Densitometric measurement of increased endothelial permeability in arteriosclerotic plaques and inhibition of permeability under the influence of two calcium antagonists.

A densitometric technique was established to investigate quantitative changes in endothelial permeability for horseradish peroxidase (HRP), mol. wt. 40,000 daltons, in rabbit carotid artery. Repeated weak electrical stimulations of rabbit carotid arterial walls with implanted electrodes lead to fibromuscular plaques mainly beneath the anode. It could be demonstrated that there exists a typical growth curve of the plaques dependent on the number of days of electrostimulation, with a fast proliferation rate of smooth muscle cells in the first 2 weeks of electrostimulation, and an increasing retardation of proliferation during the next 4 weeks. Endothelial permeability for HRP increases in close relation to the plaque development. Intravenous applications of single doses of the calcium entry blockers flunarizine or nimodipine are able to inhibit the increased permeability of the endothelial lining covering arteriosclerotic plaques. The intensity of the inhibitory action of these calcium antagonists correlates with the size of the arteriosclerotic plaques in inverse proportion, but nevertheless in large plaques an inhibitory effect is seen.

The effect of pentoxifylline on endothelial permeability of rabbit carotid artery wall.

Weak electrical stimulation of the rabbit carotid artery wall with direct current impulses over a period of 45 min increased the endothelial permeability for horseradish peroxidase (HRP), molecular weight 40,000 Da, beneath the anode region of the electrodes. The stimuli caused a massive accumulation of the reaction products of peroxidase in the subendothelial space. With a microdensitometric technique, it was possible to quantify the amount of the peroxidase reaction products in the subendothelium, and to compare the amounts in the non-stimulated regions with those in the stimulated regions. Intravenous administration of pentoxifylline before stimulation inhibited the transendothelial transport of peroxidase. Inhibition was pronounced in the stimulated regions, whereas the uptake of HRP in the non-stimulated regions was only slightly lower than that in non-treated animals. It was demonstrated that the extent of inhibition was dose-dependent (3, 10 and 30 mg pentoxifylline/kg). Electron microscopic examination showed that the main route of permeation of HRP was through the interendothelial clefts. However, electrical stimulation also caused an increase in the vesicular uptake of HRP into endothelial cells. Both pathways seem to be influenced by pentoxifylline.

Enhanced endothelial permeability and invasion of leukocytes into the artery wall as initial events in experimental arteriosclerosis.

The temporal sequence of the very early events in arteriosclerosis, as induced by electrical stimulation in carotid arteries of rabbits, was examined by combined light microscopy and transmission electron microscopy. After one session (30 min) of DC impulses, the endothelial permeability to horse-radish peroxidase was increased mainly beneath the anode, judging from the massive accumulation of reaction products of peroxidase in the subendothelium. After a further stimulation period, some of the endothelial cells displayed alterations in pattern and size and heavy cytoplasmic deposition of silver salt. However, the endothelium was maintained as a continuous lining. During this initial phase, a considerable number of granulocytes and monocytes was found adhering to the endothelium of the stimulated region and also within the subendothelial space. The invasion of the leukocytes preceded the migration of smooth muscle cells from the media into the intima, a process which began after two days of the electrical stimulation schedule. These initial phases of plaque development may represent a special form of an inflammatory response.

[Ectopic thyroid gland tissue in the liver]. / Ektopes Schilddrüsengewebe in der Leberpforte.

Heterotopia of thyroid gland can be seen very seldom in humans. Most frequently ectopic thyroid tissue is found in the middle line of neck and chest. Ectopia of thyroid tissue caudal of the diaphragm is an exceptionally rare finding. An explanation results out of the embryology of the thyroid gland development. Reviewing the literature we could find only one case of accessory thyroid tissue in the region of the hepatic porta, it was found at autopsy. In the present case report we describe a 58-year-old woman who had an indistinct tumor in the hepatic porta which was successfully removed. Histologic findings was that of aberrant normal thyroid tissue.

Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas.

Repeated weak electrical stimulations of rabbit carotid artery walls with implanted electrodes cause intimal proliferations of smooth muscle cells (SMC) and lead to fibromuscular plaques beneath the anode. If the animals receive a cholesterol-enriched diet the plaques become typical atheromas. The endothelial lining is maintained. The procedure for the production of an atheroma with 11 +/- 4 layers of SMC lasts 4 weeks. Addition of the calcium antagonist Flunarizine to the food during the stimulation periods inhibits the growth of the plaque. The inhibition is dose-dependent. Whether the drug inhibits atherogenesis by direct action on SMC or via an effect on permeation of macromolecules through the endothelium has been studied by measurement of (1) peroxidase (MW 40,000 dalton) permeability through the stimulated endothelium of the artery and (2) the inhibitory effects of Flunarizine on cultures of arterial SMC. Endothelial permeability increases for some hours after stimulation mainly beneath the anode. Flunarizine inhibits the permeation of peroxidase through the endothelial lining for the most part by its action on intercellular transport. The drug also inhibits the growth of SMC in mass cultures and clone cultures. The inhibition of proliferation is not specific for SMC. Skin fibroblasts obtained from the same animals as the artery smooth muscle cells are also inhibited in mass cultures and clone cultures. From the results it can be concluded that Flunarizine exerts its inhibitory action not only by its effect on the permeation through the endothelial lining but by a combined action on permeability and proliferation of cells in the artery wall.

[Primary osteosarcoma of the thyroid gland]. / Primäres Osteosarkom der Schilddrüse.

Primary extraosseous osteosarcoma of the thyroid gland is a very rare tumor with a poor prognosis. Only few cases have been reported in the literature. Metastases of osteosarcoma in the thyroid gland were also found but are very seldom. A primary thyroid tumor with osteosarcomatous differentiation in a 82-year-old man is presented and the histopathological classification, biological features and possible mechanisms of the occurrence of such thyroid tumors are briefly discussed.

Cytotoxicity of various crystalloid solutions to the endothelial cells of autologous grafts.

Harvesting and storage may influence the early failure of autologous grafts. We studied the endothelial layer of human saphenous veins (HSV) and bovine internal mammary arteries (BMA) after a two-hour storage period in comparison to vessel segments prepared immediately after harvesting. The storage solutions were 0.92% NaCl, 0.9% NaCL heparinized, Ringer's Lactate (RL), heparinized blood or Bretschneider's cardioplegic solution. To visualize the endothelial cell damage, specimens were stained with silver nitrate. The surface covered by morphologically intact endothelial cells was computed in percent. In a perfusion model using the carotid artery of rabbits, the increase of the permeability of the endothelium after two hours' perfusion was investigated (n = 5 each). In native HSV-segments 70.8 +/- 4.5% of the endothelium were found to be morphologically intact. The figures for stored grafts were: 14.7 +/- 4.4% (NaCl), 0.5 +/- 0.3% (hep. NaCl), 16.0 +/- 4.6% (RL), 37.2 +/- 5.3% (hep. blood) and 50.0 +/- 4.2% (Bretschneider). Comparable results were found with BMA specimens. BMA: 98.3 +/- 2.1% (native), 39.2 +/- 3.3% (NaCl), 8.4 +/- 3.5% (hep. NaCl), 11.2 +/- 4.8% (RL). 67.0 +/- 6.1% (hep. blood) and 78.1 +/- 4.7% (Bretschneider). In accordance with these results the increase of the permeability of the endothelial cell layer of the carotid artery of rabbits was significantly less after perfusion with Bretschneider's solution. The condition of the endothelial cells of autologous grafts at the time of implantation was found to be clearly dependent on the type of solutions used for perfusion and immersion. In comparison to all crystalloids studied in this series, Bretschneider's cardioplegic solution proved to be superior.

[Early and late damage in bypass vessels induced by crystalloid and cardioplegic solutions]. / Früh- und Spätschäden in Bypass-Gefässen induziert durch kristalloide und kardioplege Lösungen.

Veins and arteries grafted into the aorto-to-coronary position undergo degenerative processes, subintimal fibrous hyperplasia and diffuse atherosclerosis. We examined the influence of various crystalloid and cardioplegic solutions on the endothelium of human saphenous veins and internal mammary arteries, bovine internal mammary veins and arteries and rabbit carotid arteries, using light microscopy, TEM, SEM, immunological techniques and quantitative permeability measuring methods. Immersion or perfusion of vessel segments for various periods of time led to an ultrastructural endothelial cell damage, to an increased endothelial permeability for macromolecules, partly to an endothelial cell loss. Addition of high concentrations of heparin impaired this effect. Under influence of various solutions an increased plaque development under stimulation with atherogenic stimuli was observed.

Effect of chronic nicotine delivery on the proliferation rate of endothelial and smooth muscle cells in experimentally induced vascular wall plaques.

To study the effect of nicotine, cholesterol feeding, and their combination on endothelial and smooth muscle cells in vascular wall plaques an experimental method was established which allows the immunohistochemical detection and quantification of the fractions of endothelial and smooth muscle cells in DNA synthesis under the effect of these stimuli. For this purpose standardized fibromuscular plaques were produced by electrostimulation in the common carotid arteries of rabbits. The animals received either nicotine via implanted osmotic minipumps or a cholesterol diet or both. Plaque size was determined at the end of the experiments after 7 or 14 days as well as the fraction of endothelial and smooth muscle cells in DNA synthesis during exposure to bromodeoxyuridine (BrdU). The BrdU labeling index of endothelial cells clearly increased under chronic nicotine administration for either 7 days or 14 days compared to controls. The combination of nicotine and cholesterol diet led to a more significant increase. In contrast, the BrdU labeling index of smooth muscle cells was not increased under nicotine delivery. The combination of nicotine and cholesterol, however, led to a significant increase of the BrdU labeling index of smooth muscle cells in the plaques compared to cholesterol feeding. Measurement of the plaque size revealed no difference between controls and nicotine-treated animals after 14 days of nicotine delivery, whereas the combination of cholesterol and nicotine produced increased plaque formation compared to a group of animals which received a cholesterol diet alone.

Time course of smooth muscle cell proliferation in the intima and media of arteries following experimental angioplasty.

Smooth muscle cell (SMC) proliferation is known to be an important factor for the development of restenosis after percutaneous transluminal coronary angioplasty. To determine the time course of intimal and medial SMC proliferation and morphological changes after experimental angioplasty, an intimal atheroma was produced with repeated weak electrical stimulations in the right carotid artery of 45 male New Zealand White rabbits. Angioplasty was subsequently performed in 35 rabbits, and the proliferative responses were analyzed with histomorphological and immunohistological criteria at 3, 7, 14, 21, 28, and 42 days after intervention. A hemodynamic relevant stenosis after angioplasty was found in eight (23%) of 35 dilated arteries. In five rabbits the stenosis was due to a mural thrombus, and in three animals restenosis was caused by intimal SMC proliferation. In all dilated arteries the intimal wall thickness increased from 13 +/- 5 intimal cell layers (after electrical stimulation) to 33 +/- 14 cell layers during 28 days after angioplasty (p less than 0.05). Later than 4 weeks after angioplasty, no additional increase of intimal thickening occurred. Application of bromodeoxyuridine 18 and 12 hours before excision of the vessels allowed determination of the percent of cells undergoing DNA synthesis in the intima and media using monoclonal antibody against bromodeoxyuridine. SMCs were identified by alpha-actin staining. Immunohistological quantification of intimal SMC proliferation showed a maximum of cells undergoing DNA synthesis within the first 7 days after angioplasty (p less than 0.01). In contrast, medial proliferation of SMCs was delayed and showed a small but significant increase 21 days after dilatation (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Damage to arterial and venous endothelial cells in bypass grafts induced by several solutions used in bypass surgery.

The purpose of our study was to evaluate the cytotoxicity of incubation solutions used in heart surgery to endothelial cells. The endothelial layer of human saphenous veins (HSV) and bovine internal mammary arteries (BMA) and veins (BMV) were studied after a two-hour storage interval and compared with control vessel segments prepared immediately after harvesting. To visualize the endothelial cell damage, specimens were stained with a silver nitrate technique. The surface covered by light-microscopically intact endothelial cells was computed in percent. In the control HSV segments 70.8 +/- 4.6% of the endothelium were found to be morphologically intact. The results for stored HSV segments were 50.0 +/- 4.2% (Bretschneider's solution), 14.8 +/- 4.5% (physiological saline), 0.45 +/- 0.1% (physiological saline with heparin), 16.7 +/- 4.7% (Ringer's lactate) and 37.2 +/- 5.3% (heparinized blood). Comparable values obtained with BMA specimens were 98.3 +/- 0.7% (controls), 78.1 +/- 4.7% (Bretschneider's solution), 39.2 +/- 3.3% (physiological saline), 8.4 +/- 2.0% (physiological saline with heparin), 11.3 +/- 1.7% (Ringer's lactate) and 67.8 +/- 6.2% (heparinized blood). A similar trend was found with BMV segments: 85.2 +/- 4.7% (controls), 75.6 +/- 6.0% (Bretschneider's solution), 49.5 +/- 8.9% (physiological saline), 5.95 +/- 0.7% (physiological saline with heparin), 6.2 +/- 0.7% (Ringer's lactate) and 54.3 +/- 5.1% (heparinized blood). In conclusion, Bretschneider's solution proved to be superior for storage of bypass grafts in comparison to all other tested solutions in this series.

Influence of several solutions used in bypass surgery on the permeability of the endothelium of carotid arteries in New Zealand rabbits.

BACKGROUND: Alteration of endothelial permeability by perfusion solutions used may influence the outcome of bypass grafts. METHOD: Carotid arteries of New Zealand rabbits were locally perfused in situ for 20 or 60 min with various solutions used in bypass surgery. After restoring normal circulation, horseradish peroxidase was injected in the ear vein. Endothelial permeability was measured by electronmicroscopy as the peroxidase accumulation in the subendothelial space during 6min circulation. RESULTS: The density indices (mean standard deviation) as a parameter for permeability in comparison to the control vessels were significantly greater than 100% for all solutions: for physiological saline 254+/-22% and 358+/-15%, for Ringer's lactate 206+/-26% and 302+/-17%, for St. Thomas' Hospital solution 163+/-15 % and 252+/-29%, and for Bretschneider's HTK solution 130+/-15% (p=0.003) and 169+/-26%, after 20 and 60 min perfusion. Addition of heparin (50IU/ml) caused a significant increase in endothelial permeability (p<0.05). CONCLUSIONS: Bretschneider's is the most suitable of the solutions studied as a graft storage medium in bypass and cardiothoracic surgery, but a solution causing even less damage is desireable.