Uptake, intra-axonal transport and fate of horseradish peroxidase in embryonic spinal neurons of the chick.

Horseradish peroxidase (HRP) was injected in ovo into the ventral muscle mass of the hind limb of 5- to 7-day-old chick embryos or into the gastrocnemius muscle of 8- to 18-day embryos and localized histochemically. HRP is extensively incorporated via endocytosis into axonal growth cones or presynaptic terminals in the proximity of the injection site. Much of the tracer is taken up in vesicles and small vacuoles. Most of these are smooth-surfaced and only a few are bristle-coated. A small amount of the tracer is also incorporated into the axon terminal through the openings between the axolemma and an intricate membrane channel. The majority of the tracer-laden vesicles and vacuoles rapidly fuse with one another to become large vacuoles, some of which are transformed into multivesicular bodies (MVBs). In axon shafts, many labeled vacuoles and MVBs are transferred to tubule-like organelles, which appear to be the primary carrier for transporting the tracer back to the cell bodies in the lumbar spinal cord. HRP arrives in the sensory ganglia about 0.5-1 hour earlier than in the motoneurons of the lateral motor column. The maximal rate of the retrograde axoplasmic transport is about 3.5 mm/hour. After arriving in the cell bodies, HRP is transferred from tubule-like organelles to discrete vacuoles of various sizes and appearance. Lysosomal dense bodies and HRP-labeled vacuoles can be distinguished ultrastructurally. A fusion of HRP-labeled vacuoles with lysosomal dense bodies or Golgi vesicles was occasionally observed and the density of HRP-labeled vacuoles diminished after 2 to 3 days. Most of the HRP-labeled organelles were found to contain acid phosphatase activity. Therefore, the complete disappearance of HRP by 4 days postinjection is most likely related to lysosomal degradation. Neuronal cell bodies diffusely labeled with HRP were only observed prior to day 6. After day 6, despite various attempts to injure the peripheral axons, only granularly labeled cell bodies were found. This difference may imply that "mature" neurons have a more efficient mechanism for the sequestration of "free" HRP in the cytoplasmic matrix into membrane-bounded organelles. A mature-like retrograde transport mechanism appears to exist at the earliest stages of axonal growth in vivo.

Some aspects of synaptogenesis in the spinal cord of the chick embryo: a quantitative electron microscopic study.

We have quantitatively examined the development of synapses in the ventral part of the lumbar spinal cord of the chick from embryonic day 4 until adulthood. The first synapses occur on day 4 and are of the axo-dendritic type; they are invariably located adjacent to the border between the intermediate and marginal zones. Initially there are more synapses in the presumptive white matter than in the motoneuron neuropil, but this trend is later reversed; however, we found numerous axo-dendritic synapses throughout much of the ventrolateral white matter even in the adult stage. The first axo-dendritic synapses always contain spherical synaptic vesicles and have symmetric membrane specilizations. By day 7 a few of these synapses were found to have mixed populations of spherical and flattened vesicles and asymmetric membrane specilizations. After hatching there are still considerably more axo-dendritic synapses with symmetric membrane specializations. Axo-somatic synapses were first found on embryonic day 6 and were typically located on motoneurons lying adjacent to the marginal zone. These axo-somatic synapses contain a few spherical synaptic vesicles and have symmetric membrane densities. Flattened synaptic vesicles were first found on day 10 and increased throughout development. Although a few axo-somatic synapses with asymmetric membrane specializations were found at practically all stages, the symmetric type was always in the majority. An attempt was made to relate these observations with physiological, behavioral and neuroembryological findings from birds and other forms. For example, the fact that axo-dendritic synapses always appear prior to axo-somatic contacts would seem to rule out the role of somatic synapsesin the initial induction of dendritic growth in the spinal cord.

Platelet-derived microparticles on synthetic surfaces observed by atomic force microscopy and fluorescence microscopy.

Platelet activation on a thrombogenic surface includes the release of membrane-derived microparticles that provide catalytic sites for blood coagulation factors. Here, we describe a quantitative investigation on the production and dimensions of platelet-derived microparticles observed on glass and polyethylene under aqueous conditions, using atomic force microscopy (AFM) and complementary fluorescence microscopy. The results show that contact-activated platelet microparticles are not evenly distributed over a thrombogenic surface, but in clusters in close proximity to adherent platelets. The microparticles are localized near the platelet periphery, and in some cases appear to emanate from platelet pseudopodia, suggesting that formation may result from vesiculation of the pseudopodia. The microparticles measured 125 +/- 21 nm (n = 73) in the x-y dimensions and 5.2 +/- 3.6 nm in height. The results compared closely with 125 +/- 22 nm width and 4.1 +/- 1.6 nm height obtained for control preparations of thrombin activated microparticles, that were filtered and deposited on glass. Large differences between the measured widths and heights of adsorbed microparticles suggest that platelet microparticles may undergo spreading after attachment to a surface. The adsorbed microparticles expressed platelet membrane receptor GPIIb/IIIa, and many expressed the platelet activation marker P-selectin as determined by fluorescence microscopy. The high number distribution of procoagulant microparticles per unit area of surface compared with platelets suggests that platelet-derived microparticles provide a mechanistic route for amplifying thrombus formation on a thrombogenic surface.

Vitamin K reduces bleeding in left ventricular assist device recipients.

BACKGROUND: Despite advances in left ventricular assist device (LVAD) design that permit support without anticoagulation, LVAD recipients often suffer profound bleeding complications. This bleeding diathesis may be attributable to pre-operative right-ventricular failure with concomitant hepatic dysfunction. The purpose of this study was to characterize coagulation abnormalities in LVAD recipients and determine the impact of pre-operative vitamin K administration on the incidence of postoperative bleeding. METHODS: Hemostatic and liver function profiles were obtained in 66 recipients of the Heartmate LVAD; 39 of these patients received perioperative vitamin K. RESULTS: During LVAD support, hepatic synthetic function improved as evidenced by increases in clotting factors II, V, VII, XI. There was ongoing fibrinolysis with elevation of fibrinopeptide A and D-dimers and diminution of fibrinogen; however, plasminogen levels did not decline suggesting that systemic disseminated intravascular coagulation (DIC) did not occur. Bleeding requiring re-exploration more than 48 hours postimplantation occurred in 9 of 66 patients (13.6%). Prior to implantation, patients that bled had decreased levels of factor II (52.2 +/- 27.1% vs 69.7 +/- 26.6%; p = 0.048) and prolonged prothrombin times (16.5 +/- 2.4 seconds vs 13.8 +/- 3.1 seconds; p = 0.005) compared to patients that did not bleed. Seven of 27 patients (25.9%) not treated with vitamin K bled, while only 2 of 39 (5.1%) patients treated with vitamin K required re-exploration for bleeding (p = 0.026). CONCLUSIONS: We conclude that: (1) Liver synthetic function improves during LVAD support resulting in increased levels of circulating coagulation factors; (2) ongoing fibrinolysis occurs but likely only represents remodeling of fibrin on the LVAD surface; (3) perioperative vitamin K reduces nonsurgical bleeding in LVAD recipients.

Ultrastructure of migrating spinal motoneurons in anuran larvae.

Motoneurons in the course of migration to the lateral motor column (LMC) were identified by horseradish peroxidase (HRP) applied to lumbar ventral roots in anuran larvae (Rana catesbeiana). The ultrastructural characteristics of these migrating motoneurons were compared with motoneurons in the LMC that had completed their migration. Both migrating and LMC motoneurons tended to be bipolar with one process extending toward the ependyma and the other out towards the ventral root. Typically, centrally and peripherally directed processes in both migrating and LMC motoneurons contained many microtubules, mitochondria and rosette-like clusters of ribosomes, as did the soma. Four of the five types of synapses found on adult frog motoneurons were also found on migrating motoneurons in tadpoles. Radial glia, whose cell bodies are located in the ependyma, have processes extending to the spinal cord periphery. In less than 10% of the preparations, radial glia were labeled with the HRP reaction product. These labeled glia were further distinguished from migrating motoneurons at the ultrastructural level by the presence of abundant filaments.

Fine structure of tarsal sensory organs in the whip spider Admetus pumilio (Amblypygi, Arachnida).

The sensory organs on the tarsi of the antenniform first legs of the whip spider Admetus pumilio C. L. Koch (Amblypygi, Arachnida) were examined with the scanning and transmission electron microscope. At least four different types of hair sensilla were found: (1) thick-walled bristles, which have the characteristics of contact chemoreceptors (several chemoreceptive dendrites in the lumen plus two mechanoreceptors at the base); (2) short club sensilla, innervated by 4-6 neurons which terminate in a pore on the tip; they are possibly humidity receptors; (3) porous sensilla, which are either innervated by 20-25 neurons and have typical pore tubules, or they have 40-45 neurons but no pore tubules; both types are considered to be olfactory; (4) rod sensilla occur in clusters near segmental borders; they are innervated by only one large dendrite which branches inside the lumen. Other tarsal receptors are the claws, which correspond to contact chemoreceptors, and the pit organ which resembles the tarsal organ of spiders. Compared to other arthropod sensilla, the contact chemoreceptors are very similar to those of spiders, while the porous sensilla correspond structurally to olfactory receptors in insects; the club and rod sensilla seem to be typical for amblypygids.

Hemostatic profiles of HeartMate ventricular assist device recipients.

Candidates for ventricular assist devices often have hepatic dysfunction and concomitant coagulation abnormalities. Factors II, V, VII, XI, plasminogen, fibrinopeptide A (FpA), and D-dimers were measured in 19 HeartMate (ThermoCardiosystems, Inc., Woburn, MA) patients before device implantation; at 6 hr, 24 hr, and 2 weeks postimplantation; and before explantation. Ten patients had entry hepatic dysfunction (total bilirubin > 2 mg/dl; aspartate and alanine aminotransferases > 60 U/L); nine had normal hepatic function. All except one patient received perioperative aprotinin; all received only aspirin and dipyridamole after surgery. At preimplant, both patient groups had subnormal factor II, V, VII, XI, and plasminogen with elevated FpA and D-dimer. By 2 weeks postimplant, these factor levels had normalized, except for FpA and D-dimer levels, which suggest ongoing remodeling of fibrin deposits on the device surfaces. No statistically significant differences in the assayed hemostatic markers were observed between the two patient groups. Clinically, 15/19 (79%) patients survived to cardiac transplantation; 3/19 (16%) patients required reoperation for early bleeding. All three had low factor VII and XI; two of three also had hepatic dysfunction and subnormal levels of factor II and V. Most patients with entry hepatic dysfunction improve after device implantation; all four deaths were in patients with persistent hepatic dysfunction despite circulatory support.

Platelet-mediated adhesion of Staphylococcus epidermidis to hydrophobic NHLBI reference polyethylene.

The effects of platelets and plasma proteins on the adhesion of Staphylococcus epidermidis strain RP62A to hydrophobic NHLBI reference polyethylene was quantitatively studied using a rotating disk system to generate well-defined shear conditions simulating the hemodynamics of human blood circulation. Bacterial adhesion was quantified by adhesive coefficient, the percentage of bacteria transported to the surface that becomes adherent. The results showed that surface modification by adsorption of plasma proteins reduced the adhesion of S epidermidis as compared to the bare polymer surface. This surface modification was not sufficient to eliminate completely bacterial adhesion, even at the highest physiologic shear stress level. S epidermidis did adhere strongly to polyethylene surface modified by platelets. This is readily evident as approximately 50% of the adherent S epidermidis were bound to contact-activated platelets which occupied only 4% of the surface area. Adhesive coefficients to platelets were significantly greater than to the protein-adsorbed polyethylene surface by at least one order of magnitude (P < or = .01) across the range of physiological shear conditions investigated. These studies show that it is biologic surface modification by contact-activated platelets, and not plasma proteins, which mediates S epidermidis adhesion to polyethylene.

Staphylococcus epidermidis adhesion to hydrophobic biomedical polymer is mediated by platelets.

A quantitative investigation on the effects of plasma proteins and platelets on the adhesion of Staphylococcus epidermidis RP62A to a hydrophobic biomedical polymer (National Heart, Lung, and Blood Institute reference polyethylene) was carried out under well-defined shear conditions approximating human blood circulation by using a rotating disk system. The results showed that contact-activated platelets mediated S. epidermidis adhesion to the polymer surface. In the range of physiologic shear conditions, the adhesive coefficient (ratio of bacteria per unit area to the product of bacterial flux and the duration of the experiment) to platelets was significantly greater than to the protein-adsorbed polyethylene surface by at least one order of magnitude (P < or = .01). The presence of absorbed plasma proteins on polyethylene reduced the adhesion of S. epidermidis compared with that seen with the bare polymer surface. These studies show that S. epidermidis adhesion to polyethylene is mediated by contact-activated platelets, not absorbed plasma proteins.

Adhesion of Staphylococcus epidermidis to biomedical polymers: contributions of surface thermodynamics and hemodynamic shear conditions.

Adhesion studies of Staphylococcus epidermidis RP62A were conducted using a rotating disk system to determine the roles of surface physicochemistry and topographies under physiologic shear conditions. Six materials were investigated: biomedical reference polyethylene and polydimethylsiloxane; argon plasma-treated reference polyethylene (Ar-PE); Silastic; expanded polytetrafluoroethylene; and woven Dacron. All of the polymers except Dacron demonstrated reduced bacterial adhesion with increasing shear stress. Argon plasma treatment of polyethylene reduced the level of staphylococcal adhesion. Adsorption of human plasma proteins effected significantly lower numbers of adherent bacteria. The lowest adhesion was observed for Ar-PE in 1% human plasma protein solution, whereas Dacron had the highest number of adherent bacteria. The high adhesion on Dacron was attributed to increased bacterial flux caused by topography-induced turbulent flow and physical entrapment of the bacteria in the fiber interstices. The results indicate that the driving force for S. epidermidis adhesion is strongly influenced by substrate physicochemistry, but this may be dominated by physical forces such as shear and turbulence.

Adhesion of Staphylococcus epidermidis and transposon mutant strains to hydrophobic polyethylene.

Staphylococcus epidermidis capsular polysaccharide adhesin (PS/A) and slime were studied as possible mediators of bacterial adhesion to NHLBI polyethylene (PE) under dynamic flow. This putative interaction was examined by quantifying the adhesion of M187 (PS/A+, slime+) parent strain and isogenic transposon mutant strain sn3 (PS/A-, slime-) to polyethylene (PE) under a range of physiologic shear stress conditions in both phosphate-buffered saline (PBS) and 1% platelet poor plasma (PPP). No significant differences in adhesion were noted between the M187 and sn3 strains in either test medium. However, adhesion of both strains in 1% PPP was decreased 75-95% compared to adhesion in PBS. In PBS, adhesion was shear stress dependent from 0-15 dyne/cm2, after which adhesion was comparatively shear stress independent. Adhesion in 1% PPP was independent of shear stress. Epifluorescent imaging of both strains labeled for slime confirmed the presence of slime on the surface of M187 and suggested that PS/A and slime promote the formation of large aggregates, as aggregates were totally absent in the images of the sn3 strain. The results suggest that PS/A and slime do not mediate S. epidermidis adhesion to bare PE or PE with adsorbed plasma proteins, but may be necessary for intercellular adhesion, which is important for biofilm formation.