title: Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina.
abstract: OBJECTIVE
  Retinal neuronal cell dysfunction and even cell death are associated with increased excitotoxic glutamate (Glu) level in the retina. Our aim was to study a causative mechanism of Glu on retinal cell death and explore the neuroprotective role of erythropoietin (EPO) against Glu neurotoxicity in the diabetic retina.
  
  
  METHODS
  Male Sprague-Dawley (SD) rats and R28 cell line were employed in this study. Diabetes was induced with intraperitoneal injection of streptozotocin (STZ) in SD rats. Two weeks after diabetes onset, the intravitreal injection was performed; 4 days later, the retinas were harvested for testing. R28 cells were treated with Glu, Glu+EPO, or Glu+EPO+soluble EPO receptor (sEPOR), respectively, for 24 hours, and then the cells were collected for the following studies. Glutamate level in the retina was measured with a glutamate assay kit. Cell death was determined with TUNEL staining. The changes in glutamine synthetase (GS), glutamate-aspartate transporter (GLAST), ionotropic glutamate receptors (iGluRs), apoptosis-inducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.
  
  
  RESULTS
  In 2-week diabetic rat retinas, Glu concentration was approximately 1.21-fold that in normal control. TUNEL staining demonstrated that retinal cell death was increased. Retinal GS and GLAST expressions were decreased, while the iGluRs, for example, KA1 and NR1, and PAR polymer expression was increased. In R28 cells, 24 hours after Glu (10 mM) treatment, the cell viability was decreased by 52.7%; KA1, NR1, PAR polymer, and nuclear AIF all increased in expression. The above conditions could be largely reversed by EPO both in vivo and in vitro. The protective effect of EPO was abolished by sEPOR.
  
  
  CONCLUSIONS
  Erythropoietin showed a neuroprotective function against Glu-mediated neurotoxicity both in diabetic rat retina and in Glu-treated R28 cells. The neuroprotective mechanisms were largely through maintaining the normal expression of glutamate-glutamine cycle-related proteins and inhibiting AIF translocation and PAR polymer formation.
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.
authors.forename: Limin
authors.initials: L
authors.lastname: Gu
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Second Affiliated Hospital of Soochow University, Suzhou, China.
authors.forename: Guoxu
authors.initials: G
authors.lastname: Xu
authors.affiliation: The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
authors.forename: Debasish
authors.initials: D
authors.lastname: Sinha
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Jing-Ying
authors.initials: JY
authors.lastname: Xu
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Haibin
authors.initials: H
authors.lastname: Tian
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Furong
authors.initials: F
authors.lastname: Gao
authors.affiliation: Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States.
authors.forename: Weiye
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Lu
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China.
authors.forename: Jingfa
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology of Shanghai Tenth People's Hospital and Tongji Eye Institute, Tongji University School of Medicine, Shanghai, China Department of Regenerative Medicine and Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China Institute for Nutritional Sciences, Tongji University School of Medicine, Shanghai, China.
authors.forename: Guo-Tong
authors.initials: GT
authors.lastname: Xu
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000378:metabolism; D000818:Animals; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000188:drug therapy / Q000378:metabolism*; D004195:Disease Models, Animal; D004921:Erythropoietin / Q000502:physiology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000187:drug effects / Q000378:metabolism*; D058449:Intravitreal Injections; D008297:Male; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017467:Receptors, Erythropoietin / Q000378:metabolism; D017470:Receptors, Glutamate / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D051033:Apoptosis Inducing Factor; D018691:Excitatory Amino Acid Antagonists; D017467:Receptors, Erythropoietin; D017470:Receptors, Glutamate; D004921:Erythropoietin; D011064:Poly Adenosine Diphosphate Ribose; D018698:Glutamic Acid; D005974:Glutamate-Ammonia Ligase
keywords: cell death; diabetic retina; erythropoietin; glutamate