Effect of intravitreal and intraperitoneal cyanidin-3-glucoside injection in oxygen-induced retinopathy mouse model

Abstract

Purpose: To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model.

Methods: In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/μL), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling.

Results: The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group.

Conclusion: C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations.

Keywords: Anthocyanin; cyanidin-3-glucoside; oxygen-induced retinopathy mouse model; retinal vascular disease; retinopathy of prematurity.

Figure 1

OIR group results

Figure 2

Light micrographs of C57BL/6J mouse retina. Serial sections of the retina (4 μm thick), starting from the optic disc with the second sections after the optic disc taken for evaluation. The arrows in slides c, d, and g indicate endothelial cell proliferation, which are respectively the OIR nontreatment group, OIR DMSO-injected group and the low dose IPC group. Slides a and b have no proliferation, whereas slides e,f and h have significantly less compared with c,d and g. Original magnification ×20

Figure 3

Electron micrograph showing mitochondrial morphology. Arrows in slides c, d, and g indicate atypical mitochondria with mottled matrix and electron-dense bodies in the cristae central region surrounded by lytic matrix and extensive cristalysis. Note their increase in slide g (low dose IPC group). Slide a and b have none, whereas slides e,f and h have significantly less compared with c,d and g

Figure 4

Light micrographs showing assessed apoptotic cells using the TUNEL assay. Apoptotic cells appear distinct deep pigmented (arrow) in the outer nuclear layer and inner nuclear layer in each group. Note the high amount of apoptosis in slide f, the 600-ng/μL IVC group. Apoptotic cell counts of slides a,b,c,d,e,g and h have no statistical difference within each other. Original magnification ×100, oil immersion