Perinatal asphyxia (PA) is an obstetric complication associated with an impaired gas exchange. This health problem continues to be a determinant of neonatal mortality and neurodevelopmental disorders. Palmitoylethanolamide (PEA) has exerted neuroprotection in several models of brain injury and neurodegeneration. We aimed at evaluating the potential neuroprotective role of PEA in an experimental model, which induces PA in the immature rat brain. PA was induced by placing Sprague Dawley newborn rats in a water bath at 37°C for 19 min. Once their physiological conditions improved, they were given to surrogate mothers that had delivered normally within the last 24 h. The control group was represented by non-fostered vaginally delivered pups, mimicking the clinical situation. Treatment with PEA (10 mg/kg) was administered within the first hour of life. Modifications in the hippocampus were analyzed with conventional electron microscopy, immunohistochemistry (for NeuN, pNF-H/M, MAP-2, and GFAP) and western blot (for pNF H/M, MAP-2, and GFAP). Behavior was also studied throughout Open Field (OF) Test, Passive Avoidance (PA) Task and Elevated Plus Maze (EPM) Test. After 1 month of the PA insult, we observed neuronal nucleus degeneration in CA1 using electron microscopy. Immunohistochemistry revealed a significant increase in pNF-H/M and decrease in MAP-2 in CA1 reactive area. These changes were also observed when analyzing the level of expression of these markers by western blot. Vertical exploration impairments and anxiety-related behaviors were encountered in the OF and EPM tests. PEA treatment attenuated PA-induced hippocampal damage and its corresponding behavioral alterations. These results contribute to the elucidation of PEA neuroprotective role after PA and the future establishment of therapeutic strategies for the developing brain.

Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.

Melatonin exerts direct neuroprotection against cerebral hypoxic damage, but the mechanisms of its action on microglia have been less characterized. Using both in vitro and in vivo models of hypoxia, we here focused on the role played by silent mating type information regulation 2 homolog 1 (SIRT1) in melatonin’s effects on microglia. Viability of rat primary microglia or microglial BV2 cells and SH-SY5Y neurons was significantly reduced after chemical hypoxia with CoCl2 (250 μM for 24 h). Melatonin (1 μM) significantly attenuated CoCl2 toxicity on microglia, an effect prevented by selective SIRT1 inhibitor EX527 (5 μM) and AMP-activated protein kinase (AMPK) inhibitor BML-275 (2 μM). CoCl2 did not modify SIRT1 expression, but prevented nuclear localization, while melatonin appeared to restore it. CoCl2 induced nuclear localization of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-kB), an effect contrasted by melatonin in an EX527-dependent fashion. Treatment of microglia with melatonin attenuated potentiation of neurotoxicity. Common carotid occlusion was performed in p7 rats, followed by intraperitoneal injection of melatonin (10 mg/kg). After 24 h, the number of Iba1+ microglia in the hippocampus of hypoxic rats was significantly increased, an effect not prevented by melatonin. At this time, SIRT1 was only detectable in the amoeboid, Iba1+ microglial population selectively localized in the corpus callosum. In these cells, nuclear localization of SIRT1 was significantly lower in hypoxic animals, an effect prevented by melatonin. NF-kB showed an opposite expression pattern, where nuclear localization in Iba1+ cells was significantly higher in hypoxic, but not in melatonin-treated animals. Our findings provide new evidence for a direct effect of melatonin on hypoxic microglia through SIRT1, which appears as a potential pharmacological target against hypoxic-derived neuronal damage.