NEUROPROTECTIVE ROLE OF PALMITOYLETHANOLAMIDE IN PERINATAL ASPHYXIA. CORRELATIVE STUDIES OF IMMUNOHISTOCHEMISTRY AND WESTERN BLOT OF AXONALNEUROFILAMENTS IN RAT HIPPOCAMPUS

MARÍA I. HERRERA, JUAN.P. LUACES (*), LUCAS D. UDOVIN, CARLOS KUSNIER, NICOLÁS TORO-URREGO, RODOLFO KÖLLIKER-FRERS, MATILDE OTERO-LOSADA AND FRANCISCO CAPANI

Congreso; XIV CONGRESO CIASEM 2017 25-29 de Septiembre, Varadero.Cuba.; 2017

Perinatal asphyxia (PA) is an obstetric complication associated with an impaired gas exchange. The incidence of PA is estimated at 1/1000 live births in developed countries and 5-10/1000 live births in developing countries. This serious health problem could lead to neonatal mortality and continues to be a determinant of neurological morbidity, by inducing synaptic and cytoskeletal alterations associated with neuronal death. Cerebral palsy, epilepsy and several neurodevelopmental disorders are among the most common complications of PA. The lack of knowledge about the proper neuroprotective agents prompted us to investigate the role of Palmitoylethanolamide (PEA) in the experimental treatment of PA. This natural occurring amide has exerted protective effects in different models of brain injury and neurodegeneration. For this study, we used a well- established murine model, which induces PA at the time of delivery, mimicking relevant clinical features of the process such as delayed labor and compression of the umbilical cord. Full-term pregnant rats were rapidly decapitated and the uterus horns were replaced in a water bath at 37 °Cfor 19 minutes. When their physiological conditions improved, rats were subjected to a treatment with PEA in a dose of 10 mg/kg, which has shown the most effective results in several models of neurodegeneration and brain injury. Later, they were given to surrogate mothers. After 30 days, animals were perfused intracardiacally and coronal hippocampal sections were performed as described previously [1]. Free-floating sections were incubated overnight at 4 °C with anti- phosphorylated high and medium molecular weight neurofilaments (pNF H/Mp; 1:500, rabbit-IgG). Then, sections were incubated for 2 h at room temperature (RT) with secondary antibodies (Vector; Biotinylated anti- rabbit-IgG, 1:300, Vector). Amplification was done using avidin-biotinylated horseradish peroxidase complex (ABC; Vector) in PBS for 1 h, followed by washing in PBS before chromogen development (DAB; Vector). Light microscopic images were obtained (using a Leyca microscopy). For western blot analysis of neurofilaments (pNF-H/M) subcellular fractionation and procedure for immunoblotting was performed as it was previously described [2]. Immunocytochemistry revealed axonal alterations of pNF-H/M. Fig.1A shows a representative example of a striatum radiatum of CA1 hippocampal area immunostained for pNF-H/M. PA animals showed an increased immunostaining for pNF-H/M in comparison to the control group (CTL). This increase was reversedwhen PA rats were submitted to treatment with PEA (PA+PEA), producing a significant decrease in the percentage of pNF-H/Mreactive area by approximately 60 per cent (Fig.1B). These results were confirmed when the pNF-H/M level protein expression was analyzed by western blot. Fig.1C shows an approximate 2.5-fold increase in pNF-H/M levels in the PA rats hippocampus, which was reduced with PEA treatment, reaching values similar to CTL. In summary, after one month of PA, we observed a clear increase in pNF-HM expression both in immunohistochemistry and western blot analysis. As PA is a global event affecting the entire brain, it might result in higher concentrations of protein leaking [3]. Our results suggest that pNF-HM could be considered as a prognostic marker of PA. In addition, as pNF-HM accumulation could be reversed by PEA treatment, PEA might be a novelneuroprotective agent forPA.