J Korean Soc Neonatol.  2009 Nov;16(2):221-233.

Neuroprotection of Recombinant Human Erythropoietin Via Modulation of N-methyl-D-aspartate Receptors in Neonatal Rats with Hypoxic-ischemic Brain Injury

• Jang YJ
• Seo ES
• Kim WT

Abstract

PURPOSE
Erythropoietin (EPO) has neuroprotective effects in many animal models of brain injury, including hypoxic-ischemic (HI) encephalopathy, trauma, and excitotoxicity. Current studies have demonstrated the neuroprotective effects of EPO, but limited data are available for the neonatal periods. Here in we investigated whether recombinant human EPO (rHuEPO) can protect the developing rat brain from HI injury via modulation of NMDA receptors.
METHODS
In an in vitro model, embryonic cortical neuronal cell cultures from Sprague- Dawley (SD) rats at 19-days gestation were established. The cultured cells were divided into five groups: normoxia (N), hypoxia (H), and 1, 10, and 100 IU/mL rHuEPO-treated (H+E1, H+ E10, and H+E100) groups. To estimate cell viability and growth, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. In an in vivo model, left carotid artery ligation was performed on 7-day-old SD rat pups. The animals were divided into six groups; normoxia control (NC), normoxia Sham-operated (NS), hypoxia- ischemia only (H), hypoxia-ischemia+vehicle (HV), hypoxia-ischemia+rHuEPO before a HI injury (HE-B), and hypoxia-ischemia+rHuEPO after a HI injury (HE-A). The morphologic changes following brain injuries were noted using hematoxylin and eosin (H/E) staining. Real-time PCR using primers of subunits of NMDA receptors (NR1, NR2A, NR2B, NR2C and NR2D) mRNA were performed.
RESULTS
Cell viability in the H group was decreased to less than 60% of that in the N group. In the H+E1 and H+E10 groups, cell viability was increased to >80% of the N group, but cell viability in the H+E100 group did not recover. The percentage of the left hemisphere area compared the to the right hemisphere area were 98.9% in the NC group, 99.1% in the NS group, 57.1% in the H group, 57.0% in the HV group, 87.6% in the HE-B group, and 91.6% in the HE-A group. Real-time PCR analysis of the expressions of subunits of NMDA receptors mRNAs in the in vitro and in vivo neonatal HI brain injuries generally revealed that the expression in the H group was decreased compared to the N group and the expressions in the rHuEPO-treated groups was increased compared to the H group.
CONCLUSION
rHuEPO has neuroprotective property in perinatal HI brain injury via modulation of N-methyl-D-aspartate receptors.

Keyword

Erythropoietin; N-methyl-D-aspartate receptor; Hypoxic-ischemic brain injury

MeSH Terms

Animals
Anoxia
Brain
Brain Injuries
Carotid Arteries
Cell Culture Techniques
Cell Survival
Cells, Cultured
Eosine Yellowish-(YS)
Erythropoietin
Hematoxylin
Humans
Ischemia
Ligation
Models, Animal
N-Methylaspartate
Neurons
Neuroprotective Agents
Pregnancy
Rats
Real-Time Polymerase Chain Reaction
Receptors, N-Methyl-D-Aspartate
RNA, Messenger
Eosine Yellowish-(YS)
Erythropoietin
Hematoxylin
N-Methylaspartate
Neuroprotective Agents
RNA, Messenger
Receptors, N-Methyl-D-Aspartate