Posterior capsular opacification (PCO), caused predominately by proliferation and migration of residual lens epithelial cells (LECs), is considered the most important and common complication of contemporary cataract surgery.1,2 In vitro models of PCO have been developed,2–7 including a technique in which lens capsule explants are harvested from cadaver eyes and maintained in tissue culture.8,9
Limited information is available on the effect of cataract surgical technique on the density of residual LECs.10 In addition, limited and somewhat conflicting information is available on the benefit of techniques such as anterior capsule vacuuming or polishing, which are designed to remove LECs at the time of cataract extraction to prevent PCO.11–14 This study compared LEC regrowth rate in a lens capsule explant model after lens extraction by extracapsular cataract extraction (ECCE) or phacoemulsification with and without vacuuming of the anterior and equatorial lens capsules to remove residual LECs.
Materials and Methods
All tissues used in this study were obtained at a local humane society from healthy dogs free of ocular disease that were killed for population-control purposes. The protocol for procurement of the tissues was reviewed by the authors' Institutional Animal Use Committee and was in accordance with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research.
The eyes were examined with a focal light source and found to be free of corneal or lens opacity. Dog age, which ranged from 2 to 6 years, was estimated based on dental characteristics and density and light scatter produced by the crystalline lens. Within 2 hours after the dogs were killed, enucleations were performed and globes were transported in a povidone–iodine solution (Betadine® 2%) in an ice bath. Samples were then transferred to a sterile phosphate-buffered saline (pH = 7.4, Gibco-BRL) and refrigerated for 1 to 6 hours before capsular bag dissection.
Lens removal was performed on the cadaver eyes by an experienced ophthalmic surgeon using 1 of 3 surgical techniques, which were randomly assigned. In 1 group, ECCE was performed by removing the corneoscleral shell, followed by creation of a continuous curvilinear capsulorhexis (7.0 mm) of the axial anterior capsule and hydroexpression of the lens fiber mass with a irrigation vectus (Storz Instrument Co.) and balanced salt solution (BSS®). If present, residual lens cortical material in the equatorial region was gently removed by manual irrigation/aspiration (I/A) using a coaxial cannula (Storz Instrument Co.).
In the 2 other groups, lens removal by a 1-handed phacoemulsification technique was performed through a 2.8 mm, 2-plane limbal incision using hydrodissection and a divide-and-conquer lens fragmenting method with a 30 degree phacoemulsification tip. Irrigation fluid was BSS. Phacoemulsification power was linearly controlled with a maximum setting of 70% (Alcon Series 10,000). Phacoemulsification times ranged from 35 to 57 seconds. In both phacoemulsification groups, residual cortical fibers in the equatorial region were removed by automated I/A using a 0.5 mm port tip (Storz Instrument Co.). In 1 group a straight I/A tip, and in the superior capsular bag a curved I/A tip, were also used under low vacuum settings to gently vacuum the anterior and equatorial lens capsules to remove residual LECs. During capsule vacuuming, adherent LECs on the internal capsule surfaces were identified visually by the surgeon using retroillumination from the tapetal reflection; this maneuver typically required approximately 30 to 45 seconds.
In all 3 groups, the lens capsules were explanted by excising the ciliary zonules near the lens equator and then dissecting the lens capsule from the anterior hyaloid membrane. The capsules were removed from the globe and pinned to a poly(methyl methacrylate) petri dish to retain their normal circular shape using a previously described method.8,9 The capsule explants were then cultured in 1.5 mL of Dulbecco's minimum essential media (DMEM) supplemented with fetal calf serum 10% (FCS) or serum-free DMEM (both with 50 μg/mL gentamicin). The explants were incubated at 35°C in carbon dioxide 5%, and the culture medium was replaced every 3 to 4 days.
The surface area of the anterior and extreme equatorial lens capsule that was covered by LECs immediately after the surgery was estimated using phase contrast microscopy and a reticle and recorded as a percentage surface area. Daily phase contrast microscopy observations were made on the morphology of cells and cell proliferation along the anterior and posterior capsules, and the interval to a 100% confluence rate on the posterior capsule was determined. The initial cell density in each surgical group for capsules cultured in the 2 media conditions and the regrowth rates of the 2 culture conditions were compared using a nonpaired Student t test. The interval to confluence for each of the 3 surgical techniques was compared using an analysis of variance and Tukey test. A P value less than 0.05 was considered significant.
The percentage of the anterior lens capsule covered by residual LECs immediately after surgery was influenced by type of technique (Table 1). The ECCE, phacoemulsification alone, and phacoemulsification/capsule-vacuuming groups showed a mean 31.6%, 16.1%, and 7.7% surface area coverage, respectively. In the latter group, a small rim of LECs was consistently found only in the extreme equatorial region, particularly on the superior capsule adjacent to the limbal incision created for phacoemulsification. In all groups, LEC proliferation and migration were seen as early as 12 hours after culture with a transition to a spindle-shaped cell. When cell confluence on the posterior capsule was reached, cells assumed a cuboidal shape and aggregates of cells were associated with light scatter.
Within each surgical group, there were no significant differences in the initial percentage of cells between capsules cultured in serum-supplemented and those cultured in serum-free media. When regrowth rates of all surgery groups were considered, the mean interval to confluence on the posterior capsule was significantly shorter in the capsules cultured in FCS 10% than in those cultured in serum-free media (5.9 versus 18.5 days, P < .001). The interval to confluence was not significantly different among the 3 groups when the explants were cultured in FCS 10%. When the explants were cultured in serum-free media, the phacoemulsification/capsule-vacuuming group had a significantly longer time to confluence (mean 5.3 days longer) than the other 2 groups (P = .012).
Studies have suggested the lens capsule explant culture method used here is an excellent means of studying the in vitro development and progression of PCO as it allows monitoring of the proliferation and migration of LECs onto their natural matrix, the lens capsule. The lens capsule explants exhibited most of the features of in vivo PCO, including rapid LEC growth onto capsule surfaces, light scatter from cellular aggregates, and the development of capsule folds and wrinkling. In addition, this model provides an appropriate initial number, density, and stability of LECs to allow for cell growth, even in protein-free culture media.8,9 We recently characterized the canine lens capsule model and have documented that capsule explants collected from dogs are virtually identical to human lens explants and, as such, are an appropriate alternative tissue source for the in vitro study of human PCO (unpublished data, 1999).
Because PCO is predominately caused by residual LECs in the capsular bag after crystalline lens removal,1,2,5 several surgical techniques have been advocated to remove these cells at the time of lens extraction. These methods include aspiration of the anterior capsule with or without ultrasonic cavitation,12,15–21 pharmacological dispersion and aspiration of the anterior capsule,22–25 and manual polishing the anterior capsule, posterior capsule, or both using specially designed cannulas.11,18,26,27 However, limited and conflicting information is available on the effect of removing residual LECs on the long-term development of PCO.
Nishi and Nishi12 found that meticulous capsule vacuuming using ultrasound during endocapsular cataract surgery significantly reduced the number of patients requiring laser capsulotomy procedures (from 10.8% to 3.7%). In another study, capsule vacuuming was found to reduce, but not eliminate, PCO in experimental animals after lens removal and refilling of the capsular bag with an inflatable balloon.14 In contrast, vacuuming the posterior capsule had no effect on the long- term development of PCO in another study,11 and equatorial capsule vacuuming did not influence the severity of lens regrowth in rabbits after lensectomy by phacoemulsification.28
Although vacuuming or polishing the posterior capsule appears to have an immediate benefit on posterior capsule clarity, the long-term benefit may be limited as the cell population responsible for PCO appears to arise predominately from germinative LECs in the lens equatorial region,1,9,29 not from the displaced metaplastic LECs already present on the posterior capsule. As equatorial capsule vacuuming is associated with additional time and surgically induced trauma to the eye, as well as the risk of capsule tears,30,31 the procedure may be unwarranted if no long-term benefit is derived.
In this study, phacoemulsification and cortical I/A lead to less residual LEC concentration on the internal capsule surfaces than ECCE with manual nuclear expression. This suggests that during phacoemulsification, the ultrasonic cavitation created by the phacoemulsification tip, aspiration of lens fibers by the I/A tip, or both remove a certain percentage of LECs. However, residual LECs in both groups rapidly proliferated, and time to confluence along the posterior capsule did not differ between the 2 groups, regardless of culture conditions. These findings are similar to those reported for human lens capsules subjected to ECCE or phacoemulsification and cultured in serum-free media.10
In the study group in which capsule vacuuming was performed, LEC density was even less; 5% or less of the surface area was affected in some specimens. However, complete elimination of LECs was not possible with traditional I/A tips in any specimen, and residual cells invariably were present in the superior capsule rim adjacent to the surgical incision. Cell proliferation across the remaining anterior and axial posterior capsules proceeded even from small aggregates of these residual cells.
There were no significant differences in confluence rates among the groups when the explants were cultured in serum-supplemented media, presumably because the growth factors in the serum promoted cell proliferation to a similar extent in all groups. When the explants were cultured in serum-free media, the capsule-vacuuming group had a longer interval to LEC growth to confluence. However, the delay was only 5 to 7 days, an interval likely to be of little clinical importance. Culture conditions using serum supplementation may more accurately reflect the aqueous humor milieu immediately after intraocular surgery with disruption of the blood–ocular barrier and influx of serum proteins; serum-free culture conditions probably reflect the in vivo situation within several days after surgery, after restoration of the barrier.32
Our results suggest that near 100% elimination of residual LECs at the time of cataract extraction may be necessary to prevent LEC proliferation onto the posterior capsule and the development of PCO. As this appears to be a difficult task, at least with commonly used surgical techniques, efforts to reduce or prevent PCO may be better directed at inhibition of LEC proliferation, migration, or fibrous metaplasia.
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