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EXTENDED REPORT
Silicone oil concentrates fibrogenic growth factors in the
retro-oil fluid
R H Y Asaria, C H Kon, C Bunce, C S Sethi, G A Limb, P T Khaw, G W Aylward, D G Charteris
...............................................................................................................................
See end of article for
authors’ affiliations
.......................
Correspondence to:
Mr David Charteris,
Vitreoretinal Research
Unit, Moorfields Eye
Hospital, City Road,
London EC1V 2PD, UK;
david.charteris@
moorfields.nhs.uk
Accepted for publication
1 April 2004
.......................
Br J Ophthalmol 2004;88:1439–1442. doi: 10.1136/bjo.2003.040402
Aim: To determine whether silicone oil concentrates protein and growth factors in the retro-oil fluid.
Methods: A laboratory analysis of intraocular fluid and vitreous specimens obtained from patients
undergoing removal of silicone oil, revision vitrectomy, or primary vitrectomy for macular hole,
proliferative vitreoretinopathy (PVR), or retinal detachment. Patients were prospectively recruited from
routine vitreoretinal operating lists. Vitreous cavity fluid and vitreous samples were analysed for the
presence of transforming growth factor beta (TGF-b
2
), basic fibroblast growth factor (bFGF), interleukin 6
(IL-6), and total protein using either commercially available enzyme linked immunosorbent assays (ELISA)
or protein assay kits.
Results: The median levels of bFGF, IL-6, and protein in the retro-oil fluid were raised (p,0.05) compared
to all the other vitreous and vitreous cavity fluid samples. bFGF, IL-6, and protein levels were raised in PVR
vitreous compared to non-PVR vitreous. TGF-b
2
levels were not significantly raised in retro-oil fluid or in
PVR vitreous.
Conclusions: The concentration of fibrogenic (bFGF) and inflammatory (IL-6) growth factors and protein is
raised in retro-silicone oil fluid. This may contribute to the process of retro-oil perisilicone proliferation and
subsequent fibrocellular membrane formation.
S
ince the early use of silicone oil for the treatment of
complex retinal detachment
1
there have been concerns
about its potential retinal toxicity.
23
In general, these
have not been substantiated by clinical experience and
silicone oil has been used increasingly for the anatomical
reattachment of the retina in patients with complex retinal
detachments associated with proliferative diabetic retino-
pathy,
45
proliferative vitreoretinopathy (PVR),
6
giant retinal
tears,
7
and trauma.
8
The potential of silicone oil to promote a proliferative
response within the eye has been an additional concern and
Lewis
9
has observed that silicone oil appears to enhance
cellular proliferation and formation of preretinal membranes
in eyes treated for advanced PVR. Subsequent reports have
also noted an incidence of perisilicone proliferation.
10
An
analysis of PVR epiretinal membranes has also shown that
membranes from eyes with silicone oil contain T lymphocytes
in contrast with membranes from eyes without silicone oil
which had no T lymphocytes,
11
suggesting that an inflam-
matory reponse to silicone oil may contribute to fibrosis.
These clinical observations have been supported by analysis
of animal models.
12 13
In an experimental study Lambrou and
co-workers
14
reported that silicone oil increases the risk of
experimental PVR using an in vitro proliferation assay and
also demonstrated that vitreous cavity silicone oil had
increased mitogenic activity for retinal pigment epithelial
cells compared to gas filled or fluid filled vitreous. They
reasoned that silicone oil appears to increase proliferation by
stimulating the release of more or different mitogenic factors
as well as potentially concentrating active factors into a
smaller volume near the retina.
This study was designed to investigate the presence of
growth factors potentially able to contribute to the formation
of fibrocellular membranes in the fluid surrounding silicone
oil in human eyes that have undergone vitreoretinal surgery.
We compared this (retro-oil) fluid with vitreous cavity fluid
from patients undergoing revision vitrectomy surgery and
with vitreous from patients undergoing primary vitrectomy
surgery for (a) macular hole and (b) primary retinal
detachment with and without PVR. We analysed the
fibrogenic growth factors transforming growth factor beta
(TGF-b
2
) and basic fibroblast growth factor (bFGF) and the
inflammatory cytokine interleukim 6 (IL-6), together with
total protein levels. Where appropriate statistical comparison
was also carried out on the differing control samples.
PATIENTS AND METHODS
The study was conducted in accordance with the principles
embodied in the Declaration of Helsinki and informed
consent was obtained from all patients after the nature of
the study was explained.
Three hundred and nine patients undergoing vitreoretinal
procedures at Moorfields Eye Hospital (see table 1) were
prospectively enrolled in the study from 1995 to 1997. Retro-
oil fluid samples were taken from phakic patients undergoing
removal of silicone oil. Preoperative PVR was considered to be
present if 1 clock hour or more of grade C PVR was present
according to the Retinal Society PVR classification.
15
None of
the patients recruited had a previous history of diabetic
retinopathy, uveitis, or other ocular pathology which could
influence levels of protein or growth factors in the posterior
segment. Medical grade silicone oil (Dow-Corning), 1000
centistokes, was used in all eyes which had silicone oil
tamponade.
Collection of vitreous
At the beginning of surgery, before commencing intraocular
infusion, an undiluted sample of vitreous, retro-oil fluid, or
vitreous cavity fluid was obtained. To obtain retro-oil fluid a
20 gauge cannula attached to a syringe was introduced into
the fluid behind the oil. Fluid was then aspirated to obtain a
sample of approximately 500–750 ml. In the other cases
Abbreviations: bFGF, basic fibroblast growth factor; ELISA, enzyme
linked immunosorbent assays; IL, interleukin; PVR, proliferative
vitreoretinopathy; TGF-b
2
, transforming growth factor beta
1439
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undiluted vitreous or vitreous cavity fluid samples were
collected by aspiration through the vitreous cutter. Undiluted
samples were divided into aliquots in siliconised tubes
(Eppendorf, Freemont, CA, USA) and kept frozen at 270
˚
C
until each analysis.
Enzyme linked immunosorbent assays (ELISA)
Levels of TGF-b2, bFGF, and IL-6 were analysed using
sandwich enzyme immunoassay kits (R&D Systems, Oxon,
UK). Pilot studies were performed to determine the appro-
priate dilutions for each growth factor. The sample volumes
used were 200 ml (1:24 dilution) for TGF-b2, 150 ml (1:15
dilution) for bFGF, and 200 ml (1:75 dilution) for IL-6. The
minimum detectable concentrations (sensitivity) for the
assay kits were 2.00 pg/ml, 0.043 pg/ml, and 0.08 pg/ml for
TGF-b2, bFGF and IL-6 respectively. Since the assay for TGF-
b2 only detects its active form, samples to be analysed for this
factor were first activated by the addition of 1 M HCl (40 ml
HCl/ 200 ml sample). These were then neutralised with 40 ml
1.2 M NaOH/ 0.5 M HEPES. The assays therefore measured
the total amount of potentially active TGF-b2.
Protein analysis
The total protein concentrations of the samples were
measured using a commercial assay (Protein Microassay;
BioRad, Herts, UK). This colorimetric assay is a solution of
cupric ions that forms a copper-protein complex (coloured
compound) with protein. It allows rapid screening of multi-
ple small volume fluid and vitreous samples.
Statistical analysis
Growth factor levels were compared between retro-oil fluid
and all other vitreous and vitreous fluid samples individually
using the Wilcoxon rank sum (Mann-Whitney) test. The
retro-oil fluid samples were also compared with the control
specimens combined. Selected comparison of control speci-
mens was carried out where these were considered to be
biologically relevant: revision vitrectomy fluid was compared
to PVR and to non-PVR vitreous and PVR vitreous was
compared to non-PVR vitreous. Non-parametric methods
were used because the data showed considerable skewness.
No adjustment was made for multiple testing because the
analysis was hypothesis generated. Statistical calculations
were performed using commercial software (Stata,
StataCorp. 2003. Stata Statistical Software: Release 8.0.
College Station, TX, USA: StataCorp LP)
RESULTS
Complete data were available for all patients. Of the 13
patients undergoing removal of silicone oil from whom retro-
oil fluid was obtained two had had previous (preoperative)
PVR and seven had evidence of new proliferation (post-
operative) PVR before oil removal. In these patients the
duration of silicone oil tamponade ranged from 1 to
26 months with a median of 3 months. Two patients had
oil in situ for longer than 6 months but their protein and
growth factor levels were not markedly different from those
with lesser durations. All 13 retro-oil fluid patients had had
silicone oil injected because of clinical features suggestive of a
high risk of postoperative PVR development: two had existing
PVR, two had had previous retinal detachment surgery, one
had a giant retinal tear, and the others had large or multiple
retinal breaks.
In the revision vitrectomy fluid group three of 11 cases had
PVR; 34 of the total of 298 non-retro-oil control samples had
PVR.
The median levels of growth factors and protein for the
various groups are documented in table 1. The probability
values for the statistical analyses of comparisons of medians
using the Mann-Whitney test are presented in table 2. The
median levels of bFGF and IL-6 in the retro-oil fluid were
significantly raised compared to all the other vitreous and
vitreous fluid samples except vitreous from eyes with PVR
(table 2) and the IL-6 value in revision vitrectomy. Protein in
retro-oil fluid was raised in comparison with all other
specimens. There was little evidence of any difference,
however, between the median level of TGF-b
2
in the retro-
oil fluid compared to the other samples (tables 1 and 2).
PVR vitreous had raised levels of bFGF, IL-6, and protein
but not TGF-b
2
when compared to non-PVR vitreous (tables 1
and 2). Levels of growth factors and protein in revision
vitrectomy fluid showed little difference from vitreous from
eyes with or without PVR apart from a significantly raised
level of bFGF in eyes with PVR (tables 1 and 2).
DISCUSSION
This study demonstrates that two fibrogenic cytokines (bFGF
and IL-6) and total protein levels are significantly raised in
the retro-oil fluid compartment compared to vitreous from
eyes with macular holes and retinal detachment without PVR
and compared to vitreous cavity fluid from eyes undergoing
revision vitrectomy. Total protein was also raised when
analysed against vitreous from eyes with PVR. Selecting the
optimal control specimens for the comparison of retro-
silicone oil fluid is problematic. Vitreous from eyes with
retinal detachment, with and without PVR, or with macular
hole serves as a useful measure of growth factors levels in
more ‘‘normal’’ situations in the eye. The use of formed
vitreous itself could, however, produce a bias both in the
intraocular distribution of protein molecules and in the
ELISA assay. We have therefore additionally used vitreous
cavity fluid from eyes undergoing revision vitrectomy surgery
Table 1 Patient groups, growth factor, and protein levels
Sample type
TGF-b
2
(ng/ml) B-FGF (pg/ml) Il-6 (pg/ml) Protein (mg/ml)
Median (IQR) Median (IQR) Median (IQR) Median (IQR)
Retro-oil fluid (n = 13) 1.65 (1.03–2.30) 50.85 (28.46–60.48) 572.18 (523.81–581.37) 736.32 (343.63–1368.56)
Macular hole vitreous (n = 10) 1.07 (0.65–1.90) 0.18 (0.15–1.76) 0.98 (0.9–1.43) 20.60 (8.95–49.83)
Revision-vitrectomy fluid
(n = 11)
1.24 (0.93–1.78) 2.14 (1.05–2.48) 515.18 (78.38–615.08) 149.22 (46.77–593.8)
No preoperative PVR vitreous
(n = 244)
1.54 (0.70–3.13) 1.99 (0.72–10.77) 106.88 (31.28–384.23) 75.5 (30–151)
Preoperative PVR vitreous
(n = 31)
1.70 (1.03–2.85) 13.21 (5.11–59.19) 296.93 (63.0–628.58) 218 (112–706.92)
Non retro-oil samples
combined
1.51 (0.79–3.1) 2.23 (0.77–12.63) 110.1 (29.18–418.65) 81.5 (30.415–173.98)
IQR, interquartile range.
1440 Asaria,Kon,Bunce,etal
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to eliminate this potential bias. The use of a number of
control specimens from differing conditions strengthens the
argument that the statistically significant differences seen in
growth factor and protein levels in retro-oil fluid represent a
real pathobiological alteration in their concentration. The
consistency of the results of growth factor and protein
analyses between control specimens (and for the individual
growth factors, see tables 1 and 2) is further evidence that the
controls used are valid.
An effect of the underlying pathology on growth factor
levels cannot be discounted, again the use of multiple
controls helps to define the role of silicone oil in the altered
growth factor concentrations we have demonstrated. It is
notable that levels of growth factors were not significantly
different in retro-oil fluid from PVR vitreous suggesting that
the presence of PVR (seen in seven of 13 retro-oil fluid
specimens) has an important influence on growth factor
pathobiology.
Basic FGF has been shown to enhance the proliferation of
Muller cells, retinal astrocyes, and retinal pigment epithelial
cells in vivo,
16
and has previously been shown to be elevated
in the vitreous of eyes developing PVR
17–19
(a similar finding
to that of the analysis of controls in this study) and clearly
has the potential to have a role in mediating the proliferative
and fibrogenic responses in PVR. Raised levels in the
intraocular fluid surrounding silicone oil point to a potential
similar role in perisilicone proliferation.
IL-6 is an important mediator of the acute phase reaction
in inflammatory and immune responses. Cells with IL-6
mRNA expression have been found in PVR epiretinal
membranes
20
and previous work has also have shown that
the vitreous levels of IL-6 are significantly raised in vitreous
samples obtained from patients with PVR.
18 19 21
The consis-
tent finding of raised IL-6 levels is evidence for its role in the
marked blood-retinal barrier breakdown seen in PVR.
Both basic FGF and IL-6 have the potential to contribute to
the formation of fibrocellular epiretinal membranes and the
concentration of these growth factors found in the retro-
silicone oil fluid points to a possible pathogenic mechanism
for perisilicone membrane formation. It is notable that
animal studies have demonstrated silicone oil can have an
enhanced mitogenic effect on RPE cells and an increased PVR
rate in silicone filled vitreous.
14
The present investigation has additionally shown that
there is an increase in total protein in the retro-oil fluid. This
may be a result of a concentration effect caused by the
enhanced inflammatory response and blood-retinal barrier
breakdown seen in eyes that will generally have an existing
predisposition to fibrogenic proliferation. An increased total
protein level may represent a marker of the concentration of
inflammatory and fibrogenic mediators in the retro-oil fluid
milieu that could be central to the reproliferation which can
complicate complex retinal reattachment surgery. However, it
is notable that, compared to control samples, TGF-b levels
were not raised in the retro-oil fluid (or in the PVR vitreous or
revision vitrectomy fluid). This suggests that the increased
levels of bFGF and IL-6 may be the result of enhanced active
secretion rather than a more generalised concentration effect
of all soluble mediators. Alternatively there may be an
enhanced tissue uptake and/or clearance of TGF-b and it may
still have an important biological effect in PVR related
fibrogenesis. An additional possibility is that, since our
methodology detects total TGF-b, there may be an increase
in biologically active growth factor in the eye which we have
not detected and which could enhance PVR development.
The nature and incidence of perisilicone proliferation
remain uncertain. Previous reports have documented recur-
rent epiretinal proliferation behind silicone oil. Lewis et al
9
found that 19 of 31 eyes (61%) developed perisilicone
proliferation (and that this led to redetachment in 15 eyes)
and Zilis et al reported 21 of 55 eyes (38%) developed
perisilicone proliferation.
10
In the silicone study, however,
there was no difference in prevalence of postoperative
macular pucker between eyes randomised to gas or to
silicone oil, suggesting that both may have an effect on
postoperative epiretinal membrane formation.
22
The purity of
the silicone oil used may have an important role in the
incidence of secondary proliferation in these eyes and it has
been shown experimentally that contaminated silicone oil
has an enhanced effect on retinal pigment epithelium
proliferation compared to purified, medical grade silicone
oil.
23
Improvements in the quality of intraocular silicone oils
since their initial introduction can therefore be anticipated to
result in a decline in the incidence of perisilicone prolifera-
tion. Oil purity, however, may not be the only factor leading
to a predisposition to secondary membrane formation and
the evidence of this study is that, although purified, medical
grade silicone oil was used in all cases there remains a
concentration effect on potentially fibrogenic mediators.
Despite these caveats silicone oil remains a vital tool in the
management of complex retinal detachment. Vitreoretinal
surgeons, however, should be aware that its biological effects
will alter intraocular physiology and can potentially con-
tribute to enhanced fibrous scarring. In addition, the
presence of silicone oil will alter the pharmacodynamics of
any adjunctive treatments aimed at modifying the PVR
process
24
potentially producing a concentrated ‘‘depot’’ in the
retro-oil fluid.
Authors’ affiliations
.....................
R H Y Asaria, C S Sethi, G A Limb, P T Khaw, D G Charteris, Institute of
Ophthalmology, Bath Street, London EC1V 9EL, UK
R H Y Asaria, C S Sethi, G A Limb, P T Khaw, D G Charteris, C H Kon,
C Bunce, G W Aylward, Moorfields Eye Hospital, City Road, London
EC1V 2PD, UK
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Table 2 Probability (p) values for comparisons of specimens (Wilcoxon rank sum (Mann-
Whitney) test)
Comparison TGF-b bFGF IL-6 Protein
Retro-oil v macular hole 0.24 0.002 0.0001 0.0001
Retro-oil v revision vitrectomy 0.54 0.0007 0.32 0.005
Retro-oil v non-PVR vitreous 0.76 0.0001 0.0003 ,0.0001
Retro-oil v PVR vitreous 0.72 0.063 0.052 0.015
PVR vitreous v non-PVR vitreous 0.27 0.0004 0.035 ,0.0001
Revision vitrectomy v non-PVR vitreous 0.86 0.69 0.086 0.17
Revision vitrectomy v PVR vitreous 0.29 0.0027 0.92 0.18
Retro-oil v combined control specimens 0.75 0.0002 0.0005 ,0.0001
Retro-oil fluid 1441
www.bjophthalmol.com
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