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Caspase-3 in lens epithelium
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.ORCID iD: 0000-0001-7325-7358
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 [en]
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: urn:nbn:se:uu:diva-267543ISBN: 978-91-554-9436-0 (print)OAI: oai:DiVA.org:uu-267543DiVA: diva2:876462
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
List of papers
1. Specific spatial distribution of caspase-3 in normal lenses
Open this publication in new window or tab >>Specific spatial distribution of caspase-3 in normal lenses
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2014 (English)In: Acta Ophthalmologica, ISSN 1755-375X, E-ISSN 1755-3768, Vol. 93, no 3, 289-292 p.Article in journal (Refereed) Published
Abstract [en]

Purpose

To determine the distribution of active caspase-3 in rat eye lens epithelium.

Methods

In total, 120 sagittal sections from forty rats were assessed for active caspase-3 labelling using immunohistochemistry. Lens epithelial cells were counted, and the fraction of active caspase-3 labelled cells and their relative positions were identified in each section.

Results

Active caspase-3 is present in normal lens epithelium. The active caspase-3 expression was higher in the anterior pole of the lens. Probability of radial spatial distribution of labelling was fitted with a logistic model. The increase rate and the inflection point were estimated as CI (0.95) to 23 ± 3 cells and 114 ± 3 cells, respectively.

Conclusion

The gradually decreasing active caspase-3 labelling from the anterior pole to the periphery suggests that active caspase-3 may be involved in normal protein turnover caused by, for example, incident light.

National Category
Ophthalmology
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-226542 (URN)10.1111/aos.12501 (DOI)000353053000031 ()
Available from: 2014-06-18 Created: 2014-06-18 Last updated: 2017-12-05Bibliographically approved
2. Time evolution of active caspase-3 labelling after in vivo exposure to UVR-300 nm
Open this publication in new window or tab >>Time evolution of active caspase-3 labelling after in vivo exposure to UVR-300 nm
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.

National Category
Ophthalmology
Identifiers
urn:nbn:se:uu:diva-228214 (URN)10.1111/aos.12407 (DOI)000345342600036 ()24698086 (PubMedID)
Funder
Swedish Research Council, K2008-63X-15035-05-2
Available from: 2014-07-08 Created: 2014-07-08 Last updated: 2017-12-05Bibliographically approved
3. Modelling the time evolution of active caspase-3 protein in the rat lens after in vivo exposure to Ultraviolet radiation-B: Model for active caspase-3 expression after in vivo exposure to UVR-300 nm
Open this publication in new window or tab >>Modelling the time evolution of active caspase-3 protein in the rat lens after in vivo exposure to Ultraviolet radiation-B: Model for active caspase-3 expression after in vivo exposure to UVR-300 nm
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, e106926- p.Article in journal (Refereed) Published
Abstract [en]

Purpose: To introduce a model for the time evolution of active caspase-3 protein expression in albino rat lens up to 24 hours after in vivo exposure to low dose UVR in the 300 nm wavelength region (UVR-300 nm).

Methods: Forty Sprague-Dawley rats were unilaterally exposed in vivo to 1 kJ/m2 UVR-300 nm for 15 minutes. At 0.5, 8, 16, and 24 hours after the UVR exposure, the exposed and contralateral not-exposed lenses were removed and processed for immunohistochemistry. The differences in the probability of active caspase-3 expression at four different time points after exposure were used to determine the time evolution of active caspase-3 expression. A logistic model was introduced for the expression of active caspase-3. The parameters for the exposed and the not exposed lenses were estimated for the observation time points.

Results: The exposure to UVR-300 nm impacted on the parameters of the logistic model. Further, the parameters of the model varied with time after exposure to UVR-300 nm.

Conclusion: The logistic model predicts the impact of exposure to UVR-300 nm on the spatial distribution of probability of active caspase-3 protein expression, depending on time.

Keyword
ultraviolet radiation, caspase-3, spatial distribution, lens, time evolution, modelling
National Category
Ophthalmology
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-229465 (URN)10.1371/journal.pone.0106926 (DOI)000343679800015 ()25244366 (PubMedID)
Available from: 2014-08-07 Created: 2014-08-07 Last updated: 2017-12-05Bibliographically approved
4. Objective automated quantification of fluorescence signal in histological sections of rat lens
Open this publication in new window or tab >>Objective automated quantification of fluorescence signal in histological sections of rat lens
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Purpose: To develop an automated method to delineate lens epithelial cells and to quantify expression of fluorescent signal of biomarkers in each nucleus and cytoplasm of lens epithelial cells in a histological section.

Methods: An automated algorithm was developed in Matlab™ to localize and quantify fluorescence signal in lens epithelial cells in histological images. A region of interest representing the lens epithelium was manually demarcated in each input image. Individual cell nuclei within the region of interest were automatically delineated based on watershed segmentation and thresholding. Fluorescence signal was quantified within nuclei and cytoplasms. The classification of fluorescence signal was based on local background. Classification of cells as labelled or not labelled was thereafter optimized as compared to visual classification of a limited dataset.

The performance of the automated classification was evaluated by asking eleven independent blinded observers to classify all cells (n=395) in one lens image. Time consumed by the automatic algorithm and visual /manual classification of nuclei, was recorded.

Results: On an average, 77 % of the cells were correctly classified as compared to the majority vote of the visual observers. The average agreement among visual observers was 83 %. However, variation among visual observers was high, and agreement between two visual observers was as low as 71 % in the worst case. Automated classification was on average 10 times faster than manual scoring.

Conclusion: The presented method enables objective and fast detection of lens epithelial cells and quantification of expression of fluorescent signal in a histological section of rat lens, with accuracy comparable to the variability between different visual observers. Furthermore, automated scoring is unbiased and reproducible, and results in a 10-fold increase in throughput.

Keyword
Automatic analysis, cell counting, image analysis, lens epithelium, fluorescence
National Category
Neurosciences
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-268312 (URN)
Available from: 2015-12-03 Created: 2015-12-03 Last updated: 2017-01-25Bibliographically approved

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