1. Examination of posterior capsular opacity under intraocular tamponade using silicone oil. Norihito Gotoh, Hiroyuki Matsushima, Koichiro Mukai, Tadashi Senoo Department of Ophthalmology, Dokkyo Medical University (Japan) H. Matsushima receives research reimbursement from AMO and Hoya Vision Care. The authors have no financial interest in the subject matter of this poster.

2. Purpose Severe retinal disease patients sometimes undergo vitrectomy with intraocular tamponade using silicone oil. In these patients, posterior capsular opacity (PCO) developed markedly. However the mechanisms of PCO and effects of silicone oil to lens epithelial cells (LECs) are still unclear. In this study, we produced a novel cell culture system for analyze the effect of silicone oil to LECs experimentally. Severe proliferative diabetic retinopathy Posterior capsule fibrous opacity in silicone oil tamponade eye

3. Cultured LECs from rabbit (3.0x10 3 cells for HE- staining & 2.0x10 4 cells for immune-staining) with 10%FBS/MEM were embedded on upper chamber. Methods: Cell Culture Model Intraocular irrigating solution (Balanced salt solution) was added in lower chamber. The air group was blank in lower chamber. Silicone oil was added in lower chamber. BSS group Air group Silicone group The LECs in cell culture insert were cultured during 7days for HE-staining and 4days for immune- staining. After the selected period, the LECs were fixed with 10%formalin and were stained. The cell culture insert (12-well) has 1.0  m pores in the supporting PTE membrane for diffusion of medium between culture chambers.

4. Methods: Histological and Immune-Histological Staining Hematoxylin-eosin (HE) staining The LECs were stained by hematoxylin-eosin to examine for the morphology of cells. The aspect ratio of LEC was calculated to assess the fibrosis of LEC. Immuno-histological staining The LECs were examined by immune-staining using anti-  -Smooth Muscle Actin (  -SMA) and anti-Transforming Growth Factor-  2 (TGF-  2) to study for fibrosis of the cells. The ratio of positive cell was calculated. Aspect ratio of LEC (%) = x100 A B A B Ratio of positive cell (%) = x100 Total nuclei Immune-staining positive cells

5. 10x20x40x Results: Morphology of LEC by HE Staining BSS group Air group Silicone group The morphology of LEC in Silicone and Air group was enlarged compared with LEC in BSS group. LECs in BSS group were kept in normal cubic structure.

6. Results: Aspect Ratio of LEC The aspect ratio of LEC was 35.4% in silicone group, 65.8% in Air group, and 83.1% in BSS group. There are statistically significance (Tukey-Kramer method *P<0.05). 0 20 40 60 80 100 BSS group Air group Silicone group % *

7. Results: Immune-Histological Staining for  -SMA The LEC in Silicone group was higher expressed  -SMA than the LEC in Air and BSS group. 10x20x40x BSS group Air group Silicone group

8. Results: Ratio of Positive Cell for  -SMA The ratio of positive cell for  -SMA was 62.2% in silicone group, 28.1% in Air group, and 14.9% in BSS group. There are statistically significance (Tukey-Kramer method *P<0.05). 0 20 40 60 80 100 BSS group Air group Silicone group % * *

9. Results: Immune-Histological Staining for TGF-  2 The LEC in Silicone group was higher expressed TGF-  2 than the LEC in Air group. The LEC in BSS group was nothing TGF-  2 expression. 10x20x40x BSS group Air group Silicone group

10. Results: Ratio of Positive Cell for TGF-  2 The ratio of positive cell for TGF-  2 was 30.7% in silicone group and 5.4% in Air group. The LEC in BSS group was nothing TGF-  2 expression. There are statistically significance (Tukey-Kramer method *P<0.05). 0 10 20 30 40 50 BSS group Air group Silicone group % *

11. Hypothesis Surviving LEC Fibroblast-like cell transform EMT (epithelial-mesenchymal transition) Smads 3/4 nuclear translocation (extracellar matrix) (posterior capsular opacity) TGF-  2  -SMA(+) PCO ECM Silicone oil The silicone oil tamponade after vitrectomy may promote TGF-  2 from surviving LEC in lens capsule and transform from the LEC into fibroblast-like cell by EMT. The accretion of EMT and increasing of fibroblast-like cells may produce severe PCO.

12. Conclusions The intravitreous substituted materials have much effect on lens epithelial cell. The mechanisms of posterior capsular fibrous opacity after vitrectomy is indicated that the intravitreous materials, in particular silicone oil, undergo epithelial- mesenchymal transition.