Lens cell survival after exposure to stress in the closed capsular bag.
OBJECTIVE
Despite recent improvements in IOL design posterior capsule opacification (PCO) remains a significant clinical problem after cataract surgery. The Perfect Capsule device (Milvella, Ltd., Epping, Australia) permits the introduction and subsequent removal of potentially toxic agents into the closed capsular bag. The present purpose was to compare the relative effectiveness of exposing cells within the human capsular bag with a range of stresses with clinical potential and to compare the response to the same agents when applied to rabbit capsular bags and to cultured human lens cells.


METHODS
Human capsular bags were prepared from donor eyes and sealed with the Perfect Capsule device. Distilled water, 3 M NaCl, 250 microg/mL, 25 mg/mL 5-fluorouracil and 100 microM thapsigargin (Tg) were introduced for 2 minutes. The bags were then perfused with Eagle's minimum essential medium (EMEM) and an IOL inserted before the bags were dissected and pinned to the base of plastic culture dishes. The bags were maintained in EMEM for 28 days and phase images were acquired throughout. Rabbit eyes were prepared and cultured in a similar manner, although tests were limited to 5-FU (25 mg/mL) and Tg (30-300 microM). FHL124 cells were cultured on plastic (EMEM supplemented with 5% FCS), and serum was removed for 24 hours before exposure to the same agents as human bags. Cell survival was assessed by quantifying Coomassie blue staining after 4 days.


RESULTS
Initially, NaCl induced by far the most obvious signs of cell death, especially of anterior cells, followed by 5-FU>water>Tg. However, by 2 weeks, cell death became more apparent in the Tg-exposed bags, and, at the end of 4 weeks, there were no cells surviving. Cells on the posterior capsule were confluent in water-exposed bags (similar to unexposed controls), whereas after NaCl exposure, coverage was incomplete but greater than after 5-FU. In the rabbit bags, exposure to 25 mg/mL 5-FU totally eliminated cells, but 100 microM Tg was ineffective. At the end of a 4-day culture of FHL124 cells exposed for 2 minutes to NaCl, 5-FU or Tg, there were no cells surviving, whereas there was 50% cell survival compared with control cells after water treatment.


CONCLUSIONS
Tissue-cultured human lens cells are much more sensitive to short-term hyperosmotic than hyposmotic stress, with a rapid onset of cell death of cultured cells exposed to 3 M NaCl. This finding was confirmed in human capsular bags although adherent fibers appear to offer additional protection to 5-FU which can be overcome by the very hydrophobic Tg. The application of the Perfect Capsule system in concert with thapsigargin provides a promising means of preventing PCO.
Duncan
George
G
School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
Wang
Lixin
L
Neilson
Geoffrey J
GJ
Wormstone
Ian Michael
IM
D000199:Actins; D000818:Animals; D002386:Cataract / Q000517:prevention & control; D018929:Cell Culture Techniques; D002470:Cell Survival; D019084:Fluorescent Antibody Technique, Indirect; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007903:Lens Capsule, Crystalline / Q000502:physiology*; D019654:Lens Implantation, Intraocular; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D007910:Lenses, Intraocular; D018613:Microscopy, Confocal; D009997:Osmotic Pressure; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014746:Vimentin
D000199:Actins; D014746:Vimentin; D012965:Sodium Chloride; D019284:Thapsigargin; D005472:Fluorouracil