Tharine Dal-cim | Universidade Federal Fronteira Sul (original) (raw)

Papers by Tharine Dal-cim

Neurochemistry International, 2009

Methods in Molecular Biology, 2014

Oxygen-glucose deprivation (OGD ) is widely used as an in vitro model for stroke, showing similar... more Oxygen-glucose deprivation (OGD ) is widely used as an in vitro model for stroke, showing similarities with the in vivo models of brain ischemia. In order to perform OGD, cell or tissue cultures, such as primary neurons or organotypic slices, and acutely prepared tissue slices are usually incubated in a glucose-free medium under a deoxygenated atmosphere, for example in a hypoxic chamber. Here, we describe the step-by-step procedure to expose cultures and acute slices to OGD, focusing on the most suitable methods for assessing cellular death and/or viability. OGD is a simple yet highly useful technique, not only for the elucidation of the role of key cellular and molecular mechanisms underlying brain ischemia, but also for the development of novel neuroprotective strategies.

European neuropsychopharmacology : the journal of the European College of Neuropsychopharmacology, 2013

Atorvastatin is a synthetic and lipophilic statin that presents a good effect in decreasing chole... more Atorvastatin is a synthetic and lipophilic statin that presents a good effect in decreasing cholesterol levels and is safe and well tolerated. Population-based studies have suggested a positive role of statins in reducing depression risk. This study aimed at investigating the atorvastatin effect in the tail suspension test (TST) and in the forced swimming test (FST). The participation of NMDA receptors and L-arginine-NO-cGMP in an atorvastatin antidepressant-like effect in the TST was evaluated. Acute atorvastatin administration (0.1-30 mg/kg) reduced the immobility time both in TST and FST. A similar effect was observed by using imipramine as a positive control in the TST and FST (1 and 0.1-1 mg/kg, p.o., respectively). An atorvastatin (0.1 mg/kg) antidepressant-like effect was prevented by the pretreatment of mice with NMDA (0.1 pmol/site, i.c.v.), L-arginine (750 mg/kg, i.p.) or sildenafil (5 mg/kg, i.p.). The administration of MK-801 (0.001 mg/kg, i.p.), ketamine (0.1 mg/kg, i.p...

Pharmacology Biochemistry and Behavior, 2014

Atorvastatin is a statin largely used in the treatment of hypercholesterolemia and recently revea... more Atorvastatin is a statin largely used in the treatment of hypercholesterolemia and recently revealed as a neuroprotective agent. The antidepressant-like effect of acute atorvastatin treatment in mice has been previously demonstrated by our laboratory. The purpose of this study was to explore the contribution of the serotonergic system in the antidepressant-like effect of atorvastatin in mice. Data demonstrate that the serotonin (5-HT) depleting agent p-chlorophenylalanine methyl ester (PCPA, 100 mg/kg, i.p.) completely abolished atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. Besides atorvastatin, fluoxetine (10 mg/kg, p.o.), a serotonin selective reuptake inhibitor (SSRI) was able to exert an antidepressant-like effect, but any of them changed 5-HT content in the hippocampus or frontal cortex. The 5H-T1A (WAY100635, 0.1 mg/kg, s.c) or the 5-HT2A/2C (ketanserin, 5 mg/kg, s.c.) receptor antagonists prevented atorvastatin antidepressant-like effect. In addition, a combinatory antidepressant-like effect was observed when mice received the co-administration of sub-effective doses of atorvastatin (0.01 mg/kg, p.o.) and the SSRI fluoxetine (5 mg/kg, p.o.), paroxetine (0.1 mg/kg, p.o.) or sertraline (1 mg/kg, p.o.). Taken together, these results indicate that the antidepressant-like effect of atorvastatin depends on the serotonergic system modulation.

Neurotoxicity Research, 2014

The search for new therapeutic strategies through modulation of glutamatergic transmission using ... more The search for new therapeutic strategies through modulation of glutamatergic transmission using effective neuroprotective agents is essential. Glutamatergic excitotoxicity is a major factor common to neurodegenerative diseases and in acute events such as cerebral ischemia, traumatic brain injury and epilepsy. We have previously demonstrated that N-methyl-D-aspartate (NMDA) preconditioning in mice showed 50 % of protection against seizures and full protection against damage to neuronal tissue induced by quinolinic acid (QA). In this study, cellular and molecular mechanisms involved on NMDA preconditioning and neuroprotection were investigated in mice treated with NMDA 24 h before QA insult. Calcium uptake and D-aspartate release from hippocampal slices obtained from mice treated with NMDA plus QA and not displaying seizures (protected mice) were similar to control (saline) or NMDA preconditioned mice. Increased calcium uptake and glutamate release is evidenced in unprotected (convulsed) mice as well as QA control, demonstrating that calcium and glutamate are involved in NMDA-induced preconditioning. Increased glutamate release evoked by QA was blocked by MK-801, whereas increased calcium uptake was abolished by voltagedependent calcium channels inhibitors, but not MK-801. NMDA preconditioning is effective in normalizing the deregulation of glutamate transport and calcium homeostasis evoked by QA due to aberrant NMDA receptors activation that culminates in seizures and hippocampal cells damage.

Neuroscience, 2011

Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as... more Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as modulation of glutamate transmission and neuronal protection against excitotoxic damage. GD are spontaneously released to the extracellular space from cultured astrocytes and during oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway involvement in the mechanisms related to the neuroprotective role of guanosine in rat hippocampal slices subjected to OGD. The addition of guanosine (100 M) to hippocampal slices subjected to 15 min of OGD and followed by 2 h of re-oxygenation is neuroprotective. The presence of K ؉ channel blockers, glibenclamide (20 M) or apamin (300 nM), revealed that neuroprotective effect of guanosine was not dependent on ATP-sensitive K ؉ channels or small conductance Ca 2؉ -activated K ؉ channels. The presence of charybdotoxin (100 nM), a large conductance Ca 2؉ -activated K ؉ channel (BK) blocker, inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect was inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 M) in the re-oxygenation period. Guanosine promoted an increase in Akt protein phosphorylation. However, the presence of charybdotoxin blocked such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the increased expression of phospho-Akt protein.

Neuropharmacology, 2013

a b s t r a c t ITH33/IQM9.21 is a novel compound belonging to a family of glutamic acid derivati... more a b s t r a c t ITH33/IQM9.21 is a novel compound belonging to a family of glutamic acid derivatives, synthesized under the hypothesis implying that multitarget ligands may provide more efficient neuroprotection than single-targeted compounds. In rat hippocampal slices, oxygen plus glucose deprivation followed by reoxygenation (OGD/Reox) elicited 42% cell death. At 1 mM, ITH33/IQM9.21 mitigated this damage by 26% and by 55% at 3 mM. OGD/Reox also elicited mitochondrial depolarization, overproduction of reactive oxygen species (ROS), enhanced expression of nitric oxide synthase (iNOS) and reduction of GSH levels.

Neurochemistry International, 2012

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in seve... more Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligoinduced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1 mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A 1 or A 2A receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca 2+ -activated K + channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38 MAPK inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3b phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3b proteins and induction of the antioxidant enzyme HO-1.

Neurochemical Research, 2008

Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apop... more Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamateinduced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 lM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38 MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38 MAPK pathway.

Journal of Neurochemistry, 2013

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to ... more Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/ glucose deprivation and reoxygenation (OGD). GUO (100 lM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A 1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A 2A receptors (A 2A R), but not via A 1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.

Journal of neuroscience research, 2011

Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorde... more Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorders. We have previously demonstrated that excitotoxicity induced by millimolar concentrations of glutamate in hippocampal slices involves apoptotic features and glutamate-induced glutamate release. Guanosine, an endogenous guanine nucleoside, prevents excitotoxicity by its ability to modulate glutamate transport. In this study, we have evaluated the neuroprotective effect of guanosine against glutamate-induced toxicity in hippocampal slices and the mechanism involved in such an effect. We have found that guanosine (100 μM) was neuroprotective against 1 mM glutamate-induced cell death through the inhibition of glutamate release induced by glutamate. Guanosine also induced the phosphorylation and, thus, activation of protein kinase B (PKB/Akt), a downstream target of phosphatidylinositol-3 kinase (PI3K), as well as phosphorylation of glycogen synthase kinase 3β, which has been reported to b...

Journal of Ethnopharmacology, 2011

Ethnopharmacological relevance: Aloysia gratissima (Gill. et Hook) Tronc. (Verbenaceae) is used t... more Ethnopharmacological relevance: Aloysia gratissima (Gill. et Hook) Tronc. (Verbenaceae) is used traditionally for the treatment of headache, bronchitis, and nervous systems disorders including depression. Aim of the study: To investigate the antidepressant-like and neuroprotective effects of Aloysia gratissima aqueous extract (AE) and the involvement of l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Materials and methods: The antidepressant-like effect of AE was evaluated through behavioral despair in forced swimming test (FST) and tail suspension test (TST). Swiss albino mice were treated by oral route and after 1 h were analyzed the time of immobility in the FST and TST. In addition, the neuroprotective effect of AE against glutamate excitotoxicity was evaluate through cell viability of hippocampal slices, phosphorylation of Akt, and the immunocontent of inducible oxide nitric synthase (iNOS) were investigated by western blotting. Results: The immobility time in the FST and TST were reduced by AE (100-1000 and 10-300 mg/kg, respectively). The antidepressant-like effect of AE in the TST was prevented by the pretreatment with N-methyl-d-aspartate (NMDA), l-arginine or sildenafil. The subeffective dose of AE produced a synergistic antidepressant-like effect with MK-801 (an antagonist of NMDA receptor), methylene blue, l-NNA (an inhibitor of NO synthase) or ODQ (an inhibitor of soluble guanylate cyclase). In ex vivo experiments, pretreatment with AE prevented the loss of cell viability induced by glutamate, thus affording neuroprotection. Glutamate toxicity caused a decreased Akt phosphorylation and an increased iNOS expression. Conclusions: The present study provides convincing evidence of neuroprotection and the involvement of the l-arginine-NO-cGMP pathway in the antidepressant-like effect of AE. Therefore, AE could be of potential interest for the treatment of depressive disorders and neurological conditions associated with glutamate excitotoxicity.