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Free Radicals in Phacoemulsification and Aspiration Procedures
Hiroshi Takahashi, MD;
Atsuhiro Sakamoto, MD;
Ryoki Takahashi, PhD;
Takeo Ohmura, PhD;
Shigeto Shimmura, MD;
Kunitoshi Ohara, MD
Arch Ophthalmol. 2002;120:1348-1352.
ABSTRACT
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Objectives To detect free radicals in phacoemulsification and aspiration procedures
using electron-spin resonance and to observe the effect of ophthalmic viscosurgical
devices (viscoelastic agents) on free radical intensity.
Methods (1) A test tube containing BSS Plus (Alcon Laboratories, Inc, Fort Worth,
Tex) with 1% of the spin-trapping agent, 5,5'-dimethyl-1-pyrroline N-oxide, without irrigation and aspiration, was exposed
to ultrasound (100% for 20 seconds). A preparation of hyaluronate sodium (Healon
[a cohesive agent that contains 1% hyaluronate sodium] {Pharmacia, Uppsala,
Sweden} or Viscoat [a dispersive agent that contains 3% hyaluronate sodium
and 4% chondroitin sulfate] {Alcon Laboratories, Inc}) was added to the solution
to observe inhibitory effects. (2) To simulate a clinical procedure, an eye
model with irrigation and aspiration of a combination of 1% 5,5'-dimethyl-1-pyrroline N-oxide and BSS Plus, 25 mL/min, as the irrigating solution
was exposed to ultrasound (for 10, 20, or 30 seconds). Healon or Viscoat was
injected into the anterior chamber. Free radicals were measured by an electron-spin
resonance spectrometer.
Results (1) A characteristic signal corresponding to hydroxyl radicals was detected.
Similar inhibition by Healon and Viscoat was observed. (2) Two ophthalmic
viscosurgical devices similarly suppressed the signal at 10 seconds. The inhibition
by Healon ceased at 20 seconds, whereas Viscoat suppressed the signal throughout
the time course.
Conclusions Phacoemulsification produces hydroxyl radicals in the anterior chamber
even with irrigation and aspiration. The effect of ophthalmic viscosurgical
devices on free radicals depends on the retention of the materials within
the anterior chamber.
Clinical Relevance There are complications associated with phacoemulsification.
INTRODUCTION
PHACOEMULSIFICATION and aspiration (PEA) has become the most popular
cataract surgery owing to the establishment of safe surgical techniques and
the development of associated instruments. Corneal endothelial damage, however,
can still be a serious complication because excessive damage can lead to irreversible
bullous keratopathy. Surgeons need to be aware of this particular problem
to prevent endothelial damage, especially when functional reservoirs are poor.
Several causes that lead to damage have been documented, and include items
such as mechanical or heat injuries.
Recently, free radical formation due to ultrasound (US) has been postulated
to be another cause of the damage. In experimental situations, free radical
formation caused by US in conjunction with commercially available PEA devices
has been reported.1-3 Considering
the oxidative insult to the endothelial cells caused by free radicals, their
presence in the anterior chamber may be one of the most harmful factors during
these procedures.4-5
With regard to protecting the endothelium from mechanical injuries,
the effectiveness of ophthalmic viscosurgical devices (OVDs), the new term
recommended by the International Organization for Standardization for viscoelastic
agents,6 has been well documented. The major
ingredient of OVDs is hyaluronate sodium, which is known to be a free radical
scavenger. Several studies7-8 have
revealed that hyaluronate sodium plays an important protective role against
the oxidative damage in patients with arthritis. Hyaluronate sodium injection
therapy into the joint cavity was introduced with the expectation that it
would provide an anti–free radical effect.9 Other
ophthalmic studies10-11 have also
reported on the protective properties of hyaluronate sodium on oxidative stress
in the corneal endothelium. Thus, OVDs can be expected to provide some anti–free
radical effect during PEA procedures. One study2 has
shown that Healon (a cohesive agent that contains 1% hyaluronate sodium) (Pharmacia,
Uppsala, Sweden), when added to the irrigating solution, reduced the free
radical concentration caused by US.
To our knowledge, however, there has been no study to investigate free
radical intensity during standard PEA procedures in which the OVD is injected
into the anterior chamber followed by US with irrigation and aspiration (I/A).
It is reasonable to assume that the free radical concentration will be affected
by the continuous irrigation. In addition, because commercially available
OVDs have different properties regarding their retention in the anterior chamber
during PEA, the anti–free radical effect of OVDs is likely to depend
on their behavior during I/A.
In this study, we followed standard PEA procedures in an eye model and
measured free radical signals with the electron-spin resonance (ESR) procedure.
The kinetics of the free radical intensity and the effects of several OVDs
during clinical PEA were also demonstrated by our study.
METHODS
IN VITRO STUDY
The method for free radical detection by ESR has been described previously.12 All measurements were repeated 5 times. For a spin-trapping
agent, 5,5'-dimethyl-1-pyrroline N-oxide (DMPO)
(Sigma-Aldrich Japan, Tokyo) was used. Before each experiment, nitrogen gas
was bubbled into the solution for 15 minutes to purge oxygen and prevent nonspecific
oxidation of the DMPO.
To confirm the results of previous studies,1-3 we
first performed an in vitro experiment. In 2-mL plastic test tubes, 1.5 mL
of a combination of 1% DMPO and BSS Plus (Alcon Laboratories, Inc, Fort Worth,
Tex) solution was prepared (control). The US probe of a commercially available
PEA device (Series Ten Thousand Phacoemulsifier; Alcon Laboratories, Inc)
was placed in the center of the tube, and US was performed at a power level
of 100% for 20 seconds without I/A. Immediately after US, 300 µL of
the solution was transferred to a flat quartz ESR cuvette. The cuvette was
then placed in an ESR spectrometer (model JES-RE3X; JEOL, Tokyo), and the
signal of the spin adducts, the hydroxyl radical (· OH) trapped by
DMPO, was measured by double integration using a computer software program.
To observe the effect of an · OH-specific scavenger, 1% or 10% dimethyl
sulfoxide (Sigma-Aldrich Japan) was added to the solution before US. For observation
of the effects of the OVDs, Healon or Viscoat (a dispersive agent that contains
3% hyaluronate sodium and 4% chondroitin sulfate) (Alcon Laboratories, Inc)
was added to the solutions to achieve either a 0.1- or a 0.3-mg/mL final concentration
before US.
EYE MODEL STUDY
To detect free radicals in conditions simulating a standard clinical
PEA procedure, we used a commercially available eye model (Marty System; Iatrotech,
Menlo Park, Calif) that was developed for use as a training procedure for
various ophthalmic surgical procedures, including PEA. BSS Plus containing
1% DMPO was used as an irrigating solution. The PEA probe was inserted through
a 3.2-mm incision, and the tip was fixed at the center and on the iris plane
of the model eye. PEA was performed for 10, 20, or 30 seconds with 100% US
power level. The following experimental protocols were examined: (1) control,
no I/A and no OVD; (2) BSS Plus group, I/A (25 mL/min) and no OVD; (3) Healon
group, I/A (25 mL/min) and injection of 0.3 mL of Healon into the anterior
chamber before US; and (4) Viscoat group, I/A (25 mL/min) and injection of
0.3 mL of Viscoat into the anterior chamber before US. After PEA, 300-µL
samples of the solutions were collected from the anterior chambers, and free
radical intensity was determined as previously described.
DATA ANALYSIS
The intensities of the signals were calculated through image analysis
after standardization using the amplitudes of the Manganese signal. Statistical
analysis of the digitized data was performed by the Dunnett test and the t test, and P<.05 was considered
significant.
RESULTS
IN VITRO STUDY
The ESR spin adducts of the sample revealed the characteristic quartet
signal pattern (Figure 1), which
is specific for · OH. The hyperfine coupling constants for the spin
adduct ( N = 1.49 and H = 1.49 milli-tesla)
are consistent with those for · OH according to a previous report.13 Superoxide-related signals were not detected. There
was a dose-dependent inhibition observed with the addition of dimethyl sulfoxide
that supports the fact that the signals corresponded to · OH signals.
Ophthalmic viscosurgical devices also suppressed the signals in a dose-dependent
manner. The suppression was similar for the 2 agents tested (Figure 2).
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Figure 1. Representative signals in the
in vitro experiment. Spectrometer settings were as follows: modulation frequency,
100 kHz; microwave frequency, 9.4 GHz; microwave power, 10 mW; scan time,
120 seconds; time constant, 0.3 seconds; and receiver gain, 2500. Healon is
a cohesive agent that contains 1% hyaluronate sodium, and Viscoat is a dispersive
agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate. DMPO
indicates 5,5'-dimethyl-1-pyrroline N-oxide;
· OH, hydroxyl radical; and Mn(3) and Mn(4), the third and fourth signals,
respectively, of the manganese in the electron-spin resonance spectra.
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Figure 2. Signal intensities shown with
an arbitrary unit in the in vitro experiment. Dimethyl sulfoxide, Healon (a
cohesive agent that contains 1% hyaluronate sodium), and Viscoat (a dispersive
agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate) significantly
suppressed the signal vs control (P<.05, each
agent vs control). The difference between Healon and Viscoat was not significant
(P = .78).
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EYE MODEL STUDY
Although the change was smaller compared with the in vitro experiments,
the presence of the quartet signal was also revealed by ESR in the eye model
experiments (Figure 3). In the control,
signals increased and reached a plateau at 20 seconds. In the BSS Plus group,
signals were enhanced in a time-dependent fashion, but the intensity at 30
seconds was not significantly different from that at 20 seconds. In the Healon
group, although the signals at 10 seconds were significantly smaller than
for the BSS Plus group, signals at 20 to 30 seconds were similar to those
of the BSS Plus group. On the contrary, Viscoat significantly suppressed the
signals throughout the entire time course (Figure 4).
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Figure 3. Representative signals in the
eye model experiment. A, Control and BSS Plus groups. B, Healon (a cohesive
agent that contains 1% hyaluronate sodium) and Viscoat (a dispersive agent
that contains 3% hyaluronate sodium and 4% chondroitin sulfate) groups. Even
with irrigation and aspiration, ultrasound produced the characteristic quartet
signals in the BSS Plus group. DMPO indicates 5,5'-dimethyl-1-pyrroline N-oxide; · OH, hydroxyl radical; and Mn(3) and Mn(4),
the third and fourth signals, respectively, of the manganese in the electron-spin
resonance spectra.
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Figure 4. Signal intensities shown with
an arbitrary unit in the eye model experiment. Inhibition by Healon (a cohesive
agent that contains 1% hyaluronate sodium) was significant only at 10 seconds
(Healon vs BSS Plus group, P = .002), while suppression
by Viscoat (a dispersive agent that contains 3% hyaluronate sodium and 4%
chondroitin sulfate) was significant even at 30 seconds (Viscoat vs BSS Plus
group, P = .004). The differences at 20 and 30 seconds
were not significant for the control (P = .58), BSS
Plus (P = .13), and Healon (P =
.36) groups. Data are given as mean ± SD. The asterisk indicates P<.05.
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COMMENT
The mechanism of free radical formation by US is thought to occur as
follows. Ultrasound in aqueous solutions induces acoustic cavitation that
causes gas bubbles to collapse, leading to the thermal dissociation of water
vapor into · OH and hydrogen atoms.14 Free
radical formation associated with clinical PEA, therefore, seems inevitable.
In fact, several studies have demonstrated ophthalmic PEA device-related free
radical formation. Shimmura et al1 first described
free radical formation in vitro, and Holst et al2 demonstrated
this phenomenon in vivo. Both studies, however, used the chemiluminescence
technique that, while suitable for detecting superoxides, does not detect
· OH signals, the most potent of the free radical species.
Because · OH is highly reactive and short-lived, measurements
are achieved by using radical trap agents and the detection of these radical
adducts by ESR. Recently, Cameron et al3 reported
on the ability to detect · OH formation through ESR. They applied US
in a test chamber with a closed circulation loop in which the same solution
was recirculated. During clinical PEA, however, there is I/A of the solution
at various rates. Thus, the aqueous humor is continuously replaced by the
irrigating solution. Consequently, the actual free radical concentration in
the anterior chamber is determined by the ratio of its production and subsequent
clearance.
During clinical PEA, an OVD is injected into the anterior chamber before
US. An OVD reduces free radical concentration caused by US when added to the
test solution in vitro and in vivo.2 Furthermore,
because each of the OVDs has different properties regarding its retention
in the anterior chamber during PEA, the individual behavior of each agent
during PEA may alter the net result that occurs. Considering these factors,
we sought to simulate the clinical PEA procedures in an eye model and detect
free radicals via ESR.
In the in vitro experiment, in which the reacted solution was collected
intact, notable signals for · OH were detected, confirming the results
of a previous report.3 Ophthalmic viscosurgical
devices suppressed the free radical intensity in a dose-dependent fashion,
suggesting that the OVD, a radical scavenger itself, functions as an alternate
reactant for the radicals and consequently reduces free radical concentration
in the aqueous solution. Interestingly, there was no significant difference
between the 2 OVDs when compared at the same concentration. Healon contains
only 1% hyaluronate sodium, while Viscoat comprises 3% hyaluronate sodium
and 4% chondroitin sulfate, which is also known as a free radical scavenger.15-16 These results indicate that the anti–free
radical effects of hyaluronate sodium and chondroitin sulfate are not synergetic,
at least in the present experimental setup.
In the eye model study, the experimental conditions for the control
(no I/A and no OVD) were almost the same as for the in vitro study. Signals
increased until 20 seconds but then seemed to reach a plateau, suggesting
that the DMPO trapping mechanism was saturated. In the BSS Plus group, in
which I/A continuously replaced the reacted solution, smaller but characteristic
signals of · OH were detected. To our knowledge, this is the first
ESR evidence that during PEA there is existence of · OH in the anterior
chamber even with I/A.
Although signals increased in a time-dependent manner, the intensity
at 30 seconds was not significantly different from that at 20 seconds. Cameron
et al3 reported that, in their closed circulation
system, the · OH concentration was proportional to US duration. Present
results indicate that with I/A, free radicals may reach a stable concentration
because of a constant production and clearance ratio. In the Healon and the
Viscoat groups, similar inhibition of the signals was observed at 10 seconds.
At 20 and 30 seconds, however, the suppression compared with the BSS Plus
group was significant only in the Viscoat group. This indicates that Healon
was flushed out by I/A by the 20-second point, while Viscoat was retained
in the anterior chamber long enough to exhibit the effect for at least 30
seconds.
These data confirm the results from previous studies concerning the
retention of OVDs during PEA. Assia et al17 experimentally
compared the removal time for several OVDs from the anterior chamber due to
I/A. They found that the removal time for the cohesive agent, Healon, was
20 to 25 seconds, while that for the dispersive agent, Viscoat, was 3.5 minutes.
Poyer et al18 quantitatively measured the vacuum
levels when bolus removal of the materials occurred, and showed that such
a phenomenon was commonly observed with cohesive agents, including Healon,
but not with Viscoat. Viscoat coats the endothelial cells to a thicker degree
than any other agents after a PEA procedure.19 In
this study, the longest US duration was 30 seconds in the eye model experiment,
while it is usually longer than 30 seconds during clinical PEA. Presumably,
the longer US time would cause the more enhanced signals. Yet, it is likely
that Viscoat would remain in the anterior chamber to some extent even after
a longer duration and consequently inhibit the signals, considering the results
of the previously cited reports.17-19
The evidence so far is that the more dispersive the agent is, the more
retention that is seen in the anterior chamber during and after PEA. The present
results suggest that the anti–free radical effect of the OVD depends
on its retention in the anterior chamber during PEA. Thus, Viscoat, among
all of the commercially available OVDs, seems to be the most effective agent
for the protection of the endothelium from free radicals. Recently, a new
OVD was introduced that has similar properties to Viscoat with regard to retention
in the anterior chamber.20 Thus, it may also
have a comparable effect to Viscoat with regard to free radical concentration.
In conclusion, we demonstrated that · OH production can be documented
by ESR in conditions that simulate clinical PEA procedures and that the OVD
anti–free radical effects seen are correlated to the retention times
of the OVD within the anterior chamber during the procedure. However, many
radical scavengers exist within the anterior chamber in vivo and may play
a part in the free radical concentration.21-22 Furthermore,
during clinical PEA procedures, there are many other factors that can cause
endothelial damage, including shock wave injury, fluid-flow turbulence injury,
and thermal injury. We do not have any direct evidence as to how harmful the
radicals may actually be to the endothelial cells. Thus, further studies are
needed to elucidate the actual damage caused by the free radicals associated
with clinical PEA.
AUTHOR INFORMATION
Submitted for publication December 18, 2001; final revision received
March 27, 2002; accepted April 30, 2002.
Corresponding author and reprints: Hiroshi Takahashi, MD, Department
of Ophthalmology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602,
Japan (e-mail: tash{at}nms.ac.jp).
From the Departments of Ophthalmology (Drs H. Takahashi and Ohara)
and Anesthesiology (Dr Sakamoto), Nippon Medical School, Tokyo; Sagami Laboratory,
Wakamoto Pharmaceutical Co, Ltd, Kanagawa (Drs R. Takahashi and Ohmura); and
the Department of Ophthalmology, Tokyo Dental College, Chiba (Dr Shimmura),
Japan. None of the authors has any commercial or proprietary interest in the
products or companies described in this article.
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