Erythropoietin induces neuroprotection in experimental intracerebral hemorrhage via eNOS upregulation and STAT3 activation

Abstract

Erythropoietin (EPO) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. Both EPO and its receptor are expressed throughout the brain in glial cells, neurons and endothelial cells. In this study, we investigated whether EPO treatment reduces cerebral inflammation, edema and perihematomal cell death after experimental intracerebral hemorrhage (ICH), and whether functional recovery is enhanced. ICH was induced using stereotaxic infusion of collagenase into the left basal ganglia of adult rats. EPO (5,000 IU/kg) or PBS was administered intraperitoneally at 20 minutes after ICH induction and daily afterwards for 3 or 14 days. Seventy-two hours after ICH induction, we checked hematoma volume using spectrometric methods and water content in both hemispheres separately. TUNEL, activated caspase-3 staining and caspase-3 activity assay were done for the perihematomal cell death, and myeloperoxidase (MPO) activity was analyzed for inflammation. Expression of eNOS, pSTAT3, STAT3, and pSTAT5 was measured by western blotting. Behavioral tests were performed weekly up to 35 days after ICH. The volume of hemorrhage was decreased in EPO-treated group by 25% at 3 days after ICH (p<0.05). EPO reduced the brain water content in the lesioned hemisphere compared with ICH-only group (p<0.01). In EPO-treated group, the numbers of TUNEL+, MPO+, and OX42+ cells, activated caspase-3+ cells were decreased in the periphery of hematoma (p<0.01). Western blotting showed significant upregulation of eNOS and pSTAT3 expression in EPO-treated group compared with the ICH-only group, while protein expression of STAT3, STAT5 and pSTAT5 has not been changed. EPO-treated rats recovered better at 14 days after ICH throughout 35 days in both rotarod and limb placing tests (p<0.01), and the degree of functional improvement in EPO-treatment group was faster and better than that of ICH-only group. In this study, we provide evidences that EPO has therapeutic effects in ICH. EPO treatment enhances the functional recovery, and induces the pleiotropic neuroprotection including the reduction of hematoma volume and edema, and the inhibition of apoptosis and neuroinflammation. Both eNOS upregulation and enhanced STAT3 phosphorylation may be involved in this action of EPO.