Cardiac hypertrophy alters myocardial response to ischaemia and reperfusion in vivo

The impact of cardiac hypertrophy on myocardial biochemical and physiological responses to ischaemia-reperfusion (I-R) was investigated in vivo. Hypertrophy was produced by aortic constriction (PH) or swimming training (TH). Open-chest rat hearts in PH, TH and a sedentary control group (SC) were subjected: (1) to ischaemia, by surgical occlusion of the main descending branch of the left coronary artery for 30 min; (2) to I-R, by releasing the occluded blood vessel for 15 min; or (3) to a sham operation. Ischaemia per se had little effect on heart oxidative and antioxidant status, or lipid peroxidation. However, I-R significantly decreased glutathione (GSH) content, increased glutathione disulfide (GSSG) content, and reduced GSH/GSSG ratio in the SC hearts. These alterations were associated with decreased activities of GSH peroxidase and GSSG reductase, and an increase in lipid peroxidation. Myocardial ATP, total adenine nucleotide content and energy charge in SC were significantly decreased after ischaemia, whereas levels of purine nucleotide derivatives, particularly adenosine, were elevated. No significant alteration of GSH status or adenine nucleotide metabolism occurred after ischaemia or I-R in hypertrophied hearts. In bodi PH and TH, glutathione content was significantly higher than in SC, whereas activities of GSH peroxidase and GSSG reductase were lower. TH rats maintained a higher heart rate (HR), peak systolic pressure, and energy charge during I-R. These data indicate that hypertrophied but well-functioned hearts may be more resistant to I-R induced disturbances of myocardial oxidative and antioxidant functions.     

The protective effects of L-arginine after liver ischaemia/reperfusion injury in a pig model

Hepatic ischaemia/reperfusion is characterized by circulatory and metabolic derangement, liver dysfunction, and tissue damage. To evaluate the role of L -arginine, a substrate of nitric oxide, in ischaemia/reperfusion injury, total liver ischaemia was induced for 120 min in 22 Landrace×Large White female pigs, which were randomly assigned to a treatment group (10 animals) or a control group (12 animals). An L -arginine bolus (540 mg/kg i.v.) was administered to the treatment group 1 h before clamping the hepatic hilum, at clamping, at reperfusion, and at 1 and 2 h after reperfusion. The control animals received normal saline and an i.v. infusion. Liver function tests and analysis of serum, erythrocyte, and tissue malondialdehyde contents were performed at commencement of laparotomy, before reperfusion, and at 30 min and 7 days after reperfusion. Liver biopsies were taken at laparotomy, at 30 min, and at 7 days after reperfusion for histological and ultrastructural examination. Assessment of apoptosis included in situ end-labelling analysis and DNA gel electrophoresis. Survival at 7 days was better in the treated animals than in the controls (9/10 vs. 7/12). Tissue malondialdehyde content, aspartate aminotransferase, and lactate dehydrogenase levels were lower in the treatment group, in which morphological changes were significantly less evident than in the controls 30 min after reperfusion. At 7 days, differences between the groups with respect to cell integrity were apparent only on ultrastructural analysis. Glycogen content, 7 days after reperfusion, was higher in the treatment group than in the controls: 70·25 per cent vs. 21·66 per cent positive hepatocytes (score 3 vs. score 1). Multiparametric analysis showed fewer apoptotic cells in the treatment group at all times. Our data show that the administration of L -arginine reduces damage to liver tissue after ischaemia/reperfusion injury in a pig model. This may be explained not only by the known vasodilator, anti-aggregation, and superoxide inactivation effects of increased nitric oxide release, but possibly also by some other action of L -arginine, such as its influence on cellular metabolism. © 1997 John Wiley & Sons, Ltd.     

The effect of immobilization on myotendinous junction: an ultrastructural,histochemical and immunohistochemical study

Kannus , P., Jozsa , L., Kvist , M., Lehto , M. & Järvinen , M. 1992. The effect of immobilization on myotendinous junction: an ultrastructural, histochemical and immunohistochemical study. Acta Physiol Scand 144 , 387–394. Received 28 April 1 991 , accepted 13 October 1991. ISSN 0001–6772. Tampere Research Station of Sports Medicine, UKK-Institute, and Department of Surgery, Tampere University Central Hospital, Tampere, Finland; Department of Morphology, National Institute of Traumatology, Budapest, Hungary; and Sports Medical Research Unit, Paavo Nurmi Center, University of Turku, Turku, Finland. The effect of immobilization on the myotendinous junction of the calf muscles in the rat was studied histochemically, immunohistochemically and morphometrically with a transmission electron microscope. After 3 weeks of immobilization, the contact area between the muscle cells and tendineal collagen fibres was reduced by almost 50% in both type I (slow-twitch) and type II (fast-twitch) muscle fibres. The terminal finger-like processes of the muscle cells became shallow and cylindrical or were completely atrophied. Their basal membranes were slightly thickened. Histochemically, the most remarkable alteration in the myotendinous junction was the marked decrease in the sulphate containing glyco-saminoglycans. In the basal lamina of the muscle fibres, the glycosaminoglycan and proteoglycan content was also reduced. Immunohistochemical analyses revealed that the amount of type III collagen was markedly increased on the myotendinous interface, but the amount and distribution of type I collagen was not affected by immobilization. These findings suggest that immobilization causes degenerative changes at the myotendinous junction, which, in turn, most likely decrease its tensile strength and may predispose it to rupture during activity.     

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

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.     

Cardiac accumulation of citrate during brief myocardial ischaemia and reperfusion in the pig in vivo

Citrate is a key intermediate in energy metabolism and an inhibitor of phosphofructokinase of the glycolytic pathway. During myocardial ischaemia glycolysis is the main source of cardiac ATP. The aim of the present study was to determine if myocardial ischaemia and reperfusion alter cardiac tissue levels of citrate. Open-chest, anaesthetized pigs were subjected to 10 min of regional myocardial ischaemia by occlusion of the left anterior descending coronary artery, with and without reperfusion, and to 10 min of global ischaemia by circulatory arrest. Citrate, amino acids, glucose and NH₃ were measured in biopsies. Ischaemia, whether regional or global, caused a 60–70% increase in tissue levels of citrate. During 1 min of reperfusion following regional ischaemia the level of citrate increased 460%, to ≈600 nmol g^?1 wet weight. The level of glutamate decreased by 20–33% (corresponding to 1300–2200 nmol g^?1 wet weight), indicating net consumption of this amino acid during ischaemia. The level of aspartate decreased 50% indicating conversion of aspartate to oxaloacetate for the synthesis of citrate. Theoretically, the accumulation of myocardial citrate during brief ischaemia and early reperfusion is large enough to significantly inhibit phosphofructokinase activity and could therefore affect the ability of the myocardium to increase the glycolytic rate in response to ischaemia. This could, however, be partly compensated by the metabolism of myocardial glutamate.     

Correlation between liver cell necrosis and circulating alanine aminotransferase after ischaemia/reperfusion injuries in the rat liver

Circulating liver enzymes such as alanine transaminase are often used as markers of hepatocellular damage. Ischaemia/reperfusion (I/R) injury is an inevitable consequence of prolonged liver ischaemia. The aim of this study was to examine the correlation between liver enzymes and volume of liver cell necrosis after ischaemia/reperfusion injuries, using design‐unbiased stereological methods. Forty‐seven male Wistar rats were subjected to 1 h of partial liver ischaemia, followed by either 4 or 24 h of reperfusion. Within each group, one‐third of animals were subjected to ischaemic preconditioning and one‐third to ischaemic postconditioning. At the end of reperfusion, blood and liver samples were collected for analysis. The volume of necrotic liver tissue was subsequently correlated to circulating markers of I/R injury. Correlation between histological findings and circulating markers was performed using Pearson's correlation coefficient. Alanine transferase peaked after 4 h of reperfusion; however, at this time‐point, only mild necrosis was observed, with a Pearson's correlation coefficient of 0.663 (P = 0.001). After 24 h of reperfusion, alanine aminotransferase was found to be highly correlated to the degree of hepatocellular necrosis R = 0.836 (P = 0.000). Furthermore, alkaline phosphatase (R = 0.806) and α‐2‐macroglobulin (R = 0.655) levels were also correlated with the degree of necrosis. We show for the first time that there is a close correlation between the volume of hepatocellular necrosis and alanine aminotransferase levels in a model of I/R injury. This is especially apparent after 24 h of reperfusion. Similarly, increased levels of alkaline phosphatase and α‐2‐macroglobulin are correlated to the volume of liver necrosis.     

Clinical and experimental study of enzyme histochemical changes in the ventricular myocardium in pulmonary embolism

N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences, V. S. Savel'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 5, pp. 539–542, May, 1992.     

Role of l-arginine in preventing myocardial and endothelial injury following ischaemia/reperfusion in the rat isolated heart

The protective effect of l -arginine on ischaemia/reperfusion-induced myocardial injury was investigated in the rat isolated Langendorff perfused heart. Six groups of hearts subjected to 30 min global ischaemia and 30 min reperfusion received either vehicle, d -arginine, l -arginine, the nitric oxide (NO)-donor S-Nitroso-N-Acetyl-d, l -Penicillamine (SNAP), the inhibitor of NO formation N^G-nitro-l -arginine (l -NNA), or l -arginine plus l -NNA. The recoveries of left ventricular double product and coronary flow at the end of reperfusion were significantly higher in the l -arginine group (85±5 and 75±6%, respectively) than in the vehicle group (37±6 and 34±5%, respectively, P<0.05). During both the ischaemic and reperfusion periods, left ventricular end diastolic pressure was lower in the l -arginine group than in the vehicle group. Creatine kinase outflow and the area of no-reflow were smaller in the l -arginine treated hearts (P<0.01). There were no differences between vehicle and d -arginine treated groups. l -NNA did not affect recovery per se but abolished the protective actions of l -arginine. SNAP produced the same protective effects as l -arginine. Acetylcholine-induced endothelium-dependent vasodilation was reduced after ischaemia and reperfusion in the vehicle group but not in the l -arginine group. It is concluded that l -arginine reduces ischaemia/reperfusion-induced myocardial and endothelial injury. The results suggest that the beneficial effects of l -arginine are related to preserved synthesis and release of NO.     

Comparison of the effect of adaptation to stress and to high altitude hypoxia on resistance of the heart to reperfusion injury after total ischemia

Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 7, pp. 18–20, July, 1991.     

Pigmented granules in functional black adenoma of the adrenal gland: A histochemical and ultrastructural study

A black adenoma of the adrenal gland was laparoscopically removed from an 61-yr-old Japanese female who had clinical and laboratory findings characteristic of Cushing’s syndrome. The tumor consisted of polygonal cells that contained numerous brown pigmented granules of various sizes by routine hematoxylin-eosin staining. The histochemical study showed that the pigment had the characteristics of lipofuscin, not of melanin or neuromelanin. Electron microscopic study revealed tumor cells with two types of pigmented granules. These results show that there might be differences in the lipid metabolism of individual tumor cells in black adenomas of adrenal; which suggests an interesting histogenesis for these granules.     

Morphological analysis of the urethral muscle of the male pig with relevance to urinary continence and micturition

To investigate whether the pig could be considered a suitable model to study lower urinary tract function and dysfunction, the pelvic urethra of 24 slaughtered male pigs were collected, and the associated muscles were macroscopically, histologically and histochemically analyzed. In cross‐sections of the urethra, a muscular complex composed of an inner layer of smooth muscle and an outer layer of striated muscle that are not separated by fascial planes was observed. A tunica muscularis, composed of differently oriented smooth muscle bundles, is only evident in the proximal part of the pelvic urethra while, in the remaining part, it contributes to form the prostatic fibromuscular stroma. The striated urethral muscle surrounds the pelvic urethra in a horseshoe‐like configuration with a dorsal longitudinal raphe, extending from the bladder neck to the central tendon of perineum. Proximally to the bladder, it is constituted of slow‐twitch and fast‐twitch myofibers of very small diameter, and embedded in an abundant collagen and elastic fiber net. Moving caudally it is gradually encircled and then completely substituted by larger and compact myofibers, principally presenting circular orientation and fast‐twitch histochemical characteristics. So, like in humans, the cranial tract of the muscular system surrounding the pelvic urethra is principally composed of smooth musculature. The striated component cranially may have a role in blocking retrograde ejaculation, while the middle and caudal tracts may facilitate urine and semen flow, and seem especially concerned with the rapid and forceful urethral closure during active continence. Some differences in the morphology and structure between pigs and humans seem due to the different morphology of the ‘secondary’ sexual organs that develop from the urethral wall and to the different effect of gravity on the mechanics of the urinary system in quadruped and bipedal mammals.     

Fluorescence histochemical and electron-microscopical observations on the innervation of the atrial myocardium of the adult human heart

Summary The existence of both adrenergic and cholinergic innervation of the atrial myocardium of the adult human heart was demonstrated by means of fluorescence induced by formaldehyde or glyoxylic acid and by electron microscopy.The adrenergic fluorescing axons (1) followed the course of blood vessels as typical perivascular nerve plexuses, and (2) formed a three-dimensional fairly dense nerve net obviously not related to the blood vessels. The varicosities frequently came into close apposition on myocardial cells.Several types of nerve terminals were differentiated at electron microscopy: (1) an adrenergic type containing small (diameter 450–700 Å) dense-cored vesicles and usually (in various proportions) small empty and/or large (900–1500 Å) dense-cored vesicles, (2) a cholinergic type containing small (ca. 500 Å) empty vesicles and occasionally also some large (mean diameter ca. 1200 Å) dense-cored vesicles, (3) a pale type containing only a few or no vesicles, (4) a disintegrated type containing degenerated mitochondria, autophagic vacuoles, and occasional normal-looking mitochondria, (5) nerve terminals containing a large number of mitochondria in addition to varying vesicle populations, and (6) a (possibly baroreceptive type of) nerve terminal containing myelinlike lamellated structures. The disintegrated and the pale types of nerve terminals possibly represent different stages of axonal degeneration, or may correspond to diminution in the transmitter substance concentration under certain pathophysiologic conditions, respectively. Nerve terminals crowded with mitochondria may be sensory and involved in mechano-or chemoreceptive functions.In preliminary experiments convincing evidence was obtained that the glyoxylic acid-induced fluorescence histochemical method will be suitable for comparative studies on (human) clinical specimens, e.g., for analyzing the degree of the functional activity of the intrinsic adrenergic innervation of the myocardium under various pathophysiologic conditions. The modification which appeared most appropriate for such studies is described in detail, and is proposed for use as a standard method in other similar or related studies on human clinical series. The essential criteria for analyzing the specimens at fluorescence microscopy are suggested as well.     

Capillary supply and mitochondrial content of different skeletal muscle fiber types in untrained and endurance-trained men. A histochemical and ultrastructural study

Summary The number of capillaries per fiber, per mm², around each fiber type and relative to fiber area was determined in six untrained subjects (UT) and six elite cross-country skiers (ET). Average values for maximal oxygen uptake were 49.8 ml·kg^–1·min^–1 (UT) and 77.9 ml·kg^–1·min^–1 (ET). Type I fibers constituted 39.2% (UT) and 68.6% (ET), type II A fibers 39.6% (UT) and 19.2% (ET), while 12.8% (UT) and 6.6% (ET) of the fibers were type II B. The mean fiber area for the type II A fibers was significantly greater (p<0.01) than the areas for type I and II B in the untrained group.The average numbers of capillaries around each fiber type (CA) were 4.76-4.84-2.94 (UT) and 7.79-6.63-4.5 (ET) for type I, II A, and II B, respectively. There was a significant difference (p<0.01) in the CA values relative to fiber area for all fiber types in both groups, being highest for type I and lowest for type II B. The CA increased linearly with increasing size of the fibers for all fiber types in both groups.The mitochondrial content was determined semiquantitatively for each fiber type. The differences in capillary supply between the fiber types are accompanied by similar differences in mitochondrial content.The results indicate that endurance training increases the capillary supply of all fiber types in the human quadriceps muscle. The fact that light microscopical studies have given lower capillarization values than those obtained with the electron microscope is discussed.     

Impact of preservation solution on the extent of blood‐air barrier damage and edema formation in experimental lung transplantation

A major aim in lung transplantation is to prevent the loss of structural integrity due to ischemia and reperfusion (I/R) injury. Preservation solutions protect the lung against I/R injury to a variable extent. We compared the influence of two extracellular‐type preservation solutions (Perfadex, or PX, and Celsior, or CE) on the morphological alterations induced by I/R. Pigs were randomly assigned to sham (n = 4), PX (n = 5), or CE (n = 2) group. After flush perfusion with PX or CE, donor lungs were excised and stored for 27 hr at 4°C. The left donor lung was implanted into the recipient, reperfused for 6 hr, and, afterward, prepared for light and electron microscopy. Intra‐alveolar, septal, and peribronchovascular edema as well as the integrity of the blood‐air barrier were determined stereologically. Intra‐alveolar edema was more pronounced in CE (219.80 ± 207.55 ml) than in PX (31.46 ± 15.75 ml). Peribronchovascular (sham: 13.20 ± 4.99 ml; PX: 15.57 ± 5.53 ml; CE: 31.56 ± 5.78 ml) and septal edema (thickness of alveolar septal interstitium, sham: 98 ± 33 nm; PX: 84 ± 8 nm; CE: 249 ± 85 nm) were only found in CE. The blood‐air barrier was similarly well preserved in sham and PX but showed larger areas of swollen and fragmented epithelium or endothelium in CE. The present study shows that Perfadex effectively prevents intra‐alveolar, septal, and peribronchovascular edema formation as well as injury of the blood‐air barrier during I/R. Celsior was not effective in preserving the lung from morphological I/R injury. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

Refining murine heterotopic heart transplantation: A model to study ischemia and reperfusion injury in donation after circulatory death hearts

Heart transplantation is a lifesaving procedure, which is limited by the availability of donor hearts. Using hearts from donors after circulatory death, which have sustained global ischemia, requires thorough studies on reliable and reproducible models that developing researchers may not have mastered. By combining the most recent literature and our recommendations based on observations and trials and errors, the methods here detail a sound in vivo heterotopic heart transplantation model for rats in which protective interventions on the ischemic heart can be studied, and thus allowing the scientific community to advance organ preservation research. Knowledge gathered from reproducible animal models allow for successful translation to clinical studies.