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Abstract Ginkgo biloba extract EGb 761 was studied for its nephroprotective effects in experimentally diabetic and hypoxic rats. Duration of streptozotocin-induced diabetes was 4 months, that of respiratoric hypoxia of [...]

BACKGROUND AND AIMS: Acute hypoxia is a threatening clinical case of emergency and may result in ultrastructural damage, with complete loss of cellular and organ functions. However, little is known about the differences in hypoxia tolerance between young and old myocardia and the protective effects of radical scavenging agents in acute hypoxic stress situations. METHODS: We investigated the age-dependent differences of the myocardial ultrastructure and antioxidative status (superoxide-dismutase (SOD) activity and malondialdehyde (MDA) content) of young (6 months) and old (22-24 months) Wistar rats (Crl (Wi)Br) after acute respiratory hypoxia of 20 min at 5% v/v O2 in N2O mixture, and the protective effect of Ginkgo biloba extract (EGb 761). RESULTS: Ultrastructural-morphometric and biochemical age analysis only revealed a decrease in the sarcoplasma volume fraction, an increase in homogeneous intramitochondrial areas, significant higher SOD activity and lower MDA levels in the group of old rats. Pretreatment with EGb 761 led to a significant decrease in MDA content in both control groups. Acute hypoxic stress increased the volume fractions of sarcoplasmatic reticulum, t-tubules, vacuoles, and lipid droplets, and caused mitochondrial swelling, with a more significant increase in degenerated and homogeneous intramitochondrial areas in the old group. SOD activity decreased only in the old hypoxic group; MDA content fell in both. Pretreatment with EGb 761 reduced ultrastructural-morphometric hypoxic damage in both groups, significantly below the levels of control. Young rat myocardium showed significantly higher SOD activity after hypoxia than untreated or older specimens. CONCLUSIONS: Better hypoxia tolerance is demonstrated by the young myocardium, and an obvious hypoxia-protective effect of EGb 761 in both age groups. [ABSTRACT FROM AUTHOR]

There is a fundamental body of evidence suggesting that activated apoptosis signaling in ejaculated human sperm negatively influences their fertilization potential. However, it is still controversial whether this apoptotic signaling is a relic of an abortive apoptosis related to spermatogenesis or if it should be regarded as a functional preformed pathway in mature sperm leading to stereotypical morphological changes reflecting nuclear disassembly. To address this question, apoptosis was induced using betulinic acid in mature and immature ejaculated human sperm enriched by density gradient centrifugation. Execution of apoptosis was monitored by observing ultra-morphological changes via transmission electron microscopy. Typical morphological signs of apoptosis in somatic cells include plasma membrane blebbing with the formation of apoptotic bodies, impaired mitochondrial integrity, defects of the nuclear envelope, and nuclear fragmentation; these morphologies have also been observed in human sperm. In addition, these apoptotic characteristics were more frequent in immature sperm compared to mature sperm. Following betulinic acid treatment, apoptosis-related morphological changes were induced in mature sperm from healthy donors. This effect was much less pronounced in immature sperm. Moreover, in both fractions, the betulinic acid treatment increased the percentage of acrosome-reacted sperm. The results of our ultra-morphological study prove the functional competence of apoptosis in mature ejaculated human sperm. The theory of a sole abortive process may be valid only for immature sperm. The induction of the acrosome reaction by stimulating apoptosis might shed light on the biological relevance of sperm apoptosis.

Aim: The present study aims to analyze the differences in ultrastructural changes between right ventricular myocardium in clinically determined grades of heart failure (HF) [New York Heart Association (NYHA) classes I-IV] and their value in the routine diagnostic setting., Methods: We investigated consecutive right ventricular endomyocardial biopsies of 12 patients presenting with HF (49±11.2years; male=10) by light microscopy and ultrastructural morphometric analysis. The patients were divided into four groups according to their NYHA classes (NYHA I: n=1, II: n=2, III: n=8, IV: n=1). We used a stereological point counting method on electron micrographs to determine the volume, surface, and numerical density of cardiomyocyte myofibrils; z-lines; mitochondria; and cristae as required. Further, secondary parameters were calculated., Results: Myofibrillar parameters increased between NYHA class I and II (P<.01), which matched with more pronounced cardiomyocyte hypertrophy on the light microscopic level. In NYHA classes III and IV, the myofibrillar parameters dropped, while parameters concerning the mitochondria and their cristae rose (P<.01). This resulted in an elevated mitochondria to myofibril ratio (P<.05) and correlated with histologically evident atrophic cardiomyocytes, perinuclear loss of myofibrils and dot-like perinuclear staining positive on peroxide acid shift., Conclusion: In this present study, right ventricular myocardial ultrastructure differed between patients diagnosed with HF of different degrees in distinct subcellular changes. These findings suggest that ultrastructural analysis, while correlated with histopathological features, adds to the diagnosis in the routine diagnostic setting, specifically in lower NYHA grades, in which only minor changes are observed histologically., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Aims: Neurodegenerative processes of aging seem to be associated with oxidative stress by reactive oxygen species (ROS). This study investigates the influence of age and of acute respiratoric hypoxia on parameters of oxidative stress in different brain regions of Wistar rats and the protective effects of Ginkgo extract (EGb 761) as a radical scavenger., Methods: Biopsies of frontal and temporal cortices, the cerebellum, and the brainstem of young and old rats (each group n=6-8: normoxic - hypoxic; unprotected - EGb-protected) were analyzed for malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, glutathione (GSH) content, and creatine kinase (CK) activity. Experimental hypoxia: downregulation of oxygen partial pressure to 5 vol. % for 20 minutes. EGb administration: daily 100 mg/kg of body weight in drinking water for 3 months., Results: Effects of age: While most oxidative stress parameters in the temporal cortex, the cerebellum, and the brainstem are increased, this is not the case in the frontal cortex; after additional hypoxia SOD and GSH are diminished in the temporal cortex and the brainstem of old rats. EGb treatment causes contradictory alterations in young, old, and hypoxic brain regions. Minor effects are seen in old hypoxic brains, while there are some protective effects in old normoxic brainstems and cerebellums., Conclusions: The old brain appears to adapt appropriately to chronic oxidative stress and to the specific conditions of shortterm hypoxia. EGb's protective effect is especially notable in the brainstem and the cerebellum.

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b(-/-) mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu(2+)/H(2)O(2)/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b(-/-) mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b(-/-) mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b(-/-) mice in contrast to control with increasing age. Hepatocytes from elder Atp7b(-/-)mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Cardiovascular autonomic neuropathy causes abnormalities in the diabetic heart with various clinical sequelae, including exercise intolerance, arrhythmias and painless myocardial infarction. Little is known about (ultra)structural alterations of the myocardial nervous network. On the assumption that this diabetes-specific neuropathy develops due to permanently increased oxidative stress by liberation of oxygen-free radicals, adjuvant application of antioxidative therapeutics appears promising in preventing or delaying long-term diabetic complications. We have investigated the effects of Ginkgo biloba extract (EGb 761), a radical scavenger, against diabetes-induced myocardial nervous damage in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats. Morphological and morphometric parameters were evaluated by electron microscopy. We used immunohistochemistry to investigate protein expression of protein gene product 9.5, S100 protein, and thyroxin hydroxylase as a neuronal marker. Alterations of cardiac sympathetic activity were measured using the in vivo 123I-metaiodobenzyl-guanidine imaging, and the immunofluorescent labeling of beta1-adrenergic receptors and adenylate cyclase. Our results revealed that A) Diabetes results in slight to moderate ultrastructural alterations (hydrops, disintegration of substructure) of autonomic nerve fibers and related Schwann cells in untreated BB diabetic rats; B) Cardiac sympathetic integrity and activity is impaired due to alterations in the presynaptic nerve terminals and the postsynaptic ß1-AR-AC coupling system; C) Pre-treatment of diabetic myocardium with EGb results in an improvement of most of these parameters compared to unprotected myocardium. In conclusion, EGb may act as a potent therapeutic adjuvant in diabetics with respect to cardiovascular autonomic neuropathy, which may contribute to the prevention of late complications in diabetes.

Replication initiator 1 (Repin1) is highly expressed in liver and adipose tissue and has been suggested as candidate gene for obesity and its related metabolic disorders in congenic and subcongenic rat strains. The cellular localization and function of Repin1 has remained elusive since its discovery in 1990. To characterize the role of Repin1 in adipocyte biology, we used siRNA knockdown technology to reduce the expression of Repin1 by electroporation of semiconfluent 3T3-L1 preadipocytes. Glucose transport, palmitate uptake as well as triglyceride content were measured. In paired samples of human visceral and subcutaneous adipose tissue, we investigated whether Repin1 mRNA expression is related to measures of fat accumulation and adipocyte size. We demonstrate that Repin1 increases during adipogenesis. RNA interference based Repin1 downregulation in mature adipocytes significantly reduces adipocyte size and causes reduced basal, but enhanced insulin stimulated glucose uptake into 3T3-L1 cells. Additionally, knockdown of Repin1 resulted in reduced palmitate uptake and significantly changed the mRNA expression of genes involved lipid droplet formation, adipogenesis, glucose and fatty acid transport. Furthermore, we found significant correlations between Repin1 mRNA expression in human paired visceral and subcutaneous adipose tissue and total body fat mass as well as adipocyte size. We have identified a potential role for Repin1 in the regulation of adipocyte size and expression of glucose transporters GLUT1 and GLUT4 in adipocytes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Besides alterations in cardiomyocytes themselves, diabetic cardiopathy is characterized by interstitial and microvascular disorders. On the assumption that a specific heart muscle disease develops due to permanently increased oxidative stress on liberation of oxygen-free radicals, adjuvant application of antioxidative therapeutics appears promising in preventing or delaying long-term diabetic complications and protecting the myocardium against acute ischemia. We have investigated the effects of Ginkgo biloba extract (EGb 761), a radical scavenger, against diabetes-induced myocardial interstitium and microvasculature damage, and against additional ischemia/reperfusion injury in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats modelling diabetic cardiac infarction. Morphological and morphometric parameters in the heart muscle were evaluated by light and electron microscope. We used immunohistochemistry to investigate collagen protein expression as a marker for tissue remodelling together with endothelial nitric oxide synthase (eNOS) protein expression as a marker for endothelial-dependent vasodilation. We also evaluated inflammation response caused by neuropeptide Substance P and interacting mast cells in the diabetic heart. Our results revealed that A) Diabetic myocardium appears more vulnerable to ischemia/reperfusion injury than normal myocardium with regard to myocardial interstitium and microvessel ultrastructure, as well as eNOS protein expression; B) Inflammation response increases in diabetic animals exposed to ischemia/reperfusion injury compared to controls; C) Pre-treatment of diabetic myocardium with EGb results in an improvement of impaired endothelial-dependent vasodilation in diabetes and additional ischemia/ reperfusion, diminished mast cell and substance P accumulation, and better preserved myocardial ultrastructure compared to unprotected myocardium. In conclusion, EGb may act as a potent therapeutic adjuvant in diabetics with respect to ischemic myocardial injury, and may contribute to preventing late complications in diabetic cardiopathy.

Diabetic cardiomyopathy is known to result in increased mortality after ischemic events. Permanently increased oxidative stress with formation of oxygen-free radicals plays a key role in the development of specific heart muscle disease. Associated lesions include structural alterations to cardiomyocytes. Antioxidative treatment in addition to the usual insulin substitution would seem sensible in preventing or delaying long-term diabetic complications and protecting the myocardium against acute ischemic events. We investigated the effects of radical scavenger Ginkgo biloba extract EGb 761 against diabetes-induced damage to cardiomyocytes and additional ischemia/reperfusion injury in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats, as a model of diabetic myocardium infarction. Morphological and morphometric parameters of heart muscles were analyzed by light and electron-microscopic techniques. We used immunohistochemistry to evaluate parameters of oxidative stress (superoxide dismutase [SOD]) and inducible nitric oxide synthase (iNOS) protein expression. Our results indicated that A) Diabetic myocardium appears more vulnerable to ischemia/reperfusion damage concerning ultrastructure of cardiomyocytes (sarcomeres, vacuoles, mitochondria), expression of antioxidative enzymes (CuZnSOD, MnSOD), and iNOS than normal myocardium; B) Pre-treatment of diabetic myocardium with EGb and additional ischemia/reperfusion leads to a relative improvement in myocardial ultrastructure compared to unprotected myocardium. In summary, EGb appears to be promising as an adjuvant therapeutic drug in diabetics with respect to ischemic myocardium injury. It may contribute to the prevention of late diabetic complications in diabetic cardiomyopathy.

Spermatozoa with deteriorated plasma membranes can be separated by magnetic-activated cell sorting (MACS) after binding superparamagnetic annexin V-conjugated microbeads (ANMBs) to membrane phosphatidylserine (PS). Semen samples from 15 donors and 25 infertile patients were divided into 2 spermatozoal fractions by annexin V-MACS. Activated caspases (aCPs), which mediate degradations of cell quality, were determined by CaspaTag in the 2 subpopulations. Spermatozoa from donors showed lower levels of bound annexin V (3.6% +/- 0.5% vs 11.9% +/- 1.1%; P <.01) and aCPs (21.8% +/- 2.6% vs 43.2% +/- 2.1%; P <.01) than did spermatozoa from infertile patients. MACS resulted in a decrease of spermatozoa with aCPs from 21.8% +/- 2.6% (before separation) to 9.2% +/- 1.4% (in the ANMB-negative fraction) in donors and from 43.2% +/- 2.1% to 18.8% +/- 2.6% in infertile patients (mean +/- SEM; P <.01). Separation effects of the MACS technique were confirmed with flow cytometry using anti-annexin V antibodies and with electron microscopy. ANMB-MACS removes spermatozoa with PS-bound annexin V and produces a higher quality spermatozoal fraction. Spermatozoa with a deteriorated membrane are characterized by an increase in aCPs. A higher percentage of spermatozoa with ANMBs bound to PS and with aCPs were found in infertile patients.

Proximal (vastus lateralis) and distal (gastrocnemius) muscles of 100-day-old normal and myopathic BIO TO-2 hamsters were analysed to study the effects of myopathy on the different muscle fibre types: SO (slow oxidative), FOG (fast oxidative glycolytic) and FG (fast glycolytic). Cytophotometric measurements of enzyme activities (myofibrillic adenosine triphosphatase, succinate dehydrogenase and glycerol-3-phosphate dehydrogenase), Western blot analysis of nitric oxide synthase (NOS) I, II, III isoforms and NOS II immunohistochemistry were performed. The following alterations were found in myopathic muscle fibres: all fibre types of both proximal and distal myopathic muscles showed decreased myofibrillic adenosine triphosphatase activity indicating depressed contractility. This was associated with depressed oxidative activity of the muscle fibres. A shift to more glycolytic metabolism was observed, mainly in FG fibres of proximal muscle. We found an increased NOS II expression in both myopathic muscle types investigated. It means that increased NO production inhibits force generation in myopathic muscle. NOS II immunoreactivity was found mainly in the cytoplasm of FG fibres. NOS I and NOS III expression was not significantly effected by this form of myopathy. Our findings demonstrate that muscle fibres of proximal and distal skeletal muscles of 100-day-old cardiomyopathic BIO TO-2 hamsters are altered with respect to contractility, metabolism and NOS II expression. FG fibres of the proximal muscle were effected most strongly.

The hereditary cardiomyopathy of the Syrian hamster (strain BIO 8262) as a model of human cardiomyopathy was investigated during postnatal development using ultrastructural morphometric analysis. The aim of the study was to establish basic ultrastructural morphometric data of cardiomyocytes of this model during all stages of the disease in comparison to corresponding stages of normal development, and to compare the ultrastructural resistance of normal and myopathic myocardium against acute hypoxic stress. The postnatal development of this cardiomyopathy is characterized by focal myolytic lesions already in the first postnatal days, extended myofibrillic and mitochondrial damage developing after the 1st month, and features of hypertrophy, disarray of myofibrils, thickening, and clumping of z-lines after the 4th month. Morphometric parameters of myopathic cardiomyocytes differing from those of the control are diminished volume density of myofibrils by about 10% of the control values in middle age, slightly increased volume density of sarcoplasmic reticulum and t-tubules in the 1st months, significant increase of them after the 3rd month, and significant alterations of many mitochondrial parameters indicating degenerations (numeric density, volume density, average volume, volume density of cristae, and of destroyed areas, and ratio of surface to volume density of mitochondrial cristae). Acute isobaric hypoxia was applied to myopathic and healthy hamsters of corresponding age (at day 100). Evaluation of ultrastructural morphometric parameters of cardiomyocytes revealed that especially mitochondria were more affected by hypoxia in the myopathic animals. The average mitochondrial volume was significantly more increased in the myopathic condition, the ratio of surface to volume density of cristae significantly diminished. Our ultrastructural - morphometric study has shown, that morphometric parameters in general can be assigned to the stages of cardiomyopathy, and that the ultrastructural differences are best expressed about the 4th month.

Statistical techniques known as the analysis of variance make it possible for the morphologist to plan work in such a way as to get quantitative data with the greatest possible economy of effort. This paper explains how to decide how many measurements to make per micrograph, how many micrographs per tissue block or organ, and how many organs or individuals are necessary for getting an exactness of sufficient quality of the results. The examples furnished have been taken from measuring volume densities of mitochondria in heart muscle cells and from cell counting in lymph nodes. Finally we show, how to determine sample sizes, if we are interested in demonstration of significant differences between mean values.

The organization and structure of data masses including results of scientific research is presented on the base of the morphometric method. The data massif is realized on ESER-1056 large scale computer. Currently, all the results of the universally scientific programme "Statist", designed for mathematical and statistical morphometric data processing, are collected in this data massif. A personal computer is linked with a large-scale computer by a cable for data transfer by telecommunication, the whole system accomplishes distributed ata processing. This enables the scientist to use the data massif directly from his working site.

Tissue of myocardium from (8262) hamsters, 2, 6 and 27 weeks of age, were analyzed with quantitative microscopic techniques. Besides alterations of some other structures, we found the most evident changes in the mitochondrial abnormalities exist at all ages. They may be related to defective energetic mechanism.