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Use of a three dimensional porcine retinal explant model to detect HIF1α for understanding diabetic retinopathy
Author(s) -
Iwuagwu B. U.,
Cruickshank S. F.,
Rowe I.,
Knott R. M.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.780.3
Subject(s) - diabetic retinopathy , explant culture , retinal , agarose , hypoxia inducible factors , hypoxia (environmental) , retinal detachment , pathology , microbiology and biotechnology , biology , chemistry , ophthalmology , andrology , medicine , diabetes mellitus , in vitro , endocrinology , biochemistry , organic chemistry , oxygen , gene
Diabetic retinopathy is a major microvascular complication of diabetes mellitus and a leading cause of blindness [1]. Hypoxia is suggested to play a crucial pathophysiological role in diabetic retinopathy (DR) [2], but the exact mechanisms are complex and the precise events are still debated. An agarose‐collagen three‐dimensional (3D) porcine retinal explant model is used here to investigate the expression of the hypoxia inducible factor ‐1 alpha (HIF‐1α), a transcription factor critical for cellular response to hypoxia [2]. Aim To study the expression and localization of the hypoxia‐inducible factor 1 alpha ((HIF)‐1α, a key target in DR using a 3D porcine retinal explant model. Method Porcine retinal tissues were extracted from ocular globes, (n ≥ 6) donated by a local abattoir. Extracts were cultured in DMEM within a 3D matrix derived from 0.5% agarose + collagen (0.25mg/ml) co‐gel solution and explants were maintained in a humidified atmosphere with 5% CO 2 at 37°C. Explants were harvested at less than 2 h (T 0 ) and 24 h (T 1 ) post culture, fixed overnight in 10% neutral buffered formalin (NBF) and incubated in 30% sucrose. Serial sections (20μm) were cut using a cryostat. To ascertain tissue integrity, sections were stained with Mayer's hematoxylin and Eosin according to protocol (Sigma‐Aldrich). To detect the expression and sub‐cellular location of the hypoxia inducible factor (HIF) 1α at T 0 and T 1 , sections (20μm) were washed 2 × 15 minutes in 50mM Tris buffered saline (TBS) plus 0.1% Triton X‐100, followed with blocking in 10% normal goat serum/1% bovine serum albumin (BSA) (Santa Cruz) for 2 hours at room temperature with final incubation with primary mouse anti‐Hif1α (Thermo Fischer) (1:20) overnight at 4°C. Post‐incubation, specimens were rinsed 1 × 15 minutes with TBS 0.1% Triton. Non‐specific endogenous peroxidase reaction was blocked with 0.3% H 2 O 2 in 0.3% normal serum (goat) in TBS for 15 minutes at RT. Goat anti‐Mouse IgG (H+L) secondary antibody, DyLight 488 (Thermo Fischer scientific) (1:200) was used for detection. Results & Conclusion H & E stain demonstrated preservation of retinal integrity and does indicate that the use of agarose‐based 3D system provided structural and mechanical support which may have prevented early retinal tissue degeneration. Preliminary immunohistochemistry results revealed positive identification of HIF1α expression at less than 2 h (T 0 ) and 24 h (T 1 ). HIF1‐α expression was localized in the photoreceptor and dispersed throughout the outer and inner layers of the retina. Further investigation is needed to establish the precise cell‐specific co‐localization of HIF1α. Support or Funding Information Benedicta Iwuagwu, the presenting author is a recipient of an academic scholarship from the Robert Gordon University (RGU). This thesis is fully funded by RGU and the author receives a tax‐free monthly personal stipend in addition from the university.