losartan-potassium and Retinal-Degeneration

Erythropoietin (Epo) was once considered to be a regulator of erythropoiesis by controlling the apoptosis, proliferation and differentiation of erythroid precursor cells over an extended period of time. However, the expression of Epo and Epo receptor (Epo-R) occurs in the brain and retina in addition to the kidney. These expression behaviors lead to physiological effects in addition to hematocrit elevation. In this review we discuss the protective effect of Epo on retinal cells.

Topics: Animals; Cell Survival; Erythropoietin; Humans; Hyperoxia; Hypoxia; Retinal Degeneration

Reduced tissue oxygenation stabilizes the alpha-subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1). This leads to the induction of a number of hypoxia responsive genes. One of the best known HIF-1 targets is erythropoietin that exerts neuroprotective effects on ischemia-related injury in the brain. Thus, pre-exposure to low environmental oxygen concentrations might be exploited as a preconditioning procedure to protect tissues against a variety of harmful conditions. We present recent work on neuroprotection of retinal photoreceptors induced by hypoxic preconditioning or by systemically elevated levels of Epo in mouse plasma.

Topics: Animals; Brain; Erythropoietin; Hypoxia; Ischemia; Ischemic Preconditioning; Mice; Mice, Transgenic; Neurons; Oxygen; Retina; Retinal Degeneration; Retinal Ganglion Cells; Time Factors; Up-Regulation

Topics: Animals; Apoptosis; DNA-Binding Proteins; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Mice; Nuclear Proteins; Photoreceptor Cells, Vertebrate; Retinal Degeneration; Retinal Ganglion Cells; Transcription Factors; Up-Regulation

This study aimed to investigate the efficacy of human-derived umbilical cord mesenchymal stem cells (HDUMSC) and human-derived umbilical cord mesenchymal stem cells expressing erythropoietin (HDUMSC-EPO) to rescue total degenerated retina in a rat model.. The study included four treatment groups, namely negative control using normal saline (HBSS) injection, positive control using sodium iodide 60 mg/kg (SI), SI treated with HDUMSC, and SI treated with HDUMSC-EPO given via subretinal and intravenous routes, to test the efficacy of retinal regeneration following SI-induced retinal degeneration. Retinal function in both phases was tested via electroretinography (ERG) and histological staining examining the outer nuclear layer (ONL).. There was a statistically significant result (P < 0.05) in the SI treated with HDUMSC-EPO only when comparing day 11 (mean = 23.6 μv), day 18 (mean = 25.2 μv), day 26 (mean = 26.3 μv), and day 32 (mean = 28.2 μv) to the b-wave ERG on day 4 rescue injection day (mean = 12.5 μv). The SI treated with HDUMSC-EPO showed significant improvement in b-wave ERG readings in the Sprague-Dawley (SD) rat but did not restore baseline readings prior to degeneration (day 0). Both treated groups' ONL thicknesses did not show significant changes compared to the negative control group (HBSS) following rescue therapy.. Total retinal degeneration following intravenous SI injection was observed at 60 mg/kg. SI treated with HDUMSC and HDUMSC-EPO showed no regenerative potential compared to baseline in SI-induced total retina degeneration on ERG or histology, whereas SI treated with HDUMSC-EPO group showed a substantial increase in b-wave ERG amplitude over time.

Topics: Animals; Disease Models, Animal; Electroretinography; Erythropoietin; Humans; Mesoderm; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Stem Cells

Retinal degeneration (RD) results in photoreceptor loss and irreversible visual impairments. This study sought to alleviate the photoreceptor degeneration via the adeno-associated virus (AAV)-mediated erythropoietin (EPO) therapy. AAV-2/2-mCMV-EPO vectors were constructed and delivered into the subretinal space of a RD model. The retinal morphology, optokinetic behaviour and electrophysiological function of the treated animals were analysed. The subretinal delivery of AAV-2/2 vectors induced robust EPO gene expressions in the retinas. AAV2/2-mediated EPO therapy ameliorated the photoreceptor degeneration and visual impairments of the RD animal model. Furthermore, the multi-electrodes array (MEA) was used to detect the firing activities of retinal ganglion cells. MEA recording showed that the EPO therapy could restrain the spontaneous firing response, enhance the light-induced firing response and preserve the basic configurations of visual signal pathway in RD model. Our MEA assay provided an example to evaluate the potency of pharmacological compounds on retinal plasticity. In conclusion, AAV2/2-mediated EPO therapy can ameliorate the photoreceptor degeneration and rectify the abnormities in visual signal transmission. These beneficial results suggest the AAV vector is a viable therapeutic option for retinopathies with rapidly degenerating kinetics and lay the groundwork for future development of EPO gene therapy.

Topics: Animals; Cytomegalovirus; Dependovirus; Disease Models, Animal; Erythropoietin; Female; Genetic Therapy; Male; Mice; Mice, Inbred C57BL; Neuroprotection; Photoreceptor Cells; Retina; Retinal Degeneration

Topics: Amino Acid Transport System y+; Animals; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Erythropoietin; Glutathione; Humans; Ischemia; Male; Mice; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinal Vessels

Inherited retinopathies typically lead to photoreceptor loss and severe visual impairments in the subjects. Intranasal administration is an efficient approach to deliver therapeutic agents to the targeted tissue. The present study is designed to deliver the erythropoietin (EPO) into the N-methyl-N-nitrosourea (MNU) induced mice, a pharmacological retinopathy model via intranasal or intravenous route. The mice were then subjected to bioavailability assay and therapeutic effects evaluation. Our results showed that the intranasal delivery of EPO is effective to alleviate the morphological disruptions in the MNU induced mice. The intranasal delivery of EPO also ameliorated the visual impairments in the MNU induced mice. Immunostaining experiment showed that both the M-cone and S-cone populations in the degenerative retinas are rescued by the intranasal delivery of EPO. In particular, the M-cone photoreceptors in dorsal-temporal (DT) quadrant and the S-cone photoreceptors in ventral-nasal (VN) quadrant were preferentially preserved by the intranasal delivery of EPO. Mechanism studies showed that the intranasal delivery of EPO could the modulate apoptosis and restrict oxidation in the degenerative retina. Compared with intravenous delivery, the intranasal delivery led to the significantly higher EPO concentration in the retina. The intranasal delivery resulted in more potent protection and had less erythropoiesis-stimulating activity than the intravenous delivery. Our results suggest that the intranasal administration is a noninvasive and efficient approach to deliver EPO into the retinas. These findings lay the groundwork for further intranasal administration of EPO in ophthalmological practice.

Topics: Administration, Intranasal; Animals; Drug Delivery Systems; Erythropoietin; Female; Male; Mice; Mice, Inbred C57BL; Random Allocation; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration

Retinitis pigmentosa (RP) is a heterogeneous group hereditary retinal disease that is characterized by photoreceptor degeneration. The present study sought to explore the therapeutic effects of erythropoietin (EPO) on the N-methyl-N-nitrosourea (MNU)-induced photoreceptor degeneration. The MNU-administered mouse or normal control received a subretinal injection of EPO (at the dose of 10U). Twenty-four hours after EPO injection, the retinal EPO levels of experimental animals were quantified. Subsequently, the experimental animals were subjected to optokinetic tests, ERG examination, SD-OCT examination, histology assessment, and immunohistochemistry evaluation. The retinal superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and expression levels of several apoptotic factors were also quantified. The subretinal injection of EPO up-regulated the retinal EPO level in the retinas of MNU-administered mice. The optokinetic tests and ERG examination suggested the visual functional impairments in MNU-administered mice were ameliorated after EPO treatment. The SD-OCT and histological examination suggested the morphological devastations in MNU-administered mice were alleviated after EPO treatment. The cone photoreceptors in MNU-administered mice were protected from the MNU-induced detrimental effects. Moreover, the EPO treatment rectified the apoptotic abnormalities in MNU-administered mice, and enhanced the expression level of Foxo3, a critical mediator of autophagy. The EPO treatment also mitigated the MDA concentration and enhanced the retinal SOD activity, thereby counteracting the retinal oxidative stress in MNU administered mice. In ophthalmological practice, the subretinal delivery of EPO is a feasible therapeutic strategy to alleviate photoreceptor degeneration. These findings would enrich our pharmacological knowledge about EPO and shed light on the development of an effective therapy against RP.

Topics: Animals; Apoptosis; Erythropoietin; Methylnitrosourea; Mice; Oxidative Stress; Retina; Retinal Degeneration

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four-week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.

Topics: Animals; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Male; Peptides; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retinal Degeneration

The exogenous delivery of erythropoietin (EPO) and EPO derivatives (EPO-Ds) represents a valuable strategy to protect the retina from degeneration. In this chapter we describe a method to deliver EPO and the EPO derivative S100E in the light-damage model of induced retinal degeneration using adeno--associated viral (AAV) vectors and to evaluate the functional and morphological protection of the retina from light damage.

Topics: Animals; Dependovirus; Erythropoietin; Genetic Vectors; Mice; Photoreceptor Cells; Retinal Degeneration

Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 μm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.

Topics: Animals; Drug Delivery Systems; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Mice; Promoter Regions, Genetic; Retina; Retinal Degeneration

For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cell based gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i.e., rat mesenchymal stem cells (rMSCs) alone, erythropoietin (EPO) gene modified rMSCs (EPO-rMSCs) or doxycycline (DOX) inducible EPO expression rMSCs (Tet-on EPO-rMSCs), were transplanted into the subretinal spaces of SI-treated rats. The rMSCs were prepared for transplantation after 3 to 5 passages or modified with EPO gene. During the 8 weeks after the transplantation, the rats treated with rMSCs alone or with two types of EPO-rMSCs were all monitored with fundus examination, fundus fluorescein angiography (FFA) and electroretinogram. The transplantation efficiency of donor cells was examined for their survival, integration and differentiation. Following the transplantation, labeled donor cells were observed in subretinal space and adopted RPE morphology. EPO concentration in vitreous and retina of SI-treated rats which were transplanted with EPO-rMSCs or Tet-on EPO-rMSCs was markedly increased, in parallel with the improvement of retinal morphology and function. These findings suggest that rMSCs transplantation could be a new therapy for degenerative retinal diseases since it can protect and rescue RPE and retinal neurons, while EPO gene modification to rMSCs could be an even better option.

Topics: Animals; Cell Differentiation; Erythropoietin; Gene Transfer Techniques; Iodates; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration

Topics: Animals; Darbepoetin alfa; Electroretinography; Erythropoietin; Glycosylation; Hematinics; Intravitreal Injections; Light; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Treatment Failure

We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time.. EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate).. The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush).. A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.

Topics: Animals; Apoptosis; Biocompatible Materials; Cell Survival; Cells, Cultured; Delayed-Action Preparations; Dextrans; Disease Models, Animal; Drug Carriers; Drug Compounding; Erythropoietin; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Situ Nick-End Labeling; Lactic Acid; Male; Microspheres; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Ganglion Cells

To mimic hypoxia preconditioning by a novel specific pyruvate treatment and to study its retinal protection against white light damage.. Six-to-eight-week-old BALB/c mice were exposed to strong white light calculated to produce photoreceptor degeneration. Some were given injections of pyruvate in a preordained protocol because evidence exists that proves pyruvate can affect the concentration of hypoxia inducible factor (HIF). Western blotting and real-time PCR were used to determine the concentration of proteins and mRNAs in retinas. Morphology was analyzed with toluidine blue staining and was plotted using a spidergraph. A free nucleosome cell death assay was used to examine apoptosis. Retina explant cultures were used to investigate the background mechanism.. Pyruvate administration stabilized hypoxia inducible factor (HIF)-1α but not HIF-2α. Expression of the downstream genes hemoxygenase-1 and erythropoietin mirrored the changes of the two HIFs, respectively. Importantly, pyruvate given not only before but also after exposure to light protected photoreceptors against apoptosis. In the retinal explant system, addition or depletion of pyruvate caused only changes of HIF-1α and prolyl hydroxylase (PHD)-2, while HIF-2α and PHD1 were not affected. However, under hypoxic conditions, HIF-2α was stabilized by pyruvate but not HIF-1α.. Pyruvate evoked a hypoxia-like response under normoxic conditions and was retina-protective against strong white light. This response included stabilization of HIF-1α but not HIF-2α. This differential stabilization might be related to the distinct preference of their degrading enzyme of PHD2 and PHD1 in response to pyruvate treatment.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Erythropoietin; Heme Oxygenase-1; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Light; Mice; Mice, Inbred BALB C; Photoreceptor Cells, Vertebrate; Procollagen-Proline Dioxygenase; Pyruvic Acid; Radiation Injuries, Experimental; Retinal Degeneration; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Given the high genetic heterogeneity of inherited retinal degenerations (IRDs), a wide applicable treatment would be desirable to halt/slow progressive photoreceptor (PR) cell loss in a mutation-independent manner. In addition to its erythropoietic activity, erythropoietin (EPO) presents neurotrophic characteristics. We have previously shown that adeno-associated viral (AAV) vector-mediated systemic EPO delivery protects from PR degeneration. However, this is associated with an undesired hematocrit increase that could contribute to PR protection. Non-erythropoietic EPO derivatives (EPO-D) are available which allow us to dissect erythropoiesis's role in PR preservation and may be more versatile and safe than EPO as anti-apoptotic agents. We delivered in animal models of light-induced or genetic retinal degeneration either intramuscularly or subretinally AAV vectors encoding EPO or one of the three selected EPO-D: the mutant S100E, the helix A- and B-derived EPO-mimetic peptides. We observed that (i) systemic expression of S100E induces a significantly lower hematocrit increase than EPO and provides similar protection from PR degeneration, and (ii) intraocular expression of EPO-D protects PR from degeneration in the absence of significant hematocrit increase. On the basis of this, we conclude that erythropoiesis is not required for EPO-mediated PR protection. However, the lower efficacy observed when EPO or S100E is expressed intraocularly rather than systemically suggests that hormone systemic effects contribute to PR protection. Unlike S100E, EPO-mimetic peptides preserve PR only when given locally, suggesting that different EPO-D have a different potency or mode of action. In conclusion, our data show that subretinal delivery of AAV vectors encoding EPO-D protects from light-induced and genetic PR degeneration.

Topics: Adaptor Proteins, Signal Transducing; Animals; Dependovirus; Erythropoiesis; Erythropoietin; Gene Transfer Techniques; Genetic Vectors; Hematocrit; Intermediate Filament Proteins; Light; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Models, Animal; Mutation; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Rats; Rats, Inbred Lew; Retinal Degeneration

To observe the effects of a single intravitreal rhEPO injection on light-induced retinal damage in rats and to explore the possible mechanisms.. Male Sprague-Dawley rats were randomly divided into a normal group, a control group (PBS intravitreal injection), and a number of treatment groups (intravitreal rhEPO injection at 0.625, 1.25, 2.5, 5, 10, or 20 U). After dark adaptation for 24 h, rats were exposed to white light (2800 lx) for 5 h. Based on the effects of rhEPO on the ERG-b wave, an optimal dose (5U) was chosen for further experiments that included histopathological examination and outer nuclear layer (ONL) counting at 5 and 10 days after light exposure. Cell apoptosis and proteins (caspase-3, bcl-xL) were also examined at 3 days. Some cell signaling related proteins (AKT, ERK1/2 and STAT5) were also analyzed. In another experiment, rats received intravitreal injection of rhEPO (5U) at 30 min after light exposure. ERG and morphological changes were examined at 3 days after light exposure.. ERG-b wave amplitudes are well preserved in rats receiving 2.5, 5, or 10 U rhEPO, in comparison with the PBS control. The dose-effect relationship was bell-shaped, with the maximum effect at 5 U. Less apoptotic cells were in the ONL in the 5U group. The rats receiving 5 U rhEPO at 30 min after light exposure also had higher b-wave amplitude. Caspase-3 expression was down-regulated and Bcl-xL was up-regulated in the rhEPO group. Phosphorylated ERK(1,2) was reduced in the rhEPO group.. A single intravitreal injection of rhEPO can postpone photoreceptor light damage in rats. The optimal dose was 5 U in this study. The effective time window was from 24 h prior to light exposure to 5 h after. RhEPO may be involved in the regulation of the expression of Caspase-3 and Bcl-xL. The possible cell signal transduction maybe through the ERK1/2 pathway.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Dose-Response Relationship, Drug; Electroretinography; Erythropoietin; In Situ Nick-End Labeling; Intravitreal Injections; Light; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retina; Retinal Degeneration; STAT5 Transcription Factor

Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.. After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 μg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 μg/kg pHBSP or control peptide).. pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.. Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Topics: Animals; Animals, Newborn; Apoptosis; Cytokines; Diabetic Retinopathy; DNA Damage; Erythropoietin; Gene Expression Regulation; Ischemia; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroglia; Peptide Fragments; Protein Interaction Domains and Motifs; Random Allocation; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinal Vessels

The primary objectives of this study were to determine if erythropoietin (EPO) is neuroprotective to the photoreceptors in the retinal degeneration slow (rds) mouse in the absence of an increase in hematocrit and to determine if deglycosylated EPO (DEPO) is less neuroprotective. We performed subretinal injections of 10U EPO, DEPO or hyperglycosylated EPO (HEPO) in postnatal day 7 rds mice. Whole eye EPO levels were quantified by ELISA at specified time points post-injection. TUNEL analysis, hematocrit, and immunohistochemistry were performed at postnatal day 20. Half of the amount of EPO measured immediately after injection was detected less than 1 h later. Twenty four hours later, EPO levels were 1000 times lower than the amount originally detected. Uninjected rds mice contained 36 +/- 2 TUNEL-positive cells/mm retina and PBS injected mice contained 17 +/- 3 TUNEL-positive cells/mm retina. EPO, DEPO, and HEPO treated rds retinas contained 5 +/- 2, 9 +/- 2, and 3 +/- 1 TUNEL-positive cells/mm retina, respectively. The hematocrit was 43% in control and 41% in treated rds mice Previous studies have shown neuroprotection of the retina by treatment with as little as 24-39 mU EPO/mg total protein in the eye. In this study, we detected 40 mU/mg EPO in the eye 11 h after injection of 10 U EPO. Treatment with all forms of EPO tested was neuroprotective to the photoreceptors without a concomitant increase in hematocrit.

Topics: Animals; Animals, Newborn; Apoptosis; Darbepoetin alfa; Dependovirus; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Glycosylation; Green Fluorescent Proteins; Hematocrit; Immunohistochemistry; In Situ Nick-End Labeling; Injections; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Neuroprotective Agents; Peripherins; Photoreceptor Cells, Vertebrate; Protein Processing, Post-Translational; Retinal Degeneration; Time Factors; Transduction, Genetic

Erythropoietin (Epo) had been shown to have a neuroprotective effect independent from its erythropoietic properties. In this study, we tested whether Epo could protect the retina from damage induced by a long period of moderate light insult and how it protected. First, rats were injected intraperitoneally (i.p.) by human recombinant Epo at 5000 or 30,000U/kg to assess Epo concentration in plasma and retina. Second, rats were untreated or injected i.p. with Epo at 30,000U/kg, 1 or 4h before being placed in constant light (24h; 2200lux). Electroretinograms (ERG) were recorded before treatment, 1day and 15days (D15) after light exposure. After the last ERG, eyes were taken for histology. In parallel, we tested Epo protection against oxidative stressors on isolated retinas and its effect on caspase-9 activity. Epo injected at 30,000U/kg body weight, 4h before exposure to the damaging light, protected retinal function and structure against light damage and induced an increase in caspase-9 activity and expression. Epo had no direct or indirect protective effect against free radicals-induced death on isolated retinas. Epo protected the retina from a long period of moderate light exposure through a mechanism independent from a free radical scavenging property or an antioxidant facilitating activity. The activation of caspase-9, 4h after Epo injection, corresponding to the start of light exposure, suggests that caspase-9 plays a role in neuroprotection.

Topics: Animals; Caspase 9; Drug Evaluation, Preclinical; Electroretinography; Enzyme Activation; Erythropoietin; Free Radicals; Light; Neuroprotective Agents; Oxidative Stress; Radiation Injuries; Rats; Rats, Wistar; Recombinant Proteins; Retina; Retinal Degeneration; Tissue Culture Techniques

To study whether recombinant human erythropoietin can pass through mice blood-retina barrier and the protective role in light-induced damage in retina.. After the injection of rHEPO, the content of rHEPO in 24 BALB/c mice retina was examined by enzyme linked immunosorbent assay (ELISA). 24 BALB/c mice were used to establish a light-induced damaged model, the difference of retina in rHEPO group and control group was compared using light microscope and TdT-mediated dUTP nick end labeling (TUNEL).. The amount of retinal rHEPO in four deferent time points was (0.68 +/- 0.24) mU, (1.87 +/- 0.37) mU, (0.96 +/- 0.24) mU, (0.47 +/- 0.13) mU in 100 microg retinal total protein respectively by ELISA assay, there were statistical significances among groups. The density of rHEPO in the retina reached its peak at 4th hour after injection. Histology analysis: rHEPO group, at the 12th hour after light exposure the inner segment became condensed and disorganized. At the 36th hour the retina disorganized and vesiculated were seen in outer segments. At the 72nd hour the inner and outer segments were damaged more seriously and the outer nuclear layer became thinner and denser. On the 7th day, the retinal outer nuclear layer became thinner and condenses. rHEPO group showed a minimal damage in every time points but outer nuclear layer disorganized and vesiculated in inner and outer segments. No obvious changes in retinal thickness. The apoptotic cells were detected by TUNEL. At the 12th hour after light exposure, there were the apoptotic cells in outer nuclear layer near outer plexiform layer. At 36th hour the numbers of apoptotic cells were increased, however at the 72nd it was decreased obviously, only a few scattering apoptotic cells were revealed in the outer nuclear layer. Numbers of apoptotic cells between the rHEPO group and control group in outer nuclear layer were statistical significance (P < 0.01).. rHEPO can pass through the mice blood-retina barrier and rHEPO has neuroprotective effect on mice retina. rHEPO may be used to treat degenerative retinal diseases.

Topics: Animals; Apoptosis; Blood-Retinal Barrier; Erythropoietin; Female; Light; Male; Mice; Photoreceptor Cells, Vertebrate; Recombinant Proteins; Retina; Retinal Degeneration

Elevation of erythropoietin (Epo) concentrations by hypoxic preconditioning or application of recombinant human Epo (huEpo) protects the mouse retina against light-induced degeneration by inhibiting photoreceptor cell apoptosis. Because photoreceptor apoptosis is also the common path to cell loss in retinal dystrophies such as retinitis pigmentosa (RP), we tested whether high levels of huEpo would reduce apoptotic cell death in two mouse models of human RP. We combined the two respective mutant mouse lines with a transgenic line (tg6) that constitutively overexpresses huEpo mainly in neural tissues. Transgenic expression of huEpo caused constitutively high levels of Epo in the retina and protected photoreceptors against light-induced degeneration; however, the presence of high levels of huEpo did not affect the course or the extent of retinal degeneration in a light-independent (rd1) and a light-accelerated (VPP) mouse model of RP. Similarly, repetitive intraperitoneal injections of recombinant huEpo did not protect the retina in the rd1 and the VPP mouse. Lack of neuroprotection by Epo in the two models of inherited retinal degeneration was not caused by adaptational downregulation of Epo receptor. Our results suggest that apoptotic mechanisms during acute, light-induced photoreceptor cell death differ from those in genetically based retinal degeneration. Therapeutic intervention with cell death in inherited retinal degeneration may therefore require different drugs and treatments.

Topics: Animals; Apoptosis; Erythropoietin; Humans; Immunohistochemistry; Light; Mice; Mice, Transgenic; Mutation; Phosphoric Diester Hydrolases; Photoreceptor Cells; Protein Subunits; Recombinant Proteins; Retina; Retinal Degeneration; Rhodopsin

Molecules with neurotrophic activity are being evaluated for treatment of retinitis pigmentosa in animal models. In particular, great interest has been focused recently on erythropoietin (Epo). Evidence of its neurotrophic activity comes mainly from data demonstrating photoreceptor protection in a rodent light-damage model through systemic administration of a recombinant form of this hormone. Our goal was to test whether Epo retinal gene transfer can rescue or delay photoreceptor cell death. We delivered adeno-associated viral vectors encoding Epo intraocularly and, for comparison, intramuscularly to one light-induced and two genetic models of retinal degeneration. Intraocular Epo gene transfer resulted in sustained hormone expression in the eye, which was undetectable systemically. In contrast, Epo intramuscular gene transfer resulted in hormone secretion in the circulation, which was not detected in ocular fluids. The protein secreted from muscle and retina is of the same molecular weight as a commercial recombinant human Epo. Interestingly, following systemic but not intraocular Epo delivery, morphological photoreceptor protection was observed in the light-damage and rds/peripherin (Prph2) models of retinal degeneration. In the light-damage model, the morphological rescue was accompanied by a significant electrophysiological improvement of photoreceptor function. In contrast, no photoreceptor rescue was observed following Epo gene transfer in the rd10 model. This suggests that different apoptotic mechanisms, with varying sensitivities to Epo, occur in different retinal degeneration models. In conclusion, our data support Epo as a neuroprotective agent in some, but not all, retinal degenerations. Further, rescue is observed in specific models after systemic but not intraocular Epo gene transfer.

Topics: Animals; Anterior Chamber; Cell Nucleus; Dependovirus; Electroretinography; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Intermediate Filament Proteins; Light; Membrane Glycoproteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Peripherins; Photoreceptor Cells, Vertebrate; Rats; Rats, Inbred Lew; Retina; Retinal Degeneration

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

Topics: Animals; DNA-Binding Proteins; Electroretinography; Endothelial Growth Factors; Erythropoietin; Fibroblast Growth Factor 2; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Light; Lymphokines; Mice; Mice, Inbred BALB C; Nuclear Proteins; Retinal Degeneration; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors