Evidence of Müller cell gliosis in mature Akimba mice with a severe phenotype.

<p>In the mature wt retina GFAP expression was localised and restricted to the astrocytes that reside on the GCL (arrowheads, C-D). In Akimba mice, GFAP expression was observed as thick processes in the GCL and long radial processes in the INL/OPL, indicating Müller cell gliosis (G-H). Edn2 only co-localised with GFAP in Müller cell processes in the INL/OPL (arrows, H) but not in the astrocytes of the GCL. Sections were counterstained with DAPI (B, F). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PIS/POS: photoreceptor inner and outer segments. Scale bar: 200μm</p

Biomarkers for Diabetic Retinopathy – Could Endothelin 2 Be Part of the Answer? - Fig 1

<p><b>Edn2 (A) and Ednrb (B) fold changes in mRNA expression in young and mature retinae of wt, Akita, Kimba and Akimba mice</b>. Data represented are fold changes in expression normalised against Ppia as the housekeeping gene. Differential expression was determined using delta delta Ct according to Livak and Schmittgen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160442#pone.0160442.ref023" target="_blank">23</a>]. Fold changes in expression in Akita, Kimba and Akimba retinae represent increases or decreases in mRNA expression levels compared to wt retinae. N = 4 per group; *p<0.05 compared to wt and Akita</p

Cellular localisation of Edn2 in the mature retina.

<p>Following 20 weeks of chronic hyperglycaemia in the mature Akita retina there was increased Edn2 staining in the IPL compared to young Akita retinae (F). Particularly in the mature Akimba retina GS staining was much reduced compared to the young retina, suggesting not only loss of photoreceptors (L) but also loss of Müller cells (I, arrowheads). Müller cell loss was also evident in mature Kimba retinae (M, arrowheads; O, arrows) compared to young Kimba retinae, but the loss was less pronounced. Scale bar: 100μm</p

Cellular localisation of Edn2 in the young retina.

<p>Edn2 expression was localised to Müller cells as demonstrated by co-localisation with glutamine synthetase (GS), a Müller cell-specific enzyme, and photoreceptor inner and outer segments (PIS, POS) in wt, Akita, Akimba and Kimba retinae (C, G, K, O; arrows and arrowhead). In Akita, Edn2 expression was mostly in the GCL, where Müller cell endfeet reside, the outer plexiform layer (OPL) and the outer nuclear layer (ONL; E, G). In Akimba and Kimba, Edn2 expression was mostly in the inner retina, the GCL and the inner plexiform layer as well as the photoreceptor inner and outer segments (IPL, PIS, POS; J, K, N, O). Co-localisation of Edn2 and GS was most pronounced in the GCL and Müller cell processes in the IPL of Kimba mice (O). Scale bar: 100μm</p

Serum Edn2 concentration in young and mature wt, Akita, Kimba and Akimba mice.

<p>Serum concentrations did not differ between young wt, Akita, Kimba and Akimba mice (A). In wt mice, Edn2 serum concentration decreased with age. In mature mice (B), there was no significant difference in serum Edn2 concentration between wt, Akita and Kimba mice. However, circulating levels of Edn2 were significantly higher in mature Akimba mice (6.9pg/mg total retina protein) compared to wt (3.9pg/mg; p<0.01), Akita (3.8pg/mg; p<0.01) and Kimba (4.6pg/mg total retina protein; p<0.05) mice.</p

Physiologic and metabolic parameters of young and mature wt, Kimba, Akita and Akimba mice.

<p>Physiologic and metabolic parameters of young and mature wt, Kimba, Akita and Akimba mice.</p