Characterization of a porcine retinal explant demonstrating glucose-dependent changes in the level of factor type 1 alpha

Abstract

Diabetic retinopathy (DR) is a leading cause of blindness in the working population of the Western world. Advances in our understanding of specific variables contributing to this condition is restricted by the lack of characterization available with in vitro model systems. In this study we establish a retinal porcine model and investigate the effect of hyperglycemia on the level of hypoxia inducible factor type 1 alpha in the porcine retina.

Porcine retinae were excised from porcine eyes and cultured in 5 or 25 mM D-glucose in Dulbecco’s Minimal essential Media (DMEM), without a matrix (2D) or within a matrix (3D) consisting of a co-gel (0.5% agarose plus collagen 0.25mg/ml) in DMEM and maintained in a humidified atmosphere with 5% CO2 at 37 °C. This process took 30 min. Retinal integrity was monitored by visualization with a light microscope followed by hematoxylin and eosin staining and Gomori Trichrome. Matrix oxygen saturation (% O2) was recorded with a Microx TX3 oxygen microsensor at time zero and 24 h following incubation. Immunostaining for HIF-1α was performed using primary mouse anti-HIF-1α (1:20) followed by anti-mouse DyLight 488 goat secondary antibody (1:200) and visualized with an inverted fluorescent microscope.

Retinal tissue and vessel integrity were morphologically preserved on 3D matrices versus 2D control evidenced by hematoxylin and eosin staining. A time dependent increase in collagen incorporation within the explant was seen with the Gomori Trichrome stain. Oxygen levels significantly increased within the explant during the 23h test period from 15.5% to 22.3% (p<0.001) in media alone and from 10% to 13% (p<0.05) with the matrix (n=6) with all values relative to atmospheric pressure. HIF-1α was detected by immune-histochemical analysis in explants at t=0 in 5 and 25mM glucose concentration, with staining of serial sections with DAPi. HIF-1α levels only remained elevated at 24 h in 25 mM D-glucose and was undetected in 5 mM D-glucose.

This study presents a novel model of retinal explants demonstrating that retinal tissue can be cultured in non-hypoxic conditions and that the 3-dimensional matrix was able to sustain retinal structure. Retinal structure was, at least partially sustained by the incorporation of collagen into the retina. Differential expression of protein was identified in the different matrix environment; specifically, hyperglycemia sustained non-hypoxia induced HIF-1α expression in isolated retina.