Farmacologia

Disturbances in glutamatergic transmission and signaling pathways have been associated with temporal lobe epilepsy (TLE) in humans. However, the profile of these alterations within specific regions of the hippocampus and cerebral cortex has not yet been examined. The pilocarpine model in rodents reproduces the main features of TLE in humans. The present study aims to characterize specific alterations of the glutamatergic transmission and signaling pathways in the dorsal (DH) and ventral hippocampus (VH) and temporal cortex (Ctx) of male adult Wistar rats 60 days after pilocarpine treatment (chronic period). The western blotting analyzes show a decrease of AMPA glutamate receptor subunit (GluA1)-Ser(845) phosphorylation; reduction of ERK1 and PKA activity; up-regulation of GFAP and down-regulation of the glutamate transporter EAAT2 expression in the DH. In contrast, in the VH it was observed a decrease of GluA1-Ser(831) phosphorylation and JNKp54 and PKC activity. In the Ctx, only ERK1 phosphorylation/activity decreased. The level of GluA1-Ser(845) phosphorylation and PKA activity (DH) and the level of GluA1-Ser(831) phosphorylation and PKC activity (VH) appear to be correlated, respectively. These findings suggest a differential imbalance of the signaling pathways involved in the site-specific phosphorylation of AMPA receptor in the hippocampus. Furthermore, we suggest that dorsal hippocampus is probably more susceptible to the impairment of glutamate uptake and gliose, since only this area displayed a significant decrease of EAAT2 and increment of GFAP. Taken together, our study suggests that specific neurochemical alterations take place in hippocampal sub regions. This approach may be valuable for understanding the onset of seizures and the alterations of neuronal excitability in specific regions and may help to establish therapeutic targets for treatment of this neuropathology.

While manganese (Mn) is essential for proper central nervous system (CNS) development, excessive Mn exposure may lead to neurotoxicity. Mn preferentially accumulates in the basal ganglia, and in adults it may cause Parkinson's disease-like disorder. Compared to adults, younger individuals accumulate greater Mn levels in the CNS and are more vulnerable to its toxicity. Moreover, the mechanisms mediating developmental Mn-induced neurotoxicity are not completely understood. The present study investigated the developmental neurotoxicity elicited by Mn exposure (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 to PN27 in rats. Neurochemical analyses were carried out on PN29, with a particular focus on striatal alterations in intracellular signaling pathways (MAPKs, Akt and DARPP-32), oxidative stress generation and cell death. Motor alterations were evaluated later in life at 3, 4 or 5 weeks of age. Mn exposure (20 mg/kg) increased p38(MAPK) and Akt phosphorylation, but decreased DARPP-32-Thr-34 phosphorylation. Mn (10 and 20 mg/kg) increased caspase activity and F2-isoprostane production (a biological marker of lipid peroxidation). Paralleling the changes in striatal biochemical parameters, Mn (20 mg/kg) also caused motor impairment, evidenced by increased falling latency in the rotarod test, decreased distance traveled and motor speed in the open-field test. Notably, the antioxidant Trolox™ reversed the Mn (20 mg/kg)-dependent augmentation in p38(MAPK) phosphorylation and reduced the Mn (20 mg/kg)-induced caspase activity and F2-isoprostane production. Trolox™ also reversed the Mn-induced motor coordination deficits. These findings are the first to show that long-term exposure to Mn during a critical period of neurodevelopment causes motor coordination dysfunction with parallel increment in oxidative stress markers, p38(MAPK) phosphorylation and caspase activity in the striatum. Moreover, we establish Trolox™ as a potential neuroprotective agent given its efficacy in reversing the Mn-induced neurodevelopmental effects.

While manganese (Mn) is essential for proper central nervous system (CNS) development, excessive Mn exposure may lead to neurotoxicity. Mn preferentially accumulates in the basal ganglia, and in adults it may cause Parkinson's disease-like disorder. Compared to adults, younger individuals accumulate greater Mn levels in the CNS and are more vulnerable to its toxicity. Moreover, the mechanisms mediating developmental Mn-induced neurotoxicity are not completely understood. The present study investigated the developmental neurotoxicity elicited by Mn exposure (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 to PN27 in rats. Neurochemical analyses were carried out on PN29, with a particular focus on striatal alterations in intracellular signaling pathways (MAPKs, Akt and DARPP-32), oxidative stress generation and cell death. Motor alterations were evaluated later in life at 3, 4 or 5 weeks of age. Mn exposure (20 mg/kg) increased p38(MAPK) and Akt phosphorylation, but decreased DARPP-32-Thr-34 phosphorylation. Mn (10 and 20 mg/kg) increased caspase activity and F2-isoprostane production (a biological marker of lipid peroxidation). Paralleling the changes in striatal biochemical parameters, Mn (20 mg/kg) also caused motor impairment, evidenced by increased falling latency in the rotarod test, decreased distance traveled and motor speed in the open-field test. Notably, the antioxidant Trolox™ reversed the Mn (20 mg/kg)-dependent augmentation in p38(MAPK) phosphorylation and reduced the Mn (20 mg/kg)-induced caspase activity and F2-isoprostane production. Trolox™ also reversed the Mn-induced motor coordination deficits. These findings are the first to show that long-term exposure to Mn during a critical period of neurodevelopment causes motor coordination dysfunction with parallel increment in oxidative stress markers, p38(MAPK) phosphorylation and caspase activity in the striatum. Moreover, we establish Trolox™ as a potential neuroprotective agent given its efficacy in reversing the Mn-induced neurodevelopmental effects.

Many solutions have been examined as possible storage media for avulsed teeth. The purpose of this study was to compare the effectiveness of several storage media to preserve cultured periodontal ligament fibroblasts (PDLF) under different temperatures. The media tested were: sterile Hank's balanced salt solution (sHBSS), non-sterile HBSS (nHBSS), skimmed milk, Save-A-Tooth((R)), Minimum Essential Medium (MEM) and water (negative control). MEM at 37 degrees C was used as positive control. PDLF were obtained from explants of extracted healthy human teeth. Plates containing confluent PDLF were soaked in the various media for 3, 6, 24, 48 and 72 h at 37 degrees C and 20 degrees C. After incubation, viability of the cells was determined using the tetrazolium salt-based colorimetric (MTT) assay and the Trypan Blue exclusion test after 6, 24, 48 and 72 h of incubation at 20 degrees C. The results were analyzed statistically using Kruskal-Wallis, Scheffé and Mann-Whitney (alpha = 5%) tests. Results from the MTT assay at 37 degrees C and 20 degrees C showed that skimmed milk was the best storage medium for up to 24 and 48 h, respectively, followed by nHBSS and sHBSS. Results from the Trypan Blue exclusion test showed that the best storage media were milk, sHBSS and nHBSS, with no statistical differences, for any time period. The Save-A-Tooth((R)) had a detrimental effect on cells after 24 h. The influence of temperature on the effectiveness of the storage media tested showed at 20 degrees C a decreasing order of efficacy as follows: milk > sHBSS and nHBSS > MEM > Save-A-Tooth((R)) > water while at 37 degrees C it was: MEM > nHBSS > milk > sHBSS > Save-A-Tooth((R)) > water. In conclusion, incubation temperature altered the effectiveness of the storage media and skimmed milk at 20 degrees C was better than HBSS in maintaining PDLF viability.

Milk has been studied extensively and has gained wide acceptance as a suitable storage medium capable of maintenance of avulsed teeth that cannot be replanted immediately. The objective of this study was to evaluate whether the renewal of milk as a storage medium every 24 h for up to 120 h is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability in vitro. Plates with confluent PDLF were soaked in minimum essential medium (MEM) at 37°C (positive control) and in skimmed milk (22 wells) and water (negative control) for 24, 48, 72, 96, and 120 h at 5 and 20°C. The skimmed milk was renewed every 24 h in 11 of the wells of each plate. After these periods, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis and Scheffé tests (α = 5%). At 24 h, milk and MEM performed similarly. However, from 48 h onwards, MEM was significantly better than renewed and not renewed mi...

Statins represent a class of drugs that effectively lowers cholesterol, however they also possess pleiotropic effects, like promotion of angiogenesis, prevention of bone loss, immunomodulatory and anti-inflammatory effects. Thus, the aim of this study was to investigate the activity of simvastatin topically applied in mice in acute and chronic skin inflammation models. Skin inflammation was induced in mice ears by topical application of 12-O-tetradecanoylphorbol acetate (TPA). In the acute model, ear oedema was measured by the increase of ear thickness 6h after TPA (2.5μg/ear). The chronic inflammatory process was induced by multiple applications of TPA (2.0μg/ear) for nine alternate days, and the oedema was measured daily as the increase in ear thickness. Topical treatment was applied immediately after TPA in acute model or started at 5th day of chronic experiment. For acute model treatment was simvastatin (0.24, 0.71 and 2.40μM), dexamethasone (0.13μM), both in acetone or vehicle alone (acetone). In chronic model simvastatin (1% and 3%) and dexamethasone (0.5%) were incorporated in ointment preparations, and a group received ointment alone (vehicle). Samples of ear tissue (6mm) were taken from acute and chronic models, weighted and prepared for histological analysis and myeloperoxidase (MPO) enzymatic activity evaluation. Application of simvastatin in acetone reduced the ear oedema after a single TPA application in a dose dependent manner [ID(50) of 0.47 (0.22-1.13) μM], and the MPO enzymatic activity up to 61±10%. Also, both simvastatin ointment preparations 1% and 3% reduced acute TPA-induced ear oedema in 55±7% and 65±8%, respectively. In the chronic model, simvastatin ointment 1% was able to reduce ear oedema (25±3%) and ear weight (10±1%), though 3% formulation augmented both parameters. Histological analysis revealed a reduction of swelling and leukocyte migration in the acute model for both the formulations of simvastatin (1% and 3%), while in chronic model simvastatin 1% decreased ear swelling and epidermal thickness, but simvastatin 3% increased both parameters. The results confirm the anti-inflammatory activity of simvastatin when applied topically in both acute and chronic models of skin inflammation. Besides, the formulation of simvastatin ointment 1% shows to be a very effective formulation for a chronic usage.

A significant involvement of nitric oxide (NO) in the process of keratinocyte proliferation is reported with many divergences. To determine the involvement of NO in the hyperproliferative process of epidermis in vivo, non-selective inhibitor (N(G)-nitro-L-arginine-methyl ester.HCl: L-NAME) and selective inhibitors for inducible NO synthase (iNOS) and neuronal NO synthase (nNOS) (Aminoguanidine: AG and 7-Nitroindazole: 7-NI, respectively) and a NO-donor (Sodium nitroprusside: SNP) were topically applied twice a day in mice ear treated with multiple applications of croton oil. L-NAME and 7-NI treatments decreased and SNP increased ear edema formation. However, ear weight was reduced in groups that received L-NAME and 7-NI, while the AG and SNP groups presented an increment. The histological evaluation of epidermis thickness showed that all NOS inhibitors were able to prevent the increase in epidermis width caused by croton oil, while SNP contributed to enlargement. The same results were observed in the PCNA staining, where treatments with NOS inhibitors caused a reduction in the number of cells in the epidermis, while SNP caused an enhancement. 7-NI treatment reduced polymorphonuclear and mononuclear leukocytes migration when compared to the control group. The AG application increased the migration of polymorphonuclear and mononuclear cells, while the SNP enhanced only the polymorphonuclear cells. Therefore, in the skin NO produced by nNOS is involved in the control of keratinocyte hyperproliferation, with the contribution of iNOS. In the animal model of cutaneous chronic inflammation by croton oil, NO is involved in the exudation and leukocyte migration, with participation of all three enzymes.