REAL TIME PCR APPROACH TO STUDY THE ERYTHROPOIETIN AND ERYTHROPOIETIN RECEPTOR IN THE EPILEPTIC BRAIN

Erythropoietin (Epo) is mostly recognized for its central role in erythropoiesis. An independent system Epo/Epo receptor (EpoR) activated in response to hypoxia was detected in brain, leading to the hypothesis that it could be involved in cerebral functions such as neurotrophic and neuroprotective. Hypoxia may not be the only relevant stimulus for brain Epo production. Other stressors, such as epileptic seizures, also may stimulate upregulation of EpoR localized in the cerebral microvasculature (1). Metabolic disturbances, as hypoglycaemia or strong neuronal depolarization may increase cerebral Epo expression (1-3). Aim of this study was to accurate quantify the expression levels of Epo and EpoR in the central nervous system (CNS). The Epo and EpoR mRNA expression levels were first analysed in hematopoietic culture cells, U937 and then in surgically resected brain specimens from drug-resistant temporal lobe epilepsy (TLE) patients by PCR real time (QuantiTect SYBR-Green, iCycler-BIORAD). RNA samples of cerebral human cortex from congestive heart failure and chronic obstructive disease donors were also included as control (Ambion). The set of primers for Epo and EpoR tested in the cDNA from U937 cells allowed to validate the PCR real time system. The expression of endogen Epo in each brain tissue from TLE patients resulted under basal expression level. Furthermore the EpoR mRNA was significantly higher expressed then endogen Epo. Data obtained from the drug-resistant TLE patients were analyzed with the controls by the Mann-Whitney U-test. No significant differences between the TLE patients and the control (Ambion) were found. The real time PCR approach, set up to analyse the expression levels of Epo and its receptor in the CNS, showed a upregulation of EpoR in TL of TLE patients. These results suggest a role for EpoR in mediating the cross talk between the peripheral and central Epo systems and the translocation of Epo into the brain. Further measurement of Epo, in the same samples, by HPLC and mass spectrometry will be useful to better understand the paracrine effect of Epo on the cerebral function