Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina

Limin Gu, Hua Xu, Fang Wang, Guoxu Xu, Debasish Sinha, Juan Wang, Jing Ying Xu, Haibin Tian, Furong Gao, Weiye Li, Lixia Lu, Jingfa Zhang, Guo Tong Xu

Research output: Contribution to journalArticle

Abstract

PURPOSE. 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.

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.

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, GluþEPO, or GluþEPOþsoluble 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), apoptosisinducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.

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.

Original languageEnglish (US)
Pages (from-to)8208-8222
Number of pages15
JournalInvestigative Ophthalmology and Visual Science
Volume55
Issue number12
DOIs
StatePublished - Dec 15 2014

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Erythropoietin
Retina
Glutamic Acid
Poly Adenosine Diphosphate Ribose
Polymers
Cell Death
Ionotropic Glutamate Receptors
Glutamate-Ammonia Ligase
In Situ Nick-End Labeling
Sprague Dawley Rats
Staining and Labeling
Erythropoietin Receptors
Intravitreal Injections
Streptozocin
Intraperitoneal Injections
Fluorescent Antibody Technique
Cell Survival
Western Blotting
Cell Line
Polymerase Chain Reaction

Keywords

  • Cell death
  • Diabetic retina
  • Erythropoietin
  • Glutamate

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina. / Gu, Limin; Xu, Hua; Wang, Fang; Xu, Guoxu; Sinha, Debasish; Wang, Juan; Xu, Jing Ying; Tian, Haibin; Gao, Furong; Li, Weiye; Lu, Lixia; Zhang, Jingfa; Xu, Guo Tong.

In: Investigative Ophthalmology and Visual Science, Vol. 55, No. 12, 15.12.2014, p. 8208-8222.

Research output: Contribution to journalArticle

Gu, L, Xu, H, Wang, F, Xu, G, Sinha, D, Wang, J, Xu, JY, Tian, H, Gao, F, Li, W, Lu, L, Zhang, J & Xu, GT 2014, 'Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina', Investigative Ophthalmology and Visual Science, vol. 55, no. 12, pp. 8208-8222. https://doi.org/10.1167/iovs.14-14435
Gu, Limin ; Xu, Hua ; Wang, Fang ; Xu, Guoxu ; Sinha, Debasish ; Wang, Juan ; Xu, Jing Ying ; Tian, Haibin ; Gao, Furong ; Li, Weiye ; Lu, Lixia ; Zhang, Jingfa ; Xu, Guo Tong. / Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina. In: Investigative Ophthalmology and Visual Science. 2014 ; Vol. 55, No. 12. pp. 8208-8222.
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abstract = "PURPOSE. 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.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.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{\th}EPO, or Glu{\th}EPO{\th}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), apoptosisinducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.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.",
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T1 - Erythropoietin exerts a neuroprotective function against glutamate neurotoxicity in experimental diabetic retina

AU - Gu, Limin

AU - Xu, Hua

AU - Wang, Fang

AU - Xu, Guoxu

AU - Sinha, Debasish

AU - Wang, Juan

AU - Xu, Jing Ying

AU - Tian, Haibin

AU - Gao, Furong

AU - Li, Weiye

AU - Lu, Lixia

AU - Zhang, Jingfa

AU - Xu, Guo Tong

PY - 2014/12/15

Y1 - 2014/12/15

N2 - PURPOSE. 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.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.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, GluþEPO, or GluþEPOþsoluble 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), apoptosisinducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.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.

AB - PURPOSE. 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.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.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, GluþEPO, or GluþEPOþsoluble 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), apoptosisinducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.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.

KW - Cell death

KW - Diabetic retina

KW - Erythropoietin

KW - Glutamate

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