Erythropoietin, a Neuroprotective & Pro-Angiogenic Cytokine, and Its Receptors Are Elevated in the Neuroglial Retina Prior to Neovascularization in the Diabetic, Homozygous Ren2 Transgenic Rat

Abstract

Purpose: : Erythropoietin (Epo) is a pro-angiogenic and neuroprotective cytokine, upregulated by low oxygen. Epo/EpoR could potentially serve an early desirable tissue-protective role, and later, an undesirable pro-angiogenic role. We studied the diabetic, hypertensive homozygous mRen2(27) transgenic rat that develops ocular NV. We also compared Epo/EpoR expression against Nerve Growth Factor (NGF), the prototype neurotrophic cytokine, and its receptor, TrkA, in a rat model of low oxygen inhalation.

Methods: : Diabetes was induced in 6 week mRen2(27) rats by intra-peritoneal injection of streptozotocin (65 mg/kg). Blood glucose was measured 3 times weekly. Animals were sacrificed at 1, 3 and 5 weeks after STZ injection. In a second model, reduction in tissue oxygenation was achieved by 16 hours of exposure to 10% oxygen. Localization of Epo, NGF and their receptors was determined by epifluorescent immunohistochemistry for retinal cell-specific markers. Following total RNA isolation using the Trizol © method, mRNA levels of Epo and EpoR were quantified by real-time quantitative polymerase chain reaction (qPCR).

Results: : qPCR demonstrated a 3.5, 1.4 and 1.5-fold upregulation of Epo mRNA in STZ-treated animals compared to non-diabetic baseline, at 1, 3, and 5 weeks respectively. EpoR mRNA was decreased to 0.75 at week 1, and increased to 3.5 and 3.2, at 3 and 5 weeks respectively. Epo and EpoR protein was upregulated in all three major layers of the diabetic retina, most obviously in photoreceptor inner segments and cell bodies and in bipolar cells. This distribution was also observed in rats exposed to a low oxygen environment. Preliminary data distinguishing nascent from pre-existing iris vasculature suggest that EpoR is upregulated on these vessels, but not on retinal vessels.

Conclusions: : Our study confirms that Epo and EpoR are present early in diabetic retinopathy, prior to NV, where they could play a tissue-protective role. Their localization within the retinal parenchyma further supports this hypothesis, correlating with observations of Vascular Endothelial Growth Factor-A and NGF/TrkA upregulation in early non-PDR. We also observed pan-retinal increase in NGF/TrkA under hypoxic conditions, suggesting a potential mechanism for its upregulation in the diabetic retina.

Keywords: diabetic retinopathy • neuroprotection • retinal neovascularization