Grado de adherencia al tratamiento antivírico recomendado durante la pandemia y periodo pospandémico de gripe A (H1N1) pdm09 en 148 unidades de cuidados intensivos españolas / Degree of adherence to recommended antiviral treatment during the pandemic and post-pandemic periods of influenza A (H1N1) pdm09 in 148 intensive care units in Spain

OBJETIVO: Evaluar el grado de adherencia a las recomendaciones sobre el tratamiento antivírico y su impacto en la mortalidad de pacientes críticos afectados por gripe A (H1N1) pdm09. DISEÑO: Análisis secundario de estudio prospectivo. ÁMBITO: Medicina intensiva (UCI). PACIENTES: Pacientes con gripe A (H1N1) pdm09 en el periodo pandémico 2009 y pospandémico 2010-11. Variables La adherencia a las recomendaciones se clasificó en: total (AT), parcial dosis (PD), parcial tiempo (PT) y no adherencia (NA). La neumonía vírica, obesidad y ventilación mecánica fueron considerados criterios de gravedad para el uso de dosificaciones elevadas de antivírico (CG). Análisis mediante «chi» cuadrado y t-test. Supervivencia mediante regresión de Cox. RESULTADOS: Se incluyeron 1.058 pacientes, 661(62,5%) en pandemia y 397 (37,5%) en pospandemia. La AT global del estudio fue del 41,6% (el 43,9% y el 38%, respectivamente; p = 0,07). Los pacientes con criterios de gravedad no fueron diferentes en ambos periodos (un 68,5% y un 62,8%; p = 0,06). En estos pacientes la AT fue del 54,7% durante el 2009 y del 36,4% en pospandemia (p < 0,01). La NA (19,7% vs. 11,3%; p < 0,05) y la PT (20,8% vs. 9,9%; p < 0,01) fueron más frecuentes durante la pospandemia. La mortalidad fue mayor en la pospandemia (30% vs. 21,8%; p < 0,001). El APACHE II(HR = 1,09) y la enfermedad hematológica (HR = 2,2) se asociaron a mortalidad y la adherencia (HR = 0,47) fue un factor protector. CONCLUSIONES: Se evidencia un bajo grado de adherencia al tratamiento en ambos periodos. La adherencia al tratamiento antivírico se asocia con menor mortalidad y debería ser recomendada en pacientes críticos afectados por gripe A (H1N1) pdm09

OBJECTIVE: To determine the degree of antiviral treatment recommendations adherence and its impact to critical ill patients affected by influenza A (H1N1) pdm09 mortality. DESIGN: Secondary analysis of prospective study. SETTING: Intensive care (UCI). PATIENTS: Patients with influenza A(H1N1)pdm09 in the 2009 pandemic and 2010-11 post-Pandemic periods. Variables Adherence to recommendations was classified as: Total (AT); partial in doses (PD); partial in time (PT), and non-adherence (NA). Viral pneumonia, obesity and mechanical ventilation were considered severity criteria for the administration of high antiviral dose. The analysis was performed using t-test or «chi» square. Survival analysis was performed and adjusted by Cox regression analysis. RESULTS: A total of 1,058 patients, 661 (62.5%) included in the pandemic and 397 (37.5%) in post-pandemic period respectively. Global adherence was achieved in 41.6% (43.9% and 38.0%; P = .07 respectively). Severity criteria were similar in both periods (68.5% vs. 62.8%; P = .06). The AT was 54.7% in pandemic and 36.4% in post-pandemic period respectively (P <.01). The NA (19.7% vs. 11.3%; P <.05) and PT (20.8% vs. 9.9%, P < .01) was more frequent in the post-pandemic period. The mortality rate was higher in the post-pandemic period (30% vs. 21.8%, P <.001). APACHE II (HR=1.09) and hematologic disease (HR = 2.2) were associated with a higher mortality and adherence (HR=0.47) was a protective factor. CONCLUSIONS: A low degree of adherence to the antiviral treatment was observed in both periods. Adherence to antiviral treatment recommendations was associated with lower mortality rates and should be recommended in critically ill patients with suspected influenza A(H1N1)pdm09

Adipokine pathways are altered in hippocampus of an experimental mouse model of Alzheimer's disease.

A growing body of evidence suggests that ß-amyloid peptides (Aß) are unlikely to be the only factor involved in Alzheimer's disease (AD) aetiology. In fact, a strong correlation has been established between AD patients and patients with type 2 diabetes and/or cholesterol metabolism alterations. In addition, a link between adipose tissue metabolism, leptin signalling in particular, and AD has also been demonstrated. In the present study we analyzed the expression of molecules related to metabolism, with the main focus on leptin and prolactin signalling pathways in an APPswe/PS1dE9 (APP/PS1) transgenic mice model, at 3 and 6 months of age, compared to wild-type controls. We have chosen to study 3 months-old APP/PS1 animals at an age when neither the cognitive deficits nor significant Aß plaques in the brain are present, and to compare them to the 6 months-old mice, which exhibit elevated levels of Aß in the hippocampus and memory loss. A significant reduction in both mRNA and protein levels of the prolactin receptor (PRL-R) was detected in the hippocampi of 3 months old APP/PS1 mice, with a decrease in the levels of the leptin receptor (OB-R) first becoming evident at 6 months of age. We proceeded to study the expression of the intracellular signalling molecules downstream of these receptors, including stat (1-5), sos1, kras and socs (1-3). Our data suggest a downregulation in some of these molecules such as stat-5b and socs (1-3), in 3 months-old APP/PS1 brains. Likewise, at the same age, we detected a significant reduction in mRNA levels of lrp1 and cyp46a1, both of which are involved in cholesterol homeostasis. Taken together, these results demonstrate a significative impairment in adipokine receptors signalling and cholesterol regulation pathways in the hippocampus of APP/PS1 mice at an early age, prior to the Aß plaque formation.

Degree of adherence to recommended antiviral treatment during the pandemic and post-pandemic periods of influenza A(H1N1)pdm09 in 148 intensive care units in Spain.

OBJECTIVE: To determine the degree of antiviral treatment recommendations adherence and its impact to critical ill patients affected by influenza A(H1N1)pdm09 mortality. DESIGN: Secondary analysis of prospective study. SETTING: Intensive care (UCI). PATIENTS: Patients with influenza A(H1N1)pdm09 in the 2009 pandemic and 2010-11 post-Pandemic periods. VARIABLES: Adherence to recommendations was classified as: Total (AT); partial in doses (PD); partial in time (PT), and non-adherence (NA). Viral pneumonia, obesity and mechanical ventilation were considered severity criteria for the administration of high antiviral dose. The analysis was performed using t-test or «chi¼ square. Survival analysis was performed and adjusted by Cox regression analysis. RESULTS: A total of 1,058 patients, 661 (62.5%) included in the pandemic and 397 (37.5%) in post-pandemic period respectively. Global adherence was achieved in 41.6% (43.9% and 38.0%; P=.07 respectively). Severity criteria were similar in both periods (68.5% vs. 62.8%; P=.06). The AT was 54.7% in pandemic and 36.4% in post-pandemic period respectively (P<.01). The NA (19.7% vs. 11.3%; P<.05) and PT (20.8% vs. 9.9%, P<.01) was more frequent in the post-pandemic period. The mortality rate was higher in the post-pandemic period (30% vs. 21.8%, P<.001). APACHE II (HR=1.09) and hematologic disease (HR=2.2) were associated with a higher mortality and adherence (HR=0.47) was a protective factor. CONCLUSIONS: A low degree of adherence to the antiviral treatment was observed in both periods. Adherence to antiviral treatment recommendations was associated with lower mortality rates and should be recommended in critically ill patients with suspected influenza A(H1N1)pdm09.

Modulation of SIRT1 expression in different neurodegenerative models and human pathologies.

We examined the expression of SIRT1 in several experimental paradigms of human pathologies. We used a neuroblastoma cell line (B65), neuronal primary cultures (hippocampus and cerebellar granule cells) and in vivo approaches in rat and senescence murine models (SAM). Cell cultures and rats were treated with several well-know neurotoxins, i.e. rotenone, MPP(+), kainate and 3-nitropropionic acid. Subsequently, SIRT1 expression was compared in these different paradigms of neurotoxicity. The pattern of expression of SIRT1 in proliferating cell cultures (B65) was different to that in quiescent cell cultures. In the murine model of senescence (senescence-accelerated mice prone, SAMP8), SIRT1 expression progressively decreased, while in the control strain (senescence-accelerated mice resistant, SAMR1) it increased. Finally, we studied human samples of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Huntington's diseases (HD). SIRT1 expression decreased dramatically in HD, but there were no significant changes in Parkinson-related illnesses. In conclusion, SIRT1 expression may be a good sensor of toxic neuronal processes.

Evidence in favour of a role for peripheral-type benzodiazepine receptor ligands in amplification of neuronal apoptosis.

The mitochondrial peripheral benzodiazepine receptor (PBR) is involved in a functional structure designated as the mitochondrial permeability transition (MPT) pore, which controls apoptosis. PBR expression in nervous system has been reported in glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand fluo-FGIN-1-27 in mitochondria of rat cerebellar granule cells (CGCs). Additionally, the effect of PBR ligands on colchicine-induced apoptosis was investigated. Colchicine-induced neurotoxicity in CGCs was measured at 24 h. We show that, in vitro, PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4- benzodiazepin-2-one (Ro5-4864) and diazepam (25- 50 microM) enhanced apoptosis induced by colchicine, as demonstrated by viability experiments, flow cytometry and nuclear chromatin condensation. Enhancement of colchicine-induced apoptosis was characterized by an increase in mitochondrial release of cytochrome c and AIF proteins and an enhanced activation of caspase-3, suggesting mitochondrion dependent mechanism that is involved in apoptotic process. Our results indicate that exposure of neural cells to PBR ligands generates an amplification of apoptotic process induced by colchicine and that the MPT pore may be involved in this process.

Inhibition of cell cycle pathway by flavopiridol promotes survival of cerebellar granule cells after an excitotoxic treatment.

Kainic acid (KA)-induced neuronal damage and the protective effects of flavopiridol were studied in primary cultures of rat cerebellar granule cells (CGNs). When neurons were treated with 500 microM KA, the percentage of cells with condensed nuclei measured by nuclear counting increased by up to 55%. After flavopiridol treatment, an antitumoral drug that is a broad inhibitor of cyclin-dependent kinases, the percentage of condensed nuclei decreased by up to 26%. Furthermore, this KA-mediated cell death was only partially dependent on the activation of the initiator caspase-9 and the effector caspases-3 and -6. This argues for a minor role of caspases in the intracellular pathway leading to KA-induced programmed cell death in CGNs. We examined the possible implication of cell cycle proteins in KA-induced neurotoxicity. We found an increase in the expression of proliferating cell nuclear antigen and E2F-1, two proteins implicated in S-phase, by Western blot. KA increased bromodeoxyuridine incorporation in CGNs, a marker of cell proliferation, and flavopiridol attenuated this effect. These results indicated that flavopiridol decreased the expression of cell cycle markers in CGNs after KA treatment. Flavopiridol might thus be used as a preventive agent against neurodegenerative diseases associated with cell cycle activation.

3-Amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acid-induced apoptosis in neurons.

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.

Parameters related to oxygen free radicals in erythrocytes, plasma and epidermis of the hairless rat.

The following parameters related to oxygen free radicals (OFR) were determined in erythrocytes and the epidermis of hairless rats: catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced (GSH) and oxidized (GSSG) glutathione, glutathione S-transferase (GST), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS). GSH, GSSG and TBARS were also analyzed in plasma. In erythrocytes, the Pearson correlation coefficients (r) were significant (p < 0.001) between glutathione and other parameters as follows: GSH correlated negatively with GSSG (r = -0.665) and TBARS (r = -0.669); GSSG correlated positively with SOD (r = 0.709) and TBARS (r = 0.752). Plasma GSSG correlated negatively with erythrocytic thermostable GST activity (r = -0.608; p=0.001) and with erythrocytic total GST activity (r = -0.677; p < 0.001). In epidermis (p < 0.001 in all cases), GSH content correlated with GSSG (r = 0.682) and with GPx (r = 0.663); GSSG correlated with GPx (r = 0.731) and with GR (r = 0.794). By multiple linear regression analysis some predictor variables (R(2)) were found: in erythrocytes, thermostable GST was predicted by total GST activity and GSSG, GSSG content was predicted by GSH and by the GSH/GSSG ratio and GPx activity was predicted by GST, CAT and SOD activities; in epidermis, GSSG was predicted by GR and SOD activities and GR was predicted by GSSG, TBARS and GPx. It is concluded that the hairless rat is a good model for studying OFR-related parameters simultaneously in blood and skin, and that it may provide valuable information about other animals under oxidative stress.

C-phycocyanin protects cerebellar granule cells from low potassium/serum deprivation-induced apoptosis.

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.

Orphenadrine prevents 3-nitropropionic acid-induced neurotoxicity in vitro and in vivo.

1. Previous studies indicate that 3-nitropropionic acid (3-NPA) neurotoxicity involves the excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Thus, we examined the effect of orphenadrine (an anticholinergic drug with NMDA receptor antagonist properties) on 3-NPA neurotoxicity in both cultured rat cerebellar granule cells (CGCs) and in rats. 2. Orphenadrine protected CGCs from 3-NPA-induced mortality, as assessed by both the neutral red viability assay and laser scanning cytometry, using propidium iodide staining. 3. For rats, two indirect markers of neuronal damage were used: the binding of [(3)H]-PK 11195 to the peripheral-type benzodiazepine receptor (PBR), a microglial marker, and expression of the 27 kD heat-shock protein (HSP27), a marker of activated astroglia. Systemic administration of 3-NPA (30 mg kg(-1) per day for 3 days) induced a 170% increase in [(3)H]-PK 11195 binding, and expression of HSP27. 4. Both the increase in [(3)H]-PK 11195 and HSP 27 expression were prevented by previous administration of 30 mg kg(-1) per day of orphenadrine for 3 days. Lower doses (10 and 20 mg kg(-1)) had no protective effect. Orphenadrine also reduced 3-NPA-induced mortality in a dose-dependent manner. 5. We propose that orphenadrine or orphenadrine-like drugs could be used to treat neurodegenerative disorders mediated by overactivation of NMDA receptors.

[Endothelial function in coronary segments previously treated with balloon angioplasty]. / Función endotelial en segmentos arteriales previamente sometidos a angioplastia coronaria.

INTRODUCTION AND OBJECTIVES: Coronary angioplasty leads to endothelial disruption and a further rendotelization. The aim of our study was to determine the status of endothelial function in previously dilated coronary segments without restenosis. METHODS: Endothelium-dependent vasomotion was analysed in twelve patients with single vessel coronary disease six month after angioplasty by selective intracoronary doses of acetylcholine (10-6, 10-5, 10-4 M) in the previously treated artery. The control group was made up of seven patients with no evidence of significant coronary stenosis and without risk factors. Vasomotor response at the different doses of acetylcholine was determined by quantitative coronary angiography. RESULTS: Endothelial function showed a global vasodilator response in the dilated segment at the maximum dose of acetylcholine (increase in lumen diameter 3.6 +/- 3.5%), similar to the response observed in the control group (increase of luminal diameter 3 +/- 6%; p = NS). In particular, 8 patients (67%) showed a normal endotelial function, while 4 patients (33%) showed a vasoconstrictor response. A positive correlation was detected between the response to the maximun dose of acetylcholine and the percent of residual stenosis at 6 months of follow-up (r = 0.67; p = 0.02). CONCLUSIONS: In patients treated with coronary angioplasty without restenosis, the dilated segments frequently showed normal endothelial function. Greater residual stenosis at the dilated segment was associated with less impairment in endothelial function.

Inhibitors of NO-synthase and donors of NO modulate kainic acid-induced damage in the rat hippocampus.

The effects of nitric oxide synthase (NOS) inhibitors, N(omega)-nitro-L-arginine and 7-nitroindazole, and the NOS substrate L-arginine on kainic acid (KA)-induced microglial reactivity and stress response were studied in the hippocampus 7 and 1 days after KA, respectively. Density of peripheral-type benzodiazepine receptors was measured as an index of microglial reactivity. Histological damage in hippocampus was evaluated at 7 days by neuronal counting. KA increased the maximal number of binding sites (B(max)) versus controls. Administration of either 7-nitroindazole (25 mg/kg) or N(omega)-nitro-L-arginine (20 and 50 mg/kg) 24 hr before KA, further increased B(max). This later effect was abolished by L-arginine (1 g/kg), which given 24 hr before KA decreased B(max) to control values. Also, KA-induced HSP72 stress response was attenuated by pre-treatment with L-arginine. Histological evaluation showed reduced cell numbers in the pyramidal cell layer of the hippocampus in groups receiving KA, either alone or in combination with 7-nitroindazole. Administration of L-arginine before KA attenuated neuronal loss in CA3 but not CA1. A clear protective effect was observed, however, in CA1 and CA3, in rats receiving both L-arginine plus 7-nitroindazole before KA. The results show that the combination of a NO substrate with a NOS inhibitor reduces the neurotoxic effects of KA in the rat hippocampus. This study suggests that extremely fine regulation of NO levels in the different neural cell types can modulate excitotoxicity.

Evaluation of free radical production, mitochondrial membrane potential and cytoplasmic calcium in mammalian neurons by flow cytometry.

The overexcitation of glutamate receptors is believed to be the cause of several neurodegenerative disorders. The determination of calcium fluxes, mitochondrial membrane potential (MMP) variations or the production of reactive oxygen species (ROS) in mammalian cells are usually measured during the development of potentially useful drugs that might interfere in the events induced by glutamate receptor activation. By using flow cytometry with dissociated cerebellar granule cells, we have developed a rapid and economical method to measure changes in biochemical parameters that are involved in neuronal cell death. The formation of intracellular ROS is measured using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The mitochondrial membrane potential is assessed by the retention of rhodamine 123 (Rh123), a specific fluorescent cationic dye that is readily sequestered by active mitochondria, depending on their transmembrane potential. Finally, intracellular calcium increases are detected by using the calcium-selective indicator Indo-1. Cell viability is also assessed by using propidium iodide (PI) which stains DNA strands of permeabilized cells. This method might be useful for the screening of new drugs with potential neuroprotective activity, with improved cost/effectiveness ratio compared to other techniques.

Assessment of the adrenergic effects of orphenadrine in rat vas deferens.

The peripheral adrenergic effects of orphenadrine, an antiparkinsonian drug, have been evaluated in the rat vas deferens to investigate whether these properties are the same as those of other phencyclidine ligands. In the low micromolar range, orphenadrine enhanced electrically-evoked and exogenous noradrenaline contractile responses in the epididymal portion of rat vas deferens. It also induced spontaneous activity that was inhibited by prazosin (1 microM) but not by atropine (20 nM). It inhibited accumulation of [3H]noradrenaline in rat vas deferens (IC50 = 14.2+/-2.3 microM). Orphenadrine competitively inhibited [3H]nisoxetine binding in rat vas deferens membranes (Ki = 1.05+/-0.20 microM). It can be concluded that orphenadrine, at low micromolar concentrations, interacts with the noradrenaline reuptake system inhibiting its functionality and thus potentiating the effect of noradrenaline.

In vitro and in vivo protective effect of orphenadrine on glutamate neurotoxicity.

The anticholinergic drug orphenadrine is used in the treatment of Parkinson's disease. In this study we evaluate the neuroprotective effects of orphenadrine on excitotoxicity in vivo and in vitro. Orphenadrine prevented the mitochondrial and the cytoplasmic membrane potential decrease evoked by NMDA (100 microM) in rat dissociated cerebellar granule cells showing an IC50 value of 11.6 +/- 4.7 microM (mean +/- SEM, n = 5) and 13.5 +/- 2.3 microM (n = 3), respectively. Orphenadrine was able to protect cerebellar granule cell cultures from glutamate-induced neurotoxicity. Kainic acid (KA, 10 mg/kg)-induced excitotoxicity was evaluated in vivo using the microglial marker peripheral-type benzodiazepine receptor (PBR) and heat shock protein 72 (HSP72) expression in the hippocampus. The Bmax of PBR for control tissues was 589.1 +/- 40.0 fmol/mg protein (n = 4), increasing to 1692.5 +/- 51.6 fmol/mg protein (n = 5) after the KA treatment. Pretreatment with orphenadrine (10 mg/kg) blocked the KA-induced increase in PBR density. As expected, KA-administration induced the expression of HSP72 that was blocked in the orphenadrine + KA-treated rats. We demonstrate that orphenadrine, interacting at the NMDA receptor, is able to prevent the neurotoxicity mediated by activation at glutamate ionotropic receptors.

Effects of U-83836E on glutamate-induced neurotoxicity in dissociated rat cerebellar granule cells.

The effects of the lazaroid compound U-83836E on the glutamate-induced production of reactive oxygen species (ROS) were studied in dissociated rat cerebellar granule cells by flow cytometry. U-83836E completely inhibited ROS production with an estimated IC50 value of 21.7 +/- 9.1 nM. However, U-83836E did not inhibit the glutamate-evoked decrease in mitochondrial membrane potential (MMP). Nevertheless, U-83836E (10 nM to 10 microM) prevented cell death induced by 10 mM of glutamate. At concentrations above 10 microM, U-83836E by itself showed slight cytotoxicity, which was significant at a 100 microM concentration. U-83836E (25 to 200 microM) also increased the cytosolic calcium levels in a concentration-dependent manner. Our results indicate that the cytotoxic effects found at micromolar concentrations of U-83836E could be explained by an increase in [Ca2+]i. Finally, since U-83836E did not prevent the MMP decrease evoked by glutamate, it is suggested that antioxidant pharmacotherapy would not be sufficient to block the neurotoxic effects of glutamate.

Microgliosis and down-regulation of adenosine transporter induced by methamphetamine in rats.

Chronic administration of methamphetamine to rats induces neurotoxicity characterized by a loss of striatal dopaminergic terminals and reactive gliosis. Subcutaneous administration of methamphetamine in a scheduled procedure of four doses (10 mg/kg) at 2 h interval also induces a significant increase in the peripheral-type benzodiazepine receptor (PBR) density. This increase is maximum (76%) at 72 h post-treatment in the striatum and disappears at 7 days, suggesting that microglia may have a predominant role in necrosis-phagocytosis of neuronal debris rather than acting in a restorative manner. Microgliosis is not restricted to the striatum since it is also evident in cerebellum (75.4% of PBR increase) and hippocampus (37.2% of PBR increase). In the areas with high density of adenosine transporter, the microgliosis phenomenon correlates well with a decrease of this nucleoside transporter (about 39%). Although the microgliosis and the decrease in adenosine transporter could be parallel and not related events, we can speculate that when microglia are activated, a down-regulation of adenosine transporter occurs, playing a role in tissue homeostasis. With the same dosing schedule, methamphetamine induces HSP72 expression in both cytoplasmic and nuclear fractions of the striatum, cerebellum and hippocampus. This expression is also evident in the cerebral cortex, where adenosine transporter population did not show any variation.

Effect of PCP and sigma ligands on both noradrenaline- and electrically-induced contractions and on [3H]-noradrenaline uptake in rat vas deferens.

1. Electrically induced contractions of the epididymal portion of rat vas deferens were potentiated in concentration-dependent manner (0.1-30 microM) by different sigma and PCP receptor ligands (PCP, TCP, (+)-MK-801, dextromethorphan and (+)-3-PPP); dextrorphan did it in a minor extent. 2. Sigma and PCP receptor ligands also potentiated the effect of noradrenaline, inducing a reduction of the noradrenaline EC50 value in the rat vas deferens. The rank order of potencies was: PCP > TCP > (+)-3-PPP > (+)-MK-801 > dextrorphan > > > dextrometorphan. 3. In contrast, haloperidol (1 microM), a sigma receptor ligand, inhibited both the neurogenic and noradrenaline-induced responses in this tissue. 4. The effect of PCP and sigma receptor ligands on noradrenaline uptake was evaluated. All compounds tested, including haloperidol, inhibited the tritiated noradrenaline incorporation to the tissue. IC50 values were in the micromolar range, between 1.09 microM for dextrophan and 18 microM for dextrometorphan. 5. It is concluded that a direct interaction with the noradrenaline uptake system is involved in the potentiating effect of some sigma and PCP receptor ligands in the epididymal portion of rat vas deferens.

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation.

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.

U-83836E prevents kainic acid-induced neuronal damage.

The effect of kainic acid (KA) on mitochondrial membrane potential (MMP) and reactive-oxygen species (ROS) production was studied in dissociated cerebellar granule cells from rat pups. KA induced a maximum increase of 361%+/-35% in ROS production. The lazaroid compound U-83836E (at concentrations ranging from 10(-9) to 5x10(-6) M) completely inhibited this increase, with an IC50 value of 3.02+/-1.08x10(-7) M. KA also decreased the mitochondrial membrane potential (MMP), with a maximum decrease of about 30%. Absence of Na+ in the incubation medium did not significantly alter the effect of KA on MMP. As expected, the AMPA/kainate receptor antagonist NBQX inhibited the effects of KA on MMP with an IC50 value of 1.1+/-0.8 microM. However, the lazaroid U-83836E, indomethacin, nor-dihydroguaiaretic acid and L-nitroarginine all failed to inhibit the KA-induced decrease in the MMP. Finally, to assess the neuroprotective effect of U-83836E on KA-induced neurotoxicity in vivo, the increase in the peripheral-type benzodiazepine receptor density in rat hippocampus was measured. Treatment with KA increased the Bmax to 1341+/-192 fmol mg(-1). When U-83836E was coadministered with KA, the Bmax was reduced to 765+/-122 fmol mg(-1), which was not significantly different from the Bmax obtained from untreated rats (Bmax: 518+/-33 fmol mg(-1)). We conclude that treatment with the lazaroid U-83836E might be a suitable therapeutic strategy in neurodegenerative disorders.