Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death.

In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract EGb 761, (definition see editorial) and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death. In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death (effective concentration [EC (50)] = 5 microg/ml (12 microM) for bilobalide and 100 microg/ml for EGb 761. These results suggest that both EGb 761 and bilobalide are protective against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

BN 80933 inhibits F2-isoprostane elevation in focal cerebral ischaemia and hypoxic neuronal cultures.

Formation of the lipid peroxidation product 8-epi-prostaglandin2alpha (8-epi-PGF2alpha) a bioactive marker of oxidative stress, was quantified in in vitro and in vivo models of neuronal death. In culture media of primary rat cortical neurones exposed to hypoxia followed by reoxygenation, a 3.7-fold increase of 8-epi-PGF2alpha concentration was observed in comparison to control cells. In rats submitted to 2h middle cerebral artery occlusion followed by a 22h reperfusion period, a 27-fold increase of 8-epi-PGF2alpha was observed in the ischaemic hemisphere compared with the corresponding hemisphere of sham-operated rats. Treatment with the neuroprotective agent BN 80933 significantly reduced both 8-epi-PGF2alpha elevations in vitro and in vivo. These data suggest that 8-epi-PGF2alpha elevations might reflect the damaging free radical overproduction and subsequent lipid peroxidation during neuronal injury induced by hypoxia and ischaemia. Inhibition of 8-epi-PGF2alpha elevations participates to the neuroprotective effects of BN 80933.

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: a promising neuroprotective strategy.

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-[4-[4-[(3,4-dihydro-6-hydroxy-2, 5,7, 8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2- thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro, affords remarkable neuronal protection in vivo. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3-10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.

Synergistic protective effects of antioxidant and nitric oxide synthase inhibitor in transient focal ischemia.

Both nitric oxide synthase (NOS) inhibitors and free radical scavengers have been shown to protect brain tissue in ischemia-reperfusion injury. Nitric oxide and superoxide anion act via distinct mechanisms and react together to form the highly deleterious peroxynitrite. Therefore the authors examined the effects and the interaction between the NOS inhibitor, NG nitro-L-arginine (LNA) and the antioxidant/superoxide scavenger, di-tert-butyl-hydroxybenzoic acid (DtBHB) in the rat submitted to 2 hours of middle cerebral artery occlusion. Posttreatment was initiated 4 hours after the onset of ischemia and infarct volume was measured at 48 hours. The dose-related effect of LNA resulted in a bell-shaped curve: 15, 56, 65, and 33% reduction of total infarct for 0.03, 0.1, 0.3, and 1 mg/kg (intravenously [IV]) respectively and 11% increase in infarct volume for 3 mg/kg (IV). Whereas DtBHB (20 mg/kg; intraperitoneally [IP]) was ineffective, the dose of 60 mg/kg produced 65% protection in infarct volume. The combination of a subthreshold dose of LNA (0.03 mg/kg; IV) and DtBHB (20 mg/kg; IP) resulted in significant reduction (49%) in infarct volume. These results show that LNA and DtBHB act synergistically to provide a consistent neuroprotection against ischemic injury when administered 4 hours after ischemia. This suggests that nitric oxide and free radicals are involved and interact in synergy in ischemia-reperfusion injury.

Changes in [3H]PAF binding and PAF concentrations in gerbil brain after bilateral common carotid artery occlusion: a quantitative autoradiographic study.

This study investigated the distribution of platelet activating factor (PAF) binding sites in the brain and their involvement in global ischemia in a model of bilateral common carotid occlusion in the gerbil. In sagittal sections of gerbil brain, labeling with [3H]PAF was mainly located in the cortex, hippocampus and cerebellum. The corpus striatum, the superior and inferior colliculi showed lower binding, while the thalamus was only weakly labeled. Scatchard analysis of the data obtained from displacement curves with unlabeled PAF revealed the presence of one or two populations of binding sites with different affinity constant values depending on the brain structures. When the gerbils were submitted to 10 min ischemia, similar autoradiography with [3H]PAF demonstrated a dramatic reduction of labeling in all brain structures, particularly in the hippocampus. Immunoreactive endogenous PAF concentrations in brain tissues showed a marked increase in ischemic animals: (8977.3 +/- 1113 pg/g wet weight) as compared to sham-operated control: (997.7 +/- 77 pg/g wet weight). Endogenous PAF levels returned to basal values following 30 min reperfusion. These results indicate that PAF may be involved in the early stages of brain ischemia in the gerbil and suggest that endogenous PAF produced during ischemia may contribute to the down-regulation of [3H]PAF binding sites in the brain.

Peripheral type benzodiazepine binding sites following transient forebrain ischemia in the rat: effect of neuroprotective drugs.

Recent studies have demonstrated that measurement of peripheral type benzodiazepine binding sites (PTBBS) levels may be useful as an index for quantification of neuronal damage. In the present study, we investigated the accuracy of this index as a marker of neuronal damage induced by transient forebrain ischemia in the rat (4-vessel occlusion model). Seven days after ischemia, a good correlation was found between the increase of PTBBS levels (measured using [3H]PK 11195 as a specific radioligand) in hippocampal, striatal and cortical homogenates and the duration of ischemia. The progression of PTBBS increase was examined from 3 h to 14 days of recirculation. Increase in the maximal number of binding sites (Bmax) rather than an effect on the affinity (KD) for the radioligand was found in the 3 brain regions. Treatment of the animals with 1,3 butanediol (BD) prior to ischemia resulted in a neuroprotective effect as assessed by an improved neurological score and histological studies. The protective effect of BD was also correlated with a reduced expression of PTBBS as compared to ischemic animals not treated with the drug. No protective effects, on neurological score or PTBBS level were afforded by MK-801, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, R-phenylisopropyladenosine (RPIA), an adenosine A1 receptor agonist, or BN 52021, an antagonist of platelet-activating factor (PAF). These results suggest that PTBBS provide a useful marker of neuronal damage in a transient forebrain ischemia model and confirm the beneficial effect on ischemic damage exerted by BD.

Presence of specific binding sites for platelet-activating factor (PAF) in brain.

The existence of specific binding sites for [3H]-labelled PAF ([3H]PAF) was investigated on membrane preparations of gerbil brain. Binding assays of [3H]PAF showed a specific, saturable, reversible and time dependent binding. Scatchard analysis indicated the presence of two apparent populations of binding sites with Kd1 = 3.66 +/- 0.92 nM and Kd2 = 20.4 +/- 0.50 nM corresponding respectively to a maximum number of binding sites: Bmax1 = 0.83 +/- 0.23 pmol/mg protein Bmax2 = 1.1 +/- 0.32 pmol/mg protein. The binding of [3H]PAF was fully displaced by unlabelled PAF and partially inhibited by the PAF antagonist BN 52021 suggesting that BN 52021 interacts only with one site. Distribution of [3H]PAF specific binding revealed a maximum amount of binding in midbrain and hippocampus. These data suggest a biochemical and physiological role of PAF in brain.

Effects of a platelet activating factor antagonist (BN 52021) on free fatty acids, diacylglycerols, polyphosphoinositides and blood flow in the gerbil brain: inhibition of ischemia-reperfusion induced cerebral injury.

The effects of a platelet activating factor antagonist (BN 52021) on cerebral ischemia-reperfusion was studied in the gerbil. Following ten minutes of bilateral carotid artery ligation, gerbils were reperfused and injected intraperitoneally with either BN 52021 or vehicle-dimethylsulfoxide. Cerebral blood flow and systemic arterial pressure were monitored until 90 minutes of reperfusion. Free fatty acids, diacylglycerols and polyphosphoinositides were then analyzed in forebrains and midbrains. BN 52021 inhibited the maturation of ischemic injury. Cerebral blood flow increased following 60 to 90 minutes of reperfusion. Free fatty acid levels were reduced likely by inhibition of phospholipase A. Phospholipase activity may likely be decreased since there was a tendency to increase phosphatidylinositol-4',5'-bisphosphate and diacylglycerols in BN 52021-treated animals.

Involvement of platelet-activating factor (PAF) in cerebral post-ischemic phase in Mongolian gerbils.

Platelet-activating factor (PAF) is a potent mediator of anaphylaxis and shock. In addition, evidence for PAF participation in gastric, intestinal and heart post-ischemic phase has been recently demonstrated. Ginkgo biloba extracts improve cerebral metabolism and protect brain against hypoxic damage in various models of cerebral ischemia. Potent and specific antagonists of PAF have been found in Ginkgo biloba and termed Ginkgolides: BN 52020, BN 52021, BN 52022, BN 52024. We therefore undertook the investigation of the role of Ginkgolides in cerebral ischemia obtained by bilateral ligature of the common carotid for 10 min and 6 h of recirculation in male Mongolian adult gerbils. Given preventively (one week treatment 10 mg/kg/day orally) or at the time of clamping, BN 52021 and related Ginkgolides dose-dependently antagonize morbidity assessed by the stroke-index. Similarly the mitochondrial respiration evaluated by the respiratory control ratio is significantly improved. In both determinations, the range of activity: BN 52021 greater than, BN 52020 greater than BN 52022 greater than BN 52024 shows that the effect of Ginkgolides in cerebral ischemia are correlated with their PAF antagonistic properties. Given curatively, 1 h after declamping, BN 52021 is able to reverse the cerebral impairment trend. Kadsurenone and brotizolam, two other chemically unrelated PAF antagonists led to similar recovery. Therefore PAF appears to play an important role in the post-ischemic phase after bilateral carotid ligation in Mongolian gerbils.

[Effects of Ginkgo biloba extract on a cerebral ischemia model in gerbils]. / Effets de l'extrait de Ginkgo biloba sur un modèle d'ischémie cérébrale chez la gerbille.

Certain anatomical characteristics peculiar to the gerbil make it the animal model best adapted to experimental pathology studies of acute ischaemia. In this animal species, devoid of any substitute vertebro-basilar vascular tissue, unilateral ligature of the carotid artery produces a cerebral ischaemia with neurological signs (well quantifiable), metabolic perturbations (especially mitochondrial) and cerebral oedema development closely resembling the symptoms revealed by physiopathology in human clinical studies. Using this model and under the experimental conditions described, clear-cut, highly significant results were obtained with Ginkgo biloba, whether by oral or intravenous administration. These results were normalization of mitochondrial respiration, diminution of cerebral oedema, correction of the accompanying ionic perturbations, and practically total functional restoration revealed by a normal neurological index in the gerbils treated with Ginkgo biloba extract.

Specific inhibition of PAF-acether-induced platelet activation by BN 52021 and comparison with the PAF-acether inhibitors kadsurenone and CV 3988.

BN 52021 is a chemically defined substance extracted from Ginkgo biloba leaves. Its inhibitory potency was tested on washed human platelets prepared so as to render them specifically sensitivity either to adenosine 5'-diphosphate (ADP), arachidonic acid (AA) or PAF-acether. Its activity and specificity were compared with those of two other reported inhibitors of PAF-acether effects: Kadsurenone and CV 3988. PAF-acether-induced aggregation of washed human platelets was concentration dependently inhibited by BN 52021 (IC50: 2.22 +/- 0.79 microM against 7.5 nM PAF-acether (n = 3)). Under the same experimental conditions the aggregation triggered by ADP was not modified and that induced by AA was marginally affected. The PAF-acether EC50 in platelet-rich plasma was increased 5- and 46-fold with 1 microM and 5 microM of BN 52021 respectively. This strongly suggested that the mechanism of action of BN 52021 is of the competitive type. Analysis of [3H]PAF-acether binding showed that BN 52021 as well as unlabelled PAF-acether prevented [3H]PAF-acether binding to intact washed platelets. In washed human platelets Kadsurenone affected only PAF-acether-induced aggregation (IC50: 0.8 +/- 0.4 microM (n = 3)), whereas CV 3988 inhibited the aggregation induced by ADP, AA and PAF-acether (IC50 were 10.2 +/- 2.3 microM; 2.2 +/- 0.1 microM; 1.0 +/- 0.1 microM respectively (n = 3). In contrast, up to 30 microM, CV 3988 was a specific antagonist of PAF-acether-induced platelet aggregation in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibition of plasma protein leakage and Salmonella enteritidis-induced mortality in the rat by a specific paf-acether antagonist: BN 52021.

The effects of BN 52021, a new specific paf-acether receptor antagonist and the total Ginkgo Biloba extract (GBE 761) from which this product was isolated, were studied in the rat on paf-acether-induced permeability and cell number changes and on endotoxin-induced lethality. Their activities were compared to those of cyclooxygenase, 5-lipoxygenase and phospholipase A2 inhibitors. BN 52021 given s.c. or orally exerted a dose-related inhibition of paf-acether deleterious effects as well as of endotoxin lethality whereas the other drugs tested were poorly effective. These results strongly suggest paf-acether involvement in endotoxic and septic shock.

Effects of agents used in the pharmacotherapy of cerebrovascular disease on the oxygen consumption of isolated cerebral mitochondria.

A number of drugs used in the pharmacotherapy of cerebral metabolic and vascular disease have been studied for their effects on the respiration of mitochondria isolated from the rat brain. Some of these agents increased the respiratory control ratio by more than 5% from base-line values (at p less than 0.05), namely, aminophylline, dihydroergotoxine, ifenprodil, nicergoline, raubasine, and vincamine. The ability of these agents to increase the efficiency of mitochondrial respiration could be correlated with two other attributes peculiar to these five drugs: their ability to contract cerebrovascular smooth muscle when studied in vitro and their ability to decrease the volume of infarcted brain tissue following experimental occlusion of the middle cerebral artery in the cat. Papaverine and its derivatives (naftidrofuryl, viquidil, YC-93) decreased respiratory control, an effect that might correlate with their capacity to effect a vasodilatation of the cerebral vessels and their inefficacy in models of acute cerebral infarction. There is a considerable body of evidence suggesting that one of the earliest and most fundamental perturbations of cerebral ischaemia is a loss of respiratory control. Ifenprodil, vincamine, and some related "anti-ischaemic" compounds are capable of increasing respiratory control in normal cerebral mitochondria, and this capacity might well help to explain their therapeutic potential in cerebrovascular disorders in which energy supply to the brain is limited.