Neuroprotective effects of erythropoietin pretreatment in a rodent model of transient middle cerebral artery occlusion.
OBJECT.: There is an unmet clinical need to develop neuroprotective agents for neurosurgical and endovascular procedures that require transient cerebral artery occlusion. The aim in this study was to explore the effects of a single dose of recombinant human erythropoietin (rhEPO) before middle cerebral artery (MCA) occlusion in a focal cerebral ischemia/reperfusion model.


METHODS
Twenty-eight adult male Wistar rats were subjected to right MCA occlusion via the intraluminal thread technique for 60 minutes under continuous cortical perfusion monitoring by laser Doppler flowmetry. Rats were divided into 2 groups: control and treatment. In the treated group, rhEPO (1000 IU/kg intravenously) was administered 10 minutes before the onset of the MCA ischemia. At 24-hour reperfusion, animals were examined for neurological deficits, blood samples were collected, and animals were killed. The following parameters were evaluated: brain infarct volume, ipsilateral hemispheric edema, neuron-specific enolase plasma levels, parenchyma histological features (H & E staining), Fluoro-Jade-positive neurons, p-Akt and total Akt expression by Western blot analysis, and p-Akt-positive nuclei by immunohistochemical investigation.


RESULTS
Infarct volume and Fluoro-Jade staining of degenerating neurons in the infarct area did not vary between groups. The severity of neurological deficit (p < 0.001), amount of brain edema (78% reduction in treatment group, p < 0.001), and neuron-specific enolase plasma levels (p < 0.001) were reduced in the treatment group. Perivascular edema was histologically less marked in the treatment group. No variations in the expression or localization of p-Akt were seen.


CONCLUSIONS
Administration of rhEPO before the onset of 60-minute transient MCA ischemia protected the brain from this insult. It is unlikely that rhEPO pretreatment leads to direct neuronal antiapoptotic effects, as supported by the lack of Akt activation, and its benefits are most probably related to an indirect effect on brain edema as a consequence of blood-brain barrier preservation. Although research on EPO derivatives is increasing, rhEPO acts through distinct neuroprotective pathways and its clinical safety profile is well known. Clinically available rhEPO is a potential therapy for prevention of neuronal injury induced by transitory artery occlusion during neurovascular procedures.
Department of Neurosurgery, Hospital de São José, Centro Hospitalar de Lisboa Central;
Bernardo Oliveira
BO
Ratilal
Mariana Moreira Coutinho
MM
Arroja
Joao Pedro Fidalgo
JP
Rocha
Adelaide Maria Afonso
AM
Fernandes
Andreia Pereira
AP
Barateiro
Dora Maria Tuna Oliveira
DM
Brites
Rui Manuel Amaro
RM
Pinto
Bruno Miguel Nogueira
BM
Sepodes
Helder Dias
HD
Mota-Filipe
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism; D001921:Brain / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004921:Erythropoietin / Q000494:pharmacology / Q000627:therapeutic use*; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002546:Ischemic Attack, Transient / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use*; D010751:Phosphopyruvate Hydratase / Q000097:blood; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D018696:Neuroprotective Agents; D004921:Erythropoietin; D051057:Proto-Oncogene Proteins c-akt; D010751:Phosphopyruvate Hydratase
BBB = blood-brain barrier; BSA = bovine serum albumin; CBF = cerebral blood flow; CCA = common carotid artery; ECA = external carotid artery; EPO, EPOR = erythropoietin, erythropoietin receptor; GSK-3β = glycogen synthase kinase–3β; I/R = ischemia/reperfusion; ICA = internal carotid artery; MCA = middle cerebral artery; NF-κB = nuclear factor–kappa B; NSE = neuron-specific enolase; PBS = phosphate-buffered saline; PI3K = phosphatidylinositol 3–kinase; neuroprotection; rat; recombinant human erythropoietin; rhEPO = recombinant human EPO; transient focal cerebral ischemia; vascular disorders