Hydrogen peroxide induces endothelial cell atypia and cytoskeleton depolymerization.

Reactive oxygen intermediates induce cell injury in a variety of pathophysiological conditions. Human umbilical cord vein endothelial cell (HUVEC) cultures were exposed to 1 or 200 microM H2O2 for 15 min, and observed after 15 min, or 1, 4, 24, or 120 h. Factor VIII and the cytoskeletal proteins vimentin and tubulin were visualized immunocytochemically. Release of lactate dehydrogenase (indices of cell membrane injury) did not increase after H2O2 exposure; nor was cellular expression of factor VIII affected. 200 microM H2O2 induced cell contraction after 15 min which disappeared after 1 and 4 h, but was evident again after 24 h. Immediately after exposure, the filamentous structure of vimentin and tubulin disappeared, but normalized after 1 h. After 120 h, the cytoskeleton filaments were coarsened and disorganized, and an abundance of multinucleated giant cells were observed. Catalase (150 U/ml) abolished all effects of H2O2. One microM H2O2 did not induce any changes in HUVEC. Thus, the present concentrations of H2O2 did not induce cell necrosis or altered expression of factor VIII. Early, reversible cell contraction and depolymerization of cytoskeletal proteins were observed, followed by a delayed contraction and cell atypia after 200 microM H2O2.

Acute hemodynamic effects of induced hypothermia in hemorrhagic shock: an experimental study in the pig.

Hypothermia (HT) is used in certain surgical procedures to reduce metabolism and protect the brain, but in trauma victims accidental HT is considered harmful. Recent animal studies indicate that HT has protective effects in hemorrhagic shock. The aim of the present study was to examine how induced HT modifies the hemodynamic pattern in hemorrhagic shock. Twenty pigs with a body weight of between 17 and 24 kg (mean 20.8) were anesthetized, 50% of their blood volume was withdrawn, and hypothermia (30 degrees C) was induced in half of them (HT group) while the others served as controls. Central hemodynamics was monitored during 4 h via an arterial line and a pulmonary artery catheter. Blood samples were obtained for measurement of leukocyte and platelet counts. Three of the control pigs died while all the animals in the HT group survived the experiment. The hemorrhage resulted in a marked increase in heart rate and a drop in cardiac output and mean arterial pressure. HT slowed the heart rate and induced a further reduction of cardiac output, which parallelled the depression of the core temperature, while the stroke volume did not change in any of the groups. A significant decrease in mean arterial pressure and the leukocyte count became apparent 2 h after the induction of HT. HT aggravated the hypokinetic situation resulting from hemorrhagic shock but without increasing the mortality.

Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

Seeding of ePTFE carotid interposition grafts in sheep and dogs: species-dependent results.

Autologous endothelial seeding of thin-walled ePTFE vascular prostheses (I.D.4 mm), interposed in the carotid artery, was performed in 10 dogs and 14 sheeps. Aspirin (250 mg/day) and dipyridamole (75 mg/twice daily) were given throughout the study as antiplatelet therapy. Animals were killed 2 and 5 weeks after surgery. Patency rates for seeded grafts in dogs were 75% (6:8) and 83% (10:12) at 2 and 5 weeks, respectively. In control grafts the patency rates were identical. Patency rates for seeded grafts in sheep were 0% (0:5) and 11% (1:9) at 2 and 5 weeks, respectively. Control grafts in sheep had a patency rate of 40% (2:5) and 0% (0:9) at corresponding times. Scanning electron microscopy showed an almost complete endothelialization of seeded grafts in dogs after 5 weeks. Platelet deposition was studied in the dogs by means of chromium-51-labeled autologous platelets. Significantly fewer platelets accumulated on seeded grafts, and the ratio of 6-keto-PCF1 alpha to thromboxane B2 was significantly higher, compared with unseeded grafts, which indicated the presence of a functionally active endothelial lining in seeded grafts. Differences in the hemostatic system could account for the high clotting incidence in sheep, compared with that in dogs. Such species differences make extrapolations to the clinical situation from autologous endothelial seeding in experimental animals hazardous.

Effects of peripheral high energy missile trauma on the oxygenation of lung tissue in the pig.

The purpose of this paper was to study the effect of peripheral high energy missile trauma on oxygenation of the lung tissue. Eleven pigs were randomised to either high energy gunshot wound of the soft tissues of the thigh or sham exposure. Under anaesthesia and controlled ventilation, the carotid artery and the jugular vein were catheterised and a thoracotomy was performed. A Clark-type multiwire electrode was placed on the surface of the middle lung lobe for registration of the oxygen pressure fields of the lung tissue (PtO2). In animals subjected to missile trauma, an increased heterogeneity was seen in PtO2 1 and 2 hours after exposure, specially under hypoventilatory provocation. Furthermore, PtO2 values did not return to baseline when hypoventilation was followed by normoventilation, in contrast to what was seen in the sham exposed animals. No differences between groups were found in arterial PO2, O2-saturation, PCO2, pH or in pulse frequency. These findings of disturbance in oxygenation of pulmonary tissue, may represent an early phase in the development of a lung injury resembling the post-traumatic adult respiratory distress syndrome seen in man. The initiating events could be release of mediators from traumatised tissue and/or direct effects of the shock waves from missile impact, passing through the body.

Surgical treatment of skin lesions induced by sulfur mustard ("mustard gas")--an experimental study in the guinea pig.

Sulfur mustard has been known as a chemical warfare agent since 1915 and its biological effects, e.g. acting as an alkylating agent reacting with nucleophiles in proteins and nucleic acids, are well known. Still, there is today no specific antidote against or treatment of sulfur mustard induced cutaneous lesions. Recently, a technique of treating thermal burns with early surgical excision followed by skin grafting has been established. The present study was undertaken to evaluate, if a similar technique could be of value in the treatment of sulfur mustard lesions. Under anaesthesia, guinea pigs were exposed to sulfur mustard vapour, followed six hours later by excision of the exposed skin segment. In animals where the skin defect was primary sutured, healing time was significantly shorter and increase in body mass greater compared to conservatively treated controls. No such differences were found if excision was followed by full thickness skin grafting. Further studies in other species are suggested to clarify the possible role of surgical excision of sulfur mustard skin lesions, followed by skin transplantation, in humans.

Microvascular lung tissue oxygenation--a methodological study in the pig.

The objective of the present study was to investigate the possibility of measuring lung tissue oxygen pressure (PtO2) distributions at the microvascular level, and also if a change in the lung tissue oxygenation could be detected during hypoventilation (50% reduction in ventilatory settings). Experiments were carried out on eight mechanically ventilated ketamine-anaesthetised pigs. A thoracotomy was performed through the third right intercostal space. PtO2 measurements were made using a Clark-type multiwire microelectrode placed onto the pleural surface of the middle lobe. PtO2 was measured during normoventilation, hypoventilation (3 minutes) (reduction of respiratory volume/minute and frequency by 50%), and a second period of normoventilation. Baseline PtO2 was 5.8 (range 4.4 to 10.3) kPa and decreased to 2.9 (range 1.6 to 4.2) kPa during hypoventilation, associated with some PtO2 values close to zero. The PtO2 increased to 5.4 (range 3.6 to 8.4) kPa during the second normoventilatory period, some values still close to zero. This study demonstrates that lung tissue PO2 registrations can be made in a suitable animal (pig) model, and that hypoventilation induced an almost reversible decrease in lung tissue oxygen pressure distributions. In addition, no microscopically visible tissue damage was inflicted by the electrode on the underlying lung surface.

Acute pancreatitis after snake bite. Case report.

Gastrointestinal symptoms are the most common systemic manifestations of adder envenomation. A young, previously healthy man had severe abdominal symptoms after an adder bite. Acute pancreatitis was diagnosed at exploratory laparotomy. Adder venom contains enzymes which can, at least theoretically, cause acinar cell damage and thus trigger acute pancreatitis.

High dose corticosteroids and cell-cell interactions.

Adult Respiratory Distress Syndrome (ARDS) is a feared complication of trauma or sepsis, characterized by an interstitial and alveolar edema due to increased pulmonary microvascular permeability. In ARDS polymorphonuclear granulocytes (PMN) aggregate and accumulate in the pulmonary microvessels and activation of the complement system, especially C5a, is suggested to be of importance supporting this aggregation. Such complement activated PMN can increase vascular permeability, probably by initiating endothelial cell (EC) damage. Addition of PMN and C5a to cultured EC monolayers in vitro produced both morphological and functional EC damage. A similar EC damage could be reproduced in the absence of white cells by exposing EC monolayers to oxygen free radicals induced by xanthine and xanthine oxidase or hydrogen peroxide. High dose corticosteroid (HDC) administration has been advocated in shock and ARDS and it has been experimentally demonstrated that methylprednisolone or hydrocortisone at a concentration corresponding approximately to a dose of 30 mg/kg i.v. inhibited both PMN aggregation and adhesion to the endothelium. On the other hand, no effect of HDC on PMN thromboxane synthesis or cell membrane morphology alterations was found. It has been suggested that HDC increases PMN hydrophobicity and thus reduces the tendency of the white cells to adhere to the endothelium of the microvasculature. Furthermore, it has been demonstrated that HDC can inhibit PMN production of oxygen free radicals. Platelets seem to play a role in ARDS. Serotonin released from platelets increased the cytotoxic effect of PMN on EC more than 100% in vitro, and activated PMN seemed to recruit platelets and release vasoactive substances. On the other hand, platelet serotonin enhanced the adhesion of complement stimulated PMN to EC, thus creating a vicious circle. To conclude, complement activated PMN aggregate and adhere to the pulmonary microvascular EC which are injured by e.g. PMN-generated oxygen free radicals. Platelet aggregation and release of serotonin augments this injury and activated PMN probably stimulate platelet aggregation and release. Agents capable of diminishing PMN activation and aggregation, e.g. HDC, might be of value in attenuating these cell-cell interactions in ARDS.

A spectrophotometric method for measuring oxygen consumption in monolayers of cultured endothelial cells.

A new method for measuring oxygen consumption in monolayer cell cultures is described. Cells are cultured in shallow microchambers etched in glass chips. A haemoglobin solution is added and the microchambers are sealed airtight, the oxygenated haemoglobin solution serving as an indicator of oxygen tension within the microchamber as well as a source of oxygen. With cellular extraction of oxygen, an increasing amount of reduced haemoglobin is formed within the microchamber resulting in an increased absorbance of the haemoglobin solution at 435 nm. With a specially prepared device, it is possible to record this absorbance change with an ordinary laboratory spectrophotometer and, from this recording, it is possible to calculate the respiratory rate, provided the number of cells (or any other quantity of biological material), microchamber volume and haemoglobin concentration are known. Repeated measurements on the same undisturbed culture is possible. The system was tested on monolayer cultures of endothelial cells from the rat pulmonary artery.

Effects of 5-hydroxytryptamine and related agents on endothelial cell oxidative metabolism in vitro.

A recently described microrespirometric method was used to determine the influence of 5-hydroxytryptamine (5-HT), nialamide, imipramine and ouabain alone and in combinations on oxidative metabolism in monolayer cultures of endothelial cells from the rat pulmonary artery. It was found that exposure to 5-HT increased oxygen uptake in the cultures, while imipramine or ouabain decreased respiration; nialamide had no obvious effect. When used in combinations, the effect of ouabain or imipramine dominated over the 5-HT effect. The explanation for this 5-HT stimulating effect on endothelial cell oxidative metabolism, previously undescribed, is suggested to be that 5-HT exposure stimulated individual cells to contract. This active contraction resulted in an increased energy demand reflected in elevated oxygen consumption, an effect inhibited by ouabain or imipramine.

Comparison of oxidative metabolism in vitro in endothelial cells from different species and vessels.

Oxygen consumption was compared in confluent cultures of endothelial cells from human umbilical cord veins, rat pulmonary arteries, and bovine aortas. A microrespirometric method utilizing oxygenated hemoglobin as oxygen supply and indicator of respiration was used. Respiratory rate was equal in human and murine cells (2.0 X 10(-6) and 2.2 X 10(-6) microliters O2/cell/hour respectively), compared on a cell-to-cell basis, while respiratory rate of the bovine endothelium was significantly lower (0.4 X 10(-6) microliters O2/cell/hour) (P less than .001).

The use of lasers in vascular and cardiac surgery. Clinical review.

Use of lasers is routine in medical fields such as ophthalmology and dermatology, but in spite of intensive research in recent years, its role in cardiac and vascular surgery still remains to be determined. Laser energy can vaporize atherosclerotic plaques in human arteries obtained at necropsy. Fiberoptic catheters have been constructed to deliver the laser energy to atheromas at a distance from the arteriotomy. The healing of the arterial wall after laser treatment is rapid and results in complete reendothelialization. Lasers have been used in patients undergoing coronary surgery as well as peripheral vascular reconstructions. In some cases, the percutaneous approach has also been used. In these clinical series, the laser treatment was in almost all cases followed by either transluminal balloon angioplasty or by-pass operation. Complications after laser treatment include vessel perforation, reocclusions, thrombosis, or aneurysm formation. The development of more advanced fiberoptic catheters and better understanding of dosimetric parameters are promising features to avoid these complications as well as development of methods for steering of the laser catheter itself and means for "real time" diagnosis (e.g. angioscopes, spectroscopic diagnosis). The future use of lasers in cardiac and vascular surgery seems promising, especially as an alternative to balloon angioplasty.

Adult idiopathic enteric intussusception in the postoperative period. Case report.

Enteric intussusception developed in a 43-year-old man 13 days after operation for mechanical intestinal obstruction. No leading point of the invagination was found. The case may thus be classified as idiopathic intussusception occurring in the postoperative period. To our knowledge this condition has not previously been described.

Acute metabolic and endocrine effects of induced hypothermia in hemorrhagic shock: an experimental study in the pig.

BACKGROUND: Hypothermia is considered harmful in trauma patients. In surgery, hypothermia is occasionally used to reduce metabolism and protect the brain. Recent studies in animals have also shown protective effects of hypothermia in hemorrhagic shock. The aim of this study was to evaluate the metabolic and endocrine effects of induced hypothermia in hemorrhagic shock. METHODS: Half of the individually calculated blood volume was removed from 17 anesthetized piglets. They were then randomized to normothermia or hypothermia and followed for 4 hours after hemorrhage. RESULTS: In the hypothermic pigs, arterial PO2 increased from 10.3 +/- 0.7 to 16.4 +/- 0.9 kPa, but it remained unchanged in the normothermic group. The serum levels of potassium increased from 3.9 +/- 0.2 to 5.0 +/- 0.2 mmol/L in the normothermic group. In the hypothermic pigs, the potassium levels temporarily decreased from 3.8 +/- 0.1 to 3.0 +/- 0.1 mmol/L but then returned to baseline levels. The levels of serum catecholamines surged in both groups during hemorrhage. They remained elevated in normothermic pigs but declined in the hypothermic group. CONCLUSION: In porcine hemorrhagic shock, induced hypothermia increases arterial oxygen tension and stabilizes serum levels of potassium and catecholamines.

Pressure waves caused by high-energy missiles impair respiration of cultured dorsal root ganglion cells.

High-energy missile extremity impact causes short-lasting pressure waves which traverse the body with a velocity close to that of sound in water. In order to elucidate mechanisms for distant damage in a living body to the peripheral and central nervous system, a model system was designed aimed to create pressure waves with amplitudes, frequency spectrum, and duration fairly comparable to those recorded in situ. Our model system enabled exposure of tissue cultures of dorsal root ganglion (DRG) and endothelial cells under strictly controlled conditions to a burst of oscillating pressure waves and to determine possible influence on oxygen consumption. Oscillating pressure waves caused by high-energy missile impact (velocity 1,200 m/s) reduced the oxygen consumption by more than 80%. However, in spite of this drastic, acute effect the ganglion cells and the feeder layer cells did not reveal any immediate plasma membrane dysfunctions as revealed by cytoplasmic uptake of Evans blue protein marker complexes. It is concluded that pressure waves fairly similar to those demonstrable in vivo in the vicinity of the peripheral and central nervous system after high-energy missile extremity impact in pigs reduce the respiration of DRG cells and endothelial cells in culture. The mitochondrial impairment is not associated with concomitant plasma membrane dysfunction for macromolecules. Nerve cells seem to be more vulnerable than the other type of cultured cells examined.

Laser-induced shock wave endothelial cell injury.

BACKGROUND AND OBJECTIVE: Several laser procedures, extracorporeal lithotripsies (ESWL), and high-velocity missile trauma generate pressure transients that are transmitted through the tissues. Despite several publications demonstrating shock wave-induced tissue injury, little is known about its pathophysiology. This study introduces an in vitro model for studying shock wave effects on endothelial cell (EC) monolayers. STUDY DESIGN/MATERIALS AND METHODS: A Nd:YAG laser-driven flyer-plate technique was used to generate shock waves. Physical characteristics were determined with a pressure transducer, a high-speed video camera, and sequential photography. Biological effects were studied with phase contrast and lightfield microscopy, computerized morphometry, immunocytochemistry, spectrophotometry, and enzyme-linked immunosorbent assay (ELISA). RESULTS: The shock waves generated were highly reproducible. Cavitation was verified and quantified, and its extent could be varied in the vials. Exposed cultures exhibited areas with cell membrane damage and cell detachment. Release of LD was elevated (P < 0.01) in exposed vials. The EC lesions were larger (>P < 0.01) in cultures submitted to high vs. low extent of cavitation. CONCLUSION: The flyer-plate model can be used to subject cell monolayers to defined and reproducible shock waves causing immediate cell injury similar to the previously reported vascular lesions associated with ESWL, pulsed lasers, and blast trauma. With the flyer-plate model, such lesions may be further studied on the cellular and subcellular levels.