Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue™ and 5-carboxy-fluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm2 UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.
SivakJ.G., HerbertK.L., & FonnD. (1995). In vitro ocular irritancy measure of four contact lens solutions: damage and recovery. CLAO Journal21, 169–174.
2.
HerbertK.L., DixonD.G., & SivakJ.G. (1998). The effects of age on sensitivity of the rat lens to hexanol in vitro.Journal of Toxicology — Cutaneous and Ocular Toxicology17, 127–139.
3.
DovratA., & WeinrebO. (1999). Effects of UV-A radiations on lens epithelial NaK-ATPase in organ culture. Investigative Ophthalmology and Visual Science40, 1616–1620.
4.
PadgaonkarV.A., LeverenzV.R., FowlerK.E., ReddyV.N., & GiblinF.J. (2000). The effects of oxygen on the crystallins of cultured rabbit lenses. Experimental Eye Research71, 371–383.
5.
JohnsonI.D. (1996). Introduction to fluorescence techniques. In Handbook of Fluorescent Probes and Research Chemicals (ed. HaughlandR.P.), pp. 1–6. Eugene, OR, USA: Molecular Probes Inc.
6.
SchirmerK., ChanG.J., GreenbergB.M., DixonD.G., & BolsN.C. (1997). Methodology for demonstrating and measuring the phototoxicity of fluor-anthene to fish cells in culture. Toxicology in Vitro11, 107–119.
7.
GanassinR.C., SchirmerK., & BolsN.C. (2000). Methods for the use of fish cell and tissue cultures as model systems in basic and toxicology research. In The Laboratory Fish (ed. OstranderG.K.), pp. 631–651. San Diego, CA, USA: Academic Press.
8.
O'BrienJ., WilsonI., OrtonT., & PognanF. (2000). Investigation of the Alamar Blue™ (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry267, 5421–5426.
9.
LarsonE.M., DoughmanD.J., GregersonD.S., & ObritschW.F. (1997). A new, simple, non-radioactive, non-toxic in vitro assay to monitor corneal endothelial cell viability. Investigative Ophthalmology and Visual Science38, 1929–1933.
10.
HaughlandR.P. (1996). Assays for cell viability, proliferation and function. In Handbook of Fluorescent Probes and Research Chemicals (ed. HaughlandR.P.), pp. 365–398. Eugene, OR, USA: Molecular Probes Inc.
11.
LaycockN.L.C., SchirmerK., BolsN.C., & SivakJ.G. (2000). Optical properties of rainbow trout lenses after in vitro exposure to polycyclic aromatic hydrocarbons in the presence or absence of ultraviolet radiation. Experimental Eye Research70, 205–214.
12.
KoppS.J., GlonekT., & GreinerJ.V. (1982). Interspecies variations in mammalian lens metabolites as detected by phosphorus-31 nuclear magnetic resonance. Science, New York215, 1622–1625.
13.
VidalP., & Cabezas-CerratoJ. (1990). Column chromatography, sodium dodecyl sulphate–poly-acrylamide gel electrophoresis and two-dimensional electrophoresis of pig lens crystallins. Ophthalmic Research22, 31–38.
14.
JoblingA., StevensA., & AugusteynR.C. (1995). Distribution of proteins across the porcine lens. Clinical and Experimental Optometry78, 87–92.
15.
SivakJ.G., GershonD., DovratA., & WeerheimJ. (1986). Computer assisted scanning laser monitor of optical quality of the excised crystalline lens. Vision Research26, 1873–1879.
16.
SivakJ.G., YoshimuraM., WeerheimJ., & DovratA. (1990). Effect of hydrogen peroxide, DL-propra-nolol, and prednisolone on bovine lens optical function in culture. Investigative Ophthalmology and Visual Science31, 954–963.
17.
SivakJ.G., HerbertK.L., & BaczmanskiA. (1995). In vitro ocular irritancy measures of ketones and acetates using the cultured bovine lens. ATLA23, 689–698.
18.
SivakJ.G., HerbertK.L., & SegalL.(1994). Ocular organ culture as a measure of ocular irritancy: the effect of surfactants. Toxicology Methods4, 50–65.
19.
StuartD.D., SivakJ.G., CullenA.P., WeerheimJ.A., & MonteithC.A. (1991). UVB radiation and the optical properties of cultured bovine lenses. Current Eye Research10, 177–184.
20.
Van HornD.L., HannaC., & SchultzR.O. (1970). Corneal cryo-preservation: ultrastructural and viability changes. Archives of Ophthalmology84, 655–667.
21.
HightowerK.R., McCreadyJ.P., & BorchmanD. (1994). Membrane damage in UV-irradiated lenses. Photochemistry and Photobiology59, 485–490.
22.
MaurerH.R. (1981). Potential pitfalls of [3H]thymi-dine techniques to measure cell proliferation. Cell and Tissue Kinetics14, 111–120.
23.
OriowoO.M. (2000). A Study of Ultraviolet Radiation Effects on Porcine Crystalline Lens and Development of a New Assay Methodology for UV Cataractogenesis Investigation. PhD Dissertation. Pp. 28–54. University of Waterloo, Ontario, Canada.
24.
GoeganP., JohnsonG., & VincentR. (1995). Effects of serum protein and colloid on the Alamar Blue™ assay in cell cultures. Toxicology in Vitro9, 257–266.
25.
StuartD.D., & DoughtyM.J. (1996). In vitro UVB irradiation of bovine crystalline lens causes cell damage and reduction in leucine aminopeptidase activity in lens epithelium. Photochemistry and Photobiology B — Biology32, 81–87.
26.
VilupuruA.S., & GlasserA. (2001). Optical and bio-metric relationships of the isolated pig crystalline lens. Ophthalmic and Physiological Optics21, 296–311.
27.
PierscionekB.K. (1990). Presbyopia: the effect of refractive index. Clinical and Experimental Optometry73, 23–30.
PittsD.G., CullenA.P., & HackerP.D. (1977). Ocular effects of ultraviolet radiation from 295 to 365nm. Investigative Ophthalmology and Visual Science16, 932–939.
30.
WangG-M., RaghavachariN., & LouM.F. (1997). Relationship of protein-glutathione mixed disulfide and thiotransferase in H2O2-induced cataract in cultured pig lens. Experimental Eye Research64, 693–700.
31.
WuK-Y., WangH-Z., & HongS-J. (1998). Calcium-induced changes on crystallins in organ cultured porcine lens. Kao Hsiung I Ko Hsueh Tsa Chih (Kaohsiung Journal of Medical Sciences)14, 569–576.
