[Comparative studies on the cultivability and morphology of rat hepatocytes isolated by trypsin or collagenase]. / Vergleichende Untersuchungen zur Kultivierbarkeit und Morphologie von Rattenhepatozyten, die mittels Trypsin oder Collagenase isoliert wurden.

In the present study rat hepatocytes isolated either by trypsin or by collagenase were investigated concerning its cultivability and its morphological properties. The cultivation was carried out as monolayer for 24 h. The yields of cells prepared by trypsin or collagenase amounted to 8 x 10(7) cells per liver and 15-30 x 10(7) cells per liver, respectively, with viabilities measured by trypan blue exclusion test of 70-80 and 90-95%, respectively. Nonhepatocytes were not taken into consideration. Using the electron microscopy it could be established that both freshly isolated and 24 h cultured hepatocytes were intact. There were no morphological differences between cells isolated by trypsin and cells isolated by collagenase. After 24 h cultivation hepatocytes prepared by trypsin showed a little tendency in forming a slightly flattened appearance and in forming intercellular contacts.

Ultrastructure of the liver after hypoxia in the postnatal period.

Repeated phases of hypoxia (8 h daily for 2 to 5 days at pO2 11.33 kPa = 5.000 m in altitude) were induced to Sprague-Dawley rats in the postnatal period as well as up to the 64th day of age, and after different recovery phases the ultrastructure of hepatocytes was qualitatively and quantitatively analysed. Major results were as follows: 1. Increases in body and liver weights were delayed but were balanced off after 64 days. 2. Qualitative alterations are reversible spherical transformations of mitochondria, a degradation of lipids and a slight increase in autophagocytosis. 3. The quantitative mitochondrial parameters (volume density, number per unit area, average volume) were not even adjusted to control values after 64 days. Granular endoplasmic reticulum and ribosomes/polysomes were insignificantly reduced in comparison to control animals, structure and arrangement are regular. Lipids and glycogen were differently altered. 4. The findings of the hepatocytes after postnatal hypoxia were reversible, though the majority of parameters had not yet returned to normal after 2 months. An adaptation to repetitive hypoxic conditions is not provable.

Quantitative ultrastructure of isolated Kupffer cells of rat liver.

The average volume of isolated Kupffer cells of rat liver is 821 +/- 64 microns 3, the average surface being 423 +/- 24 microns 2 (599 microns 2, with cell processes included). The surface structure (pseudopodia, lamellipodia, filopodia, microvilli) of isolated cells is much less developed than that of Kupffer cells in situ. By morphometric characterization volume densities are 0.1264 +/- 0.0077 (SE) for mitochondria and 0.3591 +/- 0.0169 for lysosomal structures. The volume of mitochondria amount to 0.79 +/- 0.04 microns 3.

Morphometrical characterization of isolated rat hepatocytes.

Mononuclear hepatocytes account for 81% of all isolated hepatocytes of adult rats and are 3800 +/- 209 microns3 in volume with a surface of 1191 +/- 44 microns2, while the other 19% are binuclear cells, 4735 +/- 688 microns3 in volume and 1347 +/- 72 microns2 in surface. The plasma membrane contains few microvilli and different amounts of blebs. The organelle-free ectoplasmic zone beneath the plasma membrane is 1-6 microns in width. The cell organelles are centralized around the cell nucleus. The mitochondrial volume density (Vv) is 0.402 +/- 0.028; this is twice as high as that of hepatocytes in situ of adult rats. This result together with an about 50% volume decrease of isolated versus hepatocytes in situ supports the interpretation that unstructured ground plasma in the form of cytopheres is emitted into the surrounding space, probably by shedding of cytoplasmic protrusions, leading to a rise in relative mitochondrial volume levels. To avoid misinterpretation of results obtained from studies directed onto the metabolism of isolated hepatocytes, due consideration should be given to these findings.

Ultrastructure and quantitative composition of isolated endothelial cells of rat liver.

Isolated endothelial cells (n 110; 350-540 microns3 in volume) of the sinusoid of rat liver were found to differ from in situ endothelial cells by their rounded shape with the nucleus usually in the centre and fenestrae of the sieve plates across the entire cytoplasm. Their volume density is 0.2814 (SE 0.0085). Some of them tend to form complex, ramified structures as well as clefts and vacuoles up to 1 micron in size. Dense bodies are other structures quite often seen in endothelial cells (Vv 0.0189, SE 0.0085). Mitochondria, on the other hand, are extremely rare (Vv 0.0023, SE 0.0009). Both the preservation and relocation of sieve structure after isolation may probably be due to the cytoskeleton which in vivo causes changes of size and number of endothelial cell fenestrae under physiological and pathological conditions.

Ultrastructural morphometric examinations on the protection provided by prostacyclin against myocardial ischaemia.

Myocardial ischaemia in the dog lasting for three hours is followed by swellings of the mitochondria and dilatation of the sarcoplasmic reticulum. The volume density (Vv) of mitochondria in the peripheral ischaemia zone is 0.2890, that of sarcoplasmic reticulum is 0.0101. Prostacyclin was administered either i.v. or by triggering in conjunction with an intra-aortic balloon pump which led to an increase in the volume density of the mitochondria, mainly as a result of an increase in the volume of average mitochondria. The dilatation of the sarcoplasmic reticulum is reversed. It was not possible to prove in our investigations that prostacyclin provides protection against myocardial ischaemia.

Postnatal development of myocardial cells after oxygen deficiency in utero.

Pregnant rats were kept at a simulated altitude hypoxia (5000 m = pO2 11.38 kPa) for eight hours every day from the 16th to 21st day of pregnancy. The heart musculature of the left ventricle of newborn male rats underwent electron microscopic qualitative and morphometric quantitative examinations on the 2nd, 5th, 11th and 22nd day. Prenatal hypoxia leads to delayed development of the heart. The reduced weight on the 2nd day compared with that of control animals is compensated on the 22nd day. Prenatal hypoxia causes matrix disintegration and cristolysis of the mitochondria. Volume density, which is lower on the 2nd day (0.24: 0.29 (control animals], reaches that of the controls by the 22nd day. But the specific surface density of the cristae mitochondriales is markedly higher in the animals subjected to hypoxia (2nd day 31.0: 20.6; 22nd day 44.0: 38.1). Prenatal hypoxia causes an increase, in the postnatal period, in the autophagocytosis processes, which normally already exist in the heart musculature; this is recognizable by an increase in the volume density of the autophagic vacuoles (2nd day 0.0049: 0.0034; 22nd day 0.0098: 0.0045). The changes observed are regarded as reversible, i.e. as adaptive processes.

The influence of "hylase" on the ultrastructure of ischaemic heart muscle changes.

A two-hour acute ischaemia of the myocardium was induced in dogs by ligature of the coronary arteries and the effect produced on the qualitative and quantitative ultrastructure of heart muscle cells by infusing "hylase", a hyaluronidase preparation, was examined. The changes in both the central and peripheral ischaemic zones following hylase infusion are more severe than after ischaemia alone. Particularly the mitochondria showed an increased dissolution of the outer membrane. In the case of ischaemia the percentage of mitochondria in the peripheral zone is 29.6%, in the central zone 27.6%; after hylase infusion it is 25.9% in the peripheral zone and 32.3% in the central zone. After hylase infusion, the percentage of sarcoplasm in the central zone rises to 120.7% and to 186.8% in the peripheral zone. In view of the fact that macroscopic, histological and histochemical findings also show negative effects, the infusion of hyaluronidase is not recommended.

[Experimental studies on surgical therapy of acute infarct]. / Experimentelle Untersuchungen zur chirurgischen Therapie des akuten Infarktes.

The authors examined the effects of reperfusion after temporary ischemia in 50 dogs. The morphologic alterations were documented by methods of electron microscopy, fluorescence microscopy, and histochemistry. Lactate and activity of glycogen phosphorylase were assessed. According to the results, the optimal ischemia interval is 30 to 60 minutes for rational application of reperfusion, while it is just possible after 120 minutes. After a 4-hours-period of ischemia reperfusion increase morphologic damage of myocardium and impairment of myocardial metabolism. Prolonged reperfusion of 7 days resulted in a reduced extent of infarction compared with controls. In non-ischemic myocardium the morphologic and metabolic alterations were less expressed. The effects of "no-reflow"-phenomenon and conclusions for clinical practice are discussed.

[Electron microscopical findings of the myocardium in Fallot's disease and of the ventricular septal defects (author's transl)]. / Elektronenmikroskopische Befunde der Herzmuskulatur beim Morbus Fallot und Ventrikelseptumdefekt.

Ultrastructural quantitative and qualitative investigations of the myocardium of 7 children with Fallot's disease and 3 children with a ventricular septal defect (VSD) have shown bizarre nuclei and a slight increase in the myofibril share of the volume (Fallot's disease: 51.22%; VSD: 56.85%). This may be interpreted as a sign of a cellular hypertrophy. The mitochondria have a proportion of 35.07% and 33.75%, respectively. The glycogen content is relatively high. The lipofuscin bodies originate mainly from mitochondria having a percentage of 1.43 and 1.67, respectively. Particular signs of a lesion could not be observed. From the ultrastructural findings on the morphology of heart muscle cells it can be deduced that operations of vitia may be safely carried out.

[Ultrastructural and functional changes of rabbit myocardium after cholesterol feeding]. / Ultrastrukturelle und funktionelle Veränderungen des Kaninchenmyokards nach Cholesterolfütterung.

The myocardium was ultrastructurally investigated immediately after 12 weeks of feeding in one part of the animals and in the remaining animals 27--30 months after termination of the feeding period. At the same time, parameters of the myocardial contraction were measured immediately after the feeding at isolated trabeculae of the left ventricle and 27--30 months after the termination of the feeding at the left ventricle in situ. After the 12 weeks of feeding the morphometrically estimated intracellular lipid content rises from 0.23% (normal) to 5.26%. The mitochondria decline from 36.98% (normal) to 29.08%. The sarcoplasmic reticulum (SR) changes in a vesicular or vacuolar manner respectively. The contractility index Vmax of isolated trabeculae decreases insignificantly. The resting potentiation and the diastolic stiffness are increased. 27--30 months after termination of feeding the lipid content and the mitochondria are normal in comparison to controls of the same age. The vacuolar changes of SR and its cellular portion are increased. The contractility index of the heart in situ max (dp/dt)/P is decreased insignificantly. The velocity of relaxation is enhanced in comparison with controls. By comparing ultrastructural and functional findings it can be seen that variations in the contraction performance are accompanied by stronger changes of the cellular structure immediately after the feeding than 27--30 months after the termination of the feeding. At this time continuing small alterations in the contraction performance of the heart in situ are observed in addition to the reversibility of various characteristics of damage together with an increase in the Ca++-transporting SR and its alterations.

Quantitative changes of canine diaphragm muscle cells following electrostimulation.

1. In 12 dogs the right part of the diaphragm was stimulated 4, 5, 8, 11, and 14 weeks by means of a pacemaker. 2. Essential findings have been the increase of the amount of glycogen after 5 weeks, the increase of myofilaments per myofibril after 8 weeks, and the increase in the share of mitochondria in the 11th week. After 14 weeks all values have reached again their initial level as the result of an adaptation. 3. From the quantitative structural findings it can be taken that the diaphragm musculature is not changed by the electrostimulation in such a way that the functional preconditions for covering heart muscle deficiencies by diaphragm flaps are given.

[Presence of a lysis factor of mitochondria in rabbit reticulocytes]. / Uber das Vorkommen eines Lyse-Faktors der Mitochondrien in Kaninchenretikulozyten

Incubation of isolated rat liver and beef heart mitochondria together with a fraction of nonhaemoglobin proteins from rabbit reticulocytes (reti-AS) leads to drastic structural damages such as a deformation and disruption of the outer membranes as well as a disappearance of the cristae structure. The vacuolized forms obtained in this manner reveal striking similarities to the known degradation ones of reticulocyte mitochondria in situ and after isolation of them. The extent of the lysis of mitochondria depends on the amount of reti-AS and on the temperature. The lysis is caused by a protein factor (MLF) which is not identical with the respiratory inhibitor RF present in the reti-AS as well. Like RF, MLF disappears during the maturation process of reticulocytes to erythrocytes. MLF triggers the penetration of RF into the mitochondria and thus the inhibition of the succinate oxidase and the NADH oxidase activity. EDTA inactivates RF but not MLF. MLF is bound to electron transfer particles from beef heart mitochondria. The experimental conditions used are supposed to be a model for the degradation of mitochondria in situ during the maturation process of reticulocytes.