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From the Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, Minnesota.
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Abstract |
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METHODS. Anterior segments from human eyes were placed in perfusion organ culture. In cultures with initially high IOP, eyes were fixed at the high IOP level and histologic examination performed. In other cultures with high initial IOP, effluent medium was collected and subsequently reinfused after IOP had decreased to baseline. In cultures with stable baseline IOP, cell fragments from monolayer-cultured cells, or human genomic DNA, were infused at concentrations equivalent to 30,000 to 300,000 cells.
RESULTS. Electron microscopy of initially high-pressure cultures revealed scattered cell debris throughout the meshwork in greater amounts than found in eyes without initially high IOP. Reinfusion of effluent media from cultures with high initial pressures caused elevation of IOP. Centrifugation of effluent media lessened this elevation of IOP. In cultures with stable baseline IOP, infusion of cell fragments or genomic DNA raised IOP in a dose-dependent manner, with elevation of IOP for a minimum of 24 hours.
CONCLUSIONS. Cell debris can elevate IOP during the initial culture period, and after baseline pressures are established. Cell fragments and DNA increase IOP in a dose-dependent manner. The variations in baseline IOP seen during culture are probably caused by cell fragments and debris from dying cells in the meshwork, ciliary body, and other anterior segment tissues.
Changes in pressure during culture have been attributed to either transient showers of cell debris from dying cells or biochemical and metabolic cellular changes as cells adapt to the culture environment.1 2 6 Because the donor eyes used for culture are retrieved after death, blood flow through the eye and aqueous formation have ceased. Death of some cells before culture is an unavoidable consequence in this situation. These dead cells arise not only in the trabecular meshwork, but also in the ciliary body, iris, and other anterior segment tissues. After the dead cells are shed from their usual position, they ultimately are washed into the trabecular meshwork and could cause the initial elevation of IOP in cultured anterior segments. In addition to the initial cell loss, a slow, ongoing loss of trabecular cells also occurs during culture: 15% by day 7 and 25% by day 28, when compared with freshly fixed, noncultured fellow eyes.11 The extent to which debris from these dead cells can plug the outflow channels and cause variations in baseline IOP is unknown. In the present study, we examined the causes for variable IOP during culture in more detail.
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Materials and Methods |
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Anterior Segment Culture
A total of 34 pairs of fresh normal human eye bank eyes were studied (enucleated 5 ± 3 hours postmortem; cultured 11 ± 5 hours postmortem; range: 5–18). The average age of the donor eyes was 71 ± 13 years (range: 48–96). No eyes had glaucoma, uveitis, pseudoexfoliation syndrome, or treatment with ophthalmic medications. The culture technique was similar to that described previously.1 11 Eyes were bisected at the equator, and the iris, lens, and vitreous were removed. The anterior segment was clamped in a modified Petri dish and the eye perfused with Dulbecco’s modified Eagle’s medium with added antibiotics (penicillin: 10,000 U/100 mL solution, streptomycin: 10 mg, amphotericin B: 25 mg, and gentamicin: 17 mg, in 100 mL medium; Sigma-Aldrich, St. Louis, MO) at the normal human flow rate (2.5 µL/min). The anterior segments were cultured at 37°C in a 5% CO2 atmosphere. IOPs were continuously monitored with a pressure transducer connected to the second access cannula built into the dish and recorded with an automated computerized system. All dissections and culture preparations were performed by the same person (CKB).
Experimental infusions into the anterior chamber were performed with a gravity-driven, constant-pressure anterior chamber exchange over a 5-minute period. Two syringes were used, with the reservoir syringe containing the experimental solution held 14 cm (equivalent to 10 mm Hg) higher than effluent syringe containing culture medium. Fellow control eye cultures also underwent anterior chamber exchange but with vehicle or culture medium rather than the experimental solution. Pressure data from either eye were not used during the first hour after the anterior chamber exchange. A crossover design was used in experiments on some pairs of anterior segments, with the experimental solution added to one anterior segment and control solution to the fellow eye. Then, after IOP had stabilized, the previous control eye was given the experimental solution and vehicle to the previous experimental eye. Sequential doses of the experimental solutions were also used in some anterior segments after IOP had returned to baseline from the previous dose. This allowed comparisons of different doses in the same eye.
Fixation was performed either by immersion in fixative or through anterior chamber perfusion at the same pressure as present at the end of culture. Fixative was 4% paraformaldehyde in 0.1 M phosphate buffer. Wedges of tissue from the limbus were dissected, and, for routine assessment of culture survival, two quadrants 180° apart were prepared for light microscopy in embedding medium (JB-4; Polysciences Inc, Warrington, PA) and cut at 3-µm thickness. When transmission electron microscopy was required, tissue wedges were dehydrated in ascending concentrations of alcohol and embedded in epoxy resin. Routine assessment of all cultures included examination by light microscopy to examine meshwork cell shape, nuclear shape, preservation of cellular and nuclear membranes, cell–cell attachments, cytoplasmic covering of trabecular lamellae, preservation of endothelial lining of Schlemm’s canal, and integrity of trabecular lamellae. Meshworks were considered normal in appearance if trabecular cells remained in their usual position on the lamellae, normal numbers of cells were seen (subjective assessment), and little disruption of the juxtacanalicular tissue and trabecular lamellae were seen.11
Investigation of Initial Elevation of IOP.
Fifteen eyes of eight donors with an initial elevation of IOP over 35 mm Hg were studied by either histologic examination or analysis and reinfusion of effluent medium collected while the initial IOP was elevated. Four cultured anterior segments with initial pressures over 35 mm Hg were fixed at 48 hours’ time in culture, while the initial pressures remained elevated, and examined by light and electron microscopy. Two of these anterior segments had fellow eyes with cultured pressures under 30 mm Hg and were examined as controls.
Effluent medium was collected from four eyes during the initial elevation of IOP and examined by light microscopy, looking for evidence of dead cells or cellular debris. Protein levels were analyzed in effluent medium collected during the initial 3 days in 11 eyes with high IOP (>35 mm Hg) and 5 eyes with initial IOP less than 30 mm Hg (mean: 19.8 ± 5.7 mm Hg). Effluent medium was also collected from the same cultures after IOP had lowered to a stable baseline. Stable baseline IOP was considered 
25 mm Hg with variations of ±3 mm Hg over 24 hours. Medium was frozen at –70°C until analysis. Protein levels were determined with the Bradford method (BioRad, Hercules, CA).14
To determine whether debris found in the effluent medium could actually cause pressure elevation, effluent medium was collected, centrifuged, and reperfused into eyes once a stable baseline had been reached. This experiment was performed in 11 anterior segments from six pairs of eyes with initial elevation of IOP, and 5 anterior segments from three pairs of eyes with no initial elevation of IOP. Effluent medium from these eyes was collected during the time of initial IOP elevation or the comparable time if no IOP elevation occurred. Medium was centrifuged at 14,000 rpm for 5 minutes and then reinfused into the same eye after baseline IOP had been established, using an anterior chamber exchange followed by perfusion with this "old" medium for 30 hours (Table 1) . Culture medium was then switched to fresh medium and culture resumed. In 5 of the 11 anterior segments with initial IOP elevation, the effluent was spun twice: first at 14,000 rpm for 5 minutes and then again at 35,000 rpm for 30 minutes (Table 1) .
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A crossover experimental design was used in these initial IOP elevation experiments, giving sequential experimental infusions to the same anterior segment, making the total number of experimental trials greater than the total number of anterior segments listed in this portion of the study.
In addition to these experimental studies, a review of the past 125 pairs of cultured eyes used in this and other experiments was performed. Note was made of cultures with an IOP higher than 35 mm Hg during the initial time in culture. Relationships with donor age, sex, postmortem time to enucleation, and time to culture were determined and compared between eyes with pressures higher and lower than 35 mm Hg.
Investigation of Variability of Baseline IOP.
Acute loss of a large number of trabecular cells was induced by infusing the toxic compound APMA (p-aminophenylmercuric acetate) into six cultures by anterior chamber exchange at 0.6 mM in either single or sequential doses. After the final dose, DNase (1500 U) was added to both experimental and control anterior segments from four pairs of eyes, while the IOP was still elevated.
Loss of smaller numbers of cells was examined with a dose–response study using cell fragments from monolayer cultured human embryonal kidney cells (HEK293 cells). These cells were harvested and lysed by sonification. Cell fragments from 30,000 cells suspended in culture medium and mixed with 1500 U DNase were added by anterior chamber exchange. This amount of cells is approximately 6% of the total number of trabecular cells, as calculated later. After pressure returned to baseline several days later, fragments from 60,000 cells with DNase were added. Fellow control eyes received anterior chamber exchange with DNase in vehicle only. Anterior segments were fixed either at the time of maximum intraocular elevation after the infusion of the cell fragments or after IOP had returned to baseline.
The number of trabecular cells in the entire meshwork was calculated by using a circumference of 36 mm15 and 110 trabecular cells per 1-µm-thick section (actual mean ± SD: 110.4 ± 24.5; range: 82–173 cells per 1-µm section.11 16 Using nuclei as the basis for counting trabecular cells and assuming one nuclei per cell and an average nuclear size of 8 µm (range: 7–10 µm), we estimate that approximately 495,000 trabecular cells are present in the entire meshwork.
A dose–response study of human genomic DNA (Promega, Madison, WI) was performed using doses from 0.26 to 5.10 µg given in single or sequential doses (Table 1 ; the equivalent of 30,000–600,000 cells). In two pairs of eyes a repeated dose of DNA, previously found to increase IOP in that eye, was given but first digested with 1500 U DNase.
Statistical Analysis
Drug effects were expressed as the outflow facility after drug or experimental infusion (Cd) divided by the baseline facility (Co) for each anterior segment.17 Results from each pair of anterior segments were combined into a group mean for each concentration of drug or experimental agent and are expressed as the mean ± SEM. Statistical significance was tested with a paired t-test, or a Wilcoxon signed rank when data did not have a Gaussian distribution.18
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Results |
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Reinfusion of effluent medium collected from the initial culture period decreased facility 43% compared with baseline (P = 0.002; Table 1 ). This lasted a shorter period than the initial period of elevation of pressure (initial: 48.5 ± 8.7 hours; after reinfusion: 17.6 ± 3.6 hours in these same eyes). This pressure elevation did not reach as high as the initial elevation, reaching 71% of the initial pressure (initial: 49.3 ± 3.6 mm Hg; after medium reinfusion: 34.5 ± 4.1 mm Hg). High-speed centrifugation at 35,000 rpm for 30 minutes blunted the pressure elevation, with IOP reaching only 46% of the initial high pressure in these anterior segments (initial: 42.2 ± 11.7 mm Hg; after reinfusion: 19.6 ± 6.2 mm Hg). The corresponding facility of outflow decreased by 14% (not statistically significant; Table 1 ). In one anterior segment, medium collected after pressure had reached a stable baseline (day 14) was reinfused and did not cause an increase in pressure. This eye previously had an IOP increase when the initial effluent was reinfused. Five anterior segments that did not have an initial elevation of pressure had infusion of effluent medium from the initial culture period and had an 11% decrease in facility (not statistically significant; Table 1 ).
Investigation of Variability of Baseline IOP
Acute and extensive trabecular cell loss was caused by APMA, resulting in higher pressures for longer durations than seen either in the initial 3 days of culture or in the small variations of baseline IOP levels. APMA caused a rapid increase in IOP that peaked approximately 6 to 10 hours after the anterior chamber exchange. IOP then decreased, but the new stable baseline value was higher than before addition of the APMA. Facility of outflow decreased 56%, (P = 0.003; Table 1 ), whereas control cultures were relatively stable. Addition of DNase lowered IOP from this elevated level in two anterior segments (facility changed from 0.06 to 0.07 µL/min · mm Hg in one eye and 0.04 to 0.10 µL/min · mm Hg in the second eye) and had no effect in two other anterior segments. DNase did not change IOP in the fellow control anterior segments. Cell fragments and debris were found throughout the intertrabecular spaces and juxtacanalicular region. Subjective estimate of trabecular cell loss ranged from 50% to near-total loss among the six anterior segments. Greater amounts of debris were seen in cultures with higher pressure by subjective assessment. Removal of the meshwork in three experimental eyes lowered IOP from 21.7 ± 3.5 to 1.7 ± 0.6 mm Hg (P = 0.01). This indicated that the debris in the meshwork was creating most of the outflow resistance, as the remaining sclera only created a small amount of resistance.
Smaller amounts of cell loss were mimicked by the addition of cell fragments from the HEK293 monolayer-cultured cells. These cell fragments caused an increase in IOP that started immediately after the anterior chamber exchange and steadily rose over 6 hours. IOP remained maximally elevated for 6 to 20 hours, and outflow facility was reduced by 20% (P = 0.05; Table 1 ). IOP then slowly decreased over the next 48 hours (mean: 37 ± 12), but not to baseline levels (new baseline facility was 14% lower than original baseline; Fig. 1 ). An increase in number of cells (60,000 vs. 30,000) caused higher IOP levels and correspondingly lower facility of outflow (Table 1) . This IOP elevation lasted at least 48 hours, longer than with a lower number of cells, and reached a new baseline 33% lower than the previous baseline facility. Note that DNase had been added to the cell fragments before infusing into the cultures. Light and electron microscopy revealed rounded membrane spheres and cytoplasmic fragments of cells throughout the meshwork (Fig. 3) , especially under the inner wall of Schlemm’s canal in eyes fixed during maximum pressure elevation.
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Discussion |
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The initial elevation of IOP is mainly from cell debris in the meshwork. In the postmortem interval between death and culture, the lack of blood flow and aqueous flow cause death of several cell types in the eye. Vascular-dependent tissues are most susceptible, such as ciliary body and iris. Aqueous-dependent tissues, the trabecular meshwork and the cornea, can retain viability for longer periods, but are still subject to postmortem effects. Previous studies have shown that the trabecular meshwork is nonviable in approximately 25% of anterior segment cultures, presumably because of the postmortem condition of the trabecular cells at the initiation of culture.1 11 Although no significant difference between high and low initial IOP eyes for postmortem time to enucleation or time to culture, we have noted over the past 19 years that eyes placed in culture within 18 hours after death are most successful, with 24 hours the upper limit of reliable viability. Eyes beyond 24 hours have a culture success rate of only approximately 50%, versus the 78% success rate with fresher tissue (published12 and unpublished observations from our laboratory).
Given this initial load of dead cells in anterior segments, careful removal of non-necessary tissues (iris, lens, choroid, retina) is required when eyes are placed in culture. In addition, multiple rinses of the anterior segment in sterile saline before final placement in the culture dish are necessary to remove loose pigment and debris. We have removed the ciliary body in other experiments, but have found that this causes loss of the uveal meshwork layers and also appears to collapse the remaining meshwork, because of loss of countertraction on the scleral spur. Removal of ciliary processes while leaving the ciliary muscles intact could eliminate a source of dying cells, but requires meticulous dissection and also creates a pool of pigment that can enter and clog the meshwork despite saline rinses.
The reinfusion of effluent medium collected during the initial period of IOP elevation supports the idea that the cause of the pressure elevation is media borne. The finding of cellular debris in the effluent medium and the decrease in IOP-elevating effect after high-speed centrifugation are consistent with this. A significant amount of protein is present in effluent medium during the first 3 days of culture. This comes not only from the trabecular meshwork, but from other tissues throughout the anterior segment. We have found depots of protein in the sclera, cornea, and ciliary body that become depleted with culture time (data not shown). These protein stores probably arise because these tissues are bathed in plasma from their vascular supply during life. Several findings suggest this protein does not raise pressure: Protein levels in effluent medium were similar in eyes with and without elevation of pressure during the initial culture period; heat denaturation did not affect the ability of effluent medium to raise pressure; and finally, higher levels of protein from human serum (2500 µg) and fetal bovine serum (25,000 µg) did not raise pressure in previous experiments.19
The massive cell loss caused by APMA demonstrates the importance of performing histology on all drug studies in this culture system. Changes in facility may not only be due to a presumed metabolic action of an experimental drug, but could also be due to unplanned cell death caused by the drug. We have noted that several little-considered factors, such as osmolality and pH of solutions, can cause cell death in this system. Dissection and removal of the meshwork for biochemical or molecular analysis should include saving and examining two wedges of tissue, 180° apart, for histologic examination before meshworks are used in these analyses.
All culture models have strengths and limitations that must be understood in interpreting results of experiments. The elevation of IOP found in 31% of cultured anterior segments must be allowed to stabilize, as debris passes through the meshwork, before the cultures can be used. Once the initial debris and elevation of pressure have subsided and baseline pressures are present, cultures with an initial elevation of IOP can be satisfactorily used for experiments. The transient baseline pressure changes that occur in many cultures point out the necessity for the fellow eye to be used as a control eye in experiments, as these spontaneous fluctuations in pressure should not be regarded as caused by an experimental drug or agent. Given histologic verification of the health of the meshwork in each culture and knowing the limitations of the model, perfusion organ culture of the trabecular meshwork continues to have a role in glaucoma research.
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Footnotes |
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Submitted for publication February 13, 2004; revised May 7, 2004; accepted May 18, 2004.
Disclosure: C.K. Bahler, None; M.P. Fautsch, None; C.R. Hann, None; D.H. Johnson, 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.
Corresponding author: Douglas H. Johnson, Department of Ophthalmology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905; johnson.douglas{at}mayo.edu.
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  B. Bachmann, M. Birke, D. Kook, M. Eichhorn, and E. Lutjen-Drecoll Ultrastructural and Biochemical Evaluation of the Porcine Anterior Chamber Perfusion Model Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 2011 - 2020. [Abstract] [Full Text] [PDF]
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) Time of a leak in the control eye, which created low pressure. Note the effect of DNase in blunting the increase in IOP in the experimental eye. The control eye increased 2 mm Hg after infusion of DNase.