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.

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.

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.

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.