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.

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.

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.

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.