G. Fitzl

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.

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.

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.

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 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%.

Repin1 maybe involved in the regulation of cell size and glucose transport in adipocytes.

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.

Cardioprotective effect of EGb 761 on myocardial ultrastructure of young and old rat heart and antioxidant status during acute hypoxia

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.

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.

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.

The Effect of Ginkgo biloba extract (EGb 761) on parameters of oxidative stress in different regions of aging rat brains after acute hypoxia

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 short-term hypoxia. EGb’s protective effect is especially notable in the brainstem and the cerebellum.