PRECLINICAL STUDY
Serofendic Acid, a Novel Substance Extracted From Fetal Calf Serum, Protects Against Oxidative Stress in Neonatal Rat Cardiac Myocytes
Toshihiro Takeda, MD*,
Masaharu Akao, MD, PhD*,*,
Madoka Matsumoto-Ida, MD*,
Masashi Kato, MD*,
Hiroyuki Takenaka, MD*,
Yasuki Kihara, MD, PhD*,
Toshiaki Kume, PhD ,
Akinori Akaike, PhD and
Toru Kita, MD, PhD*
* Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan
Department of Pharmaceutical Science, Kyoto University Graduate School of Pharmacology, Kyoto, Japan.
Manuscript received June 19, 2005;
revised manuscript received December 14, 2005,
accepted December 19, 2005.
* Reprint requests and correspondence: Dr. Masaharu Akao, Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. (Email: akao{at}kuhp.kyoto-u.ac.jp).
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Abstract
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OBJECTIVES: We examined whether serofendic acid (SFA) has protective effects against oxidative stress in cardiac myocytes.
BACKGROUND: We previously identified a novel endogenous substance, SFA, from a lipophilic extract of fetal calf serum. Serofendic acid protects cultured neurons against the cytotoxicity of glutamate, nitric oxide, and oxidative stress.
METHODS: Primary cultures of neonatal rat cardiac myocytes were exposed to oxidative stress (H2O2, 100 µmol/l) to induce cell death. Effects of SFA were evaluated with a number of markers of cell death.
RESULTS: Pretreatment with SFA (100 µmol/l) significantly suppressed markers of cell death, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and cell viability assay. Loss of mitochondrial membrane potential (  m) is a critical step of the death pathway, which is triggered by matrix calcium overload and reactive oxygen species. Serofendic acid prevented the m loss induced by H2O2 in a concentration-dependent manner (with saturation by 100 µmol/l). Serofendic acid remarkably suppressed the H2O2-induced matrix calcium overload and intracellular accumulation of reactive oxygen species. The protective effect of SFA was comparable to that of a mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, diazoxide. Furthermore, mitoKATP channel blocker, 5-hydroxydecanoate (500 µmol/l), abolished the protective effect of SFA. Co-application of SFA (100 µmol/l) and diazoxide (100 µmol/l) did not show an additive effect. Thus, SFA inhibited the oxidant-induced mitochondrial death pathway, presumably through activation of the mitoKATP channel.
CONCLUSIONS: Serofendic acid protects cardiac myocytes against oxidant-induced cell death by preserving the functional integrity of mitochondria.
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Abbreviations and Acronyms
| | 5-HD = 5-hydroxydecanoate | | m = mitochondrial membrane potential | | [Ca2+]m = mitochondrial matrix calcium | | DAPI = 4’,6-diamidino-2-phenylindole | | DCF = chloromethyl-2,7-dichlorodihydrofluorescein diacetate | | DMEM = Dulbecco’s Modified Eagle Medium | | FACS = fluorescence-activated cell sorter | | MitoKATP = mitochondrial adenosine triphosphate-sensitive potassium | | MPTP = mitochondrial permeability transition pore | | ROS = reactive oxygen species | | SFA = serofendic acid | | TMRE = tetramethylrhodamine ethyl ester | | TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling |
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Mitochondria play critical roles in cell death in response to a variety of stresses, such as myocardial ischemia/reperfusion (1–3). Opening of the mitochondrial permeability transition pore (MPTP), a non-specific pore that opens at the contact site between outer and inner mitochondrial membranes, results in the loss of mitochondrial membrane potential (m), matrix swelling, and the release of cytochrome c and other proapoptotic factors that lead to cell death (4–6). Mitochondrial matrix calcium ([Ca2+]m) overload and reactive oxygen species (ROS) favor MPTP opening (7). Inhibition of MPTP opening by preventing [Ca2+]m overload and ROS generation will be an effective strategy for the protection of hearts from ischemia/reperfusion injury.
We have shown that adenosine triphosphate–sensitive potassium channels located in the inner mitochondrial membrane (mitoKATP channels) play a central role in the signaling cascade of protection against oxidative stress in the cardiac ventricular myocytes (8,9) and the cerebellar granule neurons (10,11). MitoKATP channels prevent [Ca2+]m overload and ROS generation, thereby inhibiting the MPTP opening in both types of cells (10,12–14). Diazoxide, a selective opener of mitoKATP channels, has been shown to have protective effects against myocardial ischemia/reperfusion both in vitro (15,16) and in vivo (17,18). Unfortunately, the clinical use of this agent has been hampered, owing to unwanted side effects, such as excessive hypotension or edema.
We previously purified a novel neuroprotective substance named "serofendic acid" (SFA) derived from the lipophilic fraction of fetal calf serum (19). The compound exhibited the ability to protect cultured cortical and striatal neurons against glutamate, nitric oxide, and H2O2 cytotoxicity (19–22). Given that H2O2 is also responsible for the tissue injury during myocardial ischemia/reperfusion, we hypothesized that SFA might have cardioprotective effects against ischemia/reperfusion injury. In the present study, we investigated whether SFA has protective effects against oxidative stress in neonatal rat cardiac myocytes.
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Methods
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All procedures were performed in accordance with the Kyoto University animal experimentation committee, which conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.
Primary culture of neonatal rat cardiac ventricular myocytes.
Cardiac ventricular myocytes were prepared from one- to two-day-old Wistar rats and cultured as previously described (8). In brief, the hearts were removed, and the ventricles were minced into small fragments, which were digested by trypsin dissociation. The dissociated cells were preplated for 1 h to enrich the culture with myocytes. The non-adherent myocytes (approximately, 30 to 50 million cells per isolation) were then plated in plating medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Kyoto, Japan) supplemented with 5% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and 2 µg/ml vitamin B12. The final myocyte cultures contained >90% cardiac myocytes at partial confluence. The cells were maintained at 37°C in the presence of 5% CO2 in a humidified incubator. Bromodeoxyuridine (0.1 mmol/l) was incubated in the medium for the first three days after plating to inhibit fibroblast growth. Cultures were then placed in serum-free DMEM containing vitamin B12 and transferrin 24 h before the drug treatment.
Experimental protocol.
Neonatal rat cardiac myocytes in primary culture were randomly assigned to one of three experimental groups: 1) control group; 2) incubation with 100 µmol/l H2O2 for 60 min; and 3) pretreated 100 µmol/l SFA for 30 min, followed by 100 µmol/l H2O2 for 60 min. At the beginning of the experiment, culture media were replaced with fresh serum-free DMEM containing those drugs, and the cells were exposed to those drugs during the entire experimental period.
The SFA was dissolved in dimethyl sulfoxide to make 100 mmol/l stock solution before being added into experimental solution. The final concentration of dimethyl sulfoxide was <0.1%.
MTS assay.
Cell viability was quantified on the basis of metabolic activity with the MTS assay (Promega, Madison, Wisconsin), according to manufacturer’s protocol.
The cultures were incubated in serum-free medium containing 20 µl/well of the MTS tetrazolium compound for 3 h at 37°C. The absorbance of formazan products was photometrically measured at 490 nm with a microplate reader, ARVOsx (PerkinElmer, Shelton, Washington). The cell viability was expressed as the percentage of the absorbance measured in the control group.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining.
The TUNEL staining was performed according to the manufacturer’s protocol (Roche, Indianapolis, Indiana). Fluorescein labels incorporated in nucleotide polymers were detected with a fluorescence microscope (Axioskop 2 plus; Zeiss, Thornwood, New York).
4',6-diamidino-2-phenylindole (DAPI) staining.
Cells were stained with the deoxyribonucleic acid binding dye DAPI (5 µmol/l; Molecular Probes, Eugene, Oregon). The nuclear morphology of cells were visualized and photographed with the fluorescence microscope.
Loading of cells with fluorescent indicator.
To monitor m, cells were loaded with tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) 100 nmol/l at 37°C for 20 min. To monitor [Ca2+]m, the cells were loaded with 2 µmol/l rhod-2 AM (Molecular Probes) at 37°C for 30 min. We assayed the intracellular ROS production with chloromethyl-2,7-dichlorodihydrofluorescein diacetate (DCF) (Molecular Probes). Cells were loaded with 4 µmol/l DCF at 37°C for 30 min, and the formation of the oxidized derivative was monitored by the increase of green fluorescence.
Fluorescence-activated cell sorter (FACS) analysis.
Cells plated on regular six-well plates (1.0 to 1.5 million cells per well) were used for the FACS analysis of m. The TMRE-loaded cells were harvested by tripsinization at the end of the experimental protocols and analyzed with FACSAria (BD Biosciences, San Jose, California) (20,000 cells/sample). The fluorescence intensity of TMRE was monitored at 582 nm (FL-2). The FACS data were analyzed with analysis software (WinMDI).
Confocal imaging.
Cells plated on 35-mm glass-bottom dishes (1.0 to 1.5 million cells per dish) were maintained at 37°C in the presence of 5% CO2 with a heater platform installed on a microscope stage and were placed in serum-free DMEM. After the desired temperature was reached, time-lapse confocal microscopy was started with 2-min intervals, with a 20x objective lens. Images were taken with laser scanning confocal microscopy (LSM510, Zeiss). The TMRE and rhod-2 AM was excited with a 543 nm line of a helium/neon laser. The DCF was excited with a 488 nm line of an argon laser.
Twenty-five cells were randomly selected in each scan by drawing regions around individual cells, and the red or green fluorescence intensity was sequentially monitored.
Image analysis.
Quantitative image analysis was performed with an image analysis software (ImageJ).
Statistical analysis.
All the quantitative data are presented as the mean ± SEM. Multiple comparisons among groups were carried out by one-way analysis of variance with Bonferroni’s post-hoc test. A level of p < 0.05 was accepted as statistically significant.
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Results
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Figure 1A demonstrates TUNEL staining and DAPI staining in each experimental group. The TUNEL staining detects nuclear deoxyribonucleic acid strand breaks, which occur during the terminal phase of apoptosis. Control cells exhibited few TUNEL-positive nuclei, but exposure to 100 µmol/l H2O2 for 16 h increased the number of TUNEL-positive nuclei, which can be seen as bright spots, indicating enhanced apoptosis under the treatment with H2O2. There were obviously fewer TUNEL-positive nuclei in the SFA-treated group, despite the similar density of cells compared with the H2O2 group. Cells incubated with 100 µmol/l H2O2 for 16 h were also stained with deoxyribonucleic acid binding dye DAPI. Fragmented or shrunken nuclei were observed in the H2O2 group, but SFA displayed a significant protective effect in the preservation of nuclear morphology. Figure 1B shows a quantitative determination of TUNEL-positive nuclei in each experimental group. The data indicate that SFA has a significant protective effect against H2O2-induced nuclear damage.
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Figure 1 (A) The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining (left panels) and the nuclear counterstaining by 4',6-diamidino-2-phenylindole (DAPI) (right panels) in neonatal rat cardiac myocytes. C = control cells; H = cells exposed to 100 µmol/l H2O2 for 16 h; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 100 µmol/l H2O2 for 16 h. Scale bars = 20 µm. (B) Quantitative determination of TUNEL-positive nuclei (n = approximately 3 to 4 for each group from two independent cultures). An average of 200 to 400 nuclei from random fields was analyzed in each sample. (C) Cellular viability evaluated by MTS assay (n = 13 for each group from two independent cultures). The H2O2 treatment was 3 h in this experiment. *p < 0.05 versus group H.
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We examined whether SFA would affect the overall viability of cultured cardiac myocytes exposed to oxidative stress. The MTS assay revealed that 100 µmol/l SFA partly but significantly protected against H2O2-induced cytotoxicity (Fig. 1C).
Loss of m is a critical event early in the process of cell death and has been linked to the opening of MPTP (3–5). To examine whether the preservation of m is associated with the cardioprotective effects of SFA, we assessed the change of TMRE fluorescence by H2O2 stimulation in each group with FACS analysis. The majority of cells in the control group (Fig. 2A, panel C) belonged to a population with a high TMRE fluorescence level (indicated by vertical dashed line). Exposure to H2O2 shifted the predominant population to a lower TMRE fluorescence (Fig. 2A, panel H). Serofendic acid protected against the H2O2-induced loss of m, preserving a population of cells with a normal m level (Fig. 2A, panel SFA). These observations were rendered quantitative by plotting the percentage of cells with high TMRE (>300, in this case), as shown in Figure 2B. Exposure to 100 µmol/l H2O2 for 1 h resulted in mitochondrial depolarization, whereas SFA prevented the loss of m in a concentration-dependent manner. The m-preserving effect of SFA reached its maximum level at 100 µmol/l. We further compared the protective effects of SFA with those of diazoxide, a mitoKATP channel opener. In isolated cardiac myocytes, we previously reported that diazoxide prevents the loss of m induced by oxidative stress in a concentration-dependent manner (8). As shown in Figure 2C, the protective effect of 100 µmol/l SFA was comparable to 100 µmol/l diazoxide (maximal protective concentration of diazoxide [8]) in preventing the loss of m induced by 100 µmol/l H2O2. The protection afforded by SFA and diazoxide was completely blocked by a mitoKATP channel blocker, 5-hydroxydecanoate (5-HD, 500 µmol/l [8]). The 5-HD alone did not aggravate the loss of m elicited by 100 µmol/l H2O2 (data not shown). Co-application of 100 µmol/l diazoxide and 100 µmol/l SFA did not exhibit an additive effect (Fig. 2C), because the combination of maximal protective concentrations of both did not exceed each single drug. In addition, SFA alone in the absence of H2O2 did not affect the control level of m (data not shown).
To further confirm the protective effect of SFA in preventing the loss of m, we examined the time-dependent changes of m on a single-cell basis (Fig. 3). Time-lapse confocal analysis of cardiac myocytes loaded with TMRE was performed at 2-min intervals. Time-lapse scanning began immediately after the application of 50 µmol/l H2O2. At first, we confirmed that TMRE fluorescence did not change during the 60 min of observation in the control group (Fig. 3A, panels C). In contrast, cells treated with H2O2 progressively lost their red fluorescence intensity, indicating the irreversible loss of m (Fig. 3A, panels H). The TMRE fluorescence was remarkably preserved in the SFA-treated group (Fig. 3A, panels SFA). Twenty-five cells were randomly selected in each group, and the TMRE fluorescence intensity from each individual cell was plotted in Figure 3B. Serofendic acid not only decreased the number of cells undergoing the dissipation of m but also delayed the onset of m loss, whereas it did not change the duration of m loss in unprotected cells. Figure 3C shows the average of TMRE fluorescence intensity from 25 randomly selected cells in each group, indicating the significant protective effects of SFA.
Reactive oxygen species is one of the most important inducers of MPTP opening. To investigate whether the suppression of ROS production is associated with the protective effects of SFA, we assessed the change of DCF fluorescence by 50 µmol/l H2O2 stimulation in each group with time-lapse confocal microscopy (Fig. 4). Cells in the control group gradually decreased the DCF fluorescence intensity (Fig. 4A, panels C). In the H2O2 group, the DCF fluorescence started to increase progressively upon H2O2 application (Fig. 4A, panels H). Serofendic acid suppressed the increase in DCF fluorescence (Fig. 4A, panels SFA). Twenty-five cells were randomly selected from each group, and the DCF fluorescence intensity from each individual cell was plotted in Figure 4B. The DCF fluorescence intensity of individual cells progressively increased in the H2O2 group, but the SFA-treated group blunted the overall increase of DCF fluorescence compared with the H2O2 group. Figure 4C shows the average of DCF fluorescence intensity from 25 randomly selected cells in each group.
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Figure 4 Time-lapse analysis of intracellular reactive oxygen species production in neonatal rat cardiac myocytes. (A) Representative sequential images of chloromethyl-2,7-dichlorodihydrofluorescein diacetate (DCF) fluorescence in each group. H = cells exposed to 50 µmol/l H2O2 for 30 min; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 50 µmol/l H2O2 for 30 min. Scale bars at 0 min frames: 20 µm. (B) Time course of DCF fluorescence of 25 cells randomly selected in each group. Similar results were obtained in three independent experiments. (C) Mean fluorescence intensity from 25 cells randomly and prospectively selected in each group. *p < 0.05 versus H at the end of the experimental period. Other abbreviations as in Figure 1. Please see the for an accompanying video to this figure.
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Calcium overload in mitochondrial matrix is one of the critical triggers of cell death and is also an important inducer of MPTP opening. To monitor the [Ca2+]m level, we performed time-lapse confocal microscopy with the [Ca2+]m sensitive dye, rhod-2, and observed the change of fluorescence due to 50 µmol/l H2O2 stimulation in each group (Fig. 5). We first confirmed that the rhod-2 fluorescence did not change during 60 min of scanning in the control group (Fig. 5A, panels C). In the H2O2 group, rhod-2 fluorescence showed pronounced elevation at approximately 20 min after H2O2 application, and the elevation persisted thereafter (Fig. 5A, panels H). Serofendic acid also partly attenuated the [Ca2+]m overload observed in the H2O2 group (Fig. 5A, panels SFA). Figure 5B shows the time course of the rhod-2 fluorescence intensity from each individual cell. Serofendic acid blunted the overall increase of rhod-2 fluorescence compared with the H2O2 group. Figure 5C shows the average of the rhod-2 fluorescence intensity from 25 randomly selected cells in each group.
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Figure 5 Time-lapse analysis of intracellular mitochondrial matrix calcium in neonatal rat cardiac myocytes. (A) Representative sequential images of rhod-2 (Molecular Probes, Eugene, Oregon) fluorescence in each group. H = cells exposed to 50 µmol/l H2O2 for 1 h; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 50 µmol/l H2O2 for 1 h. Scale bars at 0 min frames: 20 µm. (B) Time course of rhod-2 fluorescence of 25 cells randomly selected in each group. Similar results were obtained in three independent experiments. (C) Mean fluorescence intensity from 25 cells randomly and prospectively selected in each group. *p < 0.05 versus H at the end of the experimental period. Other abbreviations as in Figure 1. Please see the for an accompanying video to this figure.
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Discussion
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SFA as a cardioprotective agent.
The major findings of this study are as follows. First, in isolated cardiac myocytes, SFA suppressed the cell death induced by H2O2 by preserving the m level in a concentration-dependent manner. The preservation of mitochondrial integrity was most likely achieved by the partial inhibition of [Ca2+]moverload and ROS accumulation. Second, serofendic acid and mitoKATP opener exhibited comparable protective effects, and they did not have an additive beneficial effect on m preservation. MitoKATP channel blocker 5-HD canceled the protective effect of SFA. These observations suggest that SFA acts either directly on the mitoKATP channel or, rather, upstream of the mitoKATP channel.
We previously reported the discovery of the neuroprotective compound SFA, extracted from fetal calf serum (19,20). It is a low molecular weight substance of atisane-type diterpenoids bearing a methylsulfoxide group, a unique chemical structure among known endogenous substances. Our unpublished data (T. Kume, A. Akaike, 2005) show that SFA is contained in fetal calf serum in a considerable amount, but the content in adult bovine is below detectable level. Similarly, we were also unable to detect SFA in adult human serum, although there is a possibility that fetal human serum might contain SFA. The biosynthesis or the metabolism of SFA is unknown at present. Previous studies in vitro indicated that SFA has neuroprotective effects, as evidenced by the prevention of acute glutamate neurotoxicity in cultured cortical neurons (21) and the attenuation of ROS-induced oxidative stress in cultured striatal neurons (22). Suppression of intracellular ROS generation might constitute an important mechanism of the neuroprotective actions of SFA, because the compound exhibits hydroxyl radical-scavenging activity in electron spin resonance analysis (19).
Prevention of MPTP by SFA.
In recent studies, ROS generation and [Ca2+]m overload have been proposed to explain the pathogenesis of ischemia/reperfusion injury of the heart (3,23). Reactive oxygen species and [Ca2+]m are the most important inducers of MPTP opening. A growing body of evidence supports the concept that the inhibition of MPTP is an effective and promising strategy to prevent ischemia/reperfusion injury of the heart (3,6,24,25). We clearly showed that SFA prevents MPTP opening, as reported by the preservation of the cell population with fully-polarized (intact) m levels. Notably, SFA only partly suppressed the increases of [Ca2+]m and ROS, but this partial inhibition might decrease the number of cells that reach the threshold of the catastrophic loss of m. This is consistent with the concept that mitochondria are death signal integrators and determine the fate of cells in an all-or-none manner (26).
We have recently shown that oxidant stress produces a stereotyped progression of cellular changes in cardiac myocytes (13). The first phase we call "priming": mitochondria undergo [Ca2+]m-dependent morphological changes, but m remains unchanged. Next follows a sudden dissipation of m mediated by the opening of MPTP ("depolarization" phase); eventually, cells break up into smaller fragments ("fragmentation" phase). Serofendic acid markedly decreased the likelihood that cells would undergo priming: [Ca2+]m overload was attenuated and, consequently, many mitochondria remained fully polarized. Serofendic acid not only decreased the number of cells undergoing m depolarization but also delayed the onset of m loss, whereas it did not change the duration of depolarization in unprotected cells. This mode of action is equivalent to that of the mitoKATP channel opener diazoxide (12), raising the possibility that the cytoprotective effects of SFA are directly or indirectly mediated by the mitoKATP channel. In the present study, we used two different concentrations of H2O2:50 µmol/l in confocal time-lapse imaging, and 100 µmol/l for all the other experiments, primarily because the susceptibility of cells to H2O2 was dependent on the plating surface. We have confirmed that cells underwent similar three-step progression of cell death in an all-or-none manner, even when they were exposed to lower concentrations of H2O2 (13).
SFA and mitoKATP channel.
It is clear that a cardioprotective effect can be recruited by mitoKATP channel openers, and mitoKATP channel blockers (5-HD or glibenclamide) prevent both preconditioning and pharmacological cardioprotection (27–29). Furthermore, mitoKATP channel opening prevents mitochondrial injury, presumably by inhibiting the opening of MPTP (12,13). MitoKATP channel activation induces partial and modest m depolarization, thereby reducing the driving force for calcium uptake by mitochondria and preventing [Ca2+]m elevation (14). This is further supported by the observation that partial m depolarization elicited by the overexpression of uncoupling protein-2 also protected cardiac myocytes (30). In this study, the protective effect of SFA was comparable to that of diazoxide, and the co-application with SFA and diazoxide did not show an additive effect. Furthermore, the mitoKATP channel blocker, 5-HD, abolished the protective effect of SFA. These results strongly suggest that the protective effect of SFA might be mediated by the activation of mitoKATP channels. Nevertheless, we could not rule out the possibility that SFA might act on the surface membrane potential of cardiomyocytes and reduce cytosolic calcium overload.
Clinical implications.
Many pharmacological agents and strategies have been administered for cardiac protection during acute myocardial infarction (31); however, none have been translated into clinical practice (32). Therapeutic interventions designed to prevent MPTP opening during ischemia/reperfusion hold major promise as a novel strategy for reducing cardiac injury from ischemia/reperfusion (6). Our findings suggest SFA as a novel candidate for cardioprotective therapy against ischemia/reperfusion injury. Serofendic acid is expected to be free from unpredictable side effects, because it is an endogenous substance. Despite the positive prospect of SFA as a novel cardioprotective agent, further investigations in animal models are needed to assess the infarct size-limiting effect.
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Appendix
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For accompanying videos to Figures 3, 4, and 5, please see the online version of this article.
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References
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M. Kato, M. Akao, M. Matsumoto-Ida, T. Makiyama, M. Iguchi, T. Takeda, S. Shimizu, and T. Kita
The targeting of cyclophilin D by RNAi as a novel cardioprotective therapy: evidence from two-photon imaging
Cardiovasc Res,
July 15, 2009;
83(2):
335 - 344.
[Abstract]
[Full Text]
[PDF]
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N. Murano, M. Ishizaki, S. Sato, Y. Fukuda, and H. Takahashi
Corneal Endothelial Cell Damage by Free Radicals Associated With Ultrasound Oscillation
Arch Ophthalmol,
June 1, 2008;
126(6):
816 - 821.
[Abstract]
[Full Text]
[PDF]
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