Intravitreal Injection of Erythropoietin Protects both Retinal Vascular and Neuronal Cells in Early Diabetes
Jingfa Zhang,1,2,3 Yalan Wu,1,2 Ying Jin,2 Fei Ji,1,2 Stephen H. Sinclair,4 Yan Luo,5 Guoxu Xu,6 Luo Lu,1 Wei Dai,1 Myron Yanoff,4 Weiye Li,*,1,2,4,5 and Guo-Tong Xu*,1,2 2008
/ Abstract
PURPOSE. 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.
Diabetic retinopathy (DR) is the leading cause of blindness in patients 20 to 70 years of age.1 The direct cause of DR is still largely unknown. The notion that it is solely a microvascular complication in diabetes has been challenged in recent years.2 3 4 Evidence exists that all classes of cells within the retina are involved in a multiplicity of disease processes in the early stages of diabetes.3 4 The early clinical features of DR, such as breakdown of the blood–retinal barrier (BRB) and various visual deficits support this concept in humans.5 6 7 8 Because of gradual but accelerating deterioration, treatment should be implemented before DR progresses to the irreversible stage. Unfortunately, there are no known treatments specifically for mild to moderate DR.9 10
Growing evidence suggests that erythropoietin (EPO) has both neuroprotective and vascular protective functions.11 12 13 14 EPO and EPO receptor (EpoR) are expressed in the human retina15 and central nervous system. EPO promotes neural outgrowth from retinal ganglion cells in a dose-dependent manner and preserves their survival after axotomy.16 17 18 Hypoxia-induced retinal EPO expression appears to protect retinal neurons from transient global ischemic and reperfusion injury through an antiapoptotic pathway.19 The neurotrophic effect of EPO in the retina extends beyond damage from ischemia and axotomy. Systemic EPO administration protects retinal photoreceptors from light-induced apoptotic pathways in retinal degeneration models through a speculated interaction of EPO with EpoR in the photoreceptor inner segment.20 21 An inhibited production of systemic EPO has been clinically observed in early diabetic nephropathy that results in anemia that is associated with an aggravated course of DR.22 23 Intravenous administration of EPO to treat azotemia-induced anemia in diabetic patients demonstrated a beneficial effect on macular edema and improved visual outcome.24
In human eyes with proliferative diabetic retinopathy (PDR), elevated EPO levels were detected in the vitreous, suggesting that EPO may be produced as an endogenous neuroprotectant against ischemia.25 Meanwhile, a recent report showed that elevated EPO in the vitreous in PDR may act as an independent angiogenic factor leading to hypoxia-induced neovascularization.26 Recently, high vitreous EPO levels were also observed in patients with diabetic macular edema (DME), in which ischemia is not a predominant event.27 Apparently, the biological significance of EPO in the nonischemic retina is not completely understood. The present work was designed to study early diabetic retinas lest it be involved in angiogenic states in PDR. The STZ-induced diabetic rats were used because this animal model represents only cellular processes characteristic of human nonproliferative diabetic retinopathy (NPDR). Particularly, within 8 weeks after diabetes onset, the retina is not ischemic. By using this model, the EPO/EpoR network was studied at both the mRNA and protein levels. The upregulation of EpoR in the retina of STZ-induced diabetic rats has prompted us to explore whether exogenous EPO as a cytoprotectant, could prevent cellular injury in the early diabetic retina. The EpoR-mediated signaling pathway, ERK, was also studied. Since systemic treatment of retinopathy with EPO may be limited by EPO’s erythropoietic and angiogenic properties,20 26 intravitreal injection was the preferred approach. The protective effects of EPO on the BRB and vascular and neuronal cells were clearly demonstrated.
/ Materials and Methods
Reagents
Evans blue (30 mg/mL), streptozotocin (STZ; pH 4.5), and EPO (r-Hu-EPO, 0.1 µg/µL in normal saline, i.e., 153 U/mg) were purchased from Sigma-Aldrich (Beijing Superior Chemicals and Instruments Co., Ltd., Beijing, China). Cell-viability assay and EPO kits (In situ Cell Death Detection Kit and EPO ELISA Kit) were purchased from Roche China, Ltd. (Shanghai, China). Anti-EPO antibody (H-162) and anti-EpoR antibodies (H-194, M-20) were purchased from Santa Cruz Biotechnology (Gene Company Ltd., Shanghai, China). Phospho-STAT5 (Tyr694) antibody, STAT5 antibody, phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody, and p44/42 MAP kinase antibody were purchased from Cell Signaling Technology (Genetimes Technology, Inc. Shanghai, China). Biotinylated goat anti-rabbit IgG (BA1003) and SABC kits were purchased from Boster (Wuhan, China).
RNA Isolation and Determination of Gene Expression
Total RNA was extracted from the retinal samples. The RT product (1 µL) was then amplified by PCR. The specific primers were designed on computer with commercial (Primer Premier ver. 5.0) purchased from Shanghai DNA Biotechnology Corp., Ltd. (Shanghai, China). The primers for EpoR were 5'-CTGGGAGGAAGCGGCGAACT-3' (sense) and 5'-CGGTGGTAGCGAGGAGAT-3' (antisense), and the size of the amplified fragment was 213 bp. The primers for EPO were 5'-CTCCAATCTTTGTGGCATCT-3' (sense) and 5'-GGCTTCGTGACCCTCTGT-3' (antisense), and the size of the amplified fragment was 134 bp. PCR products for β-actin were used as a positive control and internal standard. The primers for β-actin were 5'-GTAAAGACCTCTATGCCAACA-3' (sense) and 5'-GGACTCATCGTACTCCTGCT-3' (antisense). The size of the amplified fragment was 227 bp. Amplification conditions included an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 40 seconds, an extension at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. PCR products were electrophoretically separated on 2% agarose gel in 1x TBE buffer. The optical densities of EPO and EpoR were determined by computer (Quantity One software; Bio-Rad, Hercules, CA). The densitometric values were normalized by β-actin.
Western Immunoblot Analysis for EPO/EpoR, ERK1/2, and STAT5
Individual retinas from experimental and control rats (4 single retinas from 4 rats selected randomly per group) were isolated and homogenized in ice-cold radioimmune precipitation assay (RIPA) buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, and 1% sodium deoxycholate, for Western blot analysis. RIPA buffer enables efficient retinal tissue lysis and protein solubilization while avoiding protein degradation and interference with immunoreactivity. This buffer was supplemented with the protease inhibitor PMSF (Shenergy Bicolor Bioscience Technology Company, Shanghai, China). After 15 minutes’ incubation on ice, the extracts were clarified by centrifugation at 12,000g for 15 minutes at 4°C and stored at –70°C. Protein concentrations were determined by protein assay kit (Bio-Rad). Equal amounts of protein were resolved in SDS-polyacrylamide gels and transferred electrophoretically onto a nitrocellulose membrane (Bio-Rad). The membranes were blocked for 30 minutes: for EPO and EpoR detection with 5% nonfat milk; for ERK detection with 1x gelatin; and for STAT5 detection with 5% BSA, respectively. The membranes after blocking were incubated overnight with anti-EpoR antibody (1:500; M-20; Santa Cruz Biotechnology), anti-EPO antibody (1:500; H-162; Santa Cruz Biotechnology), phospho-STAT5 (Tyr694) antibody (1:1000, Cell Signaling), phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (1:1000; Cell Signaling Technology) or anti-β-actin antibody (1:4000; Sigma-Aldrich). After they were washed with TBST, the membranes were incubated for 1 hour with horseradish peroxidase–conjugated anti-rabbit or anti-mouse antiserum in TBST and 5% nonfat milk. The membranes were washed three times with TBST, and proteins were visualized by enhanced chemiluminescence. After detection with the phospho-specific antibody of STAT5 or ERK, the blot was then stripped and reprobed successively with STAT5 antibody or p44/42 MAP kinase antibody (1:1000; Cell Signaling), and the optical density of each band was determined (Quantity One software; Bio-Rad). The densitometric values for the proteins of interest were normalized for protein loading with β-actin, and the resultant values compared statistically by Student’s t-test.
Immunohistochemistry for EpoR in Retinal Layers
Rats were killed with deep anesthesia. Slash marks were made on an enucleated eye at the 3- and 9-o’clock positions on the limbus for orientation. For preparation of cryostat sections, the eyes were fixed in PBS-buffered 4% paraformaldehyde for 24 hours and then were opened along the ora serrata, and the posterior eyecups were dehydrated through a gradient concentration of sucrose from 10% to 30%. After dehydration, the eyecups were embedded in optimal cutting temperature (OCT) compound (Tissue Tek; Sakura Finetek, Tokyo, Japan) for sectioning. Serial sections (10 µm) were cut on a cryostat microtome. The sections were thawed, washed twice in PBS for 5 minutes, and incubated with 0.3% H2O2 for 30 minutes to block endogenous peroxidase. After the sections were washed with PBS, the sections were incubated with blocking solution (10% normal goat serum in PBS) for 30 minutes at room temperature followed by overnight incubation with polyclonal rabbit anti-EpoR antibody (1:100; H-194:sc-5624; Santa Cruz Biotechnology) diluted in PBS at 4°C. A sample without the primary antibody was used as a negative control. The following day, sections were washed in PBS three times for 5 minutes and incubated with biotinylated goat anti-rabbit IgG (BA1003; Boster) for 20 minutes at room temperature. After washing in PBS, the sections were incubated with avidin-horseradish peroxidase complex (SABC Kit; Boster) for 20 minutes at room temperature. The sections were washed again in PBS, and staining was developed by 3,3'-diaminobenzidine (DAB) without nuclear staining. After reaction was terminated by water and the sections were washed, dehydrated, passed through xylene, and coverslipped. The results were evaluated by light microscopy (LM).
Experimental Animals and Intravitreal EPO Treatment
Male Sprague-Dawley rats of 150 g body weight (BW; Slaccas, SIBS, Shanghai, China) were used. The animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For diabetes induction, a single STZ intraperitoneal injection (60 mg/kg BW in citric buffer) was performed after the rats had been fasted for 24 hours. The control rats received an equal volume of citric buffer. All animals were maintained in a 12-hour alternating light–dark cycle, and allowed to eat and drink ad libitum. Animals receiving STZ were declared diabetic when their blood glucose exceeded 250 mg/dL for three consecutive days. The rats were excluded from the experiment if they failed to develop diabetes. The diabetic rats were randomly divided into two groups: EPO treated and nontreated. Their BW was recorded twice a week, and 4 units of NPH insulin were administered subcutaneously once a week to prevent ketosis. The rats were killed at 1, 2, 4, and 6 weeks after the onset of diabetes. Intravitreal injection of EPO was performed within 2 hours after administration of STZ, with a 30-gauge, 0.5-in. needle (BD Biosciences, Franklin Lakes, NJ) on a microsyringe (Hamilton, Reno, NV), using a temporal approach, 2 mm posterior and parallel to the limbus. EPO, ranging from 0.05 to 200 ng per eye, was dissolved in an equal volume of 2 µL. Sham injections (2 µL normal saline) were performed to both nondiabetic control rats as well as the untreated diabetic rats. The rats recovered spontaneously from the anesthesia and then were sent back to the animal room with food and water ad libitum.
Examination of BRB Permeability
BRB permeability was evaluated according to the method of Xu et al.28 with some modifications. Briefly, the rats were anesthetized with intraperitoneal 2% pentobarbital sodium (50 mg/kg BW). The left iliac artery and vein were cannulated with a catheter (Insyte; BD Biosciences). The Evans blue solution (30 mg/mL) was injected through the left iliac vein over 10 seconds. After 2 minutes, 0.1 mL blood was drawn from the left iliac artery. An equal volume of the blood was then drawn at 15-minute intervals up to 2 hours after the injection to obtain the time-averaged Evans blue plasma concentration. After the dye had circulated for 2 hours, the chest cavity was opened, and rats were perfused via left ventricle with 1% paraformaldehyde in 0.05 M citric acid (pH 3.5) at 37°C. Immediately after the perfusion, both eyes were enucleated. The retina was carefully separated and dried at 37°C (Speed-Vac; GMI, Ramsey, MN). The two retinas from the same animal were pooled. After the dry weight was determined, the retinas were incubated in 300 µL formamide for 18 hours at 70°C. The extract was centrifuged through a 30,000 NMWL (nominal molecular weight limit) centrifuge filter (Microcon; Millipore, Bedford, MA) at 3,000g, 4°C, for 45 minutes. The volume of the filtrate was measured. The blood samples were centrifuged at 10,000g and diluted to 1:5,000. The samples (60 µL) were used for triplicate measurements with a spectrophotometer (DU800; Beckman, Fullerton, CA) at a 5-second interval. A background-subtracted absorbance was determined by measuring each sample at both 620 nm, the maximum absorbance for Evans blue, and 740 nm, the wavelength of minimum absorbance. The concentration of the dye in the blood samples and in the retinal extracts was calculated from a standard curve of Evans blue in formamide. The result of Evans blue permeation (EBP) of the retina, as a function of BRB permeability, was calculated with the following equation and expressed in (µL plasma x g retinal dry wt–1 · h–1).