Neuroprotective role of Palmitoylethanolamide in perinatal asphyxia

HERRERA MI, LUACES JP*, UDOVIN L, KUSNIER C, TORO N, KÖLLIKER-FRERS R, OTERO-LOSADA M, CAPANI F.

Congreso; XIV Congreso Ciasem; 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 several 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 °C for 19 minutes. When their physiological conditions improved, they were given to surrogate mothers. Rats were subjected to a treatment with PEA in a dose of 10 mg/kg, which has shown effective results in several models of brain injury and neurodegeneration. After 30 days animal were perfused intracardiacally and coronal hippocampal sections were performed as described previously (Capani et al 2009). Free-floating sectionswere incubated overnight at 4 °C with anti-neuron-specific nuclear protein (NeuN; 1:1000,mouse-IgG; Millipore), anti-microtubule-associated protein 2 (MAP-2; 1:250,mouse-IgG; Sigma-Aldrich), anti-phosphorylated high and medium molecular weight neurofilaments (NF H/Mp; 1:500, rabbit-IgG; and GFAP (1: 500, Abcam) Then sections were incubated for 2 h at room temperature (RT) with secondary antibodies (Biotinylated antimouse-IgG, 1:300, 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 inPBS before chromogen development (DAB; Vector). Light microscopic images were obtained using a Leyca. For western blot analysis of neurofilaments (pNF-H/M) subcellular fractionation and procedure for immunoblotting was performed as previously described in Romero et al 2015. Western blot analyses showed an approximate 2.5-fold increase in phosphorylated neurofilaments (pNF-H/M) protein levels in the PA rats hippocampus compared to control group (CTL) (Figure 2). As PA is a global event affecting the entire brain, it might result in higher concentrations of protein leaking (Blennow et al., 2001). In addition, treatment with PEA in PA rats (PA+PEA) reduced protein expression by approximately 60% in comparison to PA rats, reaching values similar to CTL. Treatment with PEA in the control group (CTL+PA) did not produce significant changes in pNF-H/M expression levels compared to CTL in the absence of PEA. In summary, after one month of the PA insult, we observed clear alterations in hippocampus were reverted after PEA treatment. These results suggest that PEA could be a novel neuroprotective agent for PA.