|Molecular Vision 2006;
Received 2 September 2005 | Accepted 27 April 2006 | Published 30 May 2006
Simulated microgravity induced damage in human retinal pigment epithelial cells
Joan E. Roberts,1
Barbara M. Kukielczak,2
Colin F. Chignell,2
Robert H. Sik,2
Mary Ann Principato6
1Fordham University, Department of Natural Sciences, New York, NY; 2Laboratory of Chemistry and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC; 3Tissue Culture Center, Departments of 4Pathology and Laboratory Medicine and 5Ophthalmology, New York Eye and Ear Infirmary, New York, NY; 6Food and Drug Administration, Laurel, MD
Correspondence to: Joan E. Roberts, Fordham University, Department of Natural Sciences, 113 West 60th Street, New York, NY, 10023; Phone: (212) 636-6310; FAX: (212) 636-7127; email: email@example.com
Purpose: The goal of this study was to determine the potential damage to the human retina that may occur from weightlessness during space flight using simulated microgravity.
Methods: Human retinal pigment epithelial (hRPE) cells were cultured for 24 h in a National Aeronautics and Space Administration-designed rotating wall bioreactor vessel to mimic the microgravity environment of space. Single-stranded breaks in hRPE DNA induced by simulated gravity were measured using the comet assay. In addition, the production of the inflammatory mediator prostaglandin E2 (PGE2) was measured in these cells 48 h after recovery from simulated microgravity exposure.
Results: Simulated microgravity induced single-stranded breaks in the hRPE DNA that were not repaired within 48 h. Furthermore, PG E2 production was dramatically increased 48 h after the initial microgravity-induced damage, indicating the induction of an inflammatory response. There was less DNA damage and no PGE2 release in hRPE cells pretreated with the antiinflammatory agent cysteine during their exposure to microgravity.
Conclusions: We have demonstrated that the microgravity environment generated by a NASA-designed rotating wall bioreactor vessel induces an inflammatory response in hRPE cells. This system thus constitutes a new model system for the study of inflammation in the retina, a system that does not involve the introduction of an exogenous chemical agent or supplementary irradiation. This in vitro method may also be useful for testing novel therapeutic approaches for suppression of retinal inflammation. Furthermore, we suggest a safe prophylactic treatment for prevention of acute, transitory, or enhanced age-related permanent blindness in astronauts or flight personnel engaged in long-haul flights.
Space travel subjects the human eye to the stress of solar and cosmic radiation, and at the same time to microgravity. It is well established that solar and cosmic radiation induce cataracts and retinal degeneration [1-7], but the long term hazards of microgravity to ocular tissues have not been established. On earth, the human eye is constantly exposed to environmental hazards including smoke, environmental toxins, and ambient radiation. Damage is averted in the young eye by the presence of an efficient antioxidant system, including melanin (8), lutein and zeaxanthin , vitamins C  and E , and glutathione , superoxide dismutase, catalase, and the co-factors zinc and selenium . However, with age (above 40 years old), the levels of protective endogenous antioxidants decrease, and both the clarity of the lens and the function of the retina deteriorate [13,14]. Astronauts and flight personnel engaged on long-haul flights  are exposed to higher and more virulent environmental insults, and blinding disorders may appear decades after the original injury.
In the present work, we sought to examine the pathogenic mechanisms of potential damage to the retina induced by microgravity independent of radiation damage. Dutt et al.  have shown upregulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in the human retinal cell line 301-SV-40T  in a NASA Bioreactor. Barstable and Tombran-Tink have demonstrated photoreceptor rod outer segment damage in rats exposed to hypergravity and space travel [18,19]. We present here an experiment which demonstrates that microgravity (simulated using a NASA-designed rotating wall vessel (RWV) bioreactor) [20,21] can induce an inflammatory response in human retinal pigment epithelial cells. Our results have also suggested a potential method to block this damage and avoid transient or permanent damage to astronauts on long space flights.
A human retinal pigment epithelial (hRPE) cell culture derived from a human donor eye as described by Hu et al.  was used in these studies. The purity of the cell line was demonstrated by immunocytochemical methods: hRPE cells display S-100 and cytokeratin, uveal melanocytes display S-100 antigen but not cytokeratin, and fibroblasts display neither of these proteins . The cells used in the present experiments were cultured using an F12 nutrient mixture supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 μg/ml gentamicin (GIBCO, BRL Products, Rockville, MD). The cells used in these experiments were generally from passages two to three. For controls, aliquots of cells were grown in Non-Rotating Wall Vessels maintained at unit gravity.
Simulated microgravity exposure
Microgravity is simulated by using a rotating wall vessel (RWV) bioreactor. This cell culture double-walled rotating bioreactor was originally developed by NASA . The RWV bioreactor consists of a horizontally rotating cylindrical growth chamber that contains an inner corotating cylinder with a gas exchange membrane. Cells and liquid culture media are placed in the space between inner and outer cylinders. Under these conditions the fluid rotates at the same rate as its container. Chamber rotation subjects the cells to a constantly changing angular gravity vector. Constant randomization of the normal gravity vector subjects the cells to a state of simulated free fall with shear stress exerted by the fluid minimized. This system mimics microgravity conditions.
Growing hRPE were detached with trypsin-ethylenediaminetetraacetic acid [EDTA] solution (GIBCO, Worcester, MA) diluted 1:3. Detached cells were spun for 5 min at 1000 rpm at 4 °C and resuspended to 30 ml volume in RPMI-phenol red free media containing 10% fetal calf serum, 2 mM glutamine and 50 μg/ml gentamicin. The cells were counted with trypan blue and demonstrated 97-98% viability. Cells were reseeded and grown either in nonrotating wall vessels at unit gravity or in the simulated microgravity environment of the NASA-designed rotating wall vessel bioreactor (Synthecon, Houston, TX) [20,21] in the presence and absence of 1 μM cysteine.
Prior to adding the cells, each 50 ml disposable, sterile rotary cell culture vessel (Synthecon, Houston, TX) was flooded with media. All bubbles were removed by gentle flushing. Sufficient hRPE cells were added into each filter for a final concentration of 250,000 cells per ml. Each loaded cell culture filter was then attached to the baseplate drive of the RWV bioreactor. The assembled bioreactor cultures were individually rotated at 8 rpm within a humidified incubator at 37 °C under 5% CO2 for 24 h. Lewis et al.  have previously found that rotation of the RWV bioreactor at eight rpm was optimal for maintaining cells in suspension. This is equivalent to 0.01 g or 1/100 of unit gravity .
Treatments consisted of (1) cells exposed to microgravity for 24 h; (2) cells exposed to microgravity for 24 h and then incubated in media for 48 h; (3) cells pretreated with 1 μM cysteine, exposed to microgravity for 24 h, and then incubated in media for 48 h.
DNA damage (single-stranded breaks) induced by microgravity was quantified using the comet assay, in which the amount of the genetic material that migrated from the nucleus to form the comet tail was measured. Images of nuclei and migrated material were digitized so that the comet tail moment (% of pixel intensity in the tail times distance from head to tail in microns) divided by the negative control value could be calculated. This value was then used as a quantitative index of DNA single-stranded breaks and presented as the median of the comet tail moment.
The comet assay was performed essentially as described by Singh . Briefly, 85 μl of molten 1% normal agarose in PBS was dropped onto a precoated microscope slide, covered with an 18x18 mm No.1 glass coverslip, and left in ice to set. Once set, the coverslip was removed. The hRPE cells were then mixed with 85 μ 1 of 1% low melting point agar and immediately pipetted onto the layer of agarose on the slide. The coverslip was replaced and allowed to set on ice. The entrapped cells were then lysed in 150 ml of ice cold lysis buffer [2.5 M NaCl, 83 mM EDTA, 10 mM tris (hydroxymethyl) aminomethane (TRIS) and the pH was adjusted to 10 using sodium hydroxide (NaOH)]. The lysis buffer was supplemented with 1% (v/v) the nonionic detergent Triton X-100 and 10% (v/v) dimethyl sulfoxide (DMSO) prior to use. Lysis was performed at 4 °C for 60-90 min.
Following lysis, the slides were transferred to a horizontal electrophoresis apparatus. Slides were placed flat onto a gel tray and aligned equidistant from the electrodes. Electrophoresis solution (0.3 M NaOH and 1 mM EDTA) was added to the apparatus until the level just covered the samples. Samples were then incubated in this buffer for 20 min prior to electrophoresis. The power supply was set at 20 V/32 mA and this voltage was applied for 24 min. The slides were washed three times in 100 mM TRIS, pH 7.5, and exposed briefly to chilled methanol.
Comet assay data analysis
After electrophoresis, fluorescent images of the nuclei stained with ethidium bromide were captured with a video camera and digitized using the Matrox Meteor II interface (Matrox Image, Quebec, Canada) to a PC controlled by the Matrox Inspector program. A script written in BASIC was used to measure the comet tail moment (% of pixel intensity in the tail times distance from head to tail in microns) from at least 50 cells in each group. The parameter used to measure DNA damage in this study was the relative comet tail moment (i.e., the comet tail moment divided by the negative control value). Negative controls were determined using untreated cells; the negative control was employed to calculate the relative comet tail moment for each experiment.
A frequency distribution of the comet tail moment was determined for each test. The difference in distribution of comet tail moments was analyzed using the Mann-Whitney nonparametric statistical test  and presented as the median of the comet moment.
Enzyme immunoassay for prostaglandin E2
Supernatant of the conditioned medium was taken from control RPE cells or retinal pigment epithelium (RPE) cells treated with microgravity. The medium was frozen immediately in liquid nitrogen and then stored at -80 °C until analyzed. PGE2 was measured using an enzyme immunoassay (R&D Systems, Minneapolis, MN) kit.
Single-stranded DNA breaks induced by simulated microgravity
DNA damage to hRPE cells induced by microgravity was assessed by single cell gel electrophoresis (comet assay). In this assay, cleaved DNA migrates from the nucleus, forming a comet that is directly visualized as a cell with a round head and a tail (Figure 1); normal cells do not produce a tail (Figure 1A). The comet assay revealed that hRPE cells exposed to 24 h of simulated microgravity (Figure 1B) suffered significant damage in the form of single-stranded DNA breaks when compared with control cells (Figure 1A). This damage was not repaired following 48 h of post-exposure incubation in culture medium (Figure 1C). However when the hRPE cells were pretreated with 1 μM cysteine followed by 48 h of post-exposure recovery (Figure 1D), single stranded DNA breaks were diminished compared to Figure 1B-C.
The median of the relative comet tail moment for hRPE cells exposed to microgravity (Mg; Figure 2) was substantially increased compared to nonexposed controls (control; Figure 2; 44 versus 9), indicating that microgravity alone induced DNA single-stranded breaks. This microgravity damage had not been repaired after 48 h of post-exposure recovery. Less than 10% of these cells were attached (RMg.A; Figure 2) to the plates. The DNA from the attached cells had no single-stranded breaks as indicated by the median of their relative comet tail moments. The remaining 90%, which appeared as floaters (RMg.F; Figure 2), had no change in the amount of DNA damage compared to cells measured immediately after microgravity exposure (44 versus 44). When hRPE cells were pretreated with 1 μM cysteine RMg.C and subjected to microgravity and post-exposure recovery, the single-stranded DNA breaks were reduced by at least half from the levels of either hRPE cells exposed to 24 h of simulated microgravity or those subjected to microgravity and allowed post-exposure recovery (22 versus 44).
Effect of microgravity on prostaglandin E2 secretion
PGE2 secretion was measured (Figure 3) in the conditioned medium of untreated hRPE samples control. hRPE cells were subjected to 24 h simulated microgravity and then incubated in medium for 48 h RMg, and hRPE cells pretreated with 1 μM cysteine subjected to 24 h simulated microgravity and then incubated in medium for 48 h RMg.C. Microgravity treatment did not cause secretion of PGE2 immediately, suggesting that there was no acute inflammation response induced in hRPE cells directly after microgravitational stress. However, 48 h after hRPE were subjected to microgravitational stress, there was a significant secretion of PGE2, which is consistent with an acute inflammatory response. This response was blocked by pretreatment of the hRPE cells with cysteine.
Exposure to microgravity poses a unique hazard to astronauts. Our previous studies have shown that radiation exposure increases single-stranded breaks in DNA, as measured by the Comet Assay, and that this damage can be prevented by the antioxidant lutein . We have shown here using simulated microgravity that microgravity induced significant single stranded DNA breaks and that damage was not repaired within 48 h.
Furthermore, based on the secretion of PGE2, the pathogenic mechanism of microgravity damage to retinal cells appears to be, at least in part, the induction of a delayed inflammatory response in the hRPE cells. Inflammation is a serious problem in the retina. The eye is immune privileged and except during a bacterial or viral infection, the blood ocular barriers prevent most immune cells from entering the retina and releasing reactive oxygen species . Should this barrier be broken and an immune response initiated, damage can occur that can lead to retinal degeneration and/or retinal detachment [31,32]. It has been also shown that inflammation and PGE2 release in the retina are serious risk factors for the induction of proliferative vitreoretinopathy (PVR)  and macular degeneration .The recent identification of a gene variant [common variant of the complement factor H (CFH) gene] found in half of the age-related macular degeneration cases in the United States further implicates inflammation in the etiology of macular degeneration [35-37]. We have shown here that an inflammatory response is induced in hRPE cells by simulated microgravity, without the introduction of an exogenous chemical agent or supplementary irradiation. This method may be useful for testing novel therapeutic approaches that suppress retinal inflammation.
Different cell types (lymphocytes, tumor cells) cultivated in microgravity in space and on earth have altered release of cytokine and hormone secretions and this has been reviewed . The induction of an inflammatory response is not unique to retinal cells. Osteoblasts were found to have a significant increased release of PGE2, when compared to ground controls in cells grown in microgravity on earth and aboard a space shuttle . Platelet-activation factor  and IL-6  which are normally stimulated by inflammatory and stressful conditions were both activated in rat osteoblasts under microgravity.
We have succeeded in blocking much of the microgravity-induced DNA damage and all of the PGE2 release from hRPE cells with cysteine (Figure 1, Figure 2). This protective effect is not surprising as we have seen similar protection against radiation and phototoxic induced inflammation using nontoxic sulfhydryl quenchers (cysteine, N-acetyl cysteine and the radioprotector WR-77913) in clinical trials , in vivo [42,43], and in vitro . Others have found these compounds useful against X-radiation [45,46]. As these compounds are safe for human consumption, protecting against accumulated microgravity-induced damage and reducing inflammation with cysteine or another nontoxic antiinflammatory agent could be part of a treatment in preventing acute, transitory, or enhanced age-related permanent blindness in astronauts after their return to earth. This treatment may also be applicable to flight personnel on long-haul flights .
We have presented here a model system of the stress of 0.01 gravity on hRPE cells. Real reduced gravity in space is between 0.0001 and 0.000001x g . A definite answer as to whether the single-stranded DNA breaks and the induced inflammatory response of the RWV culture conditions are due to the simulated microgravity, the low-shear environment and suspension of the cells, or a combination of all will have to wait until controlled experiments can be carried out in real microgravity during space flight. However, the in vivo damage to photoreceptor rod outer segments in rats exposed to space travel argues for reduced gravity as an environmental stressor of the retina [18,19].
This work was presented at the 2005 Association for Research in Vision and Ophthalmology meeting, Fort Lauderdale, FL on May 1-6, 2005. We wish to thank Dr. Ann Motten at National Institute of Environmental Health Sciences, Research Triangle Park, NC for help in the preparation of this manuscript. This research was supported in part by the Intramural Research Program of the National Institutes of Health, and National Institute of Environmental Health Sciences. These views are independent of the views of the Food and Drug Administration.
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