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Prevention of Experimental Cataract Induced by UVR
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cataract is the leading cause of blindness in the world and is defined by opacification of the normally transparent lens of the eye. The major avoidable cause of cataract is ultraviolet radiation (UVR), but no current strategies have been developed to prevent the onset of cataract. Apoptosis and internal and external antioxidant systems that inhibit apoptosis have been shown to play a significant role in cataractogenesis.

The main purposes of this thesis were to study the time evolution of apoptosis, to develop the concept of a protection factor (PF), and to investigate the effect of thioltransferase (Grx1) and topical caffeine in UVR cataract development. Further, to elucidate pharmacokinetics and influence on iris diameter of topical caffeine.

Sprague Dawley rats were exposed to UVR and TUNEL staining of the lens sections was analysed. Grx1+/+ and Grx1-/- mice were exposed to 5 sub-doses of UVR. Based on the difference of light scattering between Grx1+/+ and Grx1-/- mice, the concept of the PF was developed. Topical caffeine and a placebo were applied to the eyes of separate groups of Sprague Dawley rats that were exposed to sub-doses of UVR and protective effect was evaluated. Penetration of topical caffeine in Sprague Dawley rats to lens and blood was analysed by high performance liquid chromatography. Pupil diameter was measured in groups of unilaterally and bilaterally caffeine-treated ketamine/xylazine anesthetized Sprague Dawley rats.

TUNEL-labeling peaked between 5 and 120 hours after UVR exposure. The PF of Grx1 was 1.3. Moreover, topically administered caffeine protected against UVR-induced cataract development with a PF of 1.23. Topical caffeine peaked at 30 min in the lens, increased up to 120 min in the blood and antagonized ketamine/xylazine-induced mydriasis.

In conclusion, UVR induces apoptosis, which is evidenced by the peak of TUNEL-labeling at 24 hours after UVR exposure. The PF is an objective relative measure of protective properties that allows the comparison of different antioxidant systems and administered antioxidant substances. Grx1 and caffeine are protective against UVR-induced cataract. Topically administered caffeine penetrates to the lens and inhibits UVR-induced apoptosis. Additionally, a miotic effect of caffeine is described for the first time.

Place, publisher, year, edition, pages
Uppsala: Uppsala universitet, 2014. , 44 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1036
Keyword [en]
cataract, ultraviolet radiation, apoptosis, thioltransferase, caffeine
National Category
Ophthalmology
Identifiers
URN: urn:nbn:se:uu:diva-232955ISBN: 978-91-554-9055-3 (print)OAI: oai:DiVA.org:uu-232955DiVA: diva2:750299
Public defence
2014-11-28, Enghoffsalen, Ing. 50, plan 1., Akademiska Sjukhuset, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-11-06 Created: 2014-09-28 Last updated: 2015-01-23
List of papers
1. Evolution of TUNEL-labeling in the Rat Lens After In Vivo Exposure to Just Above Threshold Dose UVB
Open this publication in new window or tab >>Evolution of TUNEL-labeling in the Rat Lens After In Vivo Exposure to Just Above Threshold Dose UVB
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2013 (English)In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 38, no 8, 880-885 p.Article in journal (Refereed) Published
Abstract [en]

Purpose/Aim:

To quantitatively analyse the evolution of TUNEL-labeling, after in vivo exposure to UVB.

Methods:

Altogether, 16 Sprague Dawley rats were unilaterally exposed in vivo for 15 min to close to threshold dose, 5 kJ/m(2), of ultraviolet radiation in the 300nm wavelength region. Animals were sacrificed in groups of 4 at 1, 5, 24 and 120 h after exposure. For each animal, both eye globes were removed and frozen. The frozen eye was cryo-sectioned in 10 mm thick midsagittal sections. From each globe, three midsagittal sections with at least five sections interval in between were mounted on a microscope slide. Sections were TUNEL-labeled and counter stained with DAPI. For quantification of apoptosis, a fluorescence microscope was used. In sections with a continuous epithelial cell surface, the number of lens epithelial cell nuclei and the number of TUNEL-positive epithelial cell nuclei was counted. The total number of TUNEL-positive epithelial cell nuclei for all three sections of one lens in relation to the total number of epithelial cell nuclei for all three sections of the same lens was compared between exposed and contralateral not exposed lens for each animal.

Results:

The relative difference of the fraction of TUNEL-positive nuclei between exposed and contralateral not exposed lens increased gradually, peaked in the time interval 5-120 h after exposure, and then declined.

Conclusions:

Close to threshold dose of UVB induces TUNEL-labeling that peaks in the time window 5-120 h after exposure to UVB.

Keyword
Apoptosis, cataract, in vivo exposure, Sprague Dawley rats, ultraviolet radiation
National Category
Ophthalmology
Identifiers
urn:nbn:se:uu:diva-204768 (URN)10.3109/02713683.2013.783079 (DOI)000321010100009 ()
Available from: 2013-08-15 Created: 2013-08-12 Last updated: 2017-12-06Bibliographically approved
2. Protective Effect of The Thioltransferase Gene On In Vivo UVR-300 nm Induced Cataract: In vivo protection of Grx1 against UVR in the lens
Open this publication in new window or tab >>Protective Effect of The Thioltransferase Gene On In Vivo UVR-300 nm Induced Cataract: In vivo protection of Grx1 against UVR in the lens
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2012 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 53, no 1, 248-252 p.Article in journal (Refereed) Published
Abstract [en]

Purpose. To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1−/− and Grx1+/+ mice.

Methods. Twenty Grx1+/+ mice and 20 Grx1/ mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m2 UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography.

Results. UVR-300 nm induced subcapsular and cortical cataract in Grx1−/− and Grx1+/+ mice. In both Grx1−/− and Grx1+/+, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1−/− mice than in the lenses of Grx1+/+ mice. The threshold dose for in vivo UVR-300 nm–induced cataract, expressed as MTD2.3:16, was 3.8 in the Grx1+/+ group and 3.0 in the Grx1−/− group, resulting in a PF of 1.3.

Conclusions. The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.

National Category
Medical and Health Sciences
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-163795 (URN)10.1167/iovs.11-8504 (DOI)000302694500039 ()
Available from: 2011-12-14 Created: 2011-12-14 Last updated: 2017-12-08Bibliographically approved
3. Caffeine eye drops protect against UV-B cataract
Open this publication in new window or tab >>Caffeine eye drops protect against UV-B cataract
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2013 (English)In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 113, 26-31 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study was to investigate if topically applied caffeine protects against in vivo ultraviolet radiation cataract and if so, to estimate the protection factor. Three experiments were carried out. First, two groups of Sprague-Dawley rats were pre-treated with a single application of either placebo or caffeine eye drops in both eyes. All animals were then unilaterally exposed in vivo to 8 kJ/m(2) UV-B radiation for 15 min. One week later, the lens GSH levels were measured and the degree of cataract was quantified by measurement of in vitro lens light scattering. In the second experiment, placebo and caffeine pre-treated rats were divided in five UV-B radiation dose groups, receiving 0.0, 2.6, 3.7, 4.5 or 5.2 kJ/m(2) UV-B radiation in one eye. Lens light scattering was determined after one week. In the third experiment, placebo and caffeine pre-treated rats were UV-B-exposed and the presence of activated caspase-3 was visualized by immunohistochemistry. There was significantly less UV-B radiation cataract in the caffeine group than in the placebo group (95% confidence interval for mean difference in lens light scattering between the groups = 0.10 +/- 0.05 tEDC), and the protection factor for caffeine was 1.23. There was no difference in GSH levels between the placebo- and the caffeine group. There was more caspase-3 staining in UV-B-exposed lenses from the placebo group than in UV-B-exposed lenses from the caffeine group. Topically applied caffeine protects against ultraviolet radiation cataract, reducing lens sensitivity 1.23 times.

Keyword
caffeine, cataract, ultraviolet radiation, protection factor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-208371 (URN)10.1016/j.exer.2013.04.015 (DOI)000322931700004 ()
Available from: 2013-09-30 Created: 2013-09-30 Last updated: 2017-12-06Bibliographically approved
4. Pharmacokinetics for topically applied caffeine in the rat
Open this publication in new window or tab >>Pharmacokinetics for topically applied caffeine in the rat
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2014 (English)In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 122, 94-101 p.Article in journal (Refereed) Published
Abstract [en]

Topically applied caffeine was recently identified as a promising candidate molecule for cataract prevention. Little is known about the pharmacokinetics for topically applied caffeine. Potential toxicity of 72 mM caffeine on the ocular surface and the lens was qualitatively monitored and no toxic effects were observed. The concentration of caffeine was measured in the lens and the blood after topical application of 72 mM caffeine to groups of 10 animals sacrificed at 30, 60, 90 and 120 min after topical application. The lens concentration decreased throughout the observation period while the blood concentration increased up to 120 min. Further, the concentration of caffeine in the lens and blood was measured 30 min after topical application of caffeine, the concentration of caffeine being 0.72, 3.34, 15.51 and 72 mM depending on group belonging, in groups of 10 animals. The caffeine concentration in lens and blood, respectively, increased proportionally to the caffeine concentration topically applied. The rat blood concentrations achieved were far below the equivalent threshold dose of FDA recommended daily dose for humans. This information is important for further development of caffeine eye drops for cataract prevention.

National Category
Ophthalmology
Identifiers
urn:nbn:se:uu:diva-224891 (URN)10.1016/j.exer.2014.03.009 (DOI)000336009500012 ()
Available from: 2014-05-22 Created: 2014-05-22 Last updated: 2017-12-05Bibliographically approved
5. Topically applied caffeine induces miosis in the ketamine/xylazine anesthetized rat
Open this publication in new window or tab >>Topically applied caffeine induces miosis in the ketamine/xylazine anesthetized rat
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2014 (English)In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 127, 179-183 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to examine if topically applied caffeine influences pupil size in ketamine/xylazine anesthetized animals. Two experiments were carried out. In the first experiment, caffeine was topically applied to one of the eyes of 10 ketamine/xylazine anesthetized animals, while vehicle only was topically applied to the contralateral eye. In the second experiment, caffeine was topically applied to both eyes in one group of 10 ketamine/xylazine anesthetized rats, while in another group both eyes vehicle only was topically applied to both eyes. In both experiments pupil diameter was measured at 0, 10, 20, 40 and 60 min after topical application. In three of the animals, the pupil was dilated with tropicamide 5 mg/ml at 60 min after the topical application of caffeine and the pupil diameter was measured. The first experiment showed a relative miosis in caffeine treated eyes as compared to the vehicle treated eye, that changed over time. The second experiment in line with the first experiment, also showed that topically applied caffeine causes a relative miosis as compared to vehicle only that changes over time. Eyes treated with caffeine reacted with quick dilatation after tropicamide application. Topical caffeine antagonizes ketamine/xylazine anesthesia induced mydriasis in a time dependent manner.

National Category
Ophthalmology
Identifiers
urn:nbn:se:uu:diva-229926 (URN)10.1016/j.exer.2014.07.023 (DOI)000342539900021 ()
Available from: 2014-08-18 Created: 2014-08-18 Last updated: 2017-12-05Bibliographically approved

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