Cannabinoid receptor type 1 modulates the effects of polyunsaturated fatty acids on memory of stressed rats.

Memory and GABAergic activity in the hippocampus of stressed rats improve after n-3 polyunsaturated fatty acid (PUFA) supplementation. On the other hand, cannabinoid receptor type 1 (CB1) strongly regulates inhibitory neurotransmission in the hippocampus. Speculation about a possible relation between stress, endocannabinoids, and PUFAs. Here, we examined whether the effects of PUFAs on memory of chronically stressed rats depends on pharmacological manipulation of CB1 receptors. Male Sprague-Dawley rats were orally supplemented with n-3 (fish oil) or n-6 (primrose oil) PUFAs during chronic restraint stress (CRS) protocol (6 h/day; 21 days). First, we studied if the expression of CB1 receptors in the hippocampus may be affected by CRS and PUFAs supplementation by real-time PCR and immunofluorescence. CRS up-regulated the CB1 expression compared with the non-stressed rats, while only n-3 PUFAs countered this effect. Memory was evaluated in the Morris water maze. Stressed rats were co-treated with PUFAs and/or modulators of CB1 receptor (AM251, antagonist, 0.3 mg/kg/day; WIN55,212-2, agonist, 0.5 mg/kg/day) by intraperitoneal injections. Memory improved in the stressed rats that were treated with AM251 and/or n-3 PUFAs. Supplementation with n-6 PUFAs did not affect memory of stressed rats, but co-treatment with AM251 improved it, while co-treatment with WIN55,212-2 did not affect memory. Our results demonstrate that activity of the CB1 receptors may modulate the effects of PUFAs on memory of stressed rats. This study suggests that endocannabinoids and PUFAs can both become a singular system by being self-regulated in limbic areas, so they control the effects of stress on the brain.

Development, characterization and in vitro evaluation of biodegradable rhein-loaded microparticles for treatment of osteoarthritis.

Rhein is an active metabolite of the drug diacerein, whose anti-inflammatory properties have been demonstrated in both in vitro and in vivo models. However, the low oral bioavailability of rhein has limited its utility as a potential treatment of osteoarthritis (OA), a chronic inflammatory disease. In order to overcome this limitation, the aim of this work was the development of a drug delivery system intended for intra-articular administration of rhein, based on polymeric biodegradable PLGA microparticles (MPs) loaded with the drug. The MPs, prepared by the emulsion-solvent evaporation technique were characterized in terms of several parameters including morphology, encapsulation efficiency, molecular interactions between components of the formulation and in vitro release profiling. Furthermore, cell-based in vitro studies were performed to evaluate the cytotoxicity of the formulations and their effect on the release of inflammatory markers including pro-inflammatory cytokines and reactive oxygen species (ROS). Scanning electron microscopy demonstrated that the prepared MPs exhibited an almost spherical shape with smooth surface. The size distribution of the prepared MPs ranged between 1.9 and 7.9µm, with mean diameter of 4.23±0.87µm. The optimal encapsulation efficiency of rhein was 63.8±3.0%. The results of powder X-ray diffraction and differential scanning calorimetry studies demonstrated that the active ingredient is partially the crystalline state, dispersed in the polymer matrix. This outcome is somewhat reflected in the release kinetics of rhein from the MPs. The cytotoxicity evaluation, carried out in macrophages derived from THP-1 cells, showed that both rhein-loaded MPs and unloaded MPs did not significantly affect the cell viability at MP concentrations up to 13.8µM. In lipopolysaccharide-activated macrophages, the rhein-loaded MPs significantly decreased the production of interleukin-1ß (IL-1ß) and (ROS), when compared to the unloaded MPs. In conclusion, the results of this preliminary study suggest that an MP-based formulation of rhein could be tested in animal models of inflammation, aiming for an injectable commercial product capable of providing a therapeutic solution to patients suffering from chronic joint diseases.

Expression and localization of an agmatinase-like protein in the rat brain.

Agmatinase catalyzes the hydrolysis of agmatine into putrescine and urea, and agmatine (decarboxylated L: -arginine) plays several roles in mammalian tissues, including neurotransmitter/neuromodulatory actions in the brain. Injection of agmatine in animals produces anticonvulsant, antineurotoxic and antidepressant-like actions. Information regarding the enzymatic aspects of agmatine metabolism in mammals, especially related to its degradation, is relatively scarce. The explanation for this is the lack of enzymatically active preparations of mammalian agmatinase. Recently, we have cloned a protein from a cDNA rat brain library having agmatinase activity although its amino acid sequence greatly differs from all known agmatinases, we called agmatinase-like protein. In this work, we analyzed the expression of this enzyme in the rat brain by means of RT-PCR and immunohistochemical analysis using a polyclonal antibody generated against the recombinant agmatinase-like protein. The agmatinase-like protein was detected in the hypothalamus in glial cells and arcuate nucleus neurons, and in hippocampus astrocytes and neurons, but not in brain cortex. In general, detected localization of agmatinase-like protein coincides with that described for its substrate agmatine and our results help to explain several reported effects of agmatine in the brain. Concretely, a role in the regulation of intracellular concentrations of the neurotransmitter/neuromodulator agmatine is suggested for the brain agmatinase-like protein.