Cytotoxicity of Triamcinolone Acetonide on Human Retinal Pigment Epithelial Cells

Abstract

purpose. To investigate the toxic effects of triamcinolone acetonide (TA) suspensions on human retinal pigment epithelial (RPE) cells.

methods. Cultured human RPE cells were exposed for up to 2 hours to one of seven solutions: control (balanced salt solution, BSS; Alcon Laboratories, Ft. Worth TX), commercial TA suspension (cTA), cTA from which the vehicle (which contains the preservative benzyl alcohol) had been removed (vehicle-removed TA, −vTA), vehicle of the cTA (V), or a 1:10 dilution (in BSS; Alcon) of cTA, −vTA or V. Solution effects were evaluated by phase-contrast microscopy of cells stained in situ with trypan blue and in vitro by trypan blue exclusion assay. RPE cell function was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The mechanism of TA toxicity was studied by acridine orange–ethidium bromide staining and epifluorescence microscopy, and ultrastructural changes were examined by transmission electron microscopy (TEM).

results. The effects of vehicle-removed solutions (−vTA and 1:10 −vTA) were similar to those of the control solution. Exposure for 1 hour or longer to a vehicle-containing solution (cTA and V) resulted in similar and significant degrees of cell damage that were dose and time dependent. The major mechanism of cell death was necrosis, and the early ultrastructural change was swelling of organelles in the cytoplasm.

conclusions. Preserved commercial TA suspensions damaged human RPE cells, but vehicle-free solutions did not. The authors suggest removing the vehicle as completely as possible from TA solutions before they are administered intravitreally. Furthermore, they recommend that a commercial formulation of preservative-free TA suspension be made available for intraocular use.

Because triamcinolone acetonide (TA) suspension provides longer-lasting anti-inflammatory, antiproliferative, antiangiogenesis, and antipermeability effects compared with other steroid preparations, it has recently become more widely used in treating a wide variety of vitreoretinal diseases. ^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15} In addition, because of the unique physical properties of its crystalline particles, TA in suspension is being used for visualizing the prolapsed vitreous in complicated cataract surgery ^{16 17} and visualizing the vitreous, ^{18 19 20 21 22} posterior hyaloid, ^{18 19} epiretinal membrane (ERM), ²³ or internal limiting membrane (ILM) ^{24 25} during pars plana vitrectomy and macular hole surgery. As more and more applications for TA are being identified in clinical practice, reports of new applications for it are increasing year by year. Between January 2004 and December 2006, approximately 400 articles on such topics were added to the MedLine database of the medical literature maintained by the U. S. National Library of Medicine (Bethesda, MD). 

Some reports in the literature indicate that the vehicle or preservative in TA suspensions, and not the crystalline corticosteroid drug itself, may be toxic to ocular tissues. ²⁶ Such toxicity would be of concern to ophthalmologists planning to administer TA suspension intravitreally, because if true, contact with such a solution could damage the retina—in particular, the retinal pigment epithelium (RPE) during macular hole surgery, which, to date, has not been thoroughly studied. Recently, we investigated the cytotoxicity of some agents used intracamerally, including TA, to cultured rabbit corneal endothelial cells, and we found the results to have notable implications for cataract surgery. ^{27 28 29} In the present study, we used similar methods to investigate the potential of TA to damage cultured human RPE cells. The results have implications for the safe clinical use of TA. 

Materials and Methods

The test solutions and methods used in this study were similar to those in a previously reported study of rabbit corneal endothelial cells. ²⁷  

Cell Culture

Human RPE cells (ARPE-19) ³⁰ were obtained from the American Type Culture Collection (Manassas, VA). This cell line is not transformed and has structural and functional properties characteristic of RPE cells in vivo. ³¹ The RPE cells were cultured in DMEM/Ham’s F12 medium (1:1) containing 10% fetal bovine serum (FBS; Invitrogen Corp, Grand Island, NY), and the following substances were added: 0.01 g/L transferrin, 0.01 g/L insulin, 0.91 g/L sodium bicarbonate, 100 U/mL penicillin G potassium, 0.1 mg/mL streptomycin sulfate, 5.0 mg/mL gentamicin sulfate, 3.58 g/L HEPES, and 1.75 g/L d-glucose. The cells were cultured at 37°C in 5% carbon dioxide. By passage 5, the cultures propagated rapidly and could be used for experiments. 

Preparation of Test Solutions from TA

A control and six test solutions were used (Table 1) : (1) The control was a physiologic saline solution (BSS), obtained from Alcon Laboratories (Fort Worth, TX). (2) Solution 1 was the commercial TA suspension (cTA; Kenacort-A, 40 mg/mL; Bristol-Myers Squibb, Taipai, Taiwan). (3) Solution 2 was a vehicle-removed suspension of TA (−vTA) in the saline solution. It was prepared by centrifuging 12 mL of cTA (4000 rpm for 10 minutes) to separate the TA particles from the vehicle. ¹⁸ The sediment of TA particles (approximately 1 mL) was resuspended in the saline solution to achieve the initial volume of 12 mL. Centrifugation and resuspension in the solution were repeated once to obtain solution 2 (−vTA). (4) Solution 3 was the pure vehicle of cTA (V) without any TA particles. It was prepared by passing the supernatant of vehicle (∼11 mL) from the first centrifugation of cTA (used to prepare solution 2) through 0.2-μm pore filters (Millipore Acrodisc 32-mm Syringe Filter with 0.2 μm support membrane; Pall Corp., Ann Arbor, MI). (5) Solutions 4 (1:10 cTA), 5 (1:10 −vTA), and 6 (1:10 V) were 1:10 dilutions of solutions 1, 2, and 3, respectively, in the saline solution (BSS; Alcon Laboratories). They were prepared by diluting 1 mL of solution 1, 2, or 3, respectively, with 9 mL of saline solution. Osmolality was measured by osmometer (model 3D3; Advanced Instruments, Norwood, MA); pH was measured by pH meter (model pH211; Hanna Instruments, Padua, Italy). 

Exposure to Test Solutions

Cultured RPE cells were seeded onto 24-well plates (experiments 1, 2, and 3), 3-cm culture plates (experiment 1), or four-chamber slides (experiment 4) with a cell volume of 5000 cells/mL, and were grown to 80% confluence before treatment to prevent contact inhibition. After removal of culture medium, the cells were exposed to the control or one of the six test solutions by adding sufficient solution (0.2 mL for each of 24 wells, 0.5 mL for a 3-cm plate, or 0.5 mL in a four-chamber slide) to overlay the monolayer of RPE cells. The cultures were then incubated at 37°C for 5 minutes, 30 minutes, 1 hour, or 2 hours. 

Experiment 1: Morphologic Evaluation and Quantitative Determination of Cell Damage by Trypan Blue Staining

RPE cells in 24-well chambers were exposed to test solutions for various lengths of time, gently washed with physiologic saline (BSS; Alcon Laboratories), and, without trypsinization, immediately stained in situ with trypan blue 0.2%. They were then examined under a phase-contrast microscope for size, shape, and integrity of the cell membrane, cytoplasm, and nucleus. 

Furthermore, a trypan blue exclusion assay was used, as previously described, ^{27 28 29} to determine quantitatively the percentage of cells in each RPE culture that were damaged by exposure to solutions. In brief, after exposure to a test solution for a specified length of time, the confluent layer of cells in 3-cm plates was gently washed with BSS and trypsinized. At the completion of the trypsinization process, FBS was added to the cell suspension to a final concentration of 10% to inhibit further trypsinization, which could damage cells. The cells were then exposed for 5 minutes to trypan blue 0.2% (diluted from 0.4% solution; Sigma-Aldrich, St. Louis, MO). The numbers of stain-positive (dead and dying cells) and -negative cells in each culture were counted in a hemocytometer chamber. Five cultures were exposed to each solution for each period. The means for each group of cultures were compared. 

Experiment 2: Quantitative Determination of Cell Damage by MTT Assay

Cell viability was assessed by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to determine the proportion of living cells in each culture (living cells are those with mitochondrial function of dehydrogenase). ^{32 33} The 24-well cultures, after exposure to the control or one of the six test solutions for a specific period, were incubated with 50 μg/mL MTT at a dilution of 1:10 based on the volume of culture medium for 30 minutes at 37°C. At the end of incubation, the MTT solution was removed, and the cells were dissolved in 0.5 mL 0.04 M HCl in isopropyl alcohol. The proportion of viable cells (those with mitochondria capable of cleaving the MTT molecule to produce the dark purple substance, formazan) was determined by measuring the optical density (OD) of each sample at 570 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader (GE Healthcare, Uppsala, Sweden). Five cultures were exposed to each solution for each time period. The means for each group of cultures were compared. 

Experiment 3: Determination of Mechanism of Cytotoxicity by Acridine Orange–Ethidium Bromide Staining

Acridine orange–ethidium bromide staining identifies alive, early apoptotic, late apoptotic, and necrotic cells. ³⁴ RPE cells in 24 wells were exposed to the control or a test solution for 5 minutes, 30 minutes, 1 hour, or 2 hours. The cells were then gently washed with saline solution (BSS; Alcon Laboratories) and immediately treated with acridine orange (100 μg/mL) and ethidium bromide (100 μg/mL). Each well was then examined immediately under an epifluorescence microscope (Olympus Optical, Tokyo, Japan) with a triple spectrochemical filter consisting of 4′,6-diamidino-2-phenylindole-dehydrochloride (DAPI; 400 nm), fluorescein isothiocyanate (FITC; 495 nm), and Texas red (570 nm). 

Experiment 4: Ultrastructural Changes

Ultrastructural changes were evaluated by transmission electron microscopy (TEM). ^{27 28} RPE cells cultured in four-chambers slides were exposed to BSS (control), cTA, −vTA, or V for 5 minutes or 2 hours. The slides were then gently washed with BSS and, without trypsinization, they were fixed by immersion in 2.5% glutaraldehyde-0.5% paraformaldehyde for 4 hours. After rinsing in 0.1 M PBS, the cells were osmicated, dehydrated, and embedded in epoxy resin (Epon; Electron Microscopy Sciences, Washington, PA) and examined by TEM (JEM-1200EX; JEOL, Tokyo, Japan). 

Statistical Analysis

The results of the trypan blue exclusion and MTT assays were entered into a worksheet program (Excel Office 2000; Microsoft Corp., Redmond, WA), and differences among groups were evaluated for statistical significance by using a two-tailed, unpaired Student’s t-test with the level of significance set at P ≤ 0.01. 

Results

Experiment 1: Trypan Blue Staining

Of all the RPE cells exposed to physiologic saline (BSS; Alcon Laboratories) for 5 minutes to 2 hours, only a few had trypan blue–stained nuclei (indicating cell death), and when viewed by phase-contrast microscopy, the stained cells were of the same size and shape as the unstained cells (Fig. 1) . Cultures of cells exposed to −vTA, 1:10 cTA, 1:10 −vTA, or 1:10 V for various periods showed similar percentages of stained cells and intact cellular morphology. In contrast, RPE cells exposed to cTA or V demonstrated a time-dependent increase in the percentage of trypan blue–stained cells and morphologically changed (shrunken) cells; the differences from the other solutions (−vTA, 1:10 cTA, 1:10 −vTA or 1:10 V) were particularly remarkable after 1 or 2 hours of exposure to cTA or V. 

As shown in Figure 2 , there were no significant differences in the proportions of trypan blue–stained cells in cultures exposed for 5 minutes to the control or any test solution. After 30 minutes, 1 hour, or 2 hours of exposure, the proportion of trypan blue–stained cells in most solutions did not change; however, the proportion increased for cells exposed to cTA or V for 30 minutes (cTA), 1 hour (cTA and V), or 2 hours (cTA or V). 

Experiment 2: MTT Assay

As shown in Figure 3 , after 5 or 30 minutes of exposure to a solution, there was no significant difference in optical density (an indicator of mitochondrial function) among the groups. After 1 or 2 hours of exposure, however, cultures of cells exposed to cTA or V had significantly lower optical densities than did cells exposed to BSS (control). 

Optical densities were similar for cultures of cells exposed to cTA or V for the same length of time. They were also similar for cultures of cells exposed to −vTA, 1:10 cTA, 1:10 −vTA, or 1:10 V for the same length of time. 

Experiment 3: Acridine Orange–Ethidium Bromide Staining

Figure 4shows the results of acridine orange–ethidium bromide staining of cultures exposed to the control (BSS; Alcon Laboratories) or an experimental solution for 5 minutes to 2 hours. Viable cells are those with a green nucleus and green cytoplasm. All the cultures exposed to a test solution contained at least a few cells with areas of punctate orange-red fluorescence in the cytoplasm, indicating compromise of cell membrane integrity and accumulation of acridine orange within the lysosomes. The densities of accumulations were similar for 5-minute exposures to test solutions. However, cTA or V caused time-dependent cytotoxicity, as demonstrated by the increasing number of cells with orange red-nuclei and orange cytoplasm (indicating extensive cell damage) in cultures exposed to these solutions for longer times. Nevertheless, the cells retained normal nuclear architecture, and there were no signs of apoptosis (e.g., nuclear condensation or fragmentation). 

Experiment 4: Ultrastructural Changes

On TEM (Fig. 5) , RPE cells exposed to saline control (BSS; Alcon Laboratories) or −vTA for between 5 minutes and 2 hours had intact cell membranes, cytoplasm, and nuclei. In contrast, cells exposed to cTA or V for even 5 minutes had several swollen organelles within the cytoplasm, a sign of early ultrastructural damage. Furthermore, RPE cells exposed to cTA or V for 2 hours had numerous swollen organelles, indicating extensive cellular damage. The damage was more extensive with longer exposure to vehicle-containing agents (cTA and V). 

Discussion

Because the RPE plays a crucial role in maintaining the integrity of the outer retina–blood barrier and supporting normal function of the retina, any insult to this monolayer, such as intravitreous injection of any new agent, poses the risk of RPE cytotoxicity and retinal dysfunction. Until we conducted the present in vitro study, very little had been reported regarding risks to human RPE cells of damage due to contact with a commercially available TA suspension. 

Our study showed the following: (1) vehicle-removed TA solutions (−vTA) were no more cytotoxic than was the control solution (BSS; Alcon Laboratories); (2) vehicle-containing solutions (cTA and V) caused significantly more cell damage than control or vehicle-removed solutions within the first hour of exposure; (3) solutions containing the vehicle caused similar damage (cTA versus V); (4) the damage caused by vehicle-containing solutions was dose-dependent (cTA versus 1:10 cTA, and V versus 1:10 V) and time-dependent (from 5 minutes to 2 hours), regardless of the concentrations of TA in the solutions; (5) less RPE cell damage resulted when the vehicle had been removed from the TA solution, when it had been diluted 10-fold, or both; (6) the major mechanism of cell death was necrosis rather than apoptosis; and (7) even 5 minutes of exposure of RPE cells to vehicle-containing TA could result in ultrastructural changes such as organelle swelling within the cytoplasm. 

Collectively, the results of this study indicate that it is the vehicle in which the crystalline particles of TA are suspended that is cytotoxic, not the TA particles themselves. Furthermore, the results show that to avoid early ultrastructural changes due to the TA vehicle, exposure to cTA should last no longer than 5 minutes and only vehicle-removed solutions should be used for macular hole surgery. 

Cytotoxicity of the Vehicle in Commercial TA Suspension

The commercial solution of TA used in this study (cTA) and its vehicle contain the preservative benzyl alcohol (BA). BA is rarely used as the preservative in topical ophthalmic solutions because it is irritating, acts slowly, and can dissolve polystyrene. ³⁵ Intravenous use of solutions containing BA has been reported to cause a fetal toxic syndrome, intraventricular hemorrhage, neurologic handicaps, and mortality in premature infants. ^{36 37 38}  

In a previous study in rabbits, Hida et al. ²⁶ found that preservatives in the vehicle for suspension of crystalline cortisone, rather than the cortisone itself, could be toxic to the rabbit retina and lens. We recently showed that a BA concentration of 9 mg/mL causes extensive lysis of rabbit corneal endothelial cells within the first minute of exposure. ²⁷ Intravitreous injection of BA has been shown to damage the outer segments of photoreceptors in the rabbit eye. ³⁹ Recently, Shaikh et al. ⁴⁰ showed, by MTT assay, that both the preserved TA formulation and its vehicle cause significant reductions in the viability of cultured RPE cells. Similarly, Narayanan et al. ⁴¹ investigated whether preserved TA or its vehicle was toxic to cultured RPE and retinal neurosensory cells, and they concluded that the vehicle was not totally responsible for toxicity but may initiate TA-dependent toxicity. The findings in our study, that the vehicle in cTA damages cultured human RPE cells in a time- and dose-dependent manner, are collectively supported by these experimental studies. 

Because intravitreous injection of TA started to be used in managing a wide spectrum of vitreoretinal diseases, ^{1 2 3 4 5 6 7 8 9 10 11 12 13 14 15} the incidence of postoperative noninfectious or pseudoendophthalmitis has been estimated to range from 1.1% to 6.7%. ^{42 43} The authors of these reports speculated that the preservative in the commercial TA suspension may be responsible for such cases of noninfectious endophthalmitis. In support of this conjecture, a large-scale study by Jonas et al. ⁴⁴ found that none of the 1135 intravitreous injections of 20 mg TA from which the vehicle had been removed that they administered to 915 eyes of 838 patients resulted in the development of noninfectious endophthalmitis. 

Comparison of Various Evaluations for RPE Cytotoxicity

In the present study, we used trypan blue staining, MTT assay, acridine orange–ethidium bromide staining, and TEM to evaluate the RPE toxicity of various formulations of TA. TEM demonstrated the earliest damage due to the vehicle of TA: intracytoplasmic organelle swelling of cells exposed to cTA or V for only 5 minutes, indicating that the biochemical effect of the vehicle is to change the permeability of the RPE cell membrane. As for trypan blue in situ staining, the earliest changes were cell shrinkage and nuclear staining that were first evident after 30 minutes of exposure. Furthermore, MTT assay showed that vehicle-containing solutions (cTA and V) lead to reduction of cell function within 1 hour of exposure. The results of acridine orange–ethidium bromide staining showed that cells exposed to vehicle-containing solutions died mainly by necrosis, not by apoptosis. 

All our findings indicate that ultrastructural changes could occur within as short a time as 5 minutes and irreversible cell damage—changes in cell morphology, viability, function, and switching on of the necrotic mechanism—could occur in as short an exposure time as 1 hour. Compared with two previous studies, ^{40 41} our study was designed to provide a more thorough and extensive evaluation of the cytotoxicity of commercial TA and its vehicle by evaluating damage to cultured RPE cells using four types of assay. In particular, there have been no previous reports of using acridine orange–ethidium bromide staining and TEM to evaluate this type of damage to RPE cells, so our findings from this portion of our study are particularly helpful in furthering understanding of the mechanism of cytotoxicity of TA and its vehicle. 

Clinical Relevance of Findings

Kenacort-A and Kenalog (Bristol-Myers Squibb) ^{9 16 39 42 43} are formulated BA-preserved TA suspensions, and both contain TA 40 mg/mL and BA 9 mg/mL as the preservative. They were developed for intra-articular or intramuscular injection and were only recently formulated for off-label use as ocular medications. For visualizing and removing the vitreous-posterior hyaloid-ERM-ILM, TA contacts the retina for only 10 to 30 minutes, whereas in therapeutic intravitreous TA injection, TA particles, together with their vehicle, are retained within the eye for weeks or months. Based on our finding in this study that TA is associated with subtle, early ultrastructural changes in RPE cells after only 5 minutes of exposure to the vehicle, we urge removal of the vehicle as completely as possible before the TA suspension is used in the eye, particularly during macular hole surgery. Concurring with our result that TA particles without vehicle were not toxic to RPE cells, Enaida et al. ⁴⁵ reported no apparent adverse effect in a patient with submacular deposition of TA particles after TA-assisted vitrectomy for rhegmatogenous retinal detachment. The authors concluded that the damage attributable to subretinal TA deposition was not significant either morphologically or functionally; however, they were still concerned about the potentially harmful effects on retinal structure and function of the vehicle in commercial TA suspensions. 

We recently described three methods to remove the vehicle from cTA. ²⁷ The first, described by Jonas et al., ¹ is the “standstill” method in which the TA suspension is drawn into a syringe that is held vertically for 30 minutes to allow sedimentation of TA crystals; the upper layer is ejected and the settling procedure is repeated twice more before the suspension is used. Because this procedure takes hours, it is not practical when a TA suspension is unexpectedly needed intraoperatively. Hernaez-Ortega and Soto-Pedre ⁴⁶ separated the TA particles from the vehicle by density-gradient centrifugation at 3000 rpm for 5 minutes. This method is simple and rapid, with little loss of TA particles; however, almost no operating rooms are equipped with density-gradient centrifuges. The third method for removing the vehicle from cTA, introduced by Kumagai ¹⁹ and Burk et al., ¹⁶ involves repeatedly passing the TA suspension through syringe filters (Millipore Acrodisc 32-mm Syringe Filter, Pall Corp.) and resuspending the TA particles in saline solution (BSS; Alcon Laboratories). These filters are available in most ophthalmic operating rooms, and so this method seems the most practical for routine clinical use. However, about half of TA particles would be trapped on the 0.2-μm-pore filter paper. ^{6 47} Therefore, it is necessary to titrate the solution to determine the exact concentration using this method, particularly for intravitreous injection of TA to treat various vitreoretinal diseases. 

Even though the vehicle of TA is removed, we recommend additional safety measures to prevent adverse effects of any vehicle residue on the retina. First, the intravitreous injection of TA should be directed away from the macula, particularly the fovea, unless it is being injected to enhance visualization during macular surgery. For macular hole surgery, we suggest preventing direct exposure of TA to the RPE by application of autologous blood or serum or viscoelastic substance within the macular hole. Second, when an intravitreous injection of TA is used to treat ocular inflammatory or vessel-leaking diseases, the patient should maintain a head-up position most of the time for weeks to avoid settling of the TA suspension and the residual vehicle over the macular area, particularly for the first several days in vitrectomized eyes. 

Limitations of Our Study

The first limitation of our study is that the results of our in vivo animal experiment, which has been underway for some time, are still pending. Nevertheless, using the current model, we substantiated that TA vehicle plays a role in the cytotoxicity of RPE cells. The second limitation of this study is that the in vitro cytotoxicity of BA-preserved TA suspension should not be directly extrapolated to clinical practice. In eyes, the actions of proteins and buffered ions in the vitreous and the cell rescue mechanisms may tend to decrease the toxicity of BA. Also, there is a dilution factor that occurs in vivo as the TA suspension is injected into the vitreous body. Moreover, TA suspension injected into the vitreous for a vitrectomy procedure would become diluted by continuous irrigation. The third limitation is that the longest exposure time in our study was 2 hours. It is possible that RPE cell damage due to even a low concentration of vehicle-containing solution (such as 1:10 cTA) could occur with prolonged contact (several weeks or months). This may often happen in clinical practice when ophthalmologists inject TA intravitreally. Funduscopic examinations show that it may take months for the TA agent to dissolve or even disappear. Therefore, more studies are needed to clarify whether TA suspensions are safe for the human RPE and other retinal cells when used over the long term. 

Conclusions

Our study demonstrated that the commercial TA suspension in wide clinical use is cytotoxic to human RPE cells; that cytotoxicity is dose- and time-dependent; and that the mechanism of injury is necrosis. The toxicity of the commercial TA solution is due to the vehicle and not TA itself. We found that up to 2 hours of exposure to vehicle-removed TA appeared to be safe for cultured human RPE cells. To use TA suspensions safely as a therapeutic agent through intravitreous injection or as a surgical tool in aiding visualization of the vitreous-posterior hyaloid-ERM-ILM in pars plana vitrectomy, we recommend vehicle-removed preparations for intravitreous use in human eyes and removal of the vehicle as completely as possible. Furthermore, we urge consideration of the development of a commercial formulation of a preservative-free TA suspension for intraocular use. 

 Supported by Grant NCKUH-95-016 from the National Cheng Kung University Hospital, Tainan, Taiwan, and Grant NSC 94-2314-B-006-100 from the National Science Council, Taiwan.

 Submitted for publication September 24, 2006; revised January 7, 2007; accepted April 12, 2007.

 Disclosure: Y.-S. Chang, None; C.-L. Wu, None; S.-H. Tseng, None; P.-Y. Kuo, S.-Y. Tseng, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Characteristics of Control and Test Solutions Prepared from cTA Preserved with BA

Solution	TA Concentration (mg/mL)	BA Concentration (mg/mL)	Osmolality (mOsm/kg)	pH
Control (BSS)	0	0	305	7.4
Solution 1 (cTA)	40	9	326	6.2
Solution 2 (-vTA)	∼40	0	295	7.7
Solution 3 (V)	0	9	321	6.1
Solution 4 (1/10 cTA)	4	0.9	306	7.5
Solution 5 (1/10 -vTA)	∼4	0	298	7.3
Solution 6 (1/10 V)	0	0.9	305	6.8

Figure 1.

 

Phase-contrast microscopic view of trypan blue in situ–stained RPE cells exposed for between 5 minutes and 2 hours to the control solution (BSS; Alcon Laboratories), commercial TA without processing (cTA), vehicle-removed TA in BSS (−vTA), pure vehicle (V), and 1:10 dilutions of cTA (1:10 cTA), −vTA (1:10 −vTA), or V (1:10 V). More damage is indicated by more cells with trypan blue stain in the nucleus and furthermore by morphologic changes of cells. In the microphotographs labeled−vTA 1 hour and −vTA 2 hours, the fine particles are residual TA deposits adhering to the cells after gentle washout. Trypan blue; magnification ×100.

Figure 2.

 

Trypan blue exclusion assay showing mean percentages (±SD) of trypan blue–positive (“damaged”) cells after exposures of between 5 minutes and 2 hours to control (BSS; Alcon Laboratories), commercial TA without processing (cTA), vehicle-removed TA in BSS (−vTA), pure vehicle (V), 1:10 cTA, 1:10 −vTA, or 1:10 V. Each bar, five cultures; *P < 0.01, Student’s t-test versus control; †P < 0.01, Student’s t-test for two groups.

Figure 3.

 

MTT assay showing mean optical densities (±SD) of cells after 5-minute to 2-hour exposures to control (BSS; Alcon Laboratories), commercial TA without processing (cTA), vehicle-removed TA in control saline (−vTA), pure vehicle (V), 1:10 cTA, 1:10 −vTA, or 1:10 V. Each bar, five cultures; *P < 0.01, Student’s t-test versus control; †P < 0.01, Student’s t-test for two groups.

Figure 4.

 

Acridine orange–ethidium bromide–stained cells shown by epifluorescence microscopy. Cells were exposed for between 5 minutes and 2 hours to control (BSS; Alcon Laboratories), commercial TA without processing (cTA), vehicle-removed TA in the saline solution (−vTA), pure vehicle (V), 1:10 cTA, 1:10 −vTA, or 1:10 V. Viable cells have green nuclei and green cytoplasm. Punctate orange-red fluorescence in the cytoplasm indicates compromised cell membrane integrity and accumulation of acridine orange within the lysosomes. An orange-red nucleus along with an orange cytoplasm indicates extensive cell damage. The architecture of these orange-red nuclei, however, was normal (no signs of apoptosis such as nuclear condensation or fragmentation). Acridine orange-ethidium bromide; DAPI filter; magnification ×100.

Figure 5.

 

TEM photomicrographs showing RPE cells exposed for between 5 minutes and 2 hours to control (BSS; Alcon Laboratories), commercial TA without processing (cTA), vehicle-removed TA in control (−vTA), or pure vehicle (V). Cells exposed to BSS or −vTA, either for 5 minutes or 2 hours, showed an intact cell membrane, cytoplasm, and nucleus. However, cells exposed to cTA or V for 5 minutes had several swollen organelles (arrows) in the cytoplasm, indicating early ultrastructural damage. Moreover, cells exposed to cTA or V for 2 hours showed numerous swollen organelles (arrows) in the cytoplasm, indicating an extensive cellular damage. Scale bar, 500 nm.