A new sensitive cardiac Troponin T rapid test (TROPT) for the detection of experimental acute myocardial damage in rats.

Cardiac Troponin T (cTnT) is a cardiac structural protein which is released in the circulation during myocardial cell damage. In this study we addressed the question of whether beta-sympathomimetic induced myocardial cell damage in rats can be detected in blood by the TROPT sensitive rapid test strip which is primarily manufactured for the detection of acute myocardial infarction in humans. Sixteen male rats and 16 female animals were treated once with orciprenalinesulphate s.c. to induce tachycardia. The control group which consisted of 16 rats of both sexes received vehicle (physiological saline). The heart rate of two groups of 10 rats (5 of each sex) was measured after orciprenaline or saline treatment. After 1-2 hours we could demonstrate an increased heart rate in the orciprenaline group. Furthermore in this group we could show elevated cTnT levels in peripheral blood measured with the enzyme-linked immunosorbent assay (ELISA) Enzymun-Test Troponin T for troponin T and a positive reaction of the TROPT sensitive test strip. When compared with the ELISA method, the test strip showed a positive reaction at cTnT levels of 0.64 ng/ml upwards. After 24 hours and 96 hours half of the animals were sacrificed for histological examination of the heart tissue. After 24 hours all orciprenaline treated and examined animals showed myofibrillar degeneration of the myocardial cells. The second half of the animals sacrificed 96 hours after treatment with the sympathomimetic drug showed reparative fibrosis of the myocardium. All animals with myocardial damage due to orciprenaline showed a positive test strip result 2 hours after injection. Twenty-four hours after injection only 8 of the 16 animals had a positive test strip result although histological cell damage was demonstrated. All animals treated with physiological saline had negative results of the test strip and showed no signs of myocardial cell damage. We conclude that the TROPT sensitive test strip is a rapid and reliable screening tool for detecting myocardial cell alteration in rats without the need of an expensive and time-consuming ELISA procedure if it is used in early phases of the experiment because all animals with myocardial damage were identified by the test 2 hours after induction of the damage. If the time range between the induction of the injury and the use of the test strip or the ELISA procedure is too wide, in our experiments 24 h, false negative results may occur.

Comparison of cardiac Troponin T and cardiac Troponin I concentrations in peripheral blood during orciprenaline induced tachycardia in rats.

In this study we addressed the question of whether the measurement of the cardiac structure protein cardiac Toponin I (cTnI) in serum is also able to detect marked myocardial cell damage in rats like cardiac Troponin T (cTnT). To answer this question 5 male rats and 5 female rats were injected with orciprenaline to induce myocardial cell damage. Further 5 animals of each sex received physiological saline as control. We could demonstrate that troponin I was rised significantly 6 hours after injection in serum as well as cTnT. 24 hours after injection cTnI was elevated on a lower significance level compared to cTnT. All values returned to normal 96 hours after the injection. We conclude that cTnI was also able to detect a marked myocardial cell damage in rats but we could show that cTnI was elevated on a lower significance level compared to cTnT. Furthermore we found that a cTnI increase to at least 4.1 ng/ml is necessary to detect marked myocardial cell injury in this rat tachycardia model.

Sephadex induced bronchial hyperreactivity in the rat: hematology, histology, histochemistry and immunohistology of the lung.

24 hours after an i.v. injection of 2 mg Sephadex G 200 particles ovalbumin sensitized Sprague Dawley rats show an antigen specific bronchial hyperreactivity and an unspecific hyperreactivity against serotonin. The aim of this study was to investigate the effects of Sephadex on blood parameters and lung pathology to find the morphological substrate of bronchial hyperreactivity in this animal model. In the blood neutrophilia (p < 0.01) but no eosinophilia was present. We conclude that a blood eosinophilia needs not to be necessarily correlated with hyperreactivity of the airways like claimed by other investigators for this animal model. Histologically we found that Sephadex particles are trapped in smaller-diameter arteries of the lung and lead to a granulomatous arteritis consisting mainly of ED1 positive and widely ED2 negative macrophages interspersed with eosinophils and neutrophils. Larger vessels not occluded by particles showed perivascular oedema with infiltration of eosinophils. We report here for the first time a significant hypertrophy of PAS positive goblet cells (p < 0.01) accompanied by a peribronchial infiltration with eosinophils (p < 0.01) and macrophages positive for ED1, ED2 and Ox-6 (p < 0.01) but not Ox-19 positive T-lymphocytes. The authors suggest that the peribronchial inflammation contributes importantly to the onset of bronchial hyperreactivity in this animal model and that the hypertrophy of goblet cells indicates the pathophysiological importance of peribronchial leukocytes.

Diagnostic efficiency of troponin T measurements in rats with experimental myocardial cell damage.

A cardioselective parameter has been available for about 2 years since the development by KATUS of an immunoassay for cardiac Troponin T (TnT). The major advantages of this TnT assay are its cardiospecificity and its sensitivity. The parameters usually determined in toxicity studies in rats to detect alterations in the myocardial cells, e.g. aspartate aminotransferase (ASAT), creatinine kinase (CK) and lactate dehydrogenase (LDH), are either of low sensitivity in this species or give falsely high results as the consequence of stress or haemolysis. We therefore investigated in the present study how well Troponin T, determined with the ELISA Troponin T from Boehringer Mannheim, can detect experimentally induced myocardial lesions in rats. In order to achieve hypoxic damage of the cardiomyocytes in these experiments in rats, male Sprague-Dawley rats were given two doses of 4 mg/kg isoprenaline each (Aludrin from Boehringer Ingelheim, FRG) subcutaneously. The second dose was given 7 h after the start of the experiment. Serum samples were analysed for Troponin T (TnT) levels and, for comparison, aspartate aminotransferase (ASAT), creatine kinase (CK), and lactate dehydrogenase (LDH). Histological examinations of the heart muscle were performed 24 and 96 h after the first injection. As expected, histological examinations of the isoprenaline-treated animals revealed marked myofibrillic degeneration of the myocardium 24 h after the first injection. Markedly elevated serum TnT levels (up to 7.9 ng/ml) were already evident in these animals after 6 h. TnT values decreased with time, but were still statistically significant after 48 h. Of the well-established indicators for diagnosing myocardial infarction, only ASAT showed transient statistically significant increases over 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

[Elevation of 7-dehydrocholesterol concentrations in serum and liver and pericentral peroxisome proliferation in hepatocytes of rats after inhibition of cholesterol biosynthesis by BM 15,766]. / Erhöhung der 7-Dehydrocholesterol-Konzentrationen in Serum und Leber und perizentrale Peroxisomenproliferation in Hepatozyten von Ratten nach Hemmung der Cholesterin-Biosynthese durch BM 15.766.

Sprague-Dawley rats of both sexes were treated for three months with BM 15,766, an inhibitor of cholesterol biosynthesis in conjunction with standard or high-fat and high-cholesterol diets. In serum and livers of all drug-treated rats lowered cholesterol concentration associated with an increase of 7-dehydrocholesterol (7-DHC) was found. Electron microscopy of the liver showed a distinct proliferation of peroxisomes and an increase of dumb-bell shaped mitochondria in the pericentral zone 3. Abnormal-shaped peroxisomes with DAB-negative loops attached to their membranes were found in the intermediate zone 2. These alterations were more accentuated in drug-treated rats fed standard diet, then in treated rats receiving a high-fat and high-cholesterol diet. The observations demonstrate, that the increase of 7-DHC is due to the inhibition of 7-DHC-delta 7-reductase by BM 15.766 and emphasize the zonal heterogeneity of hepatocytes. The relevance of these observations for the investigation of the human Smith-Lemli-Opitz syndrome, in which also decreased plasma-cholesterol levels and an increase of 7-DHC were reported, is discussed.

Stimulation of bone marrow by administration of excessive doses of recombinant human erythropoietin.

Recombinant human erythropoietin (rhEPO) was administered intravenously to Beagle dogs in daily doses of 100, 500 and 3000 units/kg/day for 3 months. The high dose was more than 200-fold the therapeutic human maintenance dose. Such excessive rhEPO doses elicited extreme erythropoiesis. There was a dose-dependent stimulation of bone marrow fibroblasts, leading to bone marrow fibrosis in some of the high dose animals. The extent of myelofibrosis was intra- and interindividually different in various bones. Sternebrae proved to be practical for morphometric studies. The point-counting method was used for measurement. The portion of fatty tissue, sinusoids and fibrous tissue in the medullary space as well as the number of blood vessels and megakaryocytes were calculated. Such experimental conditions are not of relevance in human patients whose rhEPO therapy is interrupted as soon as their PCV reaches 35 vol%. Experimentally induced myelofibrosis should therefore not be considered as a risk in patients receiving therapeutic doses of rhEPO.

Automatic image analysis as a valuable aid for the objective assessment of the enlargement of hepatocytes in toxicological studies.

Evaluation of histopathological slides is a subjective science; objectivity is needed, especially with minimal alterations. Quantitation using measurement procedures is of special value in toxicological studies. The present study tests a method in which the hepatocytic nuclei were measured per area of liver tissue, excluding the sinusoidal space. Different grades of hepatocellular enlargement were induced by different dose levels of a test compound (combination of two diuretics). The method demonstrates the well-established dose dependence of liver cell enlargement and permits differentiation between slight drug-induced enlargement and the normal variation in cell size. Special reference is made to avoid measurement artefacts.

Incorporation of T4 phage DNA into a specific DNA fraction from the higher plant Matthiola incana.

Isolated T4 phage DNA (sigma=1.694 g/ml) is applied to seedlings of the crucifer Matthiola incana (DNA density sigma=1.698 g/ml). The phage DNA can partly be reextracted from the plants in a specific DNA fraction, which is predominantly characterized by its unusual high density (high density complex=HDC; sigma=1.724 g/ml). DNA:DNA hybridization studies show that phage specific DNA sequences are preserved in the HDC. Results of BrdUrd labeling of the plant DNA before and during incubation with T4DNA suggest that the HDC is composed of T4DNA and a plant DNA component of high density. The analysis of ultrasonicated HDC confirms this suggestion. The ability of plant cells to recognize and handle T4 DNA specifically is discussed.