Quantitative histoenzymological characteristics of changes in the red and white fibers of skeletal muscle tissue of the extremities in temporary ischemia and postischemic recirculation

Histoenzymological study of red and white fibers of limb skeletal muscles was performed in dogs with experimental acute occlusion of aorta trifurcation during ischemic and postischemic periods. A dramatic fall in the activity of aerobic enzymes was recorded in all observation periods (3, 6, 9 and 12 h). LDH activity rose considerably during short-term and descended slowly in prolonged ischemia. Red muscle fibers showed more substantial disorders in metabolism than white fibers. Recirculation after 6, 9 and 12 h of ischemia led to an appreciable decrease in the metabolic rate in muscle tissue. Irreversibility of metabolic alterations in acute ischemia lasting over 6 h may attest to the failure of the compensatory-adaptive mechanisms.     

Quantitative changes in liver dehydrogenase activity during temporary ischemia of the extremities and in the postischemic period

Experimental temporary occlusion of the dog hind limbs was produced. Histoenzymological changes in the liver induced by the temporary ischemia of the limbs and by ischemia with a 2-hour revascularization were subjected to comparative histophotometric examination. The data obtained indicate an appreciable rise in the enzymatic activity of hepatocytes at the early stages of experiments and increasing reduction in metabolic processes at the later stages. A substantial decrease was noted in the activity of the liver enzymes under study, with that decrease being especially pronounced at the later times in experiments with limb revascularization, which is accounted for by a far greater release to the bloodstream of metabolic products from the ischemized limbs.     

Changes in acid phosphatase activity in rat liver after ischemia

The effect of ischemia on the stability, i.e. the permeability of the lysosomal membrane of rat liver has been studied using quantitative histochemical analysis of acid phosphatase activity. Ischemia in vitro was performed for 0-240 min at 37 degrees C and ischemia in vivo for 60 min was followed by 1, 5, 24 and 48 h of reperfusion. Acid phosphatase activity was demonstrated in cryostat sections using naphthol AS-BI phosphoric acid as substrate and polyvinyl alcohol was added to the incubation medium to counteract diffusion phenomena. Ischemia in vitro up to 240 min did not affect the localization nor the total activity of acid phosphatase activity. After 60-min ischemia in vivo followed by 1-h reperfusion distinct areas showed decreased acid phosphatase activity. A further decrease in activity was observed after 5 h reperfusion. Final reaction product generated by acid phosphatase activity was rather diffusely distributed in border zones between normal and damaged tissue after 24 and 48 h of reperfusion following 60 min ischemia in vivo. It is concluded that not ischemia itself but rather reperfusion affects the stability of the lysosomal membrane due to the occurrence of oxygen-derived free radicals and/or imbalanced Ca2+ concentration. Restoration of the blood flow causes leakage of acid phosphatase from the lysosomes into the cytoplasm of liver parenchymal cells and from there to the blood.     

Changes in acid phosphatase activity in rat liver after ischemia

Summary The effect of ischemia on the stability, i.e. the permeability of the lysosomal membrane of rat liver has been studied using quantitative histochemical analysis of acid phosphatase activity. Ischemia in vitro was performed for 0–240 min at 37° C and ischemia in vivo for 60 min was followed by 1, 5, 24 and 48 h of reperfusion. Acid phosphatase activity was demonstrated in cryostat sections using naphthol AS-BI phosphoric acid as substrate and polyvinyl alcohol was added to the incubation medium to counteract diffusion phenomena. Ischemia in vitro up to 240 min did not affect the localization nor the total activity of acid phosphatase activity. After 60-min ischemia in vivo followed by 1-h reperfusion distinct areas showed decreased acid phosphatase activity. A further decrease in activity was observed after 5 h reperfusion. Final reaction product generated by acid phosphatase activity was rather diffusely distributed in border zones between normal and damaged tissue after 24 and 48 h of reperfusion following 60 min ischemia in vivo. It is concluded that not ischemia itself but rather reperfusion affects the stability of the lysosomal membrane due to the occurrence of oxygen-derived free radicals and/or imbalanced Ca²⁺ concentration. Restoration of the blood flow causes leakage of acid phosphatase from the lysosomes into the cytoplasm of liver parenchymal cells and from there to the blood.     

Quantitative histophotometric study of liver dehydrogenase activity during temporary ischemia of the extremities

This prospective study depicts quantitative histophotometric characteristics of the changes in the histoenzymologic spectrum of the liver in the course of temporary ischemia of the extremities with subsequent revascularization. The increase in the activities of dehydrogenases under study in the course of a 3-hour ischemia, and the decrease of these parameters in 6- and 12-hour ischemia was discovered, that was determined as a compensatory response followed by the period of compensation exhaustion and the development of the period of complete exhaustion. The data of the study of drug correction of the postischemia syndrome confirmed the rationality of the efforts that were made. A special program developed for computer processing of histological data allows rapid adaptation of the methods for different histophotometric examinations with the use of TV analyzer of the picture, this creating wider possibilities as compared to the known analogs.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Morphological characteristics of the changes in the skeletal muscle tissue in acute experimental ischemia of the extremities

A comprehensive morphological study of the ischemic skeletal muscles of the limbs was performed in experiments on dogs. Ischemia of the muscle tissue was induced by artificial embolic occlusion of the terminal part of the aorta. A quantitative functional and morphological study revealed serious disturbances in metabolism of the skeletal muscle that was subjected to a 6-hour ischemia. Depression of aerobic metabolism, ineffectiveness of anaerobic glycolysis (a spare pathway of the synthesis of macroergic substances), a dramatic lowering of ATPase activity, and activation of acid phosphatase in experiments of such a duration are important signs of a probably compromised adaptation process and irreversibility of the lesions in the tissue. The data should be taken into consideration in determining the optimal periods of the blood flow recovery in the limbs. Morphological changes in muscle fibers under ischemia progress with an increase in the experiment duration (up to 9 and 12 h). An important morphological sign of ischemia is a disturbed typification of muscle fibers.     

Morphological characteristics of changes in the skeletal muscle tissue of the extremities after experimental post-ischemic recirculation

The canine ischemic muscle tissue was subjected to a comprehensive morphological study after the recovery of the blood flow in the limbs for 2 hours. The effectiveness of of the recovery of the blood flow after the 3-hour ischemia was supported in acute experimental occlusion of the artery. The blood flow recovery after 6 hours of ischemia was associated with appreciable structural and metabolic abnormalities in the skeletal muscle fibers. These abnormalities were more demonstrable during recirculation after 9 and 12 hours of ischemia. No morphological criteria that might indicate whether the ischemic damage to the skeletal muscle is reversible or irreversible were defined. A comprehensive morphological study with an assay of structural and metabolic alterations is required instead.     

Acid phosphatase and protease activities in immobilized rat skeletal muscles

The effect of hind-limb immobilization on selected lysosomal enzyme activities was studied in rat hind-limb muscles composed primarily of type I, IIA, or IIB fibers. Following immobilization, acid protease and acid phosphatase both exhibited significant (P less than 0.05) increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.     

Changes in myosin ATPase activity in skeletal muscles of rat during cold stress

Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats.     

Changes in the dehydrogenase activity of cardiomyocytes during acute experimental arterial occlusion of extremities

The results of quantitative histoenzymologic investigations of succinate dehydrogenase, lactate dehydrogenase and NAD-diaphorase in cardiomyocytes of dogs with acute experimental arterial occlusion in ischemic and postischemic periods are reviewed. An increased activity of dehydrogenases in the early periods (3,6 h) of ischemia and during recirculation was established, with its noticeable reduction at later terms (9,12 h). Medical correction of postischemic disorders was shown to improve cardiomyocyte metabolism.     

Para-membrane neurofilament structures of cerebral cortex synapses during ischemia and in the early postischemic period

Using PTA-method, the structure of para-membrane neurofilament density of interneuronal synapses and PTA-positive contacts in neocortical molecular layer were studied in rats during ischemia and postischemic period. Marked reduction of definite contacts (by 25.4%) was recorded by min 90 of ischemia. However, reorganization of asymmetric contacts started during ischemia and continued in postischemic period. Changes in neurofilament density of synapses (primarily, dense projections of presynaptic grid) underlie the early reorganization of synapse architectonics.     

Changes in the activity of various enzymes of carbohydrate metabolism in the liver and skeletal muscles of pigs during ontogenesis

Hexokinase, glucokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity was studied in the liver and musculus quadriceps femoris of 110-day foetuses 1, 2, 3, 30 and 60-day piglets and in adult pigs. The activity of all enzymes in the tissues of newborn piglets is considerably higher than in the tissues of foetuses. The activity of hexokinase in both tissues of piglets increases in the first days after birth and lowers by the one month age. The phosphofructokinase activity in the skeletal muscles and the glucokinase one in the pig liver increase during the postnatal development. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in both tissues of pigs increases after birth and then decreases. Glucose metabolism in the pig liver at all stages of odontogenesis proceeds more intensively by the pentose phosphate pathway, and in the skeletal muscles--by glycolytic one.