The erythropoietin receptor is not required for the development, function, and aging of rods and cells in the retinal periphery

Animals and genotyping

All animal experiments were conducted according to the regulations of the Cantonal Veterinary Authority of Zurich and the statement of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and vision research. All mice were kept at the animal facility of the University Hospital Zurich in a 12 h:12 h light-dark cycle with food and water ad libitum. Light was maintained at 60 lux at cage level during the light period. Mice were euthanized by CO₂ inhalation followed by cervical dislocation. All experiments were conducted with a minimum of n = 3 mice for each time point and condition.

Conditional EpoR knockdowns were generated by breeding EpoR^flox/flox mice [23] carrying loxP sites in exons 1 and 4 of EpoR either to mice expressing Cre recombinase under control of the α-element of the Pax6 promoter (α-Cre) [28] resulting in EpoR^flox/flox;α-Cre double mutant animals or to mice expressing Cre recombinase under control of the rhodopsin promoter (Opn-Cre) [29] resulting in EpoR^flox/flox;Opn-Cre double mutant mice. EpoR^flox/flox littermates were used as controls. All mice were on a mixed BL/6;129S6 background and homozygous for the Rpe65^450Leu allele. C57BL/6 mice were used to analyze postnatal gene expression. To investigate the spatial expression pattern of Cre recombinase in the α-Cre retina, α-Cre mice were crossed to Ai6 reporter mice (stock number: 007,906, Jackson Laboratory, Bar Harbor, ME) that express ZSGREEN upon CRE-mediated recombination of a floxed STOP cassette [30]. The retinas were cryosectioned and analyzed for native ZSGREEN expression with fluorescent microscopy. Spatial activity of CRE recombinase in the Opn-Cre mice has been previously reported [31]. The mice were genotyped using genomic DNA isolated from ear biopsies with conventional PCR using the following conditions: initial denaturation (95 °C, 5 min); 35 cycles of denaturation (95 °C, 45 s), annealing (temperature see Table 1, 45 s) and elongation (72 °C, 45 s); and final extension (72 °C, 10 min). Primer pairs and annealing conditions are shown in Table 1. PCR products were run on a 1.5% agarose gel for size detection.

RNA isolation, cDNA synthesis, and semiquantitative real-time PCR

Retinas were isolated through a corneal incision and immediately snap frozen in liquid nitrogen. Total RNA was isolated using the RNeasy isolation kit (RNeasy, catalogue number: 74,104; Qiagen, Hilden, Germany) or the High Pure RNA isolation kit (catalogue number: 11,828,665,001; Roche Diagnostics, Basel, Switzerland). Residual genomic DNA was removed by an incubation step with DNase. RNA (650 ng or 1000 ng) was reverse transcribed using oligo(dT) and M-MLV reverse transcriptase (catalogue number: M1701; Promega, Madison, WI). Ten nanograms of cDNA were used for gene expression analysis with semiquantitative real-time PCR using a LightCycler 480 instrument (Roche Diagnostics), the LightCycler 480 SYBR Green I Master kit (catalogue number: 04,887,352,001; Roche Diagnostics), and specific primer pairs (Table 2). The primer pairs were designed to span large intronic sequences or to cover exon-exon boundaries. Gene expression was normalized to actin beta (Actb), and relative quantification was calculated using the comparative threshold method (ΔΔC_T) and Light Cycler 480 software (Roche Diagnostics).

Histology and light microscopy

To analyze retinal morphology with light microscopy, the eyes were enucleated and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4 °C overnight. After fixation, the cornea and the lens were removed, and the eyecup was separated into a superior half and an inferior half by cutting through the optic nerve head. Trimmed tissue was washed in cacodylate buffer, contrasted with osmium tetroxide (1%, 1 h, room temperature), dehydrated by incubation in increasing ethanol concentrations, and embedded in Epon 812. Semithin sections (0.5 μm) were prepared and counterstained with toluidine blue. An Axioplan digitalized microscope (Zeiss Meditec, Jena, Germany) was used to examine the slides. Retinal thickness from the nerve fiber layer to the RPE was measured at fixed distances from the optic nerve head (ONH) using ImageJ (software version 1.43; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available NIH).

Immunofluorescence on retinal cryosections

For immunofluorescence, the eyes were enucleated and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. The cornea and the lens were removed, and the eyecups were incubated in 30% sucrose in phosphate buffered saline (PBS; 1X; 137 mM NaCl, 2.7 mM KCl, 81 mM Na₂HPO₄, 19 mM NaH₂PO₄, pH 7.4) for cryoprotection until the tissue sunk to the bottom of the tube. The tissue was then embedded in tissue cryoprotective medium (catalogue number: 14,020,108,926 Leica Microsystems Nussloch, Nussloch, Germany) and rapidly frozen in a 2-methylbutane bath cooled with liquid nitrogen. Twelve-micrometer-thick sections were cut through the optic nerve head on a Cryostat (Leica), air-dried, and stored at −80 °C until further use. Sections on slides were incubated with blocking solution (3% horse serum in PBS/0.3% Triton X-100) for 1 h at room temperature. Primary antibodies were applied in blocking solution overnight at 4 °C (Table 3). The slides were washed three times with PBS and incubated with Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResesarch, Soham, UK) in blocking solution (dilution 1:500) for 1 h at room temperature. To stain the blood vessels, cryosections were incubated overnight at 4 °C with G. simplicifolia isolectin IB₄-Alexa 594 (1:200; catalogue number: I21413; Invitrogen, Basel, Switzerland) in blocking solution. After three washing steps in PBS, the cell nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and the slides were mounted with antifade medium (10% Mowiol 4–88 (vol/vol); Calbiochem, San Diego, CA) in 100 mM Tris (pH 8.5), 25% glycerol (wt/vol), and 0.1% 1,4-diazabicyclo [2.2.2] octane [DABCO]). Immunofluorescently labeled proteins were visualized using an Axioplan fluorescence microscope (Zeiss).

Staining of blood vessels on retinal flatmounts

Eyes were enucleated and incubated in 2% PFA in PBS for 5 min. Subsequently, the cornea and the lens were removed, and the retina was carefully separated from the eyecup. The retina was cut into a “clover-leaf” shape, fixed in methanol (−20 °C), and post-fixed in 4% PFA in PBS for 10 min. The flatmounted retinas were washed briefly with PBS and placed in blocking solution (1% fetal calf serum, 0.1% Triton X-100 in PBS) for 1 h. The retinas were incubated overnight at 4 °C with G. simplicifolia isolectin IB₄-Alexa 594 (1:100, catalogue number: I21413; Invitrogen) in blocking solution. The retinal flatmounts were washed with PBS and mounted using antifade medium (see above). Imaging was performed using a digitalized light microscope (Axiovision, Zeiss).

Fluorescein angiography

The mice pupils were dilated with 1% Cyclogyl (Alcon, Cham, Switzerland) and 5% phenylephrine (Ciba Vision, Niederwangen, Switzerland) 1 h before angiography. Mice were anaesthetized with 2.2 µl/g bodyweight of a ketamine/xylazine mix (51/6 mix, subcutaneous injection) 5–10 min before angiography. Eyes were kept moisturized with hydroxypropylmethylcellulose (Methocel 2%, Omnivision, Puchheim, Germany). For fluorescein angiography, 50 µl of 0.2% fluorescein solution (AK Fluor, Akorn, Lake Forest, IL) were injected intraperitoneally, and the retinal vasculature was analyzed with the Micron III retinal imaging system (Phoenix Research Labs, Pleasanton, CA).

Electroretinography

Electroretinograms (ERGs) were recorded according to a previously described procedure [32,33]. The ERG equipment consisted of a Ganzfeld bowl, a direct current amplifier, and a PC-based control and recording unit (LKC UTAS-E 3000, LKC Technologies, Hellendorn, the Netherlands). Mice were dark adapted overnight and anesthetized with ketamine (66.7 mg/kg bodyweight) and xylazine (11.7 mg/kg bodyweight). The pupils were dilated, and single-flash ERG responses were obtained under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was accomplished with a background illumination of 30 cd/m² starting 5 min before recording. Single white-flash stimulus intensity ranged from –3.7 to 1.9 log cd · s/m² under scotopic and from –0.6 to 2.9 log cd · s/m² under photopic conditions, divided into ten and eight steps, respectively. Ten responses were averaged with an interstimulus interval (ISI) of either 4.95 s (for –3.7, –3, –2.6, –2, −1.6, –1, –0.6, −0.02, 0.4, and 0.9 log (cd · s/m²)) or 16.95 s (for 1.4, 1.9, 2.4, and 2.9 log (cd · s/m²)).

Laser capture microdissection

The eyes were enucleated, immediately embedded in tissue freezing medium (catalogue number: 14,020,108,926; Leica Microsystems Nussloch), and frozen in a 2-methylbutane bath cooled with liquid nitrogen. Retinal sections (20 μm) were fixed (5 min in acetone), air dried (5 min), and dehydrated (30 s in 100% ethanol, 5 min in xylene). Microdissection was performed using an Arcturus XT Laser capture device (Molecular Devices, Sunnyvale, CA). RNA was isolated using the Arcturus kit for RNA isolation (catalogue number: KIT0204; Molecular Devices) according to the manufacturer's directions including a DNase treatment to digest residual genomic DNA. Equal amounts of RNA were used for reverse transcription using oligo(dT) and M-MLV reverse transcriptase (Promega).

Hypoxic exposure

The mice were exposed to reduced oxygen levels in a hypoxic chamber (In Vivo cabinet Model 30; Coy Laboratory Products, Grass Lake, MI) equipped with an oxygen controller (Coy Laboratory Products). During hypoxia, mice had access to food and water ad libitum. By altering the O₂:N₂ ratio, oxygen levels were reduced to the desired concentration in 2% steps over 1 h. Mice were kept at 7% O₂ for 6 h. To analyze the gene and protein expression, the retinas were isolated immediately after the hypoxic period and processed for further analysis. To investigate the retinal morphology, the eyes were enucleated 12 days after hypoxic exposure.

Statistical analysis

Statistical analysis was performed using Prism 4.0 software (GraphPad, San Diego, CA). All data are presented as mean ± standard deviation (SD). The number of samples (n) used for individual experiments is given in the figure legends. Statistical differences of means were calculated using the Student t test. Differences with p values below 0.05 were considered significant.

Expression of Epo and EpoR isoforms in the retina

EpoR expression is elevated in the embryonic brain and decreases into adulthood [22]. A similar situation may be present in the retina. Thus, we analyzed the expression profile of Epo and EpoR in the newborn and adult retinas of C57BL/6 wild-type mice with semiquantitative real-time PCR. Epo was expressed in the newborn retina as early as P3 (Figure 1A). Starting at P10, the expression levels increased by about two- to threefold, reaching more or less steady-state levels in the adult retina (Figure 1A). Similarly, the mRNA levels of EpoR were maintained at moderately low levels up to P10, the time point when expression increased by sevenfold to reach a 14-fold increase at 6 months of age (Figure 1B). The EpoR gene can be expressed through alternative splicing as a membrane-bound (referred to as “EpoR” in the text) and a soluble isoform (soluble EpoR (sEpoR)), which encodes for the extracellular domain of EPOR [34-36]. The expression of this isoform has not yet been reported in the retina. To measure the expression of EpoR and sEpoR, we designed primers specific to each isoform (Appendix 1). Interestingly, the expression profile of sEpoR was similar to the membrane-bound isoform; that is, it was detectable at low levels up to P10, the time point when expression increased about tenfold and reached approximately 20-fold higher levels in the adult retina (Figure 1C). The EPOR not only forms homo-oligomers but has also been suggested to form hetero-oligomers with the beta common receptor (Csf2rb (βCR)) [37,38]. In contrast to EpoR, expression of Csf2rb (βCR) remained at steady-state levels in the retina throughout the analyzed period (Figure 1D).

In addition to elucidating the temporal expression profile of EpoR, we also analyzed the spatial expression pattern in the retinal tissue to test EpoR expression in specific retinal layers. For this purpose, we isolated individual layers (the outer nuclear layer [ONL], the inner nuclear layer [INL], the ganglion cell layer [GCL]) of 12-week-old EpoR^flox(flox mice by laser capture microdissection. Analysis of the expression of marker genes for the individual retinal layers showed high purity of the isolated tissue with only a little cross contamination (Appendix 2). EpoR transcripts were detected mainly in the GCL and the INL, with markedly lower levels in the ONL, thus suggesting low expression of EpoR in photoreceptors (Figure 2).

Knockdown of EpoR in rod photoreceptors or in cells of the retinal periphery has no effect on their development, function, and survival

To explore the possible functions of endogenous EPO-EPOR signaling in the retina, we generated two mouse strains bearing a knockdown of EpoR in specific retinal cell populations. We ablated loxP-flanked exons 1–4 of the EpoR genomic sequence either in rod photoreceptors, by generating EpoR^flox/flox;Opn-Cre mice, or in an assorted population of neurons and glia of the retinal periphery by generating EpoR^flox/flox;α-Cre mice. Note that the lack of exons 1–4 affects the expression of not only EpoR but also sEpoR (Appendix 1).

By using reporter mouse lines [30,39], we assessed the specificity and localization of CRE-mediated recombination. Although Opn-Cre-mediated recombination was specific for the ONL, expression of CRE was patchy and detected in approximately 40% of the total rod population (qualitative estimation) as previously reported [31]. CRE-mediated recombination in the α-Cre mouse was detected in a mixed cell population of the retinal periphery and a smaller subset of cells in the central retina (Appendix 3 [40]). Immunostaining with antibodies specific for different retinal cells suggested CRE activity in the Müller glia cells, rod photoreceptors, and horizontal, amacrine, and a subset of ganglion cells (Appendix 3). Whereas in Opn-Cre mice, recombination of floxed sequences was maximal only around 10 weeks of age (not shown), CRE activity in α-Cre mice showed early onset at E10.5 [41].

At the genomic level, Opn-Cre and α-Cre successfully deleted loxP-flanked exons 1–4 in the EpoR gene as evidenced by the appearance of a 220 bp fragment (Figure 3A) that was amplified from recombined genomic DNA when the respective specific primers for PCR were used (Table 1) [23]. To assess the knockdown at the mRNA level, we performed semiquantitative real-time PCR on the total retinal RNA. A statistically significant (p<0.05) 35% decrease in EpoR mRNA expression was measured in the retinas of the EpoR^flox/flox;α-Cre mice (Figure 3B). However, the decrease in EpoR mRNA was not significant in the EpoR^flox/flox;Opn-Cre mice at P84. This was unexpected, but since the total retinal RNA was used for analysis, the effect of a cell-specific knockout may have been masked by cells that did not undergo recombination, especially if those cells (e.g., in the INL and the GCL) expressed the gene at much higher levels (Figure 2). Consequently, we isolated RNA from individual retinal nuclear layers (the ONL, INL, GCL) separated by laser capture microdissection. Amplification of specific marker genes for each nuclear layer suggested high purity of the isolated layers (Appendix 2). Gene expression analysis showed an EpoR expression level in the ONL of EpoR^flox/flox;Opn-Cre mice that was reduced by about 35% compared to the control littermates, even though the knockdown did not reach statistical significance (Figure 3C). This efficiency of EpoR knockdown was similar to the fraction of photoreceptors that express Cre recombinase in the Opn-Cre mouse retina [29,31]. Although the knockdown efficiency was not high when calculated on the level of the total retina or the ONL (cells without active CRE still express EpoR), most Cre-expressing cells (e.g., 30–40% of rods) should have recombined floxed sequences on both alleles leading to many cells harboring a complete knockout and therefore lacking the EPOR protein. Due to the lack of reliable antibodies [42,43], the EPOR protein levels and tissue distribution were not analyzed with western blotting or immunofluorescence.

The absence of EPOR results in increased apoptosis of NPCs and thus affects brain development [22]. Since α-Cre mice recombine floxed sequences in embryonic retinal progenitor cells, the absence of or reduced levels of EPOR may lead to developmental defects and thus to morphological alterations in the adult retina. However, histological analysis did not reveal tangible signs of developmental defects, and normal morphology was observed up to 1 year of age in the retinal periphery of the EpoR^flox/flox;α-Cre mice (Figure 4A). Similarly, the retina and in particular the photoreceptors of the EpoR^flox/flox;Opn-Cre mice showed normal morphology up to 1 year of age (Figure 4A). Analysis of the expression of rhodopsin (Rho), guanine nucleotide binding protein, alpha transducin 2 (Gnat2), visual system homeobox 2 Vsx2 (Chx10), and POU domain, class 4, transcription factor 1 Pou4f1 (Brn3a) indicated that the number of rods, cones, bipolar cells, and RGCs, respectively, was not affected in the EpoR knockdown mice up to 7.5 months of age (Figure 4B,C). The absence of retinal degeneration in both EpoR knockdown strains was confirmed with morphometric analysis of retinal thickness in 7.5-month-old mice (Figure 4D). Furthermore, ERG recordings pointed to normal retinal function (Figure 5A,B). Neither the EpoR^flox/flox;Opn-Cre mice nor the EpoR^flox/flox;α-Cre mice showed significant changes in a- and b-wave amplitudes under scotopic or photopic conditions when compared to the control littermates (Figure 5C–E). Taken together, these results demonstrate that expression of EpoR was not necessary for the correct maturation and function of rod photoreceptors and cells in the retinal periphery. In addition, long-term survival of the retinal neurons, including photoreceptors, was not impaired by the absence of EPOR in our knockdown mice.

Lack of the erythropoietin receptor has no influence on retinal cell viability in acute hypoxia

The induction of Epo expression under hypoxic conditions suggested that this cytokine may be particularly important for preserving cell viability when oxygen is limited [9,44]. In fact, EPO was shown to support neuronal survival against hypoxia-induced cell death in vitro [45], and in an in vitro model of cerebral ischemia [46]. These findings have been corroborated in vivo in a model of brain ischemia/hypoxia [47]. In line with this notion, neural cells lacking EpoR showed increased sensitivity to hypoxia in vitro [22]. To investigate the ability of EPOR to mediate the resistance of retinal cells against hypoxia, we exposed 12-week-old EpoR knockdown mice to hypoxia (7% O₂; 6 h) and analyzed the retinal morphology 12 days after the hypoxic exposure to detect potential signs of tissue damage. Qualitative investigation of retinal morphology did not reveal signs of cell loss upon hypoxic exposure in both strains of EpoR knockdown mice when compared to the control littermates (Figure 6A, compare to Figure 4A for the retinal morphology of normoxic mice). These qualitative findings were confirmed with morphometric measurements of the retinal thickness, which was comparable to that of the normoxic mice (Figure 6B, compare to Figure 4D for retinal thickness of normoxic mice). Thus, ablation of EpoR in the retinal periphery or in rod photoreceptors does not increase retinal susceptibility to acute hypoxia.

Lack of EpoR may alter erythropoietin-erythropoietin receptor signaling in the hypoxic retina

EPOR downstream signaling activates several signal transduction pathways. They may ultimately modulate the transcription of target genes involved in various biologic processes, including cell growth or apoptosis [48]. Genes shown to be influenced by EPO signaling include the antiapoptotic genes B-cell lymphoma 2 (Bcl2) and Bcl-2-like 1 (Bcl2l1 [Bcl_XL]) [49,50], the proapoptotic genes caspase 1 (Casp1) [19] and apoptotic protease activating-factor 1 (Apaf1) [51,52], the neurotrophic factor brain-derived neurotrophic factor (Bdnf) [53], and the gliotic response gene glial fibrillary acidic protein (Gfap) [54].

To test the influence of EpoR knockdowns on EPO-EPOR signaling in the retina, we exposed mice at P84 to a short period of hypoxia (7% O₂, 6 h) and analyzed the gene expression immediately thereafter (Figure 7). As expected, expression of Epo was increased in all mouse retinas after this treatment. Expression of EpoR was not significantly affected after hypoxia in both knockdown strains. CRE-mediated deletion of exons 1–4 of the EpoR gene prevented expression of not only EpoR but also sEpoR (Appendix 1) [34-36,55]. The expression levels of this isoform were not reduced in the EpoR^flox/flox;Opn-Cre retinas when the total retinal RNA was measured. However, sEpoR transcripts were significantly (p<0.001) reduced by approximately two thirds in the retinas of the EpoR^flox/flox;α-Cre mice at P84. In both knockdown mice strains, hypoxia did not influence the expression of sEpoR. Importantly, knockdown of EpoR did not lead to compensatory upregulation of Csf2rb (βCR). Whereas expression of Bcl2l1 and Bcl2 was not affected by the lack of EPOR, Apaf1, Casp1, and Gfap were downregulated and the prosurvival factor Bdnf was upregulated specifically in the hypoxic retinas (and thus in the presence of the increased EPO) of EpoR^flox/flox;α-Cre mice (Figure 7B). This indicates that lack of EPOR influenced the gene expression profile in the hypoxic retinas in ways that suggested reduced expression of stress-related genes and increased expression of prosurvival genes. These results were unexpected since EPO-EPOR signaling is generally accepted to be prosurvival, and thus, lack of EPOR should reduce such signaling (see the Discussion section). In contrast to the EpoR^flox/flox;α-Cre mice, the lack of EPOR in the rods of the EpoR^flox/flox;Opn-Cre mice did not lead to detectable changes in the gene expression profile (Figure 7A), suggesting that EPOR signaling may affect the inner retinal layers rather than the photoreceptor layer.

Ablation of EpoR does not inhibit angiogenesis

Since evidence suggests that EPOR signaling is involved in angiogenesis [56], we investigated whether ablation of EPOR in retinal progenitors during development might affect retinal vascular development. Using retinal flatmounts and cross-sections, we did not detect any overt vascular abnormalities in the EpoR^flox/flox;α-Cre mice. In particular, the capillary coverage in the retinal periphery was not decreased (Figure 8A), and all three retinal capillary plexi developed properly at their correct location (Figure 8B). Perfusion and stability of the retinal vasculature were also not grossly affected as revealed with fluorescein angiography and the absence of signs of vascular leakage (Figure 8C). As expected, ablation of EPOR in mature rod photoreceptors did not lead to any detectable differences from the wild-type (Figure 8A–C).