Molecular views and measurements of hemostatic processes using atomic force microscopy.

Hemostasis and thrombosis are highly complex and coordinated interfacial responses to vascular injury. In recent years, atomic force microscopy (AFM) has proven to be a very useful approach for studying hemostatic processes under near physiologic conditions. In this report, we review recent progress in the use of AFM for studying hemostatic processes, including molecular level visualization of plasma proteins, protein aggregation and multimer assembly, and structural and morphological details of vascular cells under aqueous conditions. AFM offers opportunities for visualizing surface-dependent molecular and cellular interactions in three dimensions on a nanoscale and for sensitive, picoNewton level, measurements of intermolecular forces. AFM has been used to obtain molecular and sub-molecular, resolution of many biological molecules and assemblies, including coagulation proteins and cell surfaces. Surface-dependent molecular processes including protein adsorption, conformational changes, and subsequent interactions with cellular components have been described. This review outlines the basic principles and utility of AFM for imaging and force measurements, and offers objective perspectives on both the advantages and disadvantages. We focus primarily on molecular level events related to hemostasis and thrombosis, particularly coagulation proteins, and blood platelets, but also explore the use of AFM in force measurements and surface property mapping.

Ionic permeability of the opossum sciatic nerve perineurium, examined using electrophysiological and electron microscopic techniques.

A parallel electrophysiological and electron microscopic study was used to assess the ionic permeability of the sciatic nerve perineurium of the opossum Monodelphis domestica. The electrophysiological method was used to monitor permeability to K(+), followed by combined electron microscopy and X-ray probe analysis to monitor permeability to the electron-dense tracer lanthanum. Isolated but intact nerves were mounted in a 'grease gap' chamber for extracellular measurement of DC potential and compound action potential (CAP). Challenge with 100 mM [K(+)] Ringer was used to assess the K(+) permeability of the perineurium, since a change in DC potential (DeltaDC) under these conditions reflected changes in the axonal resting membrane potential. There was no detectable change in DC potential or CAP to the first K(+) challenge (n=71 nerves) indicating negligible K(+) permeability under control conditions. The inflammatory mediators histamine 0.1-40 mg/ml (1. 3-130 mM), bradykinin (0.1-4.7 mM) and 5HT (serotonin) 0.1-5.0 mg/ml (0.5-23.5 mM) caused no measurable DeltaDC on subsequent challenge with 100 mM [K(+)] Ringer, indicating no effect on perineurial K(+) permeability. In nerves exposed to the bile salt sodium deoxycholate (DOC, 6 min, 4 mM), challenge with elevated K(+) Ringer caused a dose-dependent DeltaDC in the range 10-100 mM [K(+)] (1.67+/-0.17 mV in 100 mM [K(+)], n=20), indicating increased perineurial permeability caused by DOC, but the response was smaller than that previously reported for the frog perineurium. Lanthanum was observed in the outer layers of the perineurium, but was not seen to penetrate the endoneurium in any of the nerves examined (n=51), even after DOC application. This study shows that the combined electrophysiological and electron microscopic technique for monitoring ionic permeability can be applied to mammalian nerve, and suggests that the opossum perineurium is more resistant to tight junction opening by chemical modulators than is the frog perineurium.

Ionic permeability of the frog sciatic nerve perineurium: parallel studies of potassium and lanthanum penetration using electrophysiological and electron microscopic techniques.

The isolated sciatic nerve of the frog Rana temporaria was used for a parallel electrophysiological and electron microscopic examination of the ionic permeability of the perineurium, one component of the blood-nerve barrier. Nerves mounted in a grease-gap chamber for electrophysiological recording showed negligible changes in DC potential (Delta DC) or compound action potential on challenge with 100 mM K(+) Ringer, evidence that the perineurium was tight to K(+). In preparations then fixed and exposed to 5 mM lanthanum in the fixative, and examined in the electron microscope, electron-dense lanthanum deposits were seen between perineurial lamellae, but lanthanum was not detectable within the endoneurium, confirming that the perineurium was also tight to lanthanum. Absence of lanthanum penetration was confirmed by X-ray analysis of electron microscopic sections. In nerves exposed to 2 mM sodium deoxycholate (DOC) in the recording chamber, then challenged with high [K(+)], a moderate increase in perineurial K(+) permeability (P(K)) was observed, but lanthanum was still excluded. Exposure of nerves to 4 mM DOC caused a greater increase in perineurial potassium permeability, and the two nerves with the greatest permeability (P(K) > 1 x 10(-5) cm x sec(-1)) also showed detectable lanthanum within the endoneurium. The results indicate that DOC causes a dose-dependent increase in tight junctional permeability in the perineurium, and that the electrophysiological monitoring of K(+) penetration is a more sensitive measure of small ion permeability than electron microscopical analysis using lanthanum as tracer. Vesicular profiles observed in perineurial lamellae did not form open channels for ion flux across the perineurium in control nerves, or in those exposed to DOC. In preparations where lanthanum reached the endoneurium, lanthanum was observed in dense deposits in the extracellular spaces around nodes of Ranvier, and in the outer mesaxon cleft, but did not penetrate the internodal periaxonal space, the myelin intraperiod line, or the Schmidt-Lanterman incisures, in contrast to observations in mammalian nerves. The apparent differences in accessibility of the internodal periaxonal space in frog and mammalian axons are discussed in relation to axonal physiology. The study illustrates the value of parallel electrophysiological and electron microscopic examination in elucidating the properties of extracellular ionic pathways and their role in neural function.

Early fungal endocarditis in homograft recipients.

Retrospective analysis of 200 homograft valve recipients at our institution revealed two cases of fungal endocarditis. Pathogenesis appears to be related to either recipient seeding in one elderly immunocompromised patient or a previously contaminated donor valve implanted in an otherwise healthy recipient. Therefore, our experience underscores the need for both meticulous prevention of fungal infection preoperatively in the recipient and elimination of previously contaminated homograft valves from the donor pool.

Polyurethane foams: effects of specimen size when determining cushioning stiffness.

Polyurethane (PU) foams are used as inexpensive materials for reducing interface pressure in a number of rehabilitative applications, particularly seating and prosthetic limb interfaces. Specimens of three different PU foams were cut to four different sizes and compressed according to ASTM protocols to determine their stiffness capabilities. It was found that the test results varied according to the relationship between the size of the test specimen and the test indenter. It is recommended that investigators create a testing situation that reflects their application when determining the cushioning capabilities of these materials.

Management of left ventricular assist device infection with heart transplantation.

BACKGROUND: Left ventricular assist devices (LVADs) are being used as bridges to heart transplantation (HT). Infection of the LVAD in this patient population represents a serious complication, as simple LVAD removal or delaying HT may result in death. To improve outcomes in this group of patients, we performed HT in the presence of LVAD infection. METHODS: Eighteen patients underwent LVAD implantation followed by HT. Ten underwent HT in the absence of LVAD infection (group 1); and 8, in the presence of LVAD infection (group 2). All patients were treated similarly except for modification of immunosuppression in group 2 patients. RESULTS: Infectious and noninfectious complications were equivalent between the two groups. There was no difference between groups in regard to intraoperative deaths (one versus none), long-term survival (8/10 versus 7/8), wound complications (three versus none), and mean length of hospital stay after HT (21 versus 26 days). CONCLUSIONS: Patients with LVAD infection are too seriously ill to allow LVAD removal or delay of HT. Transplantation in the face of infection is an effective treatment option.

Effects of the bile salt sodium deoxycholate, protamine, and inflammatory mediators on the potassium permeability of the frog nerve perineurium.

An electrophysiological method was used to measure the potassium permeability (PK) of the perineurium of the sciatic nerve of frogs Rana temporaria and R. pipiens. Isolated but intact nerves were mounted in a grease-gap chamber, and compound action potential and DC potential monitored. Change in the DC potential (delta DC) in response to challenge with 100 mM [K+] Ringer was used to assess the K+ permeability of the perineurium, since change in DC potential under these conditions reflected changes in the axonal resting potential. The permeability of the perineurium was calculated from the published calibration curve relating delta DC to bathing [K+] in desheathed nerves of Abbott et al. (1997). In the control condition, PK was < 1.1 x 10(-6) cm.s-1. The bile salt sodium deoxycholate (DOC, 1-4 mM) caused a dose-dependent increase in PK, which reached a maximum of 1.7 x 10(-5) cm.s-1 after 2-min exposure to 4 mM DOC, but access of K+ to the endoneurial compartment was more restricted after DOC than after desheathing. Protamine phosphate (1 mM) and protamine sulphate (0.1-5 mg/ml equals 0.125-6.25 mM) had no effect on PK. Neither histamine (0.4-40 mg/ml), bradykinin (0.1-5 mg/ml) nor serotonin (5-hydroxytryptamine, 0.1-5 mg/ml) affected PK. The frog nerve perineurium appears to be relatively insensitive to chemical agents and inflammatory mediators, in contrast to the endothelial cells forming the endoneurial blood-nerve barrier and the blood-brain barrier.

Standard criteria for an acceptable donor heart are restricting heart transplantation.

BACKGROUND: The lack of satisfactory donor organs limits heart transplantation. The purpose of this study was to determine whether the criteria for suitability of donors may be safely expanded. METHODS: One hundred ninety-six heart transplantations were performed on 192 patients at our institution from January 1992 to 1995 and were divided into two groups. Group A donors (n = 113) conformed to the standard criteria. Group B donors (n = 83) deviated by at least one factor and consisted of the following: 16 hearts from donors greater than 50 years of age, 33 with myocardial dysfunction (echocardiographic ejection fraction = 0.35 +/- 0.10, dopamine level exceeding 20 micrograms.kg-1.min-1, and resuscitation with triiodothyronine), 33 undersized donors with donor to recipient weight ratios of 0.45 +/- 0.04, 48 with extended ischemic times of 297.4 +/- 53.6 minutes, 25 with positive blood cultures, 16 with positive hepatitis C antibody titers, and 7 with conduction abnormalities (Wolff-Parkinson-White syndrome, prolonged QT interval, bifascicular block). RESULTS: Thirty-day mortality was 6.2% (7/113) in group A and 6.0% (5/83) in group B. Mortality in group A was attributed to 3 patients with myocardial dysfunction, 2 with infection, 1 with acute rejection, and 1 with pancreatitis; group B had 2 with myocardial dysfunction, 1 with infection, 1 with aspiration, and 1 with bowel infarction. At 12 months, survival and hemodynamic indices were similar between the groups. Of the 16 recipients with hepatitis C-positive hearts, 5 have become hepatitis C positive with mild hepatitis (follow up, 6 to 30 months). CONCLUSIONS: Expanding the criteria for suitability of donor hearts dramatically increases the number of transplantations without compromising recipient outcome.

Increased activation of the coagulation and fibrinolytic systems leads to hemorrhagic complications during left ventricular assist implantation.

BACKGROUND: Left ventricular assist devices (LVADs) have provided a new therapeutic option for patients with end-stage heart failure. Despite advances in device design, there remains an apparent bleeding diathesis, which leads to increased transfusion requirements and reoperative rates. The purpose of our study was to examine the abnormalities that might contribute to these clinical sequelae. METHODS AND RESULTS: To separate the effects of cardiopulmonary bypass (CPB), eight patients undergoing coronary revascularization (CABG) were compared with seven LVAD (TCI HeartMate) recipients intraoperatively and 2 hours postoperatively. We evaluated several well-characterized indexes of platelet activation: platelet count, platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), and thromboxane B2 (TXB2). We also measured activation of thrombin: thrombin-antithrombin III (TAT), prothrombin fragment 1 + 2 (F1 + 2), and fibrinopeptide A (FPA) as well as markers of fibrinolysis: plasmin-alpha 2-antiplasmin (PAP) and D-dimer. Patterns of intraoperative platelet adhesion and activation were not statistically different in the CABG control and LVAD groups. In the immediate postoperative period, however, there was significant release of PF4 and beta-TG and generation of TXB2. Compared with the CABG controls (TAT, 26 +/- 8 micrograms/L; F1 + 2, 4 +/- 1 nmol/L; mean +/- SEM), there was a significant increase in TAT (380 +/- 112 micrograms/L) and F1 + 2 (23 +/- 4 nmol/L) in LVAD patients 2 hours after surgery. Furthermore, a sharp rise in FPA was noted 20 minutes after LVAD initiation (CABG, 8 +/- 4 ng/mL; LVAD, 235 +/- 63 ng/mL; P < .05). A concomitant increase in both PAP (CABG, 987 +/- 129 micrograms/L; LVAD 3456 +/- 721 micrograms/L; P < .05) and D-dimer (CABG, 1678 +/- 416 ng/mL; LVAD, 15243 +/- 4682 ng/mL; P < .05) was observed. CONCLUSIONS: The additive effects of CPB and LVAD lead to platelet activation as well as elevation of markers of in vivo thrombin generation, fibrinogen cleavage, and fibrinolytic activity. The etiology of these findings may be secondary to the LVAD surface, flow characteristics, and/or operative procedure. Nevertheless, platelet alterations and exaggerated activation of the coagulation and fibrinolytic systems may contribute to the clinically observed hemostatic defect.

A VISUAL BASIC program to pre-process MRI data for finite element modeling.

Investigators use non-invasive imaging to collect geometric data for finite element models. A preprocessor is described to facilitate model generation of anatomical regions from serial Magnetic Resonance Imaging (MRI) data stored in a bitmap format. The MRI Data Transfer System is a stand-alone Windows-based program developed in VISUAL BASIC 3.0 which generates a NASTRAN input file. The program can be modified to generate input files for other solvers. The software will executive on any IBM-compatible computer which runs Windows Version 3.1 or higher. To demonstrate the software, model generation of a portion of a tibia is described.

Three-dimensional computer model of the human buttocks, in vivo.

In an effort to reduce the incidence of decubitus ulcers among wheelchair users, current work in cushion design concentrates on minimizing the pressure at the buttock-cushion interface. Finite element analysis can show the stress levels throughout the soft tissue between the cushion and the ischial tuberosity and give designers a better indication of the effects of a particular cushion. Finite element models were generated of the tissues around the ischial tuberosities of male and female subjects. Linear three-dimensional models were generated using a 386 computer and solved with infinitesimal deflection theory. The resulting minimal principal stresses were 17 kPa and 15 kPa at the buttock-cushion interface for seated male and female subjects, respectively. Computational results were verified experimentally with magnetic resonance imaging and interface pressure measurements.

Rotavirus infection in a small community.

Serial titres of rotavirus specific IgG and IgM have been measured in children and adults living in a small community over a 2 1/4-year period. In all age groups the mean titres of rotavirus specific IgG and IgM rose and fell in parallel with the changes in frequency of gastroenteritis symptoms in the community but after the time when respiratory symptoms reached their peak. Gastroenteritis symptoms were seen most commonly in the children but were also frequent in adults, especially the women. Titres of rotavirus specific IgG changed with age, increasing through childhood into early adult life, but decreased thereafter only to increase again in those over the age of 50 years. Females had higher levels of IgG in all age groups but especially among the children and 30-49-year-old women. The high levels of IgG did not protect the young adults from symptomatic gastroenteritis. Detectable levels of rotavirus specific IgM occurred in all age groups but more commonly in children aged under 10 years and in young adults. Raised levels of IgM were uncommon in the elderly, who rarely suffered gastroenteritis symptoms. An epidemiological model is proposed in which the older members of the community act as a reservoir of rotavirus, passing the infection to the children, who then infect the young adults.

Isolation of rotavirus from calves, foals, dogs and cats in New Zealand.

The incidence of rotaviruses in calves, foals, dogs and cats in the Dunedin urban and rural areas was investigated using electron microscopy and enzyme-linked immunosorbent assays. Of the 283 faecal specimens examined, 26% were positive for rotavirus. Comparison of the genetic electropherotypes was made by separating the viral dsRNA segments using polyacrylamide gel electrophoresis. It is possible that rotavirus infection is a zoonotic disease.

Rotavirus infection in New Zealand.

Rotavirus infection is commonly found in young infants admitted to hospital with gastroenteritis. An enzyme-linked immunosorbent assay (ELISA) for virus diagnosis is described and the results of testing stool specimens from 497 children with gastroenteritis, 192 neonates and 247 asymptomatic six month old infants are presented. Rotavirus infection was found in 45 percent of all children with gastroenteritis but only in 4.7 percent of neonates and 2 percent of asymptomatic infants. These results do not support the proposal that children in our community have a high incidence of subclinical infections.

Rotavirus infection in Otago: a serological study.

A method for measuring rotavirus antibody in human sera has been established using enzyme-linked immunosorbent assay (ELISA). A Simian strain of rotavirus (SA11) was used as the antigen. Serum eluted from dried blood spots on good quality chromatography paper was found suitable for analysis. Paired serum samples from children with gastroenteritis have shown a brisk antibody response in association with the presence of rotavirus in the faeces. Community studies indicate that although all older children and adults tested have detectable antibodies to rotavirus, there is a significant rise in the number of individuals with high titre antibody in the child bearing age group, after which the levels diminish. This finding suggests that repeated infections occur throughout childhood and early adult life.