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.

Animals↗

PubMed1987

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, I-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 Ia/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.

Animals↗

PubMed1987

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's essential role in platelet aggregation. In this study we examine platelet activation on polyetherurethaneureas (PEUUs) presently being evaluated 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's 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.

Biocompatible Materials↗

PubMed1987

Trifluoperazine inhibition of fibrinogen receptor redistribution in surface activated platelets: correlative video-enhanced differential interference contrast light microscopic, high voltage electron microscopic and scanning electron microscopic studies.

Video-enhanced differential interference contrast (VDIC) light microscopy in conjunction with fibrinogen labelled colloidal gold was employed as a probe to follow the mobility of the fibrinogen receptor on platelets. Correlative studies by both high voltage and scanning electron microscopy confirms localization of labels relative to platelet ultrastructural and surface characteristics, respectively. Treatment of platelets with trifluoperazine prior to and after incubation with fibrinogen-gold labels results in a concentration dependent inhibition of receptor movement. The results obtained from this study suggest that phosphorylation of myosin by the Ca++-calmodulin dependent enzyme, myosin-light chain kinase, is important in the fibrinogen redistribution that occurs during platelet activation.

Adult↗

PubMed1987

Colloidal gold labelling of fibrinogen receptors in epinephrine- and ADP-activated platelet suspensions.

It has been generally accepted for over twenty years that epinephrine stimulates platelet aggregation without inducing shape change. However, it has been recently reported that discoid platelets are not recruited into ADP- or epinephrine-stimulated aggregates. Previous work in our laboratory has suggested that platelet shape change is necessary for the binding of fibrinogen to its surface receptor, which is a prerequisite for platelet aggregation. These studies seem to indicate that epinephrine-induced platelet aggregation does involve shape change. To investigate this possibility, the extent of shape change and fibrinogen binding in suspensions of epinephrine- and ADP-activated and control platelets was assessed by scanning electron microscopy (SEM). Platelets were incubated with 20 microM epinephrine, 20 microM ADP, or vehicle and labelled with 18 nm gold beads conjugated to fibrinogen or to a monoclonal antibody directed against the glycoprotein IIb/IIIa complex which comprises the fibrinogen receptor. Results indicate that shape change does occur in epinephrine-activated platelets as well as ADP-activated platelets. Although GP IIb/IIIa was shown to be present on both discoid and shape-changed, pseudopodial platelets, a significant degree of fibrinogen binding did not occur earlier than the pseudopodial stage in either activated or control suspensions. Platelet aggregation studies showed that the majority of platelets involved in aggregates had changed shape in both ADP- and epinephrine-treated platelet suspensions. These studies suggest that epinephrine- and ADP-induced platelet aggregation occurs via the exposure of fibrinogen receptors on shape-changed platelets.

Adenosine Diphosphate↗

PubMed1987

Utilization of immunogold labeling to compare the adsorption behavior of fibrinogen, fibronectin and albumin on polymers.

Immunogold labeling followed by scanning electron microscopy (SEM) was used to examine the surface distribution of adsorbed plasma proteins. Adsorption was performed under various conditions on six different polymers; [low density polyethylene (PE), chromic acid-oxidized PE (OXPE), solution grade Biomer (SB), Teflon-(FEP), a laboratory synthesized polyurethane containing some zwitterions (ZW) and a polydimethylsiloxane based polyurethane (ZS) also containing zwitterions]. The proteins used were purified human and canine fibrinogen, fibronectin, and serum albumin. The immunogold staining technique was successful in the labeling of the adsorbed proteins. The adsorbed proteins were distributed differently on the polymers selected. Human and canine fibrinogen were found to cover all surfaces in a dense, uniform fashion. Albumin covered most surfaces in a less uniform fashion and on the zwitterionomers covered only a portion of the surface, leaving large bare patches. Fibronectin appeared to deposit unevenly, forming a network on part of the surface and uniformly coating other parts.

Adsorption↗