Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration

<div><p>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.</p></div

Expression of CD74, CNTF and bFGF after treatment with EPO or EPO-peptide.

<p>Four week old heterozygous or homozygous male PKD rats (each n = 5) were treated with 0.5 ml/kg body weight DynEpo three times or EPO-like peptide five days a week intraperitoneally for four weeks. At the age of eight weeks the animals were sacrificed, the eyes enucleated, retinal RNA was isolated and gene expression of CD74, CNTF and bFGF analyzed. All values are expressed as mean ± standard deviation. Significance was analyzed using Student's t-test and defined as follows: *p<0.05; **p<0.01. CD74 shows a reduction by 67% (p<0.01) in heterozygous and by 36% (n.s.). CNTF revealed no significant changes in gene expression. In heterozygous rats it decreased by 35% (n.s.), while it increased in homozygous animals by 32%. bFGF showed a reduction in both groups. It significantly decreased in heterozygous rats by 58% (p<0.01). Homozygous animals revealed 50% (p<0.01) lower expression of bFGF than heterozygous, but still achieved a reduction in gene expression by EPO-peptide of 29% (n.s.).</p

Quantification of retinal morphology after EPO and EPO-like peptide treatment.

<p>Four week old heterozygous, male PKD rats were treated with 0.5(n = 6) and homozygous rats with 0.5 ml EPO-like peptide five times a week (n = 4). After four weeks of treatment the animals were sacrificed and their eyes analyzed. Untreated animals (n = 4/5) were held as controls. All values are indicated as mean values ± standard deviation. Significance was evaluated by Student's t-test and defined as follows: *<0.05; **<0.01; ***<0.001. Quantification of endothelial cells (A) showed a decrease by 9% (p<0.001) in homozygous rats compared to heterozygous rats. Treatment with EPO or EPO-like peptide revealed no difference in endothelial cells or pericytes (A, B). Number of migrating pericytes (C) was reduced by 23% or 49% (both not significant). Quantification of acellular capillaries showed a 62% (p<0.001) increase in homozygous compared to heterozygous rats. By EPO treatment, a significant reduction of acellular capillaries by 49% (p<0.001) was achieved. EPO-like peptide reduced the number of acellular capillaries significantly by 63% (p<0.001).</p

Immunofluorescence staining of CD74 in retinal whole mounts.

<p>CD74 is stained with Fitc (green) and Lectin with Tritc (red). A shows a representative example of a superficial capillary layer with almost no CD74 positive cells. B is a magnification of CD74 positive cells with their typical ramified shape. Homozygous PKD rats without and with EPO peptide are shown in C and D. Likewise E and F represent heterozygous PKD retinae without and with EPO treatment. EPO and EPO-like peptide reduce the amount of CD74 positive cells. Arrows mark CD74 positive cells.</p

The DPP4 Inhibitor Linagliptin Protects from Experimental Diabetic Retinopathy

<div><p>Background/aims</p><p>Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in type 2 diabetes, however, their influence on the retinal neurovascular unit remains unclear.</p><p>Methods</p><p>Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated <i>C</i>. <i>elegans</i>, 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 <i>C</i>. <i>elegans</i>, neuronal function was determined using worm tracking software.</p><p>Results</p><p>Linagliptin decreased DPP4 activity by 77% and resulted in an 11.5-fold increase in active GLP-1. Blood glucose and HbA_1c 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 <i>C</i>. <i>elegans</i>.</p><p>Conclusion</p><p>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.</p></div

Quantification of central (A) and peripheral (B) retinal thickness after EPO treatment.

<p>Four week old heterozygous male PKD rats (n = 4) were treated three times a week with 0.5 ml/kg body weight DynEpo intraperitoneally for four weeks. EPO-treated SD rats (n = 6) and water-treated PKD (n = 5) or SD (n = 6) rats were held as controls. At the age of eight weeks the animals were sacrificed, their eyes enucleated and PAS-stained paraffin sections were prepared. Central, i.e. near the optic nerve, and peripheral thickness were evaluated using a Leica DMRBE microscope and Leica IM50-software. All values are expressed as mean ± standard deviation. Significance was analyzed using Student's t-test and defined as follows: *p<0.05; **p<0.01; ***p<0.001. Central (A) total thickness of PKD retinae compared to SD retinae was reduced by 14% (p<0.001), consisting of a 28% reduction in the outer nuclear layer (p<0.001) and 21% in the outer segments of the photoreceptor layer (p<0.001). EPO treatment increased total retinal thickness in PKD rats in comparison to water-treated PKD rats by 11% (p<0.05), consisting of an increase of the outer plexiforme layer by 19% (p<0.001), the outer nuclear layer by 12% (p<0.01) and the outer segments of photoreceptors by 26% (p<0.001). Total peripheral (B) thickness of PKD retinae compared to SD retinae was reduced by 14% (p<0.001), consisting of a 15% reduction in the ganglion cell, a 26% reduction in the outer nuclear layer (p<0.001) and a 19% reduction in the outer segments of the photoreceptor layer (p<0.001). EPO treatment had no significant effect on retinal thickness in SD or PKD rats. GCL ganglion cell layer, IPL inner plexiforme layer, INL inner nuclear layer, OPL outer plexiforme layer, ONL outer nuclear layer, PR outer segments of photoreceptors</p

Immunofluorescence staining of pAkt (A–D) and GFAP (E–H) in treated and untreated PKD rats.

<p>The scale bar indicates 25 µm. Four week old male PKD rats were treated with 0.5 ml/kg body weight DynEpo three times or EPO-like peptide five days a week intraperitoneally for four weeks. At the age of eight weeks the animals were sacrificed, the eyes enucleated, sections performed and stained for pAkt and GFAP. pAkt (A–D) staining in untreated hetero- (A) and homozygous (C) rats occurred spotlike in the inner plexiforme layer. Upon EPO (B) or EPO-peptide (D) administration, pAkt expression increased markedly, indicating that the protective effect of the treatment is translated via the EPO-receptor. GFAP (E–H), an activation marker for Müller cells, was regulated contrarily. Heterozygous rats (E) express GFAP in a typical Müller cell pattern, surrounding vessels (circle) and with vertical filaments (arrow head). The staining intensity in EPO-treated rats (F) was slightly increased. Homozygous rats (G) express GFAP like heterozygous rats, but with a higher intensity (G). Upon EPO-peptide treatment, GFAP staining decreases markedly in homozygous rats (H). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer.</p

Examples of central retinal sections, used to measure the thickness of retinal layers and count cell nuclei.

<p>In these examples 20fold magnification was used for a better overview. Measurement of retial layers was performed using 50fold magnification, cell counting was performed using 200fold magnification. White bars in C and D were used to illustrate the difference in thickness of the outer nuclear layer. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer.</p

Counting of cell nuclei in SD and PKD rats after EPO-treatment.

<p>Four week old heterozygous male PKD rats (n = 4) were treated three times a week with 0.5 ml/kg body weight DynEpo intraperitoneally for four weeks. EPO-treated SD rats (n = 6) and water-treated PKD (n = 5) or SD (n = 6) rats were held as controls. At the age of eight weeks the animals were sacrificed, eyes enucleated and PAS-stained paraffin sections were performed. Cell nuclei were counted in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in a central (A) and a peripheral (B) area of the retina. All values are expressed as mean ±standard deviation. Significance was analyzed using Student's t-test and defined as follows: *p<0.05; **p<0.01; ***p<0.001. Central area (A) revealed an increase in the INL by 20% (p<0.001) and a reduction in the ONL by 16% (p<0.001) of PKD rats in comparison to SD rats. EPO-treatment showed no significant effect in the central area of SD or PKD rats. The peripheral areas (B) also revealed significant reduction of 11% in the ONL (p<0.01) and an increase of 25% in the INL (p<0.001) and the GCL (p<0.05) in PKD rats compared to SD controls. EPO-treatment increased the number of cells in the ONL in SD rats by 10% (p<0.05) and decreased the number of cells in the INL of PKD rats by 11% (p<0.05). GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer</p

Effect of linagliptin on experimental diabetic retinopathy.

<p>(A) Representative images of PAS-stained retinal digest preparations. Arrowheads indicate pericytes [P] and arrows indicate acellular capillaries [AC]. (B) Numbers of acellular capillaries, and (C) pericyte numbers in capillary areas were determined by quantitative retinal morphometry (n = 7). (D) Representative images of PAS-stained paraffin sections (3 μm), (E) numbers of cell nuclei in the ganglion cell layer (GCL) (n = 5). Data are expressed as mean ± SD. **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p