Intravitreal injection of erythropoietin protects both retinal vascular and neuronal cells in early diabetes.
OBJECTIVE
To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes.


METHODS
Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC).


RESULTS
In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO.


CONCLUSIONS
These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR.
Zhang
Jingfa
J
Laboratory of Clinical Visual Sciences, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Wu
Yalan
Y
Jin
Ying
Y
Ji
Fei
F
Sinclair
Stephen H
SH
Luo
Yan
Y
Xu
Guoxu
G
Lu
Luo
L
Dai
Wei
W
Yanoff
Myron
M
Li
Weiye
W
Xu
Guo-Tong
GT
D000818:Animals; D017209:Apoptosis; D001813:Blood-Retinal Barrier / Q000187:drug effects; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D003921:Diabetes Mellitus, Experimental / Q000150:complications; D003930:Diabetic Retinopathy / Q000209:etiology / Q000378:metabolism / Q000517:prevention & control*; D004921:Erythropoietin / Q000008:administration & dosage* / Q000378:metabolism; D005070:Evans Blue / Q000378:metabolism; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D007267:Injections; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017467:Receptors, Erythropoietin / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor / Q000378:metabolism; D014822:Vitreous Body
D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; D004921:Erythropoietin; D005070:Evans Blue; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3