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

James V. Jester, Hong Fang Li, Walter M Petroll, Ron D. Parker, Harrison D Cavanagh, Gregory J. Carr, Brian Smith, James K. Maurer

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Abstract

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 μl 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 μM calcein- acetoxymethyl ester (calcein-AM) and 4 μM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 μm (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 μm in treated eyes versus 43.6 ± 3 μm in control eyes; n = 6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630 ± 203 μm2 versus 1427.2 ± 90.7 μm2). 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 μm, 47.2 ± 10.4 μm, and 764.6 ± 159.6 μm (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.

Original languageEnglish (US)
Pages (from-to)922-936
Number of pages15
JournalInvestigative Ophthalmology and Visual Science
Volume39
Issue number6
StatePublished - May 1998

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Surface-Active Agents
Cell Death
Confocal Microscopy
Epithelial Cells
Irritants
Cornea
Rabbits
Stromal Cells
Cell Size
Corneal Injuries
Corneal Stroma
Corneal Epithelium
Culture Media
Epithelium
Intravital Microscopy
Wounds and Injuries

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Area and depth of surfactant-induced corneal injury correlates with cell death. / Jester, James V.; Li, Hong Fang; Petroll, Walter M; Parker, Ron D.; Cavanagh, Harrison D; Carr, Gregory J.; Smith, Brian; Maurer, James K.

In: Investigative Ophthalmology and Visual Science, Vol. 39, No. 6, 05.1998, p. 922-936.

Research output: Contribution to journalArticle

Jester, JV, Li, HF, Petroll, WM, Parker, RD, Cavanagh, HD, Carr, GJ, Smith, B & Maurer, JK 1998, 'Area and depth of surfactant-induced corneal injury correlates with cell death', Investigative Ophthalmology and Visual Science, vol. 39, no. 6, pp. 922-936.
Jester, James V. ; Li, Hong Fang ; Petroll, Walter M ; Parker, Ron D. ; Cavanagh, Harrison D ; Carr, Gregory J. ; Smith, Brian ; Maurer, James K. / Area and depth of surfactant-induced corneal injury correlates with cell death. In: Investigative Ophthalmology and Visual Science. 1998 ; Vol. 39, No. 6. pp. 922-936.
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T1 - Area and depth of surfactant-induced corneal injury correlates with cell death

AU - Jester, James V.

AU - Li, Hong Fang

AU - Petroll, Walter M

AU - Parker, Ron D.

AU - Cavanagh, Harrison D

AU - Carr, Gregory J.

AU - Smith, Brian

AU - Maurer, James K.

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N2 - 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 μl 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 μM calcein- acetoxymethyl ester (calcein-AM) and 4 μM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 μm (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 μm in treated eyes versus 43.6 ± 3 μm in control eyes; n = 6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630 ± 203 μm2 versus 1427.2 ± 90.7 μm2). 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 μm, 47.2 ± 10.4 μm, and 764.6 ± 159.6 μm (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.

AB - 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 μl 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 μM calcein- acetoxymethyl ester (calcein-AM) and 4 μM ethidium homodimer. Using laser scanning CM, the number of dead epithelial or stromal cells in a 300 x 300 x 170 μm (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 μm in treated eyes versus 43.6 ± 3 μm in control eyes; n = 6 and 4, respectively) and a significant decrease (P < 0.001) in epithelial cell size (630 ± 203 μm2 versus 1427.2 ± 90.7 μm2). 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 μm, 47.2 ± 10.4 μm, and 764.6 ± 159.6 μm (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.

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