Cardiomyocyte apoptosis after cardioplegic ischemia: comparison to unprotected regional ischemia-reperfusion.

BACKGROUND: Cardiomyocyte apoptosis might contribute to left ventricular (LV) dysfunction following cardiac surgery. Magnetic resonance imaging is considered the most accurate method of determining LV function. We compared apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL, staining and detection of caspase 3 activation) and LV function after regional ischemia-reperfusion (I-R) and global cardioplegic ischemia. METHODS: Pigs were randomized to undergo regional myocardial I-R for 20 + 20 min, global myocardial ischemia with cardiopulmonary bypass (CPB) for 40 min or CPB without ischemia (control), followed by 274 min of reperfusion. RESULTS: Compared with the control group, the number of TUNEL-positive cardiomyocytes was higher in the global ischemia group with CPB (0.024 ± 0.014%; p = 0.02) and further increased in areas of unprotected regional I-R (0.444 ± 0.562%; p = 0.003, vs. control). Myocytes with active caspase 3 were detected after global and regional ischemia. The global ejection fraction did not differ between CPB and regional I-R groups. CONCLUSIONS: The use of cardioplegia and CPB efficiently protects the heart from global I-R-induced cardiomyocyte apoptosis during open heart surgery.

Effects of levosimendan in experimental acute coxsackievirus myocarditis.

BACKGROUND: Acute heart failure is a potentially fatal manifestation of viral myocarditis. Development of myocardial damage in myocarditis involves cardiomyocyte apoptosis. Levosimendan is a novel calcium sensitizing inotropic agent with anti-apoptotic properties. We studied the feasibility of inotropic treatment with levosimendan and its effects on apoptosis in experimental acute heart failure caused by coxsackievirus myocarditis. MATERIALS AND METHODS: Adolescent BALB/c mice were infected with myocarditic Woodruff variant of coxsackievirus B3 (2 x 10(4) plaque-forming units). Mice were randomized into those receiving levosimendan 0.33 mg kg(-1) (total dose 1 mg kg(-1) day(-1)) (n = 20) or vehicle (n = 19) given orally by gauge three times a day for 7 days after infection. Left ventricular function was evaluated by transthoracic echocardiography and the mice were euthanized on day 7. Histopathology, amount of virus in the heart (virus titration assay) and cardiomyocyte apoptosis (TUNEL assay) were studied. Uninfected untreated control mice were also studied. RESULTS: Infection resulted in histopathologically severe myocarditis and significant impairment of left ventricular function. Levosimendan treatment significantly improved ventricular function (fractional shortening 0.32 +/- 0.04 vs. 0.23 +/- 0.05, P = 0.005; contractility 0.60 +/- 0.12 vs. 0.39 +/- 0.14, P = 0.007 and myocardial performance index 0.36 +/- 0.06 vs. 0.62 +/- 0.15, P < 0.0001) compared with vehicle. Levosimendan also reduced cardiomyocyte apoptosis (0.26 +/- 0.08% vs. 0.44 +/- 0.15% in vehicle, P = 0.008), but did not have an effect on areas of myocardial necrosis or inflammation, or the amount of virus in the heart. Levosimendan treatment did not affect mortality (total mortality 63%). CONCLUSIONS; Levosimendan improves ventricular function and inhibits cardiomyocyte apoptosis; therefore, it is suggested as a potentially feasible therapy in acute heart failure caused by viral myocarditis.

Development of troponin autoantibodies in experimental coxsackievirus B3 myocarditis.

BACKGROUND: Autoantibodies against various endogenous proteins are found in myocarditis. Troponin autoantibodies are detected in patients with chronic dilated cardiomyopathy, but their presence in myocarditis remains unknown. We set out to study the presence of troponin autoantibodies in experimental viral myocarditis. MATERIALS AND METHODS: BALB/c mice infected with coxsackievirus B3 Nancy strain were followed-up at days 1-7 and 2, 4, 8 and 12 weeks after infection. Levels of circulating cardiac troponin I and circulating troponin autoantibodies were measured. Transthoracic echocardiography was performed. Myocarditis was histopathologically graded and cardiomyocyte apoptosis was quantified (TUNEL). RESULTS: Histopathologically relatively mild acute myocarditis followed by persistent cardiomyocyte damage was observed. Rate of cardiomyocyte apoptosis was the highest on day 5 (0.16 +/- 0.01% vs. 0.03 +/- 0.01% in controls, P < 0.001). Circulating troponin I levels were increased to day 5 (45.2 +/- 6.5 ng mL(-1), P < 0.005 vs. controls). Troponin autoantibodies were detected from 2 weeks after infection (20% of animals had autoantibodies at 2 weeks, 60% at 4 and 8 weeks and 20% at 12 weeks, P < 0.05 vs. controls). Fractional shortening remained decreased after acute myocarditis (0.36 +/- 0.02 at 4 weeks, 0.30 +/- 0.02 at 8 and 12 weeks vs. 0.41 +/- 0.01 before infection, P < 0.01) parallel to development of troponin autoantibodies. CONCLUSION: Troponin autoantibodies are formed in experimental virus induced myocarditis following troponin I release and cardiomyocyte apoptosis. The definite role of these autoantibodies remains to be further characterized.

Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-selectin mediate leukocyte binding to ischemic heart in humans.

OBJECTIVES: The expression of endothelial adhesion molecules and their functional significance in leukocyte adhesion to human myocardial blood vessels in acute myocardial infarction (AMI) were studied. BACKGROUND: Leukocyte extravasation, mediated by specific adhesion molecules, exacerbates tissue injury after restoration of blood supply to an ischemic tissue. Experimental myocardial reperfusion injury can be alleviated with antibodies that block the function of adhesion molecules involved in leukocyte emigration, but the relevant molecules remain poorly characterized in human AMI. METHODS: Semiquantitative immunohistochemistry and in vitro adhesion assays were used to study the expression and granulocyte binding abilities of different endothelial adhesion molecules in human AMI. Changes in the molecular nature of vascular adhesion protein-1 (VAP-1) were evaluated using immunoblotting. RESULTS: Certain endothelial adhesion molecules (intercellular adhesion molecule [ICAM-2], CD31 and CD73) were expressed in myocardial blood vessels homogeneously in normal and ischemic hearts, whereas others (E-selectin and peripheral lymph node addressin) were completely absent from all specimens. The synthesis of ICAM-1 was locally, and that of P-selectin regionally, upregulated in the infarcted hearts when compared with nonischemic controls. Vascular adhesion protein-1 showed ventricular preponderance in expression and alterations in posttranslational modifications during ischemia-reperfusion. Importantly, P-selectin, ICAM-1 and VAP-1 mediated granulocyte binding to blood vessels in the ischemic human heart. CONCLUSIONS: Human P-selectin, ICAM-1 and VAP-1 appear to be the most promising targets when antiadhesive interventions preventing leukocyte-mediated tissue destruction after myocardial ischemia are planned.

Propranolol and sotalol metabolism after a drinking party.

The effects of alcohol consumed during an evening party on the metabolism and response of beta-adrenergic blocking drugs were investigated in healthy subjects. The drugs were given 12 hr after the first drink. The plasma clearance rate of propranolol, metabolized in the liver, increased, while that of sotalol, which is eliminated unchanged, was reduced. Plasma propranolol levels, but not those of sotalol, were related to blood alcohol content. The blood pressure-reducing effect of propranolol diminished after alcohol and that of sotalol increased. Both drugs reduced the heart rate after alcohol although they were unable to cancel out entirely the alcohol-induced increase in the heart rate. The results show that drinking may alter the metabolism of beta-blocking drugs. Drinking habits must be considered in therapy with beta-blocking drugs or when seeking reasons for angina pectoris or arterial hypertension that does not respond to therapy.

Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.

The long-term behaviour of cellulose sponge implants, 10 x 10 x 5 mm in size, and tissue reactions in and around them were examined in the subcutaneous tissue of the rat from 1 to 60 weeks after implantation. The cellulose sponge used was filled up with connective tissue 4 to 8 weeks after implantation. Histologically, moderate foreign body tissue reaction inside the implant, the appearance of cracks and fissures, spotty colouration, and softening of the pore walls were observed up to 16 weeks after implantation. Later, the foreign body reaction inside the sponge became milder, the spotty colouration disappeared and micropores enlarged in the viscose cellulose matrix. Histomorphometrically, the cross-sectional area of the implants and the size of the pore wall fragments decreased, and the number of pore wall fragments increased significantly. The cellulose sponge used can be regarded as a slowly degradable implantation material. However, the time needed for the total disappearance of the cellulose sponge from subcutaneous tissue is longer than the 60 weeks.

The acute dose-related effects of ethanol on right ventricular function in anesthetized dogs.

The acute dose-related effects of small to moderate doses of ethanol on right ventricular functioning were studied on 18 anesthetized, artificially ventilated dogs in 39 sessions. Diluted ethanol (from 25-37.5%) was infused during 40 minutes, yielding total doses of 1.0 g/kg (n = 15), and 1.5 g/kg (n = 12) with corresponding venous blood ethanol peak concentrations of 1.38 +/- 0.25 and 2.41 +/- 0.31 mg/ml, respectively. Heart rate increased up to 16% in groups receiving ethanol. In the control group receiving the equivalent volume of saline (n = 12) heart rate decreased 14%. Pulmonary arterial systolic pressure increased from 24 +/- 3 to 27 +/- 3 mmHg and diastolic pressure from 11 +/- 2 to 14 +/- 4 mmHg (p less than 0.05) when the ethanol dose was 1.0 g/kg. The pulmonary arterial resistance increased from 620 +/- 135 to 805 +/- 185 dyn.s.cm-5 (p less than 0.01). The peak dP/dt decreased maximally by 20% with increasing ethanol doses. Stroke volume decreased maximally by 14% but due to the increase in heart rate, cardiac output even increased. The changes in end-diastolic volume and pressure were not significant. Hence, the ethanol increased heart rate and afterload of the right ventricle but depressed the myocardium.

Influence of ethanol on circulation in surface-induced hypothermia and subsequent rewarming.

Hypothermia and ethanol are often closely linked and in hypothermic accidents ethanol is often a contributing factor. To study the effects of ethanol on the circulation in hypothermic conditions, cardiac catheterization was carried out on 18 anaesthetized beagle dogs. They were divided into two groups. One gram of ethanol/kg of b.wt. diluted in saline was infused into the vena cava superior within 30 min to seven dogs. The dogs were then cooled between ice bags until the blood temperature in the ascending aorta was 25 degrees C and they were then rewarmed. The control group of 11 dogs was cooled and rewarmed without ethanol infusion. The heart rate first increased when cooling down to 33 degrees C and decreased thereafter in the control group. In the ethanol group heart rate increased during the ethanol infusion and remained high when cooling down to 33 degrees C and decreased thereafter. Heart rate was higher in the ethanol group throughout the experiments, and during rewarming the difference was significant. In the control group cardiac output first increased until a body temperature of 33 degrees C was achieved but then decreased. In the ethanol group cardiac output started to decrease after ethanol infusion. During rewarming there was a significantly higher cardiac output in the ethanol group, probably due to the higher heart rate. In the cardiac cycle the systolic period prolonged significantly (p < 0.001) in both groups when the body temperature decreased from 37 degrees C to 25 degrees C whereas the diastolic period remained quite stable. The contraction phase was also affected by the cooling. The changes in contraction force cannot be seen in dP/dt alone because dP/dt values first increased significantly when cooling from 37 degrees C to 33 degrees C but then decreased. Ejection fraction, systolic period, and the systemic vascular resistance increased despite the reduction of the dP/dt and thus we conclude that the contraction force is augmented in hypothermia. In the ethanol group the myocardium seems to be depressed due to ethanol. In the early phase of cooling heart rate increased but cardiac output decreased in the ethanol group, indicating the decreased ability of the heart to respond to cooling in the presence of ethanol. The time constant of exponential pressure fall (tau) increased linearly with cooling from 37 degrees C to 25 degrees C and recovered with rewarming in both groups. Changes in negative dP/dt coincided with the changes in the time constant of exponential isovolumic pressure fall. Ethanol did not influence relaxation. All the parameters we checked recovered to normal during rewarming.

The acute dose-dependent effects of ethanol on canine myocardial perfusion.

The acute effects of ethanol (1.0 g/kg and 1.5 g/kg, n = 4 and n = 5, yielding blood concentrations of 1.3 +/- 0.2 mg/ml and 2.4 +/- 0.3 mg/ml) on myocardial perfusion were studied in anesthetized, thoracotomized, artificially ventilated dogs by using a radioactive microsphere technique. The control group (n = 5) received saline. The smaller dose of ethanol decreased perfusion in the left ventricular myocardium from 0.737 +/- 0.122 to 0.555 +/- 0.122 ml/g/min (NS), whereas the greater dose nonsignificantly increased it, from 0.744 +/- 0.115 to 0.819 +/- 0.119 ml/g/min (p < 0.01 between the groups). These changes were most evident in subendocardial layers (p < 0.01 both within the groups and between the groups). The greater dose of ethanol increased systemic vascular resistance (p < 0.01 when compared to the control group). The changes in right ventricular myocardium were insignificant. The acute effect of ethanol on coronary blood flow is dose-dependent with small to moderate doses reducing demand for left ventricular flow but with increasing doses evoking not only an increase in demand for flow but also an increase in flow.

Evaluation of eosin-fluorescence in the diagnosis of sudden cardiac death.

The diagnostic significance of the eosin-fluorescence method was evaluated and compared with the enzymehistochemical beta-hydroxybutyrate-dehydrogenase method (beta-HBDH) and the degree of hyperchromasia in 568 samples from 24 bets-HBDH-negative and beta-HBDH-positive sudden cardiac deaths (SCD) and 23 non-cardiac deaths as controls. The effect of autolysis was investigated separately on isolated human hearts at +4 degrees C and +22 degrees C. All samples were examined without any knowledge of clinical or autopsy data. Normal olive-green fluorescence was observed in only five individuals of the control group and two in the beta-HBDH-negative one. The frequency of the yellow-fluorescence and hyperchromasia increased from the control subjects towards the beta-HBDH-positive-ones. The difference in frequency distribution of fluorescence between the groups was statistically highly significant (P less than 0.001). The change in fluorescence did not correlate with the loss of beta-HBDH-reaction or the distribution pattern of hyperchromasia. Postmortem autolysis did not change the fluorescence significantly but the 78% wrong positive subjects in the control group render this method too sensitive and unreliable for medicolegal purposes.

Oxidative enzyme activities and respective histochemical reactions in ischemic rat myocardium.

To evaluate the diagnostic role of histochemically demonstrated aerobic dehydrogenases in ischemic myocardial injury NADH-diaphorase, succinate dehydrogenase (SDH), beta-hydroxybutyrate dehydrogenase (HBDH) and malate dehydrogenase (MDH) were demonstrated histochemically and the corresponding enzyme activities were measured biochemically in isolated perfused rats hearts after global ischemia from 0 to 12 h. The present data show that the enzyme-histochemical methods when used properly are more sensitive indicators of early ischemic injury than classical histological staining procedures. From the enzymes tested here the histochemical demonstration of HBDH turned out to be best suited for use when suspecting ischemic myocardial injury at autopsy.

A modified method for examining the cardiac conduction system.

A revised technique is described in which the sino-atrial node, the atrio-ventricular node and the distal part of the His bundle and the bundle brances of the cardiac conduction system are demonstrated in longitudinal sections through cutting of 4-5 blocks. The longitudinal sectioning allows observation of continuity between different components of the cardiac conduction tissue. This method reduces the workload and hopefully encourages the pathologist to make the examination of the cardiac conduction system a part of the routine procedure.

Histopathological findings of cardiac conduction system of 150 Finns.

One hundred and fifty heart specimens were collected from the cases submitted for autopsy in the Department of Forensic Medicine in the University of Turku in March-May 1995 and May-July 1996, respectively. The cardiac conduction system (CCS) of these hearts were examined in order to find out the histopathological changes in the CCS of Finnish persons and their forensic pathological significance. There were 94 males and 56 females. Almost all age groups were included. The results revealed that in most of the persons the fibrous tissue and fatty tissue in the CCS increased with aging. In about half of the persons, there was deposition of calcium in the central fibrous body, pars membranacea, and the top of the musculature in the interventricular septum. In seven cases, the atrioventricular node (AVN), His bundle (HB) or bundle branches (RBB, LBB) were compressed by the calcium deposition. Hemorrhage, inflammation, amyloidosis, tumor, fatty infiltration and developmental malformations were observed in 31 cases. Twenty-eight cases died of myocardial infarct, among them, no involvement of the CCS could be observed. The authors concluded that routine examination of the CCS is helpful for revealing diseases of the CCS and improving the quality of forensic pathological diagnosis.

Alzheimer changes are common in aged drivers killed in single car crashes and at intersections.

With increasing age, diseases affecting the cognitive functions are more frequent. These diseases may increase the risk for fatal car crashes. We analyzed the frequency of neuropathological alterations characteristic of Alzheimer's disease (i.e. neuritic and diffuse plaques, and neurofibrillary tangles) in two association areas of the brain, parietal and frontal cerebral cortex, from 98 fatally injured aged drivers. In the age groups of 65-75 and over 75 years of age, 50% and 72% of the drivers, respectively, had neuritic plaques in either parietal and/or frontal cortex. In 14% of all killed drivers the number of neuritic plaques reached the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) age-related histologic score C, which indicates the diagnosis of Alzheimer's disease (AD), and an additional 33% had score B, which suggests the diagnosis of AD. Neuropathological AD changes were most common in the brains of drivers killed in single vehicle crashes, followed by multivehicle crashes at intersections and least common in multivehicle crashes elsewhere, but the differences did not reach statistical significance. In a great majority (80-85%) of cases the killed aged driver was the guilty party of the crash. The results imply, that incipient AD may contribute to fatal crashes of aged drivers, and therefore the forensic autopsy of these victims should include neuropathological examination.

Identification and quantification of polychlorinated biphenyls and some endocrine disrupting pesticides in human adipose tissue from Finland.

Organochlorine pesticides and polychlorinated biphenyls (PCBs) were analysed in extracts of human adipose tissue. The samples consisted of abdominal, mammary, and perirenal fat tissue of 27 Finnish adult males and females. Lipids were separated from the low-molecular compounds by preparative gel permeation chromatography (GPC) and the extracts were further cleaned-up using Florisil chromatography. The compounds were analysed in the extracts by gas chromatography (GC) using electron capture detection and by GC-mass spectrometry in the selected ion monitoring mode. Of the 23 analysed pesticide residues only seven could be detected in the extracts. All the extracts contained the DDT metabolite 4,4'-DDE, hexachlorobenzene, and PCBs. Other compounds found in the extracts were 4,4'-DDT, 4,4'-DDD, pentachlorobenzene, and beta- and gamma-hexachlorocyclohexane (HCH). The antiandrogenic 4,4'-DDE was the most abundant pesticide with concentration levels ranging from 3.5 to 3229 ng/g lipids (ppb). The mean concentration of DDE was 567 ppb, of pesticides in all 1008 ppb and of PCBs 504 ppb. Several of the identified compounds have been shown to exhibit endocrine disrupting effects. Statistical analysis showed a positive age correlation of DDE in females and hexachlorobenzene in males. No statistically significant differences were found for concentrations of individual compounds between sexes.

The hypotonic hemolysis and the protective action of lysophosphatidylcholine.

Lysophosphatidylcholine (LPC) protected against hypotonic hemolysis during rapid swelling of erythrocytes in spherocytic concentrations without any increase in the maximal cell volume. The total prelytic potassium release was increased, but the prelytic potassium release per cell was the same or decreased in the presence of LPC. The comparison of measured and calculated cell volume increase suggested that the prelytic potassium release must have been connected with a considerable sodium influx suggesting serious damage of the cell membrane already during swelling in the absence of LPC. Gradual swelling shifted hemolysis to more dilute solutions and LPC did not further increase the shift. We suggest that LPC protects the erythrocytes against hypotonic hemolysis during rapid swelling via a prevention of the initial membrane damage through altering the interaction between lipid bilayer and membrane cytoskeleton in the same way as occurs during incubation in a medium with low ionic strength.

[The amyloidosis in cardiac conduction system].

OBJECTIVE: To investigate the relationship between arrhythmia, cardiac failure and amyloidosis of cardiac conduction system (CCS). METHODS: CCS from 6 cases with cardiac amyloidosis were sampled using technique designed by the authors. The CCS were studied by Congo red staining and polarization microscopy. RESULTS: Pathological changes could be found in the CCS of these 6 cases. It was serious in sino-atrial node while slight in atrio-ventricular conduction system. The changes in atrio-ventricular conduction system were exacerbated from proximal part (atrio-ventricular node) to distal part (left bundle branch). In the 3 cases with sinus bradycardia and conduction blocking manifested by electrocardiogram, the pathological changes in CCS were more extensive and serious than that of other cases. CONCLUSION: The amyloidosis in CCS is one of the pathological bases of arrhythmia.

Medicolegal investigative system and sudden death in Scandinavia.

The Nordic region consists of Denmark, with the autonomous territories of the Faeroes and Greenland, Finland, with the autonomous Aland Islands, Iceland, Norway, and Sweden. However, this review deals only with the situation in Norway, Denmark, Sweden and Finland. In these four countries only a physician can confirm that a person is dead and practically any physician can certify a death due to natural causes if he knows the cause of death. A clinical autopsy can be performed to confirm the diagnosis with the permission of the relatives. The regulations concerning the reporting and investigation of sudden unexpected and non-natural deaths are very similar in all Nordic countries. In general those deaths, which are criminal, suspicious, accidental, suicidal, sudden and unexpected, unexplained or in any way not due to natural causes, cannot be certified by a doctor and must be reported to the police for medico-legal investigation. Forensic Medicine exists as a medical specialty at present only in Finland and Sweden. The total autopsy rates vary from country to country, with the lowest autopsy rate in Norway-10.6% of all deaths, followed by Denmark-16%, Sweden-22% and Finland-31.1%. Despite the apparent similarities in legislation regarding those deaths which should be referred for medicolegal examination, the practices differ so much in the different countries that it is, in fact, not possible to make many valid comparisons. The number of Group XVI-cases (symptoms and ill-defined causes of death) of the International Classification of Diseases (ICD) in the mortality statistics of each Nordic country seems to be inversely correlated to the autopsy rate. This gives some indication of the efficacy of the certification of death in a given country. Finland, having the highest autopsy rate, has the lowest number of ill-defined causes of death. The declining autopsy rate in all Nordic countries seems to be in line with the international trend. If this tendency continues, it will, without doubt, have a negative impact on the reliability of mortality statistics in general and, in the individual case, increase the possibility of incorrect classification of the mode of death. In the worst instance this might result in failure to detect homicide.

Survey of medico-legal investigation of homicides in the city of Turku, Finland.

Finland has one of the highest homicide rates in western Europe but the clear-up rate is also exceptionally high. To create a detailed analysis of homicides (n=161) in a region of western Finland during the 20-year period from 1983 to 2002 all available information was collected retrospectively from multiple sources including medico-legal reports, reports of police investigations, and court proceedings. Among the data collected and analysed were location, time, circumstances, and motives of homicides, forensic pathologists' role at the scene-of-the-crime and in court, autopsy findings, demographics and substance abuse of both victims and offenders, criminal background, forensic psychiatric examinations, and the verdicts of the offenders. A typical homicide was committed without premeditation during the weekend by a drunken male with a knife at hand and with a history of violent crimes. The perpetrator was later found guilty of manslaughter and received a prison sentence. Some noted shortcomings both in the police and the medico-legal investigation procedures are discussed, as well as the forensic pathologist's role in the judicial process in Finland.

[Sudden death caused by malignant testicular tumors]. / Plötzlicher Tod durch maligne Hodentumoren.

Sudden unexpected death of apparent healthy young males due to malignant tumours are extremely rare in forensic autopsy material. We report on three such cases dying of pulmonary embolism caused by metastazing tumour of the testis. In each subject the tumour was localized to the right testis and consisted of two types of tumour tissue: Seminoma and embryonic carcinoma, the latter of which was always the metastazing one. Each of the cases represent different aspects of forensic medicine. In the first subject the tumour had remained undiagnosed in a recent medical examination. The second one underwent surgery because of acute abdomen and died intraoperatively and the third one died suddenly during antibiotic treatment, because the tumour had been mistakenly diagnosed as orchitis.