Cellular mechanisms of the protective effect of polyphenols on the neurovascular unit in strokes.

Increasing evidence has suggested that our view of stroke should be integrative, and thus a concept of dynamic interaction between cells belonging to the neurovascular unit, such as endothelial cells, astrocytes and neurons, is emerging. The functionality of this unit is altered by the complex series of interconnected pathophysiological processes that damage the brain tissue during this kind of attack. The new strategies target both the preservation of endothelium integrity and the deleterious effects induced by ionic imbalance, excitotoxicity, and the generation of reactive oxygen species within the neurovascular unit. Polyphenols exert numerous biological effects that might participate in the protection of the neurovascular unit, including anti-aggregatory platelet activity, antioxidant and free radical scavenging properties. Moreover, polyphenols are powerful vasodilators through the generation of NO, and can act on the expression of genes protective of the cardiovascular system. Also, polyphenols contribute to the preservation of the integrity of cells belonging to the neurovascular unit, mainly the endothelium, by acting on the signaling cascades implicated in endothelial apoptosis. All these effects of polyphenols might interfere with atherosclerotic plaque development and stability, vascular thrombosis and occlusion and therefore might explain their vascular- and neuroprotective properties. In this review, we focus on the beneficial effects of polyphenols on the complex pathophysiological events of stroke and helpful indications for the design of an effective and well-tolerated therapy will be discussed.

Effect of 17 beta-estradiol on the brain damage and metabolic changes in rats.

An in vivo model of glutamate excitotoxicity in which glutamate is applied to the cortex of rats through a microdialysis probe has been used to investigate the neuroprotective processes initiated by 17 beta-estradiol. Rats were pre-treated with 17 beta-estradiol i.v. before local application of glutamate. The experimental results showed that pre-treatment with 17 beta-estradiol significantly reduced the size of the glutamate-induced lesion. In the microdialysates, the peak of lactate observed immediately after glutamate application was significantly higher and longer lasting after 17 beta-estradiol pre-treatment. The level of extracellular glucose was markedly decreased concomitantly to the increase in lactate, but no difference could be observed with and without 17 beta-estradiol pre-treatment. These suggest a new neuroprotective mechanism of 17 beta-estradiol by activating glutamate-induced lactate production. This effect on lactate production and lesion reduction is estrogen receptor dependent and is abolished totally by estrogen antagonist tamoxifen. It was also demonstrated here that high lactate subserves estrogen neuroprotection during glutamate toxicity.

17beta-Estradiol reduces cortical lesion size in the glutamate excitotoxicity model by enhancing extracellular lactate: a new neuroprotective pathway.

Estrogens play an important role in neuronal function and in protecting neurones in the cerebral cortex against pathological conditions. An in vivo model of glutamate excitotoxicity in which glutamate is applied to the cortex of rats through a microdialysis probe has been used to investigate the neuroprotective processes initiated by 17beta-estradiol. Rats were pre-treated with 17beta-estradiol (i.v.) before local application of 100 mM glutamate into the cortex through a microdialysis probe. Pre-treatment with 17beta-estradiol significantly reduced the size of the glutamate-induced cortical lesion. In the cortical microdialysates collected from the probe, a peak of lactate was observed immediately after glutamate application. After 17beta-estradiol pre-treatment this peak of lactate was significantly higher with estradiol than without 120 min after glutamate application, reaching 700% basal level at the end of measurement. The level of extracellular glucose was markedly decreased with and without 17beta-estradiol pre-treatment. Local blockage of neuronal lactate transporters with alpha-cyano-4-hydroxycinnamate (4-CIN) completely abolished the neuroprotective effect of 17beta-estradiol and induced a larger cortical lesion. An accumulation of extracellular lactate was observed after inhibition of the lactate transporters suggesting that transport of lactate into neurones is necessary for the neuroprotective effect of 17beta-estradiol. The anti-estrogen tamoxifen also abolished the neuroprotective effect of 17beta-estradiol on the lesion size and inhibited the production of lactate. These results suggest a new neuroprotective mechanism of 17beta-estradiol by activating glutamate-stimulated lactate production, which is estrogen receptor-dependent.

Characterisation of autoantibodies to peripheral myelin protein 22 in patients with hereditary and acquired neuropathies.

To investigate the possibility that an autoimmune mechanism may play a role in the hereditary neuropathy Charcot-Marie-Tooth type 1A (CMT1A), sera were analysed by Western blot for anti-peripheral myelin protein 22 (PMP22) autoantibodies. These sera were compared with sera from patients with CMT type 2 (CMT2), acquired peripheral neuropathies such as chronic inflammatory demyelinating neuropathy (CIDP), anti-MAG IgM neuropathy, Miller-Fisher syndrome (MFS), diabetic neuropathy and with control blood donors. Anti-PMP22 positive sera were detected in 70% of patients with CMT1 and unexpectedly in 60% of patients with CMT2. Interestingly, 44% of the patients with other peripheral neuropathies and 23% of the apparently healthy controls showed also anti-PMP22 antibody reactivity. Immunohistochemical analysis of the human anti-PMP22 antisera on healthy sural nerve sections and on PMP22-expressing COS cells revealed that these sera did not recognise endogenous PMP22. Our results indicate that anti-PMP22 autoantibodies are found in sera of patients with different types of peripheral neuropathies, but their role in the pathogenesis of these diseases remains to be determined.

Anti-MAG IgM penetration into myelinated fibers correlates with the extent of myelin widening.

The purpose of this study was to evaluate the relationship between immunoglobulin M (IgM) antibodies penetration into myelinated peripheral nerve fibers and the widening of the peripheral myelin sheaths in anti-myelin-associated glycoprotein (anti-MAG) demyelinating IgM monoclonal polyneuropathy. Demyelinating polyneuropathy with monoclonal IgM is often associated with anti-MAG autoantibodies, which are thought to initiate the disease with IgM deposits usually present on the myelin sheaths. We analyzed nerve biopsies from 12 patients with an anti-MAG demyelinating neuropathy by confocal and electron microscopy. The total number of nerve fibers and the proportion of IgM-associated fibers were quantified after immunohistochemical staining. The affinities of IgM were examined by analyzing the binding pattern of serum IgM on normal peripheral nerve sections. Ultrastructural examinations of the biopsies showed a good correlation between in situ widened myelin sheaths and the IgM penetration level into myelinated fibers. The terminal complement complex appears not be involved in the penetration of IgM into the myelinated fibers. Our findings suggest a causative role of the IgM anti-MAG antibodies in the ultrastructural modifications of the myelin sheaths. The basement membrane and myelin components appear to be the major targets of the IgM monoclonal antibodies. However, the pathogenic mechanism whereby IgM antibodies reach their targets and induce nerve damage are still unclear.

Intracellular acidification mediates the proliferative response of PC12 cells induced by potassium ferricyanide and involves MAP kinase activation.

Potassium ferricyanide is known to elicit cell growth and mitogenesis in various cells by stimulating a transplasma membrane electron-transport system. When serum-starved PC12 cells were treated with potassium ferricyanide, stimulation of mitogenesis was evidenced by enhanced DNA synthesis, as well as by increased cell numbers. Intracellular pH (pH(i)) of PC12 cells was measured at 37 degrees C by microfluorimetric analysis of 2',7'-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). The resting pH(i) of unstimulated cells was 7.52 (external pH 7.40). Addition of potassium ferricyanide (100 microM) decreased pH(i) by about 0.25 pH units. Lowering pH(i) to a similar extent, either by decreasing external pH (pH(o)) or by adding a weak acid, also elicited a mitogenic response, indicating that intracellular acidification by itself has growth factor-mimicking, mitogenic effects. Nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) triggered proliferation without changes in pH(i). The mitogenic treatments eliciting intracellular acidification did not activate protein kinase C (PKC) but stimulated the p42/p44 mitogen-activated protein (MAP) kinase. Our results indicate that 2 distinct mitogenic pathways are active in PC12 cells: the first is independent of pH(i) and involves activation of the PKC pathway and the second requires a permissive pH(i) value around 7.25 and involves activation of the p42/p44 MAP kinase pathway.

Impaired gene transcription and nuclear protein kinase C activation in the brain and liver of aged rats.

The expression of the hsp70 and c-fos genes and the activation of nuclear protein kinase C (PKC) were studied in young and aged whole rats under heat-shock conditions. The induction of hsp70 and c-fos genes by heat shock were decreased several fold in the brain as well as in the liver of senescent animals. Nuclear run-off transcription assay indicated that this age-related impairment could be attributed to a block at the level of transcription. Nuclear PKC activation by heat shock was not apparent in old animals. Nuclear PKC involvement in the repression of transcription during senescence is postulated.

Stimulation of nuclear protein kinase C leads to phosphorylation of nuclear inositol 1,4,5-trisphosphate receptor and accelerated calcium release by inositol 1,4,5-trisphosphate from isolated rat liver nuclei.

Rat liver nuclei inositol 1,4,5-trisphosphate receptor (Malviya, A.N., Rogue, P., and Vincendon, G. (1990) Proc. Natl. Acad. Sci U.S.A. 87, 9270-9274) is identified as a 220-kDa protein on Western blotting employing two types of antibodies (anti-goat and anti-rabbit) raised against purified rat brain inositol 1,4,5-trisphosphate receptor (IP3R). Nuclear IP3R does not seem identical with microsomal IP3R in rat liver. Treatment of isolated rat liver nuclei with 12-O-tetradecanoylphorbol-13-acetate stimulates native protein kinase C activity severalfold. Nuclear IP3R is phosphorylated by stimulated protein kinase C, with accelerated as well as enhanced maximum 45Ca2+ release by inositol 1,4,5-trisphosphate from isolated nuclei, without altering 1,4,5-trisphosphate binding characteristics (KD and Bmax). Stimulation of nuclear protein kinase C is found physiologically relevant since lamin B2, a nuclear protein, is concomitantly phosphorylated. These data deal with functional consequences of nuclear IP3R phosphorylation by native protein kinase C in isolated rat liver nuclei. It is postulated that phosphorylation of nuclear IP3R, probably dephosphorylation also, subserves a key mechanism in nuclear calcium homeostasis.

Heat stressing stimulates nuclear protein kinase C raising diacylglycerol levels. Nuclear protein kinase C activation precedes Hsp70 mRNA expression.

Protein kinase C (pKC) activity has been studied in rat liver after subjecting animals to heat shocking. Nuclear pKC activity was stimulated owing to heat shocking without any change in the cytosolic enzyme activity. The nuclear diacylglycerol levels were raised owing to heat stress along with the stimulation of polarhead phospholipid hydrolysis. Kinetically, the Vmax of nuclear pKC was enhanced as a result of heat shocking, with no change in apparent Km and with concomitant phosphorylation of nuclear lamin B2. Western blot analysis as well as phorbol dibutyrate binding indicate that pKC protein levels did not change because of heat shocking. The stimulation of nuclear pKC under heat stress conditions represents an in vivo phenomenon and the enzymes stimulation precedes Hsp70 mRNA expression.

Endothelin regulation of neuropeptide release from nerve endings of the posterior pituitary.

We have investigated the role of endothelin (ET) in the stimulus-secretion coupling mechanism in the posterior pituitary. We report that isolated nerve endings contain immunoreactive endothelin, the level of which is regulated by homeostatic mechanisms involved in control of water balance. ET-1 and ET-3 potentiate vasopressin release induced by depolarization through interaction with specific receptors of the ETA subtype and this response is antagonized by sarafotoxin S6b. The second messenger for this effect, however, remains unknown since the potentiation of depolarization-induced vasopressin release occurs in the absence of an increase in cellular calcium.