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

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Examination of posterior capsular opacity under
intraocular tamponade using silicone oil.
Department of Ophthalmology, Dokkyo Medical
University (Japan)
Norihito Gotoh, Hiroyuki Matsushima, Koichiro
Mukai, Tadashi Senoo
H. Matsushima receives research reimbursement
from AMO and Hoya Vision Care. The authors have
no financial interest in the subject matter of
this poster.
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Purpose
Severe proliferative diabetic retinopathy
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.
Posterior capsule fibrous opacity in silicone
oil tamponade eye
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Methods Cell Culture Model
Cultured LECs from rabbit (3.0x103cells for
HE-staining 2.0x104cells for immune-staining)
with 10FBS/MEM were embedded on upper chamber.
The cell culture insert (12-well) has 1.0mm pores
in the supporting PTE membrane for diffusion of
medium between culture chambers.
BSS group
Intraocular irrigating solution (Balanced salt
solution) was added in lower chamber.
Air group
The air group was blank in lower chamber.
Silicone group
Silicone oil was added in lower chamber.
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 10formalin and were
stained.
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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.
B
B
Aspect ratio of LEC () x100
A
A
Immuno-histological staining
The LECs were examined by immune-staining using
anti-a-Smooth Muscle Actin (a-SMA) and
anti-Transforming Growth Factor-b2 (TGF-b2) to
study for fibrosis of the cells. The ratio of
positive cell was calculated.
Immune-staining positive cells
Ratio of positive cell ()
x100
Total nuclei
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Results Morphology of LEC by HE Staining
10x
20x
40x
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.

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Results Aspect Ratio of LEC


100
80
60
40
20
0
BSS group
Air group
Silicone group
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 Plt0.05).
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Results Immune-Histological Staining for a-SMA
10x
20x
40x
BSS group
Air group
Silicone group
The LEC in Silicone group was higher expressed
a-SMA than the LEC in Air and BSS group.
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Results Ratio of Positive Cell for a-SMA


100

80
60
40
20
0
BSS group
Air group
Silicone group
The ratio of positive cell for a-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 Plt0.05).
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Results Immune-Histological Staining for TGF-b2
10x
20x
40x
BSS group
Air group
Silicone group
The LEC in Silicone group was higher expressed
TGF-b2 than the LEC in Air group. The LEC in BSS
group was nothing TGF-b2 expression.
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Results Ratio of Positive Cell for TGF-b2


50
40
30
20
10
0
BSS group
Air group
Silicone group
The ratio of positive cell for TGF-b2 was 30.7
in silicone group and 5.4 in Air group. The LEC
in BSS group was nothing TGF-b2 expression.
There are statistically significance
(Tukey-Kramer method Plt0.05).
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Hypothesis
Surviving LEC
Fibroblast-like cell
transform
a-SMA()
TGF-b2
Smads 3/4
ECM
(extracellar matrix)
nuclear translocation
PCO
EMT
(posterior capsular opacity)
Silicone oil
(epithelial-mesenchymal transition)
The silicone oil tamponade after vitrectomy may
promote TGF-b2 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.
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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.