The effect of GLP-1 receptor agonist lixisenatide on experimental diabetic retinopathy.

AIMS: Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC-MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. RESULTS: Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. CONCLUSIONS: Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.

Differences in junction-associated gene expression changes in three rat models of diabetic retinopathy with similar neurovascular phenotype.

Diabetic retinopathy, also defined as microvascular complication of diabetes mellitus, affects the entire neurovascular unit with specific aberrations in every compartment. Neurodegeneration, glial activation and vasoregression are observed consistently in models of diabetic retinopathy. However, the order and the severity of these aberrations varies in different models, which is also true in patients. In this study, we analysed rat models of diabetic retinopathy with similar phenotypes to identify key differences in the pathogenesis. For this, we focussed on intercellular junction-associated gene expression, which are important for the communication and homeostasis within the neurovascular unit. Streptozotocin-injected diabetic Wistar rats, methylglyoxal supplemented Wistar rats and polycystin-2 transgenic (PKD) rats were analysed for neuroretinal function, vasoregression and retinal expression of junction-associated proteins. In all three models, neuroretinal impairment and vasoregression were observed, but gene expression profiling of junction-associated proteins demonstrated nearly no overlap between the three models. However, the differently expressed genes were from the main classes of claudins, connexins and integrins in all models. Changes in Rcor1 expression in diabetic rats and Egr1 expression in PKD rats confirmed the differences in upstream transcription factor level between the models. In PKD rats, a possible role for miRNA regulation was observed, indicated by an upregulation of miR-26b-5p, miR-122-5p and miR-300-3p, which was not observed in the other models. In silico allocation of connexins revealed not only differences in regulated subtypes, but also in affected retinal cell types, as well as connexin specific upstream regulators Sox7 and miR-92a-3p. In this study, we demonstrate that, despite their similar phenotype, models for diabetic retinopathy exhibit significant differences in their pathogenic pathways and primarily affected cell types. These results underline the importance for more sensitive diagnostic tools to identify pathogenic clusters in patients as the next step towards a desperately needed personalized therapy.

Glucosamine protects against neuronal but not vascular damage in experimental diabetic retinopathy.

OBJECTIVE: Glucosamine, an intermetabolite of the hexosamine biosynthesis pathway (HBP), is a widely used nutritional supplement in osteoarthritis patients, a subset of whom also suffer from diabetes. HBP is activated in diabetic retinopathy (DR). The aim of this study is to investigate the yet unclear effects of glucosamine on DR. METHODS: In this study, we tested the effect of glucosamine on vascular and neuronal pathology in a mouse model of streptozotocin-induced DR in vivo and on cultured endothelial and Müller cells to elucidate the underlying mechanisms of action in vitro. RESULTS: Glucosamine did not alter the blood glucose or HbA1c levels in the animals, but induced body weight gain in the non-diabetic animals. Interestingly, the impaired neuronal function in diabetic animals could be prevented by glucosamine treatment. Correspondingly, the activation of Müller cells was prevented in the retina as well as in cell culture. Conversely, glucosamine administration in the normal retina damaged the retinal vasculature by increasing pericyte loss and acellular capillary formation, likely by interfering with endothelial survival signals as seen in vitro in cultured endothelial cells. Nevertheless, under diabetic conditions, no further increase in the detrimental effects were observed. CONCLUSIONS: In conclusion, the effects of glucosamine supplementation in the retina appear to be a double-edged sword: neuronal protection in the diabetic retina and vascular damage in the normal retina. Thus, glucosamine supplementation in osteoarthritis patients with or without diabetes should be taken with care.

Microglial Activation Is Associated With Vasoprotection in a Rat Model of Inflammatory Retinal Vasoregression.

Vascular dysfunction and vasoregression are hallmarks of a variety of inflammatory central nervous system disorders and inflammation-related retinal diseases like diabetic retinopathy. Activation of microglia and the humoral innate immune system are contributing factors. Anti-inflammatory approaches have been proposed as therapies for neurovascular diseases, which include the modulation of microglial activation. The present study aimed at investigating the effects of microglial activation by clodronate-coated liposomes on vasoregression in a model of retinal degeneration. Clodronate treatment over 5 weeks led to an increase in activated CD74+ microglia and completely prevented acellular capillaries and pericyte loss. Gene expression analyses indicated that vasoprotection was due to the induction of vasoprotective factors such as Egr1, Stat3, and Ahr while expression of pro-inflammatory genes remained unchanged. We concluded that activated microglia led to a shift toward induction of pleiotropic protective pathways supporting vasoprotection in neurovascular retinal diseases.

Protective effect of Soluble Epoxide Hydrolase Inhibition in Retinal Vasculopathy associated with Polycystic Kidney Disease.

Rationale: Vasoregression secondary to glial activation develops in various retinal diseases, including retinal degeneration and diabetic retinopathy. Photoreceptor degeneration and subsequent retinal vasoregression, characterized by pericyte loss and acellular capillary formation in the absence diabetes, are also seen in transgenic rats expressing the polycystic kidney disease (PKD) gene. Activated Müller glia contributes to retinal vasodegeneration, at least in part via the expression of the soluble epoxide hydrolase (sEH). Given that an increase in sEH expression triggered vascular destabilization in diabetes, and that vasoregression is similar in diabetic mice and PKD rats, the aim of the present study was to determine whether sEH inhibition could prevent retinal vasoregression in the PKD rat. Methods: One-month old male homozygous transgenic PKD rats were randomly allocated to receive vehicle or a sEH inhibitor (sEH-I; Sar5399, 30 mg/kg) for four weeks. Wild-type Sprague-Dawley (SD) littermates received vehicle as controls. Retinal sEH expression and activity were measured by Western blotting and LC-MS, and vasoregression was quantified in retinal digestion preparations. Microglial activation and immune response cytokines were assessed by immunofluorescence and quantitative PCR, respectively. 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) mediated Notch signaling, microglial activation and migration were assessed in vivo and in vitro. Results: This study demonstrates that sEH expression and activity were increased in PKD retinae, which led to elevated production of 19,20-DHDP and the depression of Notch signaling. The latter changes elicited pericyte loss and the recruitment of CD11b+/CD74+ microglia to the perivascular region. Microglial activation increased the expression of immune-response cytokines, and reduced levels of Notch3 and delta-like ligand 4 (Dll4). Treatment with Sar5399 decreased 19,20-DHDP generation and increased Notch3 expression. Sar5399 also prevented vasoregression by reducing pericyte loss and suppressed microglial activation as well as the expression of immune-response cytokines. Mechanistically, the activation of Notch signaling by Dll4 maintained a quiescent microglial cell phenotype, i.e. reduced both the surface presentation of CD74 and microglial migration. In contrast, in retinal explants, 19,20-DHDP and Notch inhibition both promoted CD74 expression and reversed the Dll4-induced decrease in migration. Conclusions: Our data indicate that 19,20-DHDP-induced alterations in Notch-signaling result in microglia activation and pericyte loss and contribute to retinal vasoregression in polycystic kidney disease. Moreover, sEH inhibition can ameliorate vasoregression through reduced activity of inflammatory microglia. sEH inhibition is thus an attractive new therapeutic approach to prevent retinal vasoregression.

N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFNγ-stimulated endothelial cells.

IFNγ enhances allograft immunogenicity and facilitates T-cell mediated rejection. This may cause interstitial fibrosis and tubular atrophy (IFTA), contributing to chronic allograft loss. We assessed if inhibition of T-cell activation by N-octanoyl dopamine (NOD) impairs adherence of activated T-cells to endothelial cells and the ability of activated T-cells to produce IFNγ. We also assessed if NOD affects IFNγ mediated gene expression in endothelial cells. The presence of NOD during T-cell activation significantly blunted their adhesion to unstimulated and cytokine stimulated HUVEC. Supernatants of these T-cells displayed significantly lower concentrations of TNFα and IFNγ and were less capable to facilitate T-cell adhesion. In the presence of NOD VLA-4 (CD49d/CD29) and LFA-1 (CD11a/CD18) expression on T-cells was reduced. NOD treatment of IFNγ stimulated HUVEC reduced the expression of MHC class II transactivator (CIITA), of MHC class II and its associated invariant chain CD74. Since IFTA is associated with T-cell mediated rejection and IFNγ to a large extent regulates immunogenicity of allografts, our current data suggest a potential clinical use of NOD in the treatment of transplant recipients. Further in vivo studies are warranted to confirm these in vitro findings and to assess the benefit of NOD on IFTA in clinically relevant models.

Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

The aim of this study is to investigate the vascular outcome after intravitreal mesenchymal stem cell (MSC) administration in rats without or with damage to the neurovascular unit [transgenic (TGR) rats]. Male Sprague-Dawley (SD) and TGR rats received an intravitreal injection of 2 × 104 rat bone marrow-derived MSCs (BMSCs) or human adipose-derived stem cells (ASCs) at postnatal d 30. After 4 wk, vasculature, neuronal function, and gene expression in the retinas were evaluated using retinal morphometry, electroretinography, immunofluorescence, Western blot, and quantitative PCR. Intravitreal administration of rat BMSCs and human ASCs in both SD and TGR eyes induced cataract, loss of pericytes, and increased formation of acellular capillaries. BMSCs remained in the vitreous cavity and did not migrate into the retinas. Intravitreal administration of BMSCs impacted retinal neuronal function in neither SD nor TGR rats. Retinal glial activation, elevation of IL-1ß, C3, arginase 1, and heat shock protein 90 were detected in BMSC-injected SD rats. Intravitreal administration of MSCs induces cataract, retinal vasoregression, activation of retinal glial cells, and inflammatory response in rat eyes.-Huang, H., Kolibabka, M., Eshwaran, R., Chatterjee, A., Schlotterer, A., Willer, H., Bieback, K., Hammes, H.-P., Feng, Y. Intravitreal injection of mesenchymal stem cells evokes retinal vascular damage in rats.

Methylglyoxal induces retinopathy-type lesions in the absence of hyperglycemia: studies in a rat model.

The aim of this study was to evaluate whether damage to the neurovascular unit in diabetes depends on reactive metabolites such as methylglyoxal (MG), and to assess its impact on retinal gene expression. Male Wistar rats were supplied with MG (50 mM) by drinking water and compared with age-matched streptozotocin-diabetic animals and untreated controls. Retinal damage was evaluated for the accumulation of MG-derived advanced glycation end products, changes in hexosamine and PKC pathway activation, microglial activation, vascular alterations (pericyte loss and vasoregression), neuroretinal function assessed by electroretinogram, and neurodegeneration. Retinal gene regulation was studied by microarray analysis, and transcription factor involvement was identified by upstream regulator analysis. Systemic application of MG by drinking water increased retinal MG to levels comparable with diabetic animals. Elevated retinal MG resulted in MG-derived hydroimidazolone modifications in the ganglion cell layer, inner nuclear layer, and outer nuclear layer, a moderate activation of the hexosamine pathway, a pan-retinal activation of microglia, loss of pericytes, increased formation of acellular capillaries, decreased function of bipolar cells, and increased expression of the crystallin gene family. MG mimics important aspects of diabetic retinopathy and plays a pathogenic role in microglial activation, vascular damage, and neuroretinal dysfunction. In response to MG, the retina induces expression of neuroprotective crystallins.-Schlotterer, A., Kolibabka, M., Lin, J., Acunman, K., Dietrich, N., Sticht, C., Fleming, T., Nawroth, P., Hammes, H.-P. Methylglyoxal induces retinopathy-type lesions in the absence of hyperglycemia: studies in a rat model.

Anti-angiogenic effects of the DPP-4 inhibitor linagliptin via inhibition of VEGFR signalling in the mouse model of oxygen-induced retinopathy.

AIMS/HYPOTHESIS: Linagliptin has protective effects on the retinal neurovascular unit but, in proliferative retinopathy, dipeptidyl peptidase 4 (DPP-4) inhibition could be detrimental. The aim of this study was to assess the effect of linagliptin on ischaemia-induced neovascularisation of the retina. METHODS: C57BL/6J and glucagon-like peptide 1 (GLP-1) receptor (Glp1r)-/- mice were subjected to a model of oxygen-induced retinopathy (OIR). Both strains were subcutaneously treated with linagliptin from postnatal days 12 to 16. Non-injected OIR and non-exposed mice served as controls. Capillary proliferations and systemic levels of active GLP-1 were quantified. The effects of linagliptin on vascular endothelial growth factor (VEGF)-induced downstream signalling were assessed in human umbilical vein endothelial cells (HUVECs) using western blot for retinal phosphorylated extracellular signal-regulated kinase (ERK)1/2 and retinal gene expression analyses. RESULTS: Linagliptin treatment led to an increase in active GLP-1 and a decreased number of neovascular nuclei in OIR mice vs controls (-30%, p < 0.05). As the reduction in neovascularisation was similar in both C57BL/6J and Glp1r-/- mice, the anti-angiogenic effects of linagliptin were independent of GLP-1R status. The expression of Vegf (also known as Vegfa) and Hif1a was increased in C57BL/6J OIR mice upon linagliptin treatment (three- vs 1.5-fold, p < 0.05, p < 0.01, respectively). In HUVECs, linagliptin inhibited VEGF-induced increases in mitogen-activated protein kinase (MAPK)/ERK (-67%, p < 0.001) and MAPK/c-Jun N-terminal kinase (JNK) (-13%, p < 0.05) pathway activities. In the retinas of C57BL/6J mice, p-ERK1/2 levels were significantly reduced upon linagliptin treatment (-47%, p < 0.05). CONCLUSIONS/INTERPRETATION: Systemic treatment with linagliptin demonstrated GLP-1R-independent anti-angiogenic effects mediated by an inhibition of VEGF receptor downstream signalling. The specific effects of linagliptin on diabetic retinopathy are of potential benefit for individuals with diabetes, independent of metabolic effects.

The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2.

Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to functionally replace retinal pericytes and normalize retinal microvasculature during disease progression. We hypothesized that Notch signaling in ASC underlies regulation and stabilization of dysfunctional retinal microvascular networks such as in DR. ASC prominently and constitutively expressed NOTCH2. Genetic knockdown of NOTCH2 in ASC (SH-NOTCH2) disturbed the formation of vascular networks of human umbilical cord vein endothelial cells both on monolayers of ASC and in organotypical three-dimensional cocultures with ASC. On ASC SH-NOTCH2, cell surface platelet-derived growth factor receptor beta was downregulated which disrupted their migration toward the chemoattractant platelet-derived growth factor beta subunits (PDGF-BB) as well as to conditioned media from EC and bovine retinal EC. This chemoattractant is secreted by pro-angiogenic EC in newly formed microvascular networks to attract pericytes. Intravitreal injected ASC SH-NOTCH2 in oxygen-induced retinopathy mouse eyes did not engraft in the preexisting retinal microvasculature. However, the in vivo pro-angiogenic capacity of ASC SH-NOTCH2 did not differ from controls. In this respect, multifocal electroretinography displayed similar b-wave amplitudes in the avascular zones when either wild type ASC or SH-NOTCH2 ASC were injected. In conclusion, our results indicate that NOTCH2 is essential to support in vitro vasculogenesis via juxtacrine interactions. In contrast, ongoing in vivo angiogenesis is influenced by paracrine signaling of ASC, irrespective of Notch signaling. Stem Cells 2018;36:240-251.

Hyperglycaemic memory affects the neurovascular unit of the retina in a diabetic mouse model.

AIMS/HYPOTHESIS: The aim of this study was to evaluate damage to the neurovascular unit in a mouse model of hyperglycaemic memory. METHODS: A streptozotocin-induced mouse model of diabetes (C57BL/6J background) received insulin-releasing pellets and pancreatic islet-cell transplantation. Damage to the neurovascular unit was studied by quantitative retinal morphometry for microvascular changes and microarray analysis, with subsequent functional annotation clustering, for changes of the retinal genome. RESULTS: Sustained microvascular damage was confirmed by persistent loss of pericytes in the retinal vasculature (PC/mm2): compared with healthy controls (1981 ± 404 PC/mm2), the pericyte coverage of the retinal vasculature was significantly reduced in diabetic mice (1571 ± 383 PC/mm2, p < 0.001) and transplanted mice (1606 ± 268 PC/mm2, p < 0.001). Genes meeting the criteria for hyperglycaemic memory were attributed to the cytoskeletal and nuclear cell compartments of the neurovascular unit. The most prominent regulated genes in the cytoskeletal compartment were Ddx51, Fgd4, Pdlim7, Utp23, Cep57, Csrp3, Eml5, Fhl3, Map1a, Mapk1ip1, Mnda, Neil2, Parp2, Myl12b, Dynll1, Stag3 and Sntg2, and in the nuclear compartment were Ddx51, Utp23, Mnda, Kmt2e, Nr6a1, Parp2, Cdk8, Srsf1 and Zfp326. CONCLUSIONS/INTERPRETATION: We demonstrated that changes in gene expression and microvascular damage persist after euglycaemic re-entry, indicating memory. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in the GEO repository, GSE87433, www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=idmbysgctluxviv&acc=GSE87433 .

The DPP4 Inhibitor Linagliptin Protects from Experimental Diabetic Retinopathy.

BACKGROUND/AIMS: Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in type 2 diabetes, however, their influence on the retinal neurovascular unit remains unclear. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats (diabetes duration of 24 weeks), DPP4 activity (fluorometric assay), GLP-1 (ELISA), methylglyoxal (LC-MS/MS), acellular capillaries and pericytes (quantitative retinal morphometry), SDF-1a and heme oxygenase-1 (ELISA), HMGB-1, Iba1 and Thy1.1 (immunohistochemistry), nuclei in the ganglion cell layer, GFAP (western blot), and IL-1beta, Icam1, Cxcr4, catalase and beta-actin (quantitative RT-PCR) were determined. In C. elegans, neuronal function was determined using worm tracking software. RESULTS: Linagliptin decreased DPP4 activity by 77% and resulted in an 11.5-fold increase in active GLP-1. Blood glucose and HbA1c were reduced by 13% and 14% and retinal methylglyoxal by 66%. The increase in acellular capillaries was diminished by 70% and linagliptin prevented the loss of pericytes and retinal ganglion cells. The rise in Iba-1 positive microglia was reduced by 73% with linagliptin. In addition, the increase in retinal Il1b expression was decreased by 65%. As a functional correlate, impairment of motility (body bending frequency) was significantly prevented in C. elegans. CONCLUSION: Our data suggest that linagliptin has a protective effect on the microvasculature of the diabetic retina, most likely due to a combination of neuroprotective and antioxidative effects of linagliptin on the neurovascular unit.

Impaired Purinergic Regulation of the Glial (Müller) Cell Volume in the Retina of Transgenic Rats Expressing Defective Polycystin-2.

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD21/703 HA). Age-matched Sprague-Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD21/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y1 or adenosine A1 receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y1 receptor antagonist was mediated by induction of oxidative-nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD21/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD21/703 rats, the gene expression levels of P2Y1 and A1 receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD21/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y1 receptor activation serve to inhibit the induction of oxidative-nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.

Systemic treatment with erythropoietin protects the neurovascular unit in a rat model of retinal neurodegeneration.

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.