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Time evolution of active caspase-3 labelling after in vivo exposure to UVR-300 nm
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
2014 (English)In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 92, no 8, 769-773 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE:To determine the time evolution of active caspase-3 protein expression in albino rat lens after in vivo exposure to low-dose UVR-300 nm, as detected by immunofluorescence.

METHODS:Forty Sprague-Dawley rats were unilaterally exposed in vivo to 1 kJ/m2 UVR-300 nm for 15 min. At 0.5, 8, 16 and 24 hr after the UVR exposure, the exposed and contralateral nonexposed lenses were removed and processed for immunohistochemistry. Three mid-sagittal sections from each lens were stained. The cells labelled for active caspase-3 in each section of both the exposed and nonexposed lenses were counted and recorded three times. The difference of the proportion of labelling between the exposed and contralateral nonexposed lenses within each animal was calculated. The differences of active caspase-3 labelling at four different time-points after exposure were used to determine the time evolution of active caspase-3 expression.

RESULTS:Caspase-3 expression was higher in the exposed than in contralateral nonexposed lenses. The mean difference between the exposed and contralateral nonexposed lenses, including all lenses from all time intervals, was 0.12 ± 0.01 (= CI 95%). The mean differences between the exposed and contralateral nonexposed lenses were 0.11 ± 0.02, 0.13 ± 0.02, 0.14 ± 0.01 and 0.09 ± 0.03 (= CI 95%) for the 0.5-, 8-, 16- and 24-hr time groups, respectively. The orthogonal comparison showed no difference in the expression of active caspase-3 between the 0.5- and the 24-hr groups (Test statistic 1.50, F1,36 = 4.11, p < 0.05) or between the 8- and the 16-hr groups (test statistic 0.05, F1,36 = 4.11, p < 0.05). There was a difference when comparing the 0.5- and 24-hr groups to the 8- and 16-hr groups (test statistic 7.01, F1,36 = 4.11, p < 0.05).

CONCLUSION:The expression of active caspase-3 in the lens epithelium increases after UVR exposure. There is a peak of expression approximately 16 hr after the exposure.

Place, publisher, year, edition, pages
2014. Vol. 92, no 8, 769-773 p.
National Category
Ophthalmology
Identifiers
URN: urn:nbn:se:uu:diva-228214DOI: 10.1111/aos.12407ISI: 000345342600036PubMedID: 24698086OAI: oai:DiVA.org:uu-228214DiVA: diva2:733108
Funder
Swedish Research Council, K2008-63X-15035-05-2
Available from: 2014-07-08 Created: 2014-07-08 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Caspase-3 in lens epithelium
Open this publication in new window or tab >>Caspase-3 in lens epithelium
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Purpose: To model the time evolution of active caspase-3 protein expression in a healthy lens, and in a lens exposed to UVR-300 nm (UVR-B). To develop an automated method to classify the fluorescent signal of biomarkers in the lens epithelial cells.

Methods: Six-week old Sprague-Dawley rats were used. Firstly, expression of active caspase-3 was studied in the lens epithelium of healthy rats. Secondly, rats were unilaterally exposed in vivo to 1 kJ/m2 UVR-B for 15 minutes. At 0.5, 8, 16, and 24 hours after the UVR-B exposure, the exposed and the contralateral non-exposed lenses were removed. Immunohistochemistry was done on three mid-sagittal sections from each lens. The florescent labelling for active caspase-3 in each lens section was counted three times. The time evolution of active caspase-3 expression in response to UVR-B exposure was modelled as a function of cell position in the lens epithelium. An automated objective method was developed to quantify the lens epithelial cells and to classify the fluorescent signal of active caspase-3. Active caspase-3 was selected as a model signal.

Results: Active caspase-3 was abundant in the anterior pole of the normal lenses. Spatial distribution of active caspase-3 labelling in the lens epithelium was fitted to a logistic model. The probability of active caspase-3 expression was higher in the UVR-B exposed lenses (95% CI = 0.12 ± 0.01). There was no difference in the expression of active caspase-3 between the 0.5 and the 24 hours groups or between the 8 and the 16 hours groups. A difference was noted, when comparing the 0.5 and 24 hours groups with the 8 and 16 hours groups (Test statistic 7.01, F1;36;0.95= 4.11). Exposure to UVR-B has an impact on the average probability of labelling for active caspase-3 as a function of cell position. The probability of labelling as a function of cell number also varied as a function of time after UVR-B exposure. The automated method counted the lens epithelial cells and estimated the proportion of active caspase-3 labelling in the lens epithelium.

Conclusions: Active caspase-3 is present in the healthy lens epithelial cells. Active caspase-3 exhibits higher expression at the anterior pole of the lens and the expression decreases towards the periphery. After UVR-B exposure, the expression of active caspase-3 in the lens epithelium increases with a peak of expression occurring around 16 hours after exposure. The average probability of labelling in the lens epithelium is dependent on both the UVR-B exposure and the time period elapsed after the exposure. The automated method enables objective and fast quantification of lens epithelial cells and the expression of fluorescent signal in the lens cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 40 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1168
Keyword
ultraviolet radiation, caspase-3, lens, cataract, apoptosis, Immunohistochemistry, spatial distribution, time evolution, modelling, automatic analysis, cell counting, image analysis.
National Category
Neurosciences
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-267543 (URN)978-91-554-9436-0 (ISBN)
Public defence
2016-02-05, Enghoffsalen, entrance 50, 1st floor, Akademiska Sjukhuset, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-01-21 Created: 2015-11-24 Last updated: 2016-02-12Bibliographically approved

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Talebizadeh, NooshinYu, ZhaohuaKronschläger, MartinSöderberg, Per

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