Experimental models of chemically induced Parkinson's disease in zebrafish at the embryonic larval stage: a systematic review.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra that results in a decrease in dopamine levels, resulting in motor-type disturbances. Different vertebrate models, such as rodents and fish, have been used to study PD. In recent decades, Danio rerio (zebrafish) has emerged as a potential model for the investigation of neurodegenerative diseases due to its homology to the nervous system of humans. In this context, this systematic review aimed to identify publications that reported the utilization of neurotoxins as an experimental model of parkinsonism in zebrafish embryos and larvae. Ultimately, 56 articles were identified by searching three databases (PubMed, Web of Science, and Google Scholar). Seventeen studies using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 4 1-methyl-4-phenylpyridinium (MPP+), 24 6-hydroxydopamine (6-OHDA), 6 paraquat/diquat, 2 rotenone, and 6 articles using other types of unusual neurotoxins to induce PD were selected. Neurobehavioral function, such as motor activity, dopaminergic neuron markers, oxidative stress biomarkers, and other relevant parameters in the zebrafish embryo-larval model were examined. In summary, this review provides information to help researchers determine which chemical model is suitable to study experimental parkinsonism, according to the effects induced by neurotoxins in zebrafish embryos and larvae.

Effects of curcumin to counteract levodopa-induced toxicity in zebrafish.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction due to the death of dopaminergic neurons in the substantia nigra pars compacta. Currently, treatment of PD has focused on increasing dopamine levels, using a dopamine precursor, levodopa (L-DOPA) or stimulation of dopaminergic receptors. Prolonged use of L-DOPA is associated with the occurrence of motor complications and dyskinesia, attributed to neurotoxic effects of this drug. The aim of this study was to investigate the effects of curcumin (CUR), a lipophilic polyphenol, to counteract L-DOPA induced toxicity. Zebrafish larvae were pre-treated with CUR (0.05 µM) or vehicle dimethyl sulfoxide (DMSO) for 24 hr and subsequently exposed to L-DOPA (1 mM) or vehicle. Immediately and 24 hr after L-DOPA exposure, spontaneous swimming and dark/light behavioral tests were performed. In addition, levels of reactive oxygen species (ROS) and lipid peroxidation products were determined at the end of treatment. CUR significantly improved the motor impairment induced by 24 hr L-DOPA treatment, and reduced levels of ROS and lipoperoxidation products in zebrafish larvae. In conclusion, our results suggest that CUR acts as a neuroprotector against toxicity initiated by L-DOPA. Evidence suggests the observed effects of CUR are associated with its antioxidant properties.

Thiazolidine-2,4-dione derivative exhibits antitumoral effect and reverts behavioral and metabolic changes in a model of glioblastoma.

The aim of the present study was to evaluate the anti-glioma activity of 3-(4-fluorobenzyl)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione (AV23) in a preclinical model of glioblastoma, as well as behavioral parameters and toxicological profile. The implantation of C6 cells in the left striatum of male Wistar rats was performed by stereotaxic surgery. After recovery, animals were treated with vehicle (canola oil) or AV23 (10 mg/kg/day) intragastrically for 15 days. It was found that AV23 reduced tumor volume by 90%. Serum biochemical parameters such as triglycerides, cholesterol, HDL-cholesterol, LDL-cholesterol, albumin, aspartate aminotransferase, urea, creatinine and total proteins were not changed; however, there was a slight increase in alanine aminotransferase. The compound AV23 reverted the hypoglycemia and the reduction in body weight caused by glioblastoma. Additionally, AV23 was able to revert the reduction of locomotion caused by the tumor implantation. Therefore, the compound AV23 can be considered a promising candidate in the treatment of glioblastoma.

Interaction Between Near-Infrared Radiation and Temozolomide in a Glioblastoma Multiform Cell Line: A Treatment Strategy?

Photodynamic therapy (PDT) is a potential therapeutic modality against cancer, resulting from the interaction of a photosensitizer (PS) and radiation that generates damage to tumor cells. The use of near-infrared radiation (IR-A) is relevant because presents recognized biological effects, such as antioxidant, neuroprotective and antitumor effects. Glioblastoma is the most aggressive central nervous system (CNS) neoplasm with high proliferation and tissue invasion capacity and is resistant to radio and chemotherapy. Here, we evaluated in vitro the possible interaction of temozolomide (TMZ) with IR-A in a glioblastoma cell line (C6) and in a human keratinocyte cell line (HaCat) how non-tumor cell model, in an attempt to search for a new treatment strategy. The effects of TMZ, IR-A and the interaction between TMZ and IR-A was evaluated by viability exclusion with trypan blue. To perform the interaction experiments, we have chosen 10 µM TMZ and 4.5 J/cm2 of IR-A. From this, we evaluated cytotoxicity, cell proliferation, intracellular reactive oxygen species levels (ROS), as well as the process of cell migration and the P-gp and MRP-1 activity. Cell death mainly due to apoptosis, followed by necrosis, decreased cell proliferation, increased ROS levels, decreased cell migration and decreased P-gp and MRP1 activity were observed only when there was interaction between TMZ and IR-A in the C6 cell line. The interaction between TMZ and IR-A was not able to affect cell proliferation in the HaCat non-tumor cell line. Our results suggest that this interaction could be a promising approach and that in the future may serve as an antitumor strategy for PDT application.

Multi-walled carbon nanotubes functionalized with pyrene-PEG via π-π interactions: toxicological assessment in zebrafish embryos.

Multi-walled carbon nanotubes (MWCNT) have many promising biological applications, even though functionalization is needed for better biocompatibility. Functionalization of MWCNT with polyethylene glycol (PEG) is a promising and widely studied approach, but the best PEGylation method is still under investigation. In this work, we have tested the biological implications of MWCNT functionalized via π-stacking with pyrene-PEG (MWCNT-Pyr-PEG) in zebrafish embryos. As Pyr toxicity is well documented and represents a major concern for the safety of the proposed approach, we have also tested the effects of the exposure to the isolated conjugate (Pyr-PEG). The resulting suspensions were stable in saline medium and well dispersed. Zebrafish embryos at 24 h post-fertilization (hpf) were dechorionated and randomly assigned to seven experimental groups (n = 50 per group): control, MWCNT-Pyr-PEG at 0.2, 2.0, and 20.0 mg l-1, and Pyr-PEG at the same concentrations, and exposures were performed in 96-well plates. Specimens were observed for heart rate, malformations, body length, mortality, traveled distance, and number of new movements. Heart rate was reduced in embryos exposed to any tested concentration of MWCNT-Pyr-PEG, while this effect was observed with Pyr-PEG from 2 mg l-1. The highest concentration of MWCNT-Pyr-PEG also led to increased occurrence of malformations, shortened body length and reduced traveled distance. The functionalization approach shows promise due to the stability in saline media, even though toxic effects were observed in the highest tested concentrations, being the MWCNT the main actors underlying these outcomes.

2-(2-Methoxyphenyl)-3-((Piperidin-1-yl)ethyl)thiazolidin-4-One-Loaded Polymeric Nanocapsules: In Vitro Antiglioma Activity and In Vivo Toxicity Evaluation.

Among gliomas types, glioblastoma is considered the most malignant and the worst form of primary brain tumor. It is characterized by high infiltration rate and great angiogenic capacity. The presence of an inflammatory microenvironment contributes to chemo/radioresistance, resulting in poor prognosis for patients. Recent data show that thiazolidinones have a wide range of pharmacological properties, including anti-inflammatory and antiglioma activities. Nanocapsules of biodegradable polymers become an alternative to cancer treatment since they provide targeted drug delivery and could overcome blood-brain barrier. Therefore, here we investigated the in vitro antiglioma activity and the potential in vivo toxicity of 2- (2-methoxyphenyl) -3- ((piperidin-1-yl) ethyl) thiazolidin-4-one-loaded polymeric nanocapsules (4L-N). Nanocapsules were prepared and characterized in terms of particle size, polydispersity index, zeta potential, pH, molecule content and encapsulation efficiency. Treatment with 4L-N selectively decreased human U138MG and rat C6 cell lines viability and proliferation, being even more efficient than the free-form molecule (4L). In addition, 4L-N did not promote toxicity to primary astrocytes. We further demonstrated that the treatment with sub-therapeutic dose of 4L-N did not alter weight, neither resulted in mortality, toxicity or peripheral damage to Wistar rats. Finally, 4L as well as 4L-N did not alter makers of oxidative damage, such as TBARS levels and total sulfhydryl content, and did not change antioxidant enzymes SOD and CAT activity in liver and brain of treated rats. Taken together, these data indicate that the nanoencapsulation of 4L has potentiated its antiglioma effect and does not cause in vivo toxicity.

In Vitro Anti/Pro-oxidant Activities of R. ferruginea Extract and Its Effect on Glioma Cell Viability: Correlation with Phenolic Compound Content and Effects on Membrane Dynamics.

Rapanea ferruginea antioxidant and antitumoral properties were not explored before in literature. This study aimed to investigate these biological activities for the R. ferruginea leaf extract and correlate them with its phenolic content and influence in biological membrane dynamics. Thus, in this study, anti/pro-oxidative properties of R. ferruginea leaf extract by in vitro DPPH and TBARS assays, with respect to the free radical reducing potential and to its activity regarding membrane free radical-induced peroxidation, respectively. Furthermore, preliminary tests related to the extract effect on in vitro glioma cell viability were also performed. In parallel, the phenolic content was detected by HPLC-DAD and included syringic and trans-cinnamic acids, quercetrin, catechin, quercetin, and gallic acid. In an attempt to correlate the biological activity of R. ferruginea extract and its effect on membrane dynamics, the molecular interaction between the extract and a liposomal model with natural-sourced phospholipids was investigated. Location and changes in vibrational, rotational, and translational lipid motions, as well as in the phase state of liposomes, induced by R. ferruginea extract, were monitored by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, differential scanning calorimetry, and UV-visible spectroscopy. In its free form, the extract showed promising in vitro antioxidant properties. Free-form extract (at 1000µ g/mL) exposure reduced glioma cell in vitro viability in 40%, as evidenced by MTT tests. Pro-oxidant behavior was observed when the extract was loaded into liposomes. A 70.8% cell viability reduction was achieved with 500 µg/mL of liposome-loaded extract. The compounds of R. ferruginea extract ordered liposome interface and disorder edits a polar region. Phenolic content, as well as membrane interaction and modulation may have an important role in the oxidative and antitumoral activities of the R. ferruginea leaf extract.

Toxicological assessment of PEGylated single-walled carbon nanotubes in early developing zebrafish.

Functionalization of single-walled carbon nanotubes (SWCNT) with polyethylene glycol (PEG) is among the most promising strategies to avoid SWCNT aggregation in aqueous media, improving its interactions with biological systems. However, the best molecular PEG weight and functionalization strategy remain under investigation. In this work we assessed the toxicological effects of SWCNT functionalized with PEG at 600 Da in zebrafish embryos. Embryos were exposed to SWCNT at 0.01, 0.1 and 1 mg/L from 3 to 96 h post-fertilization (hpf). At the highest concentration, SWCNT led to toxic effects at several endpoints, including mortality, delayed hatching, malformations, reduced body length, increased ROS production and DNA damage. Even with these effects, SWCNT could not be detected within the bodily tissues of the larvae. Our results give evidence that the tested PEGylation approach was unsuitable to avoid SWCNT aggregation in aqueous media, and that SWCNT can induce toxicity even without being absorbed by the organism by obstructing the chorion pores.

Isolation of adipose-derived stem cells: a comparison among different methods.

Adipose-derived stromal cells (ASCs) are usually isolated by digestion with collagenase. We have compared alternative methods to isolate ASCs in a more economically viable protocol. Nine protocols using red blood cells lysis buffer solution, trypsin, collagenase and centrifugation were compared; the isolation rate, cell viability, expansion rate, immunophenotype and differentiation in adipogenic and osteogenic lineages were analyzed. ASCs were isolated and successfully maintained by digestion with trypsin. Cells presented similar immunophenotypes, adipogenic differentiation and in vitro proliferation but an osteogenic differentiation capacity up to seven times higher than ASCs isolated by collagenase. This alternative protocol is thus efficient and more cost-effective than the commonly-used methods and may represent a promising protocol for obtaining ASCs for bone tissue engineering.

Effects of quercetin in preclinical models of Parkinson's disease: A systematic review.

Parkinson's disease (PD) is a neurodegenerative disease that affects dopaminergic neurons, thus impairing dopaminergic signalling. Quercetin (QUE) has antioxidant and neuroprotective properties that are promising for the treatment of PD. This systematic review aimed to investigate the therapeutic effects of QUE against PD in preclinical models. The systematic search was performed in PubMed, Scopus and Web of Science. At the final screening stage, 26 articles were selected according to pre-established criteria. Selected studies used different methods for PD induction, as well as animal models. Most studies used rats (73.08%) and mice (23.08%), with 6-OHDA as the main strategy for PD induction (38.6%), followed by rotenone (30.8%). QUE was tested immersed in oil, nanosystems or in free formulations, in varied routes of administration and doses, ranging from 10 to 400 mg/kg and from 5 to 200 mg/kg in oral and intraperitoneal administrations, respectively. Overall, evidence from published data suggests a potential use of QUE as a treatment for PD, mainly through the inhibition of oxidative stress, neuroinflammatory response and apoptotic pathways.

Combination of Curcumin and Photodynamic Therapy Based on the Use of Red Light or Near-Infrared Radiation in Cancer: A Systematic Review.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic intervention that can be applied to cancer treatment. The interaction between a photosensitizer (PS), ideal wavelength radiation, and tissue molecular oxygen triggers a series of photochemical reactions responsible for producing reactive oxygen species. These highly reactive species can decrease proliferation and induce tumor cell death. The search for PS of natural origin extracted from plants becomes relevant, as they have photoactivation capacity, preferentially targeting tumor cells and because they do not present any or little toxicity to healthy cells. OBJECTIVE: Our work aimed to carry out a qualitative systematic review to investigate the effects of curcumin (CUR), a molecule considered as PS of natural origin, on PDT, using red light or near-infrared radiation in tumor models. METHODS: A systematic search was performed in three databases (PubMed, Scopus, and Web of Science) using the PICOT method, retrieving a total of 1,373 occurrences. At the end of the peer screening, 25 eligible articles were included in this systematic review using inclusion, exclusion, and eligibility criteria. RESULTS: CUR, whether in its free state, associated with metal complexes or other PS and in a nanocarrier system, was considered a relevant PS for PDT using red light or near-infrared against tumoral models in vitro and in vivo, acting by increasing cytotoxicity, inhibiting proliferation, inducing cell death mainly by apoptosis, and changing oxidative parameters. CONCLUSION: The results found in this systematic review suggest the potential use of CUR as a PS of natural origin to be applied in PDT against many neoplasms, encouraging further search in PDT against cancer and serving as an investigative basis for upcoming pre-clinical and clinical applications.

Effect of Hydrogel Containing Achyrocline satureioides (Asteraceae) Extract-Loaded Nanoemulsions on Wound Healing Activity.

Achyrocline satureioides (Lam.) DC extract-loaded nanoemulsions have demonstrated potential for wound healing, with promising effects on keratinocyte proliferation. We carried out the first in vivo investigation of the wound healing activity of a hydrogel containing A. satureioides extract-loaded nanoemulsions. We prepared hydrogels by adding the gelling agent (Carbopol® Ultrez) to extract-loaded nanoemulsions (~250 nm in diameter) obtained by spontaneous emulsification. The final flavonoid content in formulation was close to 1 mg/mL, as estimated by ultra-fast liquid chromatography. Permeation/retention studies using porcine ear skin showed that flavonoids reached deeper layers of pig ear skin when it was damaged (up to 3.2 µg/cm² in the dermis), but did not reach the Franz-type diffusion cell receptor fluid. For healing activity, we performed a dorsal wound model using Wistar rats, evaluating the lesion size, anti-inflammatory markers, oxidative damage, and histology. We found that extract-loaded formulations promoted wound healing by increasing angiogenesis by ~20%, reducing inflammation (tumor necrosis factor α) by ~35%, decreasing lipid damage, and improving the re-epithelialization process in lesions. In addition, there was an increase in the number of blood vessels and hair follicles for wounds treated with the formulation compared with the controls. Our findings indicate that the proposed formulation could be promising in the search for better quality healing and tissue reconstruction.

Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain.

Reactive gliosis is the universal reaction to brain injury, but the precise origin and subsequent fate of the glial cells reacting to injury are unknown. Astrocytes react to injury by hypertrophy and up-regulation of the glial-fibrillary acidic protein (GFAP). Whereas mature astrocytes do not normally divide, a subpopulation of the reactive GFAP(+) cells does so, prompting the question of whether the proliferating GFAP(+) cells arise from endogenous glial progenitors or from mature astrocytes that start to proliferate in response to brain injury. Here we show by genetic fate mapping and cell type-specific viral targeting that quiescent astrocytes start to proliferate after stab wound injury and contribute to the reactive gliosis and proliferating GFAP(+) cells. These proliferating astrocytes remain within their lineage in vivo, while a more favorable environment in vitro revealed their multipotency and capacity for self-renewal. Conversely, progenitors present in the adult mouse cerebral cortex labeled by NG2 or the receptor for the platelet-derived growth factor (PDGFRalpha) did not form neurospheres after (or before) brain injury. Taken together, the first fate-mapping analysis of astrocytes in the adult mouse cerebral cortex shows that some astrocytes acquire stem cell properties after injury and hence may provide a promising cell type to initiate repair after brain injury.

Amyloid-beta neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK-3beta, tau and neuroinflammation.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid-beta (Abeta) peptide in brain regions that are important for memory and cognition. The buildup of Abeta aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Abeta-induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 microm of Abeta(25-35) in the absence or in the presence of melatonin (25, 50, or 100 microm). In addition, the authors have investigated the involvement of GSK-3beta, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Abeta-induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Abeta(25-35). In addition, melatonin significantly reduced the activation of GSK-3beta, the phosphorylation of tau protein, the glial activation and the Abeta-induced increase of TNF-alpha and IL-6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Abeta-induced phosphorylation of tau protein, and preventing GSK-3beta activation and neuroinflammation.

Hydrogels containing soybean isoflavone aglycones-rich fraction-loaded nanoemulsions for wound healing treatment - in vitro and in vivo studies.

Soybean isoflavone aglycones have been investigated as potential wound healing compounds for topical application. The aim of this study was to evaluate the wound healing properties of a soybean isoflavone aglycones-rich fraction (IAF) when incorporated into lipid nanoemulsions dispersed in acrylic-acid hydrogels. Formulations exhibited a mean droplet size in the sub 200 nm range, negative ζ-potential (-60 mV), and displayed non-Newtonian pseudoplastic behavior. The addition of a gelling agent decreased the IAF release from formulations and improved the retention of these compounds in intact porcine ear skin when compared with a control propylene glycol solution. No IAF were detected in receptor fluid of Franz-type diffusion cells. However, increasing amounts of IAF were noticed in both skin layers and the receptor fluid when the tissue was partially injured (tape stripping), or when the epidermis was completely removed. In vitro studies showed that IAF elicits an increased proliferation and migration of keratinocytes (HaCaT cell line). Subsequently, the healing effect of the formulations was evaluated in a model of dorsal wounds in rats, by assessing the size of the lesions, histology, inflammatory markers, and antioxidant activity. Overall findings demonstrated the potential of IAF-loaded formulations to promote wound healing by increasing angiogenesis by ∼200 %, reducing the lipid oxidation (TBARS) by ∼52 % and the inflammation (TNFα) by ∼35 %, while increasing re-epithelialization by ∼500 %, visualized by the epithelium thickness.

Healing activity of hydrogel containing nanoemulsified ß-caryophyllene.

ß-caryophyllene is a sesquiterpene present in the oil of many plant species, such as Copaifera sp., which has been shown to possesses potent anti-inflammatory action; however, its healing activity remains under study. The objectives of the present study were to produce a nanoemulsion containing ß-caryophyllene followed by a hydrogel containing nanoemulsified ß-caryophyllene, to evaluate the permeation profile in vitro, and to assess the in vivo healing activity, which is so far unexplored in the literature for pure ß-caryophyllene and in pharmaceutical formulation. The nanoemulsion was obtained through high-pressure homogenization and the hydrogel by direct dispersion with hydroxyethylcellulose. Both formulations were characterized according to droplet size, polydispersity index, volume-weighted mean diameters, particle distribution, droplets diameters tracking, zeta potential, viscosity and bioadhesion behavior. ß-caryophyllene content was determined by gas chromatography coupled with mass spectrometry (GC/MS). Both formulations presented a nanometric droplet size, negative zeta potential, high ß-caryophyllene content, and were stable for 60 days. In agreement with the viscosity results, the hydrogel containing the ß-caryophyllene nanoemulsion showed superior bioadhesiveness than the nanoemulsion. The skin permeation study in Franz cells demonstrated that isolated ß-caryophyllene was unable to cross the stratum corneum and that its nanoemulsification promoted its permeation. On the other hand, in the simulated deeply wounded skin (dermis), no significant differences were observed between the formulations and isolated ß-caryophyllene with respect to the amount of marker retention in the dermis, suggesting saturation of this skin layer. For the study of healing activity, the dorsal wound model was performed with an evaluation of the lesion size, anti-inflammatory markers, and antioxidant activity. The initial closure of the wound was achieved sooner in the group treated with the hydrogel containing the ß-caryophyllene nanoemulsion, indicating its anti-inflammatory effect. The histological analysis indicated that on day 12 day of the lesion, the hydrogel presented similar results to those of the positive control group (Dersani® oil), proving effectiveness in cutaneous tissue repair.

Boldine: a potential new antiproliferative drug against glioma cell lines.

Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

Conditioned medium from mesenchymal stem cells induces cell death in organotypic cultures of rat hippocampus and aggravates lesion in a model of oxygen and glucose deprivation.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

A comparative study of beta-amyloid peptides Abeta1-42 and Abeta25-35 toxicity in organotypic hippocampal slice cultures.

Accumulation of the neurotoxic amyloid beta-peptide (Abeta) in the brain is a hallmark of Alzheimer's disease (AD). Several synthetic Abeta peptides have been used to study the mechanisms of toxicity. Here, we sought to establish comparability between two commonly used Abeta peptides Abeta1-42 and Abeta25-35 on an in vitro model of Abeta toxicity. For this purpose we used organotypic slice cultures of rat hippocampus and observed that both Abeta peptides caused similar toxic effects regarding to propidium iodide uptake and caspase-3 activation. In addition, we also did not observe any effect of both peptides on Akt and PTEN phosphorylation; otherwise the phosphorylation of GSK-3beta was increased. Although further studies are necessary for understanding mechanisms underlying Abeta peptide toxicity, our results provide strong evidence that Abeta1-42 and the Abeta25-35 peptides induce neural injury in a similar pattern and that Abeta25-35 is a convenient tool for the investigation of neurotoxic mechanisms involved in AD.

Effects of chronic restraint stress and estradiol replacement on glutamate release and uptake in the spinal cord from ovariectomized female rats.

Glutamate is an excitatory neurotransmitter involved in neuronal plasticity and neurotoxicity. Chronic stress produces several physiological changes on the spinal cord, many of them presenting sex-specific differences, which probably involve glutamatergic system alterations. The aim of the present study was to verify possible effects of exposure to chronic restraint stress and 17beta-estradiol replacement on [3H]-glutamate release and uptake in spinal cord synaptosomes of ovariectomized (OVX) rats. Female rats were subjected to OVX, and half of the animals received estradiol replacement. Animals were subdivided in controls and chronically stressed. Restraint stress or estradiol had no effect on [3H]-glutamate release. The chronic restraint stress promoted a decrease and 17beta-estradiol induced an increase on [3H]-glutamate uptake, but the uptake observed in the restraint stress +17beta-estradiol group was similar to control. Furthermore, 17beta-estradiol treatment caused a significant increase in the immunocontent of the three glutamate transporters present in spinal cord. Restraint stress had no effect on the expression of these transporters, but prevented the 17beta-estradiol effect. We suggest that changes in the glutamatergic system are likely to take part in the mechanisms involved in spinal cord plasticity following repeated stress exposure, and that 17beta-estradiol levels may affect chronic stress effects in this structure.