Biomedical subjects
Publications and source records attributed to K Ramberg.
Whereas it has been commonly thought that adding polyethylene oxide PEO to a surface would diminish the capacity of the surface to cause deposition of platelets and of fibrinogen, and to activate complement C3, we present data showing exactly the opposite. These unexpected results are obtained with low molecular weight (2000) PEO, and are not found with higher molecular weight (20,000) PEO.
We investigated the role of pulmonary granulocyte sequestration in the development of early failure of the autoperfused working heart-lung preparation. A significant decline in the total circulating leukocyte count in 21 preparations at 60 minutes of perfusion (5.0 to 1.4 x 10(3)/microL; 28% of baseline; p less than 0.001) was observed. Differential cell counts in 14 of these preparations revealed a predominant decrease in granulocyte count (8.7% of baseline) and a moderate decline in lymphocyte count (46% of baseline). In study I, indium 111-labeled autologous granulocytes were injected intravenously into 10 adult New Zealand White rabbits. In group I (n = 5), an autoperfused working heart-lung preparation was harvested and perfused for 60 minutes. In group II (controls, n = 5), the heart-lung block was harvested following 60 minutes of in situ perfusion. Organ blocks were imaged before and after saline flush. There was a significant decline in granulocyte counts at 60 minutes of perfusion in group I versus no change in group II (I, 2.3 +/- 0.4 to 0.3 +/- 0.1; p less than 0.01; II, 1.7 +/- 0.2 to 2.3 +/- 0.5; not significant; x 10(3)/microL +/- standard error of the mean). Postflush lung activity was significantly increased in group I versus group II (I, 3,751 +/- 566; II, 1,867 +/- 532; p less than 0.05; counts +/- standard error of the mean). In study II, 15 autoperfused preparations were divided into two groups. Group I (n = 10) preparations were controls. Group II (n = 5) animals were depleted of leukocytes by pretreating with nitrogen mustard. Group I (controls) produced a bimodal survival distribution (Ia, 8.2 +/- 1.0; Ib, 26.4 +/- 2.0; hours +/- standard error of the mean). Group II survival was significantly longer than that of group Ia and similar to that of group Ib (II, 25.3 +/- 2.2; p less than 0.01 versus group Ia, not significant versus group Ib; hours +/- standard error of the mean). Hemodynamic profiles for group II closely paralleled those of group Ib. In conclusion, pulmonary sequestration of granulocytes occurs early in the autoperfused working heart-lung preparation (within 60 minutes of autoperfusion), and preoperative leukocyte depletion prolongs survival of the autoperfused working heart-lung preparation by eliminating the subset group Ia (short survivors) seen in untreated preparations.
Endothelial cell (EC) seeding is postulated as a mechanism of improving patency of small calibre vascular grafts. However, the majority of seeded cells are lost within hours following restoration of blood flow. We postulated that incubating EC in-vitro on a graft will improve adherence and resistance to the sheer stresses of pulsatile blood flow. Fibronectin-treated ePTFE (5 cm x 4 mm ID) seeded with Indium-111-labelled autologous canine EC (1.5 x 10(5) cells/cm2) were incubated for four different time periods; 90 min, 24 h, 72 h and 6 days. Incubated grafts were subjected to blood flow of 75 ml/min for 6 h, in a canine ex-vivo arteriovenous shunt circuit. EC retention during perfusion was studied by measuring gamma activity emitted by the grafts. Cell morphology of non-perfused control groups and perfused groups was compared using scanning electron microscopy (SEM). SEM of control grafts showed progressive EC spreading on the ePTFE surface for up to 72 h incubation. Gamma activity was significantly higher at 6 h perfusion in grafts incubated for 72 h (82 +/- 4%) and 24 h (63 +/- 6%) vs. 90 min (34 +/- 13%, p less than 0.05), and between grafts incubated for 72 h vs. 6 days (55 +/- 7%, p less than 0.05). Perfused grafts incubated for 72 h showed unaltered EC morphology on SEM, few cells remained on 90 min incubated grafts. We conclude that incubating EC on fibronectin-treated ePTFE for 72 h in-vitro after seeding improves cell retention during blood flow.
A method for communicating results of health checkups was developed in Sweden. The Diagnosis method includes seven steps, making it possible to examine the working environment. Combined information demonstrates connections between working environment and health. Involving all the employees in changes to improve the working environment simplifies the process.
Indium 111-labeled leukocyte scintigraphy provides an excellent noninvasive means for identifying and localizing infectious and inflammatory sites within the body. This article first compares gallium 67 citrate, the long-established radiopharmaceutical for infection imaging, to 111In-labeled leukocytes, particularly as applied to the chest. 111In-labeled leukocytes require time-consuming preparation, and the technical and practical aspects of cell labeling and imaging are reviewed in detail. The main focus of this article is the application of labeled leukocyte imaging to the evaluation of pleural, mediastinal, cardiovascular, and pulmonary disease.
Hydrogels are a class of synthetic material, composed of a polymer-water matrix and have been proposed as tissue substitutes and drug delivery vehicles. Polyethylene oxide (PEO) hydrogels were synthesized and used to produce coated wires and conduits for baboon blood compatibility studies. Blood-material interactions were studied both by Scanning Electron Microscopy (SEM) and 111In labeled platelet deposition. SEM processing modifications were first evaluated in order to reduce shrinkage and surface distortion incurred during sample preparation of these high water content materials. Pretreatment with 1% tannic acid reduced bulk shrinkage associated with critical point drying by 10-20%. This effect is small, nevertheless, it prevented major sample disruption. Coated guidewires were exposed to baboon blood for one hour in the inferior vena cava and conduits were placed for either 30 or 60 minutes in an ex vivo femoral arteriovenous shunt. Reference materials included Gore-tex, polyethylene and silica-free polydimethyl siloxane (PDMS). In the guidewire studies, 111In labeled platelet levels were highest on Gore-tex (6568.97 platelets/1000 microns 2) and large thrombotic deposits were well visualized by SEM. Formulations containing PEO had low levels of platelet deposition and little evidence of platelet activation was noted by SEM. Shunt studies demonstrated that materials of high PEO content and molecular weight had the lowest levels of platelet deposition. After 60 minutes of blood flow, mean platelet deposition on PDMS and Gore-tex was 50 and 1000 fold higher than on a network composed of 65% PEO 20,000 (p less than 0.05). SEM confirmed these findings.
Polyethylene oxide (PEO), because of its low levels of protein and cellular adsorption, may provide a suitable coating for synthetic small caliber vascular prostheses. PEO/polysiloxane networks were synthesized via an acid-catalyzed epoxy/hydroxyl crosslinking reaction and used to produce conduits with a 4-mm internal diameter. Three networks with nominal PEO molecular weights of 2000, 8000, and 20,000 and all 65% PEO by weight were studied. Blood compatibility was assessed by measuring 111In-platelet and 125I-fibrinogen deposition in a baboon ex vivo shunt, over a 1-hr time period and at a flow rate of 50 ml/min. Differences in material performance were noted particularly after the initial 30-min blood contact period. Materials in the mid and high PEO molecular weight range (8000 and 20,000) had significantly lower levels of platelet adsorption than networks of low PEO molecular weight (2000) at 30 min (P less than 0.005) and 60 min (P less than 0.05). The lowest level of platelet deposition was noted on networks of high PEO molecular weight (20,000). During the observation period, platelet accumulation on this surface was less than one platelet per 1000 microns. Platelet deposition on Gore-Tex was two and three orders of magnitude greater than that on the high molecular weight PEO material at 30 and 60 min, respectively (P less than 0.001). Fibrinogen adsorption was also lower on materials of mid and high PEO molecular weights, when compared with low molecular weight networks (P less than 0.05) and Gore-Tex (P less than 0.05). Scanning electron micrographs confirmed these observations. Overall, platelet and fibrinogen depositions are low for PEO networks, particularly for materials of high PEO molecular weight. This latter observation may be related to increased surface molecular mobility and a relative enhancement of PEO content at the blood-material interface.
Using 111In-labeled autologous platelets, we studied the kinetics of pulmonary platelet deposition and clearance in relation to hemodynamic and structural events during thrombin-induced pulmonary microembolism in rabbits. Autologous platelets were radiolabeled and returned to animals prior to infusion of thrombin (100 units/kg over 15 min) (n = 20) or saline (n = 6). All animals were pretreated with tranexamic acid, an inhibitor of fibrinolysis. Thrombin-treated animals manifested progressive increases in mean pulmonary platelet activity, reaching a maximum of 38% above baseline (p less than .0001), whereas no change was observed in saline-treated controls. Animals that died during, or immediately following, thrombin infusion manifested significantly greater increases in pulmonary platelet uptake (mean 1.55 +/- 0.47 times baseline), compared to surviving animals (1.14 +/- 0.16; p less than .05 survivors vs. nonsurvivors). In surviving animals, following cessation of thrombin, pulmonary platelet activity cleared gradually, with a half-time of approximately 12 min. Thrombin reduced circulating platelet counts (p less than .001), increased mean pulmonary artery pressure (13 +/- 3 mm Hg to 18 +/- 6 mm Hg; p less than .0001), and reduced mean systemic arterial pressure (55 +/- 10 mm Hg to 44 +/- 7 mm Hg; p less than .001). The time courses of these events approximated that of thrombin-induced pulmonary platelet uptake. Furthermore, the increase in pulmonary artery pressure occurred predominantly in the group of animals in which the increase in pulmonary radiolabeled platelet activity exceeded the median value of 20%. Postmortem histology showed extensive pulmonary thrombus extending from small arterial to capillary levels in animals that died during, or immediately following, thrombin infusion, but not in surviving animals. Our findings suggest that platelet aggregation plays an important role in the pathogenesis of hemodynamic change following thrombin-induced pulmonary embolization.
Advances in venous reconstruction have been limited by inherent venous thrombogenicity and the absence of a suitable prosthetic material for use in the venous system. We have designed an in vivo experimental model to evaluate early blood-material interactions within the venous system and to quantitate drug efficacy in the alteration of platelet function and fibrin deposition in the baboon. An 8F catheter was placed percutaneously in the femoral vein of an adult male baboon. Indium 111-labeled autogenous platelets or iodine 125-labeled human fibrinogen was infused before the introduction, into the inferior vena cava, of a linear array of 5 x 15 mm alternating Dacron and polytetrafluoroethylene samples attached to a benzalkonium-heparin-treated guide wire. At 60 or 120 minutes the samples were removed and a 1 ml aliquot of blood was drawn. The materials and blood samples were counted in a gamma well counter, and the material counts were normalized to the circulating label present in the 1 ml blood sample. The experiment was repeated after pretreatment with heparin, aspirin, or dextran. Whole blood clotting times and bleeding times were monitored. The results showed decreased platelet and fibrin deposition on polytetrafluoroethylene when compared with Dacron in the venous system. Aspirin, heparin, and dextran were all found to decrease platelet and fibrin deposition onto intravenously placed graft material samples (p less than 0.05, Student's t test). The data confirm the ability of the model to evaluate quantitatively anticoagulants, antiplatelet agents, and prospective graft materials for use in venous reconstructions.
Dacron (polyester fiber), a stimulus to platelet aggregation in vitro, accumulates platelets to a greater extent in vivo than autogenous artery, polytetrafluoroethylene (PTFE) or human umbilical vein (HUV). We conducted a series of experiments using the ex vivo shunt in the baboon to determine whether or not systemic activation of platelet function was produced by a Dacron graft. Two 5 centimeter segments of 4 millimeter internal diameter graft materials were placed in series in the ex vivo shunt perfused at 25 milliliters per minute flow rate for two and one-half hours. Deposition of autologous Indium 111 labeled platelets was monitored. The ex vivo shunt procedures were divided into two groups, both with PTFE as the proximal graft: one with a distal Dacron graft (n = 21), the second with PTFE or HUV distally (n = 17). In this study, an increase in platelet deposition on the proximal PTFE graft represents systemic platelet activation caused by the distal graft. Increased platelet deposition on PTFE was noted at all time points in the presence of a Dacron graft (p less than 0.05). This property of Dacron has important clinical implications, potentially accelerating the progression of vascular disease, increasing the failure rate of composite grafts and subsequent arterial reconstruction.
Low molecular weight dextran (LMD) has recently been shown in a randomized, multicenter trial to improve early patency in difficult lower extremity vascular bypass. The question remained as to whether this effect was due to plasma volume expansion by LMD or to its effects on platelets and coagulation. To analyze this we have studied the effects of LMD on platelet-graft interactions using an ex vivo baboon shunt. Indium 111-labeled autologous platelet deposition on expanded polytetrafluoroethylene and knitted Dacron grafts was monitored at a flow rate of 25 ml/min for 21/2 hours. LMD was given by intravenous bolus at a dose of 5 ml/kg and was followed by a continuous infusion of 10 ml/hr. Flow through the shunt was started after the initial dextran bolus. Control studies were performed in a similar manner without dextran. An additional series of experiments was performed with a 5 to 10 ml/kg bolus of 5% human serum albumin to analyze the contribution of plasma volume expansion to platelet deposition in this model. The results revealed a significant (p less than 0.05) reduction in platelet deposition on both graft materials with the LMD infusion. No difference was seen between the control group and the albumin group. These studies provide direct in vivo evidence of the antiplatelet effect of LMD.
Antiplatelet therapy is currently recommended in an effort to improve patency rates of small-caliber vascular grafts. The effect of aspirin and heparin on acute platelet deposition was studied in a baboon ex vivo shunt. Two grafts, expanded polytetrafluoroethylene and knitted Dacron, were exposed to a flow rate of 25 mL/min after administration of aspirin or heparin. Indium 111-labeled platelet uptake by the grafts was determined over 2 1/2 hours. The amount of platelet deposition in the treated groups was significantly less than that of controls after 2 1/2 hours. There was no difference between the aspirin and heparin groups. The finding that heparin inhibited platelet deposition to a degree comparable with aspirin suggests that it may not be necessary to start antiplatelet therapy preoperatively. Intraoperative systemic heparinization will provide sufficient inhibition of platelet deposition. A protocol for perioperative antiplatelet therapy is outlined.
Endothelial cell (EC) seeding has been proposed as a method to improve the performance of small-caliber synthetic vascular prostheses. Seeding experiments to date have all been carried out in the dog. This study investigates EC seeding of small-caliber Dacron carotid interposition grafts compared with contralateral control grafts in the baboon. Surface thrombogenicity was assessed at 24 hours, 2 weeks, and 4 weeks after implantation with indium 111-labeled autologous platelets. Morphologic and immunohistochemical techniques were used to assess the identity and homogeneity of the EC inoculum before seeding and to identify cell types on the harvested grafts. There was no significant difference in patency rates between seeded and control grafts at 5 weeks. Platelet accumulation on seeded grafts was significantly less (p less than 0.05) than on paired controls at 2 and 4 weeks after implantation. The luminal lining of seeded grafts had more cellular ingrowth, less adherent thrombus, and more surface cells with the morphologic and histochemical characteristics of EC than did the lining of controls. EC seeding reduces the platelet reactivity and accelerates EC coverage of small-caliber grafts in the baboon.
Vascular grafts lined with endothelial cells (EC) grown to confluence in culture before implantation may provide a thromboresistant flow surface. Growth of EC on and their adherence to currently available prosthetic materials under conditions of flow are two impediments remaining in the development of such a graft. To address these problems, 22 polytetrafluoroethylene grafts (PTFE) (5 cm by 4 mm inside diameter) were pretreated with collagen and fibronectin, seeded with 2 to 3 X 10(6) bovine aortic EC per graft, and placed in tissue culture (seeded grafts). Twenty-two grafts pretreated with collagen and fibronectin alone served as controls. After 2 weeks morphologic studies revealed that 20/22 seeded grafts were lined with a confluent endothelial layer. Indium 111-oxine was then used to label the EC-seeded grafts. After exposure to either low (25 ml/min) or high (200 ml/min) flow rates for 60 minutes in an in vitro circuit, examination of the luminal surface of the graft by light microscopy and scanning electron microscopy revealed minimal loss of EC. These findings were corroborated by radionuclide scans that showed an insignificant loss of the EC-associated indium label during exposure to flow (7% low flow, 11% high flow). Pretreatment of PTFE grafts with collagen and fibronectin thus promotes both attachment and adherence of EC even under flow conditions.
Surface thrombogenicity is recognized as an important factor in the failure of small caliber vascular prostheses. The baboon ex vivo shunt was developed to study small caliber grafts under controlled conditions at different flow rates. The shunt was created by percutaneous insertion of catheters into the baboon femoral artery and vein. Platelet-graft interactions were studied using autogenous indium 111 labeled platelets. Two graft materials were placed in series and exposed to blood flow for 2 1/2 hours at flow rates of 25 and 200 ml/min. At the end of this period, the grafts were removed for morphologic examination. Platelet adhesion to the grafts, especially with the less thrombogenic materials (PTFE and HUV), was found to be independent of flow rate. PTFE was found to be the least platelet-reactive material, HUV was intermediate, and knitted Dacron was the most thrombogenic surface. Platelet deposition on the flow surface was confirmed by light microscopy and scanning electron microscopy.
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The problem of surface thrombosis and subsequent embolization remains entrenched as a yet incompletely surmounted barrier to the development of truly satisfactory intravascular prosthetic devices. A baboon ex vivo shunt was used to determine the interaction of Indium-111 platelets and potential biomaterials. The uptake of Indium-111 platelets was monitored continuously by gamma camera scanning. Several of the materials tested demonstrated a saw-toothed pattern of platelet activity, with accumulation followed by rapid decline. Neither PTFE nor Dacron exhibited this pattern. Post shunt scans of the animals' chests showed discrete foci of platelet activity in the lungs, corresponding to each embolic event noted on the material's scan. In conclusion, the search for a smooth surface as a blood material interface may produce a material which accumulates and then sloughs significant platelet aggregates. It is crucial that these materials be subjected to vigorous testing to determine their safety prior to initiation of clinical trials.