A quantitative histochemical study of 5′nucleotidase activity in rat liver after ischaemia

Abstract

The lead salt method of Wachstein and Meisel15 has been applied using incubation media containing polyvinyl alcohol for the localization and quantification of 5'-nucleotidase (E.C.3.1.3.5) activity in cryostat sections from rat liver after ischaemia in vitro and ischaemia in vivo followed by different periods of re-perfusion. 5'-Nucleotidase activity at the bile canaliculi, especially in the pericentral areas, had already decreased after 60 min of ischaemia in vitro, although the total activity as measured densitometrically was not changed. After 120-240 min of ischaemia, a significant decrease of the total 5'-nucleotidase activity was found. At that stage, signs of irreversible cell damage were recognized. Short periods of re-perfusion (1 h) after ischaemia in vivo induced a decreased bile canalicular 5'-nucleotidase activity throughout the entire liver, but a restoration after longer periods of re-perfusion was observed (5, 24, and 48 h). Necrotic areas recognized by a decreased lactate dehydrogenase activity after all periods of re-perfusion showed decreased total 5'-nucleotidase activities. A correlation was observed between the decrease in bile canalicular 5'-nucleotidase activity and the disappearance of microvilli of the bile canaliculi. It is concluded that a decrease in the bile canalicular 5'-nucleotidase activity can be used as a very sensitive marker for ischaemic liver cell damage. Assessment of the irreversibility of the cell injury has to be determined using additional parameters such as a decreased lactate dehydrogenase activity.

Enzymatic heterogeneity of bovine retinal pigment epithelial cells in vivo and in vitro

“…It might be speculated that the extent of enzymatic reaction for membrane-associated enzymes is linked to the integrity of the RPE cells. Damaged cells show a decreased or absent reaction with substrate for lactate dehydrogenase in sections [13]. The lack of correlation between activities of membrane-associated proteases and lactate dehydrogenase in our study suggests that the heterogeneous pattern is not caused by the preparation of the tissue.…”

Lesion progression with time and the effect of vascular occlusion following radiofrequency ablation of the liver

“…Interference of consecutive RFA sessions within one liver due to hyperthermia in adjacent lobes raised some concern. Temperature recordings during RFA did not, however, indicate a rise in temperature in adjacent lobes, and no signs of ischaemic damage were seen in normal non-ablated liver tissue by histochemical evaluation of 5 -nucleotidase activity, a marker for ischaemic damage to plasma membranes 16 .…”

An in vitro method for comparing the efficacy of two preservation solutions in one canine liver using the 5‘-nucleotidase assay

“…5'-NT activity was demonstratcd using the metal salt method of Wachstein and Meisel (1957) as previously described [2,4]. Briefly, cryostat sections of unfixed liver specimens were cut (thickness 8 prn) and incubated for 20 min at 37°C in a medium containing 17% (wiv) polyvinyl alcohol (hot water-soluble, average molecular weight 40000 Da; Sigma, St.Louis, Mo., USA), 100 mM TRIS-maleate buffer (pH 7.2), 5 mM adenosine 5'-monophosphate (AMP; Merck, Darmstadt, FRG), 10 mM MgCI,, and 7.2 mmM lead nitrate.…”

“…5'-NT is an enzyme present in the plasma membranes of bile canaliculi and sinusoidal endothelium. Its activity and localization have been shown to be sensitive markers of ischemic injury in the rat liver [2,4]. A reliable, enzymehistochemical method has been developed in our laboratory that selectively demonstrates 5'-NT activity in cryostat sections of liver tissue and that allows direct assessment of the enzyme by light microscopy.…”