[Structure and function of suburothelial myofibroblasts in the human urinary bladder under normal and pathological conditions]. / Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen.

BACKGROUND: Myofibroblasts play a pivotal role in numerous pathological alterations. Clarification of the structure and function and of the cellular plasticity of this cell type in the bladder may lead to new insights into the pathogenesis of lower urinary tract disorders. PATIENTS AND METHODS: Bladder biopsies from patients with bladder carcinoma and interstitial cystitis were used to analyse the morphology and receptor expression using confocal immunofluorescence and electron microscopy. Cytokine effects and coupling behavior were tested in cultured myofibroblasts and detrusor smooth muscle cells. RESULTS: Myofibroblasts are in close contact with the suburothelial capillary network. They express Cx43 and form functional syncytia. The expression of muscarinic and purinergic receptors is highly variable. Dye coupling experiments showed differences to detrusor myocytes. CONCLUSIONS: Upregulation of smooth muscle cell alpha-actin and/or transdifferentiation into smooth muscle cells may contribute to the etiology of urge incontinence. A multi-step model is presented as a working hypothesis.

Ginkgo biloba extract protects rat kidney from diabetic and hypoxic damage.

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 the diabetic group 20 min. The daily dose of 100 mg EGb/kg bodyweight started 1 month after induction of the diabetes. EGb reduced diabetes-induced morphological alterations of the kidney such as increase in volume of glomeruli, capillary tufts, urinary space, and thickening of Bowman's capsule basement membrane. Diabetically increased immunostaining of interstitial collagenes of types I, III, and VI was diminished by the EGb extract. EGb reduced the relative total SOD activity from 163% in diabetic kidney to 46%. Additional hypoxia-induced ultrastructural damage was also diminished.

Ultrastructural, immunohistochemical and biochemical investigations of the rat liver exposed to experimental diabetes und acute hypoxia with and without application of Ginkgo extract.

The aim of this paper was to investigate the effect of streptozotocin-induced diabetes by i.p. bolus injection of streptozotocin at 60 mg per kg bodyweight over four months and additional acute respiratory hypoxia (20 min. duration, 5% oxygen v/v), and also the protective effect of Ginkgo biloba extract (EGb 761) on Wistar rat liver under these experimental conditions. Diabetic and additional hypoxic alterations in histology and ultrastructure were subjected to qualitative and quantitative analysis, collagen was investigated by immunohistochemistry, and some biochemical parameters of oxidative stress were determined. Diabetes caused an increase in the size of the hepatocytes and their nuclei with a decrease in nucleus-to-plasma ratio and glycogen content. Connective tissue was variably increased in individual cases as shown by routine histological staining. EGb did not influence these data. Ultrastructural morphometry revealed a significant reduction in rough endoplasmic reticulum (rER) and a significant increase in smooth endoplasmic reticulum (sER) through diabetes, an increase under EGb protection, with no significant alteration under hypoxia. The volume fraction of mitochondria was significantly increased after induction of diabetes but less increased in the protected group. Additional hypoxia reduced this parameter. The mean cross-section area of mitochondria was significantly elevated in all diabetic groups compared to controls. Volume density of mitochondrial cristae was significantly diminished in all diabetic groups; EGb could only improve this parameter in the diabetic-hypoxic group.

Effect of age and hypoxia/reoxygenation on mRNA expression of antioxidative enzymes in rat liver and kidneys.

The influence of a short-time isobaric hypoxia as well as reoxygenation on markers of oxidative stress (MDA, total SOD, GSH) and on the mRNA expression of the antioxidative enzymes (Cu/Zn-and Mn-SOD, catalase, GSH reductase and GSH peroxidase) has been studied in liver and kidneys of young (6 months) and old (22-25 months) Wistar rats. In livers of old animals, the concentration of GSH, the activity of SOD, and the mRNA expression of the antioxidative enzymes (except Mn-SOD) points to a restricted protection against oxidative stress or a lower production of ROS compared to young animals. Hypoxia resulted in a significant decrease of enzyme gene expression in both age groups. Reoxygenation caused an increase in mRNA of Cu/Zn-SOD and GPX in livers of young and of Mn-SOD in livers of old animals. In kidneys, gene expression of Cu/Zn-SOD, GSH reductase, and GPX was significantly higher in old animals compared to young animals. Whereas hypoxia caused a decrease of gene expression in the livers, it lead to a significant increase of Cu/Zn-SOD, catalase, and GSH reductase mRNA in kidneys of young rats. A reduced gene expression was observed after reoxygenation. In old kidneys, the expression of all enzymes except for catalase progressively declined in the hypoxic and reoxygenation groups. These data show that gene expression of antioxidative enzymes is affected by age and significantly differs between liver and kidney.

Experimental hypoxia of STZ-diabetic rat myocardium and protective effects of Ginkgo biloba extract. II. Ultrastructural investigation of microvascular endothelium.

Four months after induction of diabetes by intraperitonal injection of 60 mg streptozotocin/kg body weight wistar rats were exposed to an acute respiratoric hypoxia of 20 min duration. One group of the rats received daily Ginkgo biloba extract EGb 761 (100 mg/kg body weight). By means of qualitative and quantitative electron microscopic analysis we compared the hypoxia-induced ultrastructural alterations of the myocardial microvascular endothelium in normal, diabetic, and EGb-protected rats. Aim of the study was to compare the hypoxia tolerance of myocardial microvessels of normal and diabetic rats and to test the possibility of antioxidative protection. The results revealed that only some ultrastructural microvascular parameters of diabetic rats were stronger altered after acute hypoxia than normal ones, e.g. capillary dilatation, number of lysosomes, frequency of vesicles and fused vesicles, endothelial swelling, and structural state of mitochondria. Other parameters exhibited less severe alterations than in healthy rats, as luminal blebbing and protrusions, endothelial vacuoles, mitochondrial swelling, and pericapillary debris. Protective effects of EGb could be demonstrated on endothelial swelling, blebbing, vesiculation and vesicular fusioning, partly on vacuolization, but not on mitochondrial parameters. The results were discussed on pathobiochemical background. EGb 761 was estimated to be protective against hypoxic damage on myocardial microvessels also in diabetic condition, but the study should be completed by inclusion of a reoxygenation interval.

The influence of hypoxia on the myocardium of experimentally diabetic rats with and without protection by Ginkgo biloba extract. III: Ultrastructural investigations on mitochondria.

Completing our preceding ultrastructural studies on diabetes and additional acute hypoxia of rat myocardium and the protective effect of Ginkgo extract (EGb) we investigated specific ultrastructural-morphometric parameters of corresponding mitochondria. Aim of the study was to answer the question whether mitochondria of diabetic myocardium are more sensitive to hypoxia than in normal condition, and whether antioxidative protection by EGb is effective. Further we compared the ultrastructural reactions of mitochondria of different intracellular locations. Voluminal parameters of mitochondria indicated a moderate swelling after diabetes and a further slight swelling after additional hypoxia, which was slightly reduced after EGb pretreatment. Decrease of volume density of mitochondrial cristae was less expressed after diabetes and much stronger after additional hypoxia; slight protection by EGb was only visible after diabetes. Degenerative intramitochondrial areas increased significantly after diabetes and after hypoxia; EGb was protective only after additional hypoxia. The relative number of ATPase particles (F1-coupling factors) at the inner mitochondrial membranes was slightly but significantly reduced after diabetes and stronger reduced after additional hypoxia; only in the latter condition Ginkgo extract was slightly protective. The product of volume density of mitochondria x volume density of cristae x relative number of ATPase particles at the inner mitochondrial membrane (as structural equivalent of the myocardial capacity for ATP production) indicated better than single parameters the increasing mitochondrial damage after diabetes of 4 months duration and subsequent acute hypoxia of 20 min duration. After hypoxia this capacity amounted only to 46% of the normal and was improved by EGb to 53%.

The influence of hypoxia on the myocardium of experimentally diabetic rats with and without protection by Ginkgo biloba extract. I. Ultrastructural and biochemical investigations on cardiomyocytes.

The influence of acute respiratoric hypoxia in streptozotocin-diabetic rats and protective effects of Ginkgo biloba extract (EGb 761)-pretreatment were investigated by the means of ultrastructural morphometry, biochemical parameters of oxidative stress and iNOS transcription and expression. Ultrastructural parameters revealed that acute hypoxia deteriorated the morphologic condition of the diabetic cardiomyocytes: volume fractions of sarcoplasm, t-tubules, mitochondria, cytoplasmic vacuoles, and degenerative intramitochondrial areas increased after hypoxia, those of myofibrils and mitochondrial cristae decreased. Since these alterations are more striking than after hypoxia of non-diabetic animals as demonstrated in preceding studies, we regard them as indicative for reduced hypoxia tolerance of the diabetic myocardium. EGb-treatment of the diabetic animals could improve the above mentioned parameters thus indicating a gradual improvement of the hypoxia tolerance. The biochemical parameters of oxidative stress (malondialdehyde, superoxide dismutase) were decreased after hypoxia in the diabetic myocardium but increased after EGb-pretreatment. The ultrastructural damage by hypoxia and its prevention by EGb should be regarded rather as a consequence of ATP--and energy deficiency and breakdown of membrane functions and--structure resp. as membrane stabilizing and enzyme-regulating effects of EGb than as radical-related events. The hypoxia-induced deprivation of creatine kinase activity of the diabetic myocardium was not prevented by EGb-treatment. Immunohistochemical demonstration of iNOS expression was strongest in the unprotected diabetic myocardium, absent after additional hypoxia and in the controls, and very weak in the protected hypoxic specimens. Transcription of iNOS as demonstrated by RT-PCR was present in few diabetic, some of the hypoxic diabetic, in most of the EGb-treated hypoxic diabetic, and in all control animals. EGb-treatment seems to improve the hypoxia tolerance of diabetic myocardium concerning ultratructural parameters. The partly conflicting immunohistochemical and biochemical results require further investigations.

Lipoxygenase inhibitor FLM 5011, an effective protectant of myocardial microvessels against ischemia-reperfusion injury? An ultrastructural-morphometric study.

The lipoxygenase inhibitor FLM 5011 was used for protection of the coronary microcirculation against ischemia/ reperfusion injury after ligation of the left coronary artery in dogs. Epimyocardial biopsies from ischemic and non-ischemic areas of protected and unprotected areas taken before and after ischemia of 90 min duration and after 180 min reperfusion were analysed by means of electron microscopic morphometry. The ischemic injury consisted in endothelial swelling, luminal blebbing, and formation of irregular protrusions, partly occurrence of pericapillary edema and cellular debris. Plasmalemmal vesicles seemed to decrease in frequency, mitochondria showed focal or generalized degeneration of cristae and matrix. Reperfusion partly deteriorated the damage, partly restoration of ultrastructural parameters was to be observed. There were no significant differences between the infarcted and not infarcted areas. FLM 5011 treatment reduced the endothelial edema, blebbing and occurrence of pericapillary debris and stabilized the number of vesicles. The protection of the mitochondrial cristae and matrix was statistically significant. The results indicate that FLM 5011, under the condition of the experiment, effectively protects the ultrastructure of essential endothelial structures of myocardial microcirculation, explained by the blocking of the noxious leucotrienes and peptidoleucotrienes liberated by the 5-lipoxygenase pathway of the free arachidonic acid and by scavenging of oxygen free radicals. The results must be confirmed by further experiments including biochemical and functional parameters.

Protective effects of Gingko biloba extract EGb 761 on myocardium of experimentally diabetic rats. I: ultrastructural and biochemical investigation on cardiomyocytes.

Chronic diabetes in man and animal models develops cardiomyopathic alterations which cannot be absolutely avoided by insuline therapy. Since diabetic damage is partly attributed to oxidative stress antioxidative treatment could be able to reduce the alterations. Aim of this study was to investigate the cardioprotective effects of EGb 761, known as a radical scavenger, against diabetic alterations in rats. The diabetes was induced by i.p. injection of 60 mg/kg body weight streptozotocin. Duration of diabetes was 4 months, the protected group received 100 mg/kg body weight EGb 761 with the drinking water over 3 months. Electron and light microscopic morphometry of left-ventricular samples revealed typical diabetic alterations consisting in decrease of volume fraction of myofibrils, SR and t-tubules and diminishing of cardiomyocyte diameter, increase of interstitial volume, mitochondrial size and volume fraction, and of vacuoles and of lipid drops. EGb treatment could gradually prevent the loss of myofibrils and reduction of myocyte diameter but has only little influence on interstitial and mitochondria volume. The diabetic-induced increase of lipid and vacuoles and the decrease of SR and t-tubules were not influenced. Biochemical parameters of oxidative stress: malondialdehyde (MDA) was only insignificantly altered by diabetes and EGb. The superoxide dismutase (SOD) activity was increased by diabetes and more increased by EGb treatment. Creatine kinase (CK) activity was diminished by diabetes but slightly increased by EGb. The polymerase chain reaction (PCR) of i-NOS was not different between the diabetic and protected diabetic groups.

Protective effects of Ginkgo biloba extract EGb 761 on the myocardium of experimentally diabetic rats. II. Ultrastructural and immunohistochemical investigation on microvessels and interstitium.

Interstitial and microvascular disorders are known as a characteristic part of the diabetic cardiomyopathy and to resist partly insulin therapy. Aim of this study was to demonstrate structure-protecting effects of Ginkgo Extract EGb 761 known as a natural radical scavenger in streptozotocin-diabetic rats on the microvascular compartment. Wistar rats (n = 5) were made diabetical by i.p. injection of 60 mg/kg body mass streptozotocin for 4 months. Rats of the protected group (n = 5) received daily 100 mg/kg body mass EGb 761 for 3 months, starting 1 month after induction of diabetes. 5 age-matched rats served as control. The volume fraction of interstitium was slightly but significantly increased only in the unprotected diabetic group. Diminishing of the capillary to the myocyte ratio was seen in the diabetic but not in the protected group. Immunostaining of collagen revealed a slight increase of type III, type IV, and type VI fibres in the interstitium, more expressed in the unprotected group. Ultrastuctural morphometry revealed significant thickening of endothelial and muscular basement membranes in diabetic animals, less expressed in the EGb- protected group. The capillary diameter was slightly increased in the diabetic and slightly decreased in the protected group. The number of plasmalemmal vesicles was tendentially more decreased, that of lysosomes more increased in the diabetic than in the protected group. It is concluded that EGb 761 can diminish partly interstitial fibrosis and reduce endothelial and muscular basement membrane thickening of the diabetic myocardium. It may contribute to prevent late diabetic complications.

The protective effect of lipoxygenase inhibitor FLM 5011 on mitochondria ultrastructure of ischemic and reperfused cardiomyocytes.

Lipoxygenase inhibitor FLM 5011 was used in experimental ischemia and reperfusion with dogs to investigate its ultrastructure-preserving effects on the mitochondria of myocardium. Ischemic and non-ischemic areas of the heart were ultrastructural-morphometric analysed, which revealed that FLM 5011 was able to diminish ischemic damage especially of mitochondria. The protective effects on mitochondria consisted mainly in reduction of defective intramitochondrial areas and in excellent protection of the structural integrity of cristae and matrix. The injury of mitochondria by ischemia/reperfusion in unprotected condition was partly more pronounced in the non-ischemic than in the ischemic area of the hearts probably caused by compensatory overload of the residual myocardium.

The protective effect of iloprost, a PGL2 analogue, on ultratructure of ischemic and reperfused myocardial myocytes.

Iloprost was used in experiments with dogs to investigate its ultrastructure-preserving effects on the myocardium during ischemia and reperfusion, focussed especially on the mitochondria. Ischemia was performed by ligation of the left anterior descending coronary artery (LAD) for 90 min followed by reperfusion of 180 min. Samples were taken from the ischemic and non-ischemic area before ligation of LAD, immediately after ischemia and reperfusion. Iloprost was applicated into the left femoral vein after the end of LAD ligation. Ultrastructural-morphometric analysis revealed that iloprost was able to diminish damages of the mitochondria after ischemia and reperfusion as well. Mitochondrial oedema and intramitochondrial structure degenerations (destruction of cristae and matrix) were minimized in contrast to results of unprotected animals, which had to undergo the same procedure as indicated by volume, volume densities of mitochondria, and of the intramitochondrial compartments, confirmed by independent secondary morphometric parameters. There were no remarkable differences between the corresponding parameters of mitochondria in the ischemic and non-ischemic area.

N2O-induced hypoxia as a model of hypoxic stress of rat myocardium. An ultrastructural-morphometric study.

A model system consisting of a hypoxia chamber combined with a commercial narcosis apparatus adapted to small animals was used to perform a controlled acute isobaric hypoxia on rats with N2O/O2. Ultrathin sections from the left ventricular wall were analysed qualitatively and quantitatively using a computer-aided morphometric program. Compared with the control the cardiomyocytes exhibited a significant increase of volume densities of cytoplasmic vacuoles, lipid drops, sarcoplasmic reticulum, mitochondria, and degenerated intramitochondrial areas. Hypoxic alterations of microvessels consisted mainly in localized endothelial swelling and perivascular edema, protrusions of the luminal surface and moderate mitochondrial alterations similar to those of cardiomyocytes. Further, the number of plasmalemmal vesicles decreased, and the number of vacuoles increased significantly. The results were confirmed by quantitative histochemistry performed by our group in a parallel study. The model can be recommended for studies concerning protective interventions in hypoxia experiments.

Morphological investigations of the myocardium of cardiomyopathic hamsters during the postnatal development and experimental hypoxia. A quantitative ultrastructural study.

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.

Myocardium-protective effects of Ginkgo biloba extract (EGb 761) in old rats against acute isobaric hypoxia. An electron microscopic morphometric study. I. Protection of cardiomyocytes.

Ginkgo biloba extract EGb 761 was used in hypoxia experiments with old rats to investigate its ultrastructure-preserving effects on the myocardium. Hypoxia was performed by means of a hypoxia chamber combined with a commercial narcosis apparatus. N2O/O2-mixture was applied with O2 at 5 vol.% for 20 minutes under normobaric conditions. Ultrastructural-morphometric analysis revealed that EGb 761-pretreatment was able to diminish hypoxic damage at mitochondrial cristae and matrix and also distension of the sarcoplasmic reticulum during acute hypoxic stress. Whereas formation of vacuoles was depressed below the level of controls, the accumulation of lipid drops was not prevented. The preservation of mitochondrial cristae was confirmed by independent secondary morphometric parameters and by cytophotometrically measured activities of mitochondrial enzymes.

Myocardium-protective effects of Ginkgo biloba extract (EGb 761) in old rats against acute isobaric hypoxia. An electron microscopic morphometric study. II. Protection of microvascular endothelium.

Aim of this electron microscopic morphometric study was to demonstrate ultrastructure protective properties of Ginkgo biloba extract EGb 761 on myocardial microvessels of old rats during acute hypoxic stress. Hypoxia of 20 minutes duration with N2O/O2 mixture (5 vol% O2) was performed using a hypoxia chamber combined with a commercial narcosis apparatus. EGb 761-pretreatment diminished significantly the percentage of endothelial cells exhibiting edema, luminal blebs and of capillaries surrounded by pericapillary debris. Hypoxia-related decrease in plasmalemmal vesicle frequency was prevented by EGb 761, formation of vacuoles non significantly diminished against the hypoxic group. Volume density of mitochondrial cristae was significantly less diminished, the volume fraction of degenerated areas less increased in the EGb 761-protected group. The results give some evidence that EGb 761 protects endothelial cell ultrastructure of myocardial microvasculature against hypoxic alterations, probably by its radical scavenging properties.

Ultrastructural morphometric investigation of the heart of rabbits after a single administration of reserpine.

The ultrastructure of myocardial cells of chinchilla rabbits was morphometrically examined after administration of one dose of reserpine. The results show that 24 hours after administration of reserpine, there is a decrease in the number and an increase in the size of the mitochondria, a relaxation or narrowing and ramification of myofibrils, an increase in intracellular lipid content compared with control animals. These changes are the result of functional and chemical desympathization and they represent an ultrastructural-morphometric characteristic of the action on cardiomyocytes.

[Ultrastructural morphometric studies of the myocardium of cardiomyopathic hamsters of the BI0 8262 strain]. / Ultrastrukturell morphometrische Untersuchungen am Myocard cardiomyopathischer Hamster des Stammes BI0 8262.

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.

[The use of small and large computers for setting up data banks for morphometry]. / Nutzung von Klein- und Grosscomputern zum Erstellen eines Datenspeichers für die Morphometrie.

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.

Ultramicroscopic morphometric, biochemical and histophotometric investigations of myocardial biopsies taken from Fallot patients before and after cardioplegia with cold Kirsch's solution.

Biopsies taken from the myocardium of 5 patients with Morbus Fallot and from 1 patient with ventricle septum defect were investigated with a combination of morphometric, biochemical and histophotometric techniques in order to study the cardioprotective effect of cold Kirsch's solution. At the final phase of cardioplegia the cardiomyocytes reveal the following alterations: The volume densities of mitochondria, of their degenerated areas and that of cytoplasmic vacuoles show a significant increase whereas that of myofilaments decreases. Cristae and matrix mitochondriales, however, show only moderate alterations without statistical significance. Biochemically the total ATP-concentration and creatine phosphate (CP)-concentration were more or less diminished, in most of the cases the activity of the myosin ATPase was increased, that of the creatine phosphate kinase (CPK) diminished. Compared with the biochemical estimations of the ATPase activity, its histophotometric estimations yielded corresponding results in 2 of 4 cases. In general our findings confirm the cardioprotective effect of Kirsch's solution. The combination of methods used gives more reliable results than one technique alone.