Examination of posterior capsular opacity under intraocular tamponade using silicone oil.

Examination of posterior capsular Examination of posterior capsular opacity under intraocular tamponade opacity under intraocular tamponade

using silicone oil.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.

PurposePurpose

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

Cultured LECs from rabbit (3.0x103cells for HE-staining & 2.0x104cells for immune-staining) with 10%FBS/MEM were embedded on upper chamber.

Methods: Methods: Cell Culture ModelCell 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 groupBSS group

Air groupAir group

Silicone groupSilicone 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 1.0m pores in the supporting PTE membrane for diffusion of medium between culture chambers.

Methods:Methods: Histological and Immune-Histological StainingHistological and Immune-Histological Staining

Hematoxylin-eosin (HE) stainingThe 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 stainingThe 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

AB

A

B

Ratio of positive cell (%) = x100Total nuclei

Immune-staining positive cells

10x 20x 40xResults:Results: Morphology of LEC by HE StainingMorphology of LEC by HE Staining

BSS groupBSS group

Air groupAir group

Silicone groupSilicone 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.

Results: Results: Aspect Ratio of LECAspect 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 groupBSS group Air groupAir group Silicone groupSilicone group

% *

Results: Results: Immune-Histological Staining for Immune-Histological Staining for -SMA-SMA

The LEC in Silicone group was higher expressed -SMA than the LEC in Air and BSS group.

10x 20x 40x

BSS groupBSS group

Air groupAir group

Silicone groupSilicone group

Results: Results: Ratio of Positive Cell for Ratio of Positive Cell for -SMA-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 groupBSS group Air groupAir group Silicone groupSilicone group

%*

*

Results: Results: Immune-Histological Staining for TGF-Immune-Histological Staining for TGF-22

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.

10x 20x 40x

BSS groupBSS group

Air groupAir group

Silicone groupSilicone group

Results: Results: Ratio of Positive Cell for TGF-Ratio of Positive Cell for TGF-22

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 groupBSS group Air groupAir group Silicone groupSilicone group

%*

HypothesisHypothesis

Surviving LEC

Fibroblast-like cell

transform

EMTEMT(epithelial-mesenchymal transition)(epithelial-mesenchymal transition)

Smads 3/4nuclear translocation (extracellar matrix)

(posterior capsular opacity)

TGF-2

-SMA(+)

PCO

ECM

Silicone oilSilicone 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.

ConclusionsConclusions

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.