Victor Rimbau

Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas, Madrid, Madrid, Spain

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Publications (19)54.59 Total impact

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    ABSTRACT: Caspase-3 is a key protein involved in the classical apoptosis mechanism in neurons, as in many other cells types. In the present research, we describe the transcriptional activity of caspase-3 gene as a marker of acute toxicity in a primary culture model of rat cerebellar granule neurons (CGNs). CGNs were incubated for 16h in complete medium containing the chemicals at three concentrations (10, 100microM and 1mM). A total of 48 different compounds were tested. Gene transcriptional activity was determined by low-density array assays, and by single Taqman caspase-3 assays. Results from the PCR arrays showed that the caspase-3 gene was up-regulated when CGNs were exposed to neurotoxic chemicals. Significative correlations were found between the transcriptional activity of caspase-3 and the activity of some other genes related to apoptosis, cell-cycle and ROS detoxification. In our experiments, acute exposure of CGNs to well-documented pro-apoptotic xenobiotics modulated significantly caspase-3 gene expression, whereas chemicals not related to apoptosis did not modify caspase-3 gene expression. In conclusion, acute exposure of CGNs to neurotoxic compounds modulates the transcriptional activity of genes involved in the classical apoptotic pathway, oxidative stress and cell-cycle control. Transcriptional activity of caspase-3 correlates significantly with these changes and it could be a good indicator of acute neurotoxicity.
    Toxicology in Vitro 10/2009; 24(2):465-71. · 2.65 Impact Factor
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    ABSTRACT: The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.
    Toxicology in Vitro 08/2009; 23(8):1564-9. · 2.65 Impact Factor
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    ABSTRACT: Pentachlorophenol (PCP) (C(6)HCl(5)O) is a synthetic toxic organochloride fungicide for humans which exhibit neurotoxic properties. In the present research, we describe the potential pathways implicated in PCP-induced apoptosis in an acute model of toxicity in rat cerebellar granule neurons (CGNs). In our experiments, acute exposure of CGNs to micromolar concentrations of PCP induced the transcriptional activity of genes related to the classical apoptosis pathway (caspase 3, caspase 8, Bad), oxidative stress and glutathione metabolism (glutathione peroxidase-1, catalase, glutathione-S-transferase-3 and superoxide dismutase-1), and mitogenic response (cyclin D1, cdk2, cdk4, cdkn2b). Results from Western blot also shown significative increases in the expression of cyclins D1, E and A and cdk4. The mitogenic response was also related to a significative increase in the phosphorylation of retinoblastoma protein (Rb). PCP would cause apoptosis up-regulating the transcriptional activity of p53 gene and also increasing their activation by phosphorylation, concomitant with a decrease in the sirtuin 1 content. In conclusion, acute exposure of CGNs to PCP induces the classical p53 apoptotic pathway, promotes the up-regulation of several genes related to oxidative stress and the over-expression of molecules involved in the cell cycle control.
    NeuroToxicology 06/2009; 30(3):451-8. · 2.65 Impact Factor
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    ABSTRACT: The antioxidant effects of lithium and SB-415286, two glycogen synthase kinase-3 beta (GSK-3 beta) inhibitors, were studied in cerebellar granule neurons by measuring changes in 2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. GSK-3 beta inhibitors inhibit apoptosis mediated by serum and potassium withdrawal (S/K withdrawal) and GSK-3 beta activation, as measured by beta-catenin degradation. Furthermore, as both drugs prevent mitochondrial apoptosis inducing factor (AIF) release, these data indicate that GSK-3 beta inhibitors prevent caspase-independent apoptosis in cerebellar granule neurons induced by S/K withdrawal. While the most specific GSK-3 beta inhibitor, SB-415286, demonstrated antioxidant effects, Li+ 10 mM did not. These results indicate that lithium 10 mM and SB-415286 20 microM exert anti-apoptotic effects in cases of S/K withdrawal mediated by GSK-3 beta inhibition. However, these antioxidant properties are independent of GSK-3 beta inhibition and prevention of mitochondrial AIF release.
    European Journal of Pharmacology 08/2008; 588(2-3):239-43. · 2.59 Impact Factor
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    ABSTRACT: Cytoskeletal alteration is a key factor in neurodegenerative processes like Alzheimer's or Parkinson's disease. Colchicine is a microtubule-disrupting agent that binds to tubuline, inhibiting microtubule assembly, and which triggers apoptosis. The present research describes the transcriptional activation of molecules related to alternative forms of apoptosis, in an acute colchicine model of apoptosis in rat cerebellar granule neurons (CGNs). Treatment with colchicine up-regulated significantly the activity of genes related to oxidative stress: glutathione peroxidase 1 and catalase; altered significantly genes related to cell cycle control (cyclin D1 and cyclin-dependent kinase 2), genes related to classical apoptosis pathway (caspase 3) and a neuronal cell-related gene (pentraxin 1). Colchicine treatment also down-regulated the gene expression of calpain 1. In conclusion, our experiments demonstrate that the cell damage caused by exposure to colchicine activates the classical apoptosis pathway, but also promotes the up-regulation of several genes related to oxidative stress and cell cycle control. Present data may help to a better understanding of the molecular mechanisms involved in cytoskeletal degradation-induced apoptosis in neurons.
    NeuroToxicology 04/2008; 29(2):309-17. · 2.65 Impact Factor
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    ABSTRACT: Glycogen synthase kinase-3 (GSK-3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB-415286, two inhibitors of GSK-3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H2O2 decreased the inactive form of GSK-3 (phospho-GSK-3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB-415286 exerted a protective effect, lithium did not attenuate the toxic effects of H2O2 (1mM). We then examined those mechanisms implicated in the protective effects of SB-415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB-415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK-3 inhibitor SB-415286 has antioxidant properties, which may explain the cytoprotective effects against H2O2 damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect.
    International Journal of Developmental Neuroscience 01/2008; 26(3):269-276. · 2.69 Impact Factor
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    ABSTRACT: Li(+) exerts protective effect against several neurotoxins in neuronal cell preparations. Here we examined the antiapoptotic effects of GSK3beta in cerebellar granule neurons (CGNs) in the presence of several neurotoxins. Acute treatment with Li(+) protected neurons against nocodazole and serum/potassium (S/K) deprivation, but were ineffective against kainic acid and MPP(+). Li(+) 5 mM also decreased caspase-3 activation induced by nocodazole and S/K deprivation as measured by Ac-DEVD-p-nitroaniline and the breakdown of alpha-spectrin. All the neurotoxins used in the present study activated GSK3beta, evaluated with a specific antibody phospho-GSK-3beta (Ser9) by Western-blot and immunocytochemistry and were always inhibited by Li(+) 5 mM. Our results implicate Li(+) in the regulation of apoptosis mediated by caspase activation (Type I). Furthermore inhibition of GSK3beta by acute treatment with Li(+) 5 mM is not an indicator of neuroprotection. The acute antiapoptotic function of Li(+) is discussed in terms of its inhibition of Type I pathway, the intrinsic (mitochondrial) apoptotic pathway in cerebellar granule cells.
    Journal of Neural Transmission 02/2007; 114(4):405-16. · 3.05 Impact Factor
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    ABSTRACT: Experimental data implicate calpain activation in the pathways involved in neuronal apoptosis. Indeed, calpain inhibitors confer neuroprotection in response to various neurotoxic stimuli. However, the pathways involved in calpain activation-induced apoptosis are not well known. We demonstrate that apoptosis (40%) induced by serum/potassium (S/K) withdrawal on cerebellar granule cells (CGNs) is inhibited by selective calpain inhibitors PD150606 (up to 15%) and PD151746 (up to 29%), but not PD145305 in CGNs. zVAD-fmk, a broad spectrum inhibitor of caspases, attenuates apoptosis (up to 20%) mediated by S/K deprivation and protects against cell death, as measured by MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium]) assay. PD150606 and PD151746 prevented apoptosis mediated by S/K withdrawal through inhibition of calpain. Furthermore, PD151746 was able to inhibit caspase-3 activity. After S/K withdrawal, we observed an increase in cdk5/p25 formation and MEF2 phosphorylation that was prevented by 40 microM PD150606 and PD151746. This indicates that calpain inhibition may be an upstream molecular target that prevents neuronal apoptosis in vitro. Taken together, these data suggest an apoptotic route in S/K withdrawal in CGNs mediated by calpain activation, cdk5/p25 formation and MEF2 inhibition. Calpain inhibitors may attenuate S/K withdrawal-induced apoptosis and may provide a potential therapeutic target for drug treatment in a neurodegenerative process.
    British Journal of Pharmacology 09/2005; 145(8):1103-11. · 5.07 Impact Factor
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    ABSTRACT: Serum and potassium (S/K) deprivation is a well-known apoptotic model in cerebellar granule neurons (CGNs), used to study the efficacy of potential neuroprotective drugs. The objective of this study was to determine the pathways involved in the neuroprotective role of flavopiridol, a pan-inhibitor of cyclin-dependent kinases (CDKs), upon S/K withdrawal-induced apoptosis in CGNs. Cell death in primary cultures of rat CGNs was accompanied by chromatin condensation and activation of caspases-3, -6, and -9. Caspase-3 activity was also evaluated by cleavage of 120-kDa alpha-spectrin. Flavopiridol (1 microM) prevented caspase activation and abolished apoptotic features mediated by S/K withdrawal. Re-entry in the cell cycle is also involved in apoptotic neuronal cell death. Flavopiridol (1 microM) inhibited DNA synthesis as measured by BrdU incorporation, thus enhancing proliferating cell nuclear antigen expression. Serum/potassium (S/K) deprivation induced apoptotic cell death mediated by the activation of several kinases such as glycogen synthase kinase-3beta and CDK5, as well as the breakdown of p35 in the neurotoxic fragment p25; inactivation of myocyte enhancer factor-2 (MEF2) was also found. Pretreatment with flavopiridol prevented these biochemical and molecular alterations. Taken together, these findings suggest an apoptotic route in CGNs after S/K withdrawal mediated by the activation of several kinases involved in cell cycle deregulation and MEF2 inactivation. We propose that the antiapoptotic properties of flavopiridol are mediated through kinase pathway inhibition.
    Journal of Molecular Neuroscience 02/2005; 26(1):71-84. · 2.89 Impact Factor
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    ABSTRACT: Acetylcholinesterase inhibitors (AChEI) are among the drugs most widely used in the treatment of Alzheimer's disease. They increase the levels of acetylcholine and thus improve the cognitive symptoms that are impaired. We tested whether specific AChEI show additional neuroprotective properties against colchicine-induced apoptosis in cerebellar granule neurons (CGNs), a well established apoptotic model mediated by neuronal cytoskeleton alteration. Colchicine-induced apoptosis is due to an increase in the activity of GSK-3beta and CDK5, two enzymes involved in cytoskeletal alteration. Furthermore, the intrinsic apoptotic pathway is activated by colchicines, as revealed by cytochrome c release and Bax translocation. Tacrine, (-)-huperzine A and (+/-)-huprine Y, the AChEI tested in the study, did not reverse the loss of neuronal viability induced by colchicine. Moreover, the increase in apoptotic features induced by colchicine treatment, as measured by flow cytometry and nuclear chromatin condensation, was not prevented by these AChEI. Although some of these drugs are of interest to treat Alzheimer's disease, their lack of efficacy in the prevention of colchicine-induced apoptosis in CGNs suggests that they cannot prevent neuronal loss due to cytoskeleton alteration.
    Journal of Alzheimer's disease: JAD 01/2005; 6(6):577-83; discussion 673-81. · 4.17 Impact Factor
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    ABSTRACT: Kainic acid (KA) treatment induced neuronal death and apoptosis in murine cerebellar granule cells (CGNs) cultures from both wild-type and knockout p21(-/-) mice. There was not statistically significant difference in the percentage of neuronal apoptosis among strains. KA-induced neurotoxicity was prevented in the presence of NBQX (20 microM) and GYKI 52446 (20 microM), but not by z-VAD-fmk, suggesting that caspases are not involved in the apoptotic process. Data suggest that p21(WAF/Cip) was unable to modulate KA-induced apoptosis in murine CGNs.
    Brain Research 01/2005; 1030(2):297-302. · 2.88 Impact Factor
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    ABSTRACT: 1. Cyclosporin A (CsA, 1-50 microM), an immunosuppressive drug with known neurotoxic effects, did not decrease the viability of primary cultures of rat cerebellar granule neurons (CGN) or induce apoptotic features. However, CsA specifically enhanced the cytotoxicity and apoptosis induced by colchicine (1 microM). 2. Flavopiridol, an inhibitor of cyclin-dependent kinases (CDKs), prevented the neurotoxic effects of colchicine plus CsA. At 0.1-5 microM, it also showed antiapoptotic effects, as revealed by propidium iodide staining, flow cytometry and counting of cell nuclei. 3. Roscovitine (25-50 microM), a selective cdk1, 2 and 5 inhibitor, showed an antiapoptotic effect against colchicine- and colchicine plus CsA-induced apoptosis. 4. CsA increased the expression of cdk5 and cdk5/p25 mediated by colchicine, a CDK involved in neuronal apoptosis. After treatment of CGN with colchicine plus CsA, the changes in the p25/p35 ratio pointed to cdk5 activation. 5. Immunohistochemical results showed a nuclear localization of cdk5 after neurotoxic treatment, which was prevented by cdk inhibitors. Thus, we propose a new mechanism of modulation of CsA neurotoxicity mediated by cdk5.
    British Journal of Pharmacology 03/2004; 141(4):661-9. · 5.07 Impact Factor
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    ABSTRACT: Phycocyanin (Pc) is a phycobiliprotein that has been recently reported to exhibit a variety of pharmacological properties. In this regard, antioxidant, anti-inflammatory, neuroprotective and hepatoprotective effects have been experimentally attributed to Pc. When it was evaluated as an antioxidant in vitro, it was able to scavenge alkoxyl, hydroxyl and peroxyl radicals and to react with peroxinitrite (ONOO(-);) and hypochlorous acid (HOCl). Pc also inhibits microsomal lipid peroxidation induced by Fe(+2)-ascorbic acid or the free radical initiator 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Furthermore, it reduces carbon tetrachloride (CCl(4))-induced lipid peroxidation in vivo. Pc has been evaluated in twelve experimental models of inflammation and exerted anti-inflammatory effects in a dose-dependent fashion in all of these. Thus, Pc reduced edema, histamine (Hi) release, myeloperoxidase (MPO) activity and the levels of prostaglandin (PGE(2)) and leukotriene (LTB(4)) in the inflamed tissues. These anti-inflammatory effects of Pc can be due to its scavenging properties toward oxygen reactive species (ROS) and its inhibitory effects on cyclooxygenase 2 (COX-2) activity and on Hi release from mast cells. Pc also reduced the levels of tumor necrosis factor (TNF-alpha) in the blood serum of mice treated with endotoxin and it showed neuroprotective effects in rat cerebellar granule cell cultures and in kainate-induced brain injury in rats.
    Current Protein and Peptide Science 07/2003; 4(3):207-16. · 2.33 Impact Factor
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    ABSTRACT: The mechanisms underlying selective neuronal cell death in kainic acid-mediated neurodegeneration are not fully understood. We have recently demonstrated that in cerebellar granule neurons, kainic acid induces the expression of proteins associated with cell-cycle progression. In the present study we show that 3-amino thioacridone (3-ATA), a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in cerebellar granule neurons. When neurons were pre-treated with 3-ATA 10 microM for 24 h, they were less susceptible to damage induced by kainic acid 500 microM, since the number of dead cells decreased significantly. In flow cytometry studies using propidium iodide staining, 3-ATA also reduced the ratio of apoptotic cells induced by kainic acid. Moreover, 3-ATA decreased the proportion of cells with a condensed nucleus from 55% to 22%. Our data suggest that the cell cycle pathway is involved in the mechanism of apoptosis mediated by kainic acid and that cyclin-dependent kinase 4 plays a prominent role in this process. 3-ATA may to prevent the apoptosis associated with neurodegenerative disorders without the over-activation of excitatory amino acid receptors.
    Neuroscience 02/2003; 120(3):599-603. · 3.12 Impact Factor
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    ABSTRACT: We developed a method in which laser scanning cytometry (LSC) is applied to evaluate cell viability. Neuronal cell death induced by glutamic acid, serum potassium deprivation and 3-nitropropionic acid was studied in cerebellar granule cells by neutral red assay (NR) and LSC, using propidium iodide (PI) as fluorescent dye. PI labeled the nuclei of dead neurons and increased fluorescence was measured using a laser scanning cytometer. Similar levels of damage for each injury were detected by NR or LSC. The protocol presented here, provides a fast and sensitive assay for the analysis of neuronal viability using LSC, and can be used to study new neuroprotective drugs in neuronal cell cultures.
    Brain Research Protocols 03/2002; 9(1):41-8. · 1.82 Impact Factor
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    ABSTRACT: We tested the potential cytoprotective role of C-phycocyanin in rat cerebellar granule cell cultures. Cell death was induced by potassium and serum (K/S) withdrawal. Cell viability was studied using the neutral red assay and laser scanning cytometry with propidium iodide as fluorochrome. C-phycocyanin (1-3 mg/ml) showed a neuroprotective effect against 24 h of K/S deprivation in cerebellar granule cells. After 4 h K/S deprivation this compound (3 mg/ml) inhibited formation of reactive oxygen species, measured as 2',7'-dichlorofluorescein fluorescence, showing its scavenger capability. Pre-treatment with C-phycocyanin reduced thymidine incorporation into DNA below control values and reduced dramatically apoptotic bodies as visualized by propidium iodide, indicating inhibition of apoptosis induced by K/S deprivation. Flow cytometry studies, using propidium iodide in TritonX100 permeabilized cells, indicated that 24 h K/S deprivation acts as a proliferative signal for cerebellar granule cells, which show an increase in S-phase percentage and cells progressed into the apoptotic pathway. C-phycocyanin protected cerebellar granule cells from the apoptosis induced by deprivation. These results suggest that C-phycocyanin prevents apoptosis in cerebellar granule cells probably through the antioxidant activity. It is proposed that K/S deprivation-induced apoptosis could be due, in part, to an alteration in the cell cycle mediated by an oxidative stress mechanism.
    Archiv für Experimentelle Pathologie und Pharmakologie 09/2001; 364(2):96-104. · 2.15 Impact Factor
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    ABSTRACT: The neuroprotective role of C-phycocyanin was examined in kainate-injured brains of rats. The effect of three different treatments with C-phycocyanin was studied. The incidence of neurobehavioral changes was significantly lower in animals receiving C-phycocyanin. These animals also gained significantly more weight than the animals only receiving kainic acid, whereas their weight gain did not differed significantly from controls. Equivalent results were found when the neuronal damage in the hippocampus was evaluated through changes in peripheral benzodiazepine receptors (microglial marker) and heat shock protein 27 kD expression (astroglial marker). Our results are consistent with the oxygen radical scavenging properties of C-phycocyanin described elsewhere. Our findings and the virtual lack of toxicity of C-phycocyanin suggest this drug could be used to treat oxidative stress-induced neuronal injury in neurodegenerative diseases, such as Alzheimer's and Parkinson's.
    Neuroscience Letters 01/2000; 276(2):75-8. · 2.03 Impact Factor
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    V Rimbau, C Cerdan, R Vila, J Iglesias
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    ABSTRACT: Aqueous, ethanol and chloroform extracts from Corrigiliola telephiifolia, Echinops spinosus, Kundmannia sicula, Tamarindus indica and Zygophyllum gaetulum were evaluated for antiinflammatory properties in mice (ear oedema induced by arachidonic acid) and rats (subplantar oedema induced by carrageenan) after topical or i.p. administration, respectively. Our results showed that all the plants exhibit antiinflammatory activity, since at least one extract from each plant was active in one of the experimental models. Whereas all the extracts of Corrigiliola telephiifolia and Echinops spinosus were highly active on all the experimental models assayed (values of inflammation inhibition well above 50%), poorer activity profiles were recorded in Kundmannia sicula, Tamarindus indica and Zygophyllum gaetulum. These results support the traditional uses for these plants but indicate that the active principles in the chloroform extracts are probably more active and/or are contained in larger concentrations than the principles in the polar extracts used in the traditional medicine of North-African countries.
    Phytotherapy Research 04/1999; 13(2):128-32. · 2.07 Impact Factor
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    ABSTRACT: Aqueous, ethanol and chloroform extracts from five plants were administered either topically (oedema induced by arachidonic acid in mouse ear) or i.p. (subplantar oedema induced by carrageenan in rats). Our results show that Anacyclus pyrethrum, Armeria alliacea, Asphodelus ramosus, Capparis spinosa and Rhaponticum acaule possess antiinflammatory activity, since at least one extract of each plant was active in one of the experimental models. The three extracts from Anacyclus pyrethrum showed significant activity in both experimental models, but the highest antiinflammatory activity was exhibited by the polar extracts of Armeria alliacea. The ethanol extract of the latter produced 100% inhibition of the inflammation induced by carrageenan and this inhibition was highly significant (p<0.001) with reference to values found in both active (indomethacin 3 mg/kg) and vehicle administered control groups.
    Phytotherapy Research 01/1996; 10(5):421-423. · 2.07 Impact Factor

Publication Stats

296 Citations
54.59 Total Impact Points

Institutions

  • 2009
    • Centro de Investigación Biomédica en Red, Enfermedades Neurodegenerativas
      Madrid, Madrid, Spain
    • Universitat Rovira i Virgili
      Tarraco, Catalonia, Spain
  • 2008
    • Instituto de Salud Carlos III
      Madrid, Madrid, Spain
  • 2005
    • University of Leipzig
      • Rudolf-Boehm-Institut für Pharmakologie und Toxikologie
      Leipzig, Saxony, Germany
  • 2003
    • Centro Nacional de Investigaciones Cientificas
      La Habana, Ciudad de La Habana, Cuba