Area and depth of surfactant-induced corneal injury correlates with cell death.

PURPOSE In previous studies in which in vivo confocal microscopy (CM) was used, quantifiable differences were identified in the corneal epithelium and stroma for surfactants producing different degrees of ocular irritation. In the present study, in vivo confocal microscopy was used to determine area and depth of the initial corneal changes, and the correlation of the data to cell death was characterized by ex vivo live-dead assay. METHODS In four groups of rabbits (12 animals each), 10 microl surfactants known to produce slight, mild, moderate, or severe irritation was applied to the central cornea of one eye; 4 untreated rabbits served as controls. Measurements of group total mean epithelial thickness, epithelial cell area, and depth of keratocyte loss in four corneal regions were made by in vivo CM in 6 rabbits of each group and in 4 control animals at 3 hours and in the remaining rabbits at 3 hours and 1 day. Corneas were then removed and fixed for conventional histologic examination (two eyes/treatment/group), or regions were excised and placed in culture media containing 2 microM calcein-acetoxymethyl ester (calcein-AM) and 4 microM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 microm (in the x, y, and z axes, respectively) volume of the cornea was determined. RESULTS Confocal microscopy showed that application of the slight irritant resulted in decreased epithelial thickness at 3 hours (41.2+/-2.6 microm in treated eyes versus 43.6+/-3 microm in control eyes; n=6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630+/-203 microm2 versus 1427.2+/-90.7 microm2). On day 1, mild, moderate, and severe irritants caused complete loss of epithelium and disappearance of keratocytes to a depth of 30.8+/-10.7 microm, 47.2+/-10.4 microm, and 764.6+/-159.6 microm (n=6, 5, and 6), respectively. At 3 hours, live-dead assay detected more dead epithelial cells as a percentage of total surface cells (49.2+/-4.5% in slightly irritated eyes versus 20.9+/-3.2% in control eyes), significantly correlating with the measurement by in vivo CM of average epithelial cell size in each eye (r=-0.96; P < 0.005). On day 1, mild and moderate irritants showed increasing stromal cell death from 9.8+/-16.2 cells to 36.4+/-17.7 cells, which significantly correlated with the depth of stromal injury determined by in vivo CM (r=0.79; P < 0.00001). No surviving keratocytes were detected in severely irritated eyes. CONCLUSIONS The data support the hypothesis that differences in surfactant-induced ocular irritation are directly related to area and depth of acute corneal injury.

[1]  W M Petroll,et al.  Application of In Vivo Confocal Microscopy to the Understanding of Surfactant-Induced Ocular Irritation*Oryzias latipes , 1996, Toxicologic pathology.

[2]  Robert H. Webb,et al.  In-vivo confocal microscopy , 1995 .

[3]  D. J. Kain,et al.  Evaluation of seven in vitro alternatives for ocular safety testing. , 1991, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[4]  John H. Draize,et al.  METHODS FOR THE STUDY OF IRRITATION AND TOXICITY OF SUBSTANCES APPLIED TOPICALLY TO THE SKIN AND MUCOUS MEMBRANES , 1944 .

[5]  C. Parish,et al.  New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. , 1990, Journal of immunological methods.

[6]  H. D. Cavanagh,et al.  The relation between contact lens oxygen transmissibility and binding of Pseudomonas aeruginosa to the cornea after overnight wear. , 1994, Ophthalmology.

[7]  W M Petroll,et al.  Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease. , 1993, Ophthalmology.

[8]  T. Møller-Pedersen,et al.  Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy. , 1997, Ophthalmology.

[9]  H. D. Cavanagh,et al.  Assessment of f-actin organization and apical-basal polarity during in vivo cat endothelial wound healing. , 1995, Investigative ophthalmology & visual science.

[10]  D. T. Hooker,et al.  Correlation of Animal Eye Test Data with Human Experience for Household Products: An Update , 1986 .

[11]  T. Møller-Pedersen,et al.  Confocal microscopic characterization of wound repair after photorefractive keratectomy. , 1998, Investigative ophthalmology & visual science.

[12]  R. Heywood,et al.  Towards objectivity in the assessment of eye irritation , 1978 .

[13]  J. Pauluhn,et al.  Analysis of alternative methods for determining ocular irritation. , 1992, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[14]  Rowan An.  The future of animals in research and training. The search for alternatives. , 1984 .

[15]  K. Green.  A brief history of ocular toxicology. , 1992, Lens and eye toxicity research.

[16]  W M Petroll,et al.  Digital image acquisition in in vivo confocal microscopy , 1992, Journal of microscopy.

[17]  C S Weil,et al.  Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. , 1971, Toxicology and applied pharmacology.

[18]  J. Auran,et al.  Scanning slit confocal microscopic observation of cell morphology and movement within the normal human anterior cornea. , 1995, Ophthalmology.

[19]  G J Carr,et al.  Ocular Irritation: Microscopic Changes Occurring Over Time in the Rat with Surfactants of Known Irritancy , 1998, Toxicologic pathology.

[20]  J. Maurer,et al.  Light Microscopic Comparison of Surfactant-Induced Eye Irritation in Rabbits and Rats at Three Hours and Recovery/Day 35*1 , 1996, Toxicologic pathology.

[21]  W M Petroll,et al.  Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF). , 1997, Current eye research.

[22]  Marzulli Fn,et al.  Eye irritation from topically applied drugs and cosmetics: preclinical studies. , 1971 .

[23]  H. D. Cavanagh,et al.  Radial keratotomy. III. Relationship between wound gape and corneal curvature in primate eyes. , 1992, Investigative ophthalmology & visual science.

[24]  H. D. Cavanagh,et al.  Adherence of Pseudomonas aeruginosa to shed rabbit corneal epithelial cells after overnight wear of contact lenses. , 1997, The CLAO journal : official publication of the Contact Lens Association of Ophthalmologists, Inc.

[25]  G. Wilson,et al.  Apoptosis in the corneal epithelium. , 1996, Investigative ophthalmology & visual science.

[26]  G. Kennedy,et al.  Evaluation of ocular irritancy potential: intralaboratory variability and effect of dosage volume. , 1982, Toxicology letters.

[27]  F. Marks,et al.  Keratinocyte-derived proinflammatory key mediators and cell viability as in vitro parameters of irritancy: a possible alternative to the Draize skin irritation test. , 1994, Toxicology and applied pharmacology.

[28]  W M Petroll,et al.  Quantitative three-dimensional confocal imaging of the cornea in situ and in vivo: system design and calibration. , 2006, Scanning.

[29]  H. D. Cavanagh,et al.  Quantitative Assessment of Anteroposterior Keratocyte Density in the Normal Rabbit Cornea , 1995, Cornea.

[30]  H E Kaufman,et al.  Early diagnosis of infectious keratitis with in vivo real time confocal microscopy. , 1992, The CLAO journal : official publication of the Contact Lens Association of Ophthalmologists, Inc.

[31]  S D Gettings,et al.  The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II) oil/water emulsions. , 1994, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[32]  P. Toft,et al.  Recent Progress in the Eye Irritation Test , 1993, Toxicology and industrial health.

[33]  R. Folberg,et al.  Outbreak of keratitis presumed to be caused by Acanthamoeba. , 1996, American journal of ophthalmology.

[34]  J. Jester,et al.  The Fas-Fas ligand system and other modulators of apoptosis in the cornea. , 1996, Investigative ophthalmology & visual science.

[35]  G. Clover,et al.  Keratocyte networks visualised in the living cornea using vital dyes. , 1993, Journal of cell science.

[36]  I. Dubé.  [Ocular irritation]. , 1973, La Vie medicale au Canada francais.