Quantitative analysis of the effect of ATP-MgCl2 and adenosine-MgCl2 on the extent of necrosis in rat liver after ischemia.

The effect of the administration of ATP-MgCl2 and adenosine-MgCl2 on the volume density of necrosis occurring 24 hr following 60 min of ischemia in rat liver has been studied. The extent of necrosis in the lobes submitted to ischemia has been assayed by morphometric analysis of fresh liver slices incubated in tetranitro BT. The administration of ATP-MgCl2 (1.25 mumole of each solved in 0.5 ml 0.9% NaCl) reduced the volume density of necrotic areas in the liver of a fasted rat from about 15% to almost zero, provided that the compounds are given as a continuous infusion spread over a period of 15 min and the administration is started before the circulatory flow is restored following ischemia. However, the extent of necrosis was not reduced by ATP-MgCl2 administration when ischemia was induced in the liver of a fed rat which showed a more massive necrosis (about 30%). Increasing concentrations of ATP-MgCl2 to 5 mumole did not result in any improvement. Adenosine-MgCl2 reduced the extent of necrosis after ischemia in a fasted rat in the same way as ATP-MgCl2. The conclusion is drawn that ATP as a direct source of energy and adenosine as a substrate for ATP-synthesis can protect the liver against ischemic damage, whereas MgCl2 plays a supporting role.

Naphthol-yellow-S protein content of individual rat hepatocytes as related to food intake and short-term starvation.

With regard to the protein content, as analysed cytophotometrically, of hepatocytes from rats kept under a 12L 12D photoperiod (photophase 7:00-19:00), the following facts have been established: 1) Hepatocytes of different classes of ploidy all demonstrate, more or less equally, daily variations in protein content and also its reduction after 24-h fasting. 2) With computer analysis of data obtained at eight time points during a period of 24 h, a sinusoidal curve of the protein content of individual mononuclear tetraploid hepatocytes throughout the day could be demonstrated with a maximum at 6:20 and a minimum at 18:20. 3) Animals, fed with meals via a dispensing machine from 23:00 to 24:00 only, show a similar sinusoidal curve but with higher amplitude, and a virtually identical mean value as those fed ad libitum. The maximum was found at 10:40, revealing a time lag of 12 h after food intake, the minimum at 22:40. 4) Trained animals deprived of food during the standardized feeding time revealed a moderate reduction of their hepatocyte protein content in the first 6 h, then a 6-h period with a steep fall followed by a slower reduction. After 24 h, the mean hepatocyte protein mass had decreased to 72% of that at the commencement of fasting at 23:00.

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy.

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.

Influence of nutritional state on ischemic damage in rat liver.

The influence of the nutritional state of rats on the volume of necrosis in the liver at 24 h after 60 min ischemia has been estimated using fresh liver slices incubated in tetranitro BT. The volume of necrosis is about 30% in an animal fed ad libitum and about 10% in rats fasted for 24 h. Standardization of the time of feeding using a food dispensing apparatus did not reduce the relatively high individual variation in the extent of liver cell necrosis after ischemia. The beneficial effect of fasting on the extent of liver cell necrosis induced by ischemia may be due to decreased acidosis and/or a change in metabolic status.

Experiences with standardized feeding of rats with an automatic food dispensing apparatus.

A simple automatic food dispensing machine can be used to train rats to a regime of meal-feeding during one hour at night. Apart from some 20% of the animals which fail to adapt but can be recognized early during training on the basis of weight curve, a population of animals is obtained which show a high degree of standardization of rhythmic changes related to food intake and apparently have an adequate supply of food as shown by the protein mass of liver cells.

Ploidy class-dependent variations during 24 h of glucose-6-phosphate and succinate dehydrogenase activity and single-stranded RNA content in isolated rat hepatocytes.

The time-dependent variations over 24 h of glucose-6-phosphate dehydrogenase (G6PDH) activity, succinate dehydrogenase (SDH) activity and single-stranded RNA (ssRNA) content have been investigated by cytophotometric analysis of cytochemically stained isolated hepatocytes of different ploidy classes from adult male rats. A marked variation of 48% over the day in G6PDH activity of the mononuclear diploid cells was revealed, but no significant variation in the binuclear tetraploid cells. The cells of the inbetween ploidy classes showed an amplitude of variation of 38% (binuclear diploid cells) and 24% (mononuclear tetraploid cells), respectively. All cells showed a maximum activity of the enzyme at the middle of the day and a minimum during the night. The relative enzyme activity per mononuclear diploid cell was significantly higher than the relative activity in the other cells, especially at its maximum. The variation of the SDH activity in hepatocytes isolated from the same rats was similar in all cells, irrespective of their ploidy class. The activity was highest at the end of the activity phase of the animals. The SDH activity per cell was directly proportional to the quantity of genome copies. The ssRNA content of the hepatocytes showed a time-dependent variation with a maximum during the resting phase of the animals and a minimum during their activity phase. The variation was larger in the mononuclear diploid cells than in the cells of other ploidy classes and the ssRNA content was also significantly higher in these cells than in the hepatocytes of other ploidy classes when calculated on the basis of genome copies. It is concluded that the large amplitude of variation over the day and the high relative amount of G6PDH activity and ssRNA content in mononuclear diploid cells is related to the function of these cells as stem cells of the liver parenchyma.

Plasma ornithine carbamyl transferase level as an indicator of ischaemic injury of rat liver.

The activity of ornithine carbamyl transferase (OCT) and glutamate pyruvate transaminase (GPT) in serum has been correlated with the extent of necrosis 24 h after different periods of ischaemia in rat liver. The extent of necrosis has been quantified as the volume density of necrosis in the total ischaemic liver lobes using tetranitro BT. The GPT-activity in serum is maximal between 1 and 5 h after different periods of ischaemia, whereas OCT reaches its maximum between 5 and 12 h after ischaemia. The total amount of leaked enzyme-activity as well as the peak value give a linear correlation with the extent of necrosis for OCT and GPT. There is a difference between the character of these two enzymes in that a small leakage of GPT does not indicate liver cell necrosis later on. However, the appearance of OCT in the blood, an enzyme localized in the mitochondrial matrix, has a predictive value for the extent of necrosis, likely to occur later on. GPT, an enzyme from the cytoplasm, can also occur in the blood during the reversible stage of liver cell damage.

A method for quantitative analysis of the extent of necrosis in ischemic rat liver.

A method has been developed for quantitative analysis of necrotic regions in lobes of the rat liver 24 hr after temporary ischemia. Essentially the method consists of cutting the liver lobes into 2-mm-thick slices by means of a specially developed cutting apparatus. The serial slices are incubated in a special holder with tetranitro BT, which enables a clear discrimination between necrotic (unstained) and undamaged (stained) tissue. All slices from the ischemic lobes are photographed in the holder and submitted to morphometric analysis on enlarged prints. This method to obtain an estimate of the volume density of necrotic regions is rapid, simple, and reliable and compares favorably with other methods used in the literature. The extent of necrosis after 15-min clamping of the afferent vessels is virtually nil; it rises with longer periods of necrosis to reach a value of +/- 80% after 120 min.

Glucose-6-phosphate dehydrogenase activity in individual rat hepatocytes of different ploidy classes. II. Time-dependent variations during 24 h.

The time-dependent variation of glucose-6-phosphate dehydrogenase (G6PDH) activity during 24 h has been investigated by cytophotometric analysis in cytochemically stained, isolated hepatocytes of different ploidy classes from adult male rats. It was found that the amplitude of the variation in the total amount of parenchyma was 22%, with a maximum in the middle of the resting phase of the animals (13%). This small variation could not be detected subjectively in cryostat sections of liver that had been stained histochemically; nor could a metabolic zonation of the liver parenchyma be observed except the intermediate zone, which stained more strongly for G6PDH activity at 13% and 22%. The quantitative study on the isolated hepatocytes, however, revealed a relatively strong variation of 48% during the day in enzyme activity of the mononuclear diploid cells and no significant variation in the binuclear tetraploid cells. The cells of the in-between ploidy classes showed an amplitude of variation of 38% (binuclear diploid cells) and 24% (mononuclear tetraploid cells). All cells showed a maximum activity at 13% and a minimum activity during the night. It is concluded that this ploidy-dependent variation might play a role in the generation of pentoses for RNA synthesis. The findings indicate that for metabolic studies the heterogeneity of the liver parenchyma should be taken into account with respect to the different ploidy classes, as well as the well-known metabolic zonation of the acinus.

Glucose-6-phosphate dehydrogenase activity in individual rat hepatocytes of different ploidy classes. I. Developments during postnatal growth.

The glucose-6-phosphate dehydrogenase (G6PDH) activity of isolated male rat hepatocytes has been investigated in relationship to the ploidy classes of the cells during the first 20 weeks of postnatal growth. The G6PDH activity in the individual cells was measured with an improved quantitative cytochemical method. The data obtained showed that throughout the whole period of postnatal growth there existed a proportional relationship between the genome copies per cell and the amount of G6PDH activity per cell for binuclear diploid (BD), mononuclear tetraploid (MT) and binuclear tetraploid (BT) cells but not for mononuclear diploid (MD) cells. In the MD cells, which are the stem cells of the liver parenchyma, the activity measured was 1.5 times higher than expected. Furthermore, during postnatal growth, the G6PDH activity per hepatocyte was low at the age of 2 weeks, increased somewhat after weaning (5 weeks) and then more dramatically after 8 weeks to reach a maximum between 12 and 16 weeks. This development occurred in MT and BT cells at an earlier age than in MD and BD cells, in which the increase in enzyme activity followed some 3 weeks later. Castration of the rats before puberty did not influence the development of the amount of G6PDH activity per cell of any of the ploidy classes.

The value of enzyme leakage for the prediction of necrosis in liver ischemia.

Following the clamping of the afferent vessels of the left lateral and median lobes in rat liver, a considerable part of these lobes show signs of necrosis 24 h after 90 min of ischemia, whereas no necrotic areas can be detected after 30 min interruption of the blood flow. The purpose of this study was to examine the value of an analysis of the leakage of enzymes from the liver parenchyma in the early phase after restoration of the blood flow after ischemia for a prediction of the occurrence of necrosis. Leakage of the enzymes GPT, GOT and LDH can be detected in the blood plasma with a maximum activity between 1 and 5 h both following 30 and 90 min of ischemia; a considerable difference in clearance is observed, however, in the period afterwards, the normal situation being reached after 24 h with the 30-min ischemic period, but not following the 90-min period. With use of an enzyme histochemical reaction, in situ a depletion of LDH-activity in the hepatocytes could be detected within a short period of time after 30 min temporary ischemia and a restoration during the following period of 24 h; the decrease in LDH-activity persisted during 24 h with a 90-min period of ischemia. Electronmicroscopically cytoplasmic blebs arisen from hepatocytes are observed in the lumen of sinusoids immediately after 30 min of ischemia, whereas after 90 min of ischemia actual leakage of cytoplasmic material takes place through the damaged surface of the hepatocytes. Enzyme leakage probably takes place via these both types of shedding of cytoplasm. It is concluded that the enzyme leakage as such cannot be used as a discriminating test between reversible and irreversible damage of the liver parenchyma.