Lab Anim Res.  2017 Sep;33(3):216-222. 10.5625/lar.2017.33.3.216.

Early changes in retinal structure and BMP2 expression in the retina and crystalline lens of streptozotocin-induced diabetic pigs

Affiliations
  • 1Department of Ophthalmology and Inha Vision Science Laboratory, Inha University School of Medicine, Incheon, Korea. drmys@inha.ac.kr, nrkim@inha.ac.kr
  • 2Laboratory of Developmental Genetics, Department of Biomedical Sciences, Inha University School of Medicine, Incheon, Korea.

Abstract

PURPOSE
This study aims to evaluate early changes in retinal structure and BMP2 expression in the retina and crystalline lens by comparing streptozotocin-induced diabetic pigs and normal control group pigs.
METHODS
Five eye samples from five diabetic Micro-pigs (Medikinetics, Pyeongtaek, Korea) and five eye samples from five control pigs bred in a specific pathogen-free area were used. Diabetes was developed through intravenous injection of nicotinamide and streptozotocin, and the average fasting glucose level was maintained at 250 mg/dL or higher for 16 weeks. To evaluate BMP2 expression in the retina and crystalline lens, Western blotting was performed.
RESULTS
In Hematoxylin and Eosin staining, most diabetic pigs showed structural abnormalities in the inner plexiform layer. The number of nuclei in the ganglion cell layer within the range of 10⁴µm² was 3.78±0.60 for diabetic pigs and 5.57±1.07 for control group pigs, showing a statistically significant difference. In immunohistochemical staining, diabetic retinas showed an overall increase in BMP2 expression. In Western blotting, the average BMP2/actin level of diabetic retinas was 1.19±0.05, showing a significant increase compared to the 1.06±0.03 of the control group retinas (P=0.016). The BMP2/actin level of diabetic crystalline lenses was similar to the control group crystalline lenses (P=0.730).
CONCLUSIONS
Compared to control group pigs, the number of nuclei in the inner nuclear layer of retinas from streptozotocin-induced diabetic pigs decreased, while an increase in BMP2 expression was observed in the retina of diabetic pigs.

Keyword

BMP2; diabetes mellitus; lens; pigs; retina; streptozotocin

MeSH Terms

Blotting, Western
Crystallins*
Diabetes Mellitus
Eosine Yellowish-(YS)
Fasting
Ganglion Cysts
Glucose
Gyeonggi-do
Hematoxylin
Injections, Intravenous
Lens, Crystalline*
Niacinamide
Retina*
Retinaldehyde*
Streptozocin
Swine*
Crystallins
Eosine Yellowish-(YS)
Glucose
Hematoxylin
Niacinamide
Retinaldehyde
Streptozocin

Figure

  • Figure 1 Hematoxylin and Eosin staining of diabetic pig retina. Gross structural abnormalities were observed, particularly in the inner plexiform layer, compared to the normal retina. There are decreases in the numbers of nuclei in the ganglion cell layer and inner nuclear layer in the retina of diabetic subjects.

  • Figure 2 BMP2 expression in diabetic pig retinas. Immunohistochemistry of BMP2 in normal (left) and diabetic pig retinas (right). Increase in the expression of BMP2 in diabetic subjects is observed (Green stain) compared to the normal retina. BMP2 is increased especially in the photoreceptor inner segment (Arrowhead) and in relation to retinal vessels (Arrow). DAPI, 4',6'-diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate conjugated; IL B4, Isolectin B4; GSL 1, Griffonia Simplicifolia Lectin I; NFL, Nerve fiber layer; GCL, Ganglion cell layer; IPL, Inner plexiform layer; INL, Inner nuclear layer; OPL, Outer plexiform layer; ONL, Outer nuclear layer; IS, Inner segments; OS, Outer segments.

  • Figure 3 (A) BMP2 expression in diabetic pig retina. Western blot analysis of BMP2 in normal and diabetic pigs showing higher abundance of BMP2 in the retina of diabetic pigs compared to the control group. (B) BMP2 expression in diabetic pig crystalline lenses. Western blot analysis of BMP2 in normal and diabetic pigs showing similar abundance of BMP2 in the crystalline lens of diabetic pigs compared to the control group.

  • Figure 4 VEGF expression in diabetic pig vitreous. Enzyme-linked immunosorbent assay (ELISA) of VEGF in normal and diabetic pigs showing upregulation of VEGF in the vitreous of diabetic pigs compared to the control group (*P value <0.05 versus control group).


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