Leif Jørgensen

Ultrastructural changes in the myocardial myocytic mitochondria: crucial step in the development of oxygen radical-induced damage in isolated rat hearts?

SummaryThe present study focuses on the sequential development of myocardial ultrastructural changes produced by oxygen radicals. Isolated rat hearts were perfused with oxygen radicals, generated by hypoxanthine and xanthine oxidase, for 5 and 10 min followed by a 35-min recovery period. The frequency of, and the association between, ultrastructural changes were examined by semiquantitative morphometry using the micrograph as unit. In each micrograph sarcolemmal, myocytic mitochondrial and myofilamental alterations were observed and graded as slight, moderate or severe. The myocytic nucleus and the endothelial cells were scored as normal or altered.Five min group: Among the cellular organelles examined, the myocytic mitochondria showed the highest frequency of alteration (in 15.3% of the micrographs). Among the grades of myocytic mitochondrial ultrastructural changes, slight alterations predominated (12.5%). Slight myocytic mitochondrial alterations were not significantly associated with the occurrence of ultrastructural changes of other cellular organelles. Endothelial ultrastructural alterations were sparse (1.5%).Ten min group: The frequency of altered organelles was greater when compared to the 5 min group. The myocytic mitochondria were still the most frequently altered component (61.7%), and myocytic mitochondrial ultrastructural alterations of all grades were strongly associated with the occurrence of other myocytic ultrastructural changes. In conclusion, the present study showed that myocytic mitochondrial changes predominated after both 5 and 10 min of oxygen radical exposure followed by recovery. In the 5 min group slight myocytic mitochondrial changes appeared independent of other myocardial changes, but in the 10 min group, however, myocytic mitochondrial changes were strongly associated with other myocardial ultrastructural changes. These results indicate that myocytic mitochondria are especially vulnerable to oxygen radicals, and further that myocytic mitochondrial ultrastructural changes may be a crucial step in the development of oxygen radical-induced myocardial damage.

Alcohol in a series of medico-legally autopsied deaths in northern Norway 1973–1992

It is well established that use of alcohol increases the risk of fatal injuries. The presence of blood alcohol in autopsied deaths is regularly encountered in medico-legal practices. The aim of this study was to investigate the prevalence and concentration of alcohol in 1539 medico-legal autopsies in two counties in northern Norway in the period 1973-1992, and the reporting of acute alcohol influence among these deaths to the official cause-of-death statistics. Blood alcohol concentration (BAC) >/=0.5 per thousand (50 mg/100 ml) was found in 47.6% (n=456) of violent deaths tested, and in 93% (n=426) of these the BAC was >/=1.0 per thousand. In 17.4% (n=55) of tested natural deaths the BAC was >/=0.5 per thousand. Acute alcohol-influenced violent deaths were under-reported to the cause-of-death statistics. Deaths by motor vehicle traffic accidents did not differ from other violent deaths in this respect. The under-reporting among violent deaths was 41% in cases with BAC >/=0. 5 per thousand and 37% where the BAC was >/=1.0 per thousand during the whole period. It is concluded that post-mortem BAC >/=0.5 per thousand, should be regarded as a possible contributory cause in all violent deaths, and reported accordingly.

Medicolegal autopsies of violent deaths in northern Norway 1972-1992.

The aim of this paper is to describe the characteristics of medicolegal autopsies of violent deaths in northern Norway over a period of 20 years. On request by the police, 1446 violent deaths were examined--82.6% males and 17.4% females. The mean age was 40.2 years (range 0-98). The most frequent violent manners of death were suicides (24.9%), deaths caused by motor vehicle traffic accidents (18.6%), accidental poisoning (11.5%) and boating incidents (8.4%). Homicides and involuntary manslaughter came to 4.4% and 1.7%, respectively, whereas in 11.4% of cases the manner of death was unknown. The five most frequent causes of death were blunt injury (31.4%), drowning (17.4%), suffocation (11.8%), firearm (11.3%) and poisoning (10.5%). In 3.5% of the cases the cause of death was unknown. The spectrum of the manner of death and the cause of death in a subarctic population is discussed with reference to legislation, practise of request and information given by the police.

Could tendinosis be involved in osteoarthritis

Ten patients, age 60 (48–75 years), with osteoarthritis (OA) of the hip and 10 patients, age 82.5 (60–90 years), with fracture of the collum femoris (FCF; minimum Garden stage III) underwent an open biopsy procedure from the internal obturator tendon in conjunction with a total hip replacement. The histological evaluation revealed that all tendon samples in the OA group revealed scar tissue; the corresponding was found in 50% of patients in the FCF group (P=0.02). There were also more GAGs (P=0.023) and calcium deposits (P=0.001) in the samples from the OA group. The ultrastructural evaluation revealed fewer small and medium‐sized fibrils (P=0.001) and more non‐collagenous extracellular matrix (ECM) (P=0.003) in the OA group. Taken together, the samples from the internal obturator tendon in the OA group revealed a more degenerative appearance with more scar tissue, change in fibril diameter distribution and more non‐collagenous ECM. Our findings suggest that OA and periarticular tendinopathy are closely linked. Further research is needed to determine whether musculotendinous changes in the deep rotators are sequelae of joint pathology, or a contributing factor in the development of degenerative joint change.

Endothelial injury and trapping of blood cells in human myocardium following coronary bypass surgery

To investigate the focal myocytic and microvascular injury that develops during the first hour of reperfusion after hypothermic cardioplegic cardiac arrest, and to compare the influence of gentle versus more abrupt reperfusion, serial atrial biopsies were obtained from 14 patients undergoing uneventful coronary bypass surgery. The biopsies were taken before cardioplegia, at the start of reperfusion, and after 20 and 60 min of reperfusion. Transmission electron micrographs of biopsies examined by stereological techniques revealed endothelial injury. Following 20 min reperfusion there was accumulation of both red blood cells (p = 0.03) and polymorphonuclear leucocytes (p = 0.0004) were found. There was also intravascular accumulation of platelets (p = 0.008) and extravasation of red blood cells (p = 0.02), which increased throughout the observation period. If reperfusion was started with a gradual rise in temperature and pressure, the numbers of platelets in the microvessels were lower than following ordinary, abrupt reperfusion (p = 0.06). It is concluded that reperfusion injury is associated with microcirculatory disturbances with trapping of blood cells, changes which may be favourably modified by a gentle reperfusion technique.

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

SummaryThis study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of51Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When51Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of51Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.

Ultrastructural alterations during the critical phase of reperfusion: a stereological study in buffer-perfused isolated rat hearts.

The present study focuses on myocardial ultrastructural alterations during the early phase of reperfusion. Isolated buffer-perfused rat hearts were exposed to standard perfusion (control group,n = 10); 60 min of global ischemia (n = 10); 60 min of global ischemia followed by 2 min of reperfusion (n = 10); or 60 min of global ischemia followed by 10 min of reperfusion (n = 10). The hearts were perfusion-fixed for electron microscopy, and ultrastructural evaluation was performed using stereological technique in order to obtain an estimate of the volume fraction and absolute volume of different tissue components. EFFECT OF ISCHEMIA: Neither the ventricular nor the myocytic volume differed significantly from the respective control values. Both the myocytic mitochondrial volume (135+/-8 vs control 89+/-6 microl) and the volume of myocytic clear space (35+/-6 vs control 10+/-2 microl) were significantly increased. The capillary volume (22+/-4 vs control 58+/-6 microl) and the volume of the capillary lumen (15+/-3 vs control 48+/-5 microl) were significantly decreased. The volume of the capillary wall, however, was not altered after exposure to ischemia (7+/-3 vs control 10+/-1 microl). ADDITIVE EFFECT OF ISCHEMIA AND REPERFUSION: Both the ventricular volume (755+/-28 vs control 600+/-32 microl) and the myocytic volume (396+/-24 vs control 287+/-16 microl) were significantly increased after 10 min of reperfusion. EFFECT OF REPERFUSION: The ischemic-induced myocytic mitochondrial swelling and increase of clear space were not reinforced during reperfusion. Furthermore, the volume of the capillary lumen and the capillary wall did not alter significantly in the groups exposed to reperfusion compared to the ischemic hearts. In conclusion, stereological evaluation did not reveal significant aggravation of ischemic-induced myocardial injury during the early phase of reperfusion.

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

SummaryThrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of51Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the51Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells.

A clinical-pathological review of hidradenitis suppurativa: using immunohistochemistry one disease becomes two.

Fismen S, Ingvarsson G, Moseng D, Dufour ND, Jørgensen L. A clinical‐pathological review of hidradenitis suppurativa: Using immunohistochemistry one disease becomes two. APMIS 2012; 120: 433–40.

Mechanisms involved in the early interaction between HeLa cells, platelets and endothelial cells in vitro under the influence of thrombin

The aim of the study was to obtain more information about the mechanisms involved in the initial adhesion of tumour cells to endothelial cells during metastasis. In a previous paper, we found that addition of both platelets and thrombin increased the adhesion of tumour cells to cultured endothelial cells within 15 min, compared to when either one or both of the ingredients were absent. In the present study, HeLa cells, prelabelled with radioactive 51Cr, human platelets, and thrombin, were added to the medium in dishes of endothelial cells. The dishes were then shaken for 15 min at 37°C. Scanning and transmission electron micrographs showed HeLa cells adhering to the endothelium either together with platelets or without them. In other experiments, the endothelium was pretreated for 30 min with either of the following: 0.5 mM or 0.1 mM acetylsalicylic acid (ASA); 0.5 rnM or 0.1 mM Na‐salicylate (NaS). Pretreatment of the endothelium with 0.5 mM ASA significantly increased the percentage of adherent tumour cells, while 0.1 mM ASA and the two concentrations of NaS caused only minor changes. In addition, the ASA‐treatment caused more HeLa cells to adhere without platelets while NaS‐treatment caused more HeLa cells to adhere together with platelets. Release of 51Cr from HeLa cells during the experimental period was also measured; the addition of thrombin and platelets did not change the 51Cr release significantly. In separate experiments, HeLa cells and platelets were mixed without the presence of endothelial cells. Transmission electron micrographs showed that in the absence of thrombin, mixed HeLa cells and platelets did not react with each other; when thrombin was added they formed co‐aggregates. In conclusion, we show that in our experimental model HeLa cells adhere to the endothelium in two ways, both with and without platelets. The production of prostacyclin in the endothelial cells has an inhibitory effect on tumour cell adhesion. Without thrombin, the HeLa cells are not capable of activating platelets.