Anti-inflammatory α-Melanocyte-Stimulating Hormone Protects Retina After Ischemia/Reperfusion Injury in Type I Diabetes

Abstract

Retinal ischemia/reperfusion (I/R) injury is a major cause of vision loss in many ocular diseases. Retinal I/R injury is common in diabetic retinopathy, which as a result of hyperglycemia damages the retina and can cause blindness if left untreated. Inflammation is a major contributing factor in the pathogenesis of I/R injury. α-Melanocyte-stimulating hormone (α-MSH) is an anti-inflammatory peptide hormone that has displayed protective effects against I/R-induced organ damages. Here, we aimed to investigate the protective role of α-MSH on I/R-induced diabetic retinal damage using hyperglycemic C57BL/6J Ins2^Akita/+ mice. Experimental I/R injury was induced by blocking the right middle cerebral artery (MCA) for 2 h followed by 2 h or 22 h of reperfusion using the intraluminal method. Since ophthalmic artery originates proximal to the origin of the MCA, the filament also blocked blood supply to the retina. Upon treatment with α-MSH at 1 h after ischemia and 1 h after reperfusion, animals displayed significant improvement in amplitudes of b-wave and oscillatory potentials during electroretinography. α-MSH also prevented I/R-induced histological alterations and inhibited the development of retinal swelling. Loss of retinal ganglion cells as well as oxidative stress were significantly attenuated in the α-MSH-treated retinae. Level of interleukin 10 was significantly increased after α-MSH treatment. Moreover, gene expression of glutamate aspartate transporter 1, monocarboxylate transporter (MCT) 1 and MCT-2 were significantly higher after α-MSH administration. In conclusion, α-MSH mitigates the severity of I/R-induced retinal damage under hyperglycemic condition. These beneficial effects of α-MSH may have important therapeutic implications against retinal I/R injury under hyperglycemic condition.

Keywords: diabetes; inflammation; ischemia; oxidative stress; reperfusion; retina.

FIGURE 1

α-MSH preserved the retinal function after I/R injury. (A) Representative ERG waves at 2 h of reperfusion. (B) Representative ERG waves at 22 h of reperfusion. (C) The amplitude of b-wave was significantly higher in animals treated with 10, 20, 50, and 70 μg of α-MSH at 2 h of reperfusion. (D) This amplitude was significantly higher in animals treated with 10, 20, and 50 μg of α-MSH at 22 h of reperfusion. One-way ANOVA followed by Dunnett test. (E,F) OPs were significantly higher in α-MSH (50 μg) treated animals in both experimental conditions. One-way ANOVA followed by Tukey’s test. Flash 3 (P)cd.s/m². *P < 0.05, n = 5–6 for each group.

FIGURE 2

α-MSH preserved morphological integrity of retina after I/R injury. (A–D,I) Thicknesses of GCL and total retina were significantly higher in animals treated with vehicle compared with α-MSH-treated animals or vehicle-treated sham animals at 2 h of reperfusion. (E–H,J) Only thickness of GCL was significantly higher in vehicle-treated animals compared to vehicle-treated sham animals at 22 h of reperfusion. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA followed by Tukey’s test, n = 6 for each group. Scale bar = 25 μm.

FIGURE 3

α-MSH maintained the number of RGC in the GCL. (A,B) Significantly lower number of RGC was recorded in vehicle-treated animals compared with α-MSH-treated animals or vehicle-treated sham animals in both experimental conditions. *P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA followed by Tukey’s test, n = 6 for each group.

FIGURE 4

Immunoreactivities of retinal neuronal and glial cells after I/R injury. (A–D) Calretinin expression was higher in α-MSH-treated animals at 22 h of reperfusion. (E) Number of calretinin positive cell/mm. (F–I) PKC-α expression was higher in α-MSH-treated animals at 2 h of reperfusion. (J) IHC scores for PKC-α.(K–N) GFAP expression was similar between the groups in both experimental conditions. (O) IHC scores for GFAP. (P–S) α-MSH reduced the GS expression at 2 h of reperfusion. (T) IHC scores for GS. Mann-Whitney U-Test, n = 6 for each group. Scale bar = 25 μm.

FIGURE 5

α-MSH decreased the oxidative and nitrative stress after I/R injury. (A–D) Significantly weaker PAR immunoreactivity was observed in animals treated with α-MSH at 2 h of reperfusion. This immunoreactivity was comparable at 22 h of reperfusion. (E) IHC scores for PAR. (F–I) Weaker NT immunoreactivity was observed in α-MSH-treated animals in both experimental conditions. (J) IHC scores for NT. **P < 0.01, Mann-Whitney U-Test, n = 6 for each group. Scale bar = 25 μm.

FIGURE 6

α-MSH increased expressions of anti-inflammatory and pro-survival proteins after I/R injury. (A,B) Protein expressions of IL-10 was significantly higher in animals treated with α-MSH at 2 h and 22 h of reperfusion. (A,B) Although Bcl-2 expression was higher in animals treated with α-MSH in both experimental conditions, the expression was comparable. *P < 0.05, ***P < 0.001, Independent samples t-test, n = 5–6 for each group.

FIGURE 7

α-MSH increased expression of glutamate and lactate related genes after I/R injury. (A) GLAST-1 expression was significantly higher in animals treated with α-MSH at 2 h of reperfusion. (B,C) MCT-1 and MCT-2 expressions were significantly higher in animals treated with α-MSH at 22 h of reperfusion. (A–C) GLAST-1 expression at 22 h of reperfusion and expressions of MCT-1 and MCT-2 at 2 h of reperfusion were comparable. *P < 0.05, Independent samples t-test, n = 5 for each group.