Sheep primary astrocytes under starvation conditions express higher amount of LC3 II autophagy marker than neurons.

Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. Recent studies have clearly demonstrated that autophagy has a greater variety of physiological and pathophysiological roles than expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging, elimination of microorganisms, cell death, tumor suppression and antigen presentation. MAP-LC3 is one of the most common markers  to evaluate autophagic processes. In our study, the autophagic activity in neurons and astrocytes from sheep brain under starving conditions was evaluated. In order to detect LC3 immunoreactivity, confocal analysis by double immunofluorescence was performed together with the cell type markers: GFAP to identify astrocytes, ß-III tubulin to identify neurons. The results show that astrocytes are characterized by LC3-positive areas, which increase in a time-dependent manner. In contrast, LC3 immunoreactivity was very weak in neurons. Therefore, it can be assumed that astrocytes show a higher capability than neurons to cope with stress and exhibit a stronger autophagic response.

Brain aging and testosterone-induced neuroprotection: studies on cultured sheep cortical neurons.

OBJECTIVE: This research reports the expression of topoisomerase ßII in fetal sheep neuronal cells. The ß isoform of DNA topoisomerase II plays a role in DNA repair process in non proliferating cells as neurons and its expression tends to be downregulated with senescence. METHODS: Cortical neurons from 60-day-old sheep embryos underwent two protocols: the former based on rising time of culture (10, 20 and 30 days); the latter based on the 72hrs exposure to 3-nitro-L-tyrosine (oxidative/nitrosative stressor) and/or testosterone. RESULTS: Our results showed an increase in ß-galactosidase activity and, in contrast, a reduction in topoisomerase ßII expression with time (first protocol). The exposure of sheep primary neurons to 3-nitro-L-tyrosine led to an upregulation of ßII topoisomerase expression to be likely seen as a reaction to nitrosative stress. Testosterone addition to 3-nitro-L-tyrosine-exposed cells results in topoisomerase ßII decrease possibly due to the neuroprotective properties of testosterone (second protocol). No significant variations in the marker of aging ß-galactosidase were observed in the cells exposed to 3-nitro-L-tyrosine and testosterone. CONCLUSION: The protocol based on time could be of some interest as a model of neuronal senescence in vitro. Topoisomerase ßII decrease with aging likely indicates a reduced ability to repair DNA during neuronal senescence. In contrast, the second protocol may not be seen as a reliable model of aging since 3-nitro-L-tyrosine does not lead to a topoisomerase ßII decrease. Testosterone was able to cope with oxidative/nitrosative damage, allowing cells to reduce their needs in DNA repair which in turn leads to a downregulation of topoisomerase IIß expression.

Spray-dried poly(D,L-lactide) microspheres containing carboplatin for veterinary use: in vitro and in vivo studies.

The aim of this study was the development of a veterinary dosage form constituted by injectable biodegradable microspheres designed for the subcutaneous release of carboplatin, a chemotherapeutic drug. Poly(D,L-lactide) (PDLLA) microspheres were prepared by an emulsification/spray-drying method, using the drug-to-polymer weight ratios 1:9 and 1:5; blank microspheres (1% w/v) were prepared as a comparison. Microparticles were characterized in terms of morphology, encapsulation efficiency, and in vitro drug release behavior. In vivo tests were conducted on rats by subcutaneous injection of microsphere aqueous suspensions. Levels of carboplatin were evaluated both in the skin and in serum. The microparticles obtained had a spherical shape; particle size ranged from 5 to 7 microm, dependent on drug loading. Microspheres were able to control the in vitro release of the drug: approximately 90% to 100% of the carboplatin was released over 30 days. In vivo results showed that the microspheres were able to release high drug amounts locally, and sustained serum levels of drug were also achieved. Based on these results, carboplatin-loaded PDLLA microspheres may be useful for local delivery of the antineoplastic drug to the tumor, avoiding tumor recurrence in small animals, and may decrease the formation of distant metastases.